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Fractionation of nitrogen isotopes during early diagenesis
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Fractionation of nitrogen isotopes during early diagenesis
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FRACTIONATION OF NITROGEN ISOTOPES DURING EARLY DIAGENESIS by Maria Prokopenko A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements of the Degree DOCTOR OF PHILOSOPHY (GEOLOGICAL SCIENCES) December 2004 Copyright 2004 Maria Genrikh Prokopenko Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3155464 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3155464 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS The work is done and it is a surprisingly sad feeling. My travels with nitrogen isotopes through the pore waters in the labyrinths of marine sediments have been adventurous, sometimes tumultuous, but always thrilling. The person, who fearlessly led me through these “muddy” waters, taming differential equations along the way, so that they, unable to fight any longer, were giving away their secrets, was my advisor, Dr. Doug Hammond, whom I give my most grateful acknowledgement. In addition to the fundamentals of marine and stable isotope geochemistry, as well as oceanography, he taught me the very fundamentals of scientific research, from formulating a hypothesis and preparing for cruises to hunting out the most probable explanation of the most puzzling observations and finally assembling my own point of view on a problem. I also thank him for his endless support, for being always on my side, for patiently guiding me from questions to answers, and, often, back to questions. My committee deserves many thanks as well. I would like to thank Dr. Will Berelson for generosity with which he shared with me both theoretical and practical knowledge, world-class expertise on geochemical fluxes, as well as his good-natured humor in the cold room during cruises. His provocative questions, undoubtedly, made this dissertation better; his open-minded attitude encouraged many of my ideas to come to light. Dr. Lowell Stott is gratefully acknowledged for providing mass spectrometer facility and generously allocating the mass spec time for my work, as well helpful suggestions during the course of this research. I thank Drs. Robert Douglas and Donn Gorsline (who was not officially on my committee, but was always willing to help me) ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for sharing with me their extensive knowledge on the Gulf of California and complex sedimentation patterns in this basin, which provided the important context for a significant portion of this work. Dr. Doug Capone is gratefully acknowledged for very helpful discussions on the sedimentary nitrogen cycle, as well as providing equipment and lab space for amino acid HPLC analysis. The USC lab managers, Miguel Rincon and Troy Gunderson played probably, the most important role in this research. Without their help, advice and expertise with moody and grumpy mass spectrometers none of this work could have been done. I would especially like to thank Miguel for teaching me the basics mass spectrometry, which is indeed a very tricky business. Many other people helped me and contributed significantly to this work. Dr. Claire Mahaffey was extremely kind to spend significant amount of her time teaching me the HPLC technique, explaining the intricacies of DON and being my good friend. I am grateful to Gerry Smith for his invaluable help during cruises and loyalty to the “Queen of Mud”. Liz Walker, a summer REU student at USC helped substantially with amino acid work, and was a true joy to have in the lab. 1 would like to acknowledge the role of Dr. Ken Nealson for a very nice introduction into the mysterious world of geobiology and Dr. Madeiline Briskin for sparking my interest in this field. I am thankful to all the participants of ODP Leg 201 and 202 for collecting samples for me, and chief scientists of these legs: Drs. S. D’Hondt, B. Jorgensen, A. Mix and R. Tiedemann. My peers and friends at USC made these years enjoyable: Dorte Poulsen, Tonya Bunn, Steve Colbert, Nicole Fraser, Jian Ping and Madeline Worsnopp, Juliette Finzi. My dear friends in California: Diana Popa, Sangeeta iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fernandes, Katya Shelekhova, Seth Finnegan and Herb Tobias, and my family and friends in Moscow: Nelli Maximenko, Julia Romanova, Liza Orlova and Oksana Vlasenko, Genrikh and Edik Vainstein, your friendship and love carried me through many difficult times and gave me strength and courage. Finally, I thank my husband and best friend, Robert Gaines, for his everlasting love, support, tasty cooking, conversations which sparked a lot of good scientific ideas, and most importantly, his faith in me. Without him, I would not have done it. I gratefully acknowledge the financial support from ODP Schlanger Fellowship and Sonorsky Fellowship, as well NSF grants OCE-0136500 to Doug Hammond and NSF grant OCE-0002250 to Lowell Stott. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS ACKNOWLEDGEMENTS ii LIST OF TABLES xi LIST OF FIGURES xiii ABSTRACT xvi CHAPTER I: Introduction and background Nitrogen stable isotope ratio as a paleoceanographic proxy 1 Motivation for this study 2 Structure of the thesis 5 Background 7 Variations in 81 5 N observed in different environments and possible paleoceanographic interpretations; considerations of the diagenetic effect 7 Processes of bacterial remineralization of organic matter leading to diagenetic fractionation of nitrogen isotopes 11 Amino acid hydrolysis 11 Microbiological prospective: influence of newly formed bacterial biomass 11 Nitrogen in the sedimentary amino acids 15 Isotopic composition of pore water ammonium dissolved in the pore water 17 Factors which may preclude fractionation of 81 5 N during diagenesis 18 References 20 CHAPTER II: Nitrogen cycling in the sediments of Santa Barbara basin and Eastern Subtropical North Pacific - Isotopic evidence for chemosymbiosis between two lithotrophs: riding on a glider 21 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Introduction 27 Study area 30 Methods 32 Estimation of depth distribution of pore water sulfide at CALMEX stations 34 Santa Barbara Basin 34 Biogeochemistry and nitrogen isotopic composition of pore water ammonium and No rg 34 Diagenesis and 81 5 N of No rg 37 Eastern Subtropical North Pacific area: CALMEX sites 42 Biogeochemistry and 81 5 N of pore ammonium and No rg in the sediments of Mazatlan Margin 43 Isotopic composition of ammonium and stoichiometry of pore water 45 Pore water geochemistry and Thioploca metabolism 47 Mixing diagram 49 The 81 5 N of the ammonium fluxes 51 Proposed scenario: Chemosymbiosis between Thioploca and anaerobic ammonium oxidizing bacteria 54 Comparison between Santa Barbara and ESNP sites: sedimentary geochemistry, benthic microbial ecology and nitrogen cycling 57 Does the proposed symbiosis influence the 81 5 N of the oceanic nitrate? 60 Summary 62 Acknowledgements 63 References 64 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER III: Impact of long term diagenesis on §1 5 N of organic matter in marine sediments: ODP LEG 201 - Sites 1227 and 1230 70 Introduction 70 Methods 73 Site 1230 68 Site description and lithology 75 Results 77 Biogeochemistry of pore water 77 Solid phase composition; 81 5 N of pore water ammonium and sedimentary organic matter 77 Discussion 80 Diagenesis and nitrogen isotopic composition of pore water ammonium and sedimentary organic nitrogen 80 Approach I 82 Approach II 82 Site 1227 87 Site description and lithology 87 Results 88 Biogeochemistry of pore water 88 Elemental and nitrogen isotope composition of the sedimentary organic matter and pore water ammonium 90 Discussion 95 5 1 5 N and sources of pore water ammonium at Site 1227 95 Steady-state scenario 102 Non steady-state scenario 105 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Summary 107 Acknowledgements 109 References 110 CHAPTER IV: Impact of long term diagenesis on 81 5 N of organic matter in marine sediments (continued): ODP Leg 202 - Sites 1234,1235 and 1238 113 Introduction 113 Methods 114 Sites 1234 and 1235 - Central Chilean margin 119 Site description and lithology 119 Results 120 Biogeochemistry of the sediments and pore water at Sites 1234 and 1235 120 Isotopic composition of sedimentary organic matter and pore water ammonium 123 Discussion 127 Diagenesis of organic matter at Sites 1234 and 1235 127 Net loss of sedimentary nitrogen during diagenesis 129 Processes affecting the 81 5 N profiles of pore water ammonium 131 No evidence for isotopic fractionation 134 Site 1238 - Pacific equatorial waters 136 Site description, lithology and depositional history 136 Results 138 Geochemistry of sediments and pore water 138 viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 81 5 N of pore water ammonium and sedimentary organic nitrogen 141 Discussion 143 Interpretation of 81 5 N of pore water ammonium 143 Summary 148 Acknowledgements 150 References 151 CHAPTER V: Fractionation of nitrogen isotopes during incubation experiments in the presence of oxygen and under anoxic conditions 153 Introduction 153 Methods 155 Experimental set-up and sample processing 155 Analytical methods 156 THAA analysis 157 Hydrolysis 158 Fluorometric analysis 158 HPLC analysis 159 Results 160 Nitrogen inventory: Definitions and determination of various nitrogen pools 160 Initial distribution of nitrogen between different pools and 81 5 N of each pool 166 Changes in nitrogen pools through the course of the incubation 167 Anoxic incubations 167 ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Oxic incubations 169 Compositional changes in THAA 171 Pore water THAA 171 S edimentary THAA 171 81 5 N composition of the sediments and pore water ammonium 182 Discussion 185 Decomposition of different fractions of organic matter with variable lability 185 Isotopic fractionation associated with the decomposition of organic matter 189 Anaerobic decomposition - isotopic mass balance approach 189 Aerobic decomposition - isotopic fractionation in two different components 191 Processes leading to fractionation of nitrogen isotopes during organic matter decomposition 195 Summary 198 References 200 CHAPTER VI: Summary 203 BIBLIOGRAPHY 213 APPENDIX A: Methods of sample preparation 227 APPENDIX B: CALMEX-2001 cruise multicore and gravity core data 232 APPENDIX C: Wecoma-2003 cruise hydrocast, whole core squeezer, multicore and piston core data 241 APPENDIX D: Hydrolysis and HPLC analysis 251 x Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table II - 1 Station parameters. Table II - 2 Fluxes of ammonium, TCO2 and S1 5 N of the ammonium fluxes, Mazatlan margin. Table III - 1 Site 1230- Hole A (Leg 201) Sediments elemental composition, ammonium concentration and 51 5 N for sediments and pore water ammonium. Table III - 2 Parameters for modeling of ammonium profiles and modeling results, Sites 1230 and 1227. Table III - 3 Site 1227 - Hole A (Leg 201) Sediments elemental composition and 81 5 N for sediments and pore water ammonium. Table IV - 1 Ammonium concentrations measured at USC and shipboard analysis; effect of loss of ammonium during storage on 81 5 N. Table IV - 2 Site 1234, T N elemental composition, S 15N of ammonium and N 0rg. Table IV - 3 Site 1235, TN elemental composition, 81 5 N of ammonium and No rg . Table IV - 4 Summary for Site 1234 and 1235 from Leg 202 and Site 1230 from Leg 201. Table IV - 5 Site 1238, TN elemental composition, 81 5 N of ammonium and No rg . Table V - 1 Program for regulating amino acid elution time. Table V - 2 Average retention times for analyzed amino acids. Table V - 3 Initial composition of the slurry. Table V- 4 Incubation Series AN-I and OX-I. Table V - 5 N lost by washing with 2M KC1. Table V - 6 Concentrations of amino acids in AN-I and OX-I. Table V - 7 Mole fractions of THAA results in presented in Table V-6. Table V - 8 Isotopic composition of all measured nitrogen pools in AN-I, OX-I, AN-II and OX-II. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 9 Multi-G model parameters from fitting and observed distribution. Table V - 10 Mass balance calculations for AN-I series. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Fig. II - 1 Map of locations. 29 Fig. II - 2 Santa Barbara Basin: a) C/N ratios and concentrations of TOC and TN; b) Sedimentary and pore water 81 5 N, pore water ammonium concentrations. 35 Fig. II - 3 TC02/NH4+ stoichiometry in the pore water of Santa Barbara Basin and Mazatlan Margin. 39 Fig. II - 4 Site NH-29, Mazatlan Margin: a) C/N ratios and concentrations of TOC and TN in the sediments; b) Sedimentary and pore water S1 5 N, pore water ammonium concentrations. 44 Fig. II - 5 Profiles of 81 5 N of ammonium at all six CALMEX sites. 46 Fig. II - 6 TC02/NH4+ stoichiometry of pore water at all six CALMEX sites. 48 Fig. II - 7 Mixing diagram of ammonium isotopic composition. 50 Fig. II - 8 Isotopic composition of ammonium fluxes in the pore water of the Mazatlan margin sediments. 52 Fig. II - 9 Diagram of the proposed symbiosis between Thioploca and Anammox bacteria. 56 Fig. 11-10 Profiles of dissolved iron and manganese and 51 5 N of ammonium in the pore water of Mazatlan sediments. 58 Fig. Ill -1 Map of locations, ODP Leg 201, Sites 1227 and 1230. 76 Fig. Ill - 2 TOC and TN profiles in the sediments of Site 1230. 79 Fig. Ill - 3 Ammonium profile, 51 5 N of ammonium and bulk sediments, Site 1230. 81 Fig. Ill - 4 Site 1230, upper 119 mbsf - Results of modeling: Modeled and measured N wt % for the sediments. 86 Fig. Ill - 5 Pore water sulfate profile at Site 1227 (D’Hondt et al., 2003). 89 Fig. Ill - 6 Isotopic composition of pore water ammonium, bulk sediments and ammonium concentrations, Site 1227. 91 xm Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. Ill - 7 Concentrations of pore water chloride and ammonium, Site 1227. 92 Fig. Ill - 8 TOC and TN of the sediments, Site 1227. 94 Fig. Ill - 9 Ammonium vs. chloride concentrations in the pore water of Site 1227. 97 Fig. Ill - 10 Site 1227 Schematic representation of ammonium fluxes and 81 5 N of the fluxes (in pmol/cm2 yr), based on the assumption of steady state. 99 Fig. Ill -11 Site 1227. Mixing diagram. 101 Fig. Ill - 12 Site 1227, upper 36. 95 mbsf, results of modeling: modeled and measured 81 5 N of sedimentary organic matter, measured profile of N wt %, and calculated fraction of sedimentary nitrogen left after degradation. 104 Fig. IV - 1 Location map of Sites 1234, 1235 and 1238. 115 Fig. IV - 2 Storage effect on the isotopic composition of pore water ammonium for Site 1238. 118 Fig. IV - 3 Elemental composition of No rg (wt %) for Leg 202, Site 1234 and 1235 121 Fig. IV - 4 Site 1234 (a) and Site 1235 (b), pore water ammonium profile and isotopic composition of pore water ammonium and No rg . 126 Fig. IV - 5 The effect of advection on the depth profile of 51 5 N of pore water ammonium at Site 1234. 135 Fig. IV - 6 Site 1238 geochemistry: a) TN wt % concentrations in the sediments; b) depth profiles of sulfate and alkalinity, (Mix et al., 2003); c) pore water ammonium profile (USC measurements) and isotopic composition of pore water ammonium and No rg . 139 Fig. IV - 7 Mixing diagram for Site 1238 81 5 N vs. 1/[NH4+ ] 145 Fig. V - 1 A schematic representation of the nitrogen pools present in each sample. 163 xiv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. V - 2 Amount of N H / removed by five sequential washes of 0.38 g (dry weight) of sediments, which originally were in contact with 1 mM ammonium solution. 165 Fig. V - 3 Mole fractions of amino acids; a) in AN-I pore water; b) in OX-I pore water. 177 Fig. V - 4 Mole fractions of amino acids a) in AN-I sediments; b) in OX-I sediments. 179 Fig. V - 5 81 5 N of the Norg-total in AN-I and OX-I series. 183 Fig. V - 6 Kinetic of PON decomposition in a) anoxic and b) oxic incubations. 186 Fig. V - 7 Isotopic composition of PON measured and as isotopic mass balance lorg-plankton a n d N org.sed. between the 81 5N of the N o rL ,-niankton and N 0ro-sed. 193 xv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT The nitrogen isotope composition (S1 5 N) of sedimentary organic matter (No rg ) is a powerful paleoceanographic proxy. However, the processes of early diagenesis may impact the 81 5 N of preserved organic matter. This work addressed the mechanism of diagenetic fractionation of 81 5 N in anoxic marine sediments by constructing isotopic mass balances for the pore water ammonium and No rg 51 5 N on a variety of time scales, from hundreds to millions of years. If diagenesis of organic matter is the only process affecting ammonium concentration and its isotopic composition, the S1 5 N of ammonium should reflect the isotopic ratio of the No rg plus a fractionation factor, if fractionation takes place. Field studies were conducted in the Santa Barbara Basin, in the Eastern Subtropical North Pacific (ESNP) and along the Peru-Chile margin. Short time diagenesis in situ was examined in the sediments of Santa Barbara Basin. We found ammonium to be about 2 .5 %o heavier than the bulk No rg - The most likely explanation for this difference is that No rg consists of multiple fractions of organic matter with variable lability, probably a mixture of light terrestrial and light marine nitrogen. The most labile fraction must be 2 .5 %o heavier than the bulk No rg. However, because of the rapid sedimentation rate in Santa Barbara basin, only a small fraction of N org (-15%) was lost to degradation, so the diagenetic change isotopic composition of Norg was < 0 .5 %o. Sediment from the Peru-Chile margin, collected during ODP Legs 201 and 202, was used to evaluate the diagenetic impact on a time scale of millennia to millions of years. In rapidly accumulating anoxic coastal sediments that contain xvi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. organic matter that is primarily marine and has had constant §1 5 N through time, the diagenetic fractionation was less than 1 %o. Several other ODP sites are strongly influenced by non-steady state conditions, and the isotopic composition of pore water ammonium primarily reflects changes in 81 5 N of the source organic matter through time. The results of the field studies demonstrated that under anoxic conditions, no isotopic fractionation was observed during decomposition of isotopically homogeneous No rg. However, if isotopically distinct components are present and differ in their labilities, preferential degradation of the more labile fraction may alter the 51 5 N of the Norg. However, if only a small fraction of No rg is decomposed, the downcore diagenetic changes in its isotopic composition are insignificant. Laboratory incubation experiments confirmed the field observations for the anoxic organic matter degradation. By contrast, experimental results in the presence of oxygen showed that degradation of organic matter leads to enrichment of 1 5 N in the preserved No rg . The degradation process involves release of DON (Dissolved Organic Nitrogen) from organic matter and subsequent hydrolysis and deamination of DON. Fractionation, most likely, occurs at the deamination step, leaving DON heavy. Aerobic heterotrophic bacteria appear to incorporate a portion of this isotopically enriched DON pool, perhaps fractionating 81 5 N during this step as well. Anaerobic bacteria are not able to assimilate DON in the same way as aerobic bacteria do. Therefore, this type of fractionation of 81 5 N occurs only during oxic decomposition of No rg . The final conclusion of this study is that there appears to be two mechanisms of fractionation of xvii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the bulk sedimentary S1 5 N: 1) “component level” fractionation, when preferential degradation of isotopically distinct more labile fraction alters the 81 5 N of residual bulk organic matter ; 2) and “molecular level” fractionation, when isotopically heavier fraction of DON is preferentially retained by in situ growing bacteria. The first type of fractionation can occur in both oxic and anoxic settings. Due to metabolic difference between aerobic and anaerobic bacterial communities, which are involved in organic matter degradation, the second type of fractionation takes place only in the presence of oxygen. Unusual enrichment in 1 5 N of the pore water ammonium was observed in the upper 30 cm of sediments of Eastern Subtropical Pacific. Based on the field observations and the results of reaction-diffusion modeling, the pattern was interpreted as possible evidence for a novel type of chemosymbios between two chemolithotrophic bacteria, Thioploca and Anammox-like bacteria. The proposed chemosymbiosis may significantly impact sedimentary nitrogen cycling on large regional scales. These results provided new insights into the ecogeochemical role of benthic microbial communities in global biogeochemical cycles. xviii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I: Introduction and background Nitrogen stable isotope ratio as a paleoceanographic proxy Nitrogen plays a crucial role in the global ecosystem, as a vital constituent of all living organisms. As one of the limiting nutrients (Gruber, in press; Smith, 1984; Tyrell, 1999), it plays an important role in regulating global primary productivity in the oceans, which has been recognized as one of the factors regulating atmospheric C02, and consequently the climate of the planet (McElroy, 1983). Several different mechanisms linking the nitrogen cycle and atmospheric CO2 concentrations have been proposed. Me Elroy (1983) suggested that erosion from the continental shelves during glacial times made sedimentary nitrogen available to primary producers, thus increasing global productivity and contributing to the CO2 drawdown. An increase in N2 fixation during glacial periods has been suggested as another mechanism, which stimulated primary productivity during glacial times (Broecker and Henderson, 1998; Falkowski, 1997) and similarly lead to reduced CO2. Ganeshram et al. (2000) and Altabet et al. (2002), on the other hand, argued that the decrease in denitrification made more fixed nitrogen available to phytoplankton in the oceans, and led to enhanced sequestration of CO2 from the atmosphere during glacial times. The studies of the response of the nitrogen cycle to environmental changes through time are based, primarily, on the investigations of nitrogen isotope ratios, 81 5 N, of organic matter preserved in marine sediments. The 81 5 N of marine plankton often reflects oceanographic and nutrient regimes where the plankton biomass has been formed, and therefore is a powerful paleoceanographic proxy. Non-diazotrophic 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phytoplankton use nitrate as the main nitrogen source in the oceans. If utilization of nitrate is complete, the plankton acquires the nitrate isotopic signature. Thus, variations in 81 5 N of sedimentary organic matter can provide information about denitrification history through time. In settings where nitrate is highly depleted in the photic zone, the 51 5 N of phytoplankton also depends on the relative importance of various modes of incorporating nitrogen into the biomass, for instance, nitrate assimilation versus N 2 fixation (Capone et al., 1997; Haug et al., 1998), Capone since diazotrophic organisms have 51 5 N close to that of the atmospheric N2, which is 0% o. The most important process that influences the 51 5 N of the oceanic nitrate is water column denitrification, which is accompanied by a large isotopic fractionation of -20 % o (Brandes et al., 1998; Cline and Kaplan, 1975). If nitrate is only partially consumed, then, as suggested by Altabet (1991) and Francois et al. (1992), the 81 5 N of the plankton may reflect changes in the degree of nutrient utilization, which may occur as a consequence of changes in primary production in the oceans. Motivation for this study Rather than being a passive record keeper of changes in the biogeochemistry of the overlying waters, marine sediments are also an important participant in the cycling of N. Organic matter deposited on the ocean floor passes through various diagenetic zones, where it is subjected to the attack of diverse microbial communities, which feed and respire by decomposing organic compounds. Bacterially mediated early diagenesis of N-containing organic matter involves hydrolysis of the original proteins through the transformation and/or break down of 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. amino acids to ammonium (Burdige and Martens, 1988; Cowie et al., 1992; Henrichs and Farrington, 1987). In anoxic settings, the ammonium produced is largely lost from the sediments through diffusion, may be oxidized by the Anammox reaction, adsorbed on mineral surfaces, or used to form bacterial biomass and “geopolymers”. In the presence of oxygen, ammonium is also lost via aerobic ammonium oxidation A critical question is whether the process of early diagenesis alters the nitrogen isotopic ratio of preserved organic matter, and if it does, which factors control the magnitude of the diagenetically induced fractionation of 51 5 N. This problem may be of particular importance in environments where diagenetic conditions vary temporarily, such as in semi-enclosed basins, where the conditions on the sea floor fluctuate between oxygenated and anoxic, potentially imposing a variable diagenetic overprint on the 51 5 N of preserved organic matter downcore. Sigman et al. (1999), working with the sediments from the Southern Ocean, and Sachs (1997) and Sachs and Rapeta (1999), in the sapropels from Mediterranean Sea, found evidence of diagenetic alteration of 51 5 N in bulk sediment: a 2 to 5 %o positive shift relative to unaltered organic matter. On the other hand, Altabet et al. (1999) and Pride et al. (1999) studied the nitrogen isotopic composition of organic matter in California Borderland basins and in the Gulf of California, concluding that early diagenesis did not affect the bulk the 51 5 N of these sediments. The observed disparity in the apparent diagenetic effects has been attributed to the differences in the degree of oxygenation of the bottom waters, which in turn might lead to variations in the degree of organic matter preservation (Sachs and 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rapeta, 1999). However, it has been previously demonstrated (Henrichs, 1993; Hedges and Keil, 1995) that no clear relationship exists between the degree of organic matter preservation and bottom water oxygen concentrations. Altabet et al. (1999) suggested that diagenetic fractionation of nitrogen isotopes in the sediments occurs in regions with a low level of supply of organic material to the sea floor, while in highly productive regions the isotopic signal is preserved. However, the question of what factors control the occurrence and magnitude of diagenetic fractionation of 81 5 N in the sediments remains unresolved. In this study, the following questions were addressed: What diagenetic factors control the extent of nitrogen isotope fractionation during early diagenesis in marine sediments? Under which geochemical conditions does the bulk isotopic ratio in the marine sediments represent the 81 5 N of primary production, and what conditions lead to changes in this ratio? The approach employed here is to compare the isotopic composition of pore water ammonium released during diagenesis to that of the preserved organic material. This requires construction of isotopic mass balances for preserved organic matter and pore water ammonium, the final product of organic matter decomposition. This study includes results obtained in the field, where the effect of diagenesis was investigated in the natural setting. The in situ fractionation has been examined on diverse time scales. Measurements on the sediments and pore water from the Gulf of California, along the west coast of Baja California, and from Santa Barbara basin, investigated time scales of a few thousand years. For longer time scales, spanning 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hundreds of thousand to million years, material obtained during ODP Legs 201 and 202 from the Peru-Chilean margin has been used. The fractionation of 81 5 N has been also examined in an incubation experiment, where a mixture of fresh plankton and surface sediments were incubated for five months under aerobic and anaerobic conditions. Structure of the thesis One condition for successful application of the mass balance approach is that the isotopic composition of ammonium at depth is not affected by processes other than organic matter decomposition. Since the sediments investigated in this study were all characterized by anoxic diagenesis, this assumption appeared to be valid, based on previous research. However, the results of this work show that 81 5 N of the pore water ammonium from the sites in the Gulf of California is also affected by anaerobic ammonium oxidation to depth of 15-30 cm with nitrate supplied by Thioploca spp. Chapter II compares settings where this factor varies in importance: the Gulf of California, where this process was discovered, and Santa Barbara Basin, where ammonium is not oxidized. This chapter develops an argument for symbiosis between Thioploca and Anammox-like bacteria on the basis of 81 5 N of pore water ammonium, and evaluates the impact of this process on the isotopic composition of the water column nitrate in the region. Where the Thioploca is absent, in Santa Barbara Basin the effect of anoxic diagenesis on 81 5 N of preserved organic matter was evaluated on the time scale of few thousand years. The Santa Barbara data 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. indicate 2 to 3 % o fractionation between pore water ammonium and preserved organic matter, with heavy ammonium released preferentially. Chapter III compares results from two near shore sites on the Peru margin, 1227 and 1230 (ODP Leg 201). The analysis of the data from Site 1230 suggests less than 1 % o diagenetic impact on 81 5 N of the organic matter, despite a 30 % loss of organic nitrogen to decomposition. Interpretation of the results from Site 1227 is complicated by the presence of subsurface brine supplying ammonium to the pore fluids, and the possible presence of non-steady state conditions. The consequences of these two factors are considered. The possibility of a small diagenetic fractionation in the sediments of Site 1227 is discussed. Chapter IV presents data on 81 5 N of ammonium and sediments collected by ODP Leg 202 at three sites: two of them, 1234 and 1235 are continental margin localities, and one is a deep water site, 1238. The results from Sites 1234 and 1235, which are similar in their characteristics to Site 1230, show no evidence for isotopic fractionation associated with organic matter decomposition. The effect of sediment compaction on the structure of the depth profile of the ammonium 81 5 N is examined. The iso topic profile of the pore water ammonium at Site 1238 is currently not in steady state, and the consequence of this are discussed for this site. Chapter V discusses the results obtained in the incubation experiments, where 81 5 N of the organic matter decomposing under anaerobic and aerobic conditions was monitored for six months. The results show that two different mechanisms might lead to alteration of the S1 5 N of preserved organic matter. One is at work under either 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aerobic or anaerobic conditions, when sediments contain isotopically distinct fractions of organic matter that also differ in their lability. The other mechanism is associated only with decomposition of organic matter under aerobic conditions, and is related to the metabolic properties of aerobic heterotrophic bacterial communities, which degrade organic matter. Background Variations o f S1 5 N downcore observed in different environments and possible paleoceanographic interpretations; considerations o f the diagenetic effect In early studies of the natural abundances of stable isotopes, the observed variability of PON 81 5 N down core in marine sediments was explained as a result of changes in the relative contributions of terrestrial (isotopically light due to the N2 fixation) and marine (isotopically heavier due to the enriched in 1 5 N nitrate) organic matter (Wada et al., 1975, Cline and Kaplan, 1975). The progressive decrease in 81 5 N downcore has been interpreted as an indicator of increasing input of terrestrial organic matter. A new application of sedimentary 51 5 N records to paleoceanographic studies was pioneered by Altabet et al., (1991) and Francois et ah, (1992). In their search for possible mechanism linking changes in paleoproductivity in the water column of Southern ocean and fluctuations in atmospheric CO2 during the last glacial maximum , they suggested that the increase in the degree of nutrient utilization, associated with the increase in primary productivity, should be recorded as an increase in the sedimentary 61 5 N. 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Their argument was based on the results of diatom and bacterial culture studies (Wada and Hattori, 1 9 7 8 ; Mariotti, 1 9 8 1 ) . These studies experimentally demonstrated that the growth of a culture in a closed system, with nitrate as source of nitrogen, leads to Rayleigh fractionation. This process results in the increase of 8 15N in both the remaining nitrate and in newly formed biomass due to the preferential uptake of lighter nitrate by the organisms. Francois et al. ( 1 9 9 2 ) and Altabet and Francois ( 1 9 9 4 ) conducted a survey of 8 15N along the latitudinal transect from the highly productive Southern ocean towards oligotrophic regions of the Pacific. Their results conclusively showed that the process of biological utilization of nutrients in the modem oceans results in Rayleigh-type fractionation and is recorded by an increase of the sedimentary § 15N concurrent with the increase of the degree nutrient utilization. In this study, Francois et al. ( 1 9 9 2 ) reported an enrichment of 4 to 5 % of 8 15N in the sediments relative to particles from the surface water. But since the observed offset was constant, it did not affect their conclusions. Subsequently, 8 15N has become one of the major tools for deciphering the changes of paleo-productivity due to climatic change and shifts in ocean circulation. Sigman ( 1 9 9 7 ) and Sigman et al. ( 1 9 9 9 ) observed downcore enrichment in 8 15N in diatom-bound organic matter, which they interpreted as indicating an increase in the degree of nutrient utilization during the last glacial age. Comparison of bulk sediment 8 15N and that of diatom-bound organic matter protected from diagenesis by siliceous shells of diatoms, showed that the bulk 8 15N is 4 to 5%o was heavier. This 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. discrepancy was interpreted as a result of diagenetic alterations of unprotected organic material. In other settings, an increase in sedimentary 8 15N has been interpreted as an indication of increasing denitrification in the anoxic or suboxic water columns. Isotope fractionation during denitrification has a mechanism similar to assimilatory nitrate uptake, thus, the same principles are applied. Altabet et al. (1999 a, b) interpreted variations of 8 15N in the sediments of Arabian Sea and Eastern Northern Tropical Pacific region as a result of climatically linked fluctuations in water column denitrification. The absence of diagenetic alterations in the sediments was inferred from comparison between 8 15 N of sinking particles captured in the sedimentary traps through the water column and the surface sediments. Quite unexpected results were obtained by Sachs (1997) in his study of the origin of sapropel beds in the Eastern Mediterranean. It has been previously determined that bulk sediments of the non-sapropel layers have 8 15N of - 5% o, while in the sapropel layers the N isotopic ratio is about - 0.1 %o. Previous interpretation of the observed patterns invoked primary production in nutrient depleted conditions, as an explanation for heavier 8 15N in the non-sapropel layers compared to sapropel layers (Calvert et al., 1992). In his work, Sachs (1997) used preserved photosynthetic pigments as a proxy and demonstrated that 81 5 N of chlorophyll from both sapropel and non-sapropel beds is the same, about -5 %o. Through a series of incubation experiments, he showed that whole cell vs. chlorophyll fractionation factor (a) in the modem phytoplankton is 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. constant regardless of growth conditions, and is about -5 %o. These findings allowed him to conclude that 5 %o enrichment observed in 81 5 N of organic-poor layers relative to sapropel beds is of purely diagenetic origin. From the above discussion, it follows that the diagenetic alteration of 51 5 N has been documented in some environments, but not others. Sachs (1997) addressed this problem in his dissertation, compiling available data on the relationship between concentration of oxygen in the bottom waters and magnitude of diagenetic fractionation of nitrogen isotopes. According to Sachs and Rapeta (1999), the degree of diagenetically induced fractionation of 51 5 N in marine sediments is highest in well-oxygenated basins due more efficient decomposition of organic matter, when oxygen is used as an electron acceptor. However, this statement seems to be in contradiction with the findings of Altabet et al. (1999), which showed no diagenetic fractionation in both anoxic San Pedro basin and oxygenated Monterey Bay. It was also noted by Henrichs (1993) that there is no consistent relationship between oxygen availability and organic matter preservation. Another explanation proposed by Montoya et al. (1992), Altabet (1988), Altabet and Small (1990) involves the presence of isotopically heavy fecal material excreted by benthic macrofauna in the oxygenated environments. Altabet and McCarthy (1985) and Libes and Deuser (1988) hypothesized that heterotrophic bacteria preferentially remove 1 5 N-depleted N compounds, leaving behind 1 5 N enriched residual organic matter. 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Clearly, the very presence and magnitude of fractionation is likely to depend on a combination of factors influencing the decomposition of organic matter. Processes o f bacterial re-mineralization o f organic matter leading to diagenetic fractionation o f N isotopes Oxidation of organic matter involves a series of biogeochemical reactions, many of which have a potential to lead to the kinetic isotope fractionation between decomposing organic matter and products of these reactions (Macko, 1993). Amino acid hydrolysis One of the major reactions in degradation of labile proteinaceous organic matter is peptide bond rupture by hydrolysis. Silfer et al. ( 1 9 9 2 ) experimentally demonstrated that the thermally induced hydrolysis of the peptide bonds in glycylglycine (polymer of glycine) lead to 2 to 4 %o enrichment of N isotopic ratio in the residual polymer and release of isotopically lighter glycine. Macko ( 1 9 9 4 ) incubated a mixture of fresh seagrass and marine sediments for 4 weeks and observed 2 .5 %o enrichment in the residual material relative to the starting isotopic ratio. He attributed the observed shift to both peptide bond hydrolysis "with preferential bond rupture or loss of 1 4 N-enriched material". Microbiolosical prospective: influence o f newly formed bacterial/archeal biomass Decomposition of organic matter in marine sediments is performed by an array of microbial communities. Consortia of bacteria and archea use a wide variety of electron acceptors such as O2, N O ‘3 , Mn and Fe oxides and hydroxides, SO' 4 and finally CO2 in methanogenesis, (Claypool and Kaplan, 1974, Froelich et al.,1979). 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Newly formed bacterial biomass produced in the process of degradation of organic matter, if present in large amounts, might substantially alter the isotopic composition of sedimentary organic matter. Conflicting points of view exist presently on the contribution of bacterial biomass to the sedimentary organic matter. Hartgers et al. (1994) concluded on the basis of geochemical studies of sediments and oils from Western Canada and Williston basins that bacterial biomass is only a minor contribution to the sedimentary organic matter. On the other hand, microbiological studies in the sediments of the Peru margin have detected the presence of viable bacteria up to 80 meters below sea floor, mbsf (Cragg et al., 1990; Parkes et al., 1993). In the upper few meters, total count of bacteria (using an acridine orange technique) in these sediments was on the order of 1 0 9 cell/cm3. The viable cell count, determined on the basis of [H3 ]-methyl thymidine Q -5 incorporation rates, was on the order of 1 0 cell/cm , exponentially decreasing with depth. Integrating over time the bacterial carbon production rates, and taking into account sediment accumulation rates, Parkes et al. (1993) have estimated that bacterial “biomass/ necromass” might account for up to 10-16% of “uncharacterized” organic matter. The “uncharacterized” organic matter (which cannot be classified either as proteins, sugars or lipids) contributes from 20 % to 79 % of total organic carbon, increasing with depth. Therefore, according to these authors, bacterial biomass can represent between as much as 2 to 10 % o of total organic carbon at depth. These authors also reported that changes in 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. “uncharacterized” organic matter correlated well with calculated increase of bacterial necromass, confirming that part of this organic matter may contain bacterial cellular material. Even though the numbers quoted above were approximated on the basis of many assumptions (Parkes, et al., 1993), they have profound implications for the potential influence of bacterial populations 8 1 5 N on the isotope ratio of organic matter preserved in the marine sediments. Bacterial communities in the sediments use ammonium as a source of nitrogen and amino acids as sources of carbon and nitrogen. Incorporation of N-containing compounds into bacterial biomass is an enzymatically regulated process and thus, might lead to a kinetic fractionation of nitrogen isotopes between newly formed amino acids and the original sources of nitrogen. Comprehensive laboratory studies of N isotopes fractionation associated with bacterial metabolism were conducted by Macko and co-workers (Macko and Estep, 1984; Macko et al., 1983; 1987; 1994). The underlying philosophy of all these studies was that the magnitude of fractionation is controlled by ’’ key rate- determining steps and major branching points in the flow o f ... nitrogen” (Macko, et al., 1983). The growth of bacterial biomass involves production of new proteins. In the case of heterotrophic bacteria, this includes assimilation of available amino acids from decomposing organic matter, separation and transfer of the amino groups 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (deamination and transamination) during the process of biosynthesis of the new amino acids, and excretion of excessive NH4 + from the cells. Macko et al. (1987) experimentally showed that cyanobacterium Anabaena sp. (an autotrophic organism) grown on ammonium or nitrate as a nitrogen source produced biomass up to 13 %o depleted compared to the original NH4 + or nitrate (Macko, 1987). It has been also demonstrated (Goericke et al., 1994) that assimilation of NH4 + during chemosynthesis (autotrophic growth) produces biomass strongly depleted in 1 5 N (from - 9 %o to - 4 % o relative to the source). These processes may be reflected in 8 1 5 N of pore water ammonia, as well as in 8 I5 N of bulk organic matter in the sediments, if enough bacteria biomass is accumulated. Working with the heterotrophic strain Vibrio harveyii, Macko et al. ( 1 9 8 4 ) found that the magnitude and direction of fractionation of N isotopes between source amino acids and new bacterial biomass varies significantly, and depends on a particular biosynthetic pathway of incorporation of various amino acids. For example, when the cells were grown on glutamate, the observed fractionation A 15N W hoie ceii-giutamic acid was + 2 2 %o. A t the same time cells grown on alanine were 9 %o lighter than the amino acid. Macko et al. ( 1 9 8 4 ) hypothesized that the strong depletion in 1 5 N observed when alanine was used as a substrate could result from a combination of discrimination against heavy isotopes during the amino acids uptake by cells, deamination, and utilization of 15N-depleted ammonium released during deamination. The strong enrichment in 1 5 N in cells growing on glutamate might 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. results from expelling of excess 15N-depleted ammonia, released during deamination of glutamate. Macko et al. (1986, 1987) also found that strong and variable fractionation occurs between biosynthesized amino acids and glutamic acid, a common precursor in biosynthesis, which results in a significant 51 5 N difference between different amino acids. This fractionation occurs during transamination, one of the major reactions of the amino acid biosynthesis. Macko et al. (1986) and McClelland and Montoya (2002) experimentally showed that enzymatically mediated transfer of the NH2 group from glutamic acid to aspartic acid happens 1.007 to 1.0083 times faster for 14NH2 than for 15NH2. Nitrosen in the sedimentary amino acids About 80% of the organic nitrogen present in plankton is in the form of amino acids. After organisms die and become sinking particles, this percentage rapidly decreases. By the time particles are incorporated in the sediments, amino acid-bound nitrogen contributes about 30 to 40 % of total organic nitrogen (Keil et al., 2000; Cowie and Hedges, 1994; Burdige and Martens, 1988). Degradation of amino acids continues in the sediments, at rates higher then for bulk organic matter, because amino acids represent the most labile fraction of sedimentary organic material (Westrich and Berner, 1984; Burdige and Martens, 1988). Extensive studies of decomposition of amino acids during early diagenesis in Cape Lookout Bight (Burdige and Martens, 1988), in the fjords of Norway (Haugen and Lichtentaler, 1991), in Saanich Inlet (Cowie et al., 1992), sediments of Buzzards Bay (Henrichs 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and Farrington, 1987), and many others, demonstrated that 20 to 80 % of N loss during early diagenesis of organic matter is attributed to the degradation of amino acids by bacterial activity. Amino acid degradation includes structural changes, such as conversion to non-protein amino acids (P-aminoglutaric acid, a-aminobutaric acid, a-amino adiptic acid, y-amino butaric acid), possibly condensation reactions of amino acids with carbohydrates to form "geopolymers" melanoids (Millaird reaction), incorporation of amino acids into bacterial biomass and terminal degradation with formation of CO2, N H / and CH4 . It has been observed almost ubiquitously that different classes of amino acids differ substantially in their rates of degradation, with acidic amino acids such as glutamic and aspartic acids being degraded much faster than neutral and basic amino acids. Mole fractions of lysine, serine and alanine often remain constant with depth, and significant increase in glycine has been reported in many cases (for instance, Cowie et al., 1992; Burdige and Martens, 1988). Several explanations have been proposed for the commonly observed increase in glycine, such as preferential preservation of diatom cell walls, or formation of bacterial biomass enriched in peptidoglycan from bacteria cell walls (Keil et al., 2000). Differential preservation/degradation of various amino acids might affect overall changes in bulk sedimentary organic matter S15N, since amino acids account for the major portion of remineralized N. It is important to estimate in each individual case, how much nitrogen is lost from the sediments during early 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. diagenesis via terminal decomposition of organic matter and subsequent diffusion of NH4 + out of the sediments. Isotopic composition o f ammonia dissolved in the pore water If degradation of amino acids to CO2 and N H 4+ is accompanied by fractionation of nitrogen isotopes, the released ammonia should differ in its 8 15N from decomposing organic matter. Direct measurements of the nitrogen isotope ratio of pore water ammonium and comparison to the 8 15N of sedimentary organic matter potentially present a direct method to estimate the presence and magnitude of such fractionation. Very few studies have been reported on the isotopic composition of ammonia dissolved in the pore waters (Velinsky et al., 1991; Sweeney and Kaplan, 1980). The results of studies conducted by Velinsky et al. (1991) in the Great Marsh, Delaware and Framvaren fjord, Norway, indicate that ammonia iso topic composition in the upper 40 cm of the sediments is very close to the measured S 15N of the total particulate matter. Sweeney and Kaplan (1980) measured 8 I5N of dissolved ammonia and bulk sediments in Santa Barbara Basin within the upper 5 meters. They found the range of N isotope ratio in the bulk sediments to be between 3 and 9.4 % 0, with average of 6.8% o, while 8 15N of ammonium ranged from 8 to 12 % o with an average of 10.2 %o. Their interpretation of the observed difference between bulk sediments and N H 4+ was that the bulk sedimentary organic matter represents a mixture of terrestrial and marine components; terrestrially derived organic matter is very refractory, thus only marine end member is decomposing, to yield the heavier isotope values of dissolved ammonia. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, subsequent 8 15N measurements on material from the sediments traps in this basin indicate that the average isotopic composition of material reaching the sea floor in Santa Barbara Basin is 7.4% o (Emmer and Thunell, 2 0 0 0 ) . The raining material should be dominated by plankton, which suggests that their isotopic composition may be lighter than inferred by Sweeney and Kaplan ( 1 9 8 0 ) , so some fractionation may occur. On the basis of their analysis of nitrogen isotope composition of bulk sediments from ODP hole 8 9 3 - A , Emmer and Thunell ( 2 0 0 0 ) established that 8 15N was about 9%o during the last deglaciation (after the Younger Dryas event about 1 0 Kyr ago), and has gradually decreased to about 7%o by the present time. If the average isotopic composition of bulk sedimentary nitrogen is between 7 and 9 %o, the question is why the ammonium 8 15N is heavier than that of sedimentary matter? What part of sedimentary nitrogen is being converted to ammonium? What is the isotopic composition of this more labile organic fraction? What fraction of total nitrogen in the sediments is represented by this labile fraction and how significant is the isotopic shift in the bulk sedimentary nitrogen caused by degradation of the labile fraction? Factors which may preclude fractionation o f SI 5N during diagenesis Despite the multitude of processes which may lead to diagenetic alteration of original 61 5 N of preserved organic matter, it is possible that if only a small amount of No rg were lost to diagenesis, the residual fraction would retain the original isotopic composition. Altabet et al. (1999) and Pride et al. (1999) argued that in the 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. environments, where only a modest proportion of organic matter is decomposed, the original isotopic composition of No rg must be well preserved. As factors, contributing to the good preservation of original isotopic signal, these authors named rapid sediment accumulation rates, high concentration of organic matter and anoxic to suboxic bottom waters at depositional sites. All these factors usually have been known to ensure high degree of organic matter preservation in marine sediments. 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References Altabet, M.A. and Francois, R., 1994, Sedimentary nitrogen isotopic ration as a recorder for surface ocean nitrate utilization, Glob. Biochem. Cycles., 8 , 103- 116. Altabet, M.A. and McCarthy J.J., 1985, Temporal and spacial variations in the natural abundance of 1 5 N in PON from a warm -core ring, Deep Sea Research, 32 (7), 755-772. Altabet, M.A. and Small, L.F., 1990, Nitrogen isotopic ratios in fecal pellets produced by marine zooplankton, Geochimica et Cosmochimica Acta, 54, p. 155-163. 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The Gulf and Peninsular Province of the Califonias, AAPG Mem. 47, p. 555-568. Bemer, R.A., 1971, Principles of Chemical Sedimentology, McGraw-Hill Book Company, 240 p. Bemer, R.A., 1974, Kinetic fractionation for the early diagenesis of nitrogen, sulfur, phosphorus, and silicon in anoxic marine sediments, In: Goldberg, E.D. (ed)The Sea, v. 5, p. 427-450, John Wiley, New York. Brandes J.A. and Devol, A.H., 1997, Isotopic fractionation of oxygen and nitrogen in coastal marine sediments, Geochimica et Cosmochimica Acta, 61, 1793- 1801. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Brandes, J.A., Devol, A.H., Yoshinari, T., Jayakumar, D.A. and Naqvi, S.W.A., 1998. Iso topic composition of nitrate in central Arabian Sea and Eastern Tropical Pacific: A tracer of mixing and nitrogen cycles. Limnology and Oceanography, 43, 1680-1689. Broecker, W.S. and Henderson, G.M., 1998. The sequence of events surrounding Termination II and their implications for the cause of glacial-interglacial C02 changes. Paleoceanography, 13, 352-364. Burdige, D.J., 1989, The effect of sediment slurring on microbial processes, and the role of amino acids as substrates for sulfate reduction in anoxic marine sediments, Biogeochemistry, 8 , 1-23. Burdige, D.L. and Martens, C.S., 1988. Biogeochemical cycling in an organic rich coastal basin: 10. The role of amino acids in sedimentary carbon and nitrogen cycling. Geochimica et Cosmochimica Acta, 52: 1571-1584. Calvert, S.E., Nielsen, B. and Fontugne, M.R., 1992, Evidence from nitrogen isotope ratios for enhanced productivity during formation of eastern Mediterranean sapropels, Nature, 359,223-225. Christensen, C. J., Gorsline, D. S., Hammond, D. E., Lund, S.P., 1994, Non-annual laminations and expansion of anoxic basin-floor conditions in Santa Monica Basin, California borderland, over the past four centuries , Marine Geology, 116,399-418. Cline, J.D. and Kaplan, I.R., 1975. Isotopic fractionation of dissolved nitrate during denitrification in the Eastern Tropical Pacific ocean. Limnology and Oceanography, 17, 885-900. Conway, N.M., Kennicutt, M.C., Van Dover, C.L., 1994, Stable isotopes in the study of marine chemosynthetic based ecosystems, In: Lajtha, K., Michener, R.H., (eds), Stable isotopes in Ecology and Environmental Science. Blackwell, Oxford, p. 158-186. Capone, D.G., Zehr, J.P., Paerl, H.W., Bergman, B. and Carpenter, E.J., 1997. Trichodesmium, a globally significant marine cyanobacterium. Science, 276, p. 1221-1229 Cowie G.L. and Hedges, J.I., 1994, Biochemical indicators of diagenetic alteration in natural organic matter misture, Nature, 369, p. 304-307. 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cowie, G.L., Hedges, J.I. and Calvert, S.E., 1992, Sources and relative reactivities of amino acids, neutral sugars, and lignin in an intermittently anoxic marine environment, Geochimica and Cosmochimica Acta, 56,1963-1978. Cowie, G.L., Hedges, J.I. and Calvert, S.E., 1992. Sources and relative reactivities of amino acids, neutral sugars, and lignin in an intermittently anoxic marine environment. Geochimica and Cosmochimica Acta, 56, 1963-1978. Cragg, B.A., Parkes, R.J., Fry, J.C., Herbert, R.A., Wimpenny, J.W.T. and Getliff, J.M., 1990, Bacterial biomass and activity profiles within deep sediment layers, In: Suess, E., von Huene, R., et al., 1990, Proc. ODP, Sci. Results, 112: College Station, TX, (Ocean Drilling Program), 607-619. Dauwe, B. and Middleburg, J.J., 1998, Amino acids and hexoamines as indicators of organic matter degradation state in the North Sea sediments, Limnology and Oceanography, 43, 782-792. 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Gruber, N., in press. The marine nitrogen cycle. In: M. Follows, Oguz, T. (Editor), Carbon - climate interactions, NATO ASI series. John Wiley & Sons, New York. Hagadom, J.W., Stott, L.D., Sinha, A., Rincon, M., 1995, Geochemical and sedimentologic variations in inter-annually laminated sediments from Santa Monica Basin, Marine Geology, 125, p. 111-131. Hare, P.E., 1972, Ion exchange chromatography in lunar organic analyses, Space Life Science, 3, p. 354-359. Hartgers, W.A., Sinninghe Damste, JaapS., Requejo, A.G., Allan, J., Hayes, J.M. and Jan W. de Leeuw, 1994, Evidence for only minor contributions from bacteria to sedimentary organic carbon, Nature, 369, p. 224-227. Haug, G.H., Pederson, T.F., Sigman, D.M., Calvert, S.E., Nielsen, B., Peterson, L.C., 1998, Glacial/interglacial variations in production and nitrogen fixation in the Cariaco Basin during the last 580 K yr,: Paleoceanography, 13, p.427-432. 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Mariotti A., Germon J.C., Hubert P., Kaiser P., Letolle R., Tardieux P., 1981, Experimental determination of nitrogen kinetic isotope fractionation: some 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. principles; illustration for the denitrification and nitrification processes, Plant and Soil, 62,413-430. McClelland, J.W. and Montoya, J.P., 2002. Trophic relationships and the nitrogen isotopic composition of amino acids in plankton. Ecology, 83, p. 2173-2180. McElroy, M.B., 1983. Marine biological control on atmospheric C02 and climate. Nature, 302: 328-329. Montoya, J.P., Wiebe, P.H. and McCarthy, J.J., 1992, Natural abundance of 1 5 N in particulate nitrogen and zooplankton in the Gulf Stream region and warm- core ring 8 6 A, Deep-Sea Research, 39(Suppl.l), p. S363-S392. Pride, C., Thunell R., Sigman D., Keigwin, L., Altabet, M., Tappa E., 1999, Nitrogen isotopic variations in the Gulf of California since the last deglaciation: Response to global climate change, Paleoceanography, 14, 3, 397-409. Sachs, J.P. and Repeta, D.J., 1999, Oligotrophy and nitrogen fixation during Eastern Mediterranean sapropel events, Science, 286, 2485-2488. Sachs, J.P., 1997, Nitrogen isotopes in chlorophyll and the origin of Eastern Mediterranean sapropels, Ph.D. Thesis, MIT/WHOI, 97-15. Sigman, D, M, 1997, The role of biological production in Pleistocene Atmospheric Carbon Dioxide variations and the Nitrogen Isotope dynamic of the Southern Ocean, Ph.D. Thesis, MIT/WHOI, 97-28. Sigman, D.M., Altabet, M.A., Francois, R., McCorkle, C.M., and Gaillard, J.-F, 1999, The isotopic composition of diatom-bound nitrogen in the Southern Ocean sediments, Paleoceanography, 14, 118-134. Silfer, J.A., Engel, M.H., and Macko S. A., 1992, Kinetic fractionation of stable carbon and nitrogen isotopes during peptide bond hydrolysis: Experimental evidence and geochemical implications, Chemical Geology, 101, 211-221. Smith, S.V., 1984. Phosphorus versus nitrogen limitation in the marine environment. Limnology and Oceanography, 29: 1149-1160. Sweeney R.E. and Kaplan, I.R., 1980, Natural abundances of 1 5 N as a source indicator for near shore marine sedimentary and dissolved nitrogen, Marine Chemistry, 9, p. 81-94. 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thunell, R.C., 1998, Particle fluxes in coastal upwelling zone: sediment trap results from Santa Barbara Basin, California, Deep-Sea Research, Part II, 45, p. 1863-1884. Tyrell, T., 1999. The relative importance of nitrogen and phosphorus on oceanic primary production. Nature, 400(400): 525-531 Velinsky, D.J, 1991, Burdige, D.J., and Fogel, M.L., 1991, Nitrogen diagenesis in anoxic marine sediments: Isotope effect, Carnegie Inst. Washington Annu. Rep. Director 1991, p. 154-162. Wada , E., Kadonoga, and S. Matsuo, 1975,1 5 N abundance of in nitrogen of naturally occurring substances and global assessment of denitrification from isotopic point of view, Geochemical Journal, 9, p. 139-148. Wada E. and Hattori, A., 1978, Nitrogen Isotope effects in the assimilation of inorganic nitrogenous compounds by marine diatoms, Geomicrobiology Journal, 1, p. 85-101. 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER II: Nitrogen cycling in the sediments of Santa Barbara basin and Eastern Subtropical North Pacific - Isotopic evidence for chemosymbiosis between two lithotrophs: riding on a glider (to be submitted to Earth Planetary Science Letters) M.G. Prokopenko,* D.E. Hammond,* W.M. Berelson,* J.M. Bernhard, * L. Stott,* R. Douglas Introduction Recently developed global nitrogen budgets indicate that sedimentary denitrification removes from 95 (Gruber, in press; Gruber and Sarmiento, 2002), to 240 (Brandes and Devol, 2002) to 300 Tg/year (Codispoti et al., 2001) of fixed nitrogen from the oceans. It has been also argued (Brandes and D evol, 2002) that the nitrogen isotope balance in the oceans requires the sedimentary denitrification to be two to three times the magnitude of the water column denitrification. This makes sedimentary denitrification the most important single sink for the oceanic nitrate. However, a large uncertainty in the magnitude of this sink remains (Brandes and Devol, 2002). The challenge of accurate quantification of the contribution of this sink to the global nitrogen budget arises, in part, from the lack of detailed knowledge of the benthic microbial communities responsible for performing nitrogen transformations in marine sediments. Until recently, the only processes known to constitute sedimentary denitrification had been respiratory nitrate reduction (denitrification) and aerobic ammonium oxidation (nitrification) (Hattori, 1983; Kaplan et al., 1979; Nishio et al., 1982). This paradigm was challenged by a sedimentary nitrogen mass balance for San 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Clemente Basin sediments that indicated removal of nitrogen by reaction between ammonium and nitrate (Bender et al., 1989). This prediction was confirmed by Dalsgaard and Thamdrup (2002) and Thamdrup and Dalsgaard (2002), who found that in organic poor sediments underlying suboxic bottom waters, up to 67 % of N2 produced in the sediments is contributed by lithotrophic Anammox bacteria (Jetten et al., 1998; Strous et al., 1999; Vandegraaf et al., 1995), oxidizing ammonium with nitrite. Another bacterial process removing nitrate, performed by the lithotrophic gliding bacteria Thioploca, was discovered in the sediments underlying productive waters of the Oxygen Minimum Zone in the Peru upwelling region (Fossing, 1995; Gallardo, 1977; Zopfi, 2001). These bacteria reduce nitrate to ammonium (Fossing, 1995; Otte, 1999) using pore water hydrogen sulfide as an electron donor. According the estimates of Zopfi (2001), Thioploca is responsible for up to 50 % of the nitrate flux into the sediments of Peru margin. Whether this process contributes to denitrification depends on whether some of this ammonium escapes into the water column, or it is completely re-oxidized and subsequently denitrified within the sediments. In this paper, we present data on the budgets and isotopic composition of pore water ammonium from anoxic sediments of two regions, the Santa Barbara Basin and the Eastern Subtropical North Pacific (ESNP) area (Fig. II -1). We have found that the diagenetic processes affecting ammonium in these two regions are different. The isotopic composition of ammonium reflects these differences, with §1 5 N of ammonium in the ESNP sediments being significantly enriched in 1 5 N compared to that of the 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -120 -115 110 105 35 30 arrnen Soledad 25 Pescadero Magdalena Margin Mazatlan Margin 20 - 105 - 110 -115 -120 35 30 25 20 0 200 400 km Fig. II - 1 Map of locations. Symbols: black - sites discussed in this paper, grey: other CALMEX sites, white - ammonium flux is measured (Harnett and Devol, 2003), striped: site, where the 51 5 N of sedimentary denitrification was measured (Brandes and Devol, 2002). 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Santa Barbara Basin. We argue that the observed isotopic enrichment is due to chemosymbiosis between Thioploca spp. and Anammox bacteria in the sediments of ESNP. We consider specific geochemical factors which facilitate this symbiosis in the ESNP sediments, and discuss possible impact the proposed process has on the 8 1 5 N of nitrate in the water column of the ESNP. Study area Samples were collected at six sites in the Gulf of California and along the Mexican margin during the CALMEX cruise in November-December 2001 on the R/V New Horizon and in the Santa Barbara Basin in March 2003 on R/V Wecoma (Fig. II - 1, Table II - 1). CALMEX sites include two open margin locations, in the semi-silled Soledad Basin and on the Magdalena margin. Three sites lie within the Gulf of California: Alfonso Basin on the west side of the Gulf, near the city of La Paz, Carmen Basin on the east side of the Gulf of California, and Pescadero Basin, further south. The sixth site, on the Mazatlan margin, is located on the continental slope, near the site studied by Harnett and Devol (2003) and Brandes and Devol (2002). All six CALMEX sites are located within the present day Oxygen Minimum Zone, that occurs within the depth range between 400 and 700 m (table 1). The Santa Barbara Basin site was located in the middle of the basin, at a water depth of 587 m. At the time of both cruises, bottom water dissolved oxygen at all of the sampling locations was nearly undetectable (Table II - 1), except for the Magdalena margin ([0 2 ]=2 pM) . 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table II - 1 Station parameters. Site-Station ID________ Latitude Longitude Depth BW Oxygen CALMEX CRUISE-2001 (m) (pM) Soledad--10 25°12.66' 112°43.03' 541 <0.1 M agdalena -12 23°26.60' 1ir34.20' 713 1.2 Alfonso--15 24°38.40' 110°36.60' 408 <0.1 Carmen--21 26°17.4T 109°55.26' 575 <0.1 Pescadero-26 24°16.68' 108°11.70' 600 <0.1 Mazatlan--29 22°40.01' 106°28.70' 442 <0.1 WECOMA CRUISE -2003 Santa Barbara-12/14 34°15.89’ 120°04.38' 587 0.9 Table II - 2 Fluxes of ammonium, TCO2 and 51 5 N of the ammonium fluxes, Mazatlan margin FLUXES at the layer boundaries and added within each layer a N et NH4 + flux 81 5 N o f NH4+ net flux, % o b X 3 C M O O I- NH4 + flu x from Thioploca 81 b N o f NH4 + from Thioploca, % o C R atio between TC02 and NH4 + fluxes LAYER 1 Upward flux at 4.5 cm 0.118 19.8 0.52 0.049 34 4.3 4.5-32 Flux added between cm 4 . 5 and 32 cm d 0.045 19.8 0.17 0.024 30 Upward flux at at 32 LAYER 2 cm 0.073 19.9 0.36 0.032 33 4.9 32-43 Flux added between 32 cm and 42 cm 0.032 n/a e -0.04 0.032 n/a e LAYER 3 Upward flux at 43 cm 0.041 10.5f 0.4 n/a n/a 9.8 below 43 cm Fluxes at the boundaries are in bold font, fluxes added within layers are indicated by shaded background; positive values indicate upward fluxes 3 Fluxes are in mmol/m2 d b Values were found by fitting 1 5 NH4 + and 1 4 NH4 + profiles with reaction-diffusion model (Berner, 1980), see text for explanation c Uncertainty for 81 5 N of the fluxes added by Thioploca is about +5 %o calculated based on 10 % uncertainty for flux calculations d Values found by evaluating the analytical solution to the reaction-diffusion equations between 4.5 and 32 cm; TC02 flux calculated as a difference between fluxes at 4.5 and 32 cm e Layer 2 contains only three data points, S1 5 N of the fluxes has not been calculated f Isotopic composition of the source of ammonia diffusing from below_____________ Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Methods Cores were retrieved with gravity, piston and multi-corers. The detailed sampling procedure is described in Berelson et al (submitted). In brief, multicores were sectioned in a cold-room at 6 °C within a glove-bag filled with Ar, at 2 to 5.5 cm intervals. Gravity and piston cores were sectioned at 40 to 50 cm intervals on deck in a large bin filled with continuously flowing Ar under ambient temperatures. Sediment sections (3.5 cm thick) were placed in mini-squeezers, or “hockey pucks” (Berelson, et al., submitted) Pore water was extracted by squeezing, filtered (0.4pm) and stored until analysis. Separate aliquots of pore water were taken for TCO2, nutrient, ammonium §1 5 N and dissolved metal analysis. Ammonium (colorimetric analysis, (Bower and Holm-Hansen, 1980)) and TCO2 (UIC 5012 Coloumeter) concentrations were analyzed on board ship (±2% precision). Filtered and acidified splits of pore water were analyzed for calcium, iron and manganese by ICP-OES (±5% precision) at USC and at Oregon State University. Porosity was determined from weight loss after drying at 6C P C . Dissolved oxygen concentrations were determined using the microwinkler method from Niskin bottle samples. At CALMEX sites, pore water profiles in the upper interval of the gravity core overlapped the multi-core, permitting an easy alignment of two profiles. At the Santa Barbara Basin, the upper interval in the piston core was deeper than any part of the multicores. Therefore, pore water profiles (dissolved TCO2 and ammonium) were extrapolated to estimate the depth of the piston core. 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bulk sediment 51 5 N was determined on oven-dried (at 70°Q and thoroughly homogenized sediments. The §1 5 N of ammonium was determined using a passive diffusion method (Holmes et al., 1998; Sorensen and Jensen, 1991). A detailed description of sample processing is given in Prokopenko (2004). In brief, diluted pore water was placed in polypropylene centrifuge tubes, and pH was adjusted above 10 by addition of finely ground MgO, pre-combusted at 450°C. This converted N H / to uncharged NH3. A “trap” made of a Teflon™ tape envelope containing GF/C or GF/F filter wetted with 20 pi of 2M H2SO4 was then added to each tube. The uncharged NH3 diffused through the Teflon™ membrane, was protonated inside the “trap” and collected on the filter. The diffusion incubations of the CALMEX samples were initiated on board ship, usually within 24 to 48 hours after pore water retrieval. Santa Barbara samples were frozen and processed at USC. The nitrogen isotopic ratio of sediments and ammonium trapped on the filters were measured at USC on an Isoprime Micromass isotopic ratio mass spectrometer, interfaced with a Carlo Erba CHN-2500 elemental analyzer in continuous flow mode. Isotopic ratios are reported in permil (% o) relative to atmospheric N2 . The sample isotopic ratios were determined by comparing the sample gas to a reference N2 (Ultra High Purity Grade, Gilmore), which is calibrated with a set of NIST standards. The daily precision, based on internal standards run with samples, was 0 . 2 % 0 or better. Procedural standards of known isotopic composition, similar in concentration and volume to samples, were prepared and analyzed with the same extraction procedure. The sample standard deviation, based on replicate analyses of both samples and 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. standards, was typically 0.5 %o. The 8 1 5 N of procedural standards of ammonium was usually about 0.5 %o lighter than expected, likely, due to either a procedural artifact or perhaps a small reagent blank with unknown isotopic composition. The amount of nitrogen in standards was always close to that in samples, so 8 1 5 N values of samples were corrected by adding 0.5 %o to the measured values. Estimations o f depth distribution o f pore water sulfide at CALMEX stations We estimated the depth of free sulfide present in the pore water of CALMEX sites based on appearance of brown to black precipitate in pore water samples, which were amended with mercuric chloride immediately after retrieval to preserve the pore water for later isotopic analysis of §1 3 C in dissolved TCO2. The depth distribution of the horizons of the first appearance of dark precipitate varied between 15 to 40 cm at study sites. At the Mazatlan station, the dark color precipitate appeared in the pore water samples from depths 32 cm and below. Santa Barbara Basin Biogeochemistry and nitrogen isotopic composition o f pore water ammonium and N org Sediments from the Santa Barbara Basin are organic rich. Total Organic Carbon (TOC) concentrations range between 2.7 to 4.7 wt % (Fig. II - 2a), Total Nitrogen (TN) concentrations vary between 0.3 and 0.6 wt % (Fig. II - 2a). Both TOC and TN concentrations decrease downcore. The TOC/TN atomic ratio increases from 8.5 in the surface sediments to 11 at the bottom of the sediment column. The isotopic composition of sedimentary nitrogen (No rg ) gradually increases from 7.2 % o at the top 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TOC, wt % TOC/TN atomic ratio 100 200 TOC wt % £ 300 o ^ 400 ■ 0— C/N atomic * - < Q . O Q ■0— TN wt %measured TN wt %, modeled 500 600 700 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 TN wt % S15N, % > 100 200 300 [NH4+], mM 51 5 N-NH4+ 81 5 N-sed measured __81 5 N sed modeled q. 400 500 600 700 [NH4+], mM Fig. II - 2 Santa Barbara Basin; a) C/N ratios and concentrations of TOC and TN; b) Sedimentary and pore water 515N, pore water ammonium concentrations. Dashed arrows show the predicted changes in TN wt % and 5I5 N of No rg (see text for explanations). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to 9 % 0 at the bottom of the profile (Fig. II - 2a). The average 8 1 5 N of No rg is 7 .5 + 0.2 % 0 (the complete set of sedimentary and pore water data is given in Appendix C, table C-3). Results are in good agreement with analyses of bulk 8 1 5 N done by Emmer and Thunnell (2000). The organic matter raining to the sea floor is primarily marine but contains a significant terrestrial component, based on the composition of material collected during nearly 2 years of sediment trap deployments (Thunnell, 1998). Based on the C/N and S1 3 C of hypothetical end members (C/N = 2 0 , 8 1 3 C = - 2 8 %o for terrestrial, and C/N = 7 , S1 3 C = -20 % o for marine), the organic carbon is about 8 0 % marine and 2 0 % terrestrial. For nitrogen, the proportions would be 92% marine and 8% terrestrial, and the average S1 5 N = 8 .0 %o. The isotopic composition of the terrestrial end-member is unknown, but should be much lighter than the marine end-member. Assuming it is 2.5% o, the marine component should be 8.5% o. A similar result can be obtained by assuming the same end members have been mixed to create shallow sediments with C/N = 9 and SI5 N = 7 .5 %o. For this mixture, the terrestrial component of nitrogen is 15% and the estimated S1 5 N of the marine component is 8.4 %0. The highest values of S1 5 N collected in sediment traps were 8.5% o, in the late spring, when the flux should be largely marine material (Emmer and Thunnell, 2 0 0 0 ) . These observations suggest that the marine material should be approximately 8 1 5 N = 8 .5 %o.In the pore water of Santa Barbara sediments, the concentrations of NFU+ (Fig. II - 2b) and TCO2 (not shown) increase downcore, indicating continuous decomposition of organic matter through the whole length of the recovered sediment 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. column (about 7 0 0 cm). The 8 1 5 N of dissolved ammonium is nearly constant at 1 0 %0 through the depth sampled, with the exception of the shallowest interval at 1 .5 cm, where the 51 5 N is 11.8 %o (Fig. II - 2a). These results are gin good agreement with those obtained at a limited number of depths by Sweeney and Kaplan ( 1 9 8 0 ) . If 8 1 5 N from Fig. II - 2 is plotted vs. 1/N, the end member flux is 9 .5 ± 0 .7 %o (plot not shown). This indicates the isotopic composition of nitrogen released from organic matter is 9 .5 to 10.2% o (the average of ammonium S15N). This is l-3 % o heavier than the average isotopic composition ofbulkN org(Fig. II - 2b), and 1 .0 to 1.7% o heavier than the marine organic component currently arriving at the sea floor. Diagenesis and tf5 N of No rg Ammonium dissolved in the pore water of the Santa Barbara sediments is 1 to 3 % o heavier than sedimentary organic matter. We conducted a set of laboratory experiments, which demonstrated that differential diffusion of 15NH4+ and 14NH4+ cannot account for the observed enrichment of 15NFLi, because the diffusivities of 15NH4 + and 14NH4+ differ by less than 0.5 %o in sea water (Hammond and Prokopenko, in review). Ammonium is a major metabolic product of organic matter decomposition, and if it is not involved in any other diagenetic reactions, its S1 5 N should reflect the isotopic composition of decomposing organic matter, plus any fractionation that may take place during decomposition. We used the TCC> 2/NH4+ ratio in the pore water to determine whether the decomposition of organic matter is the only process affecting pore water ammonium concentrations. The TCO2 concentrations were corrected for 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. authigenic carbonate precipitation, using the deficit of Ca2 + relative to the sea water (10.4 mM). The carbonate-corrected TC0 2 c o rr values are about 15 % higher than the measured ones. The TCO2C 0rr/NH4+ ratio of 7.4 ±0.3 (Fig. II - 3) in the pore water is close to the C/N Redfield ratio of typical marine plankton (Anderson and Sarmiento, 1994; Redfield, 1963), and suggests that concentration of ammonium in the pore water of Santa Barbara Basin sediments is not affected by reactions other than marine organic matter decomposition. Consequently, we expect that SI5 N of ammonium should be determined by decomposing marine organic matter and associated fractionation. The 1-3 % o enrichment of the pore water ammonium in 1 5 N relative to No rg implies the net loss of an isotopically heavier fraction of organic matter from the sediments, unless an unknown reaction affects the isotopic composition of pore water ammonium. However, the pore water TCO2/N H / is evidence that release of ammonium from organic matter is the only significant reaction. We can evaluate the impact which the loss of isotopically heavier nitrogen has on the 8 1 5 N of preserved organic matter. This was done by calculating the difference between the diffusive ammonium fluxes at the sediment-water interface and 685 cm, using Fick’s first law. The difference is 0.15 mmol/m2 d and represents the net diagenetic loss of No rg through this interval. The flux of No rg deposited on the ocean floor, based on a sediment 2 • 2 accumulation rate of 73 mg/cm yr and 0.41 % wt of N, is equal to 0.59 mmol/m d. Therefore, through the upper 7 m of the sediment column, ~ 25 % of No rg is lost to diagenesis in Santa Barbara Basin. Fig. II - 2a shows the predicted profile of TN 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TCO„ mM 7.5 Santa Barbara, upper 70 cm, C/N -7 .4 Mazatlan, below 32 cm, C/N -9 .3 5.0 2.5 Mazatlan, above 32 cm, C/N = 4.6 0.0 1.5 1.0 0.5 NH4 + , mM Fig. II - 3 TCO2/N H / stoichiometry in the pore water of Santa Barbara basin and Mazatlan Margin. TC02 is corrected for in-situ carbonate precipitation. Correction for difference in diffusivities is applied. Symbols: black squares: Santa Barbara data in the upper 70 cm; gray diamonds: Mazatlan data, upper 32 cm; open squares: Mazatlan data, lower 4 m. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (dashed line) determined by fitting the pore water N H / profile with the reaction- diffusion model. The predicted TN wt % at depth is in reasonable agreement with the observed values. The reduced TOC burial rate calculated by Zhao et al.(2000) for the sediments older than 300 yr (corresponding to the depth of 1.1 m) is also consistent with these results. One additional remark needs to be made. The uppermost 1.3 cm of the sediments have substantially higher concentrations of TOC and TN than the sediments directly below this interval and the rest of the sediment column (Fig. II - 2a). We attribute this high organic matter anomaly to the presence of thick mats of Beggiatoa spp., covering the anoxic portions of Santa Barbara Basin (Grant, 1991; Schimmelmann and Kastner, 1993 ). The Beggiatoa mats tend to stay in the upper few cm of the sediments, so the Beggiatoa biomass is not buried and degraded at the same rate as marine organic matter from the water column. Therefore, in our calculations of the N org flux to the sediments we used TN concentrations measured in the interval below the mat (0.41 wt %) rather than the TN in the uppermost interval (0.67 wt %). If a fractionation of 1-3 %o accompanies the net release of ammonium, and the 5 15N of the material deposited on the ocean floor has remained a constant 7.5 %o (measured isotopic composition of the surface sediments, Fig. II - 2b), then, after 25 % is lost to diagenesis, the 8 1 5 N of residual No rg should be 6 . 8 to 7.2 % o (for fractionation factors 3 and 1 % o respectively). The modest calculated change supports the hypothesis of Altabet (1999 a, b) that in organic rich, rapidly accumulating sediments, good 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. preservation of organic matter precludes substantial alteration of its isotopic composition during diagenesis. However, the measured 5 15N of N org becomes heavier downcore, while diagenetic loss of isotopically heavier N at steady state predicts that the values should become lighter with depth (Fig. II - 2b, where dashed line represents the predicted 8 15N of Norg for the fractionation factor of 2.7 %o). One possibility to explain the discrepancy between calculated and observed changes is that the latter largely reflects changes in 8 15N of either or both of the terrestrial or marine end members and is not of a diagenetic nature. In addition, Schimmelmann and Tegner (1991) demonstrated that in addition to marine and terrigenous components that organic matter in the sediments could have some contribution from kelp transported to the center of the basin after major storm events. The C/N ratio in kelp is likely to be higher than that of phytoplankton (D. Capone, pers. comm.). The 8 1 5 N of kelp is not known, and could have some influence on the nitrogen isotope budget. Three mechanisms may lead to the preferential loss of isotopically heavier nitrogen from sedimentary No rg : (1 ) an assimilation of isotopically lighter ammonium into growing bacterial biomass in the sediments (Lehmann et al., 2002; Velinsky, 1999); preferential degradation of a more labile fraction of the organic matter that is isotopically heavier than the bulk 51 5 No r g, which happens (2) either due decomposition of more labile marine organic matter compared to more refractory terrestrial constituent, (as proposed for Santa Barbara sediments by Sweeney and Kaplan 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (1980)), or (3) by preferential decomposition of more labile amino acids (Burdige and Martens, 1988), that are isotopically heavier (Macko and Estep, 1984). Our laboratory experiments (Chapter V) demonstrated the absence of significance difference in rates of decomposition of individual amino acids, therefore mechanism (3) is an unlikely explanation for the observed isotopic enrichment of pore water ammonium relative to No rg . The estimate of fractionation is complicated by several unknown factors. The non-steady state in the isotopic composition of the No rg flux through time makes it difficult to determine the composition of the material that is degrading. It is likely that ammonia is oxidized near the sediment water interface, enriching the near surface pore waters slightly in the heavier isotope. The gradual convergence between 8 1 5 N of Norg and NH4 + with depth suggests that fractionation at depth may only be about l% o. It is also possible that fractionation decreases with depth, from 3 %o to 1 %o (for instance, if the enrichment of ammonium in heavy isotopes is due to bacterial uptake, it is possible that the magnitude of the uptake decreases downcore, which results in a decrease of effective fractionation factor from 3 to 1 % o. Regardless of which factor is chosen, the overall impact of diagenesis on S1 5 N of residual No rg appears to be rather small, no more than 1 % o. Eastern Subtropical North Pacific area: CALMEX sites CALMEX sites include six locations, listed in Table II - 1. Since all the sites have similar biogeochemical characteristics, and strong similarities in the nitrogen isotope patterns, we chose to focus our discussion on one of the locations, Site NH-29 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Fig.l), positioned about 50 km offshore, on the Mazatlan margin, south-east of the mouth of the Gulf of California, but we will briefly discuss the profiles from other sites as well (the data from all six sites is presented in Appendix B). Biogeochemistry and SI 5 N ofpore ammonium and No rg in the sediments o f Mazatlan Margin. Sediments collected within the Oxygen Minimum Zone of the Mazatlan margin are characterized by a high concentrations of both TOC and TN. TOC remains constant between ~ 8 and 9 wt % in the upper 2.5 m of the sediment column. Below this depth, TOC concentrations drop precipitously to 2-3 wt % (Fig. II - 4a). TN concentrations follow the pattern of TOC. In the upper 2.5 m TN is between 0.75 wt % and 0.95 wt %. Below 2.5 m, it decreases to 0.2-0.3 wt %. TOC/TN atomic ratios increase gradually from 9 to 12 until the 2.5 m horizon, where the values change to 13-14. The isotopic composition of No rg remains rather constant through the whole sediment column, averaging 8.02 + 0.09 %o (Fig. II - 4b). As in Santa Barbara Basin, pore water TCO2 and ammonium increase with depth (ammonium profile shown in Fig. II - 4b). Of particular note is the change in ammonium concentration gradient between 290 and 330 cm. This corresponds to an interval of sediments with lower porosity (0.62 vs. 0.78 right above this horizon), attributed to a turbidite deposit (Berelson et al., submitted). The 5 15N profile of pore water ammonium in Mazatlan sediments is drastically different from the Santa Barbara profile. At the Mazatlan station, the 5 15N of ammonium at depth is about 1 2 %o, which is about 3 .5 %o heavier than 5 15N of the 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TOC, wt % TOC/TN atomic ratio 100 200 E o sz Q- 300 < D Q — • — TOC, wt % □ - TOC/TN atomic — O'— t n , w t % 400 500 0.2 0.4 0.6 0.8 TN wt % 1.4 515N, % > 100 „ 200 300 400 500 0.0 1.5 2 0.5 1.0 [NH/1, mM 0 Fig. II - 4 Site NH-29, Mazatlan Margin: a) C/N ratios and concentrations of TOC and TN in the sediments; b) Sedimentary and pore water 8 15N, pore water ammonium concentrations. Dashed arrows show the predicted changes in TN wt % and 51 5 N of Norg(see text for explanations). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sediments. But, in contrast to Santa Barbara Basin, where 5 15N of ammonium remains constant through the whole sediment column, ammonium becomes strongly enriched in 15N towards the sediment - water interface. In the 4.5 to 3 2 cm interval, the 5 1SN of ammonium is between 1 9 and 2 0 %o (Fig. II - 4b). Below 3 2 cm, the 5 15N gradually decreases, reaching 1 2 %o at the bottom of the profile. Overall, at five of six CALMEX stations, pore water ammonium is strongly enriched in 15N isotopes, with 5 15N of 1 9 -2 1 %o, in the upper few cm of the sediments (Fig. II - 5). The depth at which 5 15N values start decreasing, varies among these five stations, from ~ 1 0 to ~ 3 0 cm. One exception from this pattern was observed in the 8 15N of ammonium at the sixth station, Pescadero. The pore water ammonium at this station is also enriched in 15N towards sediment - water interface, but is overall much lighter. At the surface, the § 15N is 1 3 - 1 4 %o, and decreases gradually to ~1 1 %o at the bottom of the profile (Fig. II - 5). Isotopic composition o f ammonium and stoichiometry ofpore water If the 1 5 N enrichment, of ammonium in the upper 32 cm of the Mazatlan sediments reflects a preferential release of isotopically heavier nitrogen from the decomposing organic matter (via any of the three mechanisms discussed above for Santa Barbara Basin), then an isotopic steady state mass balance requires that the 51 5 N of organic nitrogen should decrease by 18 % o at a depth of 30 cm. This is based on an accumulation rate of 0.017cm/yr (Ganeshram and Pedersen, 1998) and a N organic concentration in the sediments of about 1 wt %. No significant change in the 51 5 N of bulk sediments is observed through the sediment column analyzed (Fig. II - 4a), so 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Depth, cm E o Q . < D Q 81 5 N, % » 12 15 18 21 24 81 5 N, % o 1 1 o o L O a ! * 0 - 50 - i 100 * 100 - * 150 - NH-10 Soledad 150 - * * 2 00- ♦ 200 - 2 5 0 - 250 - * ♦I* NH-12 Magdalena E o 0 50 1 0 0 - l 150 Q 200 250 12 15 18 21 24 9 12 15 18 21 24 100 200 300 400 * * * NH-15 Alfonso ♦ 12 15 18 21 24 J___________ [ _______ L ♦ ♦ ♦ ♦ ♦ ♦ NH-26 Pescadero 1 0 0 200 300 400 1 0 0 200 300 400 * ♦ NH-21 Carmen * ♦ ♦ ♦ 12 15 18 21 24 I . A . I __________ I — ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ * NH29 Mazatlan Fig. II - 5 Profiles of S1 5 N of ammonium at all six CALMEX sites 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. another process that either adds 1 5 N enriched ammonium or consumes 1 4 N enriched ammonium in the upper 32 cm, must be responsible for the observed isotopic profile. Plotting [TCO2] vs. [NH4 + ’ ] we note that the A[TC0 2 ] / A[NH4 +] is 4.6 in the upper 32 cm (Fig. II - 3), which is significantly lower than the average C/N ratio of decomposing marine plankton predicted by Redfield stoichiometry (Anderson and Sarmiento, 1994; Redfield, 1963). This is also less than half of the AfTCCh] / A[NH4 + ] ratio below 32 cm. The change in slope requires an additional source of ammonium that is added near 32 cm. The low C/N suggests the source may not be decomposing organic matter. Similar changes are seen in plots of CO2 vs. NH4 + at other stations (Fig. II - 6 ), always near the base of the zone enriched in 51 5 N of ammonium. An exception to this pattern is observed at the Pescadero station (Fig. II - 6 ), where the isotopic enrichment of ammonium is also substantially smaller than at the other five stations (Fig. II - 5). Pore water geochemistry and Thioploca metabolism. During the CALMEX cruise in 2001 we often observed long white filaments of bacteria, which we identified as trichomes of Thioploca spp., Beggiatoaceae family. Filaments were present from the surface to a depth of ~ 20 to 30 cm. Details of Thioploca spp. occurrence have been documented in Soledad Basin (Bernhard and Buck, 2004). These bacteria base their metabolism on energy gained by oxidation of hydrogen sulfide with nitrate (Fossing, 1995; Otte, 1999). Thioploca sequester nitrate from the overlying water into large vacuoles, where concentrations can reach 550 mM (Fossing, 1995; Otte, 1999). Thioploca trichomes glide within sheaths they construct 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T C O „ m M TCO. NH10-Soledad basin 21.5-47 cm C= -\2.6*N-52 0-21.5 cm C = 2.9*N + 1.4 0.5 1 1.5 NH4 + , mM NH12-Margalena margin 21-85 cm C = 12.7*N-5.2, 0-21 cm C = 3.3*N + 1.3 1 1.5 NH4 + , mM 35 5 30 £ 25 s 20 15 10 5 0 0.0 NH15-Alfonso basin 32-273 cm C= 11.9*N - 9.9 □ 4.5-32 cm C = 3.2*N + 0.1 1.0 2.0 3.0 4.0 10 S 8 E 6 °4 0 NH21-Carmen basin 26.5-107 cm C = 9.2*N - 2.6 4.5-26.5 cm y = 4.9*N + 0.1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 NH4 + , mM NH26-Pescadero basin 20 15 4.5-91 cm C = 7.6*N + 0.85 5 0 2.0 2.5 0.0 0.5 1.0 1.5 NH4 + , mM 10 8 S £ 6 8S 4 i - 2 0 NH29-Mazatlan margin 32-218 cm C = 9.2*N - 2.2 45. cm-32 cm C = 4.6*N - 0.1 0.0 0.3 0.5 0.8 1.0 NH4 * , mM 1.3 1.5 Fig. II - 6 TCCh/NHY* stoichiometry of pore water at all six CALMEX sites. TCO2 concentrations are corrected for difference in diffusivities between HCO3’ and NH4 +. TCO2 concentrations for sites NH10, NH15, NH26 and NH29 are corrected for carbonate precipitation, based on pore water Ca 2 + profiles (Appendix B). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in the sediments, transporting nitrate from the sediment - water interface to depths of a few tens of cm. The depth of travel is determined by the depth of the horizon where Thioploca encounters free sulfide (Fossing, 1995; Zopfi, 2001). Thioploca is known to contribute significantly to the nitrogen cycling in the sediments of Peru-Chilean margin (Fossing, 1995; Gallardo, 1977; Graco, 2001; Zopfi, 2001), but its contribution to geochemistry of the sediments in the ESNP, or the influence of these bacteria on isotopic mass balances in this region has not been evaluated . A combination of reaction [1], performed by Thioploca, and reaction [2], representing oxidation of organic carbon, integrated through the sediment column, provide a mechanism for production of ammonium in excess of Redfield C/N ratio in CALMEX sediments: [1] H30 + + HS' + N O f = S 04 2 ' + NH4 + (Zopfi, 2001) [2] 2H2 0 + 2Co rg + S 04 2 ' = 2HC03 ‘ + H2S: The sum of reactions [1] and [2] is represented by equation: [3] 3H20 + 2Co rg + N 0 3 ‘ = 2HC03 ' + NH4 +, with net AC/AN = 2, if all sulfide is oxidized to sulfate. The measured TC02/NH4 + ratio of 4.6 in the pore water of Mazatlan basin indicates that about a third of the sulfide is oxidized by Thioploca in these sediments. This estimation is consistent with stoichiometry observed in the Peru-Chile margin sediments (Zopfi, 2001). Mixing diagram The activity of Thioploca spp. explains the stoichiometry observed in the pore waters at CALMEX stations, but what influences the profiles of 515N? In order to 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Heavy source ofN H t t f 5 N > 20%o Flux towards the surface at 4.5 cm Org source & 5 N ~ 10.5 % o 22.5 20.0 Diffusion towards the surface 17.5 u > X - C O 15.0 Mixing 12.5 Intercept at 10.5 % o 10.0 21 14 7 0 1/NH4+ Fig. II - 7 Mixing diagram of ammonium isotopic composition. The insert shows the sources contributing to the 51 5 N of pore water ammonium (note that diffusion does not influence the 51 5 N of ammonium. The intercept of the mixing line and Y-axis gives the 51 5 N of the end-member. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. constrain location, magnitude, and, ultimately, the nature of the source of isotopically heavy ammonium, we constructed a mixing diagram (Fig. II - 7). The diagram shows a pronounced change in slope around 32 cm depth. Above this interval, there appears to be a continuous addition of ammonium heavier than 20 % o. Below 32 cm, the linear pattern of 51 5 N vs. 1/[NH4 + ] indicates conservative mixing between two different sources: (1) the isotopically heavier ammonium produced at 32 cm and in the interval above, and (2) ammonium with 51 5 N determined by the intercept of the mixing line and Y-axis at ~ 10.5 % o (Fig. II - 6 ). Source (2) should be ammonium from decomposing No rg . Then, the following questions need to be answered: 1) What is the isotopic composition of the “heavy ammonium” source? 2) What is the nature of this source? The tfsN o f the ammonium fluxes We addressed the first question by constructing a three-layer reaction-diffusion model (Berner, 1980). The model was used to calculate diffusive fluxes of TCO2, ammonium, and S1 5 N of ammonium fluxes, at 4.5, 32 and 43 cm horizons, chosen as the boundaries for three layers (4.5-32 cm, 32-43 cm and > 43 cm) (Fig. II - 8 ). Fluxes were calculated by fitting the solutions of reaction-diffusion diagenetic equations to the profiles, taking the first derivative at the upper boundaries of each layer, and using Fick’s first law to calculate the fluxes. To find specific solutions we used the concentrations of ammonium at the specified depth horizons as the boundary conditions. The 14NH4+ and 15NH4+ profiles were fitted separately, so that fluxes of 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. F l u x o f N o rg a t 4 . 5 cm 3 Norg ~ ' 0.08 at 8 < lN = 8 Z o o Sedim ent-w ater i n t e r f a c e 4 . 5cm LAYER 1 -< 32cm LAYER 2 < 43cm LAYER 3 FLUX from Thioploca = 0.049 @34 % « ^ Thioploca = 0.017 @ 34 % 0 3 Thioploca = 0.032 @ 33 %o TOTAL NH4+ FLUX J to ta i = 0.118 @ 19.8 %o J lo ta =0.073 ©19.8% , I total " 0.041 @ f 0.5 % NET FLUX from A / decomposition = 0.069 @ 70.5 = 0.028 10.5 % o J o,y=0.00 / 3 org = < 0.041 \ @ 10.5 %o Fig. II - 8 Isotopic composition of ammonium fluxes in the pore water of the Mazatlan margin sediments. Ammonium fluxes and their isotopic composition are shown. Fluxes are in mmol/m2d. White arrows indicate the ammonium fluxes contributed by Thioploca, grey arrows indicate flux of ammonium from decomposing organic matter. Gradient arrows represent total ammonium fluxes. Dotted arrow represents the flux of organic N deposited on the ocean floor. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14NH4 + and 15NH4 + could be calculated separately as well. The 51 5 N of the ammonium fluxes was calculated as the ratio of the fluxes of individual isotopes. The results of the calculations are summarized in table 2 and Fig. II - 8 . The changes in ATOC/ANH4 + with depth (Fig. II - 3) suggest that ammonium produced by Thioploca is added only in layers 1 and 2, but not in layer 3 (Fig. II - 8 ). Therefore, the only source of ammonium in layer 3 is decomposing organic matter. The 8 1 5 N of this source must be defined by the ammonium flux from layer 3 to layer 2. The ratio of 15NH4 + to 14NH4 + at this horizon is equal to 10.5 % o. This value is identical to that obtained by extrapolating to the intercept in Fig. II - 7, as discussed above. We assume the same isotopic composition of 10.5 % o for ammonium released from the decomposing organic matter in layers 1 and 2 as well. We also assume that organic matter is decomposing with a stoichiometry of C/N = 7.3 (Anderson and Sarmiento, 1994), and any additional ammonium is produced from nitrate reduction by Thioploca. On this basis, the 6 1 5 N of the ammonium flux added by Thioploca activity was calculated to satisfy an isotopic mass balance: J t c o 2 JNH 4+ total * ^total ~ ^org * M & added = ~ t c o 2 JNH 4 +total ~ ^ where J defines flux. In Layer 2, the intermediate layer, the ammonium flux leaving is about twice the flux delivered from Layer 3. The absence of significant TCO2 production through Layer 2 indicates that the dominant source of ammonium added between 32 and 43 cm 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. is nitrate reduction to ammonium, performed by Thioploca. The 6 1 5 N of the ammonium flux generated by Thioploca in the intermediate layer, layer 2, is ~ 33 %o (Table II - 2). It is likely that most reduction of nitrate to ammonium is restricted to a narrow horizon near the base of Layer 2, but our sampling resolution precludes us from pointing to a precise location and thickness of the production horizon. In Layer 1 , two thirds of the ammonium released is produced from decomposing organic matter (calculated as TCO2 flux divided by 7.3) with an isotopic composition of 10.5 %o; the remaining one third, with S1 5 N of « 34 % o, is generated by Thioploca via reaction [1]. Proposed scenario: Chemosymbiosis between Thioploca and anaerobic ammonium oxidizing bacteria Our modeling results show that ammonium contributed by Thioploca is strongly enriched in 1 5 N isotopes, with §1 5 N of ~ 33.5 %o. These bacteria accumulate nitrate against a very large concentration gradient, requiring an active mechanism of transport across the cell membrane. Analogous to assimilation of nitrate by other bacteria (Miyazaki, 1980), the S1 5 N of intracellular nitrate stored in the vacuoles should be lighter , not heavier, than S1 5 N of bottom water nitrate, which is 14 tol5 %o in this region (Altabet et al., 1999; Sigman et al., 2003). Therefore, ammonium produced by Thioploca should be lighter than 14-15 %o. Thus, activity of Thioploca alone should not contribute isotopically heavy ammonium to the pore water. Here, we propose that the observed isotopic pattern is evidence for a special “ecogeochemical” role that Thioploca play in the sediments of ESNP. Transporting nitrate to a few tens of centimeters into severely anoxic sediments, Thioploca 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. introduces a thermodynamically potent oxidant into a strongly reducing environment, rich in ammonium. The presence of elemental sulfur globules, commonly observed in Thioploca cells (Fossing, 1995; Otte, 1999), suggests that in addition to reaction [1], Thioploca might oxidize hydrogen sulfide to elemental sulfur in a separate step, while reducing nitrate to nitrite: [5] NO3 ' + HS- + H+ = NO2' + S0 + H2O We hypothesize that nitrite is produced by this reaction and becomes available for oxidation of abundant pore water ammonium to N2 via reaction [6 ] by Anammox-like organisms (Kuenen and Jetten, 2001; Strous, 1999), which might live in close spatial (chemosymbiotic or syntrophic) relationship with Thioploca. [6 ] N 02" + NH4 + = N2 + 2H2 0 A conceptual cartoon presenting the proposed scenario is shown in Fig. II - 9. As suggested by the TC 02/NH4 + stoichiometry, less ammonium is removed by oxidation than is added by Thioploca. Oxidation of a fraction of the ammonium is likely to leave the residual ammonium pool strongly enriched in 15N, which would explain the heavy isotopic composition of ammonium contributed by the coupled production-oxidation process, performed by proposed syntrophic pair, Thioploca- Anammox (Fig. II - 9). In the laboratory studies of Thioploca spp. Otte et al. (1999) observed production of N 0 2 ", when bacteria were NO3’ limited. Based on this observation, it is likely that when Thioploca glides deep into the sediments, where free sulfide is available, but nitrate is limited, its strategy might be to quickly oxidize H2S to S°, with 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NO, Thioploca sheath ~ * n € Gliding Thioploca trichome — Symbiotic ammonium oxidizing bacteira overlying water S15N-NH4 + , % . 14 18 22 o J [1] X 4 s HS- S 0 42 - NO,- NO,’ X . HS' S° [5] NH.,1 no2 - [6] 0 0 ^ N2 + 2H2 0 $ 0 0 HoS t 16 ■ 24 0 0 32 cm - 100 Fig. II - 9 Diagram of the proposed symbiosis between Thioploca and Anammox bacteria. Numbers in parenthesis correspond to the reactions discussed in the text. The insert on the right shows 8 1 5 N of ammonium profile (note the change in depth scale). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N 0 2 - as a byproduct of the reaction. The proposed symbiosis is advantageous for both bacteria: Thioploca would benefit from the removal of potentially toxic metabolic byproduct (NO2’), and Anammox bacteria obtain both the oxidant and the reductant for their energy needs by “riding” on the Thioploca filaments. An alternative scenario is that nitrate might simply be leaking out of the cells, is reduced to nitrite by denitrifying organisms, and then is used for ammonium oxidation. However, it has been observed that the Thioploca vacuoles are relatively rigid and normally don’t leak (B. Joergensen, pers. comm.). Tantalizingly, laboratory studies of Thioploca (Schulz et al., 1996) have reported abundant epibiotic bacteria covering Thioploca filaments, although the identity of the epibionts is unknown. Presently, we cannot distinguish between the two mechanisms, but either scenario provides a possible explanation for the 15N-enriched ammonium in the upper 30 cm, where no other potential oxidants are present. Aerobic oxidation can be completely ruled out due to the anoxia in the overlying bottom water at all sites examined. Some studies (Aller, 1998; Hulth et al., 1999; Luther, 1997) suggested the possibility of ammonium oxidation with either iron or manganese oxides. However, the pore water concentrations of both of these metals in Mazatlan sediments (Fig. II - 1 0 ) indicate that metal reduction is restricted to the upper few cm of the sediments. Comparison between Santa Barbara and ESNP sites: sedimentary geochemistry, benthic microbial ecology and nitrogen cycling The dissimilarity between the 51 5 N of the pore water ammonium in Santa Barbara and ESNP indicates that the nitrogen cycling in the sediments differs 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mazatlan Margin [Fe2 + ] and [Mn2 + ] uM 0 2 4 6 8 10 0 30 60 90 120 4— 51 5 N of N H , 0 • 150 20 14 16 18 22 10 12 815N, % o Fig. 11-10 Profiles of dissolved iron and manganese and 51 5 N of ammonium in the pore water of Mazatlan sediments. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. significantly between these two regions. The floor of Santa Barbara Basin is covered with thick mats of Beggiatoa, a genus phylogenetically related and metabolically similar to Thioploca (Teske et al., 1996). Beggiatoa has a higher tolerance for sulfide than Thioploca (Huettel, 1996), and forms horizontal mats on the ocean floor (Huettel, 1996), rather than traveling vertically into the sediments, as Thioploca does. The presence of sulfide in the upper 2-4 cm of pore water in Santa Barbara Basin (Reimers, 1996) apparently creates favorable conditions for Beggiatoa, rather than Thioploca. On the contrary, in the sediments of ESNP, free pore water sulfide has not been observed shallower than few tens of cm from the sediment-water interface. We do not exclude the possibility that some sulfide is produced above the horizon of first free H2S appearance , but apparently, rapid sulfide removal into sulfide minerals does not allow accumulation of any of it above this horizon. Spatial separation between sulfide and nitrate creates an advantage for Thioploca spp. over its less motile cousin. One possible explanation for the absence of the free sulfide in the upper few decimeters in the sediments at the CALMEX sites is a titration of sulfide with dissolved Fe+2 (Canfield, 1989). The depth at which this titration is complete determines the depth, at which pore water becomes sulfidic, and should depend on the relative supply of organic matter and reactive detrital iron (Leslie et al., 1990). One factor influencing CALMEX stations might be the high iron content of the coastal rocks on the Baja Peninsula, which are mostly iron-rich basalts and andesites. Santa Barbara sediments, on the other hand, do not receive input from such iron-enriched terrestrial source. 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Does the proposed symbiosis influence the 8 1 S N of the oceanic nitrate? The depth of the shallowest horizon sampled in this study is 4.5 cm. Thus, our data is insufficient to determine whether isotopically heavier ammonium escapes from the sediments, propagating the isotopic signal of the proposed symbiotic process into the bottom water of the Eastern North Subtropical Pacific. Our location on the Mazatlan margin is within 10 km of site NH015 (Fig. II - 1) (22°41.50' N, 106° 28.20' W, water depth 420 m), where nutrient fluxes were measured by Hartnett and Devol (2003). These authors report an ammonium flux of 0.34 mmol/m d, about three times greater than the flux leaving the 4.5 cm horizon, calculated in this study (table 2). They also report a nitrate flux into the sediments of 1.1 mmol/m d. Berelson et al. (submitted) showed that at CALMEX sites, CO2 flux at sediment - water interface is two to three times greater than the flux leaving the 10 cm horizon. This implies that the rates of organic matter decomposition are highest within the upper few cm of the sediment column. Therefore, most of NH4+ released into the pore water between 4.5 cm (our topmost measured interval) and the sediment water interface is generated by the organic matter decomposition. If we assume that 8 1 5 N of this ammonium is 10.5 % o (the same as ammonium released at depth), then based on mass balance calculations, we can predict the 51 5 N of ammonium diffusing into the water column at the sediment-water interface to be around 14 %o. The diffusing ammonium should be quantitatively oxidized to nitrate within the water column to nitrate of similar isotopic composition, ~ 14 %o. 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The 51 5 N of the bottom water nitrate in the Gulf of California, just north of Mazatlan station, is 14 % 0 (Altabet et al., 1999; Pride et al., 1999; Sigman et al., 2003). The §1 5 N of nitrate to the south of Mazatlan station is 18 %o (Cline and Kaplan, 1975). Therefore, at the Mazatlan station the S1 5 N of bottom water nitrate should be between 14 and 18 %o, which is comparable to that predicted by our results, that ammonium with 51 5 N of 14 %o diffuses from the sediments in this region. It has been pointed out by Sigman et al. (2003) that the bottom water nitrate in the Gulf of California is 6%o heavier than that in Santa Barbara Basin, while the nitrate deficit is smaller. These observations are consistent with the assertion that nitrification of isotopically heavy ammonium occurs in the water column of ETSP, rather than in the sediments, and may contribute to the isotopically heavy signature of oceanic nitrate in the ETNP. Brandes and Devol (2002) measured a 3 % enrichment associated with the sedimentary denitrification in close proximity to our Mazatlan site (Fig. II - 1). On the other hand, no isotopic fractionation during sedimentary denitrification was reported by the same authors (Brandes and Devol, 1997) for Washington margin sediments. Similarly, Lehmann et al. (2004) report a fractionation factor close to 0 for sedimentary denitrification in the Bordeland Basins. These authors argued that the absence of fractionation is due to quantitative nitrate loss in the process of sedimentary denitrification. Without additional oxidants at depth, pore water ammonium is also completely oxidized to nitrate, which is subsequently completely denitrified with no isotopic fractionation. 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. It is likely that the observed small, but measurable fractionation during denitrification measured by Brandes and Devol (2002) in Mazatlan sediments, is related to the proposed symbiosis. Our results show that sedimentary denitrification should not be considered as a ’’ black box” with an identical isotopic signature everywhere. It is apparent that variations in the structure of bacterial communities can result in a variable isotopic effect associated with sedimentary nitrate reduction. Summary Two main conclusions can be drawn from the work presented here. The results from the Santa Barbara Basin demonstrate that despite a 2-3 % o fractionation, which accompanies decomposition of the bulk sedimentary organic matter, the 51 5 N of residual No rg in the sediments of this basin is not significantly altered by diagenesis . The good preservation of the original isotopic composition is facilitated by rapid sediment accumulation rates, which allows only a small fraction of organic matter to be lost to decomposition. This conclusion confirms the argument by Altabet et al. (1999a) and Pride et al. (1999), who proposed that high organic content and rapid accumulation rates are the determining factors for the preservation of original 51 5 N in sedimentary No rg . The data from the CALMEX sites, located within ESNP provides possible evidence for a chemosymbiotic relationship between Thioploca and Anammox-like bacteria. The hypothesis of symbiosis still awaits confirmation by the detailed molecular work. However, the geochemical data and the results of analytical modeling presented here make a strong case for the proposed symbiosis. 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These findings offer a novel prospective on the role of microbial diversity in sedimentary denitrification. The small, but measurable difference in fractionation factors associated with sedimentary nitrate reduction in various localities is likely to result from differences in benthic microbial communities involved in the nitrogen cycling in these areas. We suggest that it is the geochemical characteristics of the environment, perhaps the rain ratio of reactive iron to organic material, determine the structure of the bacterial communities in different areas. The metabolic diversity in bacterial communities may manifest itself on a global scale, by contributing a specific regional isotopic signature to the S1 5 N of the nitrate in the overlying water. Acknowledgements: This study has been supported by NSF grant OCE-0136500 to DH, NSF grant OCE-0002250 to LDS, WB, RGD, and an ODP Schlanger fellowship to MGP. The assistance of Pete Kalk and Chris Moser of the NORPAC coring group, as well as the personnel of R/V New Horizon and R/V “Wecoma” made field work possible. Assistance from M. Rincon, T. Gunderson and G. Smith with isotopic analysis and sample processing is greatly appreciated. We also gratefully acknowledge our discussion with D. Canfield in summer, 2002, who first pointed out the possibility of Anammox reaction occurring in the Gulf of California sediments, and thus, encouraged our search for a mechanisms to provide an oxidant for this process in the anoxic sediments. We are also thankful to C. Drennen, B.B. Jorgensen, J. Kuenen, D. Sigman, L. Stein and W. Ziebes for helpful discussions. 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References: Aller, R.C., Hall, P. O. J., Rude, P. D., Aller, J. Y., 1998. Biogeochemical heterogeneity and suboxic diagenesis in hemipelagic sediments of the Panama Basin. Deep-Sea Research Part I-Oceanographic Research Papers, 45(1): 133- 165. Altabet, M.A. et al., 1999. The nitrogen isotope biogeochemistry of sinking particles from the margin of the Eastern Tropical Pacific. Deep-Sea Research I, 46: 655- 679. Anderson, L.A. and Sarmiento, J.L., 1994. Redfield ratios of remineralization determined by nutrient data analysis. Global Biogeochemical Cycles, 8(1): 65- 80. Bender, M. et al., 1989. Organic carbon oxidation and benthic nitrogen and silica dynamics in San Clemente Basin, a continental borderland site. Geochimica et Cosmochimica Acta, 53(3): 685-697. Berner, R.A., 1980. Early Diagenesis: A theoretical approach. Princeton University Press, Princeton, NJ, 241 pp. Bernhard, J.M. and Buck, K.R., 2004. Eukaryotes of Soledad, Cariaco and Santa Barbara basins:Protists and metazoans associated with deep-water marine sulfide-oxidizing microbial mats and their possible effects on the geologic record. In: E. Amend J.P., K.J., and Lyons, T.W. (Editor), Sulfur biogeochemistry - Past and Present. Geological Society of America, pp. 35-47. 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Graco, M., Farias, L., Molina V., Gutierrez, D., Nielsen, L.P., 2001. Massive developments of microbial mats following phytoplankton blooms in a naturally eutrophic bay: Implications for nitrogen cycling. Limnology and Oceanography, 46(4): 821-832. Grant, C.W., 1991. Lateral and Vertical Distributions and Textural Features of Filamentous Bacterial (Beggiatoa Sp) Mats in Santa-Barbara Basin, California. Aapg Bulletin-American Association of Petroleum Geologists, 75(3): 585-585. Gruber, N., in press. The marine nitrogen cycle. In: M. Follows, Oguz, T. (Editor), Carbon - climate interactions, NATO ASI series. John Wiley & Sons, New York. Gruber, N. and Sarmiento, J., 2002. Biogeochemical/physical interactions in elemental cycles. In: A.R. Robinson, McCarthy, J.J., Rotschild, B.J. (Editor), The Sea: Biological-Physical Interactions in the Oceans. John Wiley & Sons, New York, pp. pp. 337-399. Hammond, D.E. and Prokopenko, M.G., in review. Differential diffusivity of light stable isotopes: 15NH3/14NH3. Geochimica et Cosmochimica Acta. 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hartnett, H.E. and Devol, A.H., 2003. Role of a strong oxygen-deficient zone in the preservation and degradation of organic matter: A carbon budget for the continental margins of northwest Mexico and Washington State. Geochimica Et Cosmochimica Acta, 67(2): 247-264. Hattori, A., 1983. Denitrification and dissimilaritory nitrate reduction. In: E. Carpenter and D. Capone (Editors), Nitrogen in the Marine Environment. Academic Press, New York, pp. 191-232. Holmes, R.M., McClelland, J.W., Sigman, D.M., Fry, B. and Peterson, B.J., 1998. Measuring N-15-NH4+ in marine, estuarine and fresh waters: An adaptation of the ammonia diffusion method for samples with low ammonium concentrations. Marine Chemistry, 60(3-4): 235-243. Huettel, M., Forster, S., Kloser, S., Fossing, H., 1996. Vertical migration in the sediment-dwelling sulfur bacteria Thioploca spp in overcoming diffusion limitations. Applied and Environmental Microbiology, 62(6): 1863-1872. Hulth, S., Aller, R.C. and Gilbert, F., 1999. Coupled anoxic nitrification manganese reduction in marine sediments. Geochimica Et Cosmochimica Acta, 63(1): 49- 66. Jetten, M.S.M. et al., 1998. The anaerobic oxidation of ammonium. Ferns Microbiology Reviews, 22(5): 421-437. Kaplan, W., Valiela, I. and Teal, J.M., 1979. Denitrification in the salt marsh ecosystem. Limnology and Oceanography, 24: 726-734. Kuenen, J.G. and Jetten, M.S.M., 2001. Extraordinary anaerobic ammonium-oxidizing bacteria. Asm News, 67(9): 456-463. Lehmann, M.F., S.M., B., Barbieri, A. and McKenzie, J.A., 2002. Preservation of organic matter and alteration of its carbon and nitrogen isotope composition during simulated and in situ early sedimentary diagenesis. Geochimica et Cosmochimica Acta, 66(20): 3573-3584. Lehmann, M.F., Sigman, D.M. and Berelson, W.M., 2004. Coupling the 15N/14N and 180/160 of nitrate as a constraint on benthic nitrogen cycling. Marine Chemistry, 88(1-2): 1-20. Leslie, B.W., Hammond, D.E., Berelson, W.M. and Lund, S.P., 1990. Diagenesis in Anoxic Sediments from the California Continental Borderland and Its Influence on Iron, Sulfur, and Magnetite Behavior. Journal of Geophysical Research-Solid Earth and Planets, 95(B4): 4453-4470. 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Luther, G.W., Sundby, B., Lewis, B. L., Brendel, P. J., Silverberg, N., 1997. Interactions of manganese with the nitrogen cycle: Alternative pathways to dinitrogen. Geochimica Et Cosmochimica Acta, 61(19): 4043-4052. Macko, S. A. and Estep, M.L.F., 1984. Microbial alteration of stable nitrogen and carbon isotopic compositions of organic matter. Organic Geochemstry, 6 : 787- 790. Miyazaki, T., Wada, E., Hattori, A., 1980. Nitrogen - isotope fractionation in the nitrate respiration by the marine bacterium Serrata marinorubra. Journal of Geomicrobiology, 2: 115-126. Nishio, T., Koike, I. and Hattori, A., 1982. Denitrification, nitrate reduction and oxygen consumption in coastal and estuarine sediments. Applied and Environmental Microbiology, 43: 648-653. Otte, S., Kuenen, J. G., Nielsen, L. P., Paerl, H. W., Zopfi, J., Schulz, H. N., Teske, A., Strotmann, B., Gallardo, V. A., Jorgensen, B. B., 1999. Nitrogen, carbon, and sulfur metabolism in natural Thioploca samples. Applied and Environmental Microbiology, 65(7): 3148-3157. Pride, C. et al., 1999. Nitrogen isotopic variations in the Gulf of California since the last deglaciation: Response to global climate change. Paleoceanography, 14(3): 397-409. Prokopenko, M.G., 2004. Fractionation of nitrogen isotopes during early diagenesis, University of Southern California, Los Angeles, 219 pp. Redfleld, A.C., Ketchum, B.H., Richards, F.A.„ 1963. The influence of organisms on the composition of of sea-water. In: M.N. Hill (Editor), The sea. Wiley- Interscience, New York, pp. 26-77. Reimers, C.E., Ruttenberg, K. C., Canfield, D. E., Christiansen, M. B., Martin, J. B., 1996. Porewater pH and authigenic phases formed in the uppermost sediments of the Santa Barbara Basin. Geochimica Et Cosmochimica Acta, 60(21): 4037- 4057. Schimmelmann, A. and Kastner, M., 1993. Evolutionary Changes over the Last 1000 Years of Reduced Sulfur Phases and Organic-Carbon in Varved Sediments of the Santa-Barbara Basin, California. Geochimica Et Cosmochimica Acta, 57(1): 67-78. 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Schimmelmann, A. and Tegner, M., 1991. Historical oceanographic events reflected in 13C/12C ratio of total organic carbon in laminated Santa Barbara sediments. Global Biochemichal Cycles, 5: 173-188. Schulz, H.N., Jorgensen, B.B., Fossing, H.A. and Ramsing, N.B., 1996. Community structure of filamentous, sheath-building sulfur bacteria, Thioploca spp, off the coast of Chile. Applied and Environmental Microbiology, 62(6): 1855-1862. Sigman, D.M. et al., 2003. Distinguishing between water column and sedimentary denitrification in the Santa Barbara Basin using the stable isotopes of nitrate. Geochemistry Geophysics Geosystems, 4(5). Sorensen, P. and Jensen, E.S., 1991. Sequential Diffusion of Ammonium and Nitrate from Soil Extracts to a Polytetrafluoroethylene Trap for N-15 Determination. Analytica Chimica Acta, 252(1-2): 201-203. Strous, M. et al., 1999. Missing lithotroph identified as new planctomycete. Nature, 400(6743): 446-449. Strous, M., Fuerst, J. A., Kramer, E. H. M., Logemann, S., Muyzer, G., van de Pas- Schoonen, K. T., Webb, R., Kuenen, J. G., Jetten, M. S. M., 1999. Missing lithotroph identified as new planctomycete. Nature, 400(6743): 446-449. Sweeney, R.E. and Kaplan, I.R., 1980. Natural abundances of 1 5 N as a source indicator for near shore marine sedimentary and dissolved nitrogen. Marine Chemistry, 9: 81-94. Teske, A., Ramsing, N.B., Kuver, J. and Fossing, H., 1996. Phylogeny of Thioploca and related filamentous sulfide-oxidizing bacteria. Systematic and Applied Microbiology, 18(4): 517-526. Thamdrup, B. and Dalsgaard, T., 2002. Production of N-2 through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments. Applied and Environmental Microbiology, 68(3): 1312-1318. Vandegraaf, A.A. et al., 1995. Anaerobic Oxidation of Ammonium Is a Biologically Mediated Process. Applied and Environmental Microbiology, 61(4): 1246- 1251. Velinsky, D., Fogel M., 1999. Cycling of dissolved and particulate nitrogen and carbon in the Framvaren Fjord, Norway: stable isotope variations. Marine Chemistry, 67: 161-180. 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Zhao, M., Eglington, G., Read, G. and Schimmelmann, A., 2000. An alkenone (UK |) quasi-annual sea surface temperature record (A.D. 1440 to 1940) using varved sediments from the Santa Barbara Basin. Organic Geochemstry, 31: 903-917. Zopfi, J., Kjaer, T., Nielsen, L.P., Jorgensen, B.B., 2001. Ecology of Thioploca spp.: Nitrate and sulfur storage in relation to chemical microgradients and influence of Thioploca spp. on the sedimentary nitrogen cycle. Applied and Environmental Microbiology, 67(12): 5530-5537. 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER III: Impact of long term diagenesis on 81 S N of organic matter in marine sediments: ODP LEG 201- Sites 1227 and 1230 (submitted to the ODP Volume 201, Scientific Results) Maria G. Prokopenko, Douglas E. Hammond, Arthur Spivack and Lowell Stott Introduction Nitrogen is one of the important limiting nutrients in the ocean (Gruber, in press; Redfield, 1963; Tyrell, 1999). The global carbon cycle, and consequently atmospheric CO2 might be tightly coupled to the nitrogen cycle (Berger and Keir, 1984; Broecker, 1982), and therefore changes in the magnitude of the sinks and sources of fixed nitrogen in the oceans can significantly influence the global climate (Falkovsky, 1997; Falkowski, 1997; Ganeshram et al., 1995; Sigman et al., 1999; Sigman and Boyle, 2000). Biological nitrogen fixation, denitrification and consumption of nitrate by phytoplankton, the major biological processes of the global nitrogen cycle, can each imprint a distinct isotopic signature, 515N, on oceanic nitrate and on the phytoplankton that assimilate this nitrate as a nitrogen source (Altabet and Francois, 1994; Haug et al., 1998). Changes in ocean circulation and nutrient supply, which occur in response to changes in environmental conditions, affect the relative importance and spatial extent of the major pathways of the nitrogen cycle, and thus, are recorded in the isotopic ratio of marine phytoplankton, making 8 1 5 N of organic 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. matter buried in marine sediments a sensitive paleoceanographic proxy (Altabet et al., 1991; Altabet and McCarthy, 1985; Francois et al., 1992). A critical question is whether diagenetic processes fractionate nitrogen isotopes in the buried organic matter, and if they do, what is the sign and magnitude of this fractionation. A few laboratory studies have previously directly addressed the effects of degradation on the isotopic composition of organic matter. Decomposition of labile proteinaceous organic matter involves peptide bond rupture by hydrolysis, which is the principal reaction in protein degradation. Silfer et al. ( 1 9 9 2 ) experimentally demonstrated kinetic fractionation of nitrogen isotopes during abiotic peptide bond hydrolysis, leading to a 2 to 4 %o enrichment of 1 5 N in the residual substrate. The experimental results of Macko and Estep ( 1 9 8 4 ) showed that both peptide bond hydrolysis and deamination result in the enrichment of 1 5 N the residual material with the fractionation factor of about 4%o. These experimental findings were supported by the field observations made a few years later. Sigman et al. (1999) working with the sediments from the Southern Ocean, and Sachs and Repeta (1999) examining the sapropels from the Mediterranean Sea, found evidence of diagenetic alteration of 6 1 5 N in bulk sediment: a 2 to 5 %o positive shift relative to unaltered organic matter. Contrary to their results, Altabet et al. (1999) and Pride et al. (1999) argued that in the rapidly accumulating organic-rich sediments of the Eastern Tropical North Pacific, early diagenesis does not affect isotopic composition of sedimentary organic matter, if a significant fraction of original sedimentary organic matter is preserved. 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In a series of incubation experiments, Lehmann et al. (2002) showed that degradation of organic matter under aerobic conditions leaves the residual biomass enriched in 1 5 N isotopes, while anoxic decomposition of organic matter results in depletion of 15N. He interpreted the latter case as evidence for bacterial growth, accompanied by the assimilation of light ammonium into newly formed bacterial biomass, since bacteria grown on ammonium as the sole nitrogen source produce biomass significantly depleted in 1 5 N compared to the original substrate (Hoch et al., 1992). To summarize, previous research has shown that under some geochemical conditions, bacterial degradation may lead to changes in nitrogen isotopic ratios of preserved organic matter. However, the degree and direction of isotopic fractionation in marine sediments, as well as factors controlling them remain poorly understood. In this study, we address the problem of diagenetic fractionation of nitrogen isotopes by constructing isotopic mass balances for the sedimentary organic nitrogen and pore water ammonium, which is a major metabolic product of organic matter decomposition. If ammonium is not involved in other diagenetic reactions, its isotopic composition should reflect the isotopic composition of organic nitrogen plus fractionation associated with diagenesis. Cores retrieved during ODP expeditions provide a unique opportunity to evaluate the effect of diagenetic processes on the nitrogen isotopic composition of sedimentary organic matter on a time scale of hundreds of thousand years to million years. Here we present the results from two 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ODP Leg 201 Sites, 1230 and 1227, which represent two geochemically distinct environments. Methods Ammonium concentrations, its 8 1 5 N and the 8 1 5 N of solid phases were determined through the sediment column recovered from Sites 1227 and 1230. Bulk sediment samples used for isotopic analyses of solids were “squeeze cakes”, kept frozen between the time of collection and analyses. Samples were oven-dried at 60°C for 24 hours and then finely ground. TN (total nitrogen) and TC (total carbon) were measured on a Carlo Erba CHN-2500 elemental analyzer. TOC (total organic carbon) was estimated from 8 1 3 C of TC by assuming, the 8 1 3 C of carbonate is 0 %o and the 8 1 3 C of organic matter is -21 % > . TOC content obtained using this approach is in relatively good agreement with the values measured during Leg 112 (Suess and von Huene, 1988); Meister et al., this volume). Pore water samples collected for isotopic analyses were acidified (to approximately pH 4 for Site 1227 samples and approximately pH 1 for Site 1230 samples) and immediately frozen on board the RVV JOIDES Resolution. Prior to isotopic analysis, samples were thawed and mixed well. Ammonium concentrations were determined at USC, and the results were compared to the shipboard measurements in order to assure the adequate preservation of ammonium in the samples. The USC and the shipboard analysis for Site 1227 agree within 2 %. However, USC analyses of samples from Site 1230 are consistently lower than shipboard measurements by 10 %. The concentrations obtained at USC agree within 2 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. % with the amount of N in each sample, measured as a peak area during isotopic analysis. Therefore, we conclude that the 10 % difference between USC and shipboard analysis stems from the discrepancies in the calibrations. The loss of ammonium during storage is unlikely, since such loss would have resulted in the large magnitude scatter in 8 15N, which has not been observed. Ammonium was extracted on acidified glass fiber filters using a method developed by Sorensen and Jensen (1991) and Holmes et al. (1998). In brief, diluted pore water was placed in polypropelene centrifuge tubes, and pH was adjusted above 10 by addition of finely ground MgO, pre-combusted at 450°C. This converted NH4 + to uncharged NH3 . A “trap” made of a Teflon™ tape envelope containing GF/C or GF/F filter wetted with 20 pi of 2M H2SO4 was then added to each tube. The uncharged NH3 diffused through the Teflon™ membrane, was protonated inside the “trap” and collected on the filter. Nitrogen isotopic ratios of sediments and of ammonium trapped on the filters were measured at USC on an Isoprime Micromass mass spectrometer, interfaced with a Carlo Erba CHN-2500 elemental analyzer in the continuous flow mode. Isotopic ratios were determined against the reference N2 (Ultra High Purity Grade, Gilmore), which has been calibrated with the set of NIST standards, routinely run in the USC stable isotope lab. NIST standards were run daily as internal standards. The daily precision, based on the internal standards was 0.2 % o or better. Procedural standards of known isotopic composition, similar in concentration and volume to samples were prepared, and carried through the whole extraction procedure with each set of samples. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The sample standard deviation based on replicate analyses of both samples and standards was typically 0.50 % o . The SI5 N of procedural standards of ammonium was usually about 0.5 % o lighter than expected, likely due to either a procedural artifact or perhaps a small reagent blank. The amount of nitrogen in standards was always close to that in samples, so 8 1 5 N values of samples were corrected by adding 0.5 % o to the measured values. Site 1230 Site description and lithology Site 1230 is located on the lower slope of the Peru Trench (9°6.7525’S, 80°35.0100’W) within 100 m of the location of Site 685 of ODP Leg 201 (Fig. Ill - 1). Water depth at this site is 5086 m, but biogeochemical processes observed in the sediments are more typical of an ocean margin than a deep sea setting. The sediments at Site 1230 have been subdivided into two lithostratigraphic units. Unit I, from 0 to 215.8 mbsf is Holocene-Pleistocene, and consists of biogenic sediments mixed with siliciclastic components (D'Hondt et al., 2003; Suess and von Huene, 1988)). The sequence is characterized by alternating layers of diatom-nannofossil ooze and marl with dark gray to olive color clays. Graded layers indicate turbidite events, originating most likely near the shelf break and incorporating material from intermediate depths along the way (Suess et al., 1988). Unit II, from 215.8 to 278.3, was deposited during the Miocene and is separated from Unit I by an unconformity. The presence of a fractured layer at this horizon suggests the unconformity is of tectonic origin. Sediments consist of diatom and silt rich deposits, interbedded with clay rich layers. 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0" •5“ Site 1 0 " -15" -15" -2 0 " -20 " -75" -70" -90" -85" -80" km 0 200 400 Fig. Ill - 1 Map of locations, ODP Leg 201, Sites 1227 and 1230. 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Estimated sediment accumulation rates for Quaternary and Miocene sequences are 100 m/m.y. and 200 m/m.y. respectively (Suess et al., 1988). Results Biogeochemistry o f pore water Concentrations of both DIC (dissolved inorganic carbon) and ammonium, the major metabolites of microbial decomposition of organic matter, are exceptionally high at this site. Concentrations of DIC reach a maximum of 162 mM at 132 mbsf, decreasing to about 130 mM below 200 mbsf. Ammonium concentrations reach a maximum value of 38 mM at about the same depth as DIC; similar to DIC concentrations, they decrease below 132 mbsf to about 25 mM at the bottom of the measured profile at 250 mbsf (D’Hondt et al., 2003). Sulfate is depleted below detection limit within the upper 7 mbsf, and methane concentrations increase steeply below this horizon, reaching values up to 7 mM at the depth of 17 mbsf, and gradually decreasing downcore. The sediments at this site also contain methane hydrates (Suess et al., 1988; D’Hondt et al., 2003). Solid phase composition; £ > 5 N ofpore water ammonium and sedimentary organic matter TOC concentrations at Site 1230 have moderate values of about 2 wt % (Meister et al., this volume), TN ranges between 0.3 and 0.4 wt % (Table III -1, Fig. Ill - 2). C/N ratios are between 8 and 9 and are relatively constant through the whole sediment column . Nitrogen isotopic ratios of the sediments vary between 5 and 7.5 % o with an average value of 5.71 + 0.19 % 0, and do not show any significant 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table III - 1 Site 1230- Hole A (Leg 201) Sediments elemental composition, ammonium concentration and 6 1 5 N for sediments and pore water ammonium. Core, section, interval (cm) Depth (mbsf) TOC (wt %) TN (wt %) C/N ratio [NH4+] mM (m eas at U S C ) Pore water 51 5 N (% o) Sed-s 81 5 N (% o) 1H-1WR, 135-150 1.4 3.22 0.37 8.7 0.78 n/a 4.17 1H-2WR, 135-150 2.9 2.07 0.34 6.1 1.34 n/a 5.72 2H-2WR, 135-150 7.7 n/a 0.33 n/a 4.32 5.19 4.31 2H-5WR, 135-150 12.2 n/a 0.31 n/a 6.85 3.67 5.49 3H-2WR, 135-150 17.2 n/a 0.27 n/a 9.57 5.84 5.00 3H-5WR, 135-150 21.7 2.26 0.26 8.7 10.25 5.72 6.24 5H-5WR, 135-150 40.7 2.56 0.3 8.5 18.47 5.88 6.18 6H-3WR, 143-158 47.2 3.01 0.31 9.7 19.95 5.57 7.73 8H-3WR, 135-150 58.7 n/a 0.29 n/a 23.31 4.32 5.73 8H-5WR, 135-150 61.7 3.51 0.39 9.0 23.99 5.33 5.78 10H-1WR, 135-150 71.7 2.53 0.33 7.7 26.65 5.22 7.96 11H-1WR, 135-150 81.2 3.44 0.41 8.4 30.50 4.78 5.93 12H-2WR, 135-150 92.2 2.15 0.23 9.4 29.98 5.33 5.05 13H-1WR, 135-150 100.2 2.44 0.33 7.4 18.42 n/a 7.53 14H-1WR, 135-150 109.7 2.96 0.37 8.0 34.35 5.37 5.33 15H-2WR, 135-150 119.5 2.65 0.3 8.8 35.43 5.52 4.61 15H-5WR, 85-98 123.5 1.81 0.22 8.2 35.87 5.03 4.98 17H-1WR, 135-150 130.7 2.93 0.34 8.6 37.84 4.67 4.50 18H-2WR, 0-20 141.7 2.98 0.34 8.8 39.41 4.79 4.81 18H-4WR, 0-20 143.3 2.54 0.35 7.3 37.88 4.78 6.07 21H-4WR, 69-84 162.5 2.83 0.35 8.1 34.99 4.80 6.33 22H-1WR, 75-90 169.1 3.83 0.45 8.5 35.79 4.91 6.17 24H-2WR, 0-98 188.8 2.56 0.29 8.8 36.43 4.38 5.18 26H-1WR, 82-97 199.6 n/a 0.33 n/a 32.96 4.98 6.20 33X-1WR, 130-150 235.7 4.45 0.44 10.1 25.41 4.90 5.89 35X-1WR, 136-156 246.4 n/a n/a n/a 23.87 5.03 n/a 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Depth, m bsf Site 1230 Wt % of TOC 100 150 UNIT 200 UNIT 250 0.6 0.8 0.4 0.2 1 Wt % Of TN -■ -T O C , wt % ■ - n wt % Fig. Ill - 2 TOC and TN profiles in the sediments of Site 1230 (dashed line represents boundary between different lithologic units). 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. change with depth (Fig. Ill - 3). Three horizons, 4 7 , 7 2 and 100 mbsf, have higher § 15N values of 7 .5 to 8 %o. The isotopic composition of ammonium is relatively constant through the whole profile, with most values between 4.6 and 5.9 %o (Fig. Ill - 3). The average 51 5 N of pore water ammonium at this site is 5.04 + 0.11 %o, which is about 0.67 + 0.22 %o lighter than the average 8 1 5 N of sediments. Discussion Diagenesis and nitrogen isotopic composition o f pore water ammonium and sedimentary organic nitrogen The ammonium and TCO2 profiles indicate that the labile organic matter in Unit I is decomposed as it is buried, releasing both metabolites. The maximum TCO2 and ammonium concentrations create downward diffusion into Unit II, which apparently had lower solute concentrations of both metabolites before its tectonic juxtaposition with Unit I. Both units have organic matter with similar 8 15N, so diffusion of ammonium across the unconformity should not strongly influence the 51 5 N of ammonium. Indeed, our measurements showed no change in S1 5 N of pore water ammonium across the boundary. Since ammonium is the product of organic matter decomposition, we can calculate the amount of organic nitrogen (No rg ) lost as a result of diagenesis as a function of depth based on the amount of amount of ammonium released through the sediment interval. Two different approaches were employed in these calculations, and the results are compared. The first approach is based on flux calculations at the 8 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Depth, mbsf -2 Site 1230 81 5 N, % o 2 4 100 150 UNIT 200 UNIT 250 30 40 50 60 70 [NH4 ], mM 51 5 N-sed 81 S N- of ammonium [NH4 + ], mM Fig. Ill - 3 Ammonium profile, 51 5 N of ammonium and bulk sediments, Site 1230 (dashed line represents boundary between different lithologic units). 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. boundaries of Unit I and estimates nitrogen loss for all of Unit I. The second approach is based on fitting a diffusion-reaction model and calculates loss as an explicit function of depth. Approach I In approach I, we determined tangents to the ammonium profile near the top (1.35 mbsf) of the sediment column and near the ammonium maximum, (119.47 mbsf). We used these tangents to calculate diffusive fluxes based on Fick’s first law. The calculated difference of 0.56 |rmol/cm2 yr represents the net nitrogen released from solid phases through this interval. The present sedimentation flux of organic nitrogen has been calculated using the average sediment accumulation rate of 1 0 0 m/m.y. (D’Hondt et al. 2003), average porosity of 0.75 and wt % of nitrogen at the topmost interval of 0.37. The No rg sedimentation flux is equal to 1.59 pmol/cm yr. The ammonium production flux constitutes about 35 % of the No rg sedimentation flux, indicating that about a third of organic nitrogen deposited on the ocean floor has been decomposed and lost as ammonium through the interval between 1.35 and 119.47 mbsf. Approach II For approach II, we calculated the loss of No rg by using a reaction-diffusion model (Berner, 1980) applied to the sediment interval between 1.35 mbsf and 119.47 mbsf. The following assumptions were made: 1) the decomposition rate of organic matter decreases exponentially with depth, 2) ammonium profile is in steady state; 3) 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. advection is not important, (Peclet number equals 3, Table III - 2); 4) porosity is constant. The specified, fitted and calculated parameters are given in Table III - 2. With the aforementioned assumptions, the general diagenetic equation (Berner, 1980) becomes: 2 dC d C [1] = d s Y + R0 exP(_A ) = 0 d t g z Where C is the dissolved ammonium in the pore water, Ro is the reaction rate per unit volume of pore water at z=0, and other parameters are defined in Table III-2. The solution to equation [1] is given by: [2] C = A + A exp(-/?z) + A z , 0 1 2 Two boundary conditions were set to find Ao and A2 (Table 2): C=Co at z= 0 and C=Ch at z=h. Values of Ai and ( 3 (Table 2 ) were found by fitting equation [2] to the profile of ammonium, and used to calculate the value of Ro. These parameters can be used to calculate the expected change in solid phase nitrogen by integrating the reaction rate deduced from pore water ammonium and requiring it to balance the loss of solid phase nitrogen (No rg ): - 14 * io~~ 6 R n d [3] A N =----------------- (exp(-A) - 1), (1 - 8 )w fip where A N is the amount of organic nitrogen (in wt %) lost via bacterial decomposition. Assuming a constant supply of No rg through time, we calculated that 0.16 wt % of nitrogen has been lost in the interval between 1.35 and 119.47 mbsf 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table III - 2 Parameters for modeling of ammonium profiles and modeling results, Sites 1230 and 1227. Site 1230 Site 1227 Specified parameters___________________________________________________ Average porosity, © 0.75 0.8 Ds, Diffusivity (cm2 /sec)a 7.5*10"6 8.12*10"' w, Accumulation rate, cm/sec 3.17*10"1 0 6.34*10'1 K, ammonium adsorption coefficientb 1.3 1.3 Peclet number = Ds *p/((1+K)*w) 3 54 C0 , pmol/cm3 (per volume of sediments) 0.78 0.68 Ch , umol/cm3 (per volume of sediments) 35.42 6.76 Upper boundary, mbsf, Z0 = 0 1.35 1.35 h, lower boundary, mbsf, Zh = Z-upper boundary 119.47 36.95 p, density of solid phase, g/cm3 2.4 2.4 Fitted parameters p, inverse of the scale distance, m"1 A1 =R0 /(Ds *p2 ), pmol/cm3 A2 =1/h*(Co-Ch +A1 *(1-exp(-p*h))), pmol/cm4 Calculated parameters 0.026827 -19.106 0.00137 0.0967 -4.34 0.000527 R0 (calculated using Ad, pmol/cm3 *sec 1.02*10"1 1 3.3*10"1 1 A N, wt % 0.16 0.71 a Corrected for tortuosity, Dm /(1-(ln(por2)), where Dm = molecular diffusion at 5°C, 11.75 cm2 /sec (Boudreau, 1997; Li and Gregory, 1974) b adsorption coefficient typical for coastal sediments (Berner, 1980) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Table III - 2). This constitutes about 42 % of the amount of No rg present at the shallowest interval, which we consider as the initial input of No rg to the ocean floor (Table III - 1, Fig. Ill - 4). The result of approach II calculations are in good agreement (within 6 %) with the 35 % loss of No rg , calculated using Approach I. Fig. Ill - 4 shows the modeled profile of residual wt % of sedimentary nitrogen at Site 1230. From Fig. Ill - 4, it is apparent that sediments deeper than 30 mbsf, when they arrived at the sea floor, must have been more enriched in organic material than those that are shallower than 30 m. Average isotopic composition of ammonium is 0.7 + 0.2 % o lighter than the 8 1 5 N of N org. One possible explanation for this difference may be a small contribution from DON in the pore water, degraded during the ammonium extraction procedure (Sigman, 1997). DOC at this site reaches concentrations of 20 mM (Smith et al., this volume). DON concentrations have not been measured during Leg 201, but the contribution from DON is probably minor (D. Smith, pers. comm.). Another possibility is that the difference is a result of fractionation associated with release of ammonium from decomposing organic matter with fractionation factor, s = -0.7 + 0.2 % o. Despite the fact that approximately a third of organic nitrogen has been lost to diagenesis, there is no pronounced change in the isotopic composition of the sediments with depth. If the small difference between 51 5 N of ammonium and No rg is indeed due to fractionation during ammonium release, the expected change in the solid phase 8 1 5 N would have been negligibly small. The absence of depth dependency of 51 5 N of the sedimentary nitrogen and close similarity between 8 1 5 N of ammonium 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. S ite 1 2 3 0 Wt % N 0 0.1 0.2 0.3 0.4 0.5 0 - T p - 20 1 V T i 40 e ■ 0 f ■ 60 © ■ ■ 0 f ■ 80 o ■ <2 ■ 100 © ■ 0 l ■ 120 8 m ■ 140 ■ N wt % measured _ -O- - N wt % modeled Fig. Ill - 4 Site 1230, upper 119 mbsf - Results of modeling: modeled and measured N wt % for the sediments. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. solid phase N, both suggest that at Site 1230, little isotopic fractionation is associated with long term diagenesis of sedimentary organic matter. One additional implication is that the §1 5 N of material reaching the sediments at this site has been rather constant through time. Site 1227 Site description and lithology Site 1227 is located on the Peru margin at 8°59.46’S and 79°57.34’W, about 100 m from the location of site 684 of ODP Leg 112. The site was drilled in the Trujillo Basin, which is a small, fault-bounded pond of sediments. Water depth at this location is 427.5 m. Presently, this site is positioned within the upwelling system. The sediment succession consists of four litholigically distinct units, and contains a mixture of marine and terrestrial components (Suess et al., 1988; D’Hondt et al., 2003). This mixture is characterized by C/N ratios of 12-18 in bulk sediments (Fig. Ill- 5) Sediments of Unit I are less than 0.9 Ma old, mostly laminated, and consist of diatom-bearing silt and clay-rich diatom ooze with occasional foraminiferal ooze layers. Unit I has been deposited under a strong upwelling regime. Unit II, of Pliocene age, depth 11.1 -34.1 mbsf, contains dark olive, bioturbated, silty sediments, with glauconite and phosphate layers, which indicate possible winnowing of sediments during the times of low sea level stand (Suess et al., 1988). Variations in the amount of terrigenous input for Unit II and evidence for sediment winnowing by bottom currents are attributed to fluctuations in sea level. Unit III, of Pliocene age (Shipboard 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Scientific Party, 1988), depth 34.1-53.1 mbsf, is characterized by reduced diatom abundance, and a high percentage of clay, feldspar and quartz, which implies the presence of higher terrigenous input. Vertically graded sequences are common. Overall, the unit exhibits a fining upwards trend; sediments are bioturbated. A strong porosity minimum, accompanied by grain density and magnetic susceptibility maxima, is observed in the interval between 40 and 50 mbsf. Unit IV, of late Miocene age, depth 53.1- 151 mbsf, is separated from the overlying Unit III by a large unconformity, which represents a time hiatus between 5.7 Ma to 8.7 Ma years (Suess et al., 1988). The sediments of Unit IV are mostly dark green clays and nanno-fossil bearing ooze. Volcanic ash layers and volcanic shards occur through the unit. Laminated dolomite layers are present. Overall, this unit is characterized by a lower terrigenous input than the three stratigraphic units above it. It is also least affected by bioturbation and reworking by currents. Estimated sediment accumulation rates are 20 m/m.y. for the Quaternary sequence, 30 m/m.y. for the Pliocene, and 50 m/m.y. for the Miocene (Suess et al., 1988). Results Biogeochemistry o f pore water The concentrations of major metabolites and electron acceptors in the pore fluids of Site 1227 indicate the presence of active microbial communities in the sediments of Site 1227 (D’Hondt et al., 2003). Sulfate concentration decreases down- core from sea water values to below detection limit at about 40 mbsf (Fig. Ill - 5). 9000icxMethane concentrations increase steeply below 38 mbsf. 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Site 1227 20 S 0 4 2 -, mM 10 15 20 25 30 W .Q E 40 60 80 o Q 100 120 140 160 Fig. Ill - 5 Pore water sulfate profile at Site 1227 (D’Hondt et al., 2003) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DIC concentrations reach a maximum of about 25 mM at 38 mbsf depth, in the sulfate-methane transition zone. Below this horizon, DIC decreases to 20 mM, and concentrations remain constant through the rest of the sediment column recovered. Concentrations of pore water ammonium increase steadily with depth (Fig. Ill- 6 and 7) reaching 23 mM at the bottom of the profile. Near the boundary between units II and III, between 36 to 38 mbsf, the gradient of ammonium concentrations shows a pronounced kink. The core recovery of the interval between 43 to 72 m was poor (D’Hondt et al., 2003), which might have led to contamination of some samples with sea water. However, sulfate concentrations (Fig. Ill- 5) show little evidence of such contamination. The ammonium profde obtained during Leg 112 (Suess et al., 1988) also exhibits a change in the gradient at the same depth horizon. In contrast to ammonium, chloride concentrations increase steadily with depth, with no significant kinks in the profile. Possible mechanisms leading to the formation of the ammonium minimum are discussed below. The location of the “kink” coincides with the interval of sulfate - methane transition, with elevated rates of microbial activity. Elemental and nitrogen isotope composition o f the sedimentary organic matter and pore water ammonium Sediments from Site 1227 are rich in organic matter, which is typical for highly productive upwelling regions (Fig. Ill - 8 and Table III - 3). The weight percent of organic carbon is 5 to 9 %, and total nitrogen 0.4 and 0.6 %. C/N ratios vary between 12 and 18. Relatively high values of sedimentary C/N ratios are consistent 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Site 1227 8 15N , %o 6 10 UNIT I UNIT UNIT < * - W n £ £ a a > Q 100 150 81 S N of sediments 81 5 N of ammonium — o —Ammonium concentration 10 20 NH4 + , mM 30 Fig. Ill - 6 Isotopic composition of pore water ammonium, bulk sediments and ammonium concentrations, Site 1227, (dashed lines represent boundaries between lithologic units) 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Site 1227 Chloride, mM 0 500 1000 1500 60 - tn X ) E 80 - § ■ 1 0 0 - Q 120 - 140 - 160 15 25 10 20 0 5 NH4 +, mM ■ 0 — Chloride, mM -♦ -N H 4 + , uM Fig. Ill - 7 Concentrations of pore water chloride and ammonium, Site 1227 (D’Hondt et al., 2003). 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table III - 3 Site 1227 - Hole A (Leg 201) Sediments elemental composition and 6 1 5 N for sediments and pore water ammonium. Core-Section, Interval (cm) Depth (mbsf) TOC (wt %) TN (wt %) C/N [NH4+], mM (meas at USC) (mM) Pore water 815N (% o) Sed-s 81 5 N (% o) 1H-2WR, 135-150 2.85 3.21 0.31 10.5 1.25 8.45 7.42 2H-3WR, 135-150 9.95 1.25 0.07 17.7 3.54 7.84 3.84 2H-5WR, 135-150 12.95 6.11 0.47 13.0 5.12 7.84 3.83 3H-1WR, 135-150 16.45 5.93 0.46 12.9 4.45 5.63 2.57 3H-5WR, 135-150 22.45 7.97 0.56 14.1 5.81 6.38 2.60 4H-5WR, 95-110 31.21 8.89 0.48 18.4 5.96 6.17 2.25 5H-2WR, 135-150 36.95 8.38 0.58 14.6 6.38 5.46 2.12 5H-4WR, 135-150 39.35 6.02 0.45 13.5 n/a 4.46 5H-5WR, 135-150 41.45 8.87 0.68 13.0 7.65 5.48 2.43 6H-4WR, 135-150 49.95 3.60 0.31 11.8 8.61 5.01 3.93 7H-2WR, 135-150 55.95 n/a n/a n/a 9.14 5.66 n/a 9H-3WR, 135-150 76.45 8.05 0.48 16.7 n/a 9.36 10H-2WR, 135-150 84.45 n/a 0.46 n/a 15.51 5.35 8.46 11H-2WR, 135-150 93.95 8.75 0.54 16.2 15.77 5.45 9.42 12H-2WR, 135-150 103.45 9.25 0.56 16.4 16.65 5.18 9.55 13H-1WR, 135-150 111.45 6.05 0.47 12.9 18.03 4.90 7.18 14H-1WR, 135-150 120.95 5.57 0.36 15.3 19.21 5.23 10.44 17H-1WR, 85-90 132.95 6.42 0.43 14.8 20.91 5.09 9.33 18H-2WR, 135-150 144.30 n/a 0.61 n/a 22.49 4.77 8.35 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Depth, mbsf Site 1227 TN, wt % 0 0.2 0.4 0.6 0.8 1 UNIT .O UNIT III 100 UNIT IV 150 TOC, wt % and C/N wt ratio B n w t % 0 TOC w t % . . C/N ratio Fig. Ill - 8 TOC and TN of the sediments, Site 1227 (dotted lines represent boundaries of lithologic units). 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with episodic input of terrestrial organic matter, suggested by the sedimentological evidence. The overall trend of C/N increasing with depth through the upper 35m may indicate possible preferential remineralization of N containing compounds. Sedimentary §1 5 N values for these lithologic units vary (Fig. Ill - 6 , Table III - 3). Nitrogen isotope ratios in Unit I change from 6 . 8 to 4 %o downcore. Sediments of Units II and III have 8 1 5 N values between 2 and 4 %o. Unit IV (Miocene sediments) is characterized by significantly heavier isotopic values, about 9 to 10 %o. Nitrogen isotope ratios of pore water ammonium show a different pattern (Fig. III - 6 , Table III - 3) and far less variation than solid phases. 8 1 5 N of ammonium is 3 to 4 % 0 heavier than sedimentary 51 5 N in the upper 36 mbsf, (Units I and II), and become progressively lighter from about 8 %o at the sediment-water interface to about 5 %o at 36 mbsf depth. The isotopic composition of ammonium in pore water of Units III and IV is lighter than sedimentary S1 5 N and decreases from 5 %o in Unit III and the upper part of Unit IV to about 4.5 % o down core. In Unit III, the ammonium isotopic composition is fairly close to the sedimentary 8 15N; in Unit IV, ammonium is about 4 %o lighter than the sedimentary 8 15N. (Fig. Ill - 6 ). Discussion S lsN and sources o f pore water ammonium at Site 1227 Pore water biogeochemistry and the vertical profile of ammonium and its isotopic composition at Site 1227 are very complex. Increasing concentrations of pore water ammonium downcore usually indicate continuous degradation of organic matter at depth. However, the pore water chemistry of Site 1227 is influenced by a 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hypersaline subsurface brine of Miocene age (Suess et al., 1988; D ’Hondt et al., 2003). The presence of the brine is manifested by increasing concentrations of Mg2 + , Ca2 + , Na+ and Cl' with depth (D’Hondt et al., 2003). A linear relationship between NH4 + and Cl' concentrations (Fig. Ill - 9) below 36.95 mbsf suggests that the predominant source of ammonium diffusing towards this horizon is the brine, rather than decomposing organic matter. The concave-down shape of the plot in the region above this horizon indicates that in this interval, ammonium is added by decomposition of organic matter. The chloride-ammonium plot also shows a pronounced “kink” at 36 to 38 mbsf, which is evidence for a net loss of ammonium at this depth. In order to calculate the magnitude of the sources and sinks of ammonium, and their isotopic contribution to the observed 8 1 5 N ammonium profile, we constructed a two-layer reaction-diffusion model, using 2.85, 36.95 and 54.45 mbsf as the boundaries. The following assumptions were made: 1) decomposition of organic matter decreases exponentially with depth, 2 ) the ammonium profile is in steady state; 3) advection is not important, (Peclet number equals 54, Table 2). The boundary conditions for 1 4 N and 1 5 N equations are the measured 1 4 N and 1 5 N ammonium concentrations at the specified boundaries of 2.85, 36.95 and 54.45 mbsf. Solutions to the diagenetic reaction-diffusion equation [2] were fit to the profiles of 1 4 N and 1 5 N profiles separately, and fluxes of 14NH4 + and 15NH4 + at the boundaries were calculated. These were used to calculate 8 1 5 N of ammonium fluxes at the boundary horizons. 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ammonium, mM Site 1227 25 20 15 10 5 ° 600 800 1000 1200 Chloride, mM Fig. Ill - 9 Ammonium vs. chloride concentrations in the pore water of Site 1227; note the “dip” at 36-38 mbsf. 97 1 — I — |— I — j— |— l l— r - ] — I — i — I — I j— ! i— i — i— j — i — ! — I — I — j i— I — i I — | I M r~ 36-38 mbfs • * • ...» 1 1 I L _ J I I I i ( I I i I I i I j ! i I I 1 I 1 I I I L_ Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The results of calculations are summarized in the Fig. 111-10. The diffusive flux of the 7 ic ammonium from the brine at 54.45 mbsf is 0.29pmol/cm yr, and it has a 8 N of 4.4 % o. The gradient steepens between 54.45 and 36.95 mbsf as a result of lower porosity in Unit III, so that the upwards flux through Unit III is approximately the same as at 54.45 mbsf. At depths around 36.95 mbsf, where the ammonium profile shows the pronounced “kink” (Fig. Ill -10), approximately 80 % of the upwards ammonium flux is removed, so the upward flux above the region of the apparent sink is only 0.05 7 ic pmol/cm yr. The 8 N of the ammonium flux leaving the “sink” region is 0.13 % o . Isotopic mass balance of the fluxes requires that S1 5 N of the ammonium sequestered in the “sink” region is 5.3 % o. This is 0.9 % 0 heavier than the 8 1 5 N of the brine and implies positive isotopic fractionation associated with the sink. Diffusive flux at the 7 1 ^ topmost horizon (2.85 mbsf) is 1.15 pmol/cm yr with 8 N of 7.3 % o. From the mass balances we calculate that 1.1 pmol/cm2 yr is added in Units I and II, with an isotopic composition of 7.6 % o. Fig. Ill - 11 presents a mixing diagram, where 8 1 5 N of ammonium is plotted against the inverse concentrations, [N H /]'1 . The diagram illustrates the conclusions obtained by modeling. Deviations from linearity reflect sources or sinks of nitrogen that differ from those defined by the end points of the diagram. If diffusion is the only mechanism of importance in transport, the y-axis intercept of a tangent to the relationship defines the isotopic composition of the nitrogen diffusing away from the source. 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. 1 81 5 N, % » 2 6 10 UNIT I Flux leaving at 2.85 _2 mbsf 1.15 @ 7.31 % o ^ _ — ^ 1 i I | I I I J I I I | 1 I 1 | I J ^ l | 1 1 1 | UNIT II Flux leaving the “ sink", 0.05 @ 0.13 >». 50 towpomsftyla^yffi''' 100 150 UNIT IV Flux from the brine, 0.29 < ® 4.4 % o Ammonium “ sink^p 4{ 0.24 @ 5.3 % « ‘ ‘ - f * t V i i ■ * \ ■ ■ ■ ■ 1 ■ ■ ■ ■ * ■ ■ ■ ■ * ■ ■ ■ ■ * ■ ■ ■ ■ * ■ ■ ■ ■ 0 10 20 NH4 + , mM 30 Fig. Ill -10 Site 1227 Schematic representation of ammonium fluxes and §1 5 N of the fluxes (in pmol/cm2yr), based on the assumption of steady state. V O VO The diagram (Fig. Ill- 11) shows the presence of three isotopically different sources: 1) ammonium diffusing upwards from the brine, with S1 5 N of about 4.4 %o; 2) ammonium that is added in Unit III, with an isotopic composition of > 6 %o; 3) ammonium with isotopic composition of about 7.6 % o in the upper portion of the profile. Source 2 is probably small and is not readily apparent in the ammonium profile (Fig. Ill - 8 ) because of the curvature created by the low porosity zone in Unit 3. The low porosity zone has little impact on the ammonium-chloride plot (Fig. Ill- 9). Two sinks are apparent. One is near 36 mbsf and removes nitrogen about 1 %o heavier than the ammonium at that horizon. The loss of isotopically heavy ammonium at 36.95 mbsf (from the modeling results) appears on the mixing diagram as the offset of the four points (inside the gray circle) to the right from the hypothetical mixing line between the ammonium of 4.4 %o and 7.6 %o. Positive isotopic fractionation associated with the ammonium sink is evidence that ammonium sequestration at this horizon is not biotic but may be abiotic, perhaps into some unknown mineral phase. However, we did not observe a corresponding increase in solid phase N wt % near this horizon. The second sink is diffusion to the sediment-water interface, where ammonium is oxidized. The average isotopic composition of the bulk sedimentary nitrogen in the upper 36.95 mbsf of the sediments is 3.8 %o. This is about 4 %o lower then the 8 1 5 N of ammonium flux (=7.6 %o) added to the pore water through this interval. So, the question arises, what is the mechanism of the apparent isotopic enrichment of pore 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Source III: isotopically enriched ammonium from Unit / " 10 Average uncertainty in 516N m easurem ents Sink II: diffusion into overlying water Hypothetical mixing line Between Source I and III to Source II: ammonium with S,5N > 6 %o~~~ Sink I Source I: ammonium 4 from the brine 0.8 1.0 0.2 0.4 0.6 0.0 ♦ Above sulfate/methane transition 1/[NH4 ], mM 1 ■ Below sulfate/methane transition o Transition zone Fig. Ill - 1 1 Site 1227. Mixing diagram (the parenthesis around two data points indicate that the measured values may be questionable). Grey circle designates the intervals, associated with the apparent sink o water ammonium relative to the organic nitrogen in the upper 36 mbsf of the sediments at Site 1227? The interpretation of the nature of the isotopically heavier source strongly depends on whether the ammonium profile at Site 1227 is in the steady state condition. Both CITNH/relationship and the mixing diagram (Fig. Ill - 9 and 11) are consistent with ammonium addition through the lithologic Units I and II, apparently from the decomposition of organic matter. If the ammonium and 8 1 5 N profiles are in steady state, then the enrichment of ammonium in heavier isotopes signifies the loss of isotopically heavier nitrogen from the organic matter through this horizon. In this case, as diagenesis progresses, the preserved organic matter should become isotopically lighter. An alternative scenario is that the ammonium profile represents a non-steady state conditions, where 8 I5 N of ammonium is strongly affected by the contribution from the decomposition of very recently deposited organic matter with 8 1 5 N > 8 % o; this would create a diffusive front that would propagate downward. In the subsequent sections, we consider each scenario and discuss the implications for the isotopic composition of the preserved organic matter. Steady-state scenario Assuming steady state, we calculated the ammonium production in the interval between 1.35 mbsf and 36.96 mbsf. Applying Approach I described for Site 1230, we find that present day ammonium flux, produced throughout this interval cannot be supported by the sedimentation flux of No rg with the N concentration equal to the topmost measured interval, which is 0.31 wt %. The ammonium production flux is 2 2 equal to 0.77 pmol/cm *yr, while No rg sedimentation flux equals to 0 . 2 2 pmol/cm yr 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (average porosity 0.8 and wt % of N = 0.31, measured in the topmost interval of this site). The unsupported ammonium flux may result from the variability of the weight percent of organic nitrogen through time, as indicated by our downcore measurements. Applying approach II, described for Site 1230, and using equation [3], we calculated that 0.71 wt % of nitrogen should have been lost at the 36.95 mbsf horizon, in order to support the ammonium flux produced within this interval (specified and calculated parameters given in Table 2). The same assumptions as described for Site 1230 were made. Currently, the measured content of sedimentary nitrogen at this depth is 0.58 wt % (Fig. Ill - 5 and table 3). This means that, in order to account for the observed rate of ammonium release to the pore water, about 55 % of original organic matter should be decomposed at the depth of 36.95 mbsf. Fig. Ill- 12 shows the modeled profile of residual organic nitrogen, presented as a fraction of original N org left after degradation. The 4 %o difference between the § 15N of pore water ammonium and sedimentary organic nitrogen represents an effective fractionation of nitrogen isotopes during the decomposition of organic matter. Applying Rayleigh fractionation expression, we can calculated the predicted change in § 15N of preserved organic nitrogen as diagenesis progresses (Fig. Ill - 1 2 ). Figure 1 2 demonstrates that the degradation of 50 % of original organic matter accompanied by fractionation of 4 % 0 should leave the preserved fraction isotopically lighter by 3 %o. Isotopically heavier ammonium might be released via one of the following mechanisms: ( 1 ) preferential degradation of isotopically heavier organic N (Macko and Estep, 1 9 8 4 ) , that might be more labile; 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (0 A a. a > a 51 S N 4 6 10 12 0 10 20 81 S N-Nsed measured 81 5 N-Nsed predicted Fraction of original N sed left Wt % of N measured 30 40 1.2 0.8 0.4 0.0 Fraction of original N left Fig IV -12 Site 1227, upper 36. 95 mbsf, results of modeling: modeled and measured 8 1 5 N of sedimentary organic matter, measured profile of TN wt %, and calculated fraction of sedimentary nitrogen left after degradation (based on rate of degradation, determined by modeling of the ammonium profile). 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (2) Preferential assimilation of isotopically lighter ammonium into bacterial biomass (Lehmann et al., 2002); or ( 3 ) preferential decomposition of an isotopically heavier, more labile marine fraction of organic matter relative to a more refractory terrestrial component (Sweeney and Kaplan, 1980). High C/N ratios in the sediments of Site 1227 imply that significant terrestrial nitrogen is present. This latter scenario is, most likely, responsible for the isotopic enrichment of pore water ammonium relative to the sedimentary nitrogen. The measured 8 1 5 N of sedimentary organic nitrogen in Units I and II (Fig. Ill - 12) become progressively lighter downcore, changing from 6 . 8 %o at the surface to about 4 %o in the lower part of Unit I, and reaching values between 2.5 and 3 %o within Unit II, which is consistent with the loss of isotopically heavier ammonium at steady state. A 3 % correction, applied to the values measured in the lowest part of Unit II, would bring 8 1 5 N of Pliocene organic matter to about 5 %o. However, this must be a mixture of marine and terrestrial components. Evaluating our assumption of the steady state condition at Site 1227, we conclude that the steady state scenario leaves three problems unresolved: ( 1 ) the apparent ammonium sink near 36 mbsf; (2) the lack of significant fractionation associated with this apparent sink (implying that the sink is due to inorganic precipitation of an unidentified and unexpected solid phase); (3) the large decrease in organic N that is predicted from the ammonium flux, but is not observed. Non-steady state scenario Altabet et al. (1995) and Ganeshram et al. (1995) have suggested that the interglacial times are characterized by globally intensified upwelling conditions. 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Biomass produced in upwelling regions typically has 8 15N around 8 - 1 0 %o or heavier (Altabet et al., 1999a; Ganeshram et al., 2 0 0 0 ) . If a pulse of organic rich material with this heavier 8 15N has been deposited within the last few thousand years, under the regime of intense post-glacial upwelling, a corresponding pulse of isotopically enriched ammonium should be released into the pore water. At present, the isotopically heavier ammonium from the pulse of organic matter deposited during the last 10 Kyr would reach a depth of about 30 mbsf (if % = 2 ^ D gt , where x = mean distance of diffusion, and Ds = 8.12*10'6 cm2 /sec is diffusion coefficient of ammonium in the sediments with porosity 0.8 and t is time). In this case, the isotopic enrichment of pore water 8 1 5 N relative to S1 5 N of Norg stems from the addition of the isotopically heavier end member, rather than fractionation during the decomposition of organic matter. Also, the curvature in ammonium profile is likely to result partially from this non-steady state condition, instead of only continuous organic matter decomposition through the whole 40 m sediment sequence. The apparent “ammonium sink” (Fig. Ill - 8 ) may reflect a non-steady state behavior of the ammonium profile as well, rather than localized consumption of ammonium at this horizon. With an average sedimentation rate of 20 m/m.y., estimated for the Site 1227, the upper 2 m of the sediment column would represent approximately 100 Kyr. Our first isotopic measurement was done at a depth of 2.85 mbsf, so 8 1 5 N of the modem surface sediments is unknown. The reported isotopic composition of modem organic matter in this region is between 6 and 8%o (Libes and Deuser, 1988). Those measurements are from a location about 2° to the south from the location of Site 1227. 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ganeshram et al. (2000) reported sedimentary values of 8 1 5 N for the last 100 Kyr to vary between 6 and 11 %o at a site located about 4° south from Site 1227. From these studies, we can infer the values of 8 1 5 N of the organic matter deposited in this region to be most likely between 8 and 10 %o or higher (Altabet et al., 1999a; Ganeshram et al., 2000) . Then, the isotopic composition of ammonium through the upper 36.95 mbsf of the sediment column at Site 1227 may be dominated by mixing between ammonium of 8 to 1 0 %o released in the upper 1 - 2 mbsf, and ammonium coming from the brine (with isotopic composition of 5 %o). Further modeling of the ammonium isotopic profile is required to test this hypothesis. Summary To summarize the previous discussion, interpretation of isotopic composition of pore water ammonium, which is required to assess the impact of diagenetic processes on 8 1 5 N of preserved organic matter, strongly depends on the presence or absence of steady state conditions. At steady state, if ammonium is not involved in any other reactions, its isotopic composition reflects 8 1 5 N of decomposing organic matter plus any fractionation associated with the process of degradation. At Site 1230, the ammonium released during diagenesis is less than 1 % 0 lighter than the bulk material, so that little fractionation is associated with diagenesis of marine organic matter. Organic matter at this site is largely of marine origin and its isotopic composition has not varied significantly through time, which is consistent with the absence of fractionation inferred from the ammonium 8 15N. 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. At Site 1227, the interpretation of 8 1 5 N of ammonium profile is complicated by several factors: addition of iso topically distinct ammonium from the brine, and variability in concentrations and isotopic composition of deposited organic matter through time, which possibly leads to the non-steady state condition of the current ammonium profile. If the ammonium profile at Site 1227 is currently in steady state, then an effective fractionation exists, about 4 % 0, between decomposing organic matter and released ammonium. In this case, 50 % loss of original organic matter should leave the 51 5 N of the residual fraction depleted in 15 N by about 3 % o. The 51 5 N of No rg does become progressively lighter with depth, which is consistent with the net loss of isotopically heavier ammonium. Organic matter at this site is a mixture of marine and terrestrial component. The net loss of an isotopically heavier marine fraction would leave the residual No rg isotopically lighter, due to the increased relative proportion of more refractory (and 1 4 N enriched) terrestrial organic matter, as suggested by Sweeney and Kaplan (1980). However, it appears that at Site 1227 several factors have varied through time including the wt % N, its lability, and the isotopic composition. If these changes have occurred in the recent past, the present profile of ammonium concentrations and its 8 1 5 N might have not reached a new steady state. The time-dependant variation in 8 1 5 N of the organic matter deposited on the ocean floor would result in mixing pattern between ammonium released in the upper few meters of the sediment column and ammonium diffusing from below. The mixing between the two end members with 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. different isotopic composition would lead to the apparent isotopic enrichment of ammonium relatively to the organic matter at depth. The mixing pattern at Site 1227 may be additionally complicated by the recent variations in the rates of decomposition (depending on the lability) and supply (wt % of N) of deposited organic matter, which lead to the non-steady state conditions in the ammonium concentration profile as well. Synthesizing results from Sites 1227 and 1230 we can conclude that no significant fractionation is associated with diagenesis of marine organic matter, even when a substantial portion of it is degraded. When a significant fraction of a terrestrial component is present, preferential decomposition of the marine fraction might lead to overall decrease in 8 1 5 N of bulk sediments down core. However, further work in a less complex system than Site 1227 is required to confirm this conclusion. Acknowledgements: This study has been supported by NSF grant OCE-0136500 to DH and an ODP Schlanger fellowship to MGP. The authors gratefully acknowledge members of the Scientific Party of Leg 201, who collected samples for us. We are also thankful to the chief-scientists of Leg 201, Drs. S. D’Hondt and B. Jorgensen for being very helpful in obtaining the samples, thus making this study possible. The technical assistance of M. Rincon and T. Gunderson at USC is greatly appreciated. 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References: Altabet, M.A., Deuser, W.G., Honjo, S. and Stienen, P., 1991. Seasonal and depth- related changes in the source of sinking particles in the N. Atlantic. Nature, 354(136-139.). Altabet, M.A. and Francois, R., 1994. Sedimentary nitrogen isotopic ration as a recorder for surface ocean nitrate utilization. Global Biochemichal Cycles, 8(103-116.). Altabet, M.A., Francois, R., Murray, D.W. and Prell, W.L., 1995. Climate related variations in denitrification in the Arabian Sea from sediment 15N/14N ratios. Nature, 373(506-509). Altabet, M.A. and McCarthy, J.J., 1985. Temporal and spacial variations in the natural abundance of 1 5 N in PON from a warm -core ring. Deep Sea Research, 32(7): 755-772. Altabet, M.A., Murray, D.W. and Prell, W.L., 1999a. Climatically linked oscillations in Arabian Sea denitrification over the past 1 M.Y.: Implications for the marine N cycle. Paleoceanography, 14(6): 732-743. Altabet, M.A. et al., 1999b. The nitrogen isotope biogeochemistry of sinking particles from the margin of the Eastern Tropical Pacific. Deep-Sea Research I, 46: 655- 679. Berger, W.H. and Keir, R.S., 1984. Glacial-Holocene atmospheric CO2 and the deep sea record. In: Hansen J.E. and T. Takahashi (Editors), Climate processes and climate sensitivity. Geophys. Monogr. Ser. AGU, Washington D.C., pp. 337- 351. Berner, R.A., 1980. Early Diagenesis: A theoretical approach. Princeton University Press, Princeton, NJ, 241 pp. Boudreau, B.P., 1997. Diagenetic models and their implementation: modeling transport and reaction in the aquatic sediments. Berlin, Heidelberg, NY, Springer, 414 pp. Broecker, W.S., 1982. Glacial to interglacial changes in ocean chemistry. Progress in Oceanography, 11: 151-197. D'Hondt, S., Jorgensen, B.B. and Miller, J., et al., 2003. Proc. ODP, Init. Repts. [CD- ROM], 201:. (Ocean Drilling Program), College Station, TX. 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Falkovsky, P.G., 1997. Evolution of the nitrogen cycle and its influence on the biological sequestration of CO2 in the oceans. Nature, 387. Falkowski, P.G., 1997. Evolution of the nitrogen cycle and its influence on the biological sequestration of CO2 in the oceans. Nature, 387: 272-275. Francois, R., Altabet, M.A. and Burke, H.L., 1992. Glacial to Interglacial changes in surface nitrate utilization in the Indian section of Southern ocean as recorded by sediment 8 15N. Paleoceanography, 7: 589-606. Ganeshram, R.S., Pedersen, T.F., Calvert, S., E., G.W., M. and M.R., F., 2000. Glacial-Interglatial variability in denitrification in the world's coeans: Causes and consequences. Paleoceanography, 15(4): 361-376. Ganeshram, R.S., Pedersen, T.F., Calvert, S., E. and J.W., M., 1995. Large changes in oceanic nutrient inventories from glacial to interglacial periods. Nature, 376: 755-758. Gruber, N., in press. The marine nitrogen cycle. In: M. Follows, Oguz, T. (Editor), Carbon - climate interactions, NATO ASI series. John Wiley & Sons, New York. Haug, G.H. et al., 1998. Glacial/interglacial variations in production and nitrogen fixation in the Cariaco Basin during the last 580 Kyr. Paleoceanography, 13(427-432.). Hoch, M.P., Fogel, M.L. and Kirchman, D.L., 1992. Isotope Fractionation Associated with Ammonium Uptake by a Marine Bacterium. Limnology and Oceanography, 37(7): 1447-1459. Holmes, R.M., McClelland, J.W., Sigman, D.M., Fry, B. and Peterson, B.J., 1998. Measuring N-15-NH4+ in marine, estuarine and fresh waters: An adaptation of the ammonia diffusion method for samples with low ammonium concentrations. Marine Chemistry, 60(3-4): 235-243. Lehmann, M.F., S.M., B., Barbieri, A. and McKenzie, J.A., 2002. Preservation of organic matter and alteration of its carbon and nitrogen isotope composition during simulated and in situ early sedimentary diagenesis. Geochimica et Cosmochimica Acta, 66(20): 3573-3584. Li, Y.-H. and Gregory, S., 1974. Diffusion of ions in sea water and deep-sea sediments. Geochimica et Cosmochimica Acta, 38(703-714). I l l Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Libes, S.M. and Deuser, W.G., 1988. The Isotope Geochemistry of Particulate Nitrogen in the Peru Upwelling Area and the Gulf of Maine. Deep-Sea Research Part a-Oceanographic Research Papers, 35(4): 517-533. Macko, S. A. and Estep, M.L.F., 1984. Microbial alteration of stable nitrogen and carbon isotopic compositions of organic matter. Organic Geochemstry, 6 : 787- 790. Pride, C. et al., 1999. Nitrogen isotopic variations in the Gulf of California since the last deglaciation: Response to global climate change. Paleoceanography, 14(3): 397-409. Redfield, A.C., Ketchum, B.H., Richards, F.A.„ 1963. The influence of organisms on the composition of of sea-water. In: M.N. Hill (Editor), The sea. Wiley- Interscience, New York, pp. 26-77. Sachs, J.P. and Repeta, D.J., 1999. Oligotrophy and nitrogen fixation during Eastern Mediterranean sapropel events. Science, 286: 2485-2488. Sigman, D.M., 1997. The role of biological production in Pleistocene Atmospheric Carbon Dioxide variations and the Nitrogen Isotope dynamic of the Southern Ocean, Ph.D. Thesis., MIT/WHOI, 97-28. Sigman, D.M., Altabet, M.A., Francois, R., McCorkle, D.C. and Gaillard, J.-F., 1999. The isotopic composition of diatom-bound nitrogen in the Southern Ocean sediments. Paleoceanography, 14: 118-134. Sigman, D.M. and Boyle, E.A., 2000. Glacial/Interglacial variations in atmospheric carbon dioxide. Nature, 407: 859-869. Silfer, J.A., Engel, M.H. and Macko, S. A., 1992. Kinetic fractionation of stable carbon and nitrogen isotopes during peptide bond hydrolysis: Experimental evidence and geochemical implications. Chemical Geology, 101: 211-221. Suess, E. and von Huene, R., et al., 1988. Proc. ODP, Init. Repts., 112. (Ocean Drilling Program), College Station, TX,. Sweeney, R.E. and Kaplan, I.R., 1980. Natural abundances of 1 5 N as a source indicator for near shore marine sedimentary and dissolved nitrogen. Marine Chemistry, 9: 81-94. Tyrell, T., 1999. The relative importance of nitrogen and phosphorus on oceanic primary production. Nature, 400(400): 525-531. 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IV: Impact of long term diagenesis on 81 5 N of organic matter in marine sediments (continued): ODP Leg 202- Sites 1234, 1235 and 1238 (submitted to ODP Volume 202, Scientific Results) Maria G. Prokopenko, Douglas E. Hammond and Lowell Stott Introduction The 8 1 5 N of organic matter preserved in marine sediments has been used over the last decade as a paleoceanographic proxy (Altabet and Francois, 1994; Altabet et al., 1999 b; Altabet et al., 1999 a; Emmer and Thunell, 2000; Ganeshram et al., 1995; Pride et al., 1999; Sachs and Repeta, 1999; Sigman et al., 1999). However, diagenetic processes may lead to alteration of the original isotopic ratios in sedimentary organic matter, No rg . The magnitude and sign of the diagenetically induced changes in the original isotopic ratios of No rg as well as geochemical conditions under which such changes might occur are currently not well understood. Previous studies addressed the problem of fractionation associated with diagenesis in sinking particles (Altabet and Francois, 1994; Altabet et al., 1999 a), in the upper few cm of the sediments (Freudenthal et al., 2001; Sigman et al., 1999) and in the laboratory experiments (Lehmann et al., 2002; Macko and Estep, 1984). The effects of the longer time scale diagenesis only recently have been investigated (Prokopenko et al., submitted). The goal of this work is to evaluate the impact of diagenesis on the isotopic composition of preserved organic matter on the time scales of 105 to 106 years. This article extends work on samples collected during the previous ODP expedition, Leg 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 201 at Sites 1227 and 1230, (Prokopenko et al, submitted). Here we discuss the results obtained from three sites: 1234,1235 and 1238 (Fig. IV -1). Our approach is to construct mass balances for nitrogen isotopic ratios of the sedimentary organic nitrogen, No rg , and pore water ammonium, which is the major metabolic product of No rg decomposition. The premise for this approach is that in the absence of any other diagenetic reactions, ammonium released from the decomposing organic matter should have 8 1 5 N of the source plus any fractionation that takes place during diagenesis (Prokopenko et al., submitted). Methods Ammonium concentrations, TN (Total Nitrogen), as well as the nitrogen isotope composition of solid phase and dissolved pore water ammonium were determined through the sediment column recovered from Sites 1234,1235 and 1238. The isotopic composition is reported in permil vs. air standard, using delta notation (S15N). The procedure of sample processing and isotopic measurements is given in Prokopenko et al. (submitted.) Here we discuss only the details specific for the samples collected during the Leg 202. In order to assure the adequate preservation of ammonium during storage, the ammonium concentrations were determined at USC, and the results were compared to the shipboard measurements. The ammonium concentrations obtained at USC for Sites 1234 and 1235 are lower than shipboard measurements by 7 to 10 %. The concentrations obtained at USC agree within 2 % with the amount of N in each sample, measured independently by elemental analysis of filters with trapped ammonium. The loss of ammonium during storage of the 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To1 ^ e o 1 Tgo1 Tqo1 ^ 7 5 " 1238 S o u t h A m e r ic a ■20 ’ - 20 ' Pacific Ocean o 1234 •40* Fig. IV - 1 Location map of Sites 1234, 1235 and 1238. The dotted line represents the backtrack of Site 1238 within the last 12 My. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. samples is unlikely, since such loss would have resulted in a positive correlation between 8 1 5 N values and the magnitude of the difference, which has not been observed. Therefore, we conclude that the 7 - 1 0 % difference between USC and shipboard analysis for Sites 1 2 3 4 and 1 2 3 5 stems from discrepancies in the ammonium calibrations. Pore water samples from Site 1238 were inadvertently defrosted during storage and remained unfrozen for about 2 weeks. Concentrations of ammonium determined at USC shortly after this were lower than the shipboard analysis by 1 0 to 4 5 % (Table IV -1). Table IV -1 shows that intervals with lowest ratios between USC and shipboard analysis ( [ N H 4 +] usc/[ N H 4+] st,iP board) have heavier 8 15N compared to neighboring data points with higher yields. Since 8 15N of ammonium at Site 1 2 3 8 shows a depth dependency (Table IV- 1), we fitted an exponential function to the depth profile of ammonium 8 15N , and compared all data points to the values predicted by this function. The plot of the 8 15Ndiffe re n c e vs. [NH4+ ]U S c/[NH4+]sh ip b o a rd revealed that samples with yields lower than 8 0 % are significantly heavier and show a large scatter in their 8 15N values (Fig. IV - 2 ) . We took this as evidence that in those samples the loss of ammonium has led to a change of original isotopic composition. Therefore, for Site 1 2 3 8 , we report ammonium 8 15N only for those samples in which USC and shipboard ammonium concentrations agreed within 2 0 %. It is possible that these samples also lost ammonium during storage, perhaps 1 0 %, if the calibration discrepancy at Site 1 2 3 8 is similar to Site 1 2 3 4 and 1 2 3 5 . Based on Fig. IV - 2 , a 1 0 % 1 1 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV -1 Ammonium concentrations measured at USC and shipboard analysis; effect of loss of ammonium during storage on S15N. Core Section Interval [NH4+] USC (mM) [NH4+] shipboard (mM) USC/ shipboard measurements3 (%) n h4 + 51 5 N (% o) Samples accepted a 001H WR 02 145-150 0.13 0.0 6.59 y 002H WR 03 145-150 0.57 0.7 81 6.43 y 003H WR 03 145-150 0.81 1.1 73 5.01 n 004H WR 02 145-150 1.46 1.8 81 5.09 y 005H WR 03 145-150 1.67 2.1 80 5.07 y 006H WR 03 145-150 1.91 2.5 76 5.18 n 007H WR 03 145-150 2.26 3 75 4.33 n 008H WR 03 145-150 2.17 2.8 77 4.09 n 009H WR 03 145-150 2.50 2.9 86 3.77 y 010H WR 03 145-150 2.11 2.9 73 4.17 n 011H WR 03 145-150 2.04 3 68 3.79 n 012H WR 03 145-150 2.15 2.9 74 4.44 n 013H WR 03 145-150 1.89 3 63 3.27 n 014H WR 03 145-150 2.48 2.9 86 3.62 y 015H WR 03 145-150 2.62 3.1 85 3.18 y 016H WR 03 145-150 2.16 2.9 74 3.41 n 017H WR 03 145-150 2.29 3.4 67 4.70 n 018H WR 03 145-150 2.45 2.9 85 2.62 y 019H WR 03 145-150 1.27 2.8 45 4.95 n 020H WR 03 145-150 2.50 2.8 89 2.56 y 021H WR 03 145-150 1.73 3 58 4.50 n 022H WR 03 145-150 3.2 024XWR03 145-150 2.53 2.9 87 1.94 y 026XWR03 145-150 1.91 2.8 68 028XWR03 145-150 2.8 030XWR03 145-150 2.43 2.9 84 1.70 y 032XWR03 145-150 2.78 3.2 87 2.04 y 034XWR03 145-150 2.13 3.2 67 4.51 n 036XWR03 145-150 2.13 3.1 69 038X WR 03 145-150 2.46 3 82 1.70 y 040X WR 03 140-150 2.57 2.8 92 1.41 y 043XWR02 90-100 2.49 3 83 1.18 y 045x WR 02 140-150 2.32 2.4 97 a all sam ples with USC/shipboard > 80 % were used 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Difference betw een m easured §1 5 N < D JZ w 0) 3 n s > T J C (0 3.5 3.0 2.5 2.0 0.5 100% -0.5 [NH4 ]usc^ [ ^ ^ 4 Ishipboard’ Fig. IV - 2 Storage effect on the isotopic composition of pore water ammonium for Site 123 8 ; Y-axis represents the difference between measured 8 1 5 N of NH4 + and the values from a smoothed fit to the profile (see explanation in the Methods section); X-axis is the ratio of USC and ship board measurements of ammonium concentration in %. Only values to the right of the dashedline were accepted and considered in this study. 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. loss may have caused 8 1 5 N measurements to be about 1 %o heavy for all the samples from Site 1238. We assume that this did not occur, but cannot be certain. Average precision of 8 15N, based on running duplicates and procedural standards, was 0 .2 0 % o for the pore water ammonium and 0 .1 5 %o for the sediments, unless indicated otherwise. Due to the high carbonate content (sediments were not acidified), the sediment samples from the lowest intervals at Site 1238 have small concentrations of No rg , often « 0.1 wt % of N. Consequently, the absolute amount of nitrogen in these samples is small. Therefore, isotopic measurements of the sediments from the lower intervals have relatively poor precision. All sediments from Site 1238 were run in duplicates. Analytical precision, calculated as standard deviation of duplicate analysis, is indicated by error bars for each individual interval (Fig. IV - 6 c). Depths for all three sites are reported in mcd (meter composite depth) (See explanation in Mix et al., 2003). Sites 1234 and 1235 - Central Chile margin Sites 1234 and 1235 are located close to each other, have similar depositiona! histories and diagenetic settings, and therefore are discussed together. Sites description and lilhology Both sites are located 60 km offshore. Site 1235 (36°9.594'S, 73.33.983'W) is 7 km north-east from site 1234 (36°13.153'S, 73°40.902'W) (Fig. IV - 1). Water depths are 1015 m at Site 1234 and 489 m at Site 1235. Sediments at both sites are characterized as a single lithologic unit, representing continuous hemipelagic 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sedimentation within the last < 260 Ka (Mix et al., 2003). The location of the sites during the Quaternary was within a strong upwelling system off Point Conception, resulting in high content of diatomaceous and calcareous nannofossils. Biogenic material is strongly diluted with high terrigenous input, the degree of dilution is higher for the shallower Site 1235 (Mix et al., 2003). The siliciclastic component is comprised mostly of silty clays and clays. The biogenic component constitutes up to 30 % of sediment material at Site 1234, but only 10 to 15 % at Site 1235. Accumulation rates are estimated to be exceptionally high, 788 m/my at Site 1234 and 696 m/my at Site 1235 (Mix et al., 2003).The lower concentration of biogenic material and lower sediment accumulation rate at the shallower Site 1235 suggests sedimentary winnowing at this location. Both sites also contain sedimentary sequences interpreted as distal turbidites (Mix et al., 2003). Results Biogeochemistry o f the sediments and pore water at Sites 1234 and 1235 Both sites are characterized by a moderate to low content of organic carbon and nitrogen, as expected due to the high degree of dilution with siliciclastic terrestrial material (Mix et al., 2003). At Site 1234, TOC ranges between 0.6 and 2.5 wt %, TN between <0.1 and 0.3 wt % (Fig. IV - 3); the TOC/TN (atomic) ratios vary between 4 and 11, with an average value of 8.3 ± 0.3. Both TOC and TN concentrations exhibit a maximum between 60 and 80 mcd. High pore water ammonium concentrations (about 10 mM) and relatively low porosity (60 %) may cause a significant amount of NH4+ to adsorb to clays. Assuming 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. ■ o o E Q . O Q 50 100 150 200 250 Site 1234 - Total Nitrogen TN, wt % 0.1 0.2 0.3 0.4 0.5 Site 1235 - Total Nitrogen i ° ° 1 < £ i° ° ° B r Diatom-rich interval 0 Shipboard measurements ■ USC measurements 0.1 TN, wt % 0.2 0.3 0.4 0.5 Corresponds to opal maximum 100 150 200 ■ °2 250 Fig. IV - 3 Elemental composition of No rg (wt %) for Leg 202, Site 1234 and 1235 h — * to an ammonium partition coefficient of 2 (Mackin and Aller, 1984), an average ammonium concentration of 9.8 + 1.9 mM and an average TN of 0.12 + 0.01 wt %, as much as 2 0 % of the total nitrogen in the bulk sediment may be the in the pool of the adsorbed ammonium. At Site 1235, both TOC and TN concentrations are lower than at Site 1234, ranging between 0.5 to 1 wt % and 0.05 to 0.12 wt % respectively; TOC/TN (atomic) ratios vary between 4 and 1 1 , with an average of 8 . 2 + 0.3. As at Site 1234, there are maxima in the TOC and TN concentration, but at depths between 30 to 40 mcd. The contribution of inorganic nitrogen to the TN pool at Site 1235 is about 20 %, comparable to that of Site 1234. This is based on the average TN of 0.08 + 0.003wt % and the average ammonium concentrations of 5.2 + 0.4 mM. The depth profiles of major metabolites (N H / and TCO2) concentrations are similar at the two sites as well (Figures IV - 4 a and b, Tables IV - 2 and IV - 3) (Mix et al., 2003). At Site 1234, DIC concentrations reach 71-72 mM at a depth of 30 to 40 mcd. Ammonium concentrations reach the maximum of ~ 12 mM at the similar depth. Sulfate is depleted below the detection limit at 9 mcd. At Site 1235, the DIC maximum of ~ 60 mM is located in the interval of 30 - 40 mcd, and ammonium concentrations reach their highest values of ~ 8 mM at the same depth interval. Sulfate concentrations go below the detection limit within the upper 19 mcd. At both sites, the DIC and ammonium maxima in pore water are roughly coincident with maxima in TN and TOC (Fig. IV - 3). 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Isotopic composition o f sedimentary organic matter and pore water ammonium The depth profiles of 51 5 N of sedimentary organic nitrogen (No rg ) are similar at Sites 1234 and 1235, although the isotopic ratios at Site 1235 are about 1 %o lighter than at Site 1234. At Site 1234, the uppermost interval measured (9.65 mcd) has 8 1 5 N of 11 %o (Table IV- 2, Fig. IV - 4). The values decrease to - 9 % o at 50 mcd and remain constant from 50 to 40 mcd. At 150 mcd, the 8 1 5 N of No rg increases to ~ 11 %o, and then gradually returns to 9.5 % o downcore. The average isotopic composition of No rg at Site 1234 is 9.8 + 0.2 % 0 (Table IV- 4). At site 1235 (Table IV- 3, Fig. IV - 4b), S1 5 N changes gradually from about 10 %o at 1.45 mcd (the uppermost interval measured) to ~ 8 %o at depth of about 70 mcd. Horizon at 9.5 mcd, a possible turbidite deposit, has anomalously low 8 15N, where N org is about 7 %o. With the exception of two horizons of small (up to -9.5 %o) enrichment, the 8 1 5 N remains constant at 8 %o through the rest of the sediment column downcore. The average 8 1 5 N of Norg at Site 1235 is 9.0 + 0.2 %o. The two sites exhibit similar trends in isotopic composition of the pore water ammonium (Fig. IV - 4 a and b) as well. At Site 1234 (Fig. IV - 4a), 8 1 5 N of ammonium decreases from 10.5 to about 9.5 %o within the upper 60-70 mcd, then increases by - 0.5 %o from 70 to 120 mcd. Between 120 mcd and the bottom of the sediment column, the 8 1 5 N declines from 10 to 8.5 %o. The average 8 1 5 N of ammonium at Site 1234 is 9.4 + 0.1 %o, which is 0.4 + 0.3 %o lighter than the solid phase isotopic composition (Table IV- 4). 123 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV - 2 Site 1234, TN elemental composition, 81 5 N o f ammonium and No r g (* n/a indicates no analysis available). Core Section Interval Depth, (mcd) Sediment TN (wt %) Sediment 81 5 N (% o) [NH4 + ], meas. at USC (mM) Pore water ammonium 81 5 N 001H WR1 145-150 1.45 n/a* n/a 1.13 n/a 002H WR3 145-150 9.65 0.11 11.11 4.78 10.41 003 H WR3 145-150 17.89 0.11 11.27 7.27 10.08 004HWR 3 145-150 29.67 n/a 10.01 9.05 9.81 005H WR3 145-150 40.37 0.13 9.94 10.94 9.61 006H WR3 145-150 51.77 0.14 9.07 10.94 9.42 007H WR3 145-150 62.87 n/a n/a 12.00 9.29 008 H WR3 145-150 74.74 0.10 9.28 n/a n/a 009H WR3 138-148 87.93 0.19 9.85 10.23 9.36 010H WR3 133-143 99.32 0.09 8.98 9.56 9.52 011H WR3 136-146 111.48 0.10 8.81 9.17 10.05 012X WR3 140-150 122.75 n/a n/a 9.27 9.92 013XWR3 140-150 132.80 0.13 9.25 9.46 9.25 014XWR3 140-150 144.11 0.12 11.39 9.78 9.47 015XWR2 140-150 153.95 n/a n/a 9.47 8.74 016XWR3 140-150 166.79 n/a n/a 9.78 9.49 017XWR1 125-135 174.95 0.16 10.26 9.91 8.95 018X WR3 140-150 189.56 n/a n/a 9.43 9.38 019XWR2 115-125 199.13 n/a 8.72 8.65 8.94 020XWR2 140-150 210.82 0.09 9.69 9.53 8.69 021X WR3 140-150 223.66 0.10 8.90 8.06 8.48 022XWR3 140-150 235.12 9.79 7.16 8.41 Average (uncertainties shown 0.12 + are stdev of the means) 0.01 9.8 + 0.2 9.4 + 1.9 9.3 + 0.2 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV - 3 Site 1235, TN elemental composition, 51 5 N o f ammonium and N o r g , (n/a same as Table IV- 2). Depth (mcd) Sediment Sediment [NH4+], Pore water Core Section Interval TN (wt %) 515N (% o) meas. at USC (mM) ammonium 81 5 N (% o) 001H WR1 145-150 1.45 0.11 10.24 0.26 n/a 002H WR3 145-150 9.49 0.06 6.99 1.34 n/a 003H WR3 145-150 19.65 0.07 9.89 5.07 9.44 004H WR3 145-150 31.04 n/a 9.63 8.18 9.20 005H WR3 145-150 42.12 0.08 9.53 8.36 8.55 006H WR3 143-148 53.55 n/a n/a 6.93 8.91 007H WR3 145-150 65.81 0.07 8.10 6.01 8.72 008H WR3 134-139 76.96 0.06 8.66 5.48 8.45 009H WR3 124134 88.03 0.08 9.48 4.86 10.22 010H WR2 140-150 96.86 0.09 9.73 4.46 8.52 011H WR3 130-140 108.49 0.07 8.69 4.69 8.63 012H WR3 140-150 120.19 n/a n/a 5.30 8.89 013H WR3 140-150 132.04 0.07 9.54 4.65 9.25 014H WR3 150-160 142.56 0.07 10.01 6.17 8.46 015H WR3 140-150 154.13 0.09 8.54 6.17 8.40 016H WR3 140-150 166.12 0.07 8.71 4.95 n/a 017H WR3 150-160 180.42 0.09 8.82 6.37 7.77 018H WR3 140-150 191.18 n/a n/a 5.84 n/a 019H WR 3 140-150 201.6 0.08 8.86 5.26 8.35 020H WR4 140-150 210.32 0.07 7.21 5.25 9.38 Average (uncertainties shown are stdev of the means) 0.083 + 0.003 8.98 + 0.2 5.2+ 0.4 8.8+ 0.1 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 815N, % o 6 8 10 12 14 0 [T&rvii^X 1 Site 1234 1015 m - water depthG 50 100 150 200 25 15 20 0 5 10 « §1 5 N- No rg ► ...51 5 N-NH4 + [NH4 + ], mM 81 5 N, % » Site 1235 489 m water depth £ ■ o 100 E £ ■ 150 Q 200 250 8 10 12 14 6 0 2 4 [NH4+ ], mM Fig. IV - 4 Site 1234 (a) and Site 1235 (b), pore water ammonium profile (USC results are plotted) and isotopic composition of pore water ammonium and No rg . Dotted lines indicate depths where sulfate is depleted below detection limit. Note the difference in scales for ammonium concentrations at the two sites. 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The shallowest interval where 8 1 5 N of ammonium has been measured at Site 1235 (Fig. IV - 4b) is 9.5 mcd (coincident with the possible turbidite deposit). At this depth, 51 5 N of ammonium is 5.3 %o. Downhole, the ammonium 51 5 N is 9.5 %o at 19.7 mcd, decreases to 8.5 % 0 at 70 mcd, increases by about 1 %o within the next 80 mcd, and oscillates between 9.3 and 7.8 %o between 150 and 210 mcd. At Site 1235, the average difference 8 1 5 N of the pore water ammonium and No rg is 0 . 2 + 0.3 %o (Table IV - 4), which is within the limits of the analytical precision. Discussion Diagenesis o f organic matter at Sites 1234 and 1235 Site 1235 is located at a shallower depth than Site 1234, which has led to a higher input of terrigenous siliciclastic component to the sediments at this site (Mix et al., 2003). However, the total sediment accumulation rates at Site 1235 are lower than at the deeper neighboring site. A likely reason for this is sediment winnowing by benthic currents, suggested by the presence of turbidite deposits. The combination of sediment winnowing and the higher degree of dilution with siliciclastic material resulted in the lower concentrations of TOC and TN at Site 1235, compared to Site 1234. As a consequence, the rate of organic matter decomposition (per volume of sediments) is lower for Site 1235 as well. This is consistent with the lower DIC and ammonium concentrations, and deeper penetration of sulfate. The average TOC/TN ratios of about 8 (atomic) at both sites indicate that the organic component does not contain significant amounts of terrestrial organic matter. 127 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV - 4 Summary for Site 1234 and 1235 from Leg 202 and Site 1230 from Leg 201 (Prokopenko et al., submitted). Site Site 1230 Site 1234 Site 1235 Average Porosity, 0 0.75 0.6 0.6 Diffusivity, Ds, (m2 /sec)a 7.46*1 O'1 0 5.81 *1 O ’1 0 5.81 *1 O'1 0 Sediment accumulation rate, (m/sec) 3.17*10"1 2 2.50*10'1 1 2.21*10"1 1 w, Pore fluid burial rate, (m/sec) (if 1.54*10'1 1 different from sediment burial rate) “ u Scaling distance'1 (m'1 ) 0.027 0.046 0.080 Peclet number = Ds *p/((1+K)*w)b 2.74 0.47 0.21 Average 81 5 N of No rg , (% > ) 5.7 + 0.9 9.8+ 0.2 9.0+ 0.2 Average 81 5 N of ammonium, (%0 ) 5.0+ 0.1 9.4+ 0.1 8.8+ 0.1 Apparent fractionation factor, e, (% o ) -0.7+ 0.2 -0.4 + 0.3 -0.2+ 0.3 a Ds=Dm/(1-ln(02 ) ) (Boudreau, 1997; Li and Gregory, 1974) b K = 2, ammonium partition coefficient, dimensionless Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The profiles of DIC and ammonium indicate that the net release of both metabolites appears to cease at depths of about 50 mcd at Site 1234 and 40 mcd at Site 1235. Below this depth, the gradient in the ammonium concentrations at Site 1234 is zero, while at Site 1235, there is a significant decrease in ammonium below 40 m (Fig. IV - 4 a and b). High sediment accumulation rates strongly influence the solute transport through the sediment column at both sites. The fast accumulation results in low (< 1) Peclet numbers for both sites (Table IV- 4). The low values of Peclet numbers indicate that diffusive transport is less significant than advection in regulating the depth distribution of solutes in the pore water, particularly at Site 1234 (Peclet number of 0.27). It is most likely, that the ammonium decreases at depth at both sites do not reflect a sink, but rather are due to higher release rates near the depths of the apparent concentration maxima, in comparison to adjacent horizons. Due to the strong influence of advection, diffusive transport is not able to homogenize the ammonium profiles at depth. Net loss o f sedimentary nitrogen during diagenesis High pore water ammonium concentrations at both sites suggest that some substantial fraction of organic matter has been degraded. We can estimate the magnitude of the lost fraction of No rg by comparing the flux of ammonium produced through the interval of active diagenesis to the time-averaged flux of No rg delivered to the sea floor. One limitation of this calculation is that steady state must be assumed. The maxima in ammonium profiles indicate this assumption is not strictly correct. 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, it provides a useful approximation. The amplitude of the maxima in pore water profiles is < 50 % greater than concentrations at depth, evidence that the steady state assumption used to calculate the N released by diagenesis should not be in error by more than 50 %. The total ammonium released to the pore fluids by the decomposition of No rg in a specified layer should be the sum of ammonium lost via diffusion plus an advective loss from this layer due to burial of pore water. The net ammonium production between two horizons (P n ) can be calculated as: 4-1 P N = ( J m - j d 2 ^ + (^j A 2 ~ j A \ > where Jdi and Jd2 are diffusive fluxes across the upper (Dl) and lower (D2) boundaries of the interval, and JA 2 and JA i represent advective transport across the lower (A2) and upper (Al) boundaries. The diffusive fluxes at the upper and lower boundaries were calculated by applying Fick’s first law. The concentration gradients were determined as tangents to the ammonium profile at the boundaries of the considered interval. Advective fluxes were calculated as follows: w 4-2 J A = C nw{\ + K - ? - ) w 0 A Pw v w Pw pw where Cp w is the concentrations at the boundaries, K is ammonium partition coefficient, 0 is porosity, wpw is advection rate of pore fluid relative to the sediment- water interface, and ws is sediment accumulation rate (Table IV - 4). In the absence of compaction, ws = wpw. 130 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The calculated ammonium production, Pn, represents the total amount of N lost from the sediments due to organic matter degradation. The total flux of No rg deposited on the ocean floor is calculated based on sediment accumulation rates and the concentrations of TN at the bottom of the zone of active diagenesis (below 62 mcd for Site 1234, and below 42 mcd for Site 1235) plus the flux of ammonium transported out of the specified interval. We calculated that at Site 1234, the total flux of ammonium produced within the upper 52 mcd is equal to 0.030 mmol/m2d. This constitutes 18 % of the 0.18 mmol/m2 d of No rg flux deposited on the ocean floor. At Site 1235, the ammonium flux produced within the upper 32 mcd is slightly lower, 0.023 mmol/m2d. This represents 19 % of the 0.12 mmol/m2 d total No rg accumulation flux. To summarize, about 20 % of No rg is lost at both sites during diagenesis of organic matter. As mentioned before, due to the possible non-steady state condition, this might be underestimated by a factor of 1.5. Processes affecting the SI5 Nprofiles o f pore water ammonium The most prominent feature of the isotopic profiles at both sites is a noticeable similarity between the 8 1 5 N of ammonium and 8 1 5 N of the No rg (Fig. IV - 4 a and b). The values of Peclet number smaller than 1 (Table IV - 4) indicate that the contribution of the advection substantially decreases the degree of openness of the diagenetic system at both sites. Thus, only a modest fraction of the ammonium released at a particular horizon is able to diffuse away (since the sink location at the sediment-water interface is moving rapidly upwards due to the sediment accumulation). 131 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. At Site 1235, the S1 5 N of ammonium follows very closely the 8 1 5 N of N o rg (Fig. IV - 4b). The averages for both isotopic ratios are very similar at this site as well, suggesting that no isotopic fractionation occurs either during ammonium release in the upper 40-50 mcd, or below, where net ammonium release ceases. At Site 1234, the shape of 51 5 N of ammonium profile bears strong resemblance to the 8 1 5 N of the No rg , but below about 60 mcd it appears to be offset upwards relative to the 8 1 5 N of N org- The thickness of offset intervals increases with depth, suggesting that the observed offset is depth and/or time dependant. From this, we conclude that the observed offset is, probably, due to the difference between the burial velocities of fluids and solids. Such difference arises from the sediment compaction, which extrudes pore fluids upwards as sediments are buried. Compaction of the sediments results in the decrease of porosity downcore within a certain interval of compaction, below which the compaction ceases and sediment porosity becomes constant. We can adjust the vertical profile of ammonium S1 5 N values for the effect of the compaction by computing the offset, yz, between the pore water and solids at each depth horizon. The offset, yz, can be calculated at each depth horizon, z, using equation 4-3: 4-3 Zz ~ vW)dt where vs = burial velocity of solids, vw = burial velocity of pore water relative to the sediment-water interface, t=time. dz The vs can be recast as — ) = v , so that equation 4-3 becomes 4-4: dt s s ’ n 132 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-4 *z = J o (!— )& V 5 where z=depth. We assume a linear decrease in porosity through an interval of thickness b, so that porosity changes downcore according to equation 4-5 : 4-5 0 =0A-mz, z 0 ’ where m = — — — , 0 Z = porosity at depth z, 0 O = porosity at sediment-water b interface, 0 b=porosity at the base of the compaction zone, b = depth of the base of the compaction zone. As shown by Berner (1980), steady state compaction requires the following equation to be true: 4-6 where F* = velocity of pore water = velocity of solids below compaction zone. For the pore water, equation 4-6 can be written as: 4-7 (1 - 0)vw = ( l - 0 b )Vb, Substituting 4-7 into equation 4-4, we obtain: 0 1 - 0 a 4-8 X, = £(1 - = £(! + « - where a - 1 - 0 /, Evaluating the integral in equation 4-8 on the interval from 0 to z, we obtain: a n r* Q dz a , . _ . 4-9 | — — = ----- ln(0 o -m z) 0 q - mz m 133 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Then the offset, x can be found as: (7j — ff]7 4-10 ^ = ( 1 + a)z + i ln(^ _ _ ) Porosity measurements indicate a systematic decrease from 0.65 near the surface to 0.55 at 250 mcd. Assuming that this depth was b, the base of the compaction zone, we can calculate the offset between solids and fluids at each depth horizon. Fig. IV - 5 shows the adjusted as well as measured profiles of the 8 1 5 N ammonium to illustrate the effect of compaction. No evidence for isotopic fractionation during diagenesis The adjusted 8 1 5 N ammonium profile in Fig. IV - 5 closely follows the 8 I5 N of No rg at each depth horizon. One implication of such close similarity between S1 5 N of ammonium and No rg is that no isotopic fractionation is associated with the advection. The slight difference between 8 1 5 N of ammonium and No rg is probably due to the diffusive mixing between the horizons with variable isotopic composition of organic matter (Fig. IV - 5), wherever local isotopic gradients are formed. Fig. IV - 5 also demonstrates that, once the impact of advection and diffusion is accounted for, the S1 5 N of ammonium reflects the isotopic composition of organic matter with no fractionation between the two ratios. It is important to note also, that no fractionation is observed through the part of the profile where ammonium is being presently released, within the upper 70 mcd. At site 1235, the ammonium S1 5 N follows the sedimentary 8 1 5 N more closely, and the effect of compaction seems to be missing. At this site, no porosity gradient 134 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 81 5 N, % o for the adjusted ammonium profile and No rg 100 100 x: a - 150 150 200 200 ~v .o r. 250 250 300 300 9 8 14 11 12 13 10 81 5 N, % > for the measured ammonium profile -©— 61 E N -N H 4 81 5 N-NH/ Fig. IV - 5 The effect of advection on the depth profile of 8 1 5 N of pore water ammonium at Site 1234. Note that the scale for 8 1 5 N measured profile is offset by 2 % 0 relative to the adjusted profile for illustrative purposes. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was observed. It is possible that higher input of siliciclastic material makes the sediments at Site 1235 less compact than at Site 1234. The close similarity between the ammonium and No rg 8 1 5 N profiles indicate that little fractionation is associated with the long-term decomposition of organic matter at Sites 1234 and 1235, where ~ 15 to ~ 25 % of organic matter has been lost to degradation. These findings support the previous inference (Altabet et al., 1999 b; Altabet et al., 1999 a; Pride et al., 1999) that at sites with the high degree of organic matter preservation, the 8 1 5 N of No rg is also well preserved. As factors controlling the extent of organic matter diagenesis, these authors considered 1 ) poor oxygenation of bottom water, 2) high concentrations of organic matter and 3) rapid sediment accumulation rates. The O2 concentration of the bottom water does not affect diagenesis in the sediments few meters below sediment-water interface, considered in this study. The factor determining the relatively high degree of organic matter preservation at both sites appears to be the rapid sediment accumulation rate, rather than organic matter concentration, since the sediments at both sites have low to moderate amounts of Co rg - Site 1238 - Pacific equatorial waters Site description, lithology and depositional history Site 1238 is located 200 km off the coast of Equador (1°52. 310'S and 82°46. 934'W). The water depth is 2203 m. The total recovered sequence of 424.7 m (467 mcd) represents continuous sedimentation for the last ~ 11 Ma (middle Miocene). During this time, the Nazca plate subduction moved the location of Site 1238 about 136 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 600 km eastward and about a degree north, relative to South America’s position (Fig. IV - 1) (Mix et al., 2003). As a result of the transit, the location might have experienced changes in sea surface temperature and nutrient availability, as well as water depth (Mix et al., 2003). The tectonic transit of this location along the equator eastward has kept Site 1238 in the region strongly influenced by the equatorial upwelling, which is reflected in the depositional history of the site. The sediment succession was described as one lithologic unit, subdivided into two subunits, based on a higher degree of diagenesis in the deeper part of the sediment column. Subunit la (0 to 400 mcd) consists of the bioturbated nannofossil ooze and diatom nannofossil ooze, with a small siliciclastic (mostly clays) component. Micritic carbonate appears at 100 mcd and gradually increases downcore, indicating continuing postdepositional carbonate precipitation. A downcore decrease in magnetic susceptibility has been interpreted as result of decrease in terrigenous component or diagenetic dissolution of magnetic minerals (Mix et al., 2003). Elevated Mass Accumulation Rates (MAR) of a biogenic component between 3.5 and 6.5 Ma (160-370 mcd), as well as between 1 and 2 Ma (50-100 mcd) suggest that these were times of increased biological productivity (Mix et al., 2003). Sediments deposited during the last 1 Ma (upper 50 mcd) show a decrease in biogenic MAR. The decrease in biogenic material in the 1 Ma has been also inferred from elevated magnetic susceptibility in the sediment deposited during this time (Mix et al., 2003). Sediments in Subunit lb (400 to 467 mcd) contain lithified diatom and nannofossil oozes interbedded with the chalk and chert horizons, which has been 137 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. interpreted as an indication of the increased lithification and diagenesis experienced by the sediments of this subunit. The accumulation rate was about 50 m/my during the Pleistocene and upper Pliocene, and about 50 m/my during the lower Pliocene and upper Miocene. Below 350 mcd, in the sediments of middle Miocene, the sedimentation rate is estimated to be only ~ 17m/m.y. Results Geochemistry o f the sediments and pore water TOC and TN are moderate to low in the sediments of Site 1238. TOC decreases gradually from 1.5 wt % near the top of the sediment column to 0.5 wt % at the bottom of the sediment sequence (Mix et al., 2003). The decrease is largest through the upper 20 m. The interval 80 to 100 mcd has elevated TOC relative to other horizons, with values 2-4 wt %. TN shows a similar pattern (Fig. IV - 6 a). Carbonate content gradually increases downcore from < 20 to 95 wt % (Mix et al., 2003). TOC/TN atomic ratios vary between 5 and 13, with an average of 9.6 + 0.4. Concentrations of pore water TCO2 and ammonium reach their respective maxima of 17.5 mM and 3 mM in the depth interval between 80 and 100 mcd, where elevated TOC and TN concentrations occur (Fig. IV - 6 b and 6 c). Below the maximum, the TCO2 concentrations decrease steadily downcore to the value of 2.3 mM. The decrease is caused by precipitation of authigenic micrite (Mix et al., 2003). From the shallowest point to the maxima, the TCO2 and ammonium profiles show curvature, which suggests the continuous decomposition of the organic matter in the upper 90 mcd of the sediments. Sulfate concentrations (Fig. IV - 6 b) decrease from 138 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TN, wt % 0.2 0.4 0.6 0 50 100 o 150 a . o a 200 250 300 350 400 450 500 • • ■r ° o ° Shipboard measurements ■ USC measurements Sulfate, mM 10 15 20 25 30 100 150 o 200 250 300 350 • Sulfate 4 S — Alkalinity 400 450 500 0 5 10 15 20 Alkalinity, mM Fig. IV - 6 Site 1238 geochemistry: a) TN wt % concentrations in the sediments; b) depth profiles of sulfate and alkalinity (Mix et al., 2003); 139 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 815N, % » -4 -2 0 2 4 6 §1 100 T 3 200 o E a. 300 0) Q Subunit la 400 Subunit lb 500 [NH4 + ], mM - ° - [N H 4 + ], mM 81 5 N- No rg ...+...81 5 N-NH4 + o in 3 < D “ D = m O t t i o a C D i< 3 8 s g n s I 3 C D C D Fig. IV - 6 (continued) c) pore water ammonium profile (USC measurements) and isotopic composition of pore water ammonium and N0rg. The time scale is based on sediment accumulation rates (Mix et al., 2003). Error bars for the sedimentary 51 5 N represent 2 a s of the replicate analysis. 140 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26.8 mM at the first interval sampled (2.95 mcd depth) to about 8 mM in the interval between 90 and 100 mcd and remain at a constant 8 mM below this depth (Mix et al., 2003). The curvature in the sulfate profile in the upper 90 m (Mix et al., 2003) indicates that sulfate reduction occurs continuously through the whole interval. SI5 N ofpore water ammonium and sedimentary organic nitrogen Isotopic ratios of No rg vary with depth through the sediment column. §1 5 N decreases from 5 %o at top of the sediment column to about 2 -3.5 %o in the interval from 20 to 110 mcd (Table IV - 5, Fig. IV - 6 c). Values are significantly lighter below this depth, 0 to 2 % 0. The overall trend of solid phase S1 5 N is that of a decrease from 5 %o to 1 %o down-core. The profile of 8 1 5 N of pore water ammonium (Fig. IV - 6 ) shows a gradual decrease from 6.7 % o at the first interval sampled (2.95 mcd), to about 1 % 0 at the bottom of the profile. The isotopic gradient is steepest in the upper 80 mcd of the profile, where values change from 6.7 to 3.8 %o. Below this depth, the 8 1 5 N decreases by another 2 %o towards the bottom of the sediment column cored (420 mcd). In the upper 75 mcd, 8 1 5 N of pore water ammonium is 2-2.5 %o heavier than sedimentary organic matter. Between 80 and 115 mcd, S1 5 N of solid phase and pore water ammonium converge, with the pore water enriched by about 1 %o relative to the solid phase. From 120 to about 180 mcd ammonium 8 1 5 N is 1 to 2 % 0 heavier than sedimentary nitrogen. From 180 mcd downwards ammonium and Norg 8 1 5 N are within 1 %o. 141 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV - 5 Site 1238, TN elemental composition, 6 1 5 N of ammonium and N org (asterisks indicate intervals, where the values were affected by the loss of ammonium during storage and, thus, were excluded from consideration), n/a same as Table IV - 2. Core, section, interval (cm) Depth (mcd) Sediment TN (wt %) Sediment 51 5 N (% o) [NH4+] meas. at use (mM) Pore water ammonium 515N (% o) 001H WR 02 145-150 2.95 0.15 4.723 0.13 6.59 002H WR 03 145-150 10.07 0.09 4.760 0.57 6.43 003H WR 03 145-150 20.83 0.12 2.501 0.81 * 004H WR 02 145-150 32.26 0.08 3.478 1.46 5.09 005H WR 03 145-150 42.77 0.14 1.923 1.67 5.07 006H WR 03 145-150 54.32 n/a n/a 1.91 * 007H WR 03 145-150 64.26 0.12 2.364 2.26 * 008H WR 03 145-150 74.47 0.10 3.17 2.17 * 009H WR 03 145-150 82.37 n/a n/a 2.50 3.77 010H WR 03 145-150 92.62 0.14 2.24 2.11 ★ 011H WR 03 145-150 103.36 0.12 3.258 2.04 * 012H WR 03 145-150 113.32 0.09 3.016 2.15 * 013H WR 03 145-150 124.72 0.13 1.724 1.89 * 014H WR 03 145-150 135.42 0.09 0.768 2.48 3.62 015H WR 03 145-150 146.71 n/a 0.878 2.62 3.18 016H WR 03 145-150 156.58 n/a n/a 2.16 * 017H WR 03 145-150 167.81 0.11 0.797 2.29 ★ 018H WR 03 145-150 177.96 n/a 1.63 2.45 2.62 019H WR 03 145-150 188.87 n/a 0.17 1.27 * 020H WR 03 145-150 198.62 n/a n/a 2.50 2.56 021H WR 03 145-150 208.93 0.06 1.910 1.73 * 022H WR 03 145-150 220.22 n/a 1.53 n/a n/a 024XWR03 145-150 238.51 0.05 2.468 2.53 1.94 026XWR03 145-150 259.31 0.06 1.902 1.91 * 028XWR03 145-150 280.52 0.04 1.162 n/a n/a 030XWR03 145-150 301.72 n/a n/a 2.43 1.65 032X WR 03 145-150 322.92 n/a n/a 2.78 1.74 034XWR03 145-150 344.12 0.07 1.14 2.13 * 036XWR03 145-150 365.22 0.03 n/a 2.13 * 038XWR03 145-150 386.27 n/a n/a 2.46 1.30 040XWR03 140-150 407.47 0.04 n/a 2.57 1.51 043XWR 02 90-100 436.26 0.01 0.32 2.49 1.18 045x WR 02 140-150 457.85 0.01 2.38 2.32 n/a Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion Pore water geochemistry indicates that decomposition of organic matter is largely completed within the upper 1 0 0 mcd of sediment column. The reason for the termination of bacterial activity below this depth is not immediately apparent. Neither sulfate nor Co rg are depleted at this depth. The termination of organic matter diagenesis occurs near the Late Pliocene/Pleistocene boundary. At this time, about 2 Ma, the tectonic movement carried Site 1238 from the High Nutrient Low Chlorophyll (HNLC) - type region into more productive coastal waters (Fig. - F7, Mix et al., 2 0 0 3 ) . In comparison to the rest of the section, sediments between 8 0 to 1 0 0 mcd (near the Pliocene-Pleistocene transition) are characterized by elevated diatom content ( 3 0 vs. 1 5 wt%, Mix et al., 2 0 0 3 ) , and substantial increases in the concentrations of TOC ( ~ 3 wt % vs. 0 .9 wt %) and TN (~ 0 .3 wt% vs. 0 .1 3 wt %) (Fig. IV - 6 a). Right below the 1 0 0 mcd horizon, a narrow peak in micritic carbonate content (Mix et al., 2 0 0 3 ) corresponds to a zone of decreased porosity (from 8 0 to 5 0 %). The combination of decreased organic content and, possibly, micrite growth on sediment surfaces might have resulted in the termination of diagenesis below 1 0 0 mcd. Interpretation o f £ > S N o f ammonium The 8 15N of ammonium is 1 to 3 %o enriched in 15N relative to N org isotopic ratios through the whole sediment column (Fig. IV - 6 c). It is possible that storage artifact may account for a systematic enrichment of up to 1 %o (See related discussion in Methods section). Unfortunately, it is not possible to either confirm or disprove this 1 4 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. possibility. However, this discrepancy cannot account for more than 1 % o of the difference. If the 8 1 5 N of organic matter reaching the sediments has become heavier with time in response to changing oceanographic conditions, as the site has been tectonically moved into the region of higher degree of nutrient utilization, then the isotopic composition of the released ammonium should have be heavier at present than in the past. Transport processes, including diffusive mixing between two isotopically different sources, might alter the isotopic composition of ammonium present at depth today from that released in the past, overprinting the signature of S1 5 N released by the sediments. This would create a difference between pore water and solid phase N, even though no fractionation accompanies ammonium release. In order to interpret the nature of 1-3 % o isotopic enrichment of the pore water ammonium, we constructed a mixing diagram (Fig. IV - 7), where 8 1 5 N of ammonium is plotted vs. inverse ammonium concentrations, [NH^]'1 . A linear relationship between these two parameters would indicate conservative mixing between two end members. At steady state, deviations from linearity signify the presence of additional sources or sinks, which contribute ammonium with isotopic composition different from the two end members. However, non-steady state conditions can result in a non linear mixing pattern as well (Prokopenko et al., submitted). Fig. IV - 7 shows that below 64 mcd, where the ammonium release has largely ceased, the 8 1 5 N profile is consistent with mixing of 2 members, one with isotopic ratios of ~ 3.5- 4 % o and the other of 1 % o . The 8 1 5 N of organic matter at 60-70 mcd 144 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. E 3 C o E E n t o T- to 7 0 ] 6.0 20 5.0 % 4.0 % < \ \ * 3.0 ♦ ♦ 2.0 | 1.0 I 0.0 Diffusive front o f 15NH/ propagating downwards £ % P Release o f ammonium stops (64 mcd) 4 6 1/[NH4+ ], mM'1 8 Isotopic ofN, org 10 Fig. IV - 7 Mixing diagram for Site 1238, 51 5 N vs. [NH4 + ], mM'1 . If isotopically heavier ammonium has been released starting 12 Kyr ago, the expected penetration depth is 20 mcd (with ammonium adsorption taken into account, see text for explanation). 4 ^ depth is 3 ± 0.6 %o and 8 1 5 N at 437 mcd (the deepest interval where 8 1 5 N of ammonium has been determined) is 0.3 + 0.7 %o. Thus, the isotopic composition of ammonium end members in the interval between 70 and 437 mcd appears to be determined (within 1 %o) by the isotopic composition of the organic matter at these respective depth horizons. Consequently, the shape of the S1 5 N ammonium profile in the lower 370 mcd is defined by diffusion along the isotopic gradient, created by the difference in 8 1 5 N of two No rg sources. This is true within the uncertainty of about 1 %o, introduced by the possible storage artifact. In the upper 70 mcd, where the continuous release of ammonium is observed, the mixing relationship is more complex. The 8 1 5 N of ammonium lies well above the hypothetical mixing line connecting the two end-members, one with 8 1 5 N of ~ 4 % 0 and another, near surface component with 8 1 5 N of > 6.5 %o (Fig. IV - 7). One explanation for the observed non-linearity is the presence of an additional source, contributing ammonium of about 6 % o through the whole interval of ammonium release, from 2.9 to 70 mcd. The isotopic composition of No rg is 4.7 ±0.1 % 0 at the top of the profile (2.95 mcd) and 2-3 %o at 65 to 75 mcd. Thus, the 8 1 5 N of Norg are about 2 % 0 lighter than the 8 1 5 N of ammonium heavier end member (-6.5 %o). Under the steady state conditions, this would imply the net loss of isotopically heavier nitrogen during organic matter diagenesis, and enrichment of residual organic matter in 14N. However, when considering diagenesis on the long time scales, thousands to millions of years, one must take into account the possibility of a non-steady state 146 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. condition, such as variations in the isotopic composition of organic matter deposited through time. The shallowest interval analyzed is at 2.95 mcd, so we lack information of the 8 1 5 N of organic matter in the upper 2.95 mcd. This represents the last 60 Kyr (based on the Pleistocene accumulation rate 50m/m.y. (Mix et al., 2003)). Pedersen et al. (1991) found that a large pulse of organic matter was deposited in this region during the Last Glacial Maximum, between 25 and 15 Kyr with 8 1 5 N of LGM No rg 2.5 to 4.5 % 0 (Farrell et al., 1995), which has been interpreted by these authors as an indicator of lower ratio of the nutrient consumption to the nutrient supply to the surface waters, due to the intensified equatorial upwelling under glacial conditions. The isotopic composition of the sediments deposited in this region within the last 12 Kyr was measured during Canadian JGOFS program in 1989 (the closest to Site 1238 JGOFS station, VNTR01- 13GC, is located at 3.092°S and 90.823°W). The results, published by Farrell et al. (1995) showed that the 51 5 N of Norg deposited within the last 12 Kyr is between 6 and 7%o, close to the ammonium 8 1 5 N in the uppermost pore waters. Ammonium released from the organic matter deposited within the last 12 Kyr would create diffusive front propagating from the surface with an isotopic composition of 6-7 % „. Using the expression for the mean diffusion distance, % = 2 i|---- — t , D s (1 + K ) -10 2 / where diffusivity, Ds, is 8.1*10" m /sec, K= 2, is ammonium partition coefficient, and t is time, we calculate that ammonium released from the organic matter deposited 1 2 Kyr ago would have diffused to 25 m depth by the present time. The profile in Fig. IV 147 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. - 6 and the mixing diagram (Fig. IV - 7) show that isotopically heavier ammonium is observed at depths between 30 to 42 mcd, which is in a relatively good agreement with the calculated mean diffusion distance of 25 m. To summarize the discussion of the results from Site 1238, the isotopic composition of ammonium primarily reflects non-steady state conditions, resulting from mixing between ammonium released from organic matter with variable 515N. Without more sophisticated modeling it is not possible to fully evaluate whether any isotopic fractionation occurs during diagenesis of organic matter at this site. Summary The results from Sites 1234 and 1235 indicate that the isotopic composition of preserved organic matter is not affected by the processes of diagenesis in these rapidly accumulating coastal sediments on the time scale of hundreds thousands of years, even though about 20 % of No rg is lost to diagenesis. We reached similar conclusions based on our results (Table IV- 4) from Site 1230 (ODP Leg 201), where despite the loss of more that 30 % of original organic matter we found no evidence for changes in 51 5 N of residual No rg (Prokopenko et al., submitted). As mentioned before, our findings are consistent with the inference of Altabet et al. (1999a, b) and Pride et al. (1999), who suggested the absence of diagenetic fractionation of nitrogen isotopes in rapidly accumulating organic rich coastal sediments, where organic matter is well preserved. However, we also found that the concentration the organic matter is not as important for the high degree of preservation as sediment accumulation rates. Sites 1234 and 1235 contain low concentrations of organic matter, but are accumulated at the very 148 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. high rates (788 m/My and 696 m/My respectively). Moreover, when the results from Site 1230 are included into consideration, it appears that the degree of organic matter preservation may not be the main factor for absence of diagenetic alteration of the sedimentary 8 15N. The close similarity between S1 5 N of ammonium and 8 1 5 N of No rg at all three sites implies that the actual process of organic matter decomposition does not have an intrinsic fractionation factor associated with it. Perhaps another important factor, shared by the three sites, determines the absence of isotopic fractionation in N org. The sediments of all three sites are characterized by relatively low atomic C/N ratios (~ 8 ). This value indicates that the sedimentary organic matter is of predominantly marine origin, which implies more or less uniform lability of such organic matter. We have shown previously (Prokopenko et al., submitted) that preferential degradation of an isotopically distinct and more labile organic fraction (such as marine vs. terrestrial organic matter) may lead to the change in the residual bulk 8 1 5 N of the sediments. Sediments at Site 1227 on Leg 201 (Prokopenko et al., submitted), may contain significant fraction of terrestrial organic matter that appears to be resistant to degradation. Terrigenous organic matter may differ isotopically from the more labile marine Norg; in this case, the preferential degradation of isotopically distinct fraction may leave the 8 1 5 N of bulk nitrogen diagenetically altered. The observed absence of fractionation between ammonium and No rg at Sites 1234 and 1235 is consistent with uniformly marine origin of organic matter at these sites. From the discussion above, we can state that the long term diagenesis of marine organic matter in rapidly accumulating sediments does not affect the isotopic composition of 149 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. preserved No rg , as long as the terrestrial component is minor. If all these factors are present, then the depth dependant variations in sedimentary 8 1 5 N should reflect changes in environmental conditions. Due to the non-steady state at Site 1238, we are presently not able to draw definite conclusion about the impact of diagenesis on sedimentary 8 1 5 N at this site. Acknowledgements: This study has been supported by NSF grant OCE-Ol36500 to DH and an ODP Schlanger fellowship to MGP. The authors gratefully acknowledge members of the Scientific Party of Leg 202, who collected samples for us. We are also thankful to the chief-scientists of Leg 202, Drs. A. Mix and and R.Tiedemann for being very helpful in obtaining the samples, thus making this study possible. The technical assistance of M. Rincon and T. Gunderson at USC is greatly appreciated. 150 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References: Altabet, M.A. and Francois, R., 1994. Sedimentary nitrogen isotopic ration as a recorder for surface ocean nitrate utilization. Global Biochemichal Cycles, 8(103-116.). Altabet, M.A., Murray, D.W. and Prell, W.L., 1999 b. Climatically linked oscillations in Arabian Sea denitrification over the past 1 M.Y.: Implications for the marine N cycle. Paleoceanography, 14(6): 732-743. Altabet, M.A. et al., 1999 a. The nitrogen isotope biogeochemistry of sinking particles from the margin of the Eastern Tropical Pacific. Deep-Sea Research I, 46: 655- 679. Boudreau, B.P., 1997. Diagenetic models and their implementation: modeling transport and reaction in the aquatic sediments. Berlin, Heidelberg, NY, Springer, 414 pp. Emmer, E. and Thunell, R.C., 2000. Nitrogen isotope variations in Santa Barbara Basin sediments: Implications for denitrification in the eastern tropical North Pacific during the last 50,000 years. Paleoceanography, 15(4): 377-387. Farrell, J.W., Pedersen, T.F., Calvert, S., E. and Nielsen, B., 1995. Glacial -interglacial changes in nutrient utilization in the equatorial Pacific Ocean. Nature, 377: 514-517. Freudenthal, T., Wagner, T., Wenzhofer, F., Zabel, M. and Wefer, G., 2001. Early diagenesis of organic matter from sediments of the eastern subtropical Atlantic: Evidence from stable nitrogen and carbon isotopes. Geochimica Et Cosmochimica Acta, 65(11): 1795-1808. Ganeshram, R.S., Pedersen, T.F., Calvert, S., E. and J.W., M., 1995. Large changes in oceanic nutrient inventories from glacial to interglacial periods. Nature, 376: 755-758. Lehmann, M.F., S.M., B., Barbieri, A. and McKenzie, J.A., 2002. Preservation of organic matter and alteration of its carbon and nitrogen isotope composition during simulated and in situ early sedimentary diagenesis. Geochimica et Cosmochimica Acta, 66(20): 3573-3584. Li, Y.-H. and Gregory, S., 1974. Diffusion of ions in sea water and deep-sea sediments. Geochimica et Cosmochimica Acta, 38(703-714). 151 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mackin, J.E. and Aller, R.C., 1984. Ammonium Adsorption in Marine-Sediments. Limnology and Oceanography, 29(2): 250-257. Macko, S.A. and Estep, M.L.F., 1984. Microbial alteration of stable nitrogen and carbon isotopic compositions of organic matter. Organic Geochemstry, 6 : 787- 790. Mix, A.C., Tiedemann, R. and Blum, P., et al., 2003. Proc. ODP, Init. Repts., 202 [CD-ROM], Available from Ocean Drilling Program. Texas A&M University, College Station, TX, 77845, USA. Pedersen, T.F., Nielsen, B. and M., P., 1991. Timing of the Late Quartemary productivity pulses in the Panama Basin and implications for atmospheric CO2 . Paleoceanography, 6 : 657-678. Pride, C. et al., 1999. Nitrogen isotopic variations in the Gulf of California since the last deglaciation: Response to global climate change. Paleoceanography, 14(3): 397-409. Prokopenko, M.G., Ftammond, D.E., Spivack, A. and Stott, L., submitted. Impact of long term diagenesis on dl5N of organic matter in marine sediments: ODP LEG 201- Sites 1227 and 1230, Proc. ODP, Scientific Results., 201. College Station, TX. Sachs, J.P. and Repeta, D.J., 1999. Oligotrophy and nitrogen fixation during Eastern Mediterranean sapropel events. Science, 286: 2485-2488. Sigman, D.M., Altabet, M.A., Francois, R., McCorkle, D.C. and Gaillard, J.-F., 1999. The isotopic composition of diatom-bound nitrogen in the Southern Ocean sediments. Paleoceanography, 14: 118-134. 152 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER V Fractionation of nitrogen isotopes during incubation experiments in the presence of oxygen and under anoxic conditions Introduction The general problem of the fractionation of nitrogen isotopes during early diagenesis is discussed in the Introduction (Chapter I) of the dissertation. Isotopic fractionation during in situ diagenesis was investigated in the field by constructing mass balances for the 8 1 5 N of ammonium, released in the decomposition of organic matter, and the 8 1 5 N of bulk sedimentary organic matter (Chapters II-IV). This chapter investigates changes in the nitrogen isotope ratios of decomposing organic matter under laboratory conditions. This study has been motivated by results obtained by in situ field measurements and presented earlier. To briefly summarize the field results, in anaerobic* sediments less than 5-7 m below the sea floor, the ammonium released during decomposition of organic matter is 2 to 3 %o heavier than the bulk sedimentary organic matter. However, deeper in the sediments, 10 - 100s of meters below sea floor, no fractionation occurs during organic matter degradation. These observations can be explained if multiple fractions of isotopically distinct organic matter are present, and these fractions differ from each other in their lability. One goal of the incubation experiment is to test this hypothesis under controlled laboratory conditions. * Geochemical literature refers to the absence of oxygen as anoxic conditions, while microbiologists commonly use the term “anaerobic”. Here, both terms will be used interchangeably. The processes related to microbial activity will be referred to as anaerobic, while in the description of the geochemistry of the setting, the term “anoxic” will be used. In the similar way, the terms “oxic” and “aerobic” will be used to describe the presence o f oxygen. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Another purpose of the incubation experiment is to determine the isotopic fractionation associated with decomposition of organic matter in the presence of oxygen. Construction of isotopic mass balances using 8 1 5 N of ammonium in oxic sediments is impeded by the large fractionation associated with aerobic ammonium oxidation, which always occurs in the presence of oxygen. Therefore, evaluation of diagenetic fractionation during oxic decomposition of organic matter in situ is virtually impossible, since downcore variations in S1 5 N of organic matter may be due to non-steady state conditions. Following changes in 8 1 5 N in a closed system in the controlled environment through time would circumvent the problem of the non-steady state and allow direct observations of 8 1 5 N as decomposition progresses. Recently, two studies addressed diagenetic fractionation of nitrogen isotopes directly. Freudenthal et al. (2001) measured the isotopic composition of ammonium adsorbed to sedimentary particles and the 8 1 5 N of No rg , and evaluated the isotopic contribution of each of these fractions to the 8 1 5 N of bulk sediments. They observed an increase with depth in 8 1 5 N of No rg degraded under oxic conditions. This finding is consistent with previous reports of isotopic enrichment in sedimentary organic matter, decomposing in the presence of oxygen (Altabet, 1988; Holmes et al., 1999; Libes and Deuser, 1988; Macko and Estep, 1984; Macko et al., 1987; Sachs and Repeta, 1999; Sigman et al., 1999). The fractionation associated with the organic matter decay under anoxic conditions was not evaluated by Freudenthal et al. The other study, by Lehmann et al. (2002), investigated changes in S1 5 N of lacustrine plankton biomass, incubated under oxic and anoxic conditions. Their results 154 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. showed that organic matter becomes progressively enriched in 1 5 N while degraded in the presence of oxygen, but becomes depleted during anoxic degradation. These authors attributed the observed differences to “the differences in types, timing and degree of microbial activity” in these two settings (Lehmann et al., 2002). In our study, we extended the work done by Lehmann et al. by measuring the isotopic composition of pore water ammonium released during degradation and determining the composition of Total Hydrolyzable Amino Acids (THAA) in the sediments. The goal of this experiment was to assess whether isotopic fractionation does occur during organic matter degradation in marine sediments and to evaluate the mechanisms, which are responsible for fractionation, if it takes place. Methods Experimental set up and sample processing Incubations were conducted under oxic and anoxic conditions. The slurry for the incubation experiments was prepared as follows. Surficial anoxic Santa Barbara Basin sediments (99 g, wet weight) from the upper 5 cm were mixed with surface sea water (200 ml) and plankton (189 g, wet weight). The plankton was a mixture of two tows, collected with mesh 200 and 400 pM nets. The final volume of the slurry mixture was 400 ml. It was divided into 4 equal parts, and each part was placed in a 100 ml HDPL bottle. Two bottles, identified as AN-I and AN-II were used for anoxic incubations. These were flushed for 30 min with Ar, placed inside glass jars and transferred into an air-tight plexiglass container. The container was flushed with Ar for about 1 hour and sealed. The flushing was repeated periodically over the course of 155 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. incubations. For sampling, the bottles were transferred into the Ar-filled glove bags, inverted several times and shaken well to ensure homogeneity. The other two bottles, OX-I and OX-II, to be incubated under oxic conditions, were covered with filter paper and placed in a horizontal shaker, to facilitate continuous aeration of the slurries. Before sampling, bottles were closed with lids, inverted, and shaken. Sample aliquots were removed at 0, 4, 7, 20, 35, 64 and 206 days, and stored frozen until analysis. The individual samples have the same nomenclature as the bottles, AN-I and AN-II for samples from anoxic incubations, and OX-I and OX-II for oxic incubations. On day 206, samples were thawed and centrifuged. The pore water was removed with a syringe and filtered through GF/C filters. The pore waters of AN-I and OX-I samples were analyzed for ammonium concentrations, 8 1 5 N of ammonium, and Total Hydrolyzable Amino Acid (THAA) concentrations. The sediment fraction of AN-I and OX-I samples was analyzed for carbon and nitrogen elemental composition (C and N wt %), bulk 8 1 5 N and THAA. For Series AN-II and OX-II samples, the same analyses were performed with the exception of dissolved and solid phase THAA and C wt %. Analytical methods Ammonium concentrations were determined using the colorimetric method of Bower and Holm-Hansen (1980). High sulfide concentrations, > ImM, interfere with color development in this procedure. I have demonstrated in a series of dilution experiments that this problem can be avoided by strong dilution of original samples (xl50 to 200) prior to addition of the color-developing reagents. The dilution 156 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. eliminates the interference. For samples run in this study, the dilutions were made accordingly. Analytical precision determined by running replicate analysis was 1-2 %. Isotopic analysis of pore water ammonium and the sediment, as well as C and N wt % concentrations in the sediments, were done at USC, using an Isoprime Micromass mass spectrometer interfaced with Carlo Erba CHN2500 or Euro Vector elemental analyzers. The precision for this analysis was 1-2 %. The detailed procedure for sediment treatment and ammonium extraction prior to isotopic analysis is described in Chapter II and III and Appendix A. Plankton biomass was prepared for 8 1 5 N analysis in two ways. A portion of thick “soup” of plankton tow material was placed on pre weighed GF/C filters, dried for 24 hours at 60°C, and scraped off the filters directly into a tin boat immediately before the isotopic analysis. Alternatively, about 2 ml of thick plankton “soup” was placed into an Eppendorf microcentrifuge tube and freeze- dried for 10 hours. The dried material was homogenized, weighed out into tin boats, and analyzed. THAA (Total Hydrolyzable Amino Acids) analysis The THAA were determined using the method by Cowie and Hedges (1992) as modified by Mahaffey (2003). The description of the step-by-step sample handling procedure is given in the Appendix D. In brief, the proteins and peptides are hydrolyzed in the presence of strong acid into individual amino acids. Individual hydrolyzed amino acids are reacted with a fluorescent compound, o-Phthalaldehyde (OP A) to form fluorescent derivatives of primary amines. The magnitude of 157 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fluorescent response is used to determine the concentrations of the individual amino acids. Hydrolysis For pore water hydrolysis, 0.9 ml of filtered pore water was combined with 1 ml of 6 N HC1 (ACS reagent grade) and 0.1 ml of 2 mM of hydroxylysine (as the internal standard) in 4 ml Weaton vials. The mixture was purged with Ar for 1 min, the vial was capped and the cap was sealed with Teflon tape. Samples were hydrolysed at 140°C for 2 hours. For sediment hydrolysis, between 0.5 and 1 g of wet sediments were combined with 0.1 ml of hydroxylysine and 1.9 ml of 6 N HC1 acid, the slurry was purged with Ar for 1 min, capped and sealed with Teflon tape. The sediments were hydrolyzed for 24 hours at 110°C. After hydrolysis, samples were filtered through a GF/C filter to remove particles. Filters were washed with 5 ml of DDIW, and the washes were combined with the samples. From this point, pore water and sediment hydrolysates were treated in the identical manner. Samples were evaporated to dryness in the rotary evaporator, re-dissolved in 2 ml of DDIW, and vortexed for 5 min each. Each sample was divided in 4 aliquots of 0.5 ml each, and stored frozen in Eppendorf microcentrifuge tubes until analysis. Flurometric analysis Prior to determination of individual concentrations of amino acids by HPLC, the concentrations of total amino acids were determined by flurometric analysis. In this method (Appendix D), 2 ml of buffered OP A reagent was mixed with 2 ml of sample. The mixture was shaken well, and fluorescence was measured exactly after 2 min on a 158 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fluorometer equipped with excitation and emission lenses. The excitation lens cuts out all the wave lengths below 360 run. The emission lens eliminates all wave lengths above 460 nm. Since the intensity of fluorescence strongly depends on the time of reaction, strict timing was ensured by using a stopwatch, as suggested by Mahaffey (2003). Glycine solutions of 1, 2 and 4 pM were used as a calibrating standard. This method does not give accurate concentrations of total amino acids because of the possible presence of interfering compounds in hydrolysate (Mahaffey, pers. comm.). Consequently, the results of these measurements were employed only to assist in designing the dilution scheme for HPLC analysis. HPLC analysis The principle of HPLC (High Pressure Liquid Chromatography) analysis is chromatographic separation of dissolved amino acids, reaction with the OPA reagent to form fluorescent derivatives of the primary amines, and determination of individual concentrations from the magnitude of fluorescence peak produced by each derivative. The instrumental set up consisted of a Waters 600 Controller pump, an Alltech Cis Adsorbosphere OPA-HR reverse phase column (5 um, 150 mm x 4.6 mm i.d.) connected with Spectrovision FD-200 fluorometer and SP 4270 data integrator. The separation of individual amino acids was achieved by gradual mixing of acetate buffer with methanol buffer (see the recipes in Appendix X) over the 50 minute time course of the run, according to the solvent program in Table V - 1 (Mahaffey, 2003). 159 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. For analysis, 75 ul of sample or standard was combined with 75 pi of mixed OPA reagent (details are given in Appendix D), shaken well, and injected after 1 min into a 2 0 pi injection loop of the pump. The determination of the individual amino acids was based on their retention times, which had been measured separately for each amino acid (Table V - 2). The total amount of 19 amino acids was identified. Concentrations of individual amino acids were determined by calibration with standards of known concentrations (5 pM, 15 pM and 30 pM. The precision of analysis based on internal standards was about 10 %. Results Nitrogen inventory: Definitions and determination o f various nitrogen pools Since a more complete set of analysis was performed on A N - I and OX-I samples, the nitrogen inventory from this series is presented here. The following 5 pools of N were present at the beginning of the incubations (Table V - 3, Fig. V -1): 1) Dissolved ammonium, NH4 +; 2) DON (Dissolved Organic Nitrogen); 3) Na d s in form of NH4 + sorbed to sedimentary particles, and finally, 4 and 5, Particulate Organic Nitrogen (PON), which encompassed two pools: 4) N org-sed present in Santa Barbara sediments prior to addition of plankton and, 5) N org-piankton from added plankton. Before establishing the inventory of each of the individual nitrogen pools, several remarks need to be made as to how these pools were determined. 160 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 1 Program for regulating amino acid elution time. Solvent A is Acetate buffer, B is methanol Time (min) Solvent A (%) Solvent B (%) 0 100 0 2 100 0 45 35 65 46 10 90 47 2 98 50 100 0 Table V - 2 Average retention times for analyzed amino acids Amino Acid Retention time (min) ASP 2.5-3 GLU 3-3.5 AAAA 7-8 SER 10-12 GLY 14-15 THR 15-17 HIS 17-18 ALA 18-19 TAU 19-20 TYR 20-21 ARG 21-22 GABA 22-23 MET 24-25 VAL 25-26 PHE 26-27 ILE 27-28 LEU 29-30 HLYS 30-31 ORN 32-33 LYS 33-34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 3 Initial composition of the slurry A. Bulk composition Dissolved N Particulate N * Average conc. of particlesA PON** Series ID DONA A [NH4+]A A A Norg- sed*** Norg- plankton *A N ads**A A Total N*a* (g/ml) (pmol/ml) (pmol/ml) (pmol/ml) (pmol/ml) (pmol/ml) (pmol/ml) AN-I 0.030 15.5 2.6 9.4 2.8 0.09 18.1 O X -1 0.034 10.2 3.6 10.6 3.3 0.11 13.8 A calculated as average of weights of dry sediments per volume (in each sample) A A calculated from THAA concentration and amino acids molecular formulas A A A initial ammonium concentration from decomposed plankton * calculated as (N wt % totaI)x( 10A 4/14) x (average concentration of particles) * * calculated as (Particulate N) - (Nads) * * * calculated as (N wt % in SBB sediments)x(10A 4/14) x (average concentration of particles) * A calculated as (PON)- (Norg-sed) * * A A calculated as ([NH4+]) x (Kd) x (average concentration of particles) * A *sum of all pools B. Isotopic composition of different N pools and components (%o) Dissolved N Particulate N Values PON DON [NH4+] Norg-sed Norg- plankton N ads Total N Measured (9.3 + 0.5) 1.8+ 0.3 6.8 + 0.2 9.3+ 0.5 1.8 + 0.3 7.4+ 0.1 Predicted* 7.4 + 0.3 'Calculated as isotopic mass balance of Norg-sed, Norg-plankton and DON and NH4+ from the pore water, which was left in the the sediments after centrifugation (estimated volume is 0.024 ml, if porosity was 0.80) 162 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DON, AN-I and OX-1 adsorbed Norgfrom plankton, 815N PORE WATER SEDIMENTS Measured pools SAMPLES THAA sedimentary, AN-I and OX-I SLURRY AN-I and II OX-I and II Legend: Calculated pools Fig. V - 1 A schematic representation of the nitrogen pools present in each sample. 8 1 5 N symbol in boxes indicates that the isotopic composition of this pool was measured or determined by mass balance calculations. Legend indicates the pools measured directly and calculated Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NH4 + was measured directly by the colorimetric technique of Bower and Holm-Hansen (1980). The DON pool was measured as concentrations of THAA, and N associated with THAA was calculated using formula weights of individual amino acids. The size of the DON pool calculated as the N-THAA is, most likely, underestimated because DON may include not only amino acids, but also pigments, amino sugars, DNA and RNA molecules, none of which were measured. Particulate nitrogen, which included PON and Na d S was determined as N wt % on the centrifuged and oven-dried particulate component of the slurry. In order to estimate the amount of adsorbed ammonium, the partition coefficient, Kd (ml/g) was determined experimentally by sequential desorption of ammonium ions with 2M KC1 solution. Fig. V-2 shows the plot of total pmol of N removed by sequential washing with KC1 vs. concentration of NH4 + present in the liquid phase. These results demonstrate non-linearity in the sorption isotherm over a range of concentrations. The non-linearity probably results from a limited number of adsorption sites at particle surfaces and variations in the sorption strength between different sites (Fetter, 1999; Mackin and Aller, 1984). The sequential desorption experiment did not cover the range of ammonium concentrations found in the slurries, (1 to l 8 mM). Therefore, the concentration dependency of Kd was found by using the following assumptions: at [NH4 + ] = 1 mM, Kd =2 ml/g and at [NH4+ ] = 10 mM, Kd = 0.5 ml/g (Mackin and Aller, 1984). The non-linear relationship fit the power function (5.1) K d = K o * C N, 164 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.45 0.40 "§ 0.35 > ® 0.30 o l. 0.25 + 3 ? 0.20 Z H- 0.15 O O 0.10 E 3 0.05 0.00 ♦ ♦ ♦ 0.000 0.010 0.020 0.030 0.040 0.050 0.060 Concentration in the KCI wash, uM Fig. V - 2 Amount of N FL*+ removed by five sequential washes of 0.38 g (dry weight) of sediments, which originally were in contact with 1 mM ammonium solution Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. where Ko = 2ml/g, C = concentration of dissolved ammonium (mM) and N=-0.6, as was found from the two boundary conditions for Kd indicated above. Applying expression 5.1, the K < j values were computed for the measured concentrations and used to calculate the size of adsorbed ammonium (Na d S pool) in the slurry. Finally, PON was determined as the difference between Particulate N and N ads- The two different components of PON, N org-sed and N org.piankton, were determined for the initial slurry only. The N org-sed was measured as wt % N in the sediments prior to slurry preparation. The other component, N org-piankton, was calculated as the difference between PON and N org-sed. The concentrations are reported as N pmol/ml, calculated as shown in the footnotes to Table V-3. Initial distribution o f nitrogen between different pools and SlsN o f each pool Table V - 3 A summarizes the distribution of various nitrogen pools at the beginning of the experiment in AN-I and OX-I series. In both settings, the largest pool of N , ~ 50 %, was DON. About 30 % of the total nitrogen was N org-sed. Nitrogen from the plankton biomass, N org-piankton, constituted about 1 0 % of the total nitrogen. Dissolved ammonium was about 10 % of total N both in AN-I and OX-I. The ammonium initially present in the slurry was, most likely, produced by the decomposition of plankton biomass in the few hours between collection and freezing of the plankton material. The adsorbed ammonium pool was very small, less than 1 % of the total in both bottles. Part B of Table V-3 summarizes isotopic composition of all the nitrogen pools at the beginning of the incubations. The values given are averages of the 8 1 5 N of 166 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. respective pools measured in A N - I and O X - I bottles. The 8 15N of the dissolved ammonium averaged 1 . 8 + 0.3 %o. N org-sed of the sediments was 6 . 8 + 0.2 %o. The 8 15N of the N org-piankton was measured on both filtered ( N org-piankton) and freeze-dried (N o rg - piankton + D O N ) samples. Results were indistinguishable, therefore, both N org-piankton and D O N were 9 .3 + 0.5 %o. The average of 8 15N of P O N , measured on the centrifuged particulate material from A N - I and O X - I slurries was 7 .4 + 0.1 %o. The last value is in excellent agreement with S 15N of 7 .3 + 0.2 %o predicted from the isotopic mass balance for the two components of P O N and adsorbed ammonium. Such consistency is an indication that the constructed inventory included most of the major pools of nitrogen present in the slurry. Changes in nitrogen pools through the course o f the incubation Anoxic incubations Tables V- 4 a and b present changes in concentrations of all nitrogen pool and particulate C through the course of the experiment. Within the first 4 days of the incubations, the distribution of nitrogen between the major nitrogen pools changed substantially. More than 90 % of DON (14 pmol/ml) pool disappeared by day 4. The rapid decrease in DON was accompanied by a sharp increase in ammonium concentration (by 12 pmol/ml). After the rapid initial increase, the ammonium concentrations increased more gradually, by 3 pmol/ml, over the course of next few months. The PON concentration decreased over the course of the incubations by 3 pmol/ml, which constituted about 25 % of the initial amount. The total loss of 167 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 4 Incubation Series AN-I and OX-I. A.Concentrations in all nitrogen pools measured directly Sample ID Days Particle conc. (mg/ml) Particulate C (wt % ) Particulate N (wt % ) C/N total (atomic) D O N * (pmol/ml) [NH4+] (pmol/ml) K d** (ml/g) Anaerobic AN-0 0 27.5 4.99 0.58 8.67 15.52 2.6 1.0 AN-2 4 38.2 4.89 0.53 9.31 1.26 14.9 0.4 AN-3 7 32.8 4.90 0.55 8.98 1.01 15.1 0.4 AN-4 20 32.5 4.70 0.50 9.49 0.63 16.1 0.4 AN-5 35 32.2 4.97 0.53 9.38 0.43 16.8 0.4 AN-6 64 21.8 4.90 0.48 10.32 0.46 17.3 0.4 AN-7 206 24.3 4.59 0.44 10.43 0.18 18.1 0.4 Average particles * A 29.9 Aerobic OX-O 0 31.9 5.06 0.58 8.72 10.21 3.6 1.2 OX-2 4 45.0 5.08 0.57 8.91 1.27 17.7 0.5 OX-3 7 28.4 5.09 0.56 9.09 1.46 18.7 0.4 OX-4 20 43.9 0.52 19.6 0.4 OX-5 35 24.2 4.69 0.54 8.76 0.53 16.7 0.4 OX-6 64 29.6 3.90 0.53 7.36 0.36 0.9 1.1 OX-7 206 33.8*** 4.05 0.51 7.94 1.0 2.0 Average particles * A 33.8 * Calculated from summing N in THAA measured, based on AA stoichiometry ** Kd=(Ko)x(CA -0.6) (see text) *** average of particle concentrations in OX-O to OX-6 *A Using all AN-I or OX-I B. Nitrogen inventory of all the pools present (measured and calculated) Sample ID Days [NH4+] (pmol/ml) Nads* (pmol/ml) PO N ** (pmol/ml) D O N (pmol/ml) N total*** (pmol/ml) Anaerobic AN-0 0 2.6 0.09 12.2 15.52 30.5 AN-2 4 14.9 0.18 11.0 1.26 27.5 AN-3 7 15.1 0.18 11.5 1.01 27.9 AN-4 20 16.1 0.18 10.4 0.63 27.4 AN-5 35 16.8 0.18 11.1 0.43 28.8 AN-6 64 17.3 0.19 10.0 0.46 28.1 AN-7 206 18.1 0.19 9.2 0.18 27.8 Aerobic OX-O 0 3.6 0.11 13.9 10.21 27.9 OX-2 4 17.7 0.21 13.6 1.27 32.9 OX-3 7 18.7 0.22 13.3 1.46 34.0 OX-4 20 19.6 0.22 11.9 0.52 32.4 OX-5 35 16.7 0.21 12.7 0.53 30.3 OX-6 64 0.9 0.07 12.7 0.36 14.2 OX-7 206 1.0 0.07 12.3 13.4 Estimated uncertainty 2% 50% 2% 10% 8% * Calculated as ([NH4+])x (Kd) ** Calculated as (wt % of N measured)x1(0A 4/14) x (average concentration of particles)-(Nads) ** * Sum of all pools 168 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. particulate C over 6 months was only 8 % of the original amount. The difference between the magnitude of the decrease in the TOC and TN pools is reflected in C/N ratio of the slurry, which changed from 8.67 to 10.43. Oxic incubations Similarly to the anoxic incubations, the DON concentration decreased by ~ 90 % within the first four days of the experiment, which resulted in sharp increase in N H / concentrations over this period of time (Table V - 4 a and b). The ammonium increase was larger than DON loss. The large uncertainty associated with THAA measurements may be partially responsible for this discrepancy. Alternatively, the presence of an unmeasured fraction of DON (other than THAA) degrading under oxic conditions may have contributed to the difference. Ammonium continued to increase for the next 20 days, but by day 35 the concentrations began to decline, perhaps reflecting ammonium oxidation and denitrification. By day 206 more 95 % of ammonium disappeared from the slurry. The PON decreased by only 12 % (1.6 pmol/ml) over the course of 206 days. C/N atomic ratio decreased slightly over the course of the incubations, from 8.7 to 7.94. A portion of dried sediments from samples AN-II and OX-II was washed with 2M KC1 solution, in order to remove adsorbed ammonium. Table V - 5 presents the wt % N in unwashed and KCl-washed sediments, and compares the difference (which was expected to be due to the adsorbed ammonium). The results shows that the fraction of nitrogen removed by KC1 treatment substantially exceed the calculated 169 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 5 N removed by washing with 2M KC1 Sample ID Days Unwashed (wt % N) WashedA (wt % N) LostA A (wt % N) NH4+ ads* (wt % N) PON leachedA * (wt % N) PON leached (pmol/g) DON** (nmol/g) Anaerobic AN-0 0 0.54 0.42 0.12 0.004 0.12 2.48 15.52 AN-2 4 0.43 0.008 0.00 1.26 AN-3 7 0.51 0.37 0.14 0.008 0.13 2.82 1.01 AN-4 20 0.39 0.008 0.00 0.63 AN-5 35 0.48 0.34 0.14 0.008 0.13 2.82 0.43 AN-6 64 0.45 0.38 0.07 0.009 0.06 1.31 0.46 AN-7 206 0.44 0.34 0.10 0.009 0.09 1.96 0.18 Aerobic OX-O 0 0.004 10.21 OX-2 4 0.52 0.4 0.12 0.007 0.11 2.73 1.27 OX-3 7 0.53 0.44 0.09 0.008 0.08 2.00 1.46 OX-4 20 0.39 0.008 0.00 0.52 OX-5 35 0.51 0.43 0.08 0.008 0.07 1.75 0.53 OX-6 64 0.49 0.43 0.06 0.004 0.06 1.36 0.36 OX-7 206 0.44 0.4 0.04 0.003 0.04 0.90 A 0.5 g of wet slurry washed with 5 ml of 2M KCI and centrifuged A A Unwashed - washed * Calculated from Kd and [NH4+] in Table V-4 A * Calculated as Lost - NH4+ads ** From Table V-4 170 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. adsorbed ammonium pool. Apparently washing with KC1 removed not only adsorbed ammonium, but also some of the organic matter adsorbed on the particle surfaces, or perhaps some low density organic particulates that did not settle during centrifugation. Compositional changes in THAA Pore water THAA The concentration of pore water THAA decreased by more than 99 % in AN-I, and by 97 % in OX-I over the whole length of incubations (Table V - 6 a). Both concentrations and mole fractions of certain individual amino acids changed notably (Table V - 6 a and 7a). The absolute concentrations of all THAA decreased, except for tyrosine, which increased in both AN-I and OX-I. By the end of the incubation of AN-I, the mole fraction of tyrosine, lysine and leucine increased substantially, when compared to the initial values (Fig. 3a, Table V - 7a), while the mole fractions of all other protein amino acids either decreased or remained the same. In oxic incubations, both the concentration and the mole fractions of tyrosine and arginine increased significantly by day 64 (the last pore water sample, analyzed for THAA), while the rest of amino acids decreased or remained the same (Fig. 3b and 6 a and 7a). Sedimentary THAA The absolute concentrations of THAA in the sediments were measured with an uncertainty of + 30 %, which arose from to the fact that only the wet weights of sediment samples were recorded before hydrolysis. The dry weights were estimated on the basis of average porosity of the surface sediments, centrifuged for 10 min at 3000 rpm. Over the course of anoxic incubations, the total concentrations and the 171 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 6 Concentrations of amino acids in AN-I and OX-I (individual amino acid concentrations are in pM; estimated uncertainty in the measurements is 1 0 %). A. Concentrations of amino acids in the pore water ANAEROBIC ___________ Sample ID Day of experiment AN-1 pw 0 AN-2 pw 4 AN-3 pw 7 AN-4 pw 20 AN-5 pw 35 AN-6 pw 64 AN-7 pw 206 Amino Acids measured ASP 834 76 95 39 37.4 41.3 16.1 GLU 1184 130 109 47 0.0 37.5 11.6 AAAA 214 82 77 72 45.6 17.5 0.0 SER 125 24 34 8 14.1 24.4 6.0 GLY 1904 83 35 32 8.7 91.5 0.0 THR 596 43 24 5 10.5 23.8 1.2 HIS 620 0 0 0 0.0 0.0 0.0 ALA 1175 37 22 42 9.7 3.3 2.2 TAU 562 17 15 8 3.9 0.0 6.6 TYR 0 28 14 0 6.3 3.9 20.4 ARG 311 0 19 14 3.3 4.7 0.0 GABA 0 0 0 0 0.0 0.0 0.0 MET 816 279 18 0 8.7 5.0 4.8 VAL 989 51 56 16 14.3 17.0 4.7 PHE 417 33 1 1 7.0 12.0 3.3 ILE 665 30 42 1 11.7 15.4 7.3 LEU 1087 25 22 0 20.2 0.0 50.5 ORN 0 0 83 0 0.0 0.0 0.0 LYS 922 161 144 149 110.9 75.6 23.7 Total cone of AA in the slurries, pM N-THAA cone in the slurry, pmol/ml* 12422 15.5 1099 1.3 809 0.9 435 0.6 312 0.4 373 0.5 158 0.2 AEROBIC Sample ID Day of experiment OX-1 pw OX-2 pw 0 4 OX-3 pw OX-4 pw 7 OX-5 pw 20 35 OX-6 pw 64 Amino Acids measured ASP 132 nd 83 47 14.1 5.4 GLU 1468 n d 139 61 37.9 4.3 AAAA 0 0 61 0 0.0 0.0 SER 176 81 42 25 16.3 3.6 GLY 2187 342 94 29 73.6 1.8 THR 471 109 52 20 13.4 0.8 HIS 126 0 0 0 0.0 0.0 ALA 1143 126 111 25 24.4 2.9 TAU 335 0 31 4 2.4 1.5 TYR 30 22 24 10 27.6 85.6 ARG 173 27 34 13 14.0 54.8 GABA 0 10 0 13 9.2 6.6 MET 197 21 20 10 2.8 0.0 VAL 798 72 75 24 6.3 4.6 PHE 175 28 2 8 2.9 5.4 ILE 507 54 51 17 3.6 1.5 LEU 705 63 60 21 138.1 2.3 ORN 0 40 0 0 0.0 0.0 LYS 410 96 239 78 49.2 7.6 Total cone of AA in the slurries, pM N-THAA cone in the slurry, pmol/ml* 9033 10.2 1092 1.2 1120 1.5 403 0.5 436 0.5 189 0.4 * Note the difference in units with the previous line 172 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 6 (continued) (Individual amino acid concentrations are in gmol/g of dry weight sediments, estimated uncertainty is 30 %.). B. Concentrations of amino acids in the sedimens ANAEROBIC__________________________________ Sample ID AN-1 sed AN-2 sed AN-3 sed AN-4 sed AN-5 sed AN-6 sed AN-7 sed Dav of experiment 0 4 7 20 35 64 206 Amino Acids measured ASP 4.5 3.5 0.0 4.0 3.5 5.4 4.5 GLU 3.9 2.7 0.0 3.1 2.7 4.0 3.2 AAAA 0.0 0.1 0.0 0.0 0.0 0.0 0.6 SER 2.5 2.4 4.5 0.1 2.1 3.3 1.9 GLY 7.4 4.7 15.3 7.8 5.5 6.7 7.1 THR 2.7 1.9 4.7 0.0 2.2 3.2 2.6 HIS 0.2 0.4 0.9 0.3 0.4 0.3 0.3 ALA 5.8 4.0 6.4 4.9 3.9 6.3 4.3 TAU 1.0 0.4 2.6 1.1 1.2 1.2 1.0 TYR 1.0 0.5 1.6 1.0 0.8 1.2 1.0 ARG 1.6 1.1 1.9 1.2 1.0 1.0 1.1 GABA 0.5 0.0 0.0 0.5 0.5 0.7 0.2 MET 0.4 0.2 0.7 0.5 0.7 0.7 0.5 VAL 3.4 1.8 4.0 2.4 2.4 2.8 2.4 PHE 1.2 1.4 2.4 1.7 1.4 1.8 1.6 ILE 1.7 1.0 3.0 1.4 1.5 1.6 1.6 LEU 2.8 1.7 4.3 2.5 2.5 3.0 2.5 ORN 0.0 1.7 2.2 0.0 0.0 0.0 0.0 LYS 0.5 0.0 4.7 1.8 2.0 0.0 2.1 Dry sediment weight, g *** 0.21 0.31 0.25 0.37 0.37 0.24 0.26 Cone of AA per, pmol/g of sed (+ 30 % of value) 41.2 29.6 29.7 34.5 34.1 43.3 38.8 Cone of N pmol/ g of sed (+ 30 % of value) 46.9 31.8 69.5 40.6 39.8 47.0 44.9 Cone of sed N-THAA in the slurry (pmol/ml) 1.4 1.0 2.1 1.2 1.2 1.4 1.3 Percent of N-THAA of PON (+ 30 % of the value) 11% 8% 18% 11% 11% 14% 14% AEROBIC Sample ID OX-1 sed OX-2 sed OX-3 sed OX-4 sed OX-5 sed OX-6 sed OX-7 sed ** Day of experiment 0 4 7 20 35 64 206 Amino Acids measured ASP 3.6 2.4 5.0 1.3 4.1 2.9 M GLU 3.3 2.3 4.6 3.9 4.3 3.9 3 2 AAAA 0.0 0.0 0.8 0.0 0.0 0.0 2 2 SER 1.7 1.4 3.5 2.4 3.2 2.9 4.5 GLY 5.2 3.1 8.0 10.8 14.0 13.5 6 ^ THR 2.5 1.6 3.4 2.9 3.2 3.1 2 2 HIS 0.0 0.3 0.0 0.0 0.5 0.7 2 2 ALA 3.9 2.5 4.8 4.0 5.4 4.4 6,5 TAU 1.3 0.0 2.0 1.2 1.7 1.7 18 TYR 1.0 0.6 1.6 0.7 1.4 1.0 0 2 ARG 1.2 0.6 0.8 1.1 1.9 1.3 2 2 GABA 0.6 0.0 3.6 0.0 0.0 0.0 0.0 MET 0.4 0.4 0.9 0.6 0.5 0.4 0 2 VAL 2.2 1.6 2.8 1.9 2.9 2.7 3 1 PHE 1.3 0.8 1.6 1.1 1.6 1.5 L I ILE 1.5 1.0 1.7 1.2 1.8 1.7 1 1 LEU 2.1 1.4 3.1 1.9 2.9 2.5 3 2 ORN 0.0 0.0 0.0 0.0 0.0 0.0 3 2 LYS 1.9 1.0 2.4 1.9 2.4 2.7 2.2 Dry sediment weight, g, por=0.8 *** 0.25 0.36 0.21 0.38 0.27 0.36 0.20 Cone of AA per, pmol/ g of sed (+ 30 % of value) 33.7 21.0 25.2 36.9 51.8 46.9 46.4 Cone of N pmol/ g of sed (+ 30 % of value) 39.1 24.3 54.9 42.0 60.9 55.0 56.8 Cone of sed N-THAA in the slurry pmol/ml 1.2 0.7 1.6 1.3 1.8 1.7 1.7 Percent of N-THAA of PON (+ 30 % of the value) 9% 6% 14% 12% 16% 15% 16% ** Suspect sample *** Dry sediments were not weighed out, instead dry weights were calculated from wet weights with assumed porosity of 80 % 173 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 7 Mole fractions o f THAA results in presented in Table V - 6. A. Mole fractions of THAA in the pore water ANAEROBIC INCUBATIONS Sample ID Days of experiment/AA pNAN-1 pw 0 pNAN-2 pw 4 pNAN-3 pw 7 pNAN-4 pw 20 pNAN-5 pw 35 NAN-6 pw 64 pNAN-7 pw 206 ASP 7% 7% 12% 9% 12% 11% 10% GLU 10% 12% 13% 11% 0% 10% 7% AAAA 2% 7% 10% 17% 15% 5% 0% SER 1% 2% 4% 2% 5% 7% 4% GLY 15% 8% 4% 7% 3% 25% 0% THR 5% 4% 3% 1% 3% 6% 1% HIS 5% 0% 0% 0% 0% 0% 0% ALA 9% 3% 3% 10% 3% 1% 1% TAU 5% 2% 2% 2% 1% 0% 4% TYR 0% 3% 2% 0% 2% 1% 13% ARG 3% 0% 2% 3% 1% 1% 0% GABA 0% 0% 0% 0% 0% 0% 0% MET 7% 25% 2% 0% 3% 1% 3% VAL 8% 5% 7% 4% 5% 5% 3% PHE 3% 3% 0% 0% 2% 3% 2% ILE 5% 3% 5% 0% 4% 4% 5% LEU 9% 2% 3% 0% 6% 0% 32% ORN 10% LYS 7% 15% 18% 34% 35% 20% 15% AEROBIC INCUBATIONS Sample ID Days of experiment/AA pNAO-1 pw 3 0 pNAO-2 pw 4 pNAO-3 pw pNAO-4 pw 7 20 pNAO-5 pw 35 pNAO-6 pw 64 ASP 1% 0% 7% 12% 3% 3% GLU 16% 0% 12% 15% 9% 2% AAAA 0% 0% 5% 0% 0% 0% SER 2% 7% 4% 6% 4% 2% GLY 24% 31% 8% 7% 17% 1% THR 5% 10% 5% 5% 3% 0% HIS 1% 0% 0% 0% 0% 0% ALA 13% 12% 10% 6% 6% 2% TAU 4% 0% 3% 1% 1% 1% TYR 0% 2% 2% 2% 6% 45% ARG 2% 3% 3% 3% 3% 29% GABA 0% 1% 0% 3% 2% 3% MET 2% 2% 2% 3% 1% 0% VAL 9% 7% 7% 6% 1% 2% PHE 2% 3% 0% 2% 1% 3% ILE 6% 5% 5% 4% 1% 1% LEU 8% 6% 5% 5% 32% 1% ORN 4% LYS 5% 9% 21% 19% 11% 4% 174 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 7 (continued). B. Mole fractions of THAA in the sediments AEROBIC INCUBATIONS Sample ID Days of experiment/AA sNAN-0 sd 0 sNAN-2 sd 4 sNAN-3 sd 7 sNAN-4 sd 20 sNAN-S sd 35 sNAN-6 sd 64 sNAN-7 si 206 ASP 11% 12% 12% 10% 13% 12% GLU 9% 9% 9% 8% 9% 8% AAAA 0% 0% 0% 0% 0% 0% 2% SER 6% 8% 8% 6% 8% 5% GLY 18% 16% 26% 23% 16% 15% 18% THR 7% 6% 8% 0% 6% 7% 7% HIS 1% 1% 2% 1% 1% 1% 1% ALA 14% 14% 11% 14% 11% 15% 11% TAU 2% 1% 4% 3% 3% 3% 3% TYR 2% 2% 3% 3% 2% 3% 2% ARG 4% 4% 3% 4% 3% 2% 3% GABA 1% 0% 0% 1% 2% 2% 1% MET 1% 1% 1% 2% 2% 2% 1% VAL 8% 6% 7% 7% 7% 6% 6% PHE 3% 5% 4% 5% 4% 4% 4% ILE 4% 4% 5% 4% 4% 4% 4% LEU 7% 6% 7% 7% 7% 7% 6% ORN 6% 4% LYS 1% 8% 5% 6% 0% 5% AEROBIC INCUBATIONS Sample ID Days of experiment/AA sNAO-1 sd 0 sNAO-2 sd 4 sNAO-3 sd 7 sNAO-4 sd 20 sNAO-5 sd 35 sNAO-6 sd 64 NAO-7 sd 206 ASP 11% 11% 10% 4% 8% 6% 11% GLU 10% 11% 9% 11% 8% 8% 8% AAAA 2% 0.0 SER 5% 7% 7% 7% 6% 6% 10% GLY 15% 15% 16% 29% 27% 29% 13% THR 7% 8% 7% 8% 6% 7% 6% HIS 0% 2% 0% 0% 1% 1% 1% ALA 11% 12% 9% 11% 10% 9% 14% TAU 4% 0% 4% 3% 3% 4% 4% TYR 3% 3% 3% 2% 3% 2% 1% ARG 3% 3% 2% 3% 4% 3% 6% GABA 2% 0% 7% 0% 0% 0% 0% MET 1% 2% 2% 2% 1% 1% 1% VAL 7% 8% 6% 5% 6% 6% 7% PHE 4% 4% 3% 3% 3% 3% 4% ILE 4% 5% 3% 3% 3% 4% 2% LEU 6% 7% 6% 5% 6% 5% 7% ORN LYS 6% 5% 5% 5% 5% 6% 5% 175 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. concentrations and mole fractions of individual amino acids remained fairly constant, with an exception of one non-protein amino acids, a-amino-adipic acid, which was absent in the initial sample, but accounted for 2 % of THAA in the sample taken at Day 206 (Fig. 4a, Table V - 6 b). The concentration of THAA ranged between 30 to 40 pmol/g (dry weight) (Table V - 6 b). N-THAA accounted on average for 13 + 4 % of PON. This value is lower than commonly reported contribution of N-THAA to total nitrogen in coastal marine sediments. Typical values reported for shallow marine sediments range between 16 to 60 % (Keil et al., 2000). Burdige and Martens (1988) found that 20 to 40 % of sedimentary nitrogen is present in the form of N-THAA in the sediments of Cape Lookout Bight. The concentrations of particulates of THAA in the oxic incubation remained fairly constant or possibly increased slightly over the course of incubation, from 33 + 10 at the beginning to 51 + 17 pmol/g by day 35, and after that remained constant through the rest of the incubation period. Among individual amino acids, the aspartate mole fraction decreased over the first 64 days from 11 to 6 mole % (Table V - 7b, Fig. 4b), and then increased to the initial value. The glutamate mole fraction decreased slightly from 10 to 8 mole %. No corresponding changes in absolute concentrations were detected for these amino acids. The glycine mole fraction increased from 15 to 29 mole % by day 64, and then decreased to the value close to initial (13 mole %). For glycine the changes in mole % were accompanied by the changes in absolute concentrations of equivalent magnitude. 176 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.35 0.30 0.25 0.20 0.15 0.10 0.05 Day 0 ■ 1 lllll iLilI Day 20 I . Day 64 ll.i I _ -■ ■ ■ Day 206 li ....I ■ ■ ■ 1 1 I Q-3^Qr>-CCf/,<Z)Q;Occt-;-lLL1iii^ 6 w CO —I 5 l i l —I I — _ J < > - Q : < L L I < x y u L| D i s _ < 0 > 0 h K h : h: < 0 5 > [ i . - - i 0 J Fig. V -3 Mole fractions of amino acids: a) in AN-I pore water; 177 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 .2 5 Day 0 0.20 I 0.15 0.10 0.05 0.00 Day 20 0.50 0.40 Day 64 X 0.30 0.20 0.10 0.00 C L =3 C O —I < 0 CO0 h_ X < | _ | _ < ^ ^ > Q _ — _JO Fig. V -3 (continued) b) in OX-I pore water. 178 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LYS A A /TH A A AA/TH A A AA/THAA Day 35 i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i Day 206 o_ 3 > ce > - a : c o < - 3 t t : O c Q f-; -j in z> £ w C O ^ W O H X C H H C O S > CL — Zi O — i Fig. V -4 Mole fractions of amino acids: a) in AN-I sediments; 179 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. AA/THAA AA/THAA AA/THAA 0.30 0 . 2 5 0.20 0 . 1 5 0.10 0 . 0 5 0 . 3 0 0 . 2 5 0.20 0 . 1 5 0.10 0 . 0 5 ■ ■ . m l I Day 20 0 . 3 0 Day 64 0 . 2 5 0.20 0 . 1 5 0.10 0 . 0 5 Q - 3 C O - I < 0 o ;> - a :m < 3 a : ( 5 in ^- J S U M j 3 Z w m -ix — _3<>-cc< w < x L Hujt£>- C O 0h- ^ <l— H < C 0 2 > 0. — _l o -I Fig. V - 4 (continued) b) in OX-I sediments. 180 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 8 Isotopic composition of all measured nitrogen pools in AN-I, OX- 1 , AN-II and OX-II. A. 815N of all N pools (%c) Series 1 (AN-I and OX-I) * Series II (AN-II and OX-II) * Sample ID Days NH4+ Particulate N PON** Particulate N NH4+ A Unwashed sediments'1 KCI washed sediments A Anaerobic AN-0 0 1.6 ± 10 7.5 + 0.3 7.6 + 0.3 [6.0] 6.8 + 0.3 [0.21 AN-2 4 8.3 + 1.0 7.8 + 0.5 7.7 + 0.5 7.0 + 0.3 [4.3] AN-3 7 8.1 + 0.6 7.3 + 1.7 7.2 + 1.7 [8.5] [6.4] ± 1.2 [7.5] AN-4 20 7.9 + 0.0 6.6 + 1.0 6.4 + 1.0 [6.4] + 1.2 AN-5 35 9.7 + 0.4 7.9 + 1.3 7.7 ± 1-3 [6.4] [6.4] ± 1-2 AN-6 64 7.9 + 1.0 7.3 + 0.3 7.3 + 0.3 [6.9] [6.1] + 1.2 [8.3] AN-7 206 8.2 + 1.9 7.7 ± 1-2 7.6 ± 1-2 7.8 7.1 + 0.3 [12.5] Aerobic OX-O 0 2.2 + 1.0 7.3 + 0.2 7.4 + 0.2 7.5 6.9 + 0.3 OX-2 4 10.5 + 2.0 8.3 + 0.2 8.0 + 0.2 [7.0] 6.9 + 0.3 [3.5] OX-3 7 11.1 + 1.3 8.5 + 0.4 8.2 + 0.4 [7.4] [5.8] + 1.2 OX-4 20 16.6 ± 1.0 9.8 + 0.7 8.9 ± 0.7 [7.4] [7.0] ± 12 OX-5 35 29.8 + 1.0 9.3 + 1.2 7.1 + 1.2 [6.8] [7.0] + 1.2 OX-6 64 6.4 + 0.7 6.0 + 0.7 [6.7] [7.5] + 1.2 OX-7 206 11.4 + 0.5 10.9 ± 0.5 [10.7] 8.7 ± 0.3 * Uncertainties are + 1 stdev of the mean of duplicate analysis; the sample stdev of replicates is used when duplicates are not available ’* Calculated from isotopic mass balance for adsorbed and pore water ammonium and Particulate N as shown in Part B A [ ] indicate suspect analysis due to problems during the run B. Mass balalance corrections for the contribution of adsorbed and pore water DIN to d15N of OX-I and AN-I PON Sample ID Days d15N of Part. N ( M N total in each sample* (pmol) Pore water present* (ml) [NH4+] (mM) DIN in each sampleA (pmol) d15N of NH4+ A A (%.) d15N Norg- total, corr for NH4+ (%.) Anaerobic AN-0 0 7.5 5.34 0.03 2.60 0.10 1.6 7.6 AN-2 4 7.8 4.88 0.03 14.91 0.45 8.3 7.7 AN-3 7 7.3 5.06 0.03 15.08 0.46 8.1 7.2 AN-4 20 6.6 4.60 0.03 16.07 0.49 7.9 6.4 AN-5 35 7.9 4.92 0.03 16.83 0.50 9.7 7.7 AN-6 64 7.3 4.41 0.03 17.34 0.48 7.9 7.3 AN-7 206 7.7 4.09 0.03 18.08 0.49 8.2 7.6 Aerobic OX-O 0 7.3 5.21 0.02 3.59 0.13 2.2 7.4 OX-2 4 8.3 5.57 0.03 17.68 0.55 10.5 8.0 OX-3 7 8.5 5.70 0.03 18.74 0.60 11.1 8.2 OX-4 20 9.8 6.02 0.03 19.61 0.75 16.6 8.9 OX-5 35 9.3 5.50 0.03 16.67 0.55 29.8 7.1 OX-6 64 6.4 4.28 0.02 0.92 0.05 {43} 6.0 OX-7 206 11.4 5.46 0.03 1.04 0.06 {54} 10.9 * Calculated from N wt % and weight of analyzed sample ** Calculated from dry weight of each sample and porosity after centrifugation A Represents N from adsorbed ammonium and DIN in pore water 181 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. S > sN composition o f the sediments and pore water ammonium The changes in 8 1 5 N of the pore water ammonium and PON over the course of incubations are summarized in Table V-8 . The 8 1 5 N data on the KCl-treated samples are presented in Table V- 8 A as well. The 8 1 5 N of PON was calculated as shown in Table V - 8 B, based on the isotopic mass balance for the ammonium and Particulate N. Non-homogenous sampling of Series I and the mass spectrometer problems during the analysis of Series II compromised the precision of the measurements on samples from the both series (Table V - 8 A). As the result of this, the standard deviation of duplicate analysis was > 1 %o for AN-I and OX-I, and the standard deviation of internal standard measurements was 1.2 %o for most of the AN-II and OX-II samples. Anoxic incubations In AN-I and AN-II series, the 8 1 5 N of the PON did not change over the 6 months of incubations (Table V - 8 A and Fig. V - 5). In the AN-I series, the PON S1 5 N at the beginning and at the end of the incubations was 7.5 and 7.7 %o respectively, with the average value of 7.5 + 0.2 %o. The average 8 1 5 N of the sediments, treated with KC1 was 0.5 + 0.5 %o lighter than untreated sediments, a difference that is not significant statistically. The S1 5 N of ammonium initially present in the slurry was 1. 6 %o. By day 4, 8 1 5 N of ammonium in AN-I increased to 8.3 and remained at a constant 8.3 + 0.3 %o for the rest of the incubations. The 8 1 5 N of ammonium from AN-II differ from the S1 5 N in AN-I. However, as indicated in Table V - 8 , the AN-II values are considered 182 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12 10 8 6 4 250 200 50 100 150 0 Days of incubations Fig. V - 5 51 5 N of the Norg-total in AN-I and OX-I series. The error bars represents precision based on duplicate analysis of the same sample 183 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. suspect due to the analytical problems. Therefore, the discussion will be based only on the ammonium 8 1 5 N obtained from AN-I samples. Oxic incubations The 8 1 5 N of PON in OX-I became overall heavier as the incubation progressed (Table V - 8 A, Fig. 5) changing from the initial value of 7.3 %o to 8.9 %o by day 20. After 2 months, the PON 8 1 5 N became lighter by about 3 %o, and afterwards, increased again to the value of 10.9 %o by day 206. The isotopic composition of ammonium was measured only on some of the samples from OX-I series. The last measurement made on the sample taken on day 35 showed the very strong enrichment in ammonium isotopic composition (29 %o). The heavy 8 1 5 N of ammonium, combined with the decrease in concentrations further indicate that a significant portion of ammonium pool has been removed by the aerobic ammonium oxidation. The fractionation factor associated with aerobic ammonium oxidation by pure bacterial cultures varies between -14 and -38 %o (Casciotti et al., 2003). Assuming a fractionation factor for ammonium oxidation of -15 %o (since environmental fractionation is often smaller than that of pure cultures), the 8 1 5 N of residual NH4+ was estimated as 54 %o by the end of the incubation (Table V - 8 B). Due to the large isotopic enrichment of the ammonium in OX series, the 8 1 5 N of Particulate N and PON differed substantially from each other (Table V - 8 A and B, Fig. 5) 184 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion Decomposition o f different fractions o f organic matter with variable lability The organic matter in the initial slurry was the mixture of fresh plankton biomass and old sedimentary organic matter. These two fractions are expected to have different labilities and, therefore, variable rate constants of decomposition. Indeed, the precipitous decrease of PON under aerobic and anaerobic conditions within the first 20 days of incubation and the substantially slower decline afterwards (Fig. V - 6 ) reflects the decomposition of more than one fraction of organic matter. The data were fitted with a double exponential decay equation (Westrich and Berner, 1984) (Fig. V - 6 ) following an approach employed by Lehmann et al. (2002) in their analysis of the incubation experiment: _ (-*l0 _ ( - * 2 0 5-2 (0 = le + 2e where G ( t) is the concentration of residual P O N i during the course of incubation, G i and G2 are fractions with different lability, and ki and k2 are the first order reaction rate constants for the decomposition of fractions Gi and G2 respectively. The sum of G i and G 2 is equal to the total concentration of P O N in the initial slurries. The G i and G2 values found by fitting the data with the equation 5-2 (Table V - 9) were comparable to the initial distribution of P O N between the N org-piankton and N org-sed fractions. However, the value for G i , the labile fraction, was smaller than the size of the N org-piankton fraction for both A N - I and O X - I series. Conversely, the refractory fraction, G 2 was bigger. This difference may imply that not all N org-piankton was labile, but some of it was refractory. 185 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14 PON in a n o x ic in cu b ation - 1 13 12 11 10 9 8 200 150 50 100 0 o E Z o 0 _ 16 PON in o x ic in cu b ation - 1 : 15 14 13 12 11 10 200 0 50 100 150 Fig. V - 6 Kinetic of PON decomposition in anoxic (a) and anoxic (b) incubations. Open diamonds are data not included in the fit calculations Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table V - 9 Multi-G model parameters from fitting and observed distribution of N between fractions with different lability. Series ID Norg-planton measured (umol/ml) Norg-sed measured (umol/ml) G 1 (umol/ml) G2 (umol/ml) k1 day'1 k2*1000 day'1 AN-I 2.8 + 0.03 9.4 + 0.1 1.8 + 0.4 10.4 + 0.4 0.13 + 0.08 0.62 + 0.31 OX-I 3.3 + 0.03 10.6 + 0.1 1.2+ 0.1 12.8 + 0.1 0.10 + 0.02 0.19 + 0.04 Table V -10 Mass balance calculations for AN-I series (concentrations are pmol/ml, 6 1 5 N are in % o). Measured Calculated from mass balance 815N of Mass balance for Concentration Concentration Concentration NH4 NH4+ over 4 days 815N initial initial 815N final final added added 1.6 + 1 2.60 8.3 + 1 14.9 12.3 9.7 + 1.2 Mass balance for S15N Of PON over 206 Concentration lost Concentration Concentration days 815N initial initial fraction lost final 815N final 7.5 + 0.3 12.2 9.7 + 1.2 3.00 9.2 6.7 + 0.6 187 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The rate constants for decomposition of the labile fractions, ki, were similar for both anaerobic and aerobic incubations, being 0 . 1 0 d' 1 for the decomposition in the presence of oxygen and 0.13 d' 1 for the decomposition under anoxic conditions (Table V - 9). This similarity is not very surprising, since there is no agreement in the current literature on whether degradation of fresh organic matter occurs faster in the presence of oxygen or under anoxic conditions (Pedersen and Calvert, 1990), Harvey et al., 1995, (Kristensen and Holmer, 2001); Lehmann et al., 2002). One peculiarity was observed in the changes of PON pool in the oxic incubations. The PON decreased over the first 20 days, but by the day 35, it increased again and remained more or less constant through the rest of incubation period. The day 20 data point lies well off the overall trend, and was not included into the fitted data. As a single data point, it cannot bear a lot of statistical significance. However, including this data point and fitting the data from the first 2 0 days of degradation only, would have resulted in 1 0 times higher rate constant, ki, for the aerobic degradation. The rate constant for degradation of the refractory fraction, k2, was slightly higher for the anaerobic decay, 0.0006 d'1 , compared to the aerobic degradation with k2 equal to 0 . 0 0 0 2 d'1 . One explanation for the observed dynamics of aerobic decay (lower rate constants for both labile and refractory fractions than for anaerobic decay) is a significant contribution of newly formed bacterial biomass to the total organic matter in the slurry. The growth of new bacterial biomass would be consistent with several other observations as well. It would explain the increase in the concentrations of particulate THAA through time (Table V - 6 b), and the increase in glycine absolute 188 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. concentrations and mole fractions (Fig. 4 b, Table V-6 b). This amino acid is present in high abundance in a peptidoglycan layer in bacterial cell walls. Bacterial growth during incubation experiments has been observed in some of the previous studies. Harvey et al. (1995) found that between the 20th and 40th days of incubations, up to 20 % of the total particulate carbon was contributed by newly formed bacterial biomass. During the incubations of marine sediments mixed with eelgrass, Pedersen et al. (1999) observed a gradual increase in THAA concentrations over a period of one month, which they attributed to protein synthesis by growing bacteria. More evidence for the contribution of bacterial biomass is the decrease in C/N ratio in the oxic incubations, starting with day 35 of the experiment (Table V-4A). Proteins, on average, have lower a C/N ratio, of about 4 to 5 (Muller, 1977), compared to bulk marine organic matter, which has C/N of 6.7 (Redfield, 1963). The decrease in C/N reflects, most likely, bacterial synthesis of proteins, associated with bacterial growth (Pedersen et al., 1999). Isotopic fractionation associated with the decomposition o f organic matter Anaerobic decomposition: isotopic mass balance apvroach In an anaerobic setting, the S1 5 N of ammonium added should be equal to the 6 1 5 N of the decomposing PON plus any fractionation, if it occurs. The initial 8 1 5 N of ammonium in AN-I was 1.6+1 % o. On day 4, the 8 I5 N of ammonium became 8.3 % o. This change was associated with the addition of 12.3 jimol/ml of ammonium from decomposed DON (Table V - 10). The isotopic mass balance requires that the 8 1 5 N of added ammonium is 9.7 ±1.6 % o. This value is in very good agreement (within the 189 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. uncertainty associated with the mass balance calculations and isotopic measurements) with the 8 15N of N org-piankton (9.3 + 0.2 %0 ). One implication of this is that the 6 15N of D O N was fairly similar to the 8 15N of particulate plankton. The ammonium pool increased by 3 pmol/ml between day 4 and 2 0 6 , without any statistically significant change in its 8 15N . This implies that the isotopic composition of the source for ammonium did not change over time. The total loss of P O N over the course of the incubations was comparable to the total increase of N H / concentration between day 4 and day 2 0 6 . The decrease in P O N reflects primarily the loss of Norg-piankton, and the 8 15N of P O N (which is a mixture of N org-sed and N org.piankton) is 7 .4 % o. The isotopic mass balance predicts that by the end of the incubations (when most of the N org-piankton is degraded) the 8 15N of P O N should be 6 .7 ± 1 . 3 %o, if no fractionation occurs during decomposition. The 8 15N of the P O N measured on day 2 0 6 was 7 .7 ± 1 . 2 % o, which is in good statistical agreement (within the precision of analysis) with the predicted value. Therefore, we can conclude that no isotopic fractionation occurs during decomposition of organic matter in the absence of oxygen. This does not mean that s is zero at each step of nitrogen transformation, but if s is zero for the release of D O N , its efficient conversion to ammonium results in no fractionation of the P O N that decomposes. However, if the P O N represents the mixture of several (two in this case) isotopically distinct fractions of different lability, the 8 15N of the residual mixture may change due to the preferential degradation of one of the fractions. 1 9 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Aerobic decomposition - fractionation in two different components The 8 15N of P O N (corrected for adsorbed and pore water ammonium) changed over the course of the incubations (Fig. 5, Table V - 8 A) from 7.4 %o on day 0 to 8.9 %o on day 20 to 10.9 on Day 206. The change in the 8 15N of the N org-totai suggests that oxic decay may be accompanied by an isotopic fractionation. As shown in the previous section, at least two different components of organic matter were present in the slurry. One possible interpretation of this data set is that Rayleigh fractionation occurs during oxic diagenesis. Because two components of P O N are present, the isotopic composition of P O N may evolve in a complex way. In order to examine the isotopic effect associated with decomposition of each fraction on the 5 15N of the P O N through time, a simple mixing model was constructed. In this model, organic matter was treated as two separate fractions, labile and refractory. The size and the isotopic composition of the fractions were those determined for the N org-piankton (more labile component) and N org-sed (more refractory component) pools respectively (Table V - 3). Each fraction was decomposing according to the first order rate law kinetics, with the rate constants determined by fitting the data with two-G model (Table V - 9). It was assumed that the 8 15N of each fraction changes during decay according to Rayleigh fractionation equation (Mariotti et al., 1981): gin f 15 io 3 -3 3 5-3 S N o r g = e LV * (10 < ? 0 + 1) - I) * 10 which can be simplified to: 191 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 15 5-4 8 N =lnf*e + S N 0 org)t where 8 15N org ) t= isotopic composition of the nitrogen in each of the two components of the P O N at time t, f = the fraction of nitrogen from each component left at time t, s is the fractionation factor and 5 15N o is the initial isotopic composition of each component (Table V - 3). The 8 15N of the mixture was calculated as the isotopic mass balance between the 8 15N of each of the components, N org-piankton and N org. sed. The parameter which is not known in this model is the fractionation factor, s. The S 15N of P O N was calculated using several different values for s. The same s was used for both labile and refractory components. Fig. V - 7 a shows the 8 15N changes during decay with no isotopic fractionation, when s=0. The decrease in the predicted isotopic ratio through time is due to the preferential loss of the labile fraction, N org-piankton with 8 15N of 9.3 % 0, so that the 8 15N of P O N is approaching the isotopic composition of the refractory N org.sed , 6 . 8 % o . The obvious disparity between measured and predicted values in Fig. 7a indicates that s * 0 during the oxic decay of organic matter. The isotopic composition of dissolved ammonium present at the beginning of the incubations was about 1.8 % o . This ammonium was released over a short period of time (less than 5 hours) between plankton collection and refrigeration. Therefore, the difference, A 8 15N piankton-NH4+ = 9.3-1.8 = 7.5 % o , may be a close approximation of the instantaneous fractionation factor for the plankton decay. Fig. 7b shows the calculated 8 1 5 N of N org-totai, decomposed with a fractionation factor of -7 % o . The predicted and measured values are in better agreement than in the previous case, but the predicted 192 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to 11 9 7 5 0 50 100 150 200 250 11 8 — ■ 7 %o 9 7 5 0 50 100 150 200 250 11 £ = - 12 % < 9 to 7 5 0 50 100 150 200 250 • Measured values Days of experiment ■ Predicted by mass - - balance, rates from fitting - Predicted by mass balance, rate for refractory fraction is 4 x rate from fitting Fig. V - 7 Isotopic composition of P O N , measured and calculated by from the 5 1 5 N of the two fractions: N org.piankton and N org-sed. Black squares indicate the isotopic composition of samples from OX-II series 193 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. values are still lower than the measured §15N. The best fit between predicted and most of the measured values was obtained with s set to - 12 %o (Fig. V - 7c, solid line). However, the very last data point is still not perfectly matched for by this fit. In order to approach the value of the last data point, the rate constant of the refractory component had to be increased four times compared to the model-derived value of k? = 0.0002 d' 1 (Table V - 9). The dashed line in Fig.7c shows the modeled values with s=-12 %o, k i = 0 .1 0 , as obtained by fitting the double exponential decay function (Table V - 1 0 and Fig. V - 6 ) and k 2 = 0 .0 0 0 8 d'1 . The 4-fold increase in the rate of decomposition requires that a larger than observed fraction PON would have been lost to degradation. A second alternative is that the fractionation factor of decomposition might be much bigger than - 1 2 %o, which seems unlikely. The value e=-12 % 0, which produced the best fit between measured and calculated values, substantially exceeds the magnitude of fractionation factors reported by previous studies of the organic matter decay. Silfer (1992) found - 4 % 0 fractionation associated with peptide bond hydrolysis. Macko et al. (1986) reported values of - 2 to - 9 %o fractionation factor for the transamination process. Lehmann observed 4 % 0 enrichment in the residual plankton material after about 80 % of it was degraded, requiring s of -2.5 % 0 according to Rayleigh calculation. However, there may have been PON with multiple components, which have not been taken into account. On the other hand, Macko and Estep ( 1 9 8 4 ) used a variety of amino acids and found great variability (from - 9 to 22 % 0 ) in isotopic fractionation between the 8 1 5 N of bacterial cells and the 6 1 5 N of these amino acids. They argued that the magnitude 194 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and direction of the fractionation depends on the specific metabolic pathway through which each amino acid is incorporated or utilized by the cell. A 6 % enrichment of the residual plankton material over 6 days of incubations was observed by Holmes et al. (1999). However these authors did not report a fractionation factor. Therefore, the -12 % o fractionation factor, estimated from this study may not be a reasonable estimate. A third, alternative to the Rayleigh model explanation, which could resolve this discrepancy, might be the ingrowth of bacterial biomass, mentioned earlier. If this is significant and isotopically distinct from Norg-piankton and Norg-sed, the 8 1 5 N of the PON could change. This idea will be explored in the next section. Processes leading to fractionation o f nitrogen isotopes during organic matter decomposition The observed changes in 8 1 5 N during organic matter decomposition suggest that two different mechanisms may be responsible for the isotopic fractionation associated with diagenesis. First, preferential degradation of the isotopically distinct, more labile fraction leads to the alteration of isotopic composition of the residual organic nitrogen. This process may occur under anoxic as well as oxic conditions, and may be important in diagenetic settings, such as coastal margin sediments, where the bulk sedimentary organic matter may be a mixture of a more labile, isotopically enriched marine fraction and a more refractory and isotopically depleted terrestrial fraction. Changes in the sedimentary 8 I5 N due to the loss of isotopically heavier marine organic matter were first proposed by Sweeney and Kaplan (1980), who observed 3 % o enrichment in pore water ammonium relative to the 8 1 5 N of bulk 195 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sediments. Our work in Santa Barbara basin (Chapter II) corroborates their observations. The result of the incubation experiments, presented here further support this scenario. The second mechanism, which leads to the isotopic enrichment of the residual organic matter, seems to be at work only in the presence of oxygen. The isotopic enrichment during oxic decay was found experimentally by Macko et al. (Macko and Estep, 1984; 1987) and Macko and Estep (1984), in the field by Sigman et al.(1999), Holmes et al. (1999), Libes and Deuser (1988), Altabet (1988), Francois and Altabet (1994). Lehmann et al. (2002) observed 4 to 5 % o positive shift in isotopic ratios of lacustrine plankton incubated in the presence of oxygen. Our incubation experiments also show enrichment in PON 8 15N. Macko et al. (1987) suggested that the observed enrichment was due to a kinetic fractionation which might take place during hydrolysis of peptide bonds of degrading proteins or during deamination and transamination of amino acids. The similarity in 8 1 5 N of PON and DON, and the light ammonium that was present in out starting incubation material suggest that fractionation should occur in the deamination and possibly, transamination steps. The question is why do these processes result in the alteration of the 8 I5 N of residual organic matter, only when organic matter decomposes in the presence of oxygen, and seem to have no effect when organic matter degrades anaerobically? The answer may lie in the difference of metabolic capabilities of aerobic heterotrophic and anaerobic bacterial communities, as they decompose organic 196 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. substrates. Most of the aerobic heterotrophs are capable of metabolizing their substrates completely (Fenchel et al., 1998). Individual aerobic bacteria can hydrolyze proteins and then consume the products of hydrolysis (short peptide chains and individual amino acids) themselves. On the other hand, in anaerobic setting, the decomposition of organic substrates is accomplished by a microbial “food web” (Fenchel et al., 1998). Fermenting microorganisms perform the hydrolysis and produce short carbon chain molecules. The products of fermentation are utilized by other members of bacterial/archeal communities, such as sulfate reducers, if sulfate is available, or, in the absence of sulfate, by methanogens. This metabolic difference between aerobic and anaerobic organisms must result in different modes of nitrogen incorporation for these groups. Since aerobic bacteria are able to metabolize the whole molecules of short peptides and amino acids, they probably fulfill their nitrogen requirements heterotrophically, from assimilating a portion of DON, affectively converting it back to PON. In the process of DON assimilation, bacteria break the longer peptide chains into shorter compounds and remove amino groups with production of ammonium. As work of Macko and Estep (1984) demonstrated, the 1 4 N containing bonds are broken down preferentially to 1 5 N containing bonds. That means that isotopically lighter DON is preferentially converted to ammonium, while isotopically heavier portion of DON has a greater chance of being incorporated in form of short peptides into growing bacterial biomass, or in other words into PON. Therefore, the isotopic enrichment of in the sedimentary organic matter, which includes some biomass of heterotrophic aerobic bacteria may, in 197 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. part, represent the trophic level effect, well known on a variety of ecological scales (Montoya, 1994; Montoya et al., 1992; WadaE. et al., 1987). On the other hand, anaerobic bacteria, such as sulfate reducers and methanogens, are able to metabolize only short chain carbon compounds, such as acetate. Consequently, they must obtain their nitrogen mostly by assimilation of ammonium, which typically present in sufficient quantities under anoxic conditions. The biomass formed with ammonium assimilation would be, if anything, isotopically light, since ammonium assimilation is accompanied by strong negative fractionation (14NH4 + is taken up preferentially (Hoch et al., 1992)). Another important factor may be the difference in growth yield between aerobic and anaerobic bacteria. Due to the higher energy yields in oxic metabolism, a much bigger proportion of organic substrates is retained by aerobic bacteria for growth, while anaerobic bacteria process a bigger proportion of organic matter as their energy source (Fenchel et al., 1998). The larger contribution of bacterial biomass to PON during oxic organic matter degradation would lead to the more pronounced net isotopic effect for oxic decay. Summary The results of the incubation experiments showed that changes in 8 1 5 N of preserved organic matter might occur via two processes. First, preferential degradation of a more labile isotopically distinct fraction can result in alteration of the §1 5 N of the bulk organic matter which survives degradation. Often, in coastal sediments, organic matter is a mixture between more labile marine organic matter, which is isotopically 198 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. heavier and terrestrial fraction, which is isotopically lighter. Preferential decomposition of the marine fraction may leave the residual bulk sedimentary organic matter isotopically depleted. The effect might be substantial, if the large fraction of terrestrial component is present. This scenario may occur under both oxic and anoxic conditions. Under oxic condition, a second mechanism of fractionation, preferential incorporation of 1 5 N enriched DON occurs. This leads to the enrichment of the residual organic matter in 1 5 N isotopes. It is likely that only aerobic bacteria are able to affectively incorporate DON into their biomass. It is possible that the first and the second mechanism may cancel each other out, if substantial a fraction of terrestrial component is present. 199 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References: Altabet, M.A., 1988. Variations in nitrogen isotope composition between sinking and suspended particles: Implications for nitrogen cycling and particle transformation in the open ocean. Deep-Sea Research, 35(535-554). Altabet, M.A. and Francois, R., 1994. Sedimentary Nitrogen Isotopic Ratio as a Recorder for Surface Ocean Nitrate Utilization. Global Biogeochemical Cycles, 8(1): 103-116. Bower, C.E. and Holm-Hansen, T., 1980. A salicylate-hypochlorite method for determining ammonia in seawater. Canadian Journal of Fisheries and Aquatic Sciences, 37(794-798.). Burdige, D.L. and Martens, C.S., 1988. Biogeochemical cycling in an organic rich coastal basin: 10. The role of amino acids in sedimentary carbon and nitrogen cycling. Geochimica et Cosmochimica Acta, 52: 1571-1584. Casciotti, K.L., Sigman, D.M. and Ward, B.B., 2003. Linking diversity and stable isotope fractionation in ammonia-oxidizing bacteria. Geomicrobiology Journal, 20(4): 335-353. Fenchel, T., King, G.M. and Blackburn, T.H., 1998. Bacterial biogeochemistry: The ecophysiology of mineral cycling. Academic Press, London. Fetter, C.W., 1999. Contaminant Hydrogeology. Prentice Hall, Upper Saddle River, NJ, 500 pp. Harvey, H.R., Tuttle, J.H. and Bell, J.T., 1995. Kinetics of phytoplankton decay during simulated sedimentation: Changes in biochemical composition adn microbial activity under oxic and anoxic conditions. Geochimica et Cosmochimica Acta, 59: 3367-3377. Hoch, M.P., Fogel, M.L. and Kirchman, D.L., 1992. Isotope Fractionation Associated with Ammonium Uptake by a Marine Bacterium. Limnology and Oceanography, 37(7): 1447-1459. Holmes, M.E., Eichner, C., Struck, U. and Wefer, G., 1999. Reconstruction of surface water nitrate utilization using stable nitrogen isotopes in sinking particles and sediments. In: G. Fischer and G. Wefer (Editors), Use of proxies in paleoceanography: Examples from the South Atlantic. Springer-Verlag, Berlin Heidelberg, pp. 447-468. 200 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Keil, R.G., Tsamakis, E. and Hedges, J.I., 2000. Early diagenesis of particulate amino acids in marine sediments. In: G.A. Goodfriend, C. M.J., M.L. Fogel, S.A. Macko and W. J.F. (Editors), Perspectives in Amino Acids and Protein Geochemistry. Oxford University Press. Kristensen, E. and Holmer, M., 2001. Decomposition of plant materials in marine sediment exposed to different electron accepters (0-2, N03-, and S042-), with emphasis on substrate origin, degradation kinetics, and the role of bioturbation. Geochimica Et Cosmochimica Acta, 65(3): 419-433. Libes, S.M. and Deuser, W.G., 1988. The Isotope Geochemistry of Particulate Nitrogen in the Peru Upwelling Area and the Gulf of Maine. Deep-Sea Research Part a-Oceanographic Research Papers, 35(4): 517-533. Mackin, J.E. and Aller, R.C., 1984. Ammonium Adsorption in Marine-Sediments. Limnology and Oceanography, 29(2): 250-257 Macko, S. A. and Estep, M.L.F., 1984. Microbial alteration of stable nitrogen and carbon isotopic compositions of organic matter. Organic Geochemstry, 6 : 787- 790. Macko, S.A., Fogel, M.L., Engel, M.H. and Hare, P.E., 1986. Kinetic isotopic fractionation during amino acid transamination. Geochimica et Cosmochimica Acta, 50: 2143-2146. Macko, S.A., Fogel, M.L., P.E., H. and Hoering, T.C., 1987. Isotopic fractionation of nitrogen and carbon in the synthesis of amino acids by microorganisms. Chemical Geology, 65: 79-92. Mahaffey, C., 2003. Biogeochemical signals for the supply of nitrogen to phytoplankton in the Atlantic Ocean, University of Liverpool, Liverpool, 224 pp. Mariotti, A. et al., 1981. Experimental determination of nitrogen kinetic isotope fractionation: some principles; illustration for the denitrification and nitrification processes. Plant and Soil(62): 413-430. Montoya, J.P., 1994. Nitrogen isotope fractionation in the modem ocean: Implications for the sedimentary records, Carbon Cycling in the Glacial Ocean: Constraints on the Ocean's Role in Global Change. NATO ASI Series, Series I: Global Environmental Change. Springer Verlag, pp. 259-280. 201 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Montoya, J.P., Wiebe, P.H. and McCarthy, J.J., 1992. Natural abundance of 1 5 N in particulat nitrogen and zooplankton in the Gulf Stream region and warm-core ring. Deep Sea Research, 39: S 363-392. Muller, P.J., 1977. C/N ratios in Pacific deep-sea sediments: effect of inorganic ammonium and organic nitrogen compounds sorbed by clays. Geochimica et Cosmochimica Acta, 41: 549-553. Pedersen, A.-G.U., Bernstein, J. and Lomstein, B.A., 1999. The effect of eelgrass decomposition on sediment carbon and nitrogen cycling: A controlled laboratory experiment. Limnology and Oceanography, 44: 1978-1992. Pedersen, T.F. and Calvert, S.E., 1990. Anoxia Vs Productivity - What Controls the Formation of Organic-Carbon-Rich Sediments and Sedimentary-Rocks. Aapg Bulletin-American Association of Petroleum Geologists, 74(4): 454-466. Redfield, A.C., Ketchum, B.H., Richards, F.A.„ 1963. The influence of organisms on the composition of of sea-water. In: M.N. Hill (Editor), The sea. Wiley- Interscience, New York, pp. 26-77. Sachs, J.P. and Repeta, D.J., 1999. Oligotrophy and nitrogen fixation during Eastern Mediterranean sapropel events. Science, 286: 2485-2488. Sigman, D.M., Altabet, M.A., Francois, R., McCorkle, D.C. and Gaillard, J.-F., 1999. The isotopic composition of diatom-bound nitrogen in the Southern Ocean sediments. Paleoceanography, 14: 118-134. Silfer, J.A., Engel, M.H. and Macko, S.A., 1992. Kinetic fractionation of stable carbon and nitrogen isotopes during peptide bond hydrolysis: Experimental evidence and geochemical implications. Chemical Geology, 101: 211-221. Sweeney, R.E. and Kaplan, I.R., 1980. Natural abundances of 1 5 N as a source indicator for near shore marine sedimentary and dissolved nitrogen. Marine Chemistry, 9: 81-94. WadaE., Terazaki, M., Kabaya, Y. andNemoto, T., 1987. 15N and 13C abundances in the Antarctic ocean with emphasis on the biogeochemical structure of the food web. Deep Sea Research, 34: 829-841. 202 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 6: Summary Fractionation of nitrogen isotopes in marine sediments: what did we learn? The work presented in this thesis covers two different themes. The first theme is fractionation of 8 1 5 N in organic matter associated with diagenesis in marine sediments. This theme was the original goal of this study. The second theme arose from the unexpected field results, which were obtained in 2001 during the CALMEX cruise to the Gulf of California. This theme represents the studies of benthic bacterial communities using S1 5 N of pore water ammonium. Theme 1 - Fractionation during organic matter diagenesis The interest in diagenetic fractionation of nitrogen isotopes in marine sediments is sparked by the wealth of oceanographic information offered by 8 1 5 N of marine organic matter as a paleoceanographic proxy. However, after the organic matter is deposited on the ocean floor, it is subject to the attack of a sedimentary microbial community. In the process of decomposition, bacterial (and archeal) metabolic activity can alter the original nitrogen isotopic ratio in the residual organic matter, thus obscuring the environmental information contained in the original 8 15N. The purpose of the present study was to evaluate the magnitude and the sign of diagenetic fractionation during organic matter decomposition. Two strategies were employed: in situ measurements and laboratory incubation experiments. The approach was to construct isotopic mass balances for the 8 1 5 N of preserved sedimentary organic matter and pore water ammonium. This approach was based on two assumptions: 1 ) systems are in steady state; 2 ) the only reaction which 203 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. determines concentration and isotopic composition of pore water ammonium is organic matter degradation. The second assumption is not valid in oxic settings, since ammonium is rapidly oxidized in the presence of oxygen with significant alterations in its S15N. Therefore, fractionation during oxic degradation of organic matter was studied under controlled conditions during laboratory incubation experiment. The in situ diagenesis in anoxic settings was investigated on short ( 1 0 to 1 0 years) and on long (1 0 5 and 1 0 6 years) time scales. Prior to the beginning of this study, three possible mechanisms were identified that might contribute to a preferential loss of isotopically heavier nitrogen: ( 1 ) sedimentary organic matter represents a mixture of more labile, isotopically heavier marine and more refractory, isotopically lighter terrestrial components, so that preferential decomposition of the labile marine fraction results in 1 5 N enrichment of the pore water ammonium; (2 ) during organic matter decomposition, a certain isotopically heavier group of amino acids is decomposed; (3) growing sedimentary bacteria incorporate lighter ammonium into their biomass, leaving the residual ammonium pool enriched in 15N. On long time scales, the diagenetic impact on the 8 1 5 N of preserved organic was evaluated using pore water ammonium and sediments, collected by two ODP expeditions, Leg 201 and 202. The expectation was that continuing loss of an isotopically distinct fraction with depth would have measurable effect on the 8 !5 N of preserved No rg , if a significant amount of sedimentary nitrogen has been lost to degradation. 204 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. At two of the five ODP sites examined (Sites 1227 and 1238), the 6 1 5 N of ammonium was 1 to 3 % o heavier than decomposing organic matter. The observed difference of 1-3 % o between the pore water and sediment 8 1 5 N was similar to that in Santa Barbara. However, examination of the depositional history at both sites indicated that the difference reflected the non-steady state in the isotopic composition of the organic matter deposited over time, rather than diagenetic fractionation. The recent deposition of isotopically enriched organic matter has resulted in the release of isotopically heavier ammonium. The 1 5 N enriched ammonium has been diffusing downward as an isotopically distinct front, different from the 51 5 N of decomposing organic matter at depth. The non-steady state hypothesis should still be tested by more rigorous numerical modeling. At three other ODP sites (Sites 1230, 1234 and 1235), the 8 1 5 N of ammonium was within < 1 % o from the 8 1 5 N of the bulk sedimentary organic matter, indicating that at depths greater than 1 0 mbsf, little fractionation is associated with organic matter decomposition. All the three sites were characterized by relatively high sediment accumulation rates and predominantly marine nature of the organic matter. None of them showed evidence for possible non-steady state conditions. The short term studies were conducted in the sediments of Santa Barbara Basin and in the Gulf of California, where low (< 2 pM) bottom water oxygen concentrations prevent bioirrigation of the sediments and result in complete anoxia in the sediments. The stoichiometric relationship of TCO2, and N H / in the pore water of the sediments in Santa Barbara Basin indicated that ammonium concentrations, and 205 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. therefore, its isotopic composition do not appear to be affected by processes other than organic matter decomposition. However, the organic matter at this site is the mixture of marine and terrestrial components. The S1 5 N of ammonium was enriched by ~ 3 %o relative to the bulk sedimentary 515N. At least half of the enrichment is attributed to the preferential degradation of the more labile and isotopically heavier marine fraction. By calculating the ammonium flux from the sediments, we determined that ~ 25 % of the sedimentary nitrogen rain was lost to diagenesis. Apparently, the bottom water anoxia, combined with rapid sediment accumulation rate prevents more complete degradation of organic matter in the Santa Barbara Basin sediments. Therefore, the loss of an isotopically heavier fraction (with fractionation factor of 1-3 %o) should have only a modest (~ - 0.7 % o) effect on the 51 5 N of preserved organic matter within the upper 7 m of the sediment column. The observed downcore increase in 8 1 5 N in the sediments of Santa Barbara Basin has been interpreted as the result of non-steady state in 8 1 5 N of Norg flux. The absence of diagenetic fractionation at depth in the sediments of the ODP sites contrasts with the results from the upper few meters in Santa Barbara Basin, where ammonium is 1-3 % o heavier than decomposing organic matter. The Santa Barbara results might be attributed to either of the two mechanisms (1) and (3) described above for isotopic fractionation. Laboratory experiments summarized below suggest mechanism (3) is not a significant factor. At the ODP sites without fractionation, the organic matter is predominantly of marine origin, as suggested by C/N ratio of the sediments, hence mechanism (1) should not occur. Mechanism (3) 206 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. should not operate at ODP sites because bacterial assimilation of ammonium is expected to decrease exponentially with depth, so that at the depth scales of ODP cores (> 1 0 mbsf) no measurable isotopic effect is present. In order to investigate the effect of differential decomposition of organic matter comprised of several fractions of variable labilities with distinct 8 I5 N of laboratory incubation experiments was conducted under anoxic conditions. In this experiment, fresh plankton material was mixed with the surficial sediments from Santa Barbara Basin. The 51 5 N of the plankton was about 9.3 %o, which was 2.5 %o heavier than the 8 1 5 N of the sediments (- 6 . 8 %o). The results of the anaerobic incubations showed that decomposition kinetics of the two-component mixture reflected preferential loss of the more labile fresh plankton. The 8 1 5 N of DON and ammonium released from this mixture was similar (within <0.5 %o) to that of the labile component, the plankton biomass. Any ammonium added from the isotopically lighter and more refractory sedimentary organic nitrogen was too small to impose a measurable effect on the 8 1 5 N of ammonium. The results of the anoxic incubations reproduced in situ observations from the Santa Barbara Basin. They also eliminated one of the alternative scenarios, proposed as the explanation for isotopic enfichment of pore water ammonium in Santa Barbara Basin: the preferential degradation of a certain class of isotopically distinct amino acids, as responsible for the difference between the 6 1 5 N of ammonium and No rg . Compositional analysis of THAA revealed no changes in the mole fractions of individual amino acids through time. An alternative explanation, which invokes 207 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ammonium assimilation into the newly formed bacterial biomass, cannot be excluded based on the results of this incubation experiment. However, no evidence supporting this scenario was found either. The incubations were also conducted under oxic conditions, since, as mentioned before, in situ investigation of oxic diagenesis is complicated by the isotopic effects of the aerobic ammonium oxidation. The results of oxic incubations of the two-component mixture showed that aerobic degradation of organic matter is accompanied by the net negative isotopic fractionation, an observation reported by a number of previous studies (see references in Background section and Chapter V). The oxic diagenesis of the organic matter also occurred according to the two-component exponential decay kinetics, similar to the anoxic decomposition. But, in contrast to the anoxic decomposition, both labile and refractory fractions were decomposed and fractionated during decomposition. A simple mixing model was constructed, which assumed Rayleigh fractionation during the decomposition of organic matter. This model incorporated the variable rates of the exponential decay for the two fractions present. A fractionation factor of about -12 % o for both fractions provided the best fit to most of the data points. The results of the incubation experiments showed that diagenetic alteration of the bulk 6 1 5 N of sedimentary organic matter may occur via two mechanisms: 1 ) in both oxic and anoxic settings, preferential degradation of an isotopically distinct fraction may result in changes of 51 5 N of the remaining mixture; 2 ) in the presence of oxygen, the decomposition of each individual fraction is 208 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. accompanied by a strong fractionation. Such fractionation does not happen when the organic matter is decomposed in the absence of oxygen. As an explanation for the difference in isotopic effect of oxic vs. anoxic organic matter decay, we hypothesized that it results from a the difference in metabolic capabilities between aerobic and anaerobic bacteria. Since aerobic bacteria are able to metabolize the whole molecules of short peptides and amino acids, they probably fulfill their nitrogen requirements heterotrophically, from assimilating a portion of DON, effectively converting it back to PON. In the process of DON assimilation, bacteria break proteins and long peptide chains into smaller fragments and remove amino groups with production of ammonium. In this process, the 1 4 N • 1S • containing bonds are broken down preferentially to N containing bonds. That means that isotopically lighter DON is preferentially converted to ammonium, while the isotopically heavier portion of DON has a greater chance of being incorporated into growing bacterial biomass, or in other words into PON. Therefore, the isotopic enrichment of in the sedimentary organic matter, which includes some biomass of heterotrophic aerobic bacteria, may, in part, represent the trophic level effect, well known on a variety of ecological scales. On the other hand, anaerobic bacteria, such as sulfate reducers and methanogens, are able to metabolize only short chain carbon compounds, for instance, acetate. Consequently, they must obtain their nitrogen mostly by assimilation of ammonium, which is typically present in sufficient quantities under anoxic conditions. The biomass formed with ammonium assimilation would be, if anything, isotopically 209 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. light, since ammonium assimilation is accompanied by strong negative fractionation, as 14NH4 + is taken up preferentially (Hoch et al., 1992). Another important factor may be the difference in growth yield between aerobic and anaerobic bacteria. Due to the higher energy yields in oxic metabolism, a much bigger proportion of organic substrates is retained by aerobic bacteria for growth, while anaerobic bacteria process a bigger proportion of organic matter as their energy source (Fenchel et al., 1998). The larger contribution of bacterial biomass to PON during oxic organic matter degradation would lead to the more pronounced net isotopic effect for oxic decay. This work has important implications for the application of organic matter 8 1 5 N as a paleoceanographic proxy. Our results confirmed previous observations that sedimentary organic matter, which is degraded in the presence of oxygen is isotopically fractionated in the process of decomposition. In oxic settings, this diagenetic alteration complicates successful interpretation of changes in the S1 5 N as reflecting changes in environmental signal. Important paleoceanographic information is recorded in the organic matter deposited along the productive coastal margins, which are also regions quite sensitive to the changes in climatic and oceanographic conditions. In such settings, the sedimentary organic matter is degraded mostly anaerobically. Our results showed that decomposition of organic matter under anoxic conditions is not accompanied by significant fractionation on the molecular level. However, the presence of isotopically distinct fractions with variable labilities might result in alteration of isotopic 210 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. composition of the residual bulk sedimentary organic matter. Such alteration is likely to occur within the first few thousand years. The possibility of the "component-level" fractionation must be taken into account in settings where significant contribution of terrestrial component is suspected. When only marine fraction is present, it is likely that the original 8 1 5 N of organic matter is well preserved. Theme II - Isotopic composition of ammonium and benthic microbial communities Ammonium in the upper 10-30 cm of pore water from six different sites in the Gulf of California was anomalously enriched in 1 5 N isotopes, by more than 10 % o relative to the 8 1 5 N of the bulk sediments. Isotopic mass balance calculations showed that the observed enrichment in ammonium is not matched by a corresponding loss of organic matter. The vertical structure of the pore water ammonium isotopic profiles, a change in reaction stoichiometry based on TCCVNH/ pore water ratios relative to the typical Redfield ratio, and the calculated isotopic composition of ammonium fluxes, all pointed towards the presence of a source of isotopically heavy ammonium. These observations were interpreted as evidence for anaerobic ammonium oxidation, which may be accomplished by a chemosymbiotic pair of lithotrophic bacteria, Thioploca spp. and Anammox - like organisms. Thioploca bacteria obtain energy by oxidizing pore water sulfide with nitrate. They take up nitrate at the sediment-water interface and transport it to depths of few tens of cm, where free pore water sulfide is encountered. We hypothesize that by transporting nitrate to the depth of a few tens of cm, the Thioploca bacteria play an unusual ecogeochemical role, introducing a thermodynamically potent oxidant into strongly reducing environments. We propose 211 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that a fraction of nitrate is converted by Thioploca into nitrite and used by anaerobic ammonium oxidizing bacteria, possibly Anammox. These bacteria may live on the surface of Thioploca filaments, where they are able to have direct access to both the oxidant, nitrite, and the reductant, ammomum, which they use to obtain necessary metabolic energy. The geochemical factors which create conditions favorable for Thioploca spp. were evaluated as well. It is likely that presence of dissolved iron in the pore water may determine the presence/absence of Thioploca by affecting the pore water sulfide profiles. It is possible that this process affects the isotopic composition of bottom water nitrate in the region. This study provided new insights into benthic microbial ecology and the role of bacterial communities in the sedimentary nitrogen cycle. It also demonstrated that variations in the species composition of sedimentary bacteria may substantially impact the bio geochemistry of the water column in the coastal waters. Reproduced with permission of the copyright owner. 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Velinsky, D.J, 1991, Burdige, D.J., and Fogel, M.L., 1991, Nitrogen diagenesis in anoxic marine sediments: Isotope effect, Carnegie Inst. Washington Annu. Rep. Director 1991, p. 154-162. Velinsky, D., Fogel M., 1999, Cycling of dissolved and particulate nitrogen and carbon in the Framvaren Fjord, Norway: stable isotope variations. Marine Chemistry, 67: 161-180. Wada , E., Kadonoga, and S. Matsuo, 1975,1 5 N abundance of in nitrogen of naturally occurring substances and global assessment of denitrification from isotopic point of view, Geochemical Journal, 9, p. 139-148. 225 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Wada E. and Hattori, A., 1978, Nitrogen Isotope effects in the assimilation of inorganic nitrogenous compounds by marine diatoms, Geomicrobiology Journal, 1, p. 85-101. Wada E., Terazaki, M., Kabaya, Y. and Nemoto, T., 1987, 1 5 N and 1 3 C abundances in the Antarctic ocean with emphasis on the biogeochemical structure of the food web. Deep Sea Research, 34: 829-841. Zopfi, J., Kjaer, T., Nielsen, L.P., Jorgensen, B.B., 2001, Ecology of Thioploca spp.: Nitrate and sulfur storage in relation to chemical microgradients and influence of Thioploca spp. on the sedimentary nitrogen cycle. Applied and Environmental Microbiology, 67(12): 5530-5537 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 226 APPENDIX A METHODS OF SAMPLE PREPARATION 227 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A -l Preparation of sediments for isotopic and elemental analysis 1. Take 2-3 g of frozen sample 2. Dry at 60-70 °C for 24 hours 3. Grind sample carefully until a homogeneous powder is formed 4. Keep ground sample in a small Wheaton vial A-2 Extraction of pore water ammonium for isotopic analysis by diffusion trap method (followed the procedure by Sorensen, 1991 and Holmes at al., 1998) 2.1 Preparation of the traps: Avoid touching any objects or part of the traps with your hand. Use gloves Material used: GF/C filters, diameter 2.5 cm, Teflon tape (3/4” wide), ruler, scissors, tweezers, 50 ml centrifuge tube 1. Cut Teflon tape into strips about 30 cm long 2. Rinse the strips in 10 % (v/v) HC1 once and two times with DDIW and allow to dry on an aluminum foil sheet (for about 30 min) 3. Cut filters in rectangular strips, ~ 2 cm long and 0.5 cm wide. Use gloves and tweezers 4. Combust GF/C filters for 4 hours at 450 °C 5. Place several layers of paper towels (4-6) on the bench, cover with 2 or 3 layers of aluminum foil and thoroughly clean it with methanol. Also clean with methanol scissors, tweezers, and all other objects which may touch the filters or traps 6 . Assisting yourself with the tweezers cut the dried tape strips into 6 cm long pieces 7. Using tweezers place a combusted filter on a piece of tape, add 20 pi of 2 M H2SO4 directly on the filter, and fold the tape, forming 3 cm long folded “envelope”. 8 . Take a 50 ml centrifuge tube with cap attached, clean the surface of the cap with methanol, and press the tube on the envelope firmly, slowly rotating and sealing all three open sides of the tape around the filter inside. Five traps at a time can be prepared. 9. Keep the prepared traps in a tightly closed Tupperware container. 2.2 Set-up of ammonium diffusion procedure Material needed: 50 ml polypropylene (PP) centrifuge tubes, MgO in powder, tweezers Set up the dilution scheme of the run, so that the samples in each particular set are as similar to each other as possible in their volumes and concentrations. Prepare standards of known isotopic composition (I used (NH4)2S0 4 as the internal standard). 228 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The volume and concentrations should be as similar as possible to the samples. If the sample volumes and concentrations vary within a considerable range, prepare several subsets of standards to cover the range. 1. Soak 50 ml PP tubes in 10 % (v/v) HC1 for a few hours, rinse once with DIW and twice with DDIW. Allow 24 hours to dry in the lab, loosely covered with paper towels. 2. Combust MgO at 650°C for 4 hours. 3. Put samples (diluted if need) or standards in tubes. The final volume should be at least 5 ml. Add ~ 20 mg of MgO with a spatula to each tube, using tweezers, drop one trap inside and cap immediately. Once MgO is added, the tube must be capped as quickly as possible to avoid ammonia loss. Visually examine the position of the trap in the tubes, making sure that it is not stuck to the sides, but is floating on the surface. 4. Incubate the tubes in a horizontal shaker at ~ 40 °C for 7 days or longer (7 days is sufficient for 5 ml samples, see Holmes et al., 1998 and Sigman, 1997 for discussion on the effect of volume on length of incubations). 5. Remove the traps with the tweezers, gently wipe with a clean Kimwipe and place each individual trap into pre-labeled compartments of a ice-cube tray, previously cleaned with methanol. 6 . Allow samples to dry in a dissecator for at least 48 hours. It’s a good practice to keep an open container with small amount of H2SO4 inside the dissecator to keep the environment ammonia-free. A-3Tips for isotopic analysis Directly before isotopic analysis, remove the Teflon tape with tweezers, and pack filters into tin boats (Costech). Fold the tin boat into a tight pellet. Make sure that each trap wrapped in tin is exposed to atmosphere for no longer than 1-2 hours. In the presence of the atmospheric moisture, tin will react with traces of acid on the filter, resulting in the poor combustion in the elemental analyzer and inaccurate measurements. If the carousel on the elemental analyzer is not isolated completely from the atmosphere, it is a good idea to pack and run no more than 5 traps at a time. If a carousel is sealed and continuously flushed with He, the tin-wrapped traps can be safely kept inside through the length of the whole daily run. A-4 Daily protocol for running elemental analyzer (EA) interfaced with isotope ratio mass spectrometer (IRMS) in a continuous flow mode It is a good idea to have a mass spec technician to introduce you to the machine 1. Replace a glass insert in the quartz tube daily, before taking the machine off the stand-by mode (up to 35 average-sized samples +15 standards can be run with one insert. This number should be smaller if running large sediment samples, > 30 mg, or large glass fiber (GF) filters.) 229 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2. Take EA off the stand-by mode. 3. After the EA reaches the working temperature, open the isolation valve (DON’T forget to turn off the source and wait till the pressure reaches the working range before turning the source back). Center N2 and CO2 beams, optimizing the peak shape and sensitivity (good sensitivity for N2 analysis is about InA per 1+ 0.3 pmol of N 4. Wait for the background to come down completely (no change in background over a minute). Operational background for Carlo Erba is 4-5 nA. Background above these values usually indicates a leak, which needs to be found and eliminated before you can start running samples. 5. Details on monitoring the beam quality and stability during the run should be discussed with the technician. 6 . Reagents in the reaction and reduction columns have to be replaced periodically. Normally, the reaction column reagents are good for 500-600 samples + standards. Reduction (copper) column should be replaced every 150-200 sample (but talk to the technician about the details). A-5 Using standards during runs The purpose of running standards is to: 1) monitor the accuracy of isotopic measurements; 2) determine elemental composition of your sample (wt % C and N).; 3) correct for “size effect”. 1) Standards to monitor accuracy and precision and drift during the run. It is best to run NIST standards for this purpose. One isotopic composition is enough for daily runs. We usually run 4 standards at the beginning of the run, and between every 5-7 samples. Once in a while it is a good practice to run one or two additional isotopic compositions of a wide range of values (for instance, from - 3 0 to + 3 0 %o). 2) Elemental standards. We normally use acetanilide (C= 70.3 wt %, N= 10.7 wt %). It is a good practice to run one acetanilide as an “elemental” at the beginning of the run (after you are satisfied with the accuracy and precision), and then run an acetanilide standard as a sample towards the end of the run. 3) Correction for “size effect”. “Size effect” is the offset between measured and true isotopic value, which is proportional to the size of the beam. Sometimes, partially, the “size effect” is due to atmospheric blank, but this is not always the case. It can be either positive or negative. The exact nature of “size effect” phenomenon is not known. In order to correct for it, we run 7-10 standards of known isotopic composition with 8 1 5 N as similar as possible to the analyzed samples, and then fit a regression (offset vs. size). The range of standard sizes should cover the expected range of samples. Size effect is often observed for nitrogen, and rarely for carbon. 230 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The same standard can be used for purpose #1 and #3. However, the precision and drift are best monitored with samples o f uniform size, which is unsuitable for the “size effect” correction. 231 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX B WECOMA 2003 CRUISE HYDROCAST, WHOLE CORE SQUEEZER, MULTICORE AND PISTON CORE DATA Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table B-l Hydrocast nutrient data, Wecoma cruise 2003 Station and sample ID N 03 ‘ + N02 ' pM detection limit = 0.5pM PO4 pM detection limit = 0 .5pM Si pM Depth m San Pedro Basin sp06-12 40.49 2.85 85.47 677.0 so06-11 40.50 2.91 89.65 698.0 sp06-10 40.13 2.92 85.19 717.0 sp06-9 40.74 2.92 93.27 738.0 sp06-8 39.88 2.89 94.07 758.0 sp06-7 40.03 2.93 93.63 778.0 sp06-6 39.41 2.91 95.95 798.0 sp06-5 39.42 2.94 95.89 818.0 sp06-4 39.28 2.93 92.68 838.0 sp06-3 38.99 2.90 98.92 858.0 sp06-2 37.24 2.93 101.26 878.0 sp06-1 37.63 2.94 100.48 888.0 Santa Monica Basin sm01-12 40.89 2.84 92.05 685.0 sm01-11 40.68 2.85 93.46 704.0 sm01-10 40.35 2.93 96.70 725.0 sm01-9 39.17 2.91 99.65 745.0 sm01-8 39.26 2.90 101.39 765.0 sm01-7 38.97 2.91 102.81 785.0 sm01-6 37.41 2.92 106.37 805.0 sm01-5 36.75 2.96 108.39 825.0 sm01-4 34.05 2.97 112.21 845.0 sm01-3 30.22 2.97 118.23 865.0 sm01-2 29.71 2.96 118.48 885.0 sm01-1 29.68 2.92 118.56 894.1 San Nicolas Basin sn18-12 BDL 0.17 3.59 5.2 sn18-11 43.72 2.96 115.41 1000.0 sn18-10 43.67 2.94 121.66 1300.0 sn18-9 43.00 2.94 124.28 1451.0 sn18-8 44.03 2.95 125.99 1520.0 sn18-7 42.26 2.99 128.61 1580.0 sn18-6 42.34 3.00 130.97 1619.2 sn18-5 42.51 3.01 129.97 1660.3 sn18-4 42.20 2.95 130.49 1699.0 sn18-3 43.00 2.98 131.33 1735.1 sn18-2 43.21 4.36 130.96 1770.0 sn18-1 43.47 2.97 131.15 1783.0 233 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table B -l (continued) Santa Barbara Basin sb09-1 21.90 3.27 115.73 581.0 sb09-2 22.05 3.30 116.20 575.0 sb09-3 22.06 3.29 116.21 570.0 sb09-4 22.06 3.27 116.54 560.0 sb09-5 22.88 3.23 114.44 550.0 sb09-6 25.79 3.24 109.94 540.0 sb09-7 25.55 3.19 108.20 529.0 sb09-8 25.81 3.18 108.86 519.0 sb09-9 27.72 3.16 105.55 510.0 sb09-10 28.79 3.13 101.08 500.0 sb09-11 35.27 2.19 50.80 300.0 sb09-12 1.63 0.19 5.92 10.5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table B-2 Whole Core Squeezer, Nutrient data Sample ID n o 3 ' + N02 ‘ pM n o 2- pM P04 pM Si pM Depth cm Santa Monica Basin We02sm-WCS-1 29.5 0.0 120.0 0.00 We02sm-WCS-2 28.9 0.0 3.8 119.8 0.00 We02sm-WCS-3 6.7 0.0 9.0 214.1 0.32 We02sm-WCS-4 252.2 3.2 24.9 272.4 0.90 We02sm-WCS-5 92.5 8.4 28.3 284.2 1.26 We02sm-WCS-6 106.7 21.4 27.4 295.3 1.63 We02sm-WCS-7 79.7 9.8 23.0 290.2 2.02 We02sm-WCS-8 131.1 9.2 23.9 225.7 2.27 San Pedro Basin We06sp-WCS-1 35.9 nd 3.3 107.7 0.00 We06sp-WCS-2 36.6 nd 2.9 108.4 0.00 We06sp-WCS-3 7.2 nd 4.2 160.2 0.31 We06sp-WCS-4 12.7 nd 12.8 190.8 0.93 We06sp-WCS-5 35.9 nd 14.7 223.1 1.50 We06sp-WCS-6 34.0 nd 12.6 227.7 1.87 We06sp-WCS-7 43.3 nd 13.4 248.7 2.47 We06sp-WCS-8 35.7 nd 14.3 259.6 2.79 Santa Barbara Basin We14sb-WCS-1 17.7 0.0 3.6 138.6 0.00 We14sb-WCS-2 17.8 0.0 3.7 139.4 0.00 We14sb-WCS-3 2.9 0.0 9.1 381.2 0.46 We14sb-WCS-4 0.0 0.0 28.6 671.7 1.12 We14sb-WCS-5 0.8 0.0 28.7 566.6 1.90 We14sb-WCS-6 630.0 2.63 We14sb-WCS-8 91.7 12.9 57.3 741.4 3.97 San Nicolas Basin We16sn-WCS-1 44.1 nd 3.0 142.5 0.00 We16sn-WCS-2 43.2 nd 3.0 134.2 0.00 We16sn-WCS-3 20.8 nd 3.1 186.2 0.39 We16sn-WCS-4 4.6 nd 3.5 205.9 1.56 We16sn-WCS-5 3.5 nd 3.7 215.0 2.13 We16sn-WCS-6 3.0 nd 4.0 224.7 2.91 We16sn-WCS-7 4.3 nd 4.2 230.8 3.49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table B-3a Santa Barbara Basin, multicore and piston core data: geochemistry of pore water, elemental composition of the sediments and 51 S N of NII4 and Nsed Pore Water Sediments Sample ID + 1 z mM + I z ■ z 10 % 0 C M O O l- mM T 3 0 ) ( 0 Z I- wt % ■ 0 o > < / > z u > " T o % 0 ■ 0 0 ) <0 O O 1 - wt % g - TOC/TN 3 sed ■ C a a > a cm We 14_1 0.168 11.77 3.51 0.64 7.2 4.67 8.51 1.3 We 14_1 c 0.186 9.92 nd nd nd nd nd 3.8 We 14_2 0.237 10.13 5.51 0.41 6.57 3.25 9.27 6.8 We 14_2 c 0.310 10.32 nd nd nd nd nd 9.3 We 14_3 0.372 10.66 6.77 0.44 6.56 3.71 9.93 11.8 We 14_4 0.440 nd 7.34 0.46 7.22 3.69 9.33 15.3 We 14_3 c 0.515 9.92 nd nd nd nd nd 18.5 We 14_5 0.552 10.13 8.60 0.42 7.05 3.36 9.39 21.8 We 14_6 0.600 10.01 9.10 0.42 7.03 3.42 9.56 25.3 We 14_4 c 0.667 10.42 nd nd nd 2.99 nd 29.0 We 14_7 0.739 10.77 10.57 0.38 7.08 3.11 33.0 We 14_8 0.840 9.68 11.53 0.37 7.29 nd nd 38.8 We 14_5 c 0.891 10.13 nd nd nd nd nd 42.0 We 14_9 0.930 9.88 12.65 0.35 7.07 2.80 9.42 45.3 We 14_10 1.030 10.67 13.90 0.44 7.36 3.54 9.49 51.8 We 14_11 1.112 10.27 14.55 0.40 7.61 3.25 9.48 57.3 We 14_12 1.180 10.22 15.77 0.44 7.57 3.61 9.48 61.8 We 12_12 2.890 10.23 39.36 nd nd nd nd 204.0 We 12_11 2.934 10.50 38.28 0.28 8.05 2.58 10.89 225.0 We 12_10 3.521 10.25 42.95 nd nd nd nd 268.0 We 12_9c 3.806 10.13 44.73 0.28 8.23 2.55 10.69 316.0 We 12_8 4.153 nd 44.89 nd nd nd nd 366.0 We 12_7c 4.251 10.33 49.19 0.29 8.63 2.74 11.09 420.0 We 12_6 4.434 9.52 46.59 nd nd nd nd 452.5 We 12_5 4.579 10.16 48.94 nd nd nd nd 486.5 We 12_4 4.625 9.96 49.06 nd nd nd nd 536.0 We 12_3 4.786 9.88 49.72 nd nd nd nd 587.0 We 12_2 4.979 10.24 48.73 0.29 8.95 2.69 10.89 637.0 We 12 1 4.987 10.07 53.42 nd nd nd nd 685.0 Notes: c=centrifuged samples. All others are squeezed in the hockey-pucks. Depth in piston core (We 12) is based on extrapolated ammonium profile from multi-core (We 14). 236 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table B-3b Santa Barbara Basin, multicore and piston core data, pore water nutrients and metals Sample ID N 03 ' + N02 ’ pM P04 3 ' pM Si pM Ca2 + mM Ba2 + pM Depth cm We 14_1 111.86 57.10 729.88 8.53 0.143 1.3 We 14_1 c nd nd nd nd nd 3.8 We 14_2 0.00 69.09 731.62 8.52 0.141 6.8 We 14_2 c nd nd nd nd nd 9.3 We 14_3 0.00 88.52 718.93 9.94 0.117 11.8 We 14_4 1.14 88.06 706.49 9.53 0.121 15.3 We 14_3 c nd nd nd nd nd 18.5 We 14_5 0.60 96.95 674.00 9.14 0.119 21.8 We 14_6 0.74 102.22 660.57 8.87 0.120 25.3 We 14_4 c nd nd nd nd nd 29.0 We 14_7 1.16 104.04 645.70 8.36 0.122 33.0 We 14_8 1.02 110.09 649.62 8.09 0.123 38.8 We 14_5 c nd nd nd nd nd 42.0 We 14_9 0.53 115.94 650.83 7.64 0.122 45.3 We 14_10 2.74 121.38 657.07 7.32 0.117 51.8 We 14_1 1 14.03 121.24 660.76 7.14 0.125 57.3 We 14_12 1.12 125.67 664.48 6.75 0.130 61.8 We 12_12 nd nd 758.82 2.96 nd 204.0 We 12_1 1 nd nd nd 0.97 nd 225.0 We 12_10 nd nd 654.85 3.47 nd 268.0 We 12_9c nd nd 626.49 3.26 nd 316.0 We 12_8 nd nd 678.69 3.21 nd 366.0 We 12_7c nd nd 641.63 2.94 nd 420.0 We 12_6 nd nd 690.02 3.02 nd 452.5 We 12_5 nd nd 619.05 2.86 nd 486.5 We 12_4 nd nd 698.64 2.73 nd 536.0 We 12_3 nd nd 522.63 2.61 nd 587.0 We 122 nd nd 523.19 2.61 nd 637.0 We 12 1 nd nd 592.08 2.53 nd 685.0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table B-4a Santa Monica Basin, multicore data: geochemistry of pore water, elemental composition of the sediments and 81 S N of NH4 + and Ns e d Sample ID Pore Water Sediments + X z mM X z 1 z IA V * C O % 0 ( N O O K - mM ■ a a » V ) z H wt % ■ c 0 ) w z t f > To % o T > 0 ) m O O 1- wt % | TOC/TN 3 sed .c a a > a cm We 02_14 0.046 nd 2.76 0.55 7.30 5.07 10.83 1.0 We 02 _14_.2 0.062 11.63 nd 0.00 nd nd nd 2.0 We 02 2 0.072 nd 3.13 0.56 7.64 5.12 10.75 3.0 We 02 3 0.091 nd 3.46 0.48 7.42 4.14 10.08 5.0 We 02_3_4 0.106 11.65 nd 0.00 nd nd nd 6.0 We 02_4 0.114 nd 3.76 0.48 7.89 4.19 10.29 7.0 We 02 5 0.140 13.27 4.12 0.52 8.08 4.65 10.52 9.0 We 02_6 0.168 9.60 4.37 0.52 8.00 4.49 9.99 11.8 We 02 7 0.197 11.66 4.93 0.54 7.75 4.60 9.90 15.3 We 0 2 8 0.217 12.93 5.09 0.51 7.69 4.26 9.81 18.8 We 02_9 0.235 12.40 5.49 0.52 7.74 4.38 9.91 22.3 We 02_10 0.258 10.53 5.87 0.47 7.69 4.21 10.42 25.8 We 02_11 0.280 11.44 6.19 0.20 5.91 1.86 11.07 29.3 We 02 12 0.308 nd 6.76 0.16 5.33 1.54 11.25 32.8 Table B-4a Santa Monica Basin, multicore data: geochemistry of pore water elemental composition of the sediments and 8 N of NH4 + and NS e d N 03 ‘ + N02 ’ pM N02 ' pM P04 3 ‘ pM Si pM Fe2 + pM Depth cm We 02_14 266.42 22.41 315.44 20.73 184.32 1.0 We 02 _14_2 nd nd nd nd nd 2.0 We 02_2 21.83 10.73 298.46 9.55 194.08 3.0 We 0 2 3 9.96 0.00 358.64 31.04 189.01 5.0 We 02_3_4 nd nd nd nd nd 6.0 We 02_4 6.96 0.00 413.26 18.01 199.14 7.0 We 02_5 2.17 0.00 379.46 16.25 205.24 9.0 We 02 6 1.78 0.00 385.96 27.27 169.49 11.8 We 02 7 0.95 0.00 395.73 22.54 207.23 15.3 We 0 2 8 1.45 0.00 423.92 32.18 180.24 18.8 We 02 9 9.89 0.00 405.15 33.85 178.65 22.3 We 02_10 nd nd nd nd 11.49 25.8 We 02 J 1 nd nd nd nd 183.23 29.3 We 02 12 1.52 nd 422.58 47.01 156.24 32.8 238 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table B-5a San Nicolas Basin, multicore and piston core data: geochemistry of pore water, elemental composition of the sediments and 81 5 N of NH4 + and Nse d Pore water Sediments Sample ID + X z mM X z 1 z in * 0 0 % 0 C M O o H mM ■ o V (0 z h- wt % T J a t u z in * 6 0 % 0 T J a > V ) O O H wt % | TOC/TN 3 sed o 3 Depth SN 16_1 0.007 nd 2.54 0.65 7.75 4.98 8.90 1.0 SN16_2 0.008 nd 2.79 nd 7.86 4.56 nd 4.0 SN 16_1 +3 0.000 11.01 nd nd nd nd nd 4.0 SN 16_3 0.031 nd 2.62 0.58 8.08 4.36 8.84 7.0 SN 16_4 0.054 17.89 2.48 0.49 8.47 3.67 8.75 10.0 SN 16_5 0.027 15.19 2.63 0.29 7.78 2.04 8.28 14.0 SN16_6 0.067 6.92 2.51 0.65 8.31 5.11 9.13 17.5 SN 16_7 0.045 15.21 2.60 0.69 8.16 5.41 9.13 21.5 SN 16_8 0.057 11.20 2.52 0.67 8.57 5.24 9.10 25.5 SN 16_9 0.072 10.77 2.56 0.53 8.14 3.96 8.76 30.5 SN16_10 0.047 nd 2.68 0.60 8.41 5.24 10.11 34.5 SN16_11 0.063 nd 2.62 0.62 8.44 4.99 9.33 38.5 SN 16_12 0.066 8.18 2.48 nd nd nd nd 42.5 SN 19_10 0.192 nd 2.99 0.38 9.20 3.70 11.25 82.0 SN 19_9 0.292 9.51 3.87 0.16 8.52 1.65 11.80 119.0 SN 19_8 0.636 10.00 7.37 0.38 8.23 3.97 12.07 170.0 SN 19_7 1.168 9.96 12.37 0.32 6.77 3.74 13.77 220.0 SN 19_6 1.675 9.91 19.78 0.34 6.84 4.01 13.93 269.0 SN 19_5 2.413 8.88 28.78 0.40 6.51 4.90 14.18 323.0 SN 19_4 2.781 9.59 31.37 0.31 6.43 3.73 14.16 369.0 SN 193 3.269 9.65 36.12 0.40 6.46 5.04 14.59 420.0 SN 19_2 3.741 9.96 42.82 0.33 6.82 4.05 14.49 466.8 SN 19 1 4.223 9.65 47.15 0.29 6.55 3.44 13.93 518.0 239 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table B-5 b San Nicolas Basin, multicore and piston core data, pore water nutrients and metals N03 ' + N02 - P04 3 ' Si Fe2 + Mn2 + Ba2 + Depth pM pM pM pM pM pM cm SN 16_1 0.832 BDL 240.01 8.41 0.00 1.267 1.0 SN16_2 1.324 6.387 276.60 46.53 0.00 1.238 4.0 SN 16_3 0.659 13.975 276.98 30.61 5.96 1.252 7.0 SN 16_4 0.723 3.072 253.84 18.81 0.00 1.252 10.0 SN 16_5 0.000 1.270 243.15 13.25 1.16 1.194 14.0 SN16_6 0.844 <0.5 232.33 11.76 0.65 1.180 17.5 SN 16_7 0.669 <0.5 232.45 13.23 0.00 1.274 21.5 SN 16_8 1.590 <0.5 222.74 13.38 0.00 1.165 25.5 SN 16_9 5.376 <0.5 222.84 7.43 0.00 1.252 30.5 SN16_10 1.286 2.822 220.42 6.13 0.00 1.151 34.5 SN16_11 1.995 0.586 218.02 5.70 0.00 1.151 38.5 SN 16_12 3.602 <0.5 210.76 3.43 0.00 1.180 42.5 SN 19_10 nd nd 270.54 nd nd nd 82.0 SN 19_9 nd nd 307.01 10.83 1.121 nd 119.0 SN 19_8 nd nd 428.69 6.02 1.231 nd 170.0 SN 19_7 nd nd 520.87 6.75 1.289 nd 220.0 SN 19_6 nd nd 570.11 9.67 1.231 nd 269.0 SN 19_5 nd nd 612.01 7.47 1.238 nd 323.0 SN 19_4 nd nd 655.81 nd nd nd 369.0 SN 19_3 nd nd 651.83 nd nd nd 420.0 SN 19_2 nd nd 672.00 nd nd nd 466.8 SN 19 1 nd nd 665.80 10.69 1.369 nd 518.0 240 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX C CALMEX - 2001 CRUISE MULTICORE AND GRAVITY CORE DATA Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table C -l a Station 10 Soledad Basin, pore water geochemistry, sediment isotopic composition, and 51 5 N of ammonium and sediments (nd= not determined, a = data provided by A.Graham, b= data provided by J. McManus) Pore water Sediments Sample ID + i z mM + * r 1 A Z O Z £ i n ■ 'lo %o mM < N o ( O < B mM 7 3 0 ) V ) z 1 - wt % 7 3 < 1 ) < A z' u > * & © %o 7 3 4 ) ( A O O 1 - W t % z t 7 3 o ® O M l- atom - E 0.0 g - O T a cm .c - M a a cm NH10_0 0.003 nd 2.37 nd 0.85 8.82 7.88 10.82 0.0 0.0 NH10_5 0.731 20.54 4.78 28.23 0.84 7.41 8.60 11.95 1.0 5.0 NH10_6 1.054 19.92 6.94 25.29 0.85 8.66 8.06 11.06 7.0 10.5 NH10_7 1.333 18.45 8.97 24.84 0.9 10.04 8.43 10.92 12.5 16.0 NH10_8 1.468 nd 10.88 22.81 0.85 9.42 8.39 11.51 18.0 21.5 NH10_9 1.628 17.03 13.41 21.68 0.81 9.90 7.98 11.50 23.5 27.0 NH10_10 1.730 nd 15.62 21.00 0.84 9.86 7.67 10.65 29.0 32.5 NH10_11 1.865 16.14 17.98 19.42 0.81 9.30 7.89 11.37 34.5 38.0 NH10_12 1.990 nd 20.50 18.97 0.79 9.96 7.91 11.68 39.0 43.5 NH10_2 2.056 15.10 21.82 18.97 0.84 9.08 8.65 12.02 44.0 47.5 NH10_G1 2.793 14.22 30.20 12.42 0.76 9.71 7.56 11.61 80.0 77.0 NH10_G2 4.633 nd 48.97 0.23 0.65 8.81 6.54 11.73 118.0 115.0 NH10_G3 5.621 13.15 55.37 0.23 0.66 10.29 6.63 11.72 158.0 155.0 NH10 G4 6.205 12.91 58.31 0.45 0.66 10.10 6.74 11.92 198.0 195.0 Table C-l b Station 10 Soledad Basin, pore water dissolved metal concentrations b Ca2 + Fe 2 + Mn 2 + S r2 + Ba 2 + Depth Sample ID mM UM pM pM pM cm NH10_0 0.0 NH10_5 9.60 43.80 1.14 93.8 1.22 5.0 NH10_6 9.11 12.60 0.16 91.5 1.29 10.5 NH10_7 8.65 4.57 0.29 92.3 1.23 16.0 NH10_8 8.33 1.95 0.99 89.4 1.17 21.5 NH10_9 8.18 7.37 0.00 90.9 1.15 27.0 NH10_10 7.85 11.20 1.27 88.0 1.18 32.5 NH10_11 8.13 11.90 0.72 86.6 1.18 38.0 NH10_12 7.38 5.44 1.52 88.6 1.22 43.5 NH10_2 7.20 8.94 0.51 87.2 1.47 47.5 NH10_G1 6.21 5.60 1.40 82.0 3.12 77.0 NH10_G2 4.66 6.75 0.90 76.3 16.50 115.0 NH10_G3 4.16 5.21 1.23 73.6 17.16 155.0 NH10 G4 3.89 11.66 0.79 75.1 15.84 195.0 242 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table C-2 Station 12 Magdalena margin, pore water geochemistry and 81 S N of pore water ammonium (nd= not determined, a = data provided by A.Graham, University of Hawaii,b = data provided by J. McManus, University of Oregon) Sample ID n h4 + mM 51 S N % 0 t c o 2 mM b Ca2 + mM a S042 ' mM Depth cm NH12_0 0 nd 2.40 nd nd 0.0 NH12_1 0.138 20.40 3.21 11.1 22.6 4.5 NH12_2 0.314 nd 4.00 11.2 26.9 10.0 NH123 0.505 19.81 4.99 9.7 27.8 15.5 NH12_4 0.637 18.33 6.28 5.5 16.3 21.0 NH12_5 0.789 17.34 8.13 10.9 25.5 26.5 NH12_6 0.917 15.84 10.48 10.8 24.8 32.0 NH12_7 1.045 15.30 13.05 10.4 20.0 37.5 NH12_8 1.138 nd 16.06 10.0 23.5 43.0 NH12_9 1.177 nd 18.10 7.7 20.8 47.0 NH12_G1 2.301 12.48 42.13 nd 3.2 85.0 NH12G2 3.212 12.54 50.24 nd 2.3 121.0 NH12_G3 4.053 12.49 54.36 nd 0.2 161.0 NH12_G4 4.845 nd 56.64 nd 0.0 202.0 NH12 G5 5.433 nd 58.82 nd 0.0 232.0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table C-3a Station 15 Alfonso Basin, pore water geochemistry, sediment elemental composition and 81 S N of pore water ammonium and Nsed (nd= not determined,a = data provided by A.Graham,b = data provided by J. McManus) Pore water Sediments Sample ID + X z mM X z i z in rr CO % 0 C M O o H mM C M * o (/) ( 0 mM ■ c 0 ) < / ) z H wt % ■ o a > (A Z m % 0 ■ o a > (0 O O H wt % Z t -O O a > O « H atom .c Q . a > a cm NH15_0 nd nd 2.38 9.69 0.66 4.69 8.24 0.0 NH15_1 0.147 21.08 3.66 27.1 11.22 0.87 6.11 8.17 4.5 NH15_2 0.396 21.27 4.54 26.7 11.11 0.91 6.33 8.10 10.0 NH15_3 0.615 20.31 5.57 26.2 11.37 0.95 6.63 8.11 15.5 NH15_4 0.821 19.78 6.56 23.9 11.82 0.89 6.24 8.17 21.0 NH15_5 0.973 20.36 7.56 23.3 11.20 0.79 5.87 8.64 26.5 NH15_6 1.140 20.11 8.32 23.5 10.82 0.77 5.67 8.60 32.0 NH15_G1 1.170 19.41 8.77 22.6 10.72 0.78 5.84 8.71 33.0 NH15_7 1.249 19.50 9.45 23.0 11.12 0.76 5.76 8.80 37.5 NH15_8 1.413 18.90 10.49 21.9 11.11 0.75 5.90 9.21 43.0 NH15_9 1.423 18.54 11.75 23.5 10.99 0.80 6.06 8.86 48.5 NH15_G2 1.858 16.94 20.90 16.0 10.94 0.70 5.44 9.09 70.0 NH15_G3 2.170 15.61 30.08 10.8 11.84 0.78 6.27 9.36 113.0 NH15_G4 2.444 15.45 42.76 4.3 10.86 0.60 5.23 10.16 155.0 NH15_G5 2.850 0.00 39.99 3.2 11.21 0.57 4.80 9.86 193.0 NH15_G6 3.152 14.76 45.22 1.4 10.92 0.40 3.85 11.19 234.0 NH15 G7 3.409 nd 47.75 2.3 11.12 0.43 3.67 10.02 273.0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table C-3 b Station 15 Alfonso Basin, pore water dissolved metal concentrations Sample ID b C a2 + mM + ® 3 . L i . Mn 2 + pM S r2 + pM Ba 2 + pM Depth cm NH15_0 0.0 NH15_1 9.70 20.60 0.41 98.7 1.12 4.5 NH15_2 9.83 14.00 0.42 97.4 1.10 10.0 NH15_3 9.47 12.00 0.34 96.8 1.43 15.5 NH15_4 9.14 7.88 0.31 96.5 1.20 21.0 NH15_5 8.80 0.00 0.25 95.8 1.14 26.5 NH15_6 nd nd nd nd nd 32.0 NH15_G1 8.26 nd 0.07 nd nd 33.0 NH15_7 8.12 0.00 0.11 93.4 1.20 37.5 NH15_8 7.78 3.17 0.15 92.3 1.11 43.0 NH15_9 7.62 10.20 0.17 91.0 1.13 48.5 NH15_G2 nd 0.00 nd 83.7 1.29 70.0 NH15_G3 5.22 nd 0.05 Nd nd 113.0 NH15_G4 nd 0.42 nd 77.7 6.94 155.0 NH15_G5 nd 8.27 nd 74.3 13.20 193.0 NH15J36 nd 2.38 nd 75.0 12.20 234.0 NH15_G7 3.31 nd 0.06 nd nd 273.0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table C-4 Station 21 Carmen Basin, pore water geochemistry and 81 5 N of pore water ammonium (nd= not determined,a = data provided by A.Graham, b = data provided by J. McManus) Sample ID n h4 + mM 81 5 N % 0 t c o 2 mM b Ca2 + mM a S042 ' mM Depth cm NH21_0 nd nd 2.37 nd nd 0.0 NH21_1 0.253 17.85 4.74 12.0 26.9 4.5 NH21_2 0.388 19.04 5.60 11.5 26.7 10.0 NH213 0.444 18.53 6.17 11.5 26.2 15.5 NH21_4 0.497 nd 6.97 11.3 26.0 21.0 NH21_5 0.564 17.71 7.23 10.9 21.2 26.5 NH21_6 0.609 nd 7.93 10.7 21.0 32.0 NH21_7 0.679 16.81 8.59 10.3 25.1 37.5 NH21_8 0.731 15.66 9.20 9.8 11.7 43.0 NH21_9 0.791 15.83 10.19 9.9 23.9 49.0 NH21_10 0.837 15.33 11.15 8.9 24.4 54.0 NH21_G1 0.689 nd 9.27 10.4 23.9 37.0 NH21_G2 0.977 14.19 13.86 9.7 22.1 75.0 NH21_G3 1.231 13.58 17.51 9.5 21.0 107.0 NH21_G4 1.491 nd 21.16 9.1 18.5 147.0 NH21_G5 1.720 nd 24.39 8.6 15.6 188.0 NH21_G6 1.888 nd 27.36 8.3 15.6 228.0 NH21_G7 2.184 12.53 31.32 7.8 12.2 279.0 NH21_G8 2.389 12.36 34.00 7.2 9.7 330.0 NH21_G9 2.587 11.66 37.64 nd 7.9 377.0 NH21 G10 2.875 12.26 40.53 6.2 5.2 427.0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table C-5a Pescadero Basin, pore water geochemistry, sediment elemental composition and 81 5 N of pore water ammonium and Ns e d (nd= not determined, a = data provided by A.Graham, b = data provided by J. McManus) Pore water Sediments Sample ID + * * ■ X z mM + X z 1 z in "lo % 0 « N O O H mM C N o w < 9 mM ■ o V ) z i- wt % ■ a a > v > z in T * ( O % 0 o 3 Depth NH26_0 nd nd nd nd nd nd 0.0 NH26_0-02 nd nd nd nd 0.52 10.49 0.2 NH26_0-1 nd nd nd nd 0.40 8.5 1.0 NH26_1 0.190 13.78 4.48 27.1 0.40 8.68 4.5 NH26_2 0.379 14.96 6.16 25.7 nd nd 10.0 NH263 0.520 14.45 7.82 25.5 nd nd 15.5 NH26_4 0.639 13.40 9.29 23.9 0.36 8.32 21.0 NH265 0.757 12.94 10.61 21.9 nd nd 27.5 NH26_6 0.880 12.33 11.94 21.7 0.45 8.53 32.0 NH267 0.970 12.98 12.95 21.2 nd nd 37.5 NH268 1.037 12.70 13.91 19.4 nd nd 43.0 NH26_9 1.153 11.87 15.26 19.7 0.40 8.37 48.5 NH26_10 1.177 12.25 16.44 18.3 nd nd 54.0 NH26_11 1.314 11.68 17.64 17.6 0.42 8.61 59.5 NH26_G1 1.255 12.18 16.04 19.0 0.41 7.93 51.0 NH26_G2 2.028 11.66 24.59 7.9 nd nd 91.0 NH26_G3 2.875 11.54 nd 5.4 0.40 8.43 131.0 NH26_G4 3.297 10.59 37.68 1.8 nd nd 171.0 NH26_G5 3.638 10.98 40.31 2.5 0.40 8.68 211.0 NH26_G6 3.957 11.27 41.98 2.0 nd nd 251.0 NH26 G7 3.986 11.03 43.12 nd 0.35 8.73 305.0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table C-5 b Station 26 Pescadero Basin, pore water dissolved metal concentrations Sample ID b C a2 + mM Fe 2 + pM Mn 2 + pM S r2 + pM Ba 2 + pM Depth cm NH26_0 nd nd nd nd nd 0.0 NH26_1 9.29 309.3 2.45 99.8 1.03 4.5 NH26_2 11.25 222.4 3.02 97.4 1.04 10.0 NH26_3 11.18 160.2 3.75 97.3 1.05 15.5 NH26_4 10.45 103.3 3.7 99.2 1.08 21.0 NH26_5 10.51 57.7 3.77 96.1 1.09 27.5 NH266 7.13 4.74 3.40 94.4 1.07 32.0 NH26_7 10.27 2.58 3.27 94.5 1.12 37.5 NH268 9.85 3.17 3.27 93.6 1.14 43.0 NH269 9.17 0.617 3.62 92.7 1.14 48.5 NH26_10 9.48 1.79 3.85 91.6 1.15 54.0 NH2611 9.18 0 3.12 92.2 1.18 59.5 NH26_G1 9.34 nd nd nd nd 51.0 NH26_G2 8.00 nd nd nd nd 91.0 NH26_G3 5.63 nd nd nd nd 131.0 NH26_G4 4.90 nd nd nd nd 171.0 NH26_G5 4.78 nd nd nd nd 211.0 NH26_G6 4.43 nd nd nd nd 251.0 NH26 G7 nd nd nd nd nd 305.0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table C-6a Station 29 Mazatlan margin, pore water geochemistry, sediment elemental composition and S1 5 N of pore water ammonium and Nsed (nd= not determined,a = data provided by A.Graham,b = data provided by J. McManus) Pore water Sediments Sample ID X z mM X z 1 z IO 't o % o C M O o H mM C M O V) R S mM ■ o a > (ft Z 1 - wt % T J a> (A z ■A r~ to % 0 ■ c 0 ) ( 0 O O H wt % g - TOC/TN 3 sed . c • *- < a a> a cm NH29_0 nd nd nd nd 0.77 7.91 6.32 9.6 0 NH29_1 0.050 19.96 2.50 29.1 0.96 8.17 8.15 9.9 4.5 NH29_2 0.130 20.18 3.07 28.0 0.96 7.80 8.40 10.3 10.0 NH29_3 0.193 20.08 3.60 28.0 0.92 8.25 8.09 10.3 15.5 NH29_4 0.278 19.84 4.14 25.7 0.94 8.24 8.24 10.2 21.0 NH29_5 0.328 19.77 4.57 26.6 0.87 7.75 7.86 10.5 26.5 NH29_6 0.384 19.88 5.04 26.9 0.90 7.84 7.69 10.0 32.0 NH29 7 0.437 19.25 5.63 27.5 0.94 8.32 8.54 10.6 37.5 NH29_8 0.495 18.43 6.01 23.7 0.87 7.96 8.17 11.0 43.0 NH29_9 0.519 nd 6.53 25.5 0.86 7.73 7.72 10.5 48.5 NH29_10 0.558 17.66 6.96 26.2 0.88 7.81 7.84 10.4 54.0 NH29_11 0.604 17.21 7.66 21.9 0.88 7.92 8.39 11.1 59.5 NH29G1 0.581 16.99 7.14 24.8 0.71 7.79 6.45 10.6 58.0 NH29G2 0.772 14.51 10.17 24.2 0.94 7.68 8.67 10.8 99.0 NH29G3 0.918 14.61 12.94 21.2 0.87 7.43 8.70 11.7 134.0 NH29_G4 1.031 14.15 14.51 nd 0.69 7.99 7.13 12.1 177.0 NH29G5 1.207 13.77 16.57 19.2 0.69 7.90 7.00 11.9 218.0 NH29_G6 1.243 13.36 18.14 16.7 0.24 8.91 2.93 14.3 257.0 NH29_G7 1.252 13.88 19.41 16.0 0.24 9.16 2.80 13.6 291.0 NH29_G8 1.386 13.80 21.33 12.9 0.1 7.37 1.23 14.3 332.0 NH29_G9 1.566 12.53 23.55 12.2 0.25 8.21 3.21 15.0 384.0 NH29 G10 1.585 12.84 24.95 12.9 0.25 8.34 3.14 14.6 431.0 249 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table C-6 b Station 29 Mazatlan margin, pore water dissolved metal concentrations Sample ID b C a2 + mM bFe 2 + pM b Mn 2 + pM Depth cm NH29_0 nd nd nd 0.0 NH29_1 9.67 2.52 0.290 4.5 NH29_2 9.68 8.98 0.271 10.0 NH29_3 10.05 2.87 0.307 15.5 NH29_4 9.50 5.43 0.274 21.0 NH29_5 9.39 2.52 0.234 26.5 NH29_6 9.34 1.47 0.262 32.0 NH29 7 9.32 0.34 0.212 37.5 NH29 8 9.16 1.25 0.251 43.0 NH29_9 9.40 0.94 0.291 48.5 NH29_10 9.62 1.78 0.274 54.0 NH29_11 9.62 1.54 0.241 59.5 NH29_G1 9.14 1.84 0.210 58.0 NH29_G2 9.04 2.08 0.117 99.0 NH29G3 8.86 0.57 0.161 134.0 NH29_G4 8.41 1.47 0.177 177.0 NH29_G5 8.19 2.74 0.134 218.0 NH29_G6 8.05 1.53 0.180 257.0 NH29_G7 7.82 3.18 0.084 291.0 NH29_G8 7.28 0.86 nd 332.0 NH29_G9 7.05 0.76 nd 384.0 NH29 G10 6.91 1.46 nd 431.0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX D HYDROLYSIS AND HPLC ANALYSIS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. All glassware used for sample processing and standards preparation was soaked for 24 hours in detergent, rinsed 3 times in DDIW and combusted for at least 1 hour at 450 °C. Sample processing 1) Thaw the samples. 2) Centrifuge, separate water from the sediment fraction, filter water immediately with GF/C using GF/C filter. 3) Freeze water till further analysis. 4) Mix the sediments (which are left after centrifugation) very well with a spatula. 5) For hydrolysis/HPLC analysis, place 0.5 to 1 g of wet sediments in pre-weighted glass test tubes, record the weights, dry at 65°C, and record the weights again. 6) Place the rest of the sediments (for isotopic/elemental analysis) in the plastic trays and dry for 48 hours at 65-70°C. Hydrolysis-Sediments 1) Add 0.1 ml of 2 mM hydroxylysine to each test tube containing dried weighed-out sediments. 2) Dry the sediments in the evaporator 3) Add 2 ml of 6 N HC1, flush with Ar for 1 min with Pasteur pipette, place Teflon tape over the top of the vials and close the screw-on caps. 4) Place in the oven at 110°C for 24 hours 5) Transfer the hydrolysate into glass centrifuge tubes 6) Centrifuge, collect supernatant using pipette, put it into 10 ml syringe and filter through the 0.4 um (Nucleopore or comparable) into a new glass test tube, then using the same syringe wash the filter with DDIW (pre-filtered as well), 5 ml volume. 7) Evaporate to dryness in a rotary evaporator 8) Re-dissolved dried hydrolysate and the in 1 ml of DDIW, vortex for 5 min and combine two parts into 1 sample (2 ml final volume) (sample and wash), split the sample into two 0.5 ml parts and one 1 ml part, and freeze at -20 °C. Hydrolysis - Pore water 1) Combine 0.9 ml of water sample, add 0.1 ml of 2 mM hydroxylysine, and 1 ml of 6NHC1. 2) Degas the mixture with Ar for 2 min using Pasteur pipette. Hydrolyse the samples for 2 hours at 140°C. Then proceed as described above for sediments. Reagent preparation OPA reagent for bulk fluoremetric analysis Combine 20 ml of OPA solution (see below) with 1 ml of mercaptoethanol and 400 ml of borate buffer (see below). The reagent is ready after 12 hours, and is stable FOR 252 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NO MORE than 2 weeks. Keep < 4°C, wrapped in aluminum foil to prevent photodegradation and autofluorescence. OPA solution: 0.2 g of dry OPA reagent (Sigma-Aldrich) in 20 ml of 95 % ethanol (HPLC grade, Sigma-Aldrich) Borate buffer: 24.7 g of ortho-boric acid ini L DDIW water, final concentration 0.4M, adjust pH to 9.5 with 5M NaOH OPA reagent for HPLC analysis In 5 ml volumetric flask, combine 0.5 ml of OPA solution (see below), 50 pi of Brij 35 solution (Sigma-Aldrich), 25 pi of mercaptoethanol and bring the volume to 5 ml with 0.8 M borate buffer OPA solution: 25 mg of OPA dry reagent in 0.5 ml of methanol Borate buffer: 49.7 g in 1 L of DDIW, adjust pH to 10.5 with 5M of NaOH Acetate buffer for HPLC analysis 5.44 g of Sodium Acetate 3-hydrate in 1 L of DDIW, final concentration is 40 mM, adjust pH to 5.8 with glacial acetic acid, filter and add 50 ml of tetrahydrofuran 253 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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Prokopenko, Maria
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Core Title
Fractionation of nitrogen isotopes during early diagenesis
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Doctor of Philosophy
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Geological Sciences
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Biogeochemistry,geochemistry,OAI-PMH Harvest
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Hammond, Douglas (
committee chair
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