Investigations of animal-sediment-microbe interactions at two different environments: coastal lagoons and methane seeps

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INVESTIGATIONS OF ANIMAL-SEDIMENT-MICROBE INTERACTIONS AT
TWO DIFFERENT ENVIRONMENTS – COASTAL LAGOONS AND
METHANE SEEPS

by

Victoria Jean Bertics

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOLOGY)

December 2009

Copyright 2009 Victoria Jean Bertics
ii
DEDICATION

To my loving parents for introducing me to the world of marine biology and for
always believing I could do this, even when I thought I could not, to Wiebke for
being not only my mentor and my advisor, but also for being a genuine friend and
continuing to have faith in me over the past 5 years, to all of my MEB friends, I
wouldn’t have made it this far if it wasn’t for you and I sincerely feel blessed to have
such a wonderful support network, and to all those in the world dealing with
adversity but still trying to make it, you can truly do it if you just work hard, believe
in yourself, and continue to have fun along the way.

iii
ACKNOWLEDGEMENTS

I would like to thank the members of Team Z: Wiebke Ziebis, Tina Treude, Randa
Abboud, and Lauren Lewis, for being the best group of people to work with both on
land and at sea. I would also like to thank my other fabulous collaborators Jill Sohm,
Cheryl Chow, Douglas Capone, and Jed Fuhrman for all of their time and energy in
regards to Chapter III. I am extremely thankful to the Fuhrman Lab at USC,
especially Ian Hewson, Josh Steele and Rohan Sachdeva for teaching and assisting
me with ARISA. I am eternally indebted to Ivona Cetinic for her help with statistical
analyses and MATLAB programing. Additional thanks goes to the captains and
crew of the Research Vessels Atlantis and Western Flyer, the pilots and crew of the
HOV Alvin and ROV Jason, Lisa Levin, Raymond Lee, Kenneth Halanych, Victoria
Orphan, Anthony Rathburn, and Joan Bernhard who graciously allowed me on their
research cruises and made sure I got the samples I needed, and Anthony Michaels,
Lauren Czarnecki, and the rest of the staff at the USC Wrigley Institute for
Environmental Sciecnes for making all of my work on Catalina Island possible. Last
but not least, I would like to thank my committee members for all of their help over
the past five years. This work was supported by the NSF grant OCE-042561 to
Wiebke Ziebis, start up funds provided to Wiebke Ziebis, and the Rose Hills
Summer Internship provided to Victoria Bertics.
iv
TABLE OF CONTENTS

DEDICATION ii

ACKNOWLEDGEMENTS iii

LIST OF TABLES vi

LIST OF FIGURES vii

ABSTRACT xi

CHAPTER I: The importance of animal-sediment-microbe interactions
throughout the world ecosystem, with an emphasis on marine sediments 1
INTRODUCTION 1
DISSERTATION AIMS 9
CHAPTER I REFERENCES 11

CHAPTER II: Biodiversity of benthic microbial communities in bioturbated
coastal sediments is controlled by geochemical microniches 15
CHAPTER II ABSTRACT 15
INTRODUCTION 16
MATERIALS AND METHODS 19
RESULTS 28
DISCUSSION 41
CHAPTER II REFERENCES 49

CHAPTER III: Burrowing deeper into benthic nitrogen fixation 57
CHAPTER III ABSTRACT 57
INTRODUCTION 58
MATERIALS AND METHODS 61
RESULTS 64
DISCUSSION 69
CHAPTER III REFERENCES 75

CHAPTER IV: Bioturbation and the role of microniches for sulfate-reduction in
coastal marine sediment 78
CHAPTER IV ABSTRACT 78
INTRODUCTION 79
MATERIALS AND METHODS 83
RESULTS 86
v
DISCUSSION 95
CHAPTER IV REFERENCES 103

CHAPTER V: When clams put their foot down: Animal-sediment-microbe
interactions at Pacific methane seeps 110
CHAPTER IV ABSTRACT 110
INTRODUCTION 111
MATERIALS AND METHODS 118
RESULTS 130
DISCUSSION 146
CHAPTER IV REFERENCES 150

CHAPTER VI: Synthesis and conclusions: The impact of bioturbation
on benthic marine ecosystem 155
INTRODUCTION 155
SUMMARY OF RESULTS 157
SYNTHESIS 161
CHAPTER V REFERENCES 166

BIBLIOGRAPHY 169

vi
LIST OF TABLES

Table 3-1: N
2
-fixation rates and NH
+
4
concentrations in a narrow aquarium 69
inhabited by a single N. californiensis.

Table 3-2: Integrated N
2
-fixation rates in Catalina Harbor sediments and a 71
comparison with other studies.

Table 4-1: Table of integrated sulfate reduction rates (SRR) from each 94
location studied.

vii
LIST OF FIGURES

Figure 1-1: Darwin’s depiction of the depth of burial of the Drudical stones 2
at Stonehedge, a process thought to be influenced by the burrowing activity
of earthworms.

Figure 1-2: A representation of the stratified structure of electron acceptors 5
found in sediment that is uninhabited and inhabited.

Figure 1-3: An example of an animal-sediment-microbe interaction at a 8
methane seep. This sketch of a clam bed and microbial mat habitat is based
on observations in the Eel River Basin.

Figure 2-1: Sampling site in Catalina Harbor, located on Santa Catalina 20
Island, which is located 35 miles off the southern coast of Los Angeles,
California, USA.

Figure 2-2: Bioturbation intensity, total organic content by the loss on 29
ignition (LOI), and porosity were determined along a 10-m transect
perpendicular to the coastline in Catalina Harbor.

Figure 2-3: Oxygen microprofiles at 3 locations in the field during low tide 32
and high tide. Additionally, contour plots of oxygen concentration are
illustrated for microprofiles measured in a narrow aquarium with and
without a burrowing shrimp.

Figure 2-4: Oxygen measured directly in a burrow opening, in ambient 33
sediment 5 cm to the left of the burrow, and oxygen concentration over time
in a shaft of a shrimp burrow at 3 cm depth, along with a 2D contour plot of
redox potential around a shrimp burrow.

Figure 2-5: Ferric iron, ferrous iron, nitrate, and ammonium concentrations 35
from sediment and pore-water samples collected at each site along the
transect down to a depth of 10 cm displayed as 2D contour plots.

Figure 2-6: Cell counts from each site down to a depth of 10 cm in 1-cm 36
vertical resolution.

viii
Figure 2-7: Microbial diversity was determined for sediment depths 0-1 cm, 38
1-2 cm, and 7-8 cm from each bioturbated site along the transect, as well as
for samples directly from the wall lining of U. crenulata and N.
californiensis burrows at these same three depths.

Figure 2-8: Bray-Curtis dendrogram combining the similarity data shown 39
in Figure 2-7 for both sediment and burrow wall samples.

Figure 2-9: An ordination diagram displaying the first and third axis of a 41
canonical correspondence analysis was created using those locations with
both species (ARISA) and environmental (NO
3
2-
, NH
4
+
, Fe (II), and Fe (III))
data.

Figure 3-1: Image of a N. californiensis burrow system. 60

Figure 3-2: Biogeochemical rate measurements, pore-water NH
+
4
66
concentrations and redox-potential of Catalina Harbor sediments.

Figure 3-3: Neighbor-joining phylogenetic tree of nifH sequences (this 68
study) and closest protein matches by tblastx.

Figure 3-4: Locations where N
2
-fixation and NH
+
4
concentration 69
measurements were taken within the narrow aquarium.

Figure 3-5: Revised view of nitrogen cycling and fluxes in bioturbated 72
coastal sediments.

Figure 4-1: Photographs of the narrow aquarium used during the laboratory 85
studies with sampling ports 1 cm apart.

Figure 4-2: Comparison of sulfate reduction rates (SRR) from a low 88
bioturbation area and a high bioturbation area.

Figure 4-3: Sulfate reduction rates measured in different burrow 89
compartments and in different types of sediment surrounding burrows.

Figure 4-4: A contour plot of sulfate reduction rates measured within an 91
aquarium inhabited with a single ghost shrimp.

Figure 4-5: The middle portion of contour plot shown in Figure 4-4. 93

ix
Figure 5-1: Simple sketch of clam bed and microbial mat habitats at methane 117
seeps.

Figure 5-2: Map of the investigated Pacific methane seep areas. 119

Figure 5-3: Typical sulfide profiles at the two microhabitats at the Eel 120
River seeps.

Figure 5-4: Methane concentrations and isotopic compositions in a 121
comparison of the two habitats.

Figure 5-5: Rates of anaerobic oxidation of methane in a comparison of the 122
two habitats determined by radiotracer incubation experiments of whole
cores from Eel River Basin.

Figure 5-6: Photograph of narrow aquaria inhabited with clams. 129

Figure 5-7: Sulfide profiles in cores taken from clam beds in the Eel 131
River Basin.

Figure 5-8: Sulfide profiles from cores taken from clam beds (top) and 133
microbial mats (bottom) found at Hydrate Ridge.

Figure 5-9: Sulfide profiles in cores collected at the ‘Clam Flats’ in the 134
Monterey Bay Canyon.

Figure 5-10: Detailed geochemical profiles and microbial abundances from 135
cores collected in clam beds found in the Eel River Basin.

Figure 5-11: Detailed geochemical profiles and microbial abundances from 137
cores collected in clam beds found at Hydrate Ridge.

Figure 5-12: Detailed geochemical profiles and microbial abundances from 139
cores collected in microbial mats found at Hydrate Ridge.

Figure 5-13: Redox potential measured in aquaria with sediment from Eel 140
River.

Figure 5-14: Redox potential measured in aquaria with sediment from 142
Hydrate Ridge.

x
Figure 5-15: Redox potential measured in aquaria with sediment from 143
Monterey Bay Canyon.

Figure 5-16: Sulfate reduction rates measured in aquaria experiments from 144
each of the three methane seeps locations.

Figure 5-17: Sulfide profiles measured in aquaria experiments from Eel 145
River (A) and Hydrate Ridge (B).

Figure 5-18: Sulfide contour plots for the aquaria experiments from Monterey 146
Bay Canyon.

xi
ABSTRACT

Understanding the interactions between macrofauna organisms and sediment
biogeochemistry and microbioloy is crucial in evaluating marine ecosystem
functioning. Most of the seafloor is influenced by bioturbation, yet macrofaunal
activity and its influences on biogeochemical processes are not well studied. The
goal of the presented research was to combine innovative approaches and modern
techniques to investigate the interactions of geochemical processes, microbial
activities, and macrofauna assemblages in two marine habitats: coastal sediments and
deep-sea methane seeps.

A coastal lagoon in Catalina Harbor, CA, was characterized by dense populations of
the ghost shrimp Neotrypaea californiensis and the fiddler crab Uca crenulata. The
shrimp lived permanently subsurface and maintained complex deep-reaching
burrows that it ventilated constantly. The crab built shallow simple burrows, mainly
for protection, and often left the burrow to forage. Differences in burrowing behavior
were reflected in contrasting microbial diversities. Shrimp burrow microbial
communities were similar to those found in surface sediments while crab burrow
communities were not significantly different from subsurface sediment communities.
Next, specific microbial activities were examined surrounding N. californiensis
burrows. High levels of sulfate reduction (SR), along with nitrogen fixation, were
xii
found in and around the burrows, supporting the idea that bioturbation can lead to the
formation of reduced microniches characterized by elevated microbial activities. 2-
dimensional mapping of these microniches showed that sulfate reduction rates (SRR)
in reduced microniches associated with burrows were 3 orders of magnitude higher
than the surrounding sediment.

At western North American methane seeps, the seepage of methane-laden fluids
supports sulfide-oxidizing microbial mats and rich communities of vesicomyid clams
(Calyptogena) harboring sulfide-oxidizing symbionts. It is assumed that the sulfide
supporting these communities is produced by the coupled reaction of SR and
anaerobic oxidation of methane. Yet, it is relatively unknown what influence the
clams have on benthic geochemical gradients and microbial processes. Three seep
locations (Hydrate Ridge, Eel River, and Monterey Bay) were examined for the
influence of clams on local biogeochemical processes. In all cases, the clams
significantly increased SRR. Microsensor measurements indicated that the clams
transported sulfate-rich overlying water deeper into the sediment where it could be
used for microbial SR.

1
CHAPTER I: The importance of animal-sediment-microbe interactions throughout
the world ecosystem, with an emphasis on marine sediments

“The plough is one of the most ancient and most valuable of man’s inventions; but
long before he existed the land was in fact regularly ploughed, and still continues to
be thus ploughed by earthworms,” (Charles Darwin, 1882).

INTRODUCTION

Macro- and microorganism interactions influence the geochemistry, microbiology,
and whole-system population ecology of most known ecosystems on Earth. These
interactions can range from commensalism and mutualism, to competition and
predation, to exo- and endosymbiosis (Kristensen et al. 2005). The impact of
animals on soil and sediment processes is traditionally believed to have been
proposed by Charles Darwin, which he later summarized in his final book entitled
The Formation of Vegetable Mould, Through the Action of Worms, With
Observations on Their Habits (1882). In this publication, Darwin described the slow
burial of stones into soil through the undermining actions of earthworms, which he
claimed were partially responsible for the sinking of such great objects as the
Druidical stones at Stonehenge (Figure 1-1) and the burial of ancient Roman villas.
Because of such burial, Darwin stated, “Archaeologist ought to be grateful to worms,
2
as they protect and preserve for an indefinitely long period every object, not liable to
decay, which is dropped on the surface of the land, by burying it beneath their
castings.” However, his analysis of worms did not stop at mere burial of objects, but
also examined the role that their burrows played on soil dynamics. He stated that
earthworm burrows allow air and roots to penetrate more easily and deeper into the
ground while also supplying the roots with nourishment via the humus that lines the
burrows. These ideas in total brought much criticism from his peers, with one critic
saying, “considering their [earthworms’] weakness and their size, the work they are
represented to have accomplished is stupendous,” (Fish, 1869). Nonetheless, some
consider Darwin’s work to be an “historical turning point” for how people, especially
farmers, gardeners, and scientists, view the importance earthworms (Brown et al.,
2003) and possibly other such burrowing animals.
Figure 1-1 Darwin’s depiction of the depth of burial of the Drudical stones at Stonehedge, a process
thought to be influenced by the burrowing activity of earthworms. Scale is ½ inch to 1 foot.

In more recent times, the study of bioturbation (the biological reworking of soils and
sediments) has gained momentum in many areas of science because burrowing
3
organisms potentially affect the majority of all surfaces on Earth (Meysman et al.,
2006). These areas are not strictly limited to studying current ecosystems but also
involve the examination of paleo-bioturbation and its impact on evolution and the
rock record. Some studies have suggested that bioturbation, along with predation,
was one of the main driving forces behind the ‘Cambrian explosion,’ during which
there was a transition from predominately worldwide microbial mats to the evolution
of complex benthic communities that lived above and below the seafloor (Thayer,
1979; Seilacher and Pflüger, 1994; Bottjer et al., 2000). However, in order to
understand these ancient marine bioturbation systems, a deeper understanding of the
way bioturbation affects today’s ecosystems is needed. Many different types of
interactions between macro- and microorganisms in marine sediments have been
examined (e.g. Aller and Aller 1986, Alongi 1985, Kristensen et al. 1991), but
studies are often limited by current sampling technology, lack of rapid sample
processing, and/or current macrobiological and microbiological knowledge gaps.
Marine microbial community structure in relation to environmental parameters alone
is still poorly understood (Suzuki and DeLong 2002) and even less so is the
microbial and environmental influences on and by macrofauna. Recent articles have
been essential for revitalizing the study of bioturbation and have stressed the
importance of revisiting these organism-sediment interactions with an emphasis on
more interdisciplinary approaches and fine scale measurements (see Interactions
Between Macro- and Microorganisms in Marine Sediments - Kristensen et al. 2005).
4
Now that better instrumentation and approaches are available, these tools can be
combined to give us a far better insight into the complexity of the burrows and their
effects on biogeochemical processes. Only upon the completion of these studies can
we truly hope to understand the complex interactions between geological processes
and macrobiological and microbiological activities.

Macrofaunal Influences on Marine Sediments: Uninhabited marine sediments are
characterized as having a stratified nature in which biogeochemical processes are
regulated by the depth sequence of electron acceptors used to mineralize organic
material (Berner 1980, Jørgensen 2000). The sequence of these electron acceptors
correlates to a decrease in redox potential and in free energy available by respiration
with each oxidant (Jørgensen 2000). However, the activity of benthic organisms can
greatly affect these biogeochemical processes (Figure 1-2). Through their activities,
these organisms increase the oxic/anoxic interface and enhance the oxidized zones
per given area of the sea floor, thus enlarging the availability of electron acceptors
for organic matter degradation and boosting microbial activity (Aller 1982,
Kristensen et al. 2005). These factors combine to make macrofauna activity an
important factor for element cycling (C, N, Fe, and S) and remineralization of
nutrients back into the water column. (Kristensen and Kostka 2005), making
burrowing macrofauna organisms important elements in benthic-pelagic coupling.
5

Figure 1-2 A representation of the stratified structure of electron acceptors found in sediment that is
uninhabited (left) and inhabited (right).

Aside from the impact that bioturbation has on the geochemical parameters of
marine sediment, there are also many questions regarding burrow-microorganism
interactions. Burrows built by different organisms often vary in size, length,
microbial composition, complexity, orientation, appearance, and/or permanence
(Griffis and Suchanek 1991, Ziebis et al. 1996a, Dworschak 2001, Papaspyrou et al.
2005). This variation makes it difficult to extrapolate from one organism to another
and from one location to another, the impact that burrowing activity has on the local
microbial communities. Furthermore, there have been relatively few studies that
have directly examined the microbial communities that form at the burrow-sediment
interface and how these communities are related to the unique biogeochemistry of
the burrows (Kristensen and Kostka 2005). This lack of knowledge arises from the
prior inability to sample burrows at the sub-millimeter scale, the scale that is relevant
6
to microbial communities. Now that current technology allows us to sample at this
sub-millimeter scale, it is important that we combine these tools with modern
approaches to examine these more complex aspects of bioturbation and its impact on
sediment biogeochemistry.

Interactions in Coastal Lagoons: In terms of the global carbon cycle, shelf sediments
are often viewed as a major sink for organic carbon (Wollast 1991). Oxygen
penetration by diffusion into these sediments has been shown to reach only a few
millimeters (Revsbech et al. 1980, Gundersen et al. 1995) yet this oxygen can
account for up to 50% of the carbon oxidation occurring in these sediments
(Thamdrup and Canfield 2000). Because oxygen is used up rather rapidly, 25-50%
of the organic carbon in these coastal sediments can be mineralized by sulfate-
reducing bacteria (Jørgensen 1982). The remaining percent of carbon is oxidized
through nitrate, Mn (IV), and/or Fe (III) reduction (Jørgensen 1996, 2000).
However, bioirrigation (the fluid transport from overlying waters deeper into
sediments via burrow-like structures, as well as the transport of fluids from the
burrow into the overlying water) can enhance the transport of dissolved substances
between the water column and the sediment (Aller 1982, Ziebis et al. 1996), causing
an increase in electron acceptors available for organic matter degradation. Benthic
macrofauna have been shown to increase the redox potential of coastal sediment by
280-614 mV to a distance of 3 mm away from the burrow-sediment interface by their
7
irrigation activities alone (Forster and Graf 1992). Redox oscillations produced by
bioturbating organisms are believed to result in faster and more complete
decomposition of organic matter (Sun et al. 1999), as well as producing a unique
animal and microbial community surrounding the bioturbated area (Meyers et al.
1988, Reichardt 1989).

Interactions at Methane Seeps: Slightly over 20 years ago, a group of researchers
found a community of organisms, reminiscent of those found at hydrothermal vents
along the East Pacific Rise, living around sulfide rich hypersaline waters seeping out
of the Florida Escarpment at near ambient temperatures (Paull et al. 1984). This type
of environment has since been termed a “cold seep,” which is defined as the release
of "cold" fluids enriched in hydrocarbons, such as methane, from the seafloor (Kulm
et al. 1986, Whiticar et al. 1995, Boetius and Suess 2004). These fluids can lead to
enhanced microbial activities, especially in regards to the coupled reactions of
sulfate reduction and anaerobic oxidation of methane (AOM) (Boetius et al. 2000,
Treude et al. 2003, Joye et al. 2004, Ziebis & Haese 2005). Microbial anaerobic
oxidation of methane (AOM) results in the formation of the hydrogen sulfide
commonly found at these seep locations. Currently it is believed that the overall
microbial reaction: CH
4
+ SO
4
2-
> HCO
3
-
+ HS
-
+ H
2
O is carried out by a syntrophic
consortium of methanotrophic archaea and sulfate-reducing bacteria (Boetius et al.
2000, Valentine & Reeburgh 2000, Orphan et al. 2001a,b, Hinrichs & Boetius 2002).
8
Figure 1-3 An example of an animal-sediment-microbe interaction at a methane seep. This sketch of
a clam bed and microbial mat habitat is based on observations in the Eel River Basin. Insert: Sulfide
concentration profiles measured in a comparison of non-seep, clam bed, and microbial mat sediment
(Levin et al. 2003, Ziebis et al. 2002, Ziebis and Hasse 2005).

The hydrogen sulfide produced by the above reaction can support large
chemosynthetic communities at these seeps, including sulfur-oxidizing bacteria and
macrofaunal species harboring thiotrophic symbionts that differ from those
communities found at hydrothermal vents (Swinbanks 1985, Suess et al. 1998).
There are indications that the vertical movement of filamentous sulfur-oxidizing
bacteria or the bioturbating activity of invertebrates harboring symbionts, such as
clams (Wallmann et al. 1997, Julian et al. 1999, Sahling et al. 2002, Treude et al.
2003), may transport sulfate rich waters deeper into the sediment where AOM is
9
occurring (Figure 1-3). However, this idea has remained relatively untested in a
laboratory setting and so it is still unknown what the exact influence of these
organisms is on the establishment of sediment microgradients. A question that
remains is do these organisms in fact transport sulfate down to areas of AOM and
actively enhance sulfide production or do they simply tap existing sulfide pools
located deep in the sediment.

Summary: While the basic effects of bioturbation are well studied, there are very few
geochemical measurements that have been done at a scale relevant to the bacterial
communities being affected. There have also been relatively few studies that have
combined small-scale measurements of chemical parameters with measurements of
microbial rates, diversity, and abundance. Overall, these knowledge gaps illustrate
the importance of performing detailed investigations using interdisciplinary
approaches to re-examine the relationship of macro- and microorganisms in sediment
ecosystems.

DISSERTATION AIMS

Two environments that are often greatly influenced by macrofauna activity are
coastal sediments and areas of methane seepage. Both environments are governed
by an interaction of fluid flow dynamics, geochemical gradients, and macrofaunal
10
and microbial activity. These processes are important factors controlling nutrient
cycling within the sediment and remineralization of these substances back into the
water column, with the potential to influence nutrient cycling throughout the world
ocean. With these ideas in mind, the overall objective of my dissertation is to
determine the influence of bioturbation activity on the biogeochemistry and
microbiology of these two differing benthic environments. This research aims to
determine 1) in what ways are biogeochemical processes and microbial activities
and community structure influenced by bioturbating activities in coastal
lagoons, 2) do bioturbation and bioirrigation form a link between the cycling of
key nutrients in coastal sediments, 3) does bioturbation lead to the formation of
microniches within the sediment and if so, how does this impact the
biogeochemical conditions of the sediment, and 4) do similar animal-sediment-
microbe interactions, as seen in coastal sediments, occur around deep-sea
methane seeps.
11
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15
CHAPTER II: Biodiversity of benthic microbial communities in bioturbated coastal
sediments is controlled by geochemical microniches

CHAPTER II ABSTRACT

We used a combination of field and laboratory approaches to address how the
bioturbation activity of two crustaceans, the ghost shrimp Neotrypaea californiensis
and the fiddler crab Uca crenulata, affects the microbial diversity of intertidal
sediments in a coastal lagoon (Catalina Harbor, Santa Catalina Island, California).
Detailed geochemical analyses, including oxygen microsensor measurements, were
performed to characterize environmental parameters. We used a whole-assemblage
fingerprinting approach (ARISA: amplified ribosomal intergenic spacer analysis) to
compare bacterial diversity along geochemical gradients and in relation to subsurface
microniches. The two crustaceans have different burrowing behaviors. The ghost
shrimp maintains complex, deep-reaching burrows and permanently lives
subterranean, supplying its burrow with oxygen-rich water. In contrast the fiddler
crab constructs simpler, J-shaped burrows, which it does not inhabit permanently and
does not actively ventilate. Our goal was to address how varying environmental
parameters affect benthic microbial communities. An important question in benthic
microbial ecology has been whether burrows support similar or unique communities
compared to the sediment surface. Our results showed that sediment surface
16
microbial communities are distinct from subsurface assemblages and that different
burrow types support diverse bacterial taxa. Statistical comparisons by canonical
correspondence analysis indicated that the availability of oxidants (oxygen, nitrate,
ferric iron) plays a key role in determining the presence and abundance of different
taxa. When geochemical parameters were alike, microbial communities associated
with burrows showed significant similarity to sediment surface communities. Our
study provides information on the community structure of microbial communities in
marine sediments and the factors controlling their distribution.

INTRODUCTION

The burrowing, ventilation and foraging activity of benthic macrofauna organisms
affects key ecosystem processes of marine sediments, including organic matter
remineralization, nutrient cycling, biogeochemical interactions and benthic-pelagic
fluxes (Rhoads, 1974; Aller, 1982; 1988; 1994; Kristensen et al., 1991, 2000; Banta,
et al., 1999; Gilbert et al., 1998, 2003). Microbial abundances and activities have
been shown to increase due to the complex biogeochemical interactions induced by
bioturbation activity (Hansen and Kristensen, 1998; Lohrer et al., 2004; Kogure and
Wada, 2005). The construction of burrows increases the sediment-water interface,
offering additional surfaces for microbial colonization and chemical reactions (Aller
and Aller, 1986; Meyers et al., 1987; Reichardt, 1989; Grossman and Reichardt,
17
1991; Marinelli et al., 2002). The transport of particles (bioturbation) and the
flushing of burrows (bioirrigation) create 3-dimensional geochemical zonation
patterns with substantial changes of redox-conditions and the formation of
temporally and spatially dynamic microenvironments. Oxidized microhabitats often
occur next to reduced sediment compartments, thus allowing a tighter coupling of
redox reactions (e.g. nitrification – denitrification) (Mayer et al., 1995; Pelegri and
Blackburn, 1996; Tuominen et al., 1999; Svensson et al., 2001). The flushing of
burrows with oxygen-rich water by a deep-burrowing thalassinidean shrimp has
been shown to transport oxygen as deep as 80 cm into the sediment (Ziebis et al.,
1996a), whereas penetration depth of oxygen by molecular diffusion is typically only
a few mm in coastal sediments (Revsbech et al., 1980; Glud et al., 1994). Only a thin
film of surface sediment is generally oxic, containing molecular oxygen, and
allowing aerobic respiration. Below this zone the sediment is anoxic. The oxidized
zone, characterized by a positive redox potential and the availability of other electron
acceptors (e.g. nitrate, ferrous iron), can extend deeper into the sediment.

One of the key questions in benthic microbial ecology is whether similar
environmental conditions support the same microbial communities. One hypothesis
is that the microbial assemblages in the oxic and oxidized zones surrounding the
burrows are similar to communities in surface sediments. In contrast, it has been
suggested that burrow walls support unique microbial communities that differ
18
considerably from those found at the surface (Kristensen and Kostka, 2005;
Papaspyrou et al., 2005; 2006). The question remains how microbial communities
within burrows compare to the ambient sediment and to the sediment surface.

There are very few studies that have addressed the impact of habitat heterogeneity on
microbial diversity, mainly because it remains a challenge to quantify the large
number of abiotic and biotic factors that shape the microbial habitat on different
spatial and temporal scales. The majority of marine studies on microbial diversity
have been performed in the water column (Hewson and Fuhrman, 2004; Hannig et
al., 2006; Pommier et al., 2007; Fuhrman et al., 2008), with a focus on whether
bacteria exhibit biogeographical patterns. However, the processes that govern these
microbial distribution patterns in the field are still poorly understood (Suzuki and
DeLong, 2002; Castro-Gonzales et al., 2005; Fuhrman et al., 2008). Sediment
systems harbor even higher abundances and a greater diversity of microorganisms
(e.g. Curtis et al., 2002; Torsvik et al., 2002), yet they remain sadly understudied
(Jørgensen and Boetius, 2007). The correlation between habitat complexity and
microbial diversity remains to be determined (Fierer, 2008). Bioturbated sediments
provide ideal opportunities to compare microbial communities in relation to
environmental parameters.

19
Fiddler crabs and ghost shrimp are among the most abundant bioturbating
macrofauna in coastal areas all over the world. Their burrows have been
acknowledged as important conduits for chemical exchange between the water
column and the sediment, linking the activity of these crustaceans to nutrient
recycling, organic matter degradation and primary productivity (e.g. Kostka et al.,
2002; Papaspyrou et al., 2005). In this study, we examined the impact of the
bioturbation activity of the ghost shrimp Neotrypaea californiensis and the fiddler
crab Uca crenulata on microbial community compositions in the sediment.

MATERIALS AND METHODS

Study Site: The investigations were carried out in a shallow lagoon located in
Catalina Harbor, Catalina Island, California (33° 25.23’ N, 118° 19.42’ W) about 35
km southwest of Los Angeles (Figure 2-1). The head of Catalina Harbor is a shallow
(< 2 m), low energy area of fine-grained sand, surrounded by beach and a gentle
plain. Below the low-water mark, the sediments become more silty. The average
tidal range is 1.1 m and tides are mixed, with the higher high water preceding the
lower low water (Colbert et al., 2008a; b). Over the course of the experiment (June-
August 2005), water temperature was typically 18ºC and salinity was 34.5 ‰. The
two most abundant burrowing macrofauna were the Mexican fiddler crab Uca
crenulata, Lockington, 1877 (Crustacea: Decapoda: Ocypodoidea) and the bay ghost
20
shrimp Neotrypaea californiensis, Dana, 1854 (Crustacea: Decapoda: Thalassinidea),
previously known as Callianassa californiensis (Manning and Felder, 1991). Both
species are typical inhabitants of intertidal areas along the west coast of North
America and occur in high abundances of greater than 200 individuals m
-2
. They are
representatives of two species-rich superfamilies, Ocypodoidea (Rafinesque, 1815)
and Thalassinoidea (Latreille, 1831) (Martin and Davis, 2001), which occur
worldwide with ~100 (Bisby et al., 2007) and more than 500 known taxa
(Dworschak, 2000), respectively.

Figure 2-1 All samples were collected in Catalina Harbor (pictured right) on Santa Catalina Island
(indicated by arrow on the left), which is located 35 miles off the southern coast of Los Angeles,
California, USA.

Ghost shrimp (Thalassinidean) have increasingly attracted attention in benthic
ecology because of their significant burrow architectures and their influence on
biogeochemical processes and microbial communities (Aller and Dodge, 1974;
Waslenchuk et al., 1983; Forster and Graf, 1995; Ziebis et al., 1996a; b; Huettel et
21
al., 1998; Kinoshita et al., 2003). Their burrows usually consist of a conspicuous
upper Y or U-shape structure, which allows for easy flushing with overlying water.
Burrows continue vertically into the sediment and often exhibit several galleries of
interconnected chambers at distinct sediment depths (e.g. Ziebis et al., 1996a). The
deepest burrows reported to date reached 3 m (Pemberton et al., 1976). Most known
species remain subsurface their entire life and maintain semi-permanent burrows
which are constantly reworked. N. californiensis inhabits intertidal and sub-tidal
areas stretching from Alaska to Baja California. Its burrow architecture is complex,
extending down to ~ 76 cm with several branches with openings to the surface
(MacGinite, 1934; Swinbanks and Murray, 1981; Brenchley, 1981).

Fiddler crabs have a distinctively different burrowing behavior. They maintain
simple J-shaped burrows, which usually have a single entrance and continue into the
sediment at a 45º angle down to a depth of ~ 20 cm, ending in a terminal chamber.
They leave their burrows frequently during low tide to forage on algae, bacteria and
detritus on the sediment surface (Zeil et al., 2006). U. crenulata is found along the
west coast of North America from Santa Barbara, California to Central Mexico.
Although these two burrowing crustaceans inhabit the same area, the difference in
behavior, burrow construction and foraging has most likely a vastly different
ecological impact and effect on benthic microbial communities.

22
At our study site, shrimp burrows reached an average depth of ~20 cm, which
seemed to be limited by a cohesive sediment layer found at that depth within the
lagoon. Shrimp burrows consisted of several branches with two openings to the
surface. Pumping activity of the permanently subterranean shrimp could be observed
in the field by sediment ejections from the burrow openings. Crab burrows extended
to a maximum of 10 cm depth and consisted of a single J- shaped tube. Fiddler crabs
were often seen at the sediment surface or sitting at their burrow opening.

Sampling: A total of six sampling sites were chosen at 2-m intervals along a transect
perpendicular to the shoreline, starting at the high water's edge (0 m) and continuing
to a distance of 10 m. Sites were named according to their distance from shore (0 m,
2 m, 4 m, 6 m, 8 m, 10 m). An additional location in the same area (2 m parallel to
the transect), but with no apparent macrofauna bioturbation activity served as a
control site (non-bioturbated zone). Except for the 0 m site there was a visible
photosynthetic mat at the sediment surface. The bioturbation intensity at each site
was determined by counting the number of burrows within a 25 cm x 25 cm frame
(10 replicates per distance). Four sediment cores were collected closely together at
each of the 6 sites and at the control site for geochemical and microbiological
analyses, using acrylic core liners (ø 5.4 cm, length 30 cm).

Microsensor Profiling: Oxygen concentrations and penetration depths were
23
determined during high and low tide conditions at the control, the 4 m and 10 m
sites. In-situ profiling was performed during low tide using a small benthic lander, to
which modified micromanipulators were attached. Microsensors were connected to
battery-powered picoammeters and signals were recorded directly on a laptop
computer. During high tide sediment cores were collected and microprofiles were
measured in intact sediment cores directly after retrieval. Microprofiles of oxygen
were measured in vertical intervals of 250 µm using Clark-type amperometric
oxygen sensors (Revsbech and Jørgensen, 1986; Revsbech, 1989; Unisense©,
Aarhus, Denmark). Microprofiles of sulfide with amperometic microelectrodes
(Jeroschewsky et al., 1996) were also performed to a depth of 5 cm, yet no sulfide
was detected and the measurements are not further discussed. Profiles of redox
potential using microsensors were performed in 250-µm steps in cores containing
burrows. For all measurements sensors were attached to motorized
micromanipulators (Märzhäuser, Wetzlar, Germany), and driven vertically into the
sediment in µm to mm steps, controlled by a computer. Signals were amplified and
transformed to millivolt (mV) by a 2-channel picoammeter (PA 2000) (Unisense©),
and directly recorded on a computer using the software Profix® (Unisense©).
Redox-potential sensors were connected to portable or table-top pH/mV meter
(WTW-pH340, WTW GmbH, Weilheim, Germany; pH-m210 Meter Lab,
Radiometer Analytical, Lyon, France). Oxygen measurements were also performed
directly inside burrows, by positioning the tip of the electrode inside a burrow and
24
recording the change in oxygen concentration over time.

Aquarium Setup: Narrow aquaria were set up to observe the burrowing behavior and
to perform detailed measurements in and around burrows. Two narrow aquaria
(dimensions: 40 cm x 30 cm x 3 cm) were filled with sieved sediment (500 µm) from
the study site. The first aquarium served as a control and did not contain a ghost
shrimp, whereas to the second aquarium one adult shrimp was added. The aquaria
were kept in the laboratory under running seawater and a simulated natural light
cycle for 6 weeks. At this time the shrimp had established a burrow system and
oxygen profiles were measured in vertical profiles (250 µm depth increments) in 2
cm horizontal intervals along the width of both aquaria. Fiddler crabs were also kept
in aquaria. After they had established a simple burrow, they usually stayed at the
surface or at the entrance of their burrows. Oxygen profiles measured in and around
crab burrows revealed no oxygen transport into the burrows.

Geochemical and Microbiological Analyses: All four cores were sliced in 1-cm
intervals under nitrogen atmosphere and each section was sub-sampled for further
processing. All sub-samples for one site (pore-water and sediment) were taken from
the exact same core. A second core was taken as a back up. Small samples of
sediment (0.5 cm
3
) were frozen at -20ºC for the analyses of iron, a second sub-
sample (0.5 cm
3
) was immediately frozen at –80ºC for molecular biological studies.
25
Exactly one cm
3
of sediment was fixed in 9 ml of filter sterilized (0.2 µm)
formaldehyde/seawater solution (4 % volume/volume) for the enumeration of
microorganisms. Pore-water was collected using a pore-water press (KC Denmark,
Silkeborg, Denmark) and samples (3 ml) for ammonium and nitrate analyses were
frozen immediately. The remainder of the sediment section was used for the
determination of porosity and organic content (Loss on ignition, LOI). Two sediment
cores were taken directly targeting burrows of U. crenulata and N. californiensis for
direct sampling of burrow linings for microbiological analyses.

Porosity was determined by drying a known volume of sediment at 65°C for 24 hrs.
The loss on ignition (LOI) was determined after combusting samples at 450°C for 24
hrs. Ferric and ferrous iron concentrations in sediment samples were analyzed
following procedures described by Lovley and Philips (1987), with modifications
described by Kostka and Luther III (1994) and Thamdrup et al. (1994). This
extraction procedure allows for the quantification of microbially reducible ferric iron
in aquatic sediments (Lovley and Philips, 1987). Pore-water ammonium
concentrations were determined by flow injection analysis modified for small sample
volumes (100 µl sample) (Hall and Aller, 1992). The sum of nitrate and nitrite was
determined spectrophotometrically after reduction of samples with spongy cadmium
(Jones, 1984). The procedure was modified for the analyses of small sample volumes
26
(0.5 – 1 ml). The small sample sizes allowed relatively high spatial resolution of the
analyses.

Microbial abundances in the sediment were determined by direct counts of stained
cells (acridine orange) using epifluorescence microscopy following the enumeration
protocol by Epstein and Rossel (1995). For comparing microbial community
structures the PCR-based whole-community fingerprinting approach ARISA
(amplified ribosomal intergenic spacer analysis) (Fisher and Triplet, 1999; Hewson
and Fuhrman, 2004; Brown et al., 2005) was applied. ARISA amplifies the
intergenic spacers between the 16S and 23S rRNA genes using a fluorescent primer.
Results are displayed as the amount of different PCR products of specific fragment
length, which correspond to “operational taxonomic units” (OTUs) (Brown and
Fuhrman, 2005). DNA was extracted from 100 mg of sediment using the Qbiogene
(Bio101) Soil DNA kit (Qbiogene Inc., Carlsbad, CA, U.S.A). Extracted DNA was
quantified using PICO Green fluorescence (Molecular Probes Inc., Eugene, OR,
U.S.A.) and diluted to 10 ng cm
-3
. PCR was performed using the universal primer
16S-1392F (5’-G[C/T]ACACACCGCCCGT-3’) and the bacterial primer 23S-125R
labeled with a 5’ TET (5’-GGGTT[C/G/T]CCCCATTC[A/G]G-3’). The 50-µl PCR
combined 200 nM of each primer with 1 x PCR buffer, 2.5 mM MgCl
2
, 250 µM of
each deoxynucleotide, 2.5 units of Taq polymerase (Promega, Madison, WI, U.S.A.)
and 40 nM BSA (Sigma, St. Louis, MO, catalog no. A-7030). Thermocycling
27
consisted of a 5-min heating step at 94°C, followed by 30 cycles of denaturing at
94°C for 30 s, annealing at 56°C for 30 s, and extending at 72°C for 45 s, and
finished with a 10-min extension step at 72°C. The products were purified using a
Clean & Concentrator kit (Zymo Research Corp., Orange, CA, U.S.A.), quantified
using PICO Green fluorescence, and duplicates of 10 ng of purified products from
each sample were run on an ABI 377XL automated slab gel sequencer (Applied
Biosystems, Foster City, CA, U.S.A.). Results were analyzed using the ABI Peak
Scanner software where each peak displayed in an electropherogram represented one
OTU and the peak area represented the amount of the OTU present. Peak Scanner
outputs were transferred to Microsoft Excel (Seattle, WA, U.S.A.) spreadsheets for
subsequent analysis and binning as described by Hewson and Fuhrman (2006). The
program Primer
6
(PRIMER-E Ltd, Lutton, UK) was used to further analyze the data
sets and to perform a cluster analysis comparing the similarity (Bray-Curtis) of
microbial communities found at different locations. Prior to clustering, OTU data
was logarithmically transformed (log (x+1)) to achieve a normal distribution. PC-
ORD (MJM Software Design, Gleneden Beach, OR, U.S.A.) was used to perform a
canonical correspondence analysis (CCA) to determine the relationships between
microbial communities and environmental parameters (ter Braak, 1986; 1995;
Cetinic′ et al., 2006).

28
RESULTS

Bioturbation Activity: Based on the average number of burrow openings m
-2
,
bioturbation intensity increased with distance from the shoreline (R
2
= 0.9478, p <
0.001) to a total of 870 burrow openings m
-2
at 10 m distance (Figure 2-2 A). We
distinguished three general areas of varying bioturbation intensity: 1. Low
bioturbation, corresponding to ~100 burrow openings m
-2
at 0 m distance, 2.
Moderate bioturbation, corresponding to a number between 260 and 400 burrow
openings m
-2
at the 2 m and 4 m sites, and 3. High bioturbation, reaching 560 to 870
burrow openings m
-2
at the 6 m, 8 m and 10 m sites. U. crenulata burrows remained
constant with distance from shore (~170 burrow openings m
-2
) except for the
shoreline where only ~42 burrow openings m
-2
were counted. In contrast, the
abundance of N. californiensis burrows increased linearly with distance from 60
burrows m
-2
at the 0 m site to 640 burrows m
-2
at the distance of 10 m (Figure 2-2 A).
Using a simple calculation, based on the number of burrows for each species and the
dimensions of the burrows (crab burrow: ~10 cm deep, ø 2.5 cm) this leads to an
increase of the sediment-water interface by ~ 600%. The tidal range and dry
exposure of the near shore sites might be an explanation for the increase of
bioturbation intensity.

29

Figure 2-2 (A) Bioturbation intensity determined along a 10-m transect perpendicular to the coastline
in Catalina Harbor. Number of burrow openings was counted in 2-m intervals (0 m, 2 m, 4 m, 6 m, 8
m, 10 m) and compared to a non-bioturbated control site. 10 frames were counted per site, the bars
indicate the mean number of burrows per m
2
and error bars represent the standard error of the mean.
There was a linear increase in total number of burrows with distance. Burrows created by the ghost
shrimp N. californiensis (gray bars) were distinguished from burrows by the fiddler crab U. crenulata
(white bars). (B) As a measure of organic content the loss on ignition (LOI) was determined in 1-cm
depth intervals in sediment cores collected at the 6 sites and the control area. LOI is illustrated as the
percentage of the sediment dry weight. (C) On the same cores sediment porosity was determined as
the weight loss after drying (65ºC, 24 h) and is expressed as parts of 1.
30
Organic Content and Porosity: LOI (in % dry weight) varied little (0.84% to 2.8%)
with depth or along the transect, except for the 8 m and 10 m sites, where in the top 2
centimeters values reached 17.5% of the dry weight (Figure 2-2 B). The
photosynthetic mat at the sediment surface appeared to be thicker in this region,
possibly contributing to a higher organic content. At the other sites LOI was slightly
higher in the top 2 cm (2.8 %) than deeper in the sediment. Porosity was highest at
the sediment surface (0.3 – 0.55) at all sites except for the control site where porosity
stayed constant (0.2) throughout the sediment column (Figure 2-2 C). Porosity
decreased below the surface and stayed at values between 0.22 and 0.25.

Geochemical Zonation
Microsensensor Studies: During both high and low tide conditions, oxygen
penetration into the sediment increased from the non bioturbated zone at the 0 m site
(~ 1 mm) to deepest oxygen penetration (> 6 mm) within the area of high
bioturbation (10 m distance) (Figure 2-3 A, B). Oxygen concentrations and
penetration depths were slightly higher during low tide, decreased rapidly within the
first mm at all sites but stayed at elevated concentrations deeper into the sediment at
the bioturbated sites. During high tide the oxygen profile measured at the 10 m site
showed subsurface maxima indicating oxygen transport deeper into the sediment
probably within burrows. Detailed oxygen measurements in narrow aquaria (Figure
2-3 C, D) showed deepest oxygen penetration to a depth of 3 – 4 mm in the sediment
31
without shrimp (Figure 2-3 C). The contour lines describe the heterogeneity of the
sediment surface and show photosynthetic activity at the sediment-water interface
with highest oxygen concentrations at the interface. In the second narrow aquarium
deeper oxygen transport was documented within burrows and across burrow walls
(Figure 2-3 D, 25 cm width) creating radial gradients of oxygen concentration to a
distance of ~ 2mm. Vertical oxygen profiles measured directly within a burrow
opening of the ghost shrimp N. califoniensis compared to the sediment 5 cm away
from the burrow (Figure 2-4 A) illustrated also deep penetration (> 2 cm) of oxygen-
rich water within the burrow compared to oxygen penetration into the sediment by
molecular diffusion (3 mm). Oxygen concentrations measured at a depth of 3 cm
inside the burrow over a period of 35 minutes (Figure 2-4 B) illustrated that oxygen
concentrations were maintained at constant high levels (150 µM -250 µM). These
measurements were repeated for several burrows showing the similar pumping
activity. Vertical profiles of redox-potential (mV) measurements, depicted as a
contour plot (Figure 2-4 C), showed an oxidized zone (positive redox-potential) of ~
2 cm surrounding a burrow. Similar measurements were performed on fiddler crab
burrows, yet we were not able to document or measure an intrusion of oxygen-rich
water into the crab burrow. No dissolved sulfide was detected in any of the field or
laboratory measurements, despite the fact that sulfate reduction has been previously
measured (unpublished data) in the exact same area. One explanation might be a
precipitation of dissolved sulfide as iron sulfide.
32

Figure 2-3 Oxygen microprofiles at 3 locations in the field, in the non-bioturbated control area, at the
4 m site (moderate biotruabtion) and at the 10 m site (high bioturbation). In-situ microprofiles were
measured during low tide (A) using a battery powered, portable microsensor system. During high tide
cores were collected and profiles were performed in intact cores immediately after retrieval (B).
Contour plots of oxygen concentration are illustrated for microprofiles measured in a narrow
aquarium without a burrowing shrimp (C) and in a second aquarium that contained one adult shrimp
(D). The contour lines represent concentration intervals of 20 µM.
33

Figure 2-4 (A) Oxygen was measured directly in a burrow opening and compared to a profile in
ambient sediment 5 cm to the left of the burrow. (B) The change of oxygen concentration over time
was monitored in a shaft of a shrimp burrow at 3 cm depth. (C) A 2D contour plot of redox potential
was created from profiles performed around a shrimp burrow. The dashed lines depict the position of
the burrow. The contour lines represent increments of 50 mV.

Distribution of Redox Species: The distributions of the redox couples ferric/ferrous
iron and nitrate/ammonium are illustrated as contour plots along the transect (Figure
2-5 A-D). The profiles measured at the control site are not included in the contour
plots. Here the oxidized form of iron (Fe III) was only present in the top 2 cm,
whereas reduced iron (Fe II) increased with depth to concentrations of 1200 µM cm
-3

wet sediment. Similarly, nitrate was only present just below the sediment surface (20
µM), whereas ammonium showed a typical increase with depth starting at 7 µM at 1
34
cm sediment depth and increased to 110 µM at 10 cm sediment depth. In contrast, a
comparison of oxidized and reduced species of iron and nitrogen along the transect
showed a patchy distribution reflecting the presence of burrows and bio-irrigation
activity. The distribution of ferric iron at the 0 m – 4 m sites was similar to the
control site, with high concentrations occurring in the top 2 cm. With increasing
distance (6 m, 8 m and 10 m) and increasing bioturbation activity, this sharp surface
peak disappears and ferric iron is present in fairly high concentrations also at greater
depths. A deeper reaching zone of oxidized iron is especially apparent at the 6 m site.
In a similar pattern the subsurface (2- 3 cm) peak in ferrous iron concentration,
which is still evident at the 0 – 2 m sites, disappeared along the transect. The
subsurface decrease in ferrous iron at the 6 – 10 m sites seemed to mirror the zones
with an increase in ferric iron. Similarly a decrease of ammonium at the 6 – 8 m sites
was reflected in an increase of subsurface nitrate concentrations, especially at 6 m
distance. Oxidation effects are primarily evident at the 6 m to 10 m sites, which were
characterized by dense populations of mainly ghost shrimp (Figure 2-2 A).

35
Figure 2-5 Ferric iron (A), ferrous iron (B), nitrate (C), and ammonium (D) concentrations from
sediment and pore-water samples collected at each site along the transect down to a depth of 10 cm
displayed as 2D contour plots. Contour lines represent increments of 45 µM cm
-3
wet sediment (ferric
iron), 100 µM cm
-3
wet sediment (ferrous iron), 5 µM (nitrate), and 10 µM (ammonium) respectively.
36

Figure 2-6 Cell counts (Acridine Orange Direct Counts) were performed for each site down to a
depth of 10 cm in 1-cm vertical resolution. Cell abundances per cm
3
of sediment were compared for
the non-bioturbated control site (A), the sites characterized by low to moderate bioturbation (0 m, 2 m,
4 m) (B) and the highly bioturbated sites (6 m, 8 m, 10 m) (C). Triplicate filters were prepared and
counted for each sample and error bars indicate the standard deviation for each sample.

Microbiology
Cell Counts: Cell numbers were comparably low at the control site (Figure 2-6 A)
and remained rather constant (1.5 x 10
9
cells cm
-3
sediment) throughout the 10 cm.
In contrast, microbial abundances were up to a magnitude higher in the bioturbated
areas (Figure 2-6 B, C), reaching highest values at 10 m distance (20 x 10
9
cells cm
-3

sediment). At the 0 – 4 m sites the depth profiles of cell showed highest values
toward the sediment surface and a decrease with depth. At the 6 m – 10 sites cell
37
numbers were generally highest at the surface and decreased rapidly to a depth of 5
cm, except for the 6 m site, where cell numbers were highest at 3 cm depth.
Abundances remained more or less constant below 5 cm depth at values higher than
at the control site (3 – 5 x 10
9
cells cm
-3
sediment).

Clustering and Similarity Analysis of Community Fingerprints: Statistical analyses
based on a Bray-Curtis similarity comparison of bacterial-community fingerprints as
measured by ARISA in 1 cm, 2, cm and 8 cm sediment depths at all sites along the
transect, showed a very distinct cluster of all surface microbial communities (Figure
2-7 A). This cluster was distinct from the communities found at 2 cm and 8 cm,
which showed a similarity of 40 – 65 % to one another. A separate cluster for
communities found at 2 cm and 8 cm depth showed a high similarity (>80%)
between both depths. Highest similarity was found between samples collected in the
bioturbated areas 2 m – 10 m distance, whereas the communities at 0 m were less
similar.

38

Figure 2-7 Microbial diversity was determined for sediment depths 0-1 cm, 1-2 cm, and 7-8 cm from
each bioturbated site along the transect (A), as well as for samples directly from the wall lining of U.
crenulata and N. californiensis burrows at these same three depths (B). A cluster analysis was
performed for all of these communities using the Bray-Curtis similarity index after log (x+1)
transformation, and is represented as a dendrogram. Bold lines highlight sample clusters that are not
statistically different. The samples are named according to the distance in m (x) and the depth in cm
(y) where they were taken (xmycm). CB stands for ‘Crab Burrow’ and SB stands for ‘Shrimp Burrow’
and the number indicates the depth in cm.

A comparison of the microbial communities sampled directly at the burrow walls of
the two species U. crenulata and N. californiensis at the three depths (1 cm, 2 cm
39
and 8 cm) showed separate clusters for the two burrow systems with less than 30%
similarity between them (Figure 2-7 B). The communities which formed at all three
depths at the wall of the mud shrimp burrow showed highest similarity to the surface
communities (Figure 2-8). In contrast, the communities in the crab burrow at all
depths were more closely related to deeper sediment communities, although the
communities at 1 cm and 2 cm depth appear to form a separate cluster.

Figure 2-8 The Bray-Curtis dendrogram combines the similarity data shown in Figure 2-7 for both
sediment and burrow wall samples. Samples clustered with bold lines are not statistically different.

A CCA biplot was used to compare the community fingerprinting data with
environmental parameters. The CCA technique creates an ordination diagram where
axes are created by a linear combination of environmental variables (ter Braak, 1986,
1995). The eigenvalue for each axis generated by CCA indicates how much of the
variation seen in species data can be explained by that axis. In this case, Axis 1
40
explained 34.3% of the variance in species data (eigenvalue (e.v.) = 0.473) and Axis
3 explained 4.9% of the variance (e.v. = 0.067). While Axis 2 explained 11.4% of
the variance the p value was > 0.05 and so was not displayed. The right side of the
ordination diagram is predominately occupied by surface samples with the exception
of two samples taken at 2 cm depth at the 6 and 10 m sites. Whereas on the left side
of the plot all remaining samples from depths 2 cm and 8 cm were located.
Therefore, the CCA biplot supported the distinction between communities from the
surface sediment and deeper sediment layers (Figure 2-9). The length of an
environmental line (ferric iron, ferrous iron, nitrate, ammonium) represents the
extent of species distributions change along that environmental variable. The
orientation of the line represents the gradient of the environmental variable. The
closer an environmental arrow is to an axis, the stronger the correlation between that
variable and the axis. There was a strong positive correlation of Fe (III) with Axis 1
(r = 0.899) where the majority of surface samples were found. The plot also
indicated a strong negative correlation of Fe (II) and ammonium with Axis 1 (r = -
0.764 and r = -0.580 respectively) into the direction of the deeper samples. Nitrate
correlated strongest with Axis 3 but also had a stronger correlation with Axis 1 (r =
0.292) than all other environmental variables, which might be an indication that
different environmental parameters contribute to the effect of nitrate on local
microbial communities.

41
Figure 2-9 An ordination diagram displaying the first and third axis of a canonical correspondence
analysis was created using those locations with both species (ARISA) and environmental (NO
3
2-
,
NH
4
+
, Fe (II), and Fe (III)) data. Samples are grouped according to their depth (1 cm, 2 cm, 8 cm) and
displayed with different symbols as indicated in the legend.

DISCUSSION

Bioturbation Activity and Microbial Communities: Despite the regional and global
importance of macrofauna sediment reworking, reasonable estimates of bioturbation
exist only for a limited set of conditions and regions of the World (Henderson et al.,
1999; Teal et al., 2008). Although it has been known that burrowing organisms affect
sediment biogeochemistry, the interactions are often complex and detailed
investigations on the effect of geochemical microzonations on microbial diversity are
scarce (Kostka et al., 2002; Lucas et al., 2003; Matsui et al., 2004; Papaspyrou et al.,
2006). This is not surprising because studies on microbial diversity in marine
sediments have also only just begun (Mussman et al., 2005; Wilms et al., 2006; Wu
et al., 2008). The impact of bioturbation is species-specific and activity dependent
42
(e.g. Pelegri and Blackburn, 1996; Christensen et al., 2000; Marinelli et al., 2002;
Solan & Kennedy, 2002; Mermillod-Blondin et al., 2004), therefore microbial
communities associated with specific burrow structures most likely also show
variability.

Our observations demonstrate that the two species of sediment dwelling crustaceans
in the shallow lagoon of Catalina Harbor exhibit different bioturbation activities,
which in turn have contrasting effects on the subsurface microbial communities
associated with the burrows. While the ghost shrimp lives permanently subterranean,
is constantly reworking its burrow, and flushing it, to maintain a certain oxygen level
within the burrow; the fiddler crab builds a simple burrow in which it resides only
occasionally and does not actively ventilate it. Oxygen transport into the shrimp
burrow as well as into the surrounding sediment led to oxidation effects that were
documented also in the field studies by a decrease of reduced compounds and
extension of oxidized conditions deeper into the sediment (Figure 2-5).

A key question in benthic microbial ecology has been whether distinct microhabitats
harbor distinct microbial assemblages. This issue has been much discussed in
microbial ecology and in the emerging field of microbial biogeography. Do habitats
with similar environmental conditions promote similar microbial communities? It
has been suggested (Kristensen and Kostka, 2005), that even if geochemical
43
conditions in burrows are equivalent to the sediment surface, the microbial
communities in and around burrow walls are most likely unique. This hypothesis has
been explained by the great temporal variability of environmental conditions inside
the burrows, but a greater physical stability of the burrow itself compared to a
frequently disturbed sediment surface.

Based on our investigations we suggest that the geochemical conditions at and
around the burrow walls of N. californiensis burrows were very similar to the
sediment surface (Figure 2-3 D, 2-4 C). Our community fingerprinting results
(Figure 2-8) indicate that the oxic and oxidized zones associated with the shrimp
burrow support microbial communities that are very similar to those found in the top
1 cm of the sediment (similarity of ~ 55-70%). At all sample depths (1 cm, 2 cm, 8
cm) inside the burrows, the ARISA fingerprints clustered within the surface
communities found at all sites and are very different from communities deeper in the
sediment.

In contrast to the constantly bio-irrigating ghost shrimp, fiddler crabs may be called
“temporary” bioturbators. They establish a burrow but do not live below the surface.
In the field, they are most often seen at the sediment surface or guarding the entrance
of their burrow and do not seem to spend long periods within the burrows. We were
not successful in measuring oxygen in any of the crab burrows and assume that they
44
are not actively ventilating their burrows. The communities found within the crab
burrows differed from the ones in the shrimp burrows (Figure 2-7 B), showing more
similarity to subsurface communities (Figure 2-8). At 8 cm depth the crab burrow
communities did not differ significantly from any of the communities found at that
the same depth from all other sites. Interestingly, the crab burrow communities at 1
cm and 2 cm depth seemed to form a unique cluster, although showing closer
similarity to subsurface than surface communities. This might support the hypothesis
by Kristensen and Kostka (2005), who suggested that burrows support unique
communities. Burrowing behavior is species-specific and to understand the effect of
bioturbation on microbial communities one has to take into account the ecology of
the macrofauna organisms. Papaspyrou and co-workers (2005) studied the bacterial
communities in burrows of the ghost shrimp Pestarella tyrrhena (Decapoda:
Thalassinidea). They found a 10-fold increase of bacterial abundance associated with
the burrow wall, which they attributed to the higher organic content compared to the
surrounding sediment. They did not measure oxygen at the burrow wall or the
surrounding sediment. Their study by molecular fingerprints (DGGE) of the bacterial
communities suggested that the bacterial composition of the burrow wall was more
similar to the ambient anoxic sediment. The authors therefore suggested that burrow
walls have distinct properties and should not be considered merely as a simple
extension of the sediment surface. Organic content and the quality thereof is
certainly a structuring component for microbial communities. Oxygen is one of the
45
strongest parameters affecting microbial communities in sediments, as it is the most
favorable electron acceptor in the oxidation of carbon. It divides the communities
into assemblages that vary in their oxygen requirements and tolerances (aerobes,
anaerobes). The question is what are the key parameters structuring microbial
communities. Another question is how to define ‘similar’. In our example we define
the similarity between burrow walls and their surrounding as the similarity in the
oxic and oxidized conditions. We do not dispute that burrow walls may represent
unique environments. It depends on a combination of factors and may vary among
different burrow types, depths and compartments as well as oxidation events. Our
study suggests that burrows can support similar microbial populations compared to
the surface if the environmental parameters, e.g. redox conditions, are equal.

The argument that free-living microbes are ubiquitously (Finlay, 2002) and
environmental parameters determine their presence has been suggested by the ‘Baas-
Becking’ hypothesis (Baas-Becking, 1934: ‘everything is everywhere and the
environment selects’). This assumption has been controversial for much of the last
century (e.g. reviewed in Hedlund and Staley, 2003; Ge et al., 2008) and recent
studies have shown that the richness or diversity of microorganisms might be more
complex (McArthur et al., 1988; Horner-Devine et al., 2004). Salinity, temperature
or nutrient gradients have been identified to change aquatic microbial communities
(Ward et al., 1998; Crump et al., 2004) and a more recent hypothesis has been that
46
microbial composition and diversity patterns reflect the influences of both past
events and contemporary environmental variations (Martiny et al., 2006, Ge et al.,
2008). Hewson and Fuhrman (2004) found, by comparing the diversity and richness
of bacterio-plankton along a salinity gradient, that some taxa (or OTUs) are specific
to distinct environments while others have a ubiquitous distribution from river to sea.
So both might be true, there are microorganisms that are everywhere and others
which are endemic. A lot more work needs to be done to address this.

Few studies on microbial diversity have been performed in benthic systems (Llobet-
Brossa et al., 1998; Köpke et al., 2005; Córdova-Kreylos et al., 2006; Wilms et al.,
2006, Hewson and Fuhrman, 2007; Liang et al., 2007) and studies on the effect of
bioturbation on microbial community structure remain extremely scarce (Plante and
Wilde, 2004; Papaspyrou et al., 2005, 2006). Environmental parameters vary
quickly, especially in coastal sediments. To adequately address the factors
controlling biodiversity in sediments, the environmental parameters have to be
measured at a resolution that accounts for this variability, which is unfortunately not
always possible. Some laboratory and field studies (Korona et al., 1994; Haubold and
Rainey, 1996; Rainey and Travisano, 1998; Zhou et al., 2002; Treves et al., 2003;
Horner-Devine et al., 2004) suggested that environmental ‘patchiness’ played a role
in the maintenance of the microbial diversity in soils and sediments. Patchiness
comprises the complexity of environmental conditions that can vary along multiple
47
spatial and temporal scales. Our examples show that the stability of environmental
conditions over time supports the establishment of specific microbial assemblages.
One remaining question is how quickly these communities develop as a result of
varying redox conditions.

There are a number of different approaches to study the types of microbes present in
an environment and their abundance. We used ARISA (Fisher and Triplet, 1999;
Hewson and Fuhrman, 2004; Brown et al., 2005; Fuhrman, 2008) as a PCR based
community finger printing method to characterize and compare the richness of
different taxa (OTU’s). This method results in data of discrete numbers, such as the
total fluorescence of a specific fragment length, thus permitting statistical
comparisons of species (OTU) richness. ARISA is a relatively inexpensive and fast
way to compare bacterial community structures by analyzing the lengths of the
intergenic spacers between 16S and 23S rRNA genes present in almost all bacteria.
Therefore, archaea are not considered in this approach and some groups like the
Planktomycetes might not be included in the analyses, if they lack linked 16S-23S
genes (Fuhrman, 2008). Nevertheless, this assemblage fingerprinting approach
allows comparison of microbial richness along environmental gradients with a
phylogenetic resolution, that is within 98% of 16S rRNA sequence identity, near the
range widely considered to be bacterial “species” or “ecotypes” (Fuhrman, 2008).

48
Effect of Bioturbation on Biogeochemical Cycles: The combined effect of the
bioturbation activity of the two investigated crustaceans on the biogeochemistry in
the shallow lagoon of Catalina Harbor showed an increase of microbial abundances
(Figure 2-6 A-C) at all sediment depths as bioturbation intensity increased (Factor 10
or higher). The overall increase of oxidized chemical compounds (ferric iron and
nitrate, Figure 2-4 A, C) reflects a significant increase of oxidation-reduction
potential within the sediment. The statistical analyses (Figure 2-9) suggested that the
availability of ferric iron correlated with all surface (1 cm) microbial fingerprints.
Whereas the abundance of reduced nitrogen and iron species correlated with
assemblages found deeper in the sediment. More investigations are needed to
correlate environmental parameters with microbial diversity and activity. Another
important next step in our investigations will be to determine the metabolic activities
associated with distinct microniches. Microbial processes enhanced or induced by
bioturbation activity may significantly contribute to element cycling at the seafloor.
49
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57
CHAPTER III: Burrowing deeper into benthic nitrogen fixation

CHAPTER III ABSTRACT

Biologically available nitrogen (N) limits marine productivity, and microbial
processes leading to its loss (denitrification) and gain (dinitrogen (N
2
)-fixation into
ammonium) are essential for global biogeochemical cycles. Bioturbation by benthic
organisms can affect key ecosystem functions, including N cycling, most often
reported as enhancement of denitrification and a subsequent loss of N
2
from the
system. Sedimentary N
2
-fixation has been considered important only in relatively
rare, localized habitats such as rhizosphere and phototrophic microbial mat
environments. However, the potential for N
2
-fixation in marine sediments may be far
more widespread. Sulfate-reducing bacteria (SRB) are ubiquitous and abundant in
marine sediments, with many possessing the genetic capacity to fix N
2
. We show
here that N
2
-fixation occurs at high rates (up to 0.8 mmol N m
-2
d
-1
) in coastal
sediments primarily bioturbated by the ghost shrimp Neotrypaea californiensis and
at depths below 5 cm. Inhibition experiments and genetic analysis showed that N
2
-
fixation was mainly linked to sulfate reduction. Integrated subsurface N
2
-fixation
rates dwarfed those previously found for un-vegetated estuarine sediments and were
comparable to the highest values previously recorded, those from photosynthetic
microbial mats and rhizospheres. Our results show that N
2
-fixation by SRB in

58
bioturbated sediments is an important process leading to significant new N input into
marine sediments. A re-evaluation of N budgets and the processes controlling N
cycling in benthic environments is thus called for. Given the ubiquity of bioturbation
and of SRB in marine sediments, this overlooked benthic N
2
-fixation may play a
significant role in global nitrogen and carbon cycles.

INTRODUCTION

Temperate coastal and estuarine sediments are generally considered to be areas of
net nitrogen (N) loss through the consumption of combined N by denitrification and
efflux of dinitrogen gas (N
2
) (Gilbert et al., 1997), with N
2
-fixation rarely considered
in N budgets of such systems. Nonetheless, there have been numerous studies of
benthic N
2
-fixation, most focusing on photosynthetic microbial mats or sediments
vegetated by seagrasses and marsh plants, demonstrating that not all N is lost from
this system (Capone, 1983; Carpenter and Capone, 2008). Almost no attention has
been given to deeper sediment layers, perhaps because of generally increasing
concentrations of ammonium (NH
+
4
), a known inhibitor of N
2
-fixation, with depth
(Yoch and Whiting, 1986). However, N
2
-fixation has been shown, enigmatically, to
occur in sediments even at elevated NH
+
4
(700 µM) concentrations (McGlathery et
al., 1998). Many coastal sediments have a high abundance of burrowing infauna
(Teal et al., 2008) with bioturbation (movement of particles) and bioirrigation

59
(movement of fluids) having direct consequences on organic matter degradation,
biogeochemical processes, and nutrient cycling (Rhoads, 1974; Aller, 1982;
Kristensen, 2000). However, macrofauna burrows have rarely been considered as
sites for N
2
-fixation because of the general view that bioirrigation leads to the
transport and presence of high levels of oxygen, an inhibitor of the nitrogenase
protein (Stewart, 1969; Postgate 1982). Because oxygen transport across burrow
walls is limited by molecular diffusion and because oxygen is rapidly consumed,
oxygen penetration into the surrounding sediment is only a few mm (Ziebis et al.,
1996). Beyond this oxic zone, bioturbation induces the formation of oxidized zones
several cm in diameter (Figure 3-1) characterized by a positive redox-potential,
reflecting the enhanced availability of other electron acceptors (Forster and Graf,
1992; Ziebis et al., 1996). Within these zones, the availability of labile organic
carbon may also be enhanced (Aller, 1982), creating ‘hot spots’ of elevated
microbial activity, such as sulfate reduction (Goldhaber et al., 1977). Because many
sulfate-reducing bacteria (SRB) have the genetic ability to fix N
2
(Zehr et al., 1995)
and have been shown to fix N
2
in other benthic environments (Capone, 1988;
Nielsen et al., 2001; Steppe and Paerl, 2002), these subsurface habitats associated
with macrofaunal burrows might also be ideal environments for N
2
-fixation.

60

Figure 3-1 Image of a N. californiensis burrow system. A ghost bay shrimp can be seen in its burrow
(1), with an oxidized, light colored region adjacent to the burrow (2) and black microniches in the
surrounding sediment (3).

To test the idea that N
2
-fixation may occur in and around macrofaunal burrow
systems, we carried out experiments in Catalina Harbor, a shallow intertidal lagoon
on Catalina Island, California. The study site is characterized by intense bioturbation
by the bay ghost shrimp Neotrypaea californiensis Dana 1854 (Crustacea; Decapoda;
Thalassinidea) (Bertics and Ziebis, in press), previously known as Callianassa
californiensis (Manning and Felder, 1991), with up to ~1800 burrow openings m
-2
.
N. californiensis belongs to a cosmopolitan group of decapods known to maintain
deep-reaching burrows, some as deep as 3 m (Pemberton et al., 1976), which
significantly impact geochemical gradients and influence biogeochemical processes
world-wide (Ziebis et al., 1996; Dworschak, 2000). At our study site N.
californiensis burrows generally reached 20 cm deep into the sediment and had 3 to

61
4 openings at the surface. The burrows consists of shafts (~ 1-cm diameter) and
chambers (~ 2-cm diameter) that the shrimp maintain and frequently flush with
oxygen-rich water. Different microniches can be associated with the burrow system,
which are evident from the coloration of the sediment (Figure 3-1). Burrow walls
are generally light colored in contrast to the surrounding sediment, but dark reduced
microniches may also occur where burrows have been abandoned or organic material
might be stored (Figure 3-1).

MATERIALS AND METHODS

Field Sampling: Investigations were carried out in a shallow intertidal lagoon
located in Catalina Harbor, Catalina Island, California (33° 25.23’ N, 118° 19.42’
W). Sediment cores were collected over four years (2005-2008) at various locations
within this lagoon, with respect to bioturbation intensity (defined as number of
burrow openings m
-2
). Cores were sliced in 1-cm intervals and pore-water samples
were collected from these slices using a nitrogen gas pore-water press (KC Denmark,
Silkeborg, Denmark). Pore-water NH
+
4
concentrations were measured by flow
injection analysis (Hall and Aller, 1992) and averaged over this four-year period.
Three areas were then chosen for further investigation: non-bioturbated (NON),
medium bioturbation (MED) and high bioturbation (HI) with 0, ~600 and, ~1800
burrow openings m
-2
respectively. The MED and HI areas also had a visible

62
microbial mat at the surface. Redox-potential measurements using a redox-potential
microsensor (Unisense, DK) were performed in-situ (Bertics and Ziebis, in press) at
all three locations. Subsequently, cores were collected at each area and taken back to
the laboratory for microbial rate measurements.

Rate Measurements and Inhibition Experiment: Sediment cores were sliced in 1-cm
intervals, placed in serum vials flushed with N
2
and assayed by acetylene reduction
in triplicate for nitrogenase activity (NA) in the dark at in-situ temperature (Capone,
1993). N
2
-fixation rates were calculated from the NA results using a conversion
factor of 3 C
2
H
4
: 1 N
2
. DNA samples were concurrently taken and stored at -80°C.
Inhibition experiments were carried out on sediment slurries from the 0-5 and 5-10
cm horizons, using 20 mM sodium molybdate (Oremland and Capone, 1988), to look
for possible linked N
2
-fixation/sulfate reduction. Sulfate reduction rates (SRR),
applying radiotracer techniques with radio-labeled sulfate (
35
S) (Kallmeyer et al.,
2004), and NA were measured in triplicate for inhibited and non-inhibited samples,
with non-inhibited samples being treated the same as the inhibited samples minus
sodium molybdate.

nifH Genetic Analysis: Nitrogenase genes (nifH) from sediment DNA (20 ng per
reaction) were amplified by nested PCR using fluorescently-labeled degenerate
primers (Hewson and Fuhrman, 2006). Amplicons (~ 370 bp) were gel-purified and

63
quantified before digestion. For TRFLP, 200 ng of amplified product was digested
with HaeII to determine overall nifH community structure (Hewson and Fuhrman,
2006). TRFLP profiles were used to select three samples for cloning and sequencing.
These samples came from an area of low SRR/high NA (HI 1 cm), an area of high
SRR/high NA (MED 8 cm), and an area of high SRR/low NA (NON 8 cm).
Sequences were aligned with closest GenBank tblastx matches from cultured
representatives to construct a neighbor-joining tree with Kimura correction in ARB.

Laboratory Experiments: Detailed investigations of a N. californiensis burrow
system were performed using a narrow aquarium (40 cm x 30 cm x 3 cm) that was
kept in the laboratory at the Wrigley Institute for Environmental Studies located on
Catalina Island. The aquarium was filled with sediment collected from the study site
that was sieved through a 500-µm sieve to remove large sediment particles and
sediment-dwelling macrofauna. The aquarium was populated with one shrimp from
our field site and placed in a larger aquarium that was continuously flushed with
fresh seawater so that the surface of the narrow aquarium was supplied with oxygen
rich water. This setup was maintained for several months, allowing the shrimp to
establish an elaborate burrow system. The development of the burrow was
documented by digital photography (Olympus Stylus digital camera model
u10D,S300D,u300D) and tracked on transparences placed against the aquarium wall.
The front wall of the aquarium was perforated with silicon-filled holes in a 1-cm grid

64
that served as ports for NH
+
4
sample collection targeting specific burrow
compartments. At the end of the experiment the front wall of the aquarium was
carefully removed to allow for sampling in selected microniches for N
2
-fixation
measurements using the acetylene reduction method. Because of the small sample
size only single incubations could be performed.

RESULTS

In Catalina Harbor, NH
+
4
concentrations increased with depth in a non-bioturbated
area, whereas areas of increased bioturbation resulted in a complex geochemical
zonation pattern (Figure 3-2 A) with subsurface patches of decreased NH
+
4

concentrations. These results indicate enhanced nitrification activity associated with
the burrows. Profiles of redox-potential (mV) at our three experimental locations
(non-bioturbated (NON), medium bioturbated (MED) and high bioturbated (HI))
showed higher oxidation potential at depth with increasing bioturbation activity
(Figure 3-2 B). Sulfate reduction rates (SRR) were higher in the deeper sediment (5-
10 cm) when compared to the surface layer (0-5 cm) at all three locations (Figure 3-2
C). Notably, SRR were highest in the MED area as compared to rates measured at
the NON and HI areas. We hypothesize that the medium level of bioturbation
enhanced microbial activity through a combination of increased access to organic
carbon without the introduction of inhibitory levels of oxygen or accumulation of

65
NH
+
4
. Although our study did not focus on sediment surface activity of N
2
-fixation,
we measured the highest rates of nitrogenase activity (NA) within the top 1 cm of
sediment (35.1 nmol C
2
H
4
cm
-3
hr
-1
) where a visible mat was present. These rates
are comparable to rates seen in other studies that have focused on photosynthetic
mats (Capone, 1983; Howarth et al., 1988). Below the mat, NA paralleled the pattern
of SRR, with higher activity at greater depth (below 5 cm) and highest rates in the
MED area (Figure 3-2 D). It is still not clear why bacteria would carry out N
2
-
fixation in the presence of appreciable NH
+
4
because N
2
-fixation is an energy
intensive process relative to NH
+
4
assimilation. Two possibilities that have been
discussed are that N
2
-fixation can serve as a sink for excess electrons (Tichi and
Tabita, 2000) or that natural organic compounds can decouple nitrogenase
expression from NH
+
4
inhibition (Capone, 1988), as occurs with the inhibitor
methionine sulfoximine (Yoch and Whiting, 1986).

66

Figure 3-2 Biogeochemical rate measurements, pore-water NH
+
4
concentrations and redox-potential of
Catalina Harbor sediments. (A) NH
+
4
concentrations (µmol l
-1
) at different bioturbation intensities (x-
axis shows increasing bioturbation intensity) and averaged over a four-year period. All sample
locations (indicated by black dots) showed less than 20% variation over this time period. (B) In-situ
redox-potential at the NON (orange), MED (green) and HI (blue) locations on 09Jun07. (C) Sulfate
reduction rates in sediment slurries from the 0-5 and 5-10 cm horizon on 27Sept07. (D) N
2
-fixation
rates from 08June07. Error bars shown are standard error.

Based on the similar vertical distributions of NA and SRR, we undertook
experiments to quantify the amount of N
2
-fixation associated with sulfate reduction.
Using MoO
4
as a specific inhibitor of sulfate reduction, we measured the inhibition

67
of SRR and NA in sediment slurry incubations. SRR were essentially eliminated
(99.4 ± 1.5%), when compared to controls, at all three locations, at 0-5 and 5-10 cm
depths. NA was decreased 88% between 0-5 cm and 97% between 5-10 cm at the
NON area, confirming a direct coupling between SRR and NA, which increased with
depth. At the HI area, NA was reduced by 77 ± 3.4% throughout the sediment
column, indicating that some of the observed NA may be linked to other processes.
At the MED area, NA was reduced 95 ± 1.6%, between 0-10 cm, suggesting that
almost all NA at this location was linked to SRR. These findings show that there is a
close coupling between sulfate reduction and N
2
-fixation and that the degree of
coupling varies with bioturbation activity.

To determine the identity and diversity of potential N
2
-fixers, we assessed the
presence of a gene involved in N
2
-fixation (nifH) in our three contrasting habitats
(Figure 3-3). As expected, we found cyanobacterial nifH genes within the
phototrophic microbial mat, consistent with the high rates observed. Within the
surface sample, as well as in the two deep samples, we detected nifH genes most
closely related to those from various SRB, including Desulfovibrio spp. and
Desulfobacter spp., two SRB that have been shown to fix N
2
(Sisler and Zobell,
1951; Widdel, 1987). These results corroborate our findings that NA below the
surface sediment layer is largely carried out by SRB.

68

Figure 3-3 Neighbor-joining phylogenetic tree of nifH sequences (this study) and closest protein
matches by tblastx. The number in parentheses indicates the number of sequences present for the
designated species or location within the grouping. nifH cluster designations assigned according to
(Zehr et al., 2003).

Further investigations were done to determine N
2
-fixation activity in specific micro-
environments associated with bioturbation activity. Using sediment collected from a
narrow aquarium inhabited by a single ghost shrimp, we found that N
2
-fixation was
highest in deep burrow chambers in addition to reduced areas at the same depth (2.0
– 3.3 nmol N cm
-3
d
-1
, Figure 3-4, Table 3-1). Much lower rates were seen the

69
vertical shaft of the burrow and at the burrow opening (< 0.8 nmol N cm
-3
d
-1
). NH
+
4

concentrations, in general, were much lower in all measured locations when
compared to the bulk sediment values.

Figure 3-4 Locations where N
2
-fixation and NH
+
4
concentration measurements were taken within the
narrow aquarium. Numbering starts with 4 so as not to be confused with Figure 3-1.

Location N
2
-fixation rate
(nmol N cm
-3
d
-1
)
NH
+
4
concentrations
(µM)
4 0.4 4.69 ± 1.01
5 3.3 10.74 ± 0.77
6 2.9 9.54 ± 1.03
7 2.9 -
8 2.0 -
9 0.8 5.19 ± 0.21

Table 3-1 N
2
-fixation rates and NH
+
4
concentrations in a narrow aquarium inhabited by a single N.
californiensis. Location numbers refer to those seen in Figure 3-4.

DISCUSSION

Our field studies revealed that bioturbation enhances benthic N
2
-fixation coupled to
sulfate reduction. Further detailed laboratory experiments (narrow aquaria)

70
demonstrated that this activity occurs mainly in microniches associated with burrow
compartments, as well as in highly reduced microzones at depth. Although the
observed experimental rates were lower than in the field, they confirmed that
bioturbation activity induced N
2
-fixation. We conclude that laboratory rates were
lower because they showed the effect of only one individual shrimp, whereas
subsurface microniches in the field are the result of a whole community.

Overall, our findings stand in contrast to previous findings suggesting that
bioturbation leads to an overall N
2
loss from sediments through increased
nitrification/denitrification (Gilbert et al., 1997). These authors only analyzed
denitrification rates, which did not allow for a calculation of net N
2
flux. However, a
recent study demonstrated that shallow estuarine sediments are capable of taking up
N
2
gas, indicating net N
2
-fixation (Fulweiler et al., 2007). While we did not measure
denitrification rates, our study shows that N
2
-fixation must be considered in
sedimentary N budgets. Rates of benthic denitrification in bioturbated sediments
have been found in the range of 5.64 mmol N m
-2
d
-1
(Gilbert et al., 1998). Based on
our areal rates of subsurface N
2
-fixation (Table 3-2), some to all of the N lost
through denitrification in the mentioned study could have been fixed in the
sediments. If N
2
-fixation was occurring at the rate seen in our MED area (including
the microbial mat), the N cycle could be closed, with inputs balancing losses, or even
result in a net fixed N gain (Figure 3-5). Further studies are needed to directly

71
determine the relative importance of sedimentary N
2
-fixation with respect to
denitrification.

Environment N
2
-fixation
(mmol N m
-2
d
-1
)
Integration Depth
(cm)
Study
Catalina Harbor
Non-Bioturbated 0.8 0-10
1
Non-Bioturbated 0.78 1-10
1
Med. Bioturbation 8.05 0-10
1
Med. Bioturbation 2.43 1-10
1
High Bioturbation 2.54 0-10
1
High Bioturbation 0.42 1-10
1
Other Recent Studies

Lagoon w/o Mat 0.03 0-2
2
Intertidal Microbial Mat 1.63±1.15 Mat
3
Averages By Environment

Lake Sediments

Heterotrophic 0.02±0.03 -
4
Phototrophic 0.03±0.02 -
4
Atlantic Ocean (2,800 m) 0.00008 -
4
< 200 m Sediments 0.02±0.01 -
5
Bare Estuarine Sediments 0.08±0.03 -
5
Zostera Estuarine Sediments 0.39 -
5
Coral Reef Sediments 6.09±5.62 -
5
Mangrove Rhizosphere 0.56 -
5
Mangrove Mat 1.66 -
5
Salt Marsh Rhizosphere 5.27±3.64 -
5
Salt Marsh Surface Sediment 0.38±0.41 -
5

Table 3-2 Integrated N
2
-fixation rates in Catalina Harbor sediments. Values integrated over the 0-10
cm horizon include the cyanobacterial mat, while those integrated over 1-10 cm exclude it. Examples
of rates from recent studies and average rates for different types of sediments are shown for
comparison (
1
This Study;
2
Charpy-Roubaud et al., 2001;
3
Steppe and Paerl, 2002;
4
Howarth et al.,
1988;
5
Capone, 1983).

72

Figure 3-5 Revised view of nitrogen cycling and fluxes in bioturbated coastal sediments. (Left) Figure
showing the vertical depletion of electron acceptors in non-bioturbated coastal sediments. We
hypothesize that bioturbation (Right) leads to a 3-dimensional chemical zonation pattern that enhances
zones of nitrification and denitrification as well as creating microniches of subsurface N
2
-fixation
carried out by SRB.

Our calculated areal N
2
-fixation rates in the investigated bioturbated sediments were
one to two orders of magnitude higher than previous studies of bare (without
vegetation or microbial mats) estuarine sediments (Table 3-2). Interestingly, N
2
-
fixation in our NON area was also an order of magnitude higher than those
previously reported, possibly because we integrated N
2
-fixation through the sediment
column. The total areal N
2
-fixation rate of the MED area, including the

73
photosynthetic microbial mat activity, rivals the highest rates ever measured in
benthic environments (Capone, 1983; Carpenter and Capone, 2008).

While this study was carried out in a selected estuarine mudflat, N. californiensis
inhabits similar environments along the west coast of North America from Alaska to
Baja California (MacGinitie, 1934). An estimation of the areal extent of estuarine
habitat along the North American coast, from Washington to California (a fraction of
this shrimps’ range), is 4641 km
2
(Emmet et al., 2000). We calculated that 4.6 × 10
6
mol N d
-1
could be fixed in bioturbated sediments inhabited with N. californiensis,
assuming that first, 41% of the estuarine area represents mudflat possibly inhabited
by the N. californiensis (Van Dyke and Wasson, 2005), second, the depth of N
2
-
fixation is 10 cm, and third, the average bioturbation intensity is similar to our MED
area (Griffis and Suchanek, 1991). This calculation only uses our sub-1 cm values of
N
2
-fixation and therefore does not take into account the potential fixation rates of
any photosynthetic microbial mat associated with the sediment surface. One estimate
of annual global benthic N
2
-fixation is 15.4 Tg N yr
-1
over an area of 363 × 10
6
km
2

(Capone, 1983), whereas our estimate in N. californiensis habitats is 0.024 Tg N yr
-1

over an area of 1.9 × 10
3
km
2
. Considering an estimate of >20,700 km
3
bioturbated
sediments worldwide (Teal et al., 2008), as well as the fact that there are many types
and sizes of burrowing organisms, some capable of creating deep and elaborate
burrows like the cosmopolitan Thalassinidean shrimp, we assert that N
2
-fixation is

74
important, but has been largely overlooked in these extensive benthic environments.
This discovery is significant because it demands a re-evaluation of the role of benthic
N
2
-fixation in N cycling and fluxes across the sediment-water interface.
Additionally, the contribution of benthic fixed N to the global N budget may be far
greater than previously thought.

75
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Forster, S. and Graf, G. 1992. Continuously measured changes in redox potential influenced by
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Hall, P. O. and Aller, R. C. 1992. Rapid small-volume flow injection analysis for ΣCO
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Kallmeyer, J., Ferdelman, T. G., Weber, A., Fossing, H., and Jørgensen, B. B. 2004. A cold chromium
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Nielsen, L. B., Finster, K., Welsh, D. T., Donelly, A., Herbert, R. A., de Wit, R., and Lomstein, B. A.
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Yoch, D. C. and Whiting, G. J. 1986. Evidence for NH
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78
CHAPTER IV: Bioturbation and the role of microniches for sulfate-reduction in
coastal marine sediments

CHAPTER IV ABSTRACT

The effects of bioturbation in marine sediments are mainly associated with an
increase in oxic and oxidized zones below the sediment-water interface through an
influx of oxygen-rich water deeper into the sediment and the rapid transport of
particles between oxic and anoxic conditions. However, sediment-dwelling
macrofauna activity can also increase the occurrence of reduced microniches and
anaerobic processes of organic matter degradation, such as sulfate reduction. Our
goal was to determine the 2-dimensional distribution of reduced microniches
associated with burrows of a ghost shrimp (Neotrypaea californiensis). Sulfate
reduction rates (SRR) were measured by injecting, in a 1-cm grid, radiolabeled
sulfate into a narrow aquarium (40 cm x 30 cm x 3 cm) containing the complex
burrow system of an actively burrowing ghost shrimp. Light-colored oxidized
burrow walls, as well as black reduced microniches, were clearly visible through the
aquarium walls. The direct injection of radiotracers allowed for whole-aquarium
incubation to obtain two-dimensional documentation of sulfate reduction activity.
Results indicated SRR were up to three orders of magnitude higher (140 - 790 nmol
SO
4
2-
cm
-3
d
-1
) in reduced microniches associated with burrows when compared to

79
the surrounding sediment. We show that bioturbation activity has a direct impact on
the distribution of microbial communities and the remineralization of organic matter
via sulfate reduction in marine sediments.

INTRODUCTION

Biogeochemical processes in surface (50 cm) marine sediments are characterized by
interactions between macrofauna organisms, microbial communities, and
geochemistry. Understanding benthic biogeochemistry is crucial in evaluating
marine ecosystem functioning and nutrient cycling. In a simplified way, marine
sediments are stratified such that biogeochemical processes are regulated by the
depth sequence of electron acceptors (O
2
> NO
3
-
>MnO > Fe (III) > SO
4
2-
> CO
2
)
used to remineralize organic material (Berner, 1980; Jørgensen, 2000). Although
sulfate is not the most favorable electron acceptor energetically, it is extremely
abundant in seawater, making sulfate reduction one of the most important processes
in marine sediments. Sulfate reduction alone can account for a large portion (up to
50 %) of benthic organic carbon remineralization, especially in organic rich coastal
sediments (Jørgensen, 1982; Canfield, 1989). As a consequence marine sediments
harbor a ubiquitous and diverse number of sulfate reducers.

80
The availability of organic matter in sediments also allows for the presence of
macrofauna organisms that effectively add to a more complex transport of particles
(bioturbation) and fluids (bioirrigation) and a three-dimensional zonation pattern of
geochemical processes (Rhoads, 1974; Aller, 1982, 2001; Kristensen, 2000).
Surface sediments receive an input of organic material and oxygen, the driving
forces for microbial processes, from the overlying water. Labile organic matter
reaching the seafloor is, in general, rapidly subducted below the sediment interface
by macrofauna organisms and quickly degraded by microbial communities (Graf,
1989; Jørgensen, 1996; Witte et al., 2003). It has long been recognized that benthic
communities directly contribute to the flux of material across the sediment-water
interface, the remineralization of organic matter and the recycling of nutrients
(Rhoads, 1974; Aller, 1994; Snelgrove and Butman, 1994; Hulthe et al., 1998;
Kristensen, 2000; Kristensen and Kostka, 2005). Models of early diagenesis have
tried to integrate macrofauna organisms in reactive transport models of aquatic
sediments (Berner, 1980; Boudreau, 1997; Soetaert et al., 2002; Meysman et al.,
2003) but the way biological processes are represented in diagenetic models is still
very abstract (Meysman et al., 2005). This problem arises from the complexity to
include macrofauna dynamics in present models and the necessity to rely on a
number of steady state assumptions. There is also a need for more detailed
investigations of animal-microbe-sediment interactions, on spatial and temporal
scales, to understand how macrofauna organisms influence the processing of organic

81
matter in marine sediments. The effect of bioturbation and bioirrigation is dependent,
not only on the abundance or biomass of sediment-dwelling organisms (e.g. Glud et
al., 2003), but on the degree or intensity (e.g. depth, frequence) of bioturbation and
bioirrigation, as well as on the type of species-specific interaction between
macrofauna organisms and their environment (Kristensen and Kostka, 2005).
Benthic ecologists are faced with the challenge to estimate the type and intensity of
animal-sediment-microbe interactions and their effects on geochemical processes to
better integrate the ‘biology’ into models of early diagensis, especially in coastal
sediments.

A number of bioturbation studies have focused on high-resolution and two-
dimensional mapping of oxic and oxidized microniches in sediments and around
burrow systems through the use of microsensors and planar optrodes (Glud et al.,
1996; Wenzhöfer and Glud, 2004; Zorn et al., 2006; Stockdale et al., 2009).
Additionally, pH planar fluorosensors have also been used in bioturbated sediments
to study the two-dimensional redoxclines that surround burrow walls (Zhu et al.,
2006). While oxygen penetration by diffusion into coastal sediments is only a few
millimeters deep (Revsbech et al., 1980; Gundersen and Jørgensen, 1990), oxygen
can be transported by benthic activity > 50 cm deep (e.g. Ziebis et al., 1996). The
interest in oxygen and a 2-dimensional documentation of its distribution is not
surprising as oxygen is not only the most important electron acceptor for benthic

82
respiration and microbial degradation of organic matter (Canfield et al., 1993), but
the infusion of oxygen into deeper sediment layers also leads to an important re-
oxidation of reduced compounds and increase of microbial activity (e.g. sulfide
oxidation). A close proximity of reduced and oxic/oxidized microenvironments
increases the oxidation potential of the sediment stimulating microbial activity.
Bioturbation can create oxic microniches within anoxic sediment layers that display
high metabolic activities (Nielsen et al., 2004) and additionally, due to the increase
in organic matter associated with bioturbation, can induce anoxic microniches within
the sediment that display an increase in sulfate reduction rates (Goldhaber et al.,
1977). By modeling biological interactions in aquatic sediments as coupled reactive
transport, Meysman et al. (2005) found that the rate of sulfate reduction increases
with bioturbation activity.

The aim of this study was to provide a two-dimensional mapping of microniches and
variations in sulfate reduction rates in sediments inhabited by the bay ghost shrimp
Neotrypaea californiensis Dana, 1854 (Crustacea: Decapoda: Thalassinidea),
previously known as Callianassa californiensis (Manning and Felder, 1991).
Thalassinidean shrimp are known to inhabit most of the world ocean’s sediments,
except for polar regions, with more than 500 known taxa worldwide (Dworschak,
2000), making them extremely important organisms in terms of benthic processes.
Thalassinidean burrow morphology is typically species-specific (Dworschak, 1983,

83
2001; Griffis and Chavez, 1988), with some thalassinids capable of creating 3 m
deep burrows (Pemberton et al., 1976). Our overall goal was to locate and measure
the intensity of sulfate reduction microniches surrounding an individual bay ghost
shrimp burrow and to compare these results with those obtained from our field site, a
shallow intertidal lagoon in Catalina Harbor, California, U.S.A.

MATERIALS AND METHODS

Field Studies and Whole-Core Sulfate Reduction Incubation Experiments: All field
sampling was conducted in a shallow intertidal lagoon located in Catalina Harbor,
Catalina Island, California (33° 25.23’ N, 118° 19.42’ W) from May through July
2008. Two sampling areas in Catalina Harbor were chosen based on differing levels
of bioturbation intensity (number of burrow openings m
-2
). The bioturbation intensity
at each site was determined by counting the number of burrow openings within a 25
cm x 25 cm frame ten times. These two areas, low bioturbation and high
bioturbation, were characterized as having ~ 120 burrow openings m
-2
and ~ 320
burrow openings m
-2
, respectively. From each location, triplicate cores were
collected for determination of sulfate reduction rates through whole-core incubation
experiments (Jørgensen, 1978) using the radioactive tracer
35
SO
4
2-
. Each core was
injected with
35
SO
4
2-
(6 µl, 200 kBq ) at 1-cm depth intervals throughout the length of
the core (length 13-14 cm) and incubated at in situ temperatures (18˚C) for 24 h in

84
the dark. Sulfate reduction rates were determined using the method of Fossing and
Jørgensen (1989) modified to a cold single step distillation after Kallmeyer et al.
(2004).

Laboratory Experiments: Detailed examination of N. californiensis burrows was
performed using narrow aquaria (Figure 4-1 A) that were kept in a wet lab at the
Wrigley Institute for Environmental Studies located on Catalina Island. Aquaria
were constructed to be 40 cm tall x 30 cm wide x 3 cm deep to allow enough space
for each ghost shrimp to build a complete burrow system. Each aquarium was filled
with sediment collected from the study site that was sieved through a 500-µm sieve
to remove large sediment particles and sediment-dwelling macrofauna. After the
addition of the sediment, the aquaria were allowed to sit for 24 hrs so that all
sediment could settle and compact. After this time, each aquarium was populated
with one shrimp and placed in a larger aquarium that was continually under a
flowing seawater system. The entire setup was allowed to sit untouched for several
months, allowing the shrimps to establish a burrow system. Once a burrow was
established, transparences were placed against the aquaria so that the visible burrow
structures could be traced and exact sampling locations recorded. Photographs of the
aquaria were also taken using an Olympus Stylus digital camera model
u10D,S300D,u300D with daylight lighting (Figure 4-1). The front wall of each
aquarium was perforated with holes in a 1 cm grid that were filled with aquarium

85
silicone. These holes served as ports through which sampling could be done from
the side, directly targeting specific burrow structures. Furthermore, this wall could
be removed entirely so that larger sediment samples could be collected from areas of
interest.

Figure 4-1 Photographs of the narrow aquarium used during the laboratory studies with sampling
ports 1 cm apart. (A) Shows the full view of the narrow aquarium while occupied by one ghost
shrimp. (B) A close-up view of a portion of a ghost shrimp burrow system with key burrow features
identified: the burrow opening (1), a burrow shaft (2), a burrow chamber (3), ambient brown sediment
(4), and black niches associated with the burrow (5).

Sulfate Reduction Rates From Different Burrow Compartments: Duplicate samples
of 0.5 cm
3
of sediment were collected from nine burrow features (Figure 4-1 B)
using 1-ml cut syringes and placed in 5 ml serum vials that were flushed with N
2
.
Each sample was injected with radiolabeled sulfate (
35
SO
4
2-
, 5 µl, 200 kBq), incubated at
in situ temperatures (18˚C) for 24 h in the dark, and analyzed for sulfate reduction
rates using the method previously described.

86
Whole-Aquarium Incubation and 2-Dimensional Mapping of Sulfate Reduction
Rates: To determine the location of sulfate reduction microniches around an
individual shrimp burrow,
35
SO
4
2-
(5 µl, 160 kBq) was injected through the ports in an
aquarium wall in a 15 cm wide and 20 cm deep portion of an aquarium in a 1-cm
grid resolution. The aquarium was allowed to incubate for 24 hr in the dark and in
an aerated water bath that kept the aquarium at in situ temperature and with steady
levels of oxygen in the overlying water. To sample the sediment, the aquarium was
laid on a slight incline so that all sediment and pore-water would remain undisturbed
while the front wall of the aquarium was removed. The sediment column was then
cut into cubes following the 1-cm resolution grid that was used for the radiotracer
injections. Samples were then assayed for sulfate reduction rates using the same
protocol as previously mentioned. These rates were then plotted on a 2-D contour
graph and the location of the burrow (as traced on transparencies) was overlaid on
this graph so that direct comparisons between rates and burrow locations could be
made.

RESULTS

Field Sulfate Reduction Rates Measured in Whole Cores: The Catalina Harbor
lagoon is inhabited by large numbers of the bay ghost shrimp N. californiensis. N.
californiensis inhabitants intertidal areas along the west coast of North America from

87
Alaska to Baja California (MacGinitie, 1934) and spends the majority of its life
subsurface, constructing its burrow while depositing feeding (MacGinitie, 1934;
Brenchley, 1981). N. californiensis is known to constantly rework its deep reaching
(as great as 76cm) and highly branching burrow; adding new tunnels, creating new
surface openings, extending burrow depth, or closing off old chambers (MacGinitie,
1934; Swinbanks and Murray, 1981). In Catalina Harbor, the abundance of N.
californiensis increases with distance from shore, sometimes reaching a density of up
to ~1800 burrow openings m
-2
(Bertics and Ziebis, in press). During this field study,
two areas of differing bioturbation intensity, termed ‘low bioturbation’ and ‘high
bioturbation’ with ~ 120 burrow openings m
-2
and ~ 320 burrow openings m
-2

respectively, were chosen for comparison of sulfate reduction rates (SRR) with
depth.

Average sulfate reduction rates (SRR, Figure 4-2 A, B) from the low bioturbation
area were typically lower than the average SRR found at the location with higher
bioturbation. However, the top 7 cm of both areas displayed the highest and the most
variable rates, with the high bioturbation area showing extreme variation in SRR at
the 6 cm depth (2.4 - 140 nmol SO
4
2-
cm
-3
d
-1
). Typically one would expect to see an
increase in SRR with depth into the sediment (Berner, 1980; Jørgensen, 2000), yet at
both areas affected by bioturbation, this typical profile is not seen. This finding may
suggest that bioturbation activity enhances sulfate reduction in the upper layers of

88
the sediment column. These rates were highly variable, possibly indicating the
presence of microniches with enhanced sulfate reduction activity scattered
throughout an individual 1-cm sediment layer. Traditional coring and whole-core
incubation techniques average typically through 1-cm segments and may under
represent macrofauna burrows and associated anoxic microniches.

Figure 4-2 Comparison of sulfate reduction rates (SRR) measured in 1-cm intervals from two
differing field locations, (A) the low bioturbation area and (B) the high bioturbation area.

89

Figure 4-3 Sulfate reduction rates measured from aquaria in different burrow compartments (white
bars) and in different types of sediment surrounding burrows (gray bars). Numbers in parenthesis
state the depth at which those samples were taken. Error bars indicate standard deviation.

Sulfate Reduction Rates in Selected Burrow Compartments: In aquarium setups,
several features associated with N. californiensis’ burrows were identified for
targeted sampling and determination of SRR (Figure 4-1 B). These compartments
consisted of the burrow opening to the surface, round chambers where the shrimp
could easily turn around, and burrow shafts or the narrow vertical portions of the
burrow that led from shallow chambers to the burrow openings. In addition to these
burrow features, several areas from the surrounding sediment were also selected,
namely brown ambient sediment that did not appear to be directly associated with the

90
burrow and black sediment niches that were found in association with the burrow.
SRR measured in sediments collected from the aquaria ranged from 0 to 42 nmol
SO
4
2-
cm
-3
d
-1
(Figure 4-3). The area that displayed the highest rates (25.2 ± 23.7
nmol SO
4
2-
cm
-3
d
-1
) was a black sediment niche that was found next to a burrow
chamber, indicating the burrow may have supplied large amounts of organic matter
to the surrounding sediment, thus enhancing sulfate reduction. This black niche was
located at the same depth (6 cm) as the sediments that showed the highest SRR in the
high bioturbation field location. Furthermore, this black sediment niche area, like
most of the others with the exception of a chamber located 2 cm deep and ambient
sediment located 3 cm deep, showed a high degree of variation between replicates.
This variation suggests that despite the appearance of homogeneity within a burrow
compartment, each burrow feature is heterogeneous within itself.

In non-bioturbated sediments, SRR typically increase with depth (Berner, 1980;
Jørgensen, 2000), however, in our study there appeared to be no correlation between
depth and SRR, regardless of burrow compartment type. Instead, SRR seemed to be
tied to the type of burrow feature the samples were taken from. The black sediment
niche displayed highest rates while the burrow shaft (a vertical portion of the burrow
that led from a shallow chamber to the burrow opening) displayed the lowest rates
(0.44 ± 0.62 SO
4
2-
cm
-3
d
-1
). This lack of activity may be due to the fact that the
shrimp does not reside in this portion of the burrow but instead uses these shafts to

91
facilitate the flushing of its burrow by producing a ventilation current (MacGinitie,
1934). Water is pumped up to the surface through a thin shaft and the smaller
diameter of this ejection shaft compared to the rest of the burrow induces an
accelerated outflow (Bernoulli effect). The outflow is compensated by an inflow of
water at the second burrow opening, providing not only oxygen but also particulate
organic matter suspended in the bottom current which is transported deeper into the
burrow, potentially enhancing aerobic and anaerobic remineralization processes.
Figure 4-4 A contour plot of sulfate reduction rates measured within an aquarium inhabited with a
single ghost shrimp. The smaller figure in the upper right shows the area of the aquarium that was
sampled and the lower right figure shows those portions of the burrow (in dark gray) that were visible
at the aquarium wall at the time of sampling.

92

Whole-Aquarium Incubation Approach for a 2 –D Mapping of Sulfate Reduction: To
determine the distribution of sulfate-reducing microenvironments in relation to N.
californiensis burrows, a narrow aquarium (40 cm tall x 30 cm wide x 3 cm deep)
was injected with radiolabeled sulfate in a 1 cm vertical and horizontal resolution
grid. Through 300 silicone filled ports,
35
S sulfate was injected and the whole
aquarium containing an actively burrowing shrimp was incubated. Sulfate reduction
was subsequently mapped in 2 dimensions and 1-cm resolution. To avoid edge
effects, we only present here the center portion (20 cm tall x 15 cm wide). The
burrow structure was documented by tracing visible portions of the burrow onto
transparences that were placed against the aquarium. Those visible portions of the
burrow, for the most part, ran parallel to the sediment surface at multiple depths and
the sediment surrounding these portions were light in color, suggesting that the
sediment was oxidized. SRR in specific zones associated with the burrow showed
clear formation of sulfate reduction microniches throughout the entire system (Figure
4-4). The highest SRR activity (790 nmol SO
4
2-
cm
-3
d
-1
) was found several cm
below a visible portion of the burrow and exhibited SRR three orders of magnitude
higher than that found throughout the rest of the aquarium. Another area of high
activity (~ 140 nmol SO
4
2-
cm
-3
d
-1
) appeared directly below the chamber connected
to the burrow opening to the surface. Interestingly, the maximum SRR found at 6 cm
in the high bioturbation area (Figure 4-2 B) was the same high rate (140 nmol SO
4
2-

93
cm
-3
d
-1
)

measured at 5 cm depth within the aquarium below the burrow entrance
(Figure 4-4). This result may further confirm that sulfate-reducing microniches may
often occur in the upper few cm below burrow entrances.

Figure 4-5 A closer view of the middle portion of contour plot shown in Figure 4-4. The smaller
figure in the upper right shows the area of the aquarium that was sampled and the lower right figure
shows those portions of the burrow (in dark gray) that were visible at the aquarium wall at the time of
sampling.

94
While the center portion of the burrow system appeared to be low in activity, closer
inspection (Figure 4-5) revealed distinct sulfate-reducing microniches on the order of
12 – 50 nmol SO
4
2-
cm
-3
d
-1
in and around the burrow. These rates are in the same
order of magnitude as the average SRR seen in the whole-core measurements (Figure
4-2 A, B). In total, the aquarium displayed an intricate distribution pattern of sulfate-
reducing microniches throughout the entire sediment column, which supported the
maximum rates of the one-dimensional profiles measured in whole-core incubations
from the field.

Sample Location Avg. Integrated SRR
(nmol SO
4
2-
cm
-2
d
-1
)
Max. Integrated SRR
(nmol SO
4
2-
cm
-2
d
-1
)
Min. Integrated SRR
(nmol SO
4
2-
cm
-2
d
-1
)
High Bioturbation 363.3 580.2 146.6
Low Bioturbation 177.5 324.2 67.1
Aquarium 175.8 807.6 0

Table 4-1 Table of integrated sulfate reduction rates (SRR) from each location studied. Integrations
were done by adding up all of the SRR for the given sample location down to a depth of 11cm.
Maximum and minimum integrated rates were calculated from the highest and lowest rates,
respectively, found at each depth from a given sample location.

Comparison of Field and Aquarium Sulfate Reduction Rates: When integrating the
average SRR at each location down to a depth of 11cm (Table 4-1), the average
integrated SRR fell into two groups. The high bioturbation area had a high average
integrated SRR and the low bioturbation area and the aquarium had average
integrated SRR roughly half of those seen in the high bioturbation area. This finding
is not surprising because abundances of shrimp are much higher in the field
compared to our experiments where a single shrimp inhabited the aquarium, making

95
the formation of microniches underrepresented and subsequent SRR underestimated.
The high bioturbation area had the highest average integrated SRR, supporting the
hypothesis that bioturbation increases sulfate reduction rates, probably due to an
increase of organic matter availability, microniche formation, and coupled
oxidation/reduction activity.

DISCUSSION

Sediment Heterogeneity: The idea that sediments, and in particular bioturbated
sediments, exhibit varying degrees of heterogeneity is not a new concept, with some
of the earliest studies occurring in the 1800’s (ex. Buckland, 1835; Buchanan, 1890).
However, with the advancement of technology that allows researchers to sample at a
finer resolution comes a corresponding demand to reevaluate these benthic systems
for the importance of microniches in benthic biogeochemical processes (Stockdale et
al., 2009). Sulfate reduction microniches within oxidized regions of the sediment
have been suggested as important places for the formation of pyrite in oxidized
sediments (Emery and Rittenberg, 1952) and sulfate reduction microniches can form
around detrital particles as small as 50 – 200 µm in diameter in oxidized portions of
the sediment, including the top 1 cm of the sediment column (Jørgensen, 1977a).
Complex sediment heterogeneity also has been seen when looking at pH and O
2

distributions within bioturbated sediment systems (Wenzhöfer and Glud, 2004; Zhu

96
et al., 2006; Zorn et al., 2006), supporting the idea that burrowing organisms often
impact micro-scale distributions of microbial activities. In this experiment, as
bioturbation intensity was increased, the amount of variation seen in SRR also
increased (Figure 4-2). Large variations in SRR were also seen when looking
directly at specific burrow features, including the burrow opening, several burrow
chambers, and the black sediment niche (Figure 4-3). Because these features
generally cover several cm of surface area and because sulfate-reducing microniches
have been found on as small as 50 - 200 µm in diameter (Jørgensen, 1977a) and oxic
microniches have been found ranging from 0.5 – 2 mm in diameter (Fenchel, 1996a),
it is possible for several of these anoxic and oxic microniches to occur within the
same burrow structure, causing this high degree of SRR variability. These findings
support the notion that the sampling resolution of bioturbated sediemnts needs to be
reduced in order to detect the distribution of small microniches in and around burrow
systems.

Sulfate Reduction in Bioturbated Sediments: Previous studies have shown that
burrowing activity can produce oxic and oxidized microenvironments throughout the
sediment column (Fenchel, 1996a, b; Nielsen et al., 2004; Zorn et al., 2006).
However, next to oxic respiration, sulfate reduction is the most important process
accounting for a large portion of benthic organic carbon remineralization, especially
in organic rich coastal sediments (Jørgensen, 1982; Canfield, 1989). As organisms

97
move through the sediment, particles are rapidly transported between oxic and
anoxic conditions, causing redox oscillations that disrupt the local redox succession
with depth (Jumars et al., 1990; Aller, 1994) and frequently result in the formation of
a “halo” surrounding the burrow that displays the same redox sequence typically
seen with increasing depth in the sediment (Aller and Yingst, 1985; Boudreau and
Marinelli, 1994; Zorn et al., 2006). Those redox oscillations produced by
bioturbating organisms are believed to result in faster and more complete
decomposition of organic matter (Sun et al., 1999), as well as producing a unique
animal and microbial community surrounding the bioturbated area (Meyers et al.,
1987; Reichardt, 1989). Our results provide a 2-dimensional documentation of the
formation of ‘hot spots’ characterized by extremely high rates of sulfate reduction
(up to 790 nmol SO
4
2-
cm
-3
d
-1
) associated with the burrow system (Figure 4-4, 4-5).
The increase in biomass associated with burrow formation (Aller and Aller 1986,
Branch and Pringle 1987), the death of organisms in or around these burrows, and/or
the production of fecal pellets by macrofauna inhabiting burrows can provide a
substantial quantity of organic matter for sulfate reduction reactions occurring in the
anoxic portions of the sediment (Goldhaber et al. 1977). Even burrow walls are
often rich in organic matter due to the excretion of a mucus used to stabilize burrow
walls (Aller and Aller, 1986; de Vaugelas and Buscail, 1990), leading to the
development of dense bacterial communities along the wall that use this mucus as a
substrate (Aller and Aller, 1986; Branch and Pringle, 1987; Dworschak, 2001,

98
Papaspyrou et al., 2006). In addition, the irrigation activity of macrofauna can
transport suspended organic material deeper into the burrow to compartments or
chambers where microbial degradation via sulfate reduction can take place.

N. californiensis belongs to the group of deposit-feeding thalassinidean shrimp that
feed on detritus that is deposited on the sediment surface, as well as ‘wall grazing’
where sediment is removed from the burrow wall and a portion of it is ingested
(MacGinitie, 1934; Griffis and Suchanek, 1991). N. californiensis alone can display
extremely high rates of sediment processing, turning over 2700 ml (wet sed) m
-2
d
-1

(Swinbanks and Luternauer, 1987). Because they feed on detritus, N. californiensis
often lives in areas of high detrital deposition, making the upper areas of their
burrow systems generally the mostly fertile in organic matter (MacGinitie, 1934).
Others have also suggested that members of the deposit-feeding thalassinidean
shrimp may ‘garden’ burrow walls to cultivate microbes for later consumption
(Griffis and Suchanek, 1991), an idea that has also been presented for other
bioturbating organisms like Abarenicola pacifica (Hylleberg, 1975). A relative of N.
californiensis, Callianassa trilobata, which produces the same type of burrow
(Griffis and Suchanek, 1991), was shown to create burrow walls that supported a
microbial biomass four times greater than the sediment surface (Dobbs and Guckert,
1988). In addition, N. californiensis occasionally stores seagrass and algae in burrow
chambers if these materials enter the burrow system (Griffis and Chavez, 1988).

99
Because of these trophic modes, N. californiensis’ burrows can be high in organic
matter, allowing for the support of high rates of sulfate reduction in those areas
where organic material collects.

The detrital material that gets funneled into N. californiensis’ burrows most likely
collects in those burrow chambers directly associated the burrow opening. The
possible high organic loading into these chambers due to their direct connection to
the sediment surface, may be responsible for the development of highly active
sulfate-reducing microniches directly below the burrow opening and chamber in the
2-D aquarium SRR plot (Figure 4-4). Previous studies have found that highest
benthic sulfate reduction rates (SRR) are often found directly below the oxic-anoxic
boundary (Jørgensen and Bak, 1991; Minz et al., 1999). These burrow chambers
represent such an oxic-anoxic boundary and, in conjunction with the high levels of
organic carbon, are reasonable areas to expect elevated SRR. The high SRR (140
nmol SO
4
2-
cm
-3
d
-1
) seen within the aquarium (Figure 4-4) is the same rate as was
seen in the high bioturbation field area at roughly the same depth, ± 1 cm (Figure 4-
2), suggesting that same process may also be occurring in the field and not just
within the aquarium setup. In addition, comparing the high and low bioturbation
locations, it is much more probable to sample one of these entrances in the high
bioturbation area through random core sampling than in the low bioturbation area,
which is perhaps why this same peak was not seen in low bioturbation samples.

100

While sulfate reduction is typically considered to be anaerobic (Postgate, 1984),
studies have shown that sulfate reduction can occur in the presence of oxygen
(Canfield and Des Marais, 1991; Marschall et al., 1993). Other studies have also
shown that some sulfate-reducing bacteria (SRB) can withstand periodic oxygen
exposure (Abdollahi and Wimpenny, 1990; Jørgensen and Bak, 1991; Fründ and
Cohen, 1992; Marschall et al., 1993; van Niel and Gottschal, 1998) even if they
cannot perform sulfate reduction under oxic conditions. Because of this capacity to
tolerate oxygen, at least periodically at low levels, it is not surprising to find SRB in
association with macrofaunal burrow systems. It has been shown that A. ornata (a
marine polychaete) burrows can support higher sulfate reduction rates (SRR) than
the surrounding sediment (Aller and Yingst, 1978) and several studies have detected
the presence of SRB within other macrofaunal burrows (Dobbs and Guckert, 1988;
Steward et al., 1996; Matsui et al., 2004). However, the ability for SRB to protect
themselves from O
2
exposure is often energy intensive and can lead to the depletion
of energy reserves (van Niel et al., 1996). Therefore, it is to be expected that the
burrow features with the least amount of sulfate reduction would be the areas that are
constantly subjected to high levels of O
2
, as was seen with the low levels of sulfate
reduction in the burrow shaft of the N. californiensis’ aquarium burrow (Figure 4-3).
As was mentioned earlier, the ghost shrimp uses such shafts to facilitate their
pumping-induced respiratory current (MacGinitie, 1934), bringing in seawater high

101
in O
2
and potentially inhibiting sulfate reduction. Surprisingly, the burrowing
opening, an area that also should be subjected to high levels of O
2
, displayed
moderate levels of sulfate reduction (Figure 4-3). Because the burrow opening is
often shaped like a funnel (see Figure 4-1 B), possiblly to allow for the capture of
falling detritus, it is probable that some of this organic matter gets stuck along the
burrow funnel and supports the development of sulfate-reducing communities at the
burrow opening.

Use of Novel Approaches in the Study of Microniches: Many studies have suggested
that sampling resolution, modeling, and methodology can play a large role in the
ability to detect and predict the occurrence of microenvironments within sediments
(Blomqvist, 1991; Brendel and Luther, 1995; Morse et al., 2003; Zorn et al., 2006;
Stockdale et al., 2009). This lack of ability to detect such microenvironments
stresses the importance in developing novel methods for studying microniches in
marine sediments. In this study, average SRR in the field (Figure 4-2) were the same
as those seen throughout most of the aquarium (Figure 4-4, 4-5). However, the
aquarium also showed zones of extremely high SRR (up to 790 nmol SO
4
2-
cm
-3
d
-1
)
that were not detected in the core samples. These rates, which spanned over several
sampling points, represent SRR higher than those seen in several studies of coastal
sediments (Jørgensen, 1977a, b; Rysgaard et al., 1998; Leloup et al., 2009) and
comparable to those seen in coastal sediments with high organic matter content

102
(Thode-Andersen and Jørgensen, 1989) and at deep-sea methane seeps (Treude et
al., 2003; Omoregie et al., in press). Without the ability to look at the N.
californiensis burrow system in such detail, these extremely high SRR may have
been completely overlooked. Similar errors will persist in whole system
biogeochemical studies until the advent of sampling techniques that allow for the
determination of microbial activities and geochemical processes at a scale relevant to
the microbiology.

Conclusions: Bioturbation by N. californiensis led to the formation of sulfate-
reducing microniches that enhanced integrated SRR of the sediment column. 2-
dimensional and high resolution mapping of biogeochemical processes gave insight
into the factors controlling microbial activity and will help us to understand and
model organic carbon degradation and nutrient cycling in marine sediments.

103
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CHAPTER V: When clams put their foot down: Animal-sediment-microbe
interactions at Pacific methane seeps

ABSTRACT

There is a close relationship between fluid flow regime, sediment and pore-water
geochemistry, biota, and microbial communities in seep environments. Pacific
methane seep environments are generally characterized by two different
microhabitats, thick microbial mat communities of filamentous sulfide-oxidizing
bacteria and clam beds with dense aggregations of vesicomyid clams. These habitats
often occur in close proximity (meters) to one another but are characterized by very
different sedimentary biogeochemical parameters. Microbial mats occur in areas of
intense seepage where methane and sulfide-rich fluids are transported to the
sediment surface. The clams prefer areas of lower sulfide concentrations, however
they have to provide their thiotrophic symbionts with sulfide to gain energy. Clams
bridge the gap between available oxygen in the overlying water and sulfide flux from
below by extending their foot into the sediment. Seep fluids are depleted in sulfate,
which potentially limits sulfate reduction. We hypothesized that the clam
bioturbation activity enhances solute exchange (sulfate transport) across the sediment
water interface and thus increases microbial activity of sulfate reduction coupled to
methane oxidation. During three cruises onboard the R/V Atlantis in 2006 and 2007

111
(AT 15-7, 15-11, and 15-20) we visited three different well studied methane seep
areas: Eel River Basin off Northern California (~ 500 m water depth), Hydrate Ridge
off Oregon (~ 750 m) and Monterey Bay ( ~ 1000 m). Sediment cores in different
microhabitats were collected with the submersible Alvin (AT 15-7 and AT 15-11)
and with the ROV Jason II (AT 15-20). Microprofiles were immediately measured in
the ship’s cold room in intact cores and sediments were further processed for
geochemical and microbiological analyses, as well as for microbial rate
measurements (sulfate reduction, methane oxidation, methanogenesis). On all three
cruises, experiments were set up to directly study the effects of the three different
clams Calyptogena pacifica (Eel River), Calyptogena kilmeri (Hydrate Ridge) and
Calyptogena gigas (Monterey Bay) on geochemical gradients and microbial activity.
Although there are differences between clam species and seep environments, we
could demonstrate that the clams transport sulfate into the sediment, thereby
enhancing microbial sulfate reduction and methane oxidation rates and greatly
influencing the d
13
C values of methane and dissolved inorganic carbon (DIC) on
small spatial scales.

INTRODUCTION

Biogeochemical processes at seeps: Numerous cold seeps have been discovered and
investigated along both active and passive continental margins (Sibuet and Olu,

112
1998). At cold seeps, methane is transported by advective forces to the sediment
surface where microbial communities of methane-oxidizing archaea, in syntrophy
with sulfate-reducing bacteria, facilitate methane consumption, thus potentially
controlling methane efflux from sediments (Boetius et al., 2000; Hinrichs and
Boetius, 2002; Orphan et al., 2001a, b, 2002). In contrast to methane, sulfate is
devoid in the ascending fluid and is supplied to the sediment from the bottom water
to the zone of anaerobic methane oxidation. Because the original cold seeps fluid is
not only depleted in sulfate, but typically also in sulfide, it is the process of sulfate
reduction that provides sulfide for subsequent sulfide-based chemoautotrophic
communities.

Upwelling of methane and sulfide-rich fluids supports characteristic benthic
communities at seep environments of sulfur-oxidizing bacterial mats and abundant
macrofauna species (clams, mussels, tubeworms) harboring thiotrophic or
methanotrophic symbionts. Variations in fluid flow, and thus methane supply and
hydrogen sulfide concentrations, are key factors controlling the occurrence and
community structure of benthic communities (reviewed in Levin et al., 2003). When
gas hydrates are present at seeps, dissolved methane can be expected to reach its
saturation concentration, which is dependent on the local pressure and temperature
conditions, as well as on the fluid composition, e.g. the salinity (Zatsepina and
Buffett, 1997). Methane seeps are localized extreme environments where sulfide

113
concentration can reach maxima of 20 mM and methane concentrations of up to 80
mM (Boetius et al., 2000; Ziebis et al., 2002; Levin et al., 2003; Ziebis and Haese,
2005). As a comparison hydrothermal vent fluids, which are strongly enriched in
sulfide, have maximum sulfide concentrations of typically 1.5 mM – 8 mM and
methane concentrations of < 1mM (Herzig and Hannington, 2000).

The isotopic carbon composition of methane can have a wide range depending on the
degree of mixing from different sources (biogenic/ thermogenic) and the degree of
microbial alteration. A study of the molecular and isotopic characteristics of
hundreds of natural gas samples from a region of the Gulf of Mexico has identified
the presence of biogenic and thermogenic gas, but most samples revealed an
intermediate signature between the two end members (Bissada et al., 2002).
Similarly, 28 fluid samples from seeps of the Eel River Basin revealed a range of
d
13
C CH
4
between –63 and –35‰ (PDB) (C. Paull, pers. comm., cited in Orphan et
al., 2001b), which indicated both mixing of methane from different sources as well
as microbial production and consumption processes (methanogenesis, aerobic and
anaerobic oxidation of methane). Thermogenic methane generated at temperatures
of above ~120 °C as a consequence of thermocatalytic degradation of kerogen
(Tissot and Welte, 1984) has a resulting d
13
C CH
4
-value equal to or more positive
than -55‰ (PDB) (Bernard et al., 1977). In contrast, non-thermophilic archaea are
restricted to temperatures below 60˚C and biogenic methane typically has

114
distinctively lighter d
13
C CH
4
values between -90 and -55 ‰ (PDB) (Bernard et al.,
1977). Therefore thermogenic and biogenic methane-derived carbon is distinctively
depleted in
13
C.

During the anaerobic oxidation of methane (AOM), which is proposed as a process
of ‘reverse methanogensis’ carried out by archaea in consortia with sulfate-reducing
bacteria (Zehnder and Brock, 1979; Boetius et al., 2000), methane is converted to
CO
2
(Overall reaction AOM/SR: CH
4
+ SO
4

2-
=> HCO
3
-
+ HS
-
+ H
2
O). This process
leads to an enrichment of the lighter isotopes in the resulting bicarbonate and to a
heavier isotopic composition in the residual methane. The overall reaction of
AOM/SR leads to an increase in alkalinity and the precipitation of authigenic
carbonate. Anomalously light carbon isotopic composition of preserved carbonate
suggests it is methane-derived (e.g. Stakes et al., 1999; Aloisi et al., 2002). Negative
excursions of the d
13
C of benthic and planktonic foraminiferal skeletons have been
used as a geologic recorder for methane release from hydrate reservoirs, which then
triggered rapid global climate change (Dickens et al., 1995; Hesselbo et al., 2000;
Kennett et al., 2000). High-resolution stable isotopic analyses at seeps are rare and
interpretations are often not so straight-forward. Differences in stable isotopic
carbon composition have been a useful tool in depicting trophic structures of benthic
communities. By stable carbon isotope analyses, it is possible to trace the flow of
methane-derived carbon through anaerobic methane-oxidizing archaea into SRB, as

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well as into aerobic methane-oxidizing bacteria and its incorporation into eukaryotic
biomass through heterotrophy A limited number of quantitative sampling combined
with stable isotope analyses of seep associated macro- and meiofauna clearly
indicated the incorporation of methane derived carbon into benthic biomass (Werne
et al., 2002; Levin and Michener, 2002; Levin et al., 2003). Limited measurements
of sulfate reduction rates at methane seeps have found extremely high activities
(Boetius et al., 2000; Treude et al., 2003) leading to high sulfide fluxes, which are
the basis of the chemosynthetic communities thriving in these locations. The
availability of methane, sulfate, and sulfide in surface sediments is an essential
prerequsite for biological activity at cold seeps, and the supply of these constituents
may control the rate of metabolic activity and biomass accumulation as well as
carbon isotopic compositions.

Microhabitats: Pacific methane seeps are generally characterized by two different
kind of microhabitats: microbial mats of sulfide oxidizing filamentous sulfur bacteria
and clam beds of dense assemblages of vesicomyid clams that harbor sulfide-
oxidizing symbionts. Tubeworm aggregations, which are typical for hydrocarbon
seeps of the Gulf of Mexico, are usually not found at Pacific methane seeps.
Although there are regional differences, sulfide seems to be the key factor structuring
these communities. Extremely high sulfide concentrations are found in areas of

116
highest methane seepage. Where hydrogen sulfide reaches the sediment-water
interface, it fuels chemosynthetic communities of free-living sulfide-oxidizing
bacteria that live at the sediment surface and require sulfide and oxygen
simultaneously (Fig 5-1). Hence, areas of intensive seepage are colonized by thick
mats of filamentous sulfur bacteria (Beggiatoa, Thioploca) that form visible patches
that can range from several centimeters to several meters in diameter. Studies of
fluid flow in different environments (Tryon et al., 1999, 2002; Levin et al., 2003)
tend to indicate that microbial mat environments were generally characterized by a
strong upward flow, whereas in clam bed areas there either was no net flow or an
irregular downward directed flow. In areas of focused seepage, a distinct benthic
community structure often occurs in concentric zones around a central source of
fluid. This has been observed in Monterey Bay (Barry et al., 1996, 1997; Goffredi
and Barry, 2002; Rathburn et al., 2003), and similarly at Hydrate Ridge (Sahling et
al., 2002; Sommer et al., 2002; Treude et al., 2003). Vesicomyid clams generally
occur in areas where sulfide concentrations are lower and do not reach the sediment
surface (Fig 5-1). By extending their foot into the sediment, the clams tend to bridge
the gap between oxygen availability and sulfide availability deeper in the sediment to
supply their symbionts with both to meet their energy demand and provide their host
with nutrients. Sulfide is acquired through their highly vascularized foot, using a
unique extracellular sulfide-binding component (Childress and Fisher, 1986). The

117
clams themselves cannot withstand high sulfide concentrations and need oxygen for
respiration.

Figure 5-1 Simple sketch of clam bed and microbial mat habitats at methane seeps (redrawn from
Ziebis and Haese, 2005).

Microbial mats and clam beds vary not only in sulfide concentrations, but also in the
location and intensity of microbial processes (Fig 5-1). By reaching deeper into the
sediment the clams are not only tapping the local sulfide source but the bioturbation
activity greatly influences the geochemical gradients in the upper sediment layer,
depending on the size of clams and the vertical extension of their foot. Transport of
solutes between the water column and the sediment is most likely enhanced,
impacting microbial, abundances, distribution and activities. The goal of this study
was to examine and compare the biogeochemical properties of the two microhabitats,

118
microbial mats and clam beds, at three different Pacific methane seeps (Eel River,
Hydrate Ridge, Monterey Bay) and specifically investigate the impact of vesicomyid
clams on microbial processes, geochemical gradients and stable isotope
compositions.

MATERIALS AND METHODS

Sampling: During two cruises onboard the R/V Atlantis (AT 15-7, AT 15-11) in July
and September/ October of 2006 sediment cores were collected with the submersible
Alvin at the Hydrate Ridge area of Oregon (~ 34º34.2 N, 124º36.6 W; water depth ~
700 – 800 m) and Eel River Basin off Northern California (~ 40º48.7 N, 124º36.6 W;
water depth 500 – 560 m). In July 2007, we were invited on a short cruise onboard
the Atlanits with the new ROV Jason and obtained sediment cores from the
Monterey Bay seep site ‘Clam Flats’ (~36º44.7N, 122º16.6 W, water depth: ~1000
m). Preliminary data for this study was also obtained in the Eel River area during
two cruises in October 2000 and April 2001 onboard the R/V Thompson using the
ROVs Jason I and Oceanic Explorer, respectively.

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Study sites

Figure 5-2 Map of the investigated Pacific methane seep areas.

EEL RIVER
The Eel River basin is located on the northern California margin (USA) at 525 m off
shore of the mouth of the Eel River (47.1°N 135.7°W). The area is characterized by
dense aggregation of clams (Calyptogena pacifica), which occur in beds of ca. 15 to
100 cm
2
in diameter interspersed within an area of active seepage of ~ 1 km
2
(Levin
et al., 2000, 2003; Gieskes et al., 2005). In 2000 and 2001, the area was also
characterized by a conspicuous microbial mat that spread out ~ 100 m in diameter.

120
Although these microhabitats occurred in close vicinity to one another, they
exhibited fundamental differences in geochemical parameters. Microbial mats were
characterized by an upward flow of seep fluids (Tryon and Brown, 2001) and
extremely high sulfide concentrations (up to 20 mM) reaching the sediment surface
supporting the thick mats of sulfide oxidizing bacteria (Beggiatoa). Clam beds had
lower sulfide concentrations, peaking at 2.5 mM at a depth of ~ 6 – 8 cm, indicating
a local source of sulfide right below the depth that is reached by the activity of the
clams (Levin et al., 2006) (Fig 5-3).

Figure 5-3 Typical sulfide profiles at the two microhabitats at the Eel River seeps.

Methane concentrations were extremely high in microbial mats (up to 10 mM),
indicating the upward flow of methane-rich fluids, whereas methane concentrations
were below 1 mM in clam beds (Fig 5-4) (Ziebis and Haese, 2005). When comparing
the carbon isotopic composition of methane between the two habitats, there was a

121
striking difference of up to 40‰ at ~10 cm sediment depth. The enrichment of the
lighter isotope in the microbial mat and a heavier isotopic composition in the clam
beds suggests AOM at that depth, which leads to a heavier isotopic composition of
the residual methane (Fig 5-4). Methane turnover rates were also highest at that 10
cm in the clam bed, whereas in microbial mats, highest concentrations were found at
the sediment surface (Fig 5-5).

Figure 5-4 Methane concentrations and isotopic compositions in a comparison of the two habitats.

122

Figure 5-5 Rates of anaerobic oxidation of methane in a comparison of the two habitats determined
by radiotracer incubation experiments of whole cores from Eel River Basin (Ziebis and Haese, 2005).

Preliminary investigations in 2000 and 2001 also showed that the two
microenvironments differed greatly in methane concentrations, isotopic composition
and turnover rates. Orphan et al. (2001a) confirmed these results by combining
phylogenetic information and FISH experiments of whole cells looking for the
presence of the ANME-2/Desulfosarcinales consortia (Boetius et al., 2000) at the
Eel River methane seeps. In addition, they suggested that additional groups might be
involved in the process of AOM (Orphan et al., 2001b). When we revisited the exact
same location in 2006, there were only very small patches of microbial mats visible

123
in the same area. We did not succeed in sampling an intact microbial mat habitat
therefore we took sediment cores at different clam bed locations.

HYDRATE RIDGE
Hydrate Ridge on the Cascadia convergent margin is characterized by methane
hydrates situated directly below the seafloor. The dissolution of the hydrates and
upward flow of methane rich fluids induces some of the highest methane oxidation
rates reported for the marine environment (Boetius et al., 2000; Boetius and Suess,
2004). Sulfate reduction rates (SRR) of hydrate-bearing sediments are concurringly
high and extremely elevated compared to the surrounding sediments (Treude et al.,
2003). Sulfide-rich fluids support chemosynthetic communities composed of sulfide-
oxidizing bacteria and vesicomyid clams. Comprehensive studies (Sahling et al.,
2002; Sommer et al., 2002) suggest that the distribution of benthic communities is
mainly related to the sulfide flux and availability (Treude et al., 2003). In sediments
colonized by dense mats of Beggiatoa, sulfide concentrations in the underlying
sediment were as high as 26 mM and hydrogen sulfide reached the sediment–water
interface. The vesicomyid clams (Calyptogena kilmeri) occur in adjacent areas, but
where sulfide concentrations are much lower and built up only below 5 cm sediment
depth. Fluid flow rates can vary over six orders of magnitude between different sites
(Boetius and Suess, 2004). AOM rates and the number of consortia were found to be
highest in sediment layers where the supply of both methane and sulfate was optimal

124
(Treude et al., 2003). During cruises AT 15-7 and AT15-11, we mainly sampled
clam bed and microbial mat environments of the well studied area located on the
southern summit of Hydrate Ridge.

MONTEREY BAY
Chemosynthetic communities associated with fluid seepage at water depths ranging
from 600 to 3,000 m have been documented at several additional locations in the
Monterey Canyon (Barry et al., 1996). At these sites, individual seeps vary in size (5
cm to 0.5 m in diameter) and configuration, but usually include a central barren zone
characterized by high levels of dissolved sulfide and by the absence of vesicomyid
clams (Barry et al., 1997). Mats of sulfide-oxidizing bacteria occur in the center,
where seepage is highest and sulfide reaches the sediment surface. With increasing
distance from the center, the area is populated by different species of clams that
dominate at different distances from the seep source according to their sulfide
tolerances and sulfide-uptake mechanisms (Barry et al., 1996, 1997; Goffredi and
Barry, 2002). The predominant clam species are Calyptogena kilmeri and C.
pacifica. Investigations showed that C. pacifica inhabits areas further away from the
seep source, with lower sulfide levels, whereas C. kilmeri prefers the areas of higher
sulfide levels. Physiological experiments with the two species showed that C.
pacifica is physiologically prepared for the uptake and transport of sulfide;
furthermore, their endosymbionts have a higher sulfide-binding ability as well as an

125
increased sulfide oxidation potential. In contrast, C. kilmeri has a less effective
sulfide-uptake mechanism and therefore needs higher environmental sulfide levels.
Bacterial mats and clam beds vary greatly in geochemical parameters (Barry et al.,
1996; Rathburn et al., 2003), which effect benthic community structures such as
benthic foraminfera (Rathburn et al., 2003). During the cruise (AT 15-20) in 2007
(chief scientist: Anthony Rathburn) we revisited and sampled the ‘Clam Flat’ seep
area (Barry et al., 1996). Surprisingly, during this cruise it was found that these clam
beds were also inhabited by the vesicomyid clam C. gigas. Over the 4 days that the
ROV Jason stayed at the bottom, only clam beds were found but no microbial mats
were visible. Cores were brought up to the ship by means of an elevator and were
processed immediately in the ship’s cold room.

Micoelectrode Studies: Immediately after retrieval, sediment cores were subjected to
microprofiling (µm–mm intervals) of pH, redox-potential, H
2
S and O
2
, as well as H
2
.
The chemical sensors were commercially available (Unisense) Clark-type
amperometric electrodes (Revsbech and Jørgensen, 1986; Revsbech, 1989;
Jeroschewsky et al., 1996). For redox-potential measurements and pH profiles we
used miniaturized redox platinum electrodes (Unisense) and long needle combination
electrodes (Diamond General), which were connected to a high-impedance mV-
meter. The electrodes were driven vertically into the sediment (µm –mm intervals)
by a motorized micromanipulator attached to a heavy stand. Signals were amplified

126
and transformed to mV by a picoammeter (Unisense PA 2000) and data was
collected directly on a computer. The microgradients provide detailed information to
guide sampling for subsequent biological analyses. Chemical microprofiling in intact
sediment cores has been performed on a number of cruises and was essential for the
study of infauna organisms along geochemical gradients (Ziebis et al., 2002; Levin
et al., 2003; Rathburn et al., 2003).

Geochemical Analyses: Pore-water and solid-phase geochemical parameters were
analyzed in sediment cores at 0.5-cm or 1-cm intervals (CH
4
, SO
3
2-
, total N, C).
Pore-water was gained from 0.5 cm or 1 cm sections by using a pore-water pressing
bench. Exactly 1 ml of pore-water was preserved with 1 ml of zinc acetate (2%w/v)
for the analyses of sulfate. Concentrations of methane were analyzed by the
headspace method. 5 cm
3
of sediment were transferred to 25 ml NaOH (2.5% w/w)
in 40 ml serum bottles and aliquots of the headspace were analyzed by gas
chromatography (Shimadzu Model 2014). Hydrocarbon concentrations were
quantified from injection of known standards. In situ CH
4
concentrations were
calculated using the solubility coefficient of methane (Yamamoto et al., 1976). The
remaining sediment was transferred to 20 ml zinc acetate (20%) and stored frozen
until further analyses of sulfate and sulfide was performed using ion chromatography
and spectrophotometry (Cline, 1969), respectively. Total carbon and nitrogen
content, as well as their isotopic composition, were analyzed in the laboratory of R.

127
Lee (School of Biological Sciences, Washington State University). Sediment
samples were dried at 80°C for 24 hr, then milled to a fine powder. 10 mg of dry
material was placed into a tin capsules and combusted in a Costec (Valencia, CA)
elemental analyzer. The resulting N
2
and CO
2
gases were separated by gas
chromatography and admitted into the inlet of a GV Instruments (Manchester, UK)
Isoprime isotope ratio mass spectrometer (IRMS) for determination of
15
N/
14
N and
13
C/
12
C ratios. Typical precision of analyses was ±0.5 ‰for δ
15
N and ±0.2 ‰ for δ

13
C where δ = 1000 x (R
sample
/R
standard
)-1 ‰, where R=
15
N/
14
N, the standard for δ
15
N
is atmospheric nitrogen and Peedee belemnite (PCB) for δ
13
C. Delta values correlate
with
15
N and
13
C content of samples. Higher δ values correspond with higher
15
N and
13
C contents. Egg albumin was used as a working standard.

Microbial rates: Measurements of the anaerobic oxidation of methane (AOM) and
sulfate reduction were performed in whole-core incubation experiments (Jørgensen,
1978) using radioactive tracers.
14
CH
4
and
35
SO
4
, was injected at 1-cm depth
intervals into replicate sub-cores. The cores were incubated at in situ temperatures
for 12 - 24 hr in the dark. Sulfate reduction was analyzed using a cold single step-
distillation with reduced chromium (Fossing and Jørgensen, 1989; Kallmeyer et al.,
2004). Methane oxidation rates were analyzed after Iversen and Blackburn (1981)
and Iversen and Jørgensen (1985) modified after Treude et al. (2003) and Joye et al.
(2004).

128

Bacterial counts: Samples were taken as sub-samples from the 1cm core sections
and exactly 1-cm
3
of each depth interval was transferred into vials filled with 9 ml
formaldehyde (2% in seawater, 0.22 µm filtered) and stored at 4°C for subsequent
staining and bacterial counting according to Meyer-Reil (1983) as modified by
Boetius and Lochte (1996).

Microprofiling, geochemical analyses (sulfate, C and N content and isotopic
compositions), as well as bacterial counts were performed on the exact same cores.
Methane analyses and determination of methane isotopic composition were done on
a parallel core taken directly next to the first core. Whole-core incubations were
performed on sub-cores taken from a second set of two push cores taken in the same
cluster of cores.

Experimental Set-up for Detailed Investigations in Laboratory Aquaria: During the
two cruises on RV Atlantis in 2006 (July and September) to the Eel river and
Hydrate ridge seeps sites and during the short cruise in 2007 to the Monterey Bay
area, we were able to keep clams alive in specialized aquaria in the cold room to
perform detailed measurements and sampling (Fig 5-6). For these laboratory
investigations, we used narrow aquaria that were built of acrylic glass, had the
dimensions 15 cm x 20 cm x 5 cm (H x W x D), and had the front side perferated

129
with holes in a grid of equal distances of 1cm. These holes were filled with aquarium
silicone and served as sampling ports or for the introduction of microsensors. The
narrow aquaria were filled with sieved sediment from each of the sampling sites.
Within each aquarium, we placed 5-7 clams of same length while keeping one
aquarium without clams to serve as a control. 3 parallel aquaria (2 with clams and 1
without) for each site were kept at in-situ temperature and under flowing seawater in
a larger tank in the ship's cold room. After incubating for 1-2 weeks, vertical and
horizontal microprofiles were measured and pore-water samples were extracted to
gain a 2-dimensional distribution pattern of geochemical gradients (contour lines). At
the end of the experiment, small sediment cores (ø 1cm) were taken along the length
of the aquaria and directly below the clams for microbial rate measurements (sulfate
reduction), as well as for chemical and microbiological analyses (see methods
above).
Figure 5-6 Photograph of narrow aquaria inhabited with clams.

130
RESULTS

Sulfide profiles: During the time between first cruises in 2000/2001 and the two
revisits to the Eel River methane seeps in July and September/October in 2006, there
were some drastic changes in the distribution and intensity of microhabitats.
Whereas during our earlier investigations, there was a large area (~ 100 m
2
)
characterized by upward seepage of methane and sulfide-rich fluids and covered by
dense communities of filamentous sulfur bacteria (Beggiatoa), we could hardly find
any microbial mats in 2006. We went back to the same targets, which had been
marked in 2001, but despite intensive searches we only found very small areas that
had filaments and were not characterized by high sulfide concentrations. Fluid flow
had obviously ceased and was no longer supporting extensive microbial mats in this
area. Clam beds (C. pacifica) were still abundant and several different areas were
cored for detailed analyses and experiments. Representative sulfide profiles (Fig 5-7)
were measured in two clam beds that were cored during Alvin dives AD 4210 (July)
and AD 4259 (Sept./Oct.). They supported previous measurements of a distinct
sulfide peak (2-4 mM) between 4 and 6 cm depth. Above this zone, the layer
inhabited by the clams, sulfide concentrations decrease rapidly and the top 2 cm
were devoid of sulfide Deeper into the sediment, sulfide concentrations also
decreased. The average length of the clams was 3-4 cm. One half of the shell length
is usually within the sediment and the foot is extended to a depth of 4-5 cm. This

131
distinct peak indicates a source of sulfide just below the depth that is reached by the
clams. By introducing sulfate rich water deeper into the sediment by the movement
of the foot they might stimulate sulfate reduction, which would otherwise be limited
by the availability of sulfate.

Figure 5-7 Sulfide profiles in cores taken from clam beds in the Eel River Basin.

On the southern summit of Hydrate Ridge clam beds (C. kilmeri), as well as
microbial mats, were still abundant. Extensive mats were present that were
pigmented from white to yellow or orange. Representative profiles (Fig 5-8) are
shown for 2 clam beds sampled during dives AD 4212 and AD 4214, as well as for
two microbial mat habitats, one orange (AD 4213) and a white mat (AD 4216).

132
Sulfide concentrations in clam beds were lower than in the microbial mat and
decreased towards the surface. There was obviously heterogeneity in the two clam
beds, possibly depending on the location where the cores were taken, closer to the
source or at the edge of the seepage. The core taken during dive AD 4212 exhibited a
similar profile to the Eel River sulfide profiles with a peak of ~ 2mM at ~ 4 cm
depth, indicating increased sulfide production at this depth. The clams in this area
(C. kilmeri) were smaller than the C. pacifica with an average shell length of 3 cm.
The sulfide profile measured in the second core (AD 4214) suggested that the core
was taken at the edge of an upward seeping fluids area, with higher sulfide
concentrations from below that gradually decreased towards the sediment surface. In
contrast, the microbial mats (one orange, one white) both had higher sulfide
concentrations (5 mM) that reached the sediment surface supporting the filamentous
sulfur oxidizing bacteria at the sediment-water interface. The linear increase of
sulfide with depth indicates upward advection of the reduced fluids.

133

Figure 5-8 Sulfide profiles from cores taken from clam beds (top) and microbial mats (bottom) found
at Hydrate Ridge.

At the ‘Clam Flats’ seep site in the Monterey Bay area, the clam aggregations
consisted of the large vesicomyid clam C. gigas occurring in localized clusters. The

134
sediment they inhabited was characterized by active outgassing of methane and very
reduced sediment. These larger species were obviously more tolerant to higher
sulfide concentrations and with their large body size (8 – 10 cm shell length) they
protruded ~ 5 cm out of the sediment, gaining access to oxygen in the overlying
water. Their foot extended up to 7 cm or more into the sediment, tapping a rich
sulfide source. High sulfide concentrations in core 52 (4 mM) almost reached the
sediment surface (Fig 5-9). In core 68, sulfide concentrations were comparably lower
(< 1 mM), and the sulfide profile suggests a supply from below and a depletion in the
layer inhabited by the clam. However, there is also a peak of elevated sulfide
concentration at the base of the clam, also suggesting a local source.
Figure 5-9 Sulfide profiles in cores collected at the ‘Clam Flats’ in the Monterey Bay Canyon.

135
Geochemical Data and Cell Abundances: Geochemical data and microbial
abundances for each of the investigated habitats in the Eel River and Hydrate Ridge
areas are shown in Fig 5-10 – 5-12. We were not able to obtain sufficient cores
during the short cruise in Monterey Bay to produce the same data set. Also, the
degassing of the sediment taken at ‘Clam Flats’ probably altered the geochemical
conditions while the cores were being stored in the cold room.

Figure 5-10 Detailed geochemical profiles and microbial abundances from cores collected in clam
beds found in the Eel River Basin. Cores were collected during two different Alvin dives (AD 4210
and AD 4256).

136
In the Eel river clam beds (Fig. 5-10) sulfate concentrations indicated a supply of
sulfate from the overlying water that decreased rapidly with sediment depth and was
not detectable below 9 cm sediment depth. Methane concentrations almost mirrored
those of sulfate concentrations with higher concentrations below 9 cm (0.5 – 1.3
mM) and a rapid decrease towards the surface. The co-occurring decrease of
methane and sulfate in a narrow transition zone (4 – 10 cm) below the surface
suggests a coupling between sulfate reduction and methane oxidation. Sulfide
concentrations (Fig 5-7) were highest at this depth interval and isotopic signatures of
methane and dissolved inorganic carbon supported the supposition that AOM is
taking place at this specific depth. Methane isotopic values were ~ -60 ‰ in the first
clam bed and varied between –42 and –69 ‰ in the second clam bed (AD 4259).
DIC became enriched in the lighter isotope by up to 10 ‰ (-25‰ - -35 ‰) at a
distinct depth (4 – 10 cm) in both cores. This negative excursion in isotopic
composition was reflected by a heavier isotopic signature of methane (AD 4256).
There is a significant change of isotopic carbon composition on a small spatial scale
reflecting microbial alteration processes at distinct sediment depths. Cell numbers
were high throughout the sediment core (15 and 17 cm sediment depth) with a slight
decrease within the top 3 cm. A peak in cell numbers coincided with the peak in the
negative excursion in δ
13
C
DIC
values, (AD 4210). δ
15
N values varied in the first
clam bed (AD 4210) between 5 and 1 ‰ with a trend towards lighter isotopic
composition with depth. Carbon content showed a peak between 6 and 9 cm

137
sediment depth and C/N values values varied between 10 and 15 and increased in the
top 5 cm. In the second clam bed (AD 4256) δ
15
N were fairly constant with depth
around 2 ‰ and C/N ratios showed minor changes varying between 5 and 10.
Carbon content slightly decreased with depth from 1 to 0.5 %.

Figure 5-11 Detailed geochemical profiles and microbial abundances from cores collected in clam
beds found at Hydrate Ridge. Cores were collected during two different Alvin dives (AD 4212 and
AD 4214).

At the Hydrate Ridge clam beds (Fig 5-11) sulfate decreased with depth, but not as
rapidly as at the Eel River sites. Sulfate stayed at values of ~ 10 mM throughout the

138
depth of the cores, potentially indicating sulfate transport from the overlying water
deeper into the sediment. Methane samples were limited for the first clam bed (AD
4212), but values ranged between 1 and 3 mM. The depth profile shows a subsurface
peak at 4 cm sediment depth and a rapid decrease below. In the second clam bed
(AD 4214), methane concentrations were low in the top 4 cm and showed a peak in
concentration at ~ 10 cm depth. At both sites δ
13
C
DIC
values again showed an
enrichment of the lighter isotope by up to 15 ‰ at a specific depth between 6 and 10
cm sediment depth. The limited methane samples for the first clam bed (AD 4210)
indicated a change to a heavier isotopic composition by 8 ‰ at the same depth. Cell
numbers were higher in the top 10 cm and there was an increase in cell numbers with
depth, with a peak in abundances at 7 cm depth in the first clam bed, coinciding with
the zone of methane decrease and negative excursion of δ
13
C
DIC
values. There was
an interesting enrichment in lighter isotopes with depth for δ
15
N values, which
varied between values of ~6 ‰ in the top 1 cm and decreased to values of 1 ‰ and
0 ‰, respectively for the two clam beds. Carbon contents varied around 1 % and
C/N values were between 6 and 10, with little variations throughout the core.

139

Figure 5-12 Detailed geochemical profiles and microbial abundances from cores collected in
microbial mats found at Hydrate Ridge. Cores were collected during two different Alvin dives (AD
4213 and AD 4216).

Geochemical profiles in the Hydrate Ridge mat cores (Fig 5-12) showed decreased
sulfate concentrations of 15 and 20 mM in the top 1 cm and rapidly decreasing
values with depth. At 6 and 8 cm depth, respectively for the two cores, sulfate
concentrations were not detectable. Methane reached the sediment surface and there
was a decrease of methane co-occurring with the decrease in sulfate concentrations
in the top 4 – 5 cm. In both cores, methane concentrations peaked at ~ 5 cm and were

140
decreasing with depth below 5 cm. The decrease of both methane and sulfate in the
top ~ 3 – 4 centimeters was reflected in a change in carbon isotopic composition.
δ
13
C
DIC
values became lighter and δ
13
C
methane
became heavier. Cell numbers were
higher in the top ~ 7 cm with a sub-surface peak around 7 cm for the first core and 3
cm for the second core. δ
15
N values ranged between values of –1 ‰ and 3 ‰.

Figure 5-13 Redox potential measured in aquaria with sediment from Eel River. One aquarium was
inhabited with seven clams (A), while the other aquarium remained uninhabited (B). The white clam-
like objects represent the location of the clams at the time of the measurements.

141
Experiments: During all three cruises we were able to collect live clams for the
experiments in the narrow aquaria and were able to keep the clams alive for several
weeks. In a comparison of sediment without clams and seep sediments inhabited by
the respective Calyptogena species, we compared the effect on the 2-dimensional
variation in redox-potential of the sediment (Fig 5-13 – 5-15). The clams (6 – 7
individuals) added to the sediment for the Eel River (C. pacifica) and Hydrate ridge
(C. kilmeri) experiments (Fig 5-13 and 5-14) quickly established themselves in the
aquaria. Within the time of the experiment (~ 1 week) they created localized reduced
zones within the sediment of negative Eh values of –300 mV in the sediment
surrounding them, while in the control aquarium redox conditions below the surface
stayed relatively unchanged and uniform at values around +/- 100 mV. The reduced
zone was deeper within the sediment for the Eel River sediment (4-5 cm) and the top
2 cm showed a more oxidized condition compared to the control. Whereas in the
Hydrate Ridge experiment, the reduced zone started at 1 cm depth and there was no
oxidation effect compared to the control in the top centimeters. This might be
explained by size and the behavior of the 2 clams. C. pacifica is slightly bigger than
C. kilmeri. Also, C. pacifica was observed to constantly move around at the surface
of the aquarium, whereas C. kilmeri remained more or less in one location.

142

Figure 5-14 Redox potential measured in aquaria with sediment from Hydrate Ridge. One aquarium
was inhabited with six clams (A), while the other aquarium remained uninhabited (B). The white
clam-like objects represent the location of the clams at the time of the measurements.

The sediment collected at ‘Clam Flats’ in Monterey Bay was more reduced (~ -200 -
- 300 mV) than the first two environments (Fig 5-15). We collected relatively small
representatives of C. gigas to add to the aquaria but they were still almost 9 cm in
length, so only one specimen was added to each aquarium. In comparison to the

143
control, the clam induced more zones of more positive redox-potential in the
locations it occupied for a certain time.

Figure 5-15 Redox potential measured in aquaria with sediment from Monterey Bay Canyon. One
aquarium was inhabited with one clam (A), while the other aquarium remained uninhabited (B). The
white clam-like objects represent the location of the clams at the time of the measurements.

Sulfate reduction rates (Fig 5-16) were significantly (p < 0.05) increased in all three
aquaria containing the clams compared to the controls. Rates were higher in the Eel
River setup compared to the Hydrate Ridge experiments and were highest between 2

144
and 4 cm sediment depth. In the Hydrate ridge sediments, rates were only slightly
higher in the aquarium containing the clams compared to the control. Highest values
were measured in the Monterey Bay experiment. A subsurface peak in sulfate
reduction rate of 200 nmol SO
4
2-
cm
-3
d
-1
occurred at 2 cm depth in the clam
aquarium and is 100 fold higher than the rates measured in the Eel River and Hydrate
ridge experiments.

Figure 5-16 Sulfate reduction rates measured in aquaria experiments from each of the three methane
seeps locations. Red lines represent rates measured in aquaria without clams and blue lines represent
rates measured in aquaria with clams.

Sulfide microprofiles were measured along the length of the aquaria to document the
effect on sulfide production in the Eel River and Hydrate ridge experiments (Fig 5-
17). For the Monterey Bay experiments measurements were performed in a similar
manner as the redox measurements were done, to allow a 2-dimensional

145
documentation of sulfide distribution as contour plots (Fig 5-18). Sulfide profiles in
the Eel River clam aquarium increased below a depth of 4 cm whereas they remained
low and constant with depth in the control. In the Hydrate Ridge experiment sulfide
profiles of both the clam aquarium and the control, showed subsurface peaks around
3 cm sediment depth of up to 5 mM. The contour plots generated from the data of the
Monterey Bay experiment showed a localized increase in sulfide concentration of up
to 8 mM close to the clam compared to a more uniform distribution of lower sulfide
concentrations in the control.

Figure 5-17 Sulfide profiles measured in aquaria experiments from Eel River (A) and Hydrate Ridge
(B). Red lines represent rates measured in aquaria without clams and blue lines represent rates
measured in aquaria with clams.

146

Figure 5-18 Sulfide contour plots for the aquaria experiments from Monterey Bay Canyon. One
aquarium was inhabited with one clam (A), while the other aquarium remained uninhabited (B).

DISCUSSION

Vesicomyid Clam Beds: Aggregations of vesicomyid clams are a characteristic
feature of the unique benthic communities at Pacific methane seeps (Barry et al.,
1996, 1997; Levin et al., 2003). Despite their key presence and their obvious
perseverance after the methane seepage has ceased, their interactions with the seep

147
environment, the ‘animal-sediment-microbe’ interactions, are not well studied. In our
investigations, bioturbation by the clams greatly influenced microbial activities and
abundances. We could show that vesicomyid

clams have a major impact on the
geochemical gradients and microbial activities that lead to a shift in isotopic
composition at seeps. Small-scale differences not only influence microbial
communities, but also the isotopic signature of methane and DIC by at least 10 ‰ on
a spatial scale of cm. This finding is important because it will aid in isotopic
signature interpretation of methane seep biota, e.g. foraminifera that are used for the
documentation of recent and passed events of methane seepage (Dickens et al., 1995;
Hesselbo et al., 2000; Kennett et al., 2000).

Significance: Cold seeps found along active and passive continental margins
represent links between geologic processes, geochemical reactions, biogeochemical
processes, and associated microbial communities, as well as benthic community
structure and functions. Vesicomyid clams harboring thiotrophic symbionts live in
association with sites of sulfide seepage. The distribution of specific populations is
aligned closely with the sulfide concentrations, suggesting specific capabilities of
sulfide tolerance for the different vesicomyid species (e.g. Goffredi and Barry,
2002). Shells of vesicomyid clams have served as fossil records for seep activity
along active and passive continental margins. Carbon and oxygen isotope records of
calcareous macro- and microfossils have been used to obtain information on the

148
biology of living and fossil specimens and on the physical environment in which
they lived. Yet, we still lack a clear understanding of the spatial and temporal
variability of these systems.

The clams tap a sulfide source deeper in the sediment by extending their foot to
provide their symbionts with the electron donor for their energy metabolism. This
bioturbation activity has an impact on the geochemical gradients and microniches in
the surrounding sediment and possibly enhances solute exchange across the
sediment-water interface, thus increasing microbial activity. Smaller organisms
living in the sediment, e.g. foraminifera, may be directly affected by these small-
scale environmental factors that differ in geochemical character from the
surrounding sediments. Characterizing the microenvironments using microelectrode
techniques will provide information about the potential small-scale distribution of
microhabitats available to the foraminifera. Equally important for the clustering of
individuals in microenvironments may be the information of increased biomass and
the change in carbon isotopic composition of particulate and dissolved substances
related to the microbial activity. This presented study provides detailed information
on animal-sediment-interactions, ecosystem functioning and biogeochemical
processes at methane seeps, and has direct relevance to microniche formation and
carbon isotopic equilibria found at these extreme environments. This research
reveals how macrofauna organisms influence biogeochemical processes at methane

149
seeps, with relevance not only to geochemical gradient and microbial activity but
also to stable isotope gradients.

150
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CHAPTER VI: Synthesis and conclusions: The impact of bioturbation on benthic
marine ecosystems

INTRODUCTION

Organic matter produced in the photic zone of the ocean ultimately settles out of the
water column to the sea floor where it plays a major part in the formation of
sediments. These sediments are not only a record of paleooceanographic processes,
but also play a major role in the global carbon and nutrient cycles. In a simplified
manner, marine sediments are stratified such that biogeochemical processes are
regulated by the depth sequence of electron acceptors (O
2
> NO
3
-
>MnO > Fe (III) >
SO
4
2-
> CO
2
) (Berner, 1980; Jørgensen, 2000). The accumulated organic matter is
degraded by this sequence of aerobic and anaerobic processes (e.g. aerobic
respiration, sulfate reduction), leading to the remineralization of organic matter and
the recycling of nutrients. The availability of organic matter also allows for the
presence of benthic macrofauna organisms that can structure the seafloor in a 3
dimensional way (Rhoads, 1974; Aller, 1982, 2001; Kristensen, 2000), a process
generally termed ‘bioturbation’. More specifically, these organisms move particles
between oxic and anoxic regions of the sediment (bioturbation) and facilitate the
transport of oxygen rich water deeper into the sediment (bioirrigation).

156
The study of animal-sediment-microbe interactions and the environmental factors
controlling these interactions is of great interest to many benthic ecologists.
Bioturbation, and thus the interactions between macrofauna organisms and microbial
communities, plays a major role in structuring benthic biogeochemical processes.
Macrofaunal burrows often display unique microbial communtities, species-specific
architecture, and varying size and permanence (Griffis and Suchanek 1993, Ziebis et
al. 1996, Dworschak 2001, Papaspyrou et al. 2005), making it difficult to extrapolate
between different organisms and habitats, the impact that bioturbation or
bioirrigation has on the geochemistry and microbial ecology of inhabited sediments.
The aim of the presented work was to study animal-sediment-microbe interactions in
two relatively dissimilar benthic environments, namely coastal lagoons and deep-sea
methane seeps. It is important to study these interactions in a variety of
environments because not only can bioturbation affect the local biogeochemistry, but
also the pre-existing geochemical gradients can potentially influence the macro and
micro community structure of growing populations. Overall, these processes are
important factors controlling nutrient cycling within the sediment and
remineralization of these substances back into the water column, with the potential to
influence nutrient cycling throughout the world ocean.

157
SUMMARY OF RESULTS

Chapter II investigated the impact that two burrowing crustaceans, the ghost shrimp
Neotrypaea californiensis and the fiddler crab Uca crenulata, had on the local
biogeochemical processes of a shallow intertidal lagoon found in Catalina Harbor,
CA. A wide range of interdisciplinary approaches, including oxygen and redox
potential microsensor measurements, detailed pore-water and solid phase
geochemistry analyses, and whole-community fingerprinting (ARISA), were
combined to examine how the change in biogeochemistry associated with
bioturbation directly influences the unique microbial communities that form at the
burrow-sediment interface. Results showed that different burrow types supported
discrete microbial communities and that these communities reflected the burrowing
behavior of the macrofauna organism. The ghost shrimp, which permanently
occupies its deep-reaching, highly branching burrow and constantly supplies its
burrow with oxygen-rich water, created a burrow that supported microbial
communities similar to those found at the sediment surface. In contrast, the fiddler
crab, which often leaves its simple J-shaped burrow to forage and does not actively
ventilate its burrow, constructed a burrow that supported microbial communities
similar to those found in deeper sediment layers. Results indicated that the
availability of key electron acceptors determined the microbial community
composition of these two different burrow systems.

158

Chapter III examined the linkage of sulfate reduction and nitrogen fixation in a
coastal lagoon heavily bioturbated by the ghost shrimp Neotrypaea californiensis.
Acetylene reduction was used to measure nitrogen fixation rates with increasing
depth in three differing bioturbation locations: a non-bioturbated area, a moderate
bioturbation area, and a high bioturbation area. Results showed that nitrogen
fixation is occurring at depths greater than 5 cm, with substantial activity (up to 0.8
mmol N m
-2
d
-1
) occurring in the moderate bioturbation area. Inhibition experiments
using sodium molybdate and genetic analysis of nifH indicated that nitrogen fixation
was primarily linked to sulfate reduction. When nitrogen fixation rates were
integrated from a depth of 1 cm down to 10 cm, it was found that the moderate
bioturbation area displayed rates several orders of magnitude higher than previously
reported rates for un-vegetated estuarine sediments and were comparable to the
highest benthic rates ever recorded. This study showed that nitrogen fixation by
sulfate-reducing bacteria in bioturbated sediments is an important process leading to
significant new nitrogen input into marine sediments, a process that had previously
been overlooked in such systems.

Chapter IV explored the occurrence of sulfate-reducing microniches in sediments
bioturbated by Neotrypaea californiensis. While bioturbation is typically thought of
as increasing the occurrence of oxidized microniches in sediments, it can increase the

159
occurrence of reduced microniches, such as those carrying out sulfate reduction, as
well. To determine the distribution of sulfate-reducing microniches, radiolabeled
sulfate was injected in a 1-cm grid pattern into an aquarium inhabited by a single
ghost shrimp. Results indicated that sulfate reduction rates were up to three orders of
magnitude higher (140 - 790 nmol SO
4
2-
cm
-3
d
-1
) in reduced microniches associated
with the burrow when compared to those rates found in the surrounding sediment.
One of these high sulfate-reducing microniches (140 nmol SO
4
2-
cm
-3
d
-1
) was found
directly below the burrow entrance chamber at a depth of approximately 5-6 cm. In
a bioturbated field location, a similar rate was seen at approximately the same depth.
This result indicated that the burrow entrance chamber might often support the
development of sulfate-reducing microniches, possibly through the funneling of
organic particles settling on the sediment surface into this chamber via the burrow
opening. Chapter IV showed that bioturbation activity directly influences the
distribution of sulfate-reducing microniches and can play a key role in the
remineralization of organic matter in marine sediments.

Chapter V focused on a different benthic system, namely deep-sea methane seeps,
and looked to examine the impact that three vesicomyid clams, Calyptogena
pacifica, C. kilmeri, and C. gigas, have on the biogeochemistry of seep sediments.
Three different seep locations along the western coast of North America, Hydrate
Ridge, Eel River Basin, and Monterey Bay Canyon, were sampled on a series of

160
cruises from 2006-2007. Areas around the seeps with dense microbial mats were
compared to those seep areas that were inhabited by the vesicomyid clams. Push
cores collected at each environment were profiled for O
2
, H
2
S, and pH using
microsensors and then sliced to obtain samples for detailed measurements of
sediment and pore-water parameters, including porosity, total organic carbon, CH
4
,
as well as sampling for analyses of microbial abundance and diversity along vertical
and horizontal geochemical gradients. Parallel cores were used to conduct incubation
experiments to measure rates of sulfate reduction and anaerobic oxidation of
methane. It was found that microbial mats differed from clam beds, not only in their
vertical profiles of geochemical gradients, but also in the microbial community
composition of the sediments. Additional laboratory experiments were conducted to
determine if the bioturbating activity of clams, through the extension of their foot to
tap local sulfide producing sources, plays a role in influencing benthic
biogeochemical processes. It was hypothesized that this activity led to the transport
of overlying water deeper in the sediment, bringing sulfate to areas of enhanced
sulfate reduction and anaerobic oxidation of methane and thus affecting the overall
sediment biogeochemistry. Results indicated that the clams do in fact transport
sulfate deeper into the sediment and impact sulfate reduction rates occurring in
deeper sediment layers. The degree to which vesicomyid clams influence these
sulfate-reducing communities appeared to be dependent on the size of the clam
bioturbating. Overall, the introduction of sulfate into the sediment through the clam

161
bioturbating activity enhanced the microbial activity of sulfate reduction and the
locally produced sulfide could then serve as an electron donor for the clam
thiotrophic symbionts.

SYNTHESIS

The study of bioturbation has come a long way since Darwin proposed it back in the
1800’s (e.g. Darwin, 1882). However, there are still many knowledge gaps,
questions to be answered, and areas to be explored. Large regions of the ocean floor,
along with whole-system benthic biogeochemistry, remain ruefully understudied.
The study of the effects of bioturbation on biogeochemical processes, as well as on
the abundance, diversity, and function of microbial communities, is a rapidly
evolving field as approaches in molecular ecology are advancing and new tools of
detailed in-situ measurements become available. Currently, we know relatively little
about how the mechanisms of different types of bioturbation influence specific
microbial functional groups (Solan and Wigham, 2005). Furthermore, there have
been relatively few studies that have directly examined the microbial communities
that form at the burrow-sediment interface and how these communities are related to
the unique biogeochemistry of the burrows (Kristensen and Kostka, 2005). Much of
our understanding surrounding bioturbation dynamics is still in the beginning phases
of exploration. Only by conducting detailed and quantitative studies of bioturbation

162
behavior using novel tools and modeling systems can we hope to understand the
impact that bioturbation has on the entire world ecosystem (Meysmen et al., 2003,
2006).

Aside from the obvious local implications of bioturbation, bioturbation also has the
ability to affect processes thought to be removed from the sediment. For example,
most phyto- and zooplankton have a dormant benthic stage (Fryxell, 1983; Gyllström
and Hansson, 2004), which allows bioturbation to play a big role in the burial,
survival, and hatching of these organisms. This interaction can be either negative,
through the ingestion of diapausing eggs (Cáceres and Hairston, 1998; Viitasalo et
al., 2007) or positive, by introducing dormant stages to environmental conditions
necessary for hatching and development (Hairston and Kearns, 2002; Gyllström et
al., 2008). Perhaps by studying the interactions between bioturbation, benthic
geochemistry, and plankton dormant benthic stages, we can gain a deeper
understanding for the mechanisms behind phyto- and zooplankton recruitment to the
water column. However, before we can hope to truly understand systems this
complex, we need to gain a better understand of the small-scale impacts that
bioturbation has on sediment processes on a day to day basis.

The work presented in this thesis focused on learning more about the influence
bioturbation has on local biogeochemical processes, especially in terms of microbial

163
activity. Traditionally, bioturbation is thought to increase the oxic and oxidized
regions of the seafloor. It is for this reason that many studies have focused on
exploring the relation between bioturbation and oxic or oxidized microenvironments
(Fenchel, 1996a, b; Glud et al., 1996; Wenzhöfer and Glud, 2004; Zorn et al., 2006;
Stockdale et al., 2009). In chapter II, we saw that bioturbation, especially by
organisms that actively ventilate their burrow such as Neotrypaea californiensis, can
result in the transport of oxidants deeper into the marine sediment, thus affecting the
redox potential of the entire sediment column. The availability of these oxidants
directly impacted the microbial community composition of the entire burrow system
and thus the overall nutrient cycling within Catalina Harbor.

In chapters III – V we saw that bioturbation can also have an important influence on
anaerobic processes occurring in marine sediments. Sulfate reduction rates are
known to be high in areas that experience high organic loading, such as whale falls
(Treude et al., 2009) and microbial mats (Visscher et al., 1992; Dillon et al., 2007).
However, organic matter does not need to be visible to be in high amounts. Coastal
burrows can increase the sediment biomass during formation (Aller and Aller 1986,
Branch and Pringle 1987), by using mucus to stablilize burrow walls (Aller and
Aller, 1986; de Vaugelas and Buscail, 1990), through release of fecal pellets, and
through irrigation techniques that bring in organic particles from the overlying water.
In the studies involving Neotrypaea californiensis, sulfate reduction rates were

164
comparable to those seen in the previously mentioned high organic loading areas,
supporting the idea that bioturbation can cause enhance sulfate reduction rates in
marine sediments. This increase in sulfate reduction does not just impact carbon and
sulfur cycling, but can possibly impact other elemental cycling as well, such as
nitrogen cycling (see chapter III).

It has been suggested that some bioturbating organisms can serve as ‘ecosystem
engineers,’ or organisms that significantly modify the environment in such a way as
to greatly change the availability for resources for other organisms (Jones et al.,
1994; Meysmen et al., 2006). A perfect example of this idea is seen in chapter V
when looking at the interactions of clams, benthic sulfate reducers, and symbiotic
sulfide oxidizers. The clam supplied the sulfate reducers with high levels of sulfate,
the sulfate reducers supplied the symbiotic sulfide oxidizers with sulfide, and the
sulfide oxidizers in turn supplied the clams with nutrition. Moreover, the clams
supplied the sulfide oxidizers with the oxygen needed for the oxidation reaction,
meaning that this overall interaction is not simply a one-way circle. However,
without studies looking at small-scale benthic interactions, such as those involving
the vesicomyid clams or Neotrypaea californiensis, we would not have the slightest
idea of how these ecosystems truly function.

165
Taking into account all of the information provided in this work, there still remain
many unanswered questions. Those future research questions include:
• How do temporal and seasonal variations in bioturbation influence the overall
impact that bioturbation has on sediment biogeochemistry?
• When dealing with an individual bioturbating organism, which is more
important in terms of impact: a large abundance of small organisms or a
small abundance of larger organisms?
• What is the succession of bacteria within a burrow during its formation and
over time since what we usually see is just a snap shot in time?
• Looking specifically at the Chapter V, how would the addition of methane to
the clam experiments, making the aquaria more like in-situ conditions, affect
the overall outcome of the experiment? Would we still see the same results?

166
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Abstract (if available)

Abstract Understanding the interactions between macrofauna organisms and sediment biogeochemistry and microbioloy is crucial in evaluating marine ecosystem functioning. Most of the seafloor is influenced by bioturbation, yet macrofaunal activity and its influences on biogeochemical processes are not well studied. The goal of the presented research was to combine innovative approaches and modern techniques to investigate the interactions of geochemical processes, microbial activities, and macrofauna assemblages in two marine habitats: coastal sediments and deep-sea methane seeps.

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University of Southern California Dissertations and Theses

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Creator Bertics, Victoria Jean (author)

Core Title Investigations of animal-sediment-microbe interactions at two different environments: coastal lagoons and methane seeps

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Biology

Publication Date 10/11/2009

Defense Date 05/27/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag benthic microbial ecology,bioturbation,crustaceans,marine sediments,microbial diversity,microniches,nitrogen fixation,OAI-PMH Harvest,sulfate reduction,vesicomyid clams

Place Name California (states), hydrographic features: Catalina Harbor (geographic subject), hydrographic features: Eel River Basin (geographic subject), hydrographic features: Hydrate Ridge (geographic subject), hydrographic features: Monterey Bay Canyon (geographic subject), Oregon (states)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Ziebis, Wiebke (committee chair), Capone, Douglas G. (committee member), Fuhrman, Jed Alan (committee member), Hammond, Douglas E. (committee member), Michaels, Anthony (committee member)

Creator Email bertics@usc.edu,vbertics@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2657

Unique identifier UC1441909

Identifier etd-Bertics-3294 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-269149 (legacy record id),usctheses-m2657 (legacy record id)

Legacy Identifier etd-Bertics-3294.pdf

Dmrecord 269149

Document Type Dissertation

Rights Bertics, Victoria Jean

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu