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Preservation of gas-related textures in microbialites: Evidence for ancient metabolisms and environments

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Preservation of gas-related textures in microbialites: Evidence for ancient metabolisms and environments

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Content i

Preservation of Gas-Related Textures in Microbialites: Evidence for
Ancient Metabolisms and Environments

by
Dylan T. Wilmeth

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
in
GEOLOGICAL SCIENCES

August 2018

ii

Table of Contents
List Of Figures .............................................................................................................................................. v
List Of Tables & Supplementary Information ............................................................................................. vi
Acknowledgements ..................................................................................... Error! Bookmark not defined.

Introduction ................................................................................................... Error! Bookmark not defined.
Preservation Of Fenestral Cones Within Cambrian Oncoids .................................................................... 1
Examining Oxygenic Photosynthesis Within Neoarchean Lacustrine Stromatolites Of The Ventersdorp
Supergroup ................................................................................................................................................ 2
Rates Of Microbial Mat Growth And Lithification Within A Modern Microbial Mat ............................. 4
References ................................................................................................................................................. 6

Chapter 1: Punctuated Growth Of Microbial Cones Within Early Cambrian Oncoids, Bayan Gol
Formation, Western Mongolia ................................................................................................................. 12
Abstract ................................................................................................................................................... 12
Introduction ............................................................................................................................................. 13
Geological Setting ................................................................................................................................... 15
Methods .................................................................................................................................................. 16
Results ..................................................................................................................................................... 16
Oncoid Morphology ............................................................................................................................ 16
Discussion ............................................................................................................................................... 19
Laminae............................................................................................................................................... 19
Fenestrae ............................................................................................................................................. 21
Processes Of Oncoid Formation ......................................................................................................... 23
Conclusions ............................................................................................................................................. 25
Acknowledgements ................................................................................................................................. 26
References ............................................................................................................................................... 27
Figures .................................................................................................................................................... 34
Supplementary Information .................................................................................................................... 40

Chapter 2: Neoarchean (2.7 Ga) Lacustrine Stromatolite Deposits In The Hartbeesfontein Basin,
Ventersdorp Supergroup, South Africa: Implications For Oxygen Oases .......................................... 59
Abstract ................................................................................................................................................... 59
Introduction ............................................................................................................................................. 60
Geologic Setting...................................................................................................................................... 61
iii

Depositional Setting And Location ..................................................................................................... 61
Methods .................................................................................................................................................. 64
Results ..................................................................................................................................................... 64
Sedimentology & Field Observations ................................................................................................. 64
Stromatolite Petrography .................................................................................................................... 68
Discussion ............................................................................................................................................... 72
Depositional Setting ............................................................................................................................ 72
Stromatolite Biogenicity ..................................................................................................................... 74
Origin And Significance Of Rounded Fenestrae In The Hartbeesfontein Stromatolites .................... 75
Conclusions ............................................................................................................................................. 78
Acknowledgements ................................................................................................................................. 79
References ............................................................................................................................................... 79
Figures .................................................................................................................................................... 90
Supplementary Information .................................................................................................................. 101

Chapter 3: Rapid Rates Of Oxygenic Photosynthesis Within Neoarchean Stromatolites ............... 125
Abstract ................................................................................................................................................. 125
Introduction ........................................................................................................................................... 126
Yellowstone Stromatolites: Modern Analogues For Bubble Formation And Lithification .............. 128
Preservation Of Bubble Fenestrae Within 2.7 Ga Stromatolites, South Africa ................................ 130
Methods ................................................................................................................................................ 132
Estimating Gas Production Within Archean Microbial Mats ........................................................... 132
Gas Production Rates In Modern Microbial Mats ............................................................................ 133
Time Required to Produce Fenestrae ................................................................................................ 134
Geochemical Analysis of Stromatolites ............................................................................................ 135
Results ................................................................................................................................................... 136
Discussion ............................................................................................................................................. 137
Potential For Modern Metabolisms To Produce Hartbeesfontein Textures ...................................... 137
Acknowledgements ............................................................................................................................... 141
References ............................................................................................................................................. 141
Figures .................................................................................................................................................. 150
Tables And Supplementary Information ............................................................................................... 154

iv

Chapter 4: Environmental And Biological Influences On Carbonate Precipitation Within Hot
Spring Microbial Mats In Little Hot Creek, CA .................................................................................. 166
Abstract ................................................................................................................................................. 166
Introduction ........................................................................................................................................... 167
Site Description And Previous Work .................................................................................................... 170
Methods ................................................................................................................................................ 171
Field Geochemical Analysis ............................................................................................................. 171
Mat Extraction And Description ....................................................................................................... 172
Incubation Experiments .................................................................................................................... 173
DNA And Community Composition Analysis ................................................................................. 176
Results ................................................................................................................................................... 178
Environmental Characterization ....................................................................................................... 178
A Layered Mat With Extensive Carbonate Precipitation .................................................................. 178
Production Rates Of Organic And Inorganic Carbon ....................................................................... 179
Mat Communities With Depth .......................................................................................................... 181
Discussion ............................................................................................................................................. 182
Correlating Carbon Fixation With Carbonate Precipitation Within Mat Layers .............................. 182
Precipitation Rates Compared With Microbialite Formation ........................................................... 184
Acknowledgments ................................................................................................................................. 187
References ............................................................................................................................................. 188
Figures: ................................................................................................................................................. 199
Tables: ................................................................................................................................................... 207
Supplementary Information: ................................................................................................................. 208

v

List of Figures
Chapter 1 ................................................................................................................................................... 34
Figure 1: Location of Salaany Gol within Mongolia and stratigraphic column of oncolite beds ......... 34
Figure 2: Field photos of oncoid beds. ................................................................................................. 35
Figure 3: Petrographic details of oncoidal laminae. ............................................................................. 36
Figure 4: Cross-sections through oncoids, highlighting bubble fenestrae and conical growth.. .......... 37
Figure 5: Petrographic details of fenestrae.. ......................................................................................... 38
Figure 6: Simplified growth pattern within a conical oncoid. .............................................................. 39
Chapter 2 ................................................................................................................................................... 90
Figure 1: Generalized stratigraphy of the Ventersdorp Supergroup, geological map and locations of
stratigraphic sections in Hartbeesfontein Basin. .................................................................................... 90
Figure 2: Measured stratigraphic sections within the Hartbeesfontein Basin. ....................................... 91
Figure 3: Stromatolitic cherts. ................................................................................................................ 92
Figure 4: Stromatolitic dolomites .......................................................................................................... 93
Figure 5: Non-stromatolitic sedimentary features.. ................................................................................ 94
Figure 6: Petrographic textures in stromatolitic cherts. ......................................................................... 95
Figure 7: Fenestral textures within stromatolitic cherts.. ....................................................................... 96
Figure 8: Filamentous textures in stromatolitic cherts.. ......................................................................... 97
Figure 9: Raman Spectroscopy of fenestrally-associated opaque minerals. .......................................... 98
Figure 10: Petrographic textures of stromatolitic dolomites.. ................................................................ 99
Figure 11: Gas-related textures within stromatolites. .......................................................................... 100
Chapter 3 ................................................................................................................................................. 150
Figure 1: Laminae and filaments in Yellowstone and Hartbeesfontein stromatolites. ........................ 150
Figure 2: Fenestral textures within Yellowstone and Hartbeesfontein stromatolites. .......................... 151
Figure 3: Morphological evidence of coalescing gas bubbles within microbial mats.. ....................... 152
Figure 4: A survey of gas production rates within modern microbial mats. ........................................ 153
Chapter 4 ................................................................................................................................................. 199
Figure 1: Location of Little Hot Creek (LHC) microbial mats. ........................................................... 199
Figure 2: Structure of LHC microbial mats. ........................................................................................ 200
Figure 3: SEM images of carbonate precipitates. ................................................................................ 201
Figure 4: Percent organic and inorganic carbon of mat layers. ............................................................ 202
Figure 5: δ
13
C of organic carbon of mat layers after incubation experiments. .................................... 203
Figure 6: δ
13
C of inorganic carbon of mat layers after incubation experiments.. ................................ 204
Figure 7: Rates of calcium carbonate precipitation and autotrophic organic carbon production. ........ 205
Figure 8: Major orders in LHC mat layers…….......………………………………………………….206
vi

List of Tables & Supplementary Information
Chapter 1 ................................................................................................................................................... 40
Supplementary Appendix: Oncoid Photomosaics ....................................................................................... 40

Chapter 2 ................................................................................................................................................. 101
Supplementary Appendix: Stromatolite Photomosaics ............................................................................. 101

Chapter 3 ................................................................................................................................................. 154
Table 1: Comparison of surveyed metabolic rates and limiting reactants for different gas-producing
metabolisms .............................................................................................................................................. 154
Supplementary Table 1: Compiled gas production rates measured in situ within microbial mats: .......... 154
Supplementary Table 1 References........................................................................................................... 157
Supplementary Table 2: Calculated aqueous gas saturation concentrations in Archean environments: .. 162
Supplementary Table 2 References........................................................................................................... 162
Supplementary Table 3: Time for microbial metabolisms to saturate Archean lakes with gas ................ 164
Supplementary 4: Raman spectra of fenestrally-associated opaque minerals ........................................... 165

Chapter 4 ................................................................................................................................................. 207
Table 1: Field measurements of stream water and pore water at LHC taken in June 2015 ...................... 207
Supplementary Information: Production Rates of Carbonate and Organic Carbon by Volume: .............. 208
Supplementary Table 1: Percent organic and inorganic carbon in all mat layers. .................................... 209
Supplementary Table 2: δ
13
C values for inorganic and organic carbon during incubation experiments. . 210
Supplementary Table 3: Differences in organic and inorganic carbon triplicate averages. ...................... 214

vii

ACKNOWLEDGMENTS
Few students could dream of a more positive, encouraging group of mentors and peers
than the professors, students and staff in USC Earth Sciences. I could not have finished this
degree without their scientific and emotional guidance, as well as the support I received from
collaborators, friends, and family outside of USC. The amount of gratitude I feel would itself
comprise a sizeable thesis, but the condensed lines below are no less heartfelt.
First and foremost, I thank Frank Corsetti for mentoring me these last five years. Frank
has been and will continue to be one of the most important people in my life, giving me patience
and encouragement, even at times when I had none for myself. Through lesson and through
example, Frank has shown me how to be a good scientist, a good science communicator, a good
mentor, and most importantly, a good human being. Thank you for everything.
I thank Will Berelson for raising me up from a student skittish about geochemistry to a
researcher who knows that omega matters, and for excellent music conversations. I thank Dave
Bottjer for five years of traversing the dry landscapes of the Southwest and the culinary horizons
of Los Angeles, as well as building my appreciation for a well-placed pun. I thank them and the
rest of my dissertation and qualifying committees- Josh West, Naomi Levine, and Dave Caron-
for ensuring that my research was as rigorous as possible.
I thank my field assistants for trekking with me across three countries - Vicky Petryshyn
and Alyssa Bell in South Africa, Carlie Pietsch in Nova Scotia, and Liz Petsios in Virginia. I also
thank the landowners who gave permission to collect samples, particularly the Venter Family of
Hartbeesfontein, who were essential in finding both local contacts and outcrops.
I thank my co-authors for guidance in the laboratory and field, and for countless
revisions. I thank Stephen Dornbos for preparing me as an undergraduate for the larger scientific
world, Nicolas Beukes for opening the door into Hartbeesfontein, and Stanley Awramik for
giving me Tumbiana samples as well as Frank Corsetti himself. I thank the leaders and students
of the International Geobiology Course 2015 for teaching me how to roll with it- in particular,
my co-authors John Spear, Hope Johnson, Bradley Stevenson, and Blake Stamps.
I thank the Corsetti Lab throughout my time at USC, my academic brothers and sisters
who made the lab brighter- Olivia Piazza, Scott Perl, Reena Joubert, Joyce Yager, and Yadi
Ibarra. I thank the undergraduates I have mentored for teaching me how to be a better advisor
and putting up with my mistakes along the way- Taleen Mahseredjian and Nemanja Bisenic.
I thank Jeff Thompson, my best friend in the department, and in the world. Without him,
the past five years would have been far less fun. With him, that fun will last the rest of my life.
Finally, and most importantly, I thank my family for their love and support over 27 years
of life. Mom, Dad, Cindy, and Grandpa Orlo- thank you all for giving me a curious mind, a song
in my heart, and two feet that won’t stop moving forward, no matter how rocky the road.
“Love is like oxygen: You get too much, you get too high. Not enough, and you’re gonna die.”
-Sweet, “Love Is Like Oxygen”, 1978
1

INTRODUCTION
Distinguishing the specific processes and rates of processes that shape modern and
ancient microbialites remains an enduring challenge within the field of geobiology. Microbialites
are defined as “organosedimentary deposits that accrete as a result of a benthic microbial
community trapping and binding detrital sediment and/or forming the locus of mineral
precipitation” (Burne and Moore, 1987). This definition encompasses a diverse variety of
structures observed in modern and ancient environments, including laminated stromatolites,
oncoid grains, and clastic wrinkle structures, among many others (Kalkowsky, 1908; Pia, 1927;
Logan, 1964; Schieber, 1986). Different forms of microbialites result from various combinations
of biological, chemical, and physical processes which can be challenging to specify in ancient
deposits. Parsing out different factors and rates of formation and preservation can help determine
which microbialite features serve as biogenicity indices, which represent chemical conditions
within microbial mats, and which are governed by physical parameters of the surrounding
environment. This dissertation examines the interactions and rates of biological, chemical and
physical processes within ancient microbialites and modern microbial mats. Chapters 1 through 3
focus on the conditions required for preservation of gas production within microbial mats as
fenestral textures in ancient microbialites, while Chapter 4 compares rates of organic carbon
production and carbonate precipitation within a modern microbial mat.
PRESERVATION OF FENESTRAL CONES WITHIN CAMBRIAN ONCOIDS
Phototropic growth and nutrient limitation within cyanobacterial mats results in the
formation of filamentous cones (Bosak et al., 2009; 2010; Petroff et al., 2010; Sumner et al.,
2011). Oxygenic photosynthesis within cyanobacterial cone tips produces mm-scale bubbles
which remain stable in unagitated environments (Bosak et al., 2009; 2010). Rounded fenestrae
2

within ancient conical stromatolites closely resemble modern cone-associated bubbles, and have
been used as biogenicity indicators as well as morphological evidence for oxygenic
photosynthesis (Bosak et al., 2009). Chapter 1 provides the first description of fenestral cones
within oncoids, occurring within the Cambrian Bayan Gol Formation of Mongolia (~525 Ma)
(Wilmeth et al., 2015). Oncoids are microbialite grains that form in shallow, agitated
environments as biofilms encapsulate and mineralize around a nucleus (Logan, 1964; Peryt,
1981). The presence of fenestral cones within oncoids is unusual, as oncoid formation requires
frequent agitation for biofilms to completely surround grains, while grain motion should
hypothetically perturb bubbles within cones and prevent preservation. Chapter 1 investigates the
rates of metabolic activity, carbonate precipitation, and environmental perturbation required to
form fenestral cones within oncoids. Our results indicate rapid metabolic and precipitation rates
in conjunction with limited grain agitation to produce and preserve observed oncoid
morphologies (Wilmeth et al., 2015).
EXAMINING OXYGENIC PHOTOSYNTHESIS WITHIN NEOARCHEAN
LACUSTRINE STROMATOLITES OF THE VENTERSDORP SUPERGROUP
The evolution of oxygenic photosynthesis dramatically altered Earth’s biosphere,
geosphere, and atmosphere, yet the initial timing of oxygen production remains contested. Two
competing hypotheses exist regarding the origins of oxygenic photosynthesis. One suggests that
the Great Oxidation Event (GOE) ~2.3 Ga represents the rapid oxidation Earth’s atmosphere and
marine environments after oxygenic photosynthesis evolved (Fischer et al., 2016; Ward et al.,
2016; Soo et al., 2017). Alternatively, oxygenic photosynthesis potentially evolved hundreds of
millions of years earlier, but abundant reduced chemical species acted as redox buffers,
preventing atmospheric oxygen accumulation until the GOE (Anbar et al., 2007; Kendall et al.,
3

2010, Crowe et al., 2013; Riding et al., 2014). Stromatolite-rich Archean lakes serve as excellent
locations to test for the presence or absence of oxygenic photosynthesis prior to the GOE.
Smaller, restricted lake basins are candidate “oxygen oases” where oxygen production could
briefly overwhelm limited concentrations of redox buffers (Lalonde & Konhauser, 2015; Sumner
et al., 2015). While stromatolites are often interpreted as shallow, photosynthetic microbial mats,
abiogenic mineralization can produce similar macroscale textures (Grotzinger & Knoll, 1999),
and both biogenic and abiogenic stromatolites can form at various water depths (Frantz et al.,
2014). Therefore, stromatolite biogenicity and depositional environment must be determined
before investigating their merits as Archean oxygen sources. Chapter 2 describes extensive
lacustrine stromatolites within the Neoarchean Ventersdorp Supergroup of South Africa (~2.7
Ga), and investigates biogenicity and depositional environments using petrography and
stratigraphy. Petrographic analysis of Ventersdorp stromatolites reveals well-preserved microbial
textures, such as fenestrae recording the former presence of mm-scale gas bubbles entrained
within filamentous mats. Sedimentary and petrographic evidence, such as detrital grains trapped
within stromatolite laminae, indicate relatively high-energy depositional environments.
Bubble-related fenestrae present within Ventersdorp stromatolites indicate the production
and entrainment of gas within microbial mats, as observed in several modern environments
(Bosak et al., 2009; Mata et al., 2012; Hoehler et al., 2001; Burow et al., 2012). In particular,
stromatolites from Yellowstone National Park form excellent textural analogues, with abundant
fenestrae entrained by mat filaments and accompanied by iron oxides (Berelson et al., 2011;
Mata et al., 2012; Pepe-Ranney et al., 2012). Since modern metabolisms can produce oxygen,
hydrogen sulfide, hydrogen, and methane, among other gases (Revsbech et al., 1983; Canfield &
Des Marais, 1993; Hoehler et al., 2001; Burow et al., 2012), determining specific gases present
4

within ancient microbial mats remains enigmatic. Identifying gas-producing metabolisms within
Archean stromatolites can help constrain our knowledge of early microbial ecosystems, and
potentially provide evidence for oxygen production prior to the GOE. Chapter 3 examines
modern rates of gas production and bubble preservation to determine which microbial
metabolisms were most likely to produce fenestral textures observed within Ventersdorp
stromatolites. Yellowstone stromatolites preserve bubbles as fenestral textures on a quasi-daily
basis. While multiple metabolisms can produce Ventersdorp fenestral volumes within
appropriate preservational windows, oxygenic photosynthesis is orders of magnitude faster than
any other metabolism, and would not have been reactant-limited in Archean environments.
Oxides associated with fenestrae also support the presence of oxygen bubbles over more
reducing gases. Therefore, oxygenic photosynthesis has the highest potential for producing
Hartbeesfontein fenestrae. The model provides petrographic support for the geochemical
hypothesis of oxygen “whiffs” prior to 2.4 Ga.
RATES OF MICROBIAL MAT GROWTH AND LITHIFICATION WITHIN A
MODERN MICROBIAL MAT
Microbialites are found throughout Earth’s history, and microbial mats have been
observed in a diverse array of modern environments. However, relatively few modern mats are
actively precipitating minerals. The scarcity of observed mat mineralization has resulted in
various hypotheses regarding the processes that eventually preserve mats as microbialites.
Investigations of biogenicity determine whether precipitation is induced by microbial
metabolisms (biogenic), or is influenced by aspects of water chemistry independent of microbial
communities (abiogenic) (Trichet & Defarge, 1995; Frankel & Bazylinski, 2003; Dupraz et al.,
2009). Further examination of biogenic precipitation attempts to discern which specific
5

metabolisms induce mat mineralization (Pentecost & Riding, 1986; Visscher et al., 2000, Dupraz
et al., 2004; Michaelis et al., 2002). For example, decreases in TCO2 associated with autotrophy
hypothetically favor precipitation of carbonate minerals, though observed precipitation in mats
suggests an additional dependency on local hydrochemistry and exopolymeric substances
produced by mats (De Philippis et al., 1998; Arp et al., 1999). Layered mats from Little Hot
Creek (LHC), California, contain diverse microbial communities and exhibit nascent carbonate
precipitation, and thus constitute a good test case to investigate the relationship between rates of
mat growth and carbonate precipitation. Chapter 4 describes novel tracer experiments within
LHC mat layers to quantify rates of carbon fixation and calcium carbonate precipitation, as well
microbial communities present in each layer. Comparing precipitation rates between control vs.
poisoned samples allowed for differences in biogenic and abiogenic precipitation to be
determined. Carbon fixation rates were highest in the top layer of the mat, which also contained
the most phototrophs. Conversely, carbonate precipitation within the top layer of living mat
samples was negligible, though abiogenic precipitation was observed. In contrast, the bottom
three layers exhibited biogenic carbonate precipitation. The lack of correlation between rates of
carbon fixation and biogenic carbonate precipitation suggests more significant roles for
processes other than autotrophy to preserve mats as microbialites.

6

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Punctuated growth of microbial cones within early Cambrian oncoids, Bayan Gol
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12

CHAPTER 1:
PUNCTUATED GROWTH OF MICROBIAL CONES WITHIN EARLY CAMBRIAN
ONCOIDS, BAYAN GOL FORMATION, WESTERN MONGOLIA
ABSTRACT
Oxygen bubbles produced during photosynthesis internally deform filamentous
cyanobacterial mats, producing distinctive fenestral patterns. Similar textures preserved in
ancient microbialites are useful biosignatures when filaments are no longer preserved, but have
typically been observed within stromatolites. This study describes bubble-associated fenestrae
within oncoids from the early Cambrian Bayan Gol Formation of Mongolia. Fenestrae appear in
mm-scale micritic laminae which contain dense accumulations of large (10 x 300 μm)
filamentous Girvanella microfossils. Many laminae are not spherical, often with one flat side
opposite a conical peak. Up to six generations of conical geometry are present, with each cone
rotated with respect to the previous peak. We hypothesize that the oncoids experienced
intermittent disturbances followed by periods of stasis and vertical growth. During resting
periods, we hypothesize that flat areas formed the oncoids’ resting base, and the peaked areas the
top. The presence of bubble laminae within peaks implies formation in part via entrapment of
microbially-produced gases. Examples of resting oncoids growing into stromatolites are well
known, as well as irregularly laminated oncoids with no cones; the Bayan Gol Formation
samples are intermediate between typical spherical oncoids and stromatolites. The preservation
of cones also provides evidence for relatively rapid mineralization in the Cambrian ocean, as
antecedent microbial tufts would likely have collapsed if disturbed before calcification.

13

INTRODUCTION
Despite the widespread absence of microfossils in most ancient microbialites, diagnostic
mat-specific fabrics from modern microbial mats have been successfully identified in ancient
stromatolites, and are commonly used as a proxy for microbial involvement. Oxygen bubbles
formed within cyanobacterial mats have been observed to create surficial cones and pillars as the
upward buoyant movement of the bubbles is hindered by a meshwork of filaments (Bosak et al.,
2010; Mackey et al., 2015). Bubbles in cyanobacterial mats can remain stable for months without
disturbance under laboratory conditions (Bosak et al., 2010), and potentially in low-energy
environments such as lake beds (Mackey et al., 2015). A particularly striking example of gas-
related structures was reported from a cyanobacterially-constructed stromatolite found in a hot
spring from Yellowstone National Park (Pepe-Ranney et al., 2012). The Yellowstone examples
contain distinct laminae featuring rounded fenestral pores separated by narrow vertical filament
bundles termed “hourglass structures” by Mata et al., (2012). Individual filaments within the
stromatolites were typically oriented either normal or parallel to laminae instead of vertically,
indicating that phototaxis was not a driver of stromatolite fabric. However, hourglass bundles
were almost always oriented vertically alongside rounded fenestrae, though filaments within
these bundles showed no preferential orientation. The fenestrae were interpreted as oxygen
bubbles that exerted enough upward buoyancy on the surrounding mat to vertically orient
bounding filament bundles (Mata et al., 2012). Though initially described as a passive process
where filaments were pushed aside by bubbles, there is the possibility that cyanobacteria were
able to actively glide over and colonize the same bubbles, as seen in modern mats (Bosak et al.,
2010). In the Yellowstone example, the bubbles were composed of oxygen produced during
photosynthesis, but any buoyant gas bubbles produced by microbial metabolism have the
14

potential to form such structures in a filamentous mat. The location of hourglass-associated and
cone-specific bubbles solely within cyanobacterial mats makes these features excellent
biosignatures for ancient microbialites. Rounded pore spaces have been found concentrated at
the apexes of Archean and Proterozoic conical stromatolites (Bosak et al., 2009; 2013), as well
as hourglass-associated fenestrae along specific laminae (Mata et al., 2012; Knoll et al., 2013).
The distinct morphologies and mat-limited distribution of bubble fenestrae make biological
interpretations feasible even after micritization or diagenetic destruction of filaments (Mata et al.,
2012).
Studies using carbonate mesoscale textures as biosignatures have focused primarily on
stromatolites, revealing both abiogenic and biogenic processes of formation (Grotzinger and
Knoll, 1999). In contrast, oncoids are typically interpreted as biogenic in origin, forming from
the calcification of cyanobacteria, eukaryotic algae or encrusting foraminifera around a nucleus,
forming spheroidal mobile grains that often preserve microfossils of calcifying organisms
(Gradziński et al., 2004; Flügel, 2010). Oncoids that lack microfossils are also interpreted as
biogenic features, either forming through micritic adhesion to sticky exopolymers instead of
rapid calcification (Flügel, 2010), or through diagenesis of oncoids that originally preserved
microfossils (Catalov, 1983). However, the only support for biogenicity of oncoids without
microfossils is their occasional association with oncoids that preserve microfossils (Catalov,
1983). Accurately assessing oncoid formation requires the examination of mesoscale textures
that are preserved despite micritization of microfossils. This study describes conical structures
and hourglass-associated fenestrae within early Cambrian oncoids from the Bayan Gol
Formation of Mongolia, features that have been more commonly described in stromatolites and
modern microbialites.
15

GEOLOGICAL SETTING
The Bayan Gol Formation is an Early Cambrian (Stage 2) mixed carbonate-siliciclastic
deposit located in western Mongolia. Samples were collected from the Salaany Gol Valley of the
Khasagt Khairkhan Range, Gobi-Altai Province (Fig. 1). The valley has been extensively studied
for its continuous sedimentary record across the Precambrian-Cambrian boundary, with
stratigraphy well-constrained through sedimentology, paleontology and geochemistry (Voronin
et al., 1982; Brasier et al., 1996; Khomentovsky & Gibsher, 1996; Lindsay et al., 1996). The
Bayan Gol Formation consists of intercalated carbonate and siliciclastic layers, with a gradual
shift in dominance from limestone to siltstone and sandstone (Khomentovsky & Gibsher, 1996).
The environment is interpreted as a shallow sea between the Siberian craton and the Baydaric
microcontinent with an encroaching shoreline increasing siliciclastic influx over time (Lindsay et
al., 1996).
Oncoids are found near the top of the formation, in a 4.6-meter thick limestone marker
bed (Fig. 1) under- and overlain by extensive siltstone and sandstone units (marker bed R,
Voronin et al., 1982). The matrix surrounding oncoid grains is rich in ooids (Fig. 2B), which
occasionally form cm-thick oolite beds in the absence of oncoids. Oncolite units vary in texture
between ooid-supported to oncoid-supported. The lowermost meter of beds grades upward from
ooid- to oncoid-supported oncolites, overlain by a thin (3 cm) oolite. The oolite is followed by
three meters of grain-supported oncolites, starting as massive beds up to half a meter thick and
thinning upwards, each retaining a consistent texture (Fig. 2A). The top two beds exhibit reverse
grading, topped by a rippled oolite bed to form the uppermost carbonate layer. The oolite bed is
overlain by a meter of cross-bedded, medium to coarse sandstone, which is in turn overlain by
16

siltstone and very fine sandstone. Maximum oncoid size increases upward in section, starting at 3
cm in the lower ooid-supported oncolite beds and eventually reaching up to 7 cm in the massive
oncolite unit.
METHODS
Forty oncoids were thin-sectioned to examine interior mesoscale and microscale textures.
Oncoids were collected from grain-supported oncolite beds 60, 240 and 380 cm above the base
of the Salaany Gol section (Fig. 1). Photomosaics were composed using a Zeiss Imager.M2m
microscope with Axiovision to obtain whole-slide images and better visualize mesoscale
features. White-card technique (Folk, 1959) was used to discern the presence of microfossils and
subtle mesoscale features in thin section.
RESULTS
Oncoid Morphology
Oncoids studied range from 0.9 to 5.6 cm in diameter. Shapes are spherical to sub-
spherical, with length to width ratios between 1.1 and 1.28. External textures of the loose
oncoids are micritic and generally smooth, with minor cm-scale ridges and knobs. The outermost
layer appears as a ~1-2 mm peloid-rich crust, distinct from the outermost laminae of the cortex
(Fig. 3A). Peloids are typically ~100 μm in diameter, and are cemented with equant calcite spar.
All oncoids are composed of calcite, with no signs of conversion from aragonite and no
secondary silicification. Distinct nuclei are not apparent in any sample.
17

Cortices are composed of alternating dark and light gray micritic laminae (Fig. 3B). Dark
laminae are 500 to 1000 μm thick, with individual layers retaining relatively consistent
thicknesses around the oncoids. Most dark laminae can be traced continuously, though some
neighboring layers intersect and merge. Merging laminae show no cross-cutting relationships,
and diverge again into two distinct layers after several millimeters (Fig. 3E). Dark laminae often
preserve abundant Girvanella microfossils, with individual fossils averaging 300 μm long by 10
μm wide (Fig. 3C). Microfossils are usually oriented with long axes of Girvanella parallel to the
laminae. Apart from the layer parallel orientation, filaments typically show no preferential
alignment within laminae. Light gray laminae are generally thicker than darker layers (1000 to
2500 μm), but tend to pinch out when two dark layers intersect and thus exhibit greater
variability in thickness. Girvanella microfossils are less abundant within lighter laminae, but the
lower fossil density often enables longer filaments to be viewed in thin section (Fig. 3D).
Microfossil preservation is not continuous throughout individual light or dark laminae, with
Girvanella often appearing in small mm-scale patches. Quality of microfossil preservation
increases towards the edges of the oncoids, with more well-defined filament edges.
The variable thicknesses of light micritic laminae within Bayan Gol oncoids often creates
asymmetrical morphologies, forming large protrusions that comprise up to half the laminar
circumference (Fig. 4). The major asymmetries in light laminae are hereafter described as
“cones”, though a variety of morphologies are observed, from sub-conical to sub-rectangular.
Smaller protrusions up to 5 mm in height and width, described hereafter as “nodes” are also
commonly seen in dark laminae (Fig. 4), though these irregularities do not greatly impact
oncoidal shape. Cone-opposing hemispheres are generally rounded, but also show “flattening”
normal to cone apices. Light micritic layers directly underneath cone-bearing laminae are
18

relatively thicker at cone apices and thinner on the opposing rim, especially when flattened.
Cones of successive layers often change orientation and point in different directions (sometimes
as much as 180 degrees opposite the previous cones). Changes in cone orientation are less
dramatic with increasing distance away from the nucleus, with increasing uniformity of shape.
One sample (SG1-III, see bottom right of Fig. 4) bore no distinctive cones or nodes, but had
several successive laminae form distinctive rounded, sub-rectangular patterns.
All forty oncoids examined contain extensive fenestral fabrics along various laminae.
Fenestrae are filled with equant calcite spar forming granular and drusy mosaics, occasionally
accompanied with micritic geopetal structures, with no original pore space remaining. Fenestrae
are present in light and dark laminae as well as peloidal crusts, but are most prevalent in thick,
light gray micritic laminae. Many fenestrae are separated into multiple chambers by thin micritic
pillars which usually extend parallel or sub-parallel to oncoidal radii (Figs. 5A-D). Pillars range
between 50 and 200 μm in width, while fenestrae vary between 200 and 2000 μm in diameter.
While some dividing pillars preserve faint Girvanella filaments (Fig. 5E), most consist of non-
fossiliferous micrite. Girvanella within pillars typically align along the lengths of the pillars,
similar to the filament orientation noted in the Yellowstone hot spring stromatolites (e.g., Mata et
al., 2012). Pillar-separated fenestrae show a wide range of morphologies, most exhibiting
rounded edges. Many fenestrae are vertically aligned with respect to oncoidal laminae, forming
series of thin finger-like spaces between pillars. Pillar-related textures do not extend completely
around the circumferences of individual laminae. Instead, the fenestrae are found in isolated
patches or in galleries of up to nine pockets extending up to several mm.
19

Dark Girvanella-rich layers often form upper or lower bounding surfaces for fenestrae,
though some larger voids cut across dark laminae. Laminae crossing is more common in non-
pillar-separated fenestrae. Fenestrae without pillars are often larger than individual fenestrae in
pillar-separated galleries, extending several mm in diameter (Fig. 5F). Margins of large
undivided fenestrae are often irregular and jagged, rarely exhibiting the smooth, rounded edges
seen in pillar-related textures. Smaller undivided fenestrae that do not cross-cut laminae
boundaries have similar margins, and are more commonly observed than their larger
counterparts.
DISCUSSION
Laminae
Individual oncoids show varying levels of microfossil preservation within laminae, with
all samples studied bearing zones of well-preserved Girvanella. When preserved, microfossils
form dense aggregates, with greater densities in darker micritic laminae. Individual laminae with
various states of microfossil preservation do not significantly change in mesoscale fabric
between Girvanella-rich and barren textures. Despite extensive micritization, the preservation of
dense Girvanella populations in multiple layers strongly supports a microbial origin for the
Bayan Gol oncoids. Similar conclusions have been made from other Phanerozoic oncoids with
patterns of micritization and fossil preservation (Stel & de Coo, 1977; Peryt, 1981). Alternation
between dark and light micritic laminae and increased microfossil preservation within dark
laminae is typical of Girvanella¬-rich oncoids (Peryt, 1981). Light micritic laminae are often
described as “barren” regarding microfossils, but Girvanella filaments have been described in
light laminae of well-preserved specimens (Peryt, 1981). Similar light-dark laminae patterns are
20

also known from ancient stromatolites, with varying degrees of filament preservation (Monty,
1976; Awramik & Semikhatov, 1979).
In ancient stromatolites, thick, light micritic laminae have been previously interpreted as
preserving vertical phototaxis during daylight, with intervening thin dark laminae representing
horizontal repose (Golubic, 1973). However, more recent studies indicate that many factors
impact filament density within modern microbial mats, including diffusion limitation of key
nutrients (Petroff et al., 2011; Petroff et al., 2013) and local oxygenation (Sim et al., 2012).
Unlike the strictly vertical phototactic filament orientations hypothesized for light micritic
laminae formation, filaments within thicker, more porous laminae of modern mats exhibit
generally random and non-linear orientations (Sim et al., 2012). In their investigation of more
recent Yellowstone stromatolites, Mata et al., (2012) demonstrated that cyanobacterial filament
orientation was surface normal, rather than vertical, in light layers and more randomly packed in
dark layers, with hourglass fenestrae found in the light layers.
While discontinuous laminae and non-spherical growth patterns are commonly observed
oncoidal features (Logan et al., 1964; Flügel, 2010), the conical structures seen within the Bayan
Gol samples have not previously been described from any oncoids, ancient or modern. In
contrast, conical textures are commonly reported from ancient stromatolites. Conical growth is
interpreted as a macroscale biosignature, as modern cyanobacterial mats often form visible cones
and tufts in field and laboratory conditions (Walter et al., 1976). Along with light-dark laminae
couplets, stromatolite cones have been classically interpreted as evidence for phototaxis (Walter
et al., 1976). However, more recent studies have shown that diffusive gradients of nutrients also
enhance mat growth and carbonate precipitation of microbial cones (Batchelor et al., 2004;
21

Petroff et al., 2013). If phototaxis was a factor in cone formation, filamentous textures should
have vertical alignments, particularly along steeper oncoid margins. However, filaments within
cones show no preferential orientation, vertical or otherwise, suggesting that cone formation was
not due to phototropic growth (e.g., Mata, 2012).
Fenestrae
The pillar-separated fenestrae in Bayan Gol oncoids are similar to textures observed in
modern and ancient stromatolites known as “hourglass-associated” fenestrae (Mata et al., 2012).
Modern textures are formed in a similar fashion to cone-associated bubbles, with oxygen
produced by photosynthesis contorting cyanobacterial filaments around growing bubbles (Bosak
et al., 2009; Mata et al., 2012). These bubbles remain within their original laminae, often
forming galleries of multiple fenestrae along a single layer. Filament bundles caught between
two bubbles are thicker near over- and underlying laminae, pinching out to a few filaments in
between, forming a distinctive hourglass structure. Filaments within bubble-separated bundles
have no preferred alignment, which distinguishes hourglass structures from bundles of
phototactic filaments. Similar hourglass structures are also observed in Proterozoic stromatolites,
composed of micrite instead of microfossils (Mata et al., 2012; Knoll et al., 2013). Frequent
partitioning by thin micritic columns distinguishes hourglass-associated fenestrae from abiotic
fabrics, where fenestrae extend undivided along individual laminae.
Within the Bayan Gol oncoids, fenestrae-dividing pillars rarely exhibit the flared
hourglass shapes observed in modern stromatolites, more commonly appearing as vertical or
curved pillars (Figs. 5A-D). This deviation is also seen in ancient stromatolites, and is likely due
to degradation of filaments before lithification, as well as subsequent diagenesis (Mata et al.,
22

2012). While some oncoidal pillars show the presence of filaments, the majority of fenestrae are
divided by micritic structures similar to those seen in ancient stromatolites. Those filaments that
are preserved within the oncoids do not preferentially show vertical orientations, although this
observation could be affected by relatively poor preservation within pillars. Unlike ancient or
modern stromatolites, pillar-associated fenestrae within oncoids occasionally break through
overlying laminae, forming large pockets in light micritic layers above (Fig 5F). It is possible
that agitation of oncoid grains caused remnant air pockets to partially escape upwards due to
buoyancy before complete lithification. Within modern cyanobacterial mats, increased bubble
size often decreases stability within the mat, forming less spherical, vertically-oriented columns
(Bosak et al., 2010). The irregular, more consistently cross-cutting morphologies of non-divided
fenestrae, as well as their presence in non-Girvanella bearing peloidal crusts and fossil-rich
laminae, likely represent different processes than the distinct pillar-related textures.
Gas-related fenestrae have also been described within cone-forming cyanobacterial
colonies and ancient stromatolites. In modern mats, photosynthesis occurs at higher rates within
cone tips, creating bubbles at these apices through increased oxygen production (Bosak et al.,
2009). It is important to note that modern cone growth occurs before bubble formation, and is
distinct from structures formed through gas buoyancy (Bosak et al., 2010). Bubble fenestrae have
also been described in the cone tips of ancient stromatolites (Bosak et al., 2009), providing
evidence for early photosynthesis. Bubble fenestrae concentrated at cone tips are less common in
the Bayan Gol oncoids than hourglass-related fenestrae, but their presence strengthens the
hypothesis that the oncoids were formed by photosynthetic organisms.

23

Processes of Oncoid Formation
Despite complete bisection through the centers of all samples, none of the oncoids
examined show a distinct nucleus in thin-section, only amorphous micritic masses surrounded by
irregular light and dark laminae. It is possible that these masses represent clasts of microbially-
bound muds, or perhaps rolled-up mats later lithified and modified via diagenesis, though poor
microfossil preservation deeper within the oncoids makes this hypothesis difficult to test.
Dissolution of an original nucleus is unlikely due to the lack of secondary collapse structures in
surrounding laminae.
Oncoids are typically viewed as proxies for higher-energy marine and freshwater settings,
with concentric laminae requiring frequent movement to expose fresh surfaces for precipitation
(Peryt, 1983). More recent surveys of asymmetric growth suggest that oncoids can form in
waters of intermediate energy (Flügel, 2010), and can even develop into stromatolites when
sedentary for longer periods of time (Martin-Algarra and Vera, 1994). The asymmetrical nature
of initial oncoid growth suggests longer periods of rest and unimpeded mat growth in a relatively
low-energy environment, while the prevalence of cyanobacterial microfossils implies
calcification within the photic zone. The depositional environment is interpreted as a shallow
lagoon below fair-weather wave base. A shallow setting is also favored by deposition late within
the Bayan Gol Formation, which is interpreted as increasingly proximal to the paleo-shoreline
with time (Lindsay et al., 1996). After unknown nuclei fell to rest on the seabed, Girvanella mats
encrusted exposed surfaces (Fig. 6). Diffusive gradients at surface irregularities of the mat would
have fostered increased carbonate and biomass accumulation at cone tips. During photosynthesis,
oxygen production created bubbles which became trapped within the mats, contorting
24

surrounding filaments into curved hourglass structures that formed extensive galleries along
specific laminae. Increased photosynthesis at cone tips also fostered bubble formation. After
calcification, storm events would have disturbed the oncoids, tumbling the grains over the seabed
until once again coming to rest. After such disturbances, the cycle of microbial colonization and
precipitation began once again. The increase in concentric laminae towards the oncoids’ outer
edges is potentially due to increased rounding as microbial overgrowth smoothed irregularities
over time. Alternatively, as oncoids increased in mass over time, more time would have been
spent in sedentary phases, and exposed portions of the oncoid would have continued to develop
thicker laminae, while opposing hemispheres consistently precipitated thinner laminae.
The punctuated repetition of mobility and calcification inherent in oncoid formation
provides finer-scale estimates for calcification rates than in sedentary structures such as
stromatolites. While bubble-related cones can remain entrained within mats for several months in
ideal laboratory conditions (Bosak et al., 2010), the active overturning and compression of
microbial colonies during oncoidal motion are not conducive to preserving these delicate features
without relatively rapid mineralization. Timing of calcification can therefore be narrowed down
to the length of intervals between perturbations. Early non-spherical laminae in Bayan Gol
oncoids indicate longer sedentary periods favorable for mat growth and calcification, punctuated
with periodic disturbances by storm activity below fair-weather wave base. Paleomagnetic
studies place deposition of the Bayan Gol Formation between 10° N and 10° S paleolatitude in
the early Cambrian (Wood et al., 1993; Kravchinsky et al., 2010). Modern tropical storm
frequencies range between 6.4 and 27.7 storms per year depending on location (Vitart, 2006). If
similar frequencies existed in the early Cambrian, disturbances could have occurred at rates at
one storm every 15 and 60 days. Thicknesses are variable between cone-shifting laminae,
25

typically ranging from 1.5 to 3.5 mm between bounding dark laminae. Multiplying the number
of tropical storms in 365 days by the average lamina thickness precipitated between storms
provides a potential range of calcification between 9 and 85 mm per year for Bayan Gol oncoids.
Even a conservative estimate of 9 mm accretion per year is higher than calculated rates of 3.1
mm per year for recent oncoids (Garcia-Pichel et al., 2004) and 0.05 to 5 mm per year for
stromatolites and microbialites (Rasmussen et al., 1993; Brady et al., 2009; Petryshyn et al.,
2010; Power et al., 2011). Given that Cambrian CO2 concentrations were substantially higher
than present (Berner, 2006), warmer climates could have increased tropical storm frequency,
which would imply precipitation rates even higher than 9 mm per year.
CONCLUSIONS
While oncoids are commonly interpreted as biogenic features, direct microfossil evidence
is frequently missing. Within oncoids of the early Cambrian Bayan Gol Formation, similar
mesoscale fabrics in both fossil-rich and barren textures, as well as the high density of
Girvanella preserved, strongly implies a biological origin. Mesoscale fabrics include galleries of
rounded fenestrae separated by thin micritic pillars, concentrated within specific laminae. Pillar-
separated fenestrae resemble those seen in modern stromatolites, where oxygen produced
through photosynthesis becomes entrained within the mat, shifting surrounding cyanobacterial
filaments to form thin, flared columns between bubbles. The consistent roundness of the air
pockets and the separation by micritic columns distinguishes bubble-associated pores from
abiotic, laterally extensive fenestrae commonly seen in carbonates. The distinctness of bubble
fenestrae renders the structures useful biosignatures within the Bayan Gol oncoids despite the
26

absence of fossils within many separating pillars, similar to previous studies of ancient
stromatolites.
Several layers within the oncoids deviate from typical sphericity, forming cone-shaped
features generally opposing flat sides. Irregular growth patterns are commonly seen within
oncoids, but distinctly conical growth has only been documented from stromatolites and modern
cyanobacterial mats. The absence of consistently vertically-aligned filaments within cones (sub-
parallel to oncoidal radii), as well as the occasional presence of rounded bubble fenestrae
underneath cones indicates formation through gas entrainment rather than purely phototactic
growth. Cone growth requires time for surface colonization, followed by production and
subsequent entrapment of photosynthetic oxygen. The presence of multiple cones of different
orientations within individual oncoids suggests longer sedentary periods punctuated by storm
events, as opposed to the more consistent agitation which forms spherical oncoids. In addition to
providing useful biosignatures, this periodic exposure of fresh surfaces for mineral growth can
theoretically provide better constraints on local precipitation rates than purely sedentary
structures, though precise values are beyond the scope of the current study.

ACKNOWLEDGEMENTS
The authors would like to thank the Agouron International Geobiology Summer Course
and the NASA Wisconsin Space Consortium for funding this research, as well as two
anonymous reviewers for their insightful commentary.

27

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34

FIGURES: CHAPTER 1

FIGURE 1: Location of Salaany Gol within Mongolia and stratigraphic column of oncolite beds,
with the right-hand margin equivalent to weathering profile. Salaany Gol map modified from
Khomentovsky & Gibsher, 1996.

35

FIGURE 2: Field photos of oncoid beds. A) Matrix-supported oncolite overlying grain-
supported oncolite (hammer lying on boundary), ~ 4 m from base of section. Hammer = 30 cm.
B) Grain-supported oncolite, scale bar in cm. C) Oncoid surrounded by oolitic matrix, scale bar
in cm.
36

FIGURE 3: Petrographic details of oncoidal laminae. All images ppl, C & D with white card
technique (Folk, 1987) for better visualization of microfossils. A) Outer peloidal layer. B)
Alternation between dark laminae (at top and bottom of photograph) and light laminae. C & D)
Girvanella preservation within dark and light laminae, respectively. E) Two dark laminae
merging together from right to left, pinching out a light lamination in between.
37

FIGURE 4: Cross-sections through oncoids, highlighting bubble fenestrae and conical growth.
A & B represent the same oncoid. A) Locations of bubble fenestrae. B-D) Growth patterns based
off of light-dark laminae couplets and hourglass fenestrae. Laminae with bubble fenestrae
present around the entire circumference indicate conical growth into/out of the plane of
dissection, represented by bulls-eye structures.
38

FIGURE 5: Petrographic details of fenestrae. All images ppl with white card technique. A-D)
Bubble-associated fenestrae, defined by rounded edges and division by thin micritic pillars. E)
Girvanella filaments lining bottom of dividing pillar. F) The fenestrae on the right breaks
through a dark laminae that apparently bounds similar fenestrae on the left.

39

FIGURE 6: Simplified growth pattern within a conical oncoid. Motion is portrayed as a 2-
dimensional rolling pattern, while in reality, disturbances would have occurred in all directions.
Grey circles = lithified bubbles.

40

SUPPLEMENTARY APPENDIX: ONCOID PHOTOMOSAICS
Bayan Gol Formation:
Location: Salaany Gol Valley, Mongolia
Age: Cambrian Stage 2 (~525 Ma)
Paleoenvironment: Marine lagoon, below fair-weather wave base
p. Thin Section Label μm Morphology Notes
41 SG1-I 40 Oncoid Hourglass structures, cones, surrounding
oolite
42 SG1-I (detail) Hourglass structures
43 SG1-I (detail) Girvanella
44 SG1-II 40 Oncoid Hourglass structures, cones, surrounding
oolite
45 SG1-II (detail) Hourglass structures
46 SG1-III 40 Oncoid Hourglass structures
47 SG1-III (detail) Hourglass structures
48 SG1-III (detail) Girvanella
49 SG1-IV 40 Oncoid Hourglass structures
50 SG1-IV (detail) Hourglass structures
51 SG14-2a 40 Oncolite Multiple fenestral oncoids with ooid
matrix
52 SG14-2a (detail) Hourglass structures
53 SG14-2b 40 Oncolite Multiple fenestral oncoids with ooid
matrix
54 SG14-2b (detail) Hourglass structures
55 SG14-2c 40 Oncolite Multiple fenestral oncoids with ooid
matrix
56 SG14-2c (detail) Hourglass structures and ooid matrix
57 SG14-3 40 Oncolite Multiple fenestral oncoids with ooid
matrix
58 SG14-3 (detail) Oncoid laminae

Notes: All samples are in cross-sectional view. Arrows adjacent to scale bars indicate up
directions. Individual oncoid samples SG1-I through SG1-IV were collected from float, and
therefore have no up direction indicated.

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

-
58

59

CHAPTER 2:
NEOARCHEAN (2.7 GA) LACUSTRINE STROMATOLITE DEPOSITS IN THE
HARTBEESFONTEIN BASIN, VENTERSDORP SUPERGROUP, SOUTH AFRICA:
IMPLICATIONS FOR OXYGEN OASES
ABSTRACT
The Hartbeesfontein Basin contains the most extensive deposits of Archean lacustrine
stromatolites on the Kaapvaal Craton, with stromatolitic facies occurring over ~100 km
2
in beds
up to 7 m thick. Stromatolitic dolostones and cherts exhibit evidence for biogenicity. Dolomitic
stromatolites have grumelous microspar textures between organic-rich laminae suggestive of
carbonate precipitation within microbial mats. Stromatolitic laminae within chert preserve
detrital material beyond the angle of repose, indicating the presence of microbial mats either
trapping and binding grains or adhesion via extra-polymeric substances. Stromatolitic cherts also
preserve fenestral and filamentous textures. Many fenestrae have rounded shapes surrounded by
filamentous laminae and appear to have formed in situ within stromatolite fabrics prior to
lithification. Fenestrae within stromatolitic chert resemble hourglass structures noted from recent
silica stromatolites and are interpreted to originate from gas bubbles within the stromatolite-
building mat. The preservation of delicate structures (e.g., gas-related fenestrae and filaments)
implies rapid lithification of microbial mats. The mm-scale gas structures preserved in
Hartbeesfontein cherts suggest the presence of metabolisms capable of producing abundant
quantities of gas and conditions that promote lithification before disturbance of gas bubbles
within the mat.

60

INTRODUCTION
Archean lacustrine environments are currently best known from two ~2.7 Ga sedimentary
successions: the Fortescue Group on the Pilbara Craton, Australia (Bolhar & van Kranendonk
2007; Awramik & Buchheim, 2009; Flannery et al., 2016) and the Ventersdorp Supergroup on
the Kaapvaal Craton, South Africa (van der Westhuizen et al., 1991; Altermann and Lenhardt,
2012). Both regions preserve abundant and diverse stromatolite morphologies (de la Hunty,
1963; Winter, 1963; Grobler & Emslie, 1976; Buck, 1980; Walter, 1983; Karpeta, 1989; Buick,
1992 Awramik & Buchheim, 2009) that have been subjected to relatively low-grade, prehnite-
pumpellyite to greenschist facies metamorphic alteration (Smith et al., 1982; Cornell, 1987;
Crow & Condie, 1988). The abundance, diversity, and low metamorphic grade of Archean
lacustrine stromatolites provide unique opportunities to investigate multiple aspects of deep-time
geobiology. In particular, the limited metamorphism of Archean lacustrine sediments increases
the preservation potential of stromatolite fabrics which provide evidence for biogenicity, patterns
of lithification, and microbial mat textures. Archean lakes are ideal candidates to search for
oxygen oases prior to the Great Oxidation Event, where the appropriate microbial metabolisms,
if present, could lead to the oxidation of smaller bodies of water prior to more extensive marine
oxygenation (Lalonde & Konhauser, 2015; Sumner et al., 2015). Additionally, the sedimentology
of lake basins prior to metazoan colonization of non-marine environments provides an analogue
to ancient lacustrine deposits on Mars (Malin & Edgett, 2000; Cabrol & Grin, 2002; Grotzinger
et al., 2014).
The most extensive outcrops of Ventersdorp lacustrine stromatolites occur in the Rietgat
Formation within the Hartbeesfontein Basin, an intracratonic half-graben 150 km west of
61

Johannesburg in South Africa (Fig. 1B). Stromatolites within the basin form laterally-extensive
facies ~100 km
2
in area in beds up to 7 m thick (Karpeta, 1989; 1993). Unlike many Ventersdorp
or Fortescue locations, most Hartbeesfontein stromatolites are preserved entirely as chert, which
has the potential to preserve microfossils and detailed microbial mat textures. Well-preserved
mesoscale textures can also distinguish biogenic stromatolites from abiogenic structures that
share similar macroscale morphologies (Buick et al., 1981; Grotzinger & Knoll, 1999; Riding,
1999; Awramik & Grey, 2005; Bosak et al., 2013). To date, petrography has not been reported
on stromatolites from the Hartbeesfontein Basin, despite the relative abundance and high
preservation potential of stromatolite samples. Here, we present the sedimentology and
stratigraphy of stromatolitic facies in the Rietgat Formation of Hartbeesfontein Basin, as well as
the first detailed petrography of Hartbeesfontein stromatolites, describing exquisitely preserved
microbial mat textures with implications for ancient microbial metabolisms.
GEOLOGIC SETTING
Depositional Setting and Location
The Ventersdorp Supergroup (Fig. 1A) is a Neoarchean volcano-sedimentary succession
within the Kaapvaal Craton of northern South Africa and southern Botswana, deposited between
the underlying Witwatersrand and overlying Transvaal Supergroups between 2.78 to 2.71 Ga
(van der Westhuizen et al., 1991; de Kock et al., 2012). The Ventersdorp Supergroup is
subdivided into the Klipriviersberg, Platberg and Pniel Groups, which are separated from each
other by unconformities (Winter, 1976). The Klipriviersberg Group is comprised of flood basalts
limited to the central Kaapvaal Craton (Winter, 1976; van der Westhuizen et al., 1991). The
Platberg Group consists of chert, carbonate, and clastic sediments, as well as mafic and felsic
62

volcanic deposits (van der Westhuizen et al., 1991). A lacustrine interpretation for Platberg
sediments is supported by deposition in isolated intracratonic grabens within the central
Kaapvaal Craton (Grobler & Emslie, 1976; Buck, 1980; Eriksson et al., 2002), intercalated with
predominantly subaerial volcanic flows (van der Westhuizen et al., 1991; Altermann & Lenhardt,
2012). The overlying Pniel Group consists of Bothaville Formation conglomerates and
Allanridge Formation mafic-intermediate lavas (Clendenin et al., 1988).
Ventersdorp Supergroup stromatolites predominantly occur within the Rietgat Formation
and correlative strata of the Platberg Group (Winter, 1963; Grobler & Emslie et al., 1976; Buck,
1980; Karpeta, 1989; 1993; Altermann & Lenhardt, 2012). Rietgat Formation stromatolites were
first described from a subsurface borehole northwest of Wesselbron, South Africa (Winter,
1963), with subsequent samples examined from nearby Platberg and Pniel boreholes from the
Welkom Goldfield (Buck, 1980). Surficial outcrops of Ventersdorp stromatolites in South Africa
are currently known from the Rietgat Formation of the Hartbeesfontein Basin (Karpeta, 1989;
1993), and the Rietgat-equivalent Omdraaivlei Formation in the T’Kuip Hills (Grobler and
Emslie, 1976; Altermann & Lenhardt, 2012), with a potential third area of exposure in the Mohle
Formation near Taung (Liebenberg, 1977; de Kock et al., 2012). Of the known surface exposures
of Ventersdorp stromatolites, the most extensive occur within the Hartbeesfontein Basin
(Karpeta, 1989).
The Hartbeesfontein Basin is a 400 km
2
half-graben within the North West Province of
South Africa (Fig. 1B). The Platberg Group of the Hartbeesfontein Basin includes the
Kameeldoorns Conglomerate, followed by volcanic deposits of the intermediate-felsic
Makwassie and mafic Rietgat Formations (Winter, 1976). The Rietgat Formation also includes
63

intercalated chert, dolostone, and shale beds at the end of volcanic deposition, including
extensive stromatolitic facies (Karpeta, 1989). Pniel Group deposits of the Bothaville and
Allanridge Formations unconformably overlie Rietgat Formation strata (Karpeta, 1993). The
interpreted tectonic history of the Hartbeesfontein Basin is similar to other Ventersdorp
Supergroup localities (Eriksson et al., 2002). Crustal extension opened the basin through listric
faulting, followed by Kameeldoorns alluvial deposition, Makwassie ashes and Rietgat basalt
flows (Karpeta, 1993, Tinker et al., 2002). As volcanism diminished in the Rietgat Formation,
sediments such as dolomites and cherts began to fill the basin. After basin infill, fluvial systems
deposited Bothaville Formation conglomerates (Karpeta, 1993). Arguments for lacustrine
sedimentation within the Hartbeesfontein Basin include 1) sedimentary facies restricted to an
isolated intracratonic graben within a continental rift, 2) frequent lateral and vertical facies shifts,
indicative of smaller basins influenced by changes in precipitation and evaporation, and 3)
abundant subaerial volcanism associated with sedimentation (Karpeta, 1989; 1993).
Karpeta, 1989 described laterally persistent silicified stromatolite beds up to 2 m thick in
the Hartbeesfontein Basin, including domal and laterally-linked morphologies. Carbonate
stromatolites were also briefly described in dolostone beds intercalated between upper Rietgat
Formation basalt flows (Karpeta, 1993). Stromatolites were interpreted as “algal bioherms”
forming within the photic zone along lake margins, with associated desiccation cracks as
evidence for shallow, periodically exposed deposition (Karpeta, 1989). This study performs the
first petrographic examination of stromatolitic cherts and dolostones from the Rietgat Formation
of the Hartbeesfontein Basin, along with sedimentology and stratigraphy of stromatolitic facies.

64

METHODS
Five stratigraphic sections were measured and described around the Hartbeesfontein
Basin (Figs. 1, 2). To account for lateral facies variability, a key component in establishing
lacustrine depositional environments, three southern sections (N1-SC1, N1-SC2, and VV) were
measured across 0.2 km of exposure (Fig. 1C), and two eastern sections (BR & LP) were
measured 0.9 km apart (Fig. 1B). Sedimentary samples, including 24 stromatolites, were
collected for laboratory analyses. Hand-sized samples were sectioned with a water-cooled saw,
polished, and scanned prior to petrographic analysis. Thin sections were made between 30 and
300 microns thick to search for microfossils and microbially-influenced mesoscale features.
White-card technique was used to differentiate microbial fabrics from secondary crystal
boundaries in carbonate samples (Folk, 1987). Photomosaics of entire thin sections were
composed using a Zeiss Imager M2m microscope with Axiovision to obtain whole-slide images
and characterize mesoscale morphology. Geochemical analysis of organic carbon and opaque
minerals was performed using a Horiba XGT-7200 micro-XRF analyzer and a Horiba XploRa+
micro-Raman spectrometer at the Natural History Museum in Los Angeles, California.
RESULTS
Sedimentology & Field Observations
Rietgat Formation sediments within the Hartbeesfontein Basin are predominantly chert,
with less extensive dolostone beds. Five sedimentary facies are identified: stromatolitic chert,
stromatolitic dolostone, thinly-bedded chert, vuggy chert, and thinly-bedded dolostone. Both
65

individual beds and total stratigraphic exposure of each facies are typically thicker in eastern
than southern sections.
1) Stromatolitic Chert
Stromatolitic chert beds are 1.5 to 3 m thick in southern exposures, and 4 to 7 m thick in
eastern outcrops (Fig. 2). Beds are highly resistant to weathering, and thicker outcrops form
laterally-extensive cliff faces (Fig. 3). Fresh surfaces are light or dark gray, weathering orange or
tan. Stromatolites form laterally-linked domes 5 to 10 cm wide with synoptic relief between 1
and 2 cm (Fig. 3A-C). Exposed surfaces show patterns of resistant laminae 2-5 mm thick
regularly separated by cuspate voids 5-10 mm thick, showing both convex- and concave-up
curvatures (Fig. 3A-C). Stromatolite laminae have high degrees of inheritance at the outcrop
scale, even when accounting for void space, though no fans or isopachous cements are present.
Where lower contacts are visible, stromatolitic chert overlies thinly-bedded or vuggy chert.
Irregular, interconnected patches of massive chert commonly obscure the lamination patterns of
the stromatolitic chert and contacts between vuggy chert beds and stromatolites. (Fig. 3D).
Stromatolitic chert can be distinguished from vuggy chert facies by the prevalence of laterally-
linked domes over meters of exposure (Fig. 3D). No evidence of cross-bedding or other
sedimentary structures were observed in association with the stromatolitic chert facies, either in
vertical section or on bedding planes (Fig. 3).
2) Stromatolitic Dolostone
Dolomitic stromatolites occur in one four-meter dolostone bed in the BR stratigraphic
column of the eastern Hartbeesfontein Basin (Figs. 2). Dolostone exposures are less resistant to
66

weathering than chert exposures, and do not form the same distinctive cliff faces as silicified
stromatolites. Fresh surfaces are black to dark gray, weathering to brown on exposed surfaces.
Stromatolites form laterally-linked domes and cones 5 to 10 cm in diameter, with synoptic relief
between 1 and 3 cm (Fig. 4). The degrees of inheritance between laminae in dolomitic
stromatolites are moderate. Individual stromatolites grade upward from cones to low-relief
domes within several centimeters; the synoptic relief varies between 1 and 3 cm within adjacent
domes (Fig. 4). The stromatolitic dolostone bed in column BR is conformably overlain by thinly-
bedded chert, while the lower contact is obscured by cover.
3) Thinly-Bedded Chert
Thinly-bedded chert is 5 to 40 cm thick in southern sections, and are 0.5 to 6.5 m thick in
eastern exposures (Fig. 2). The cherts are dark gray on fresh surfaces, and weather to tan, orange,
and occasionally red or yellow (Fig. 5A-B). Thinly-bedded chert is less resistant to weathering
than massive or stromatolitic chert, and is often associated with covered portions of stratigraphic
sections. Individual layers are 0.5 to 1 cm thick, with no lateral pinching, swelling or truncation.
No evidence of cross-bedding is apparent in vertical section or plan view. In one measured
eastern section (Fig. 2, column BR), desiccation cracks and pustular textures resembling
microbially-induced sedimentary structures (Hagadorn & Bottjer, 1997; Noffke et al., 2001)
were observed on two bedding planes separated by 15 cm (Fig. 5A-B). In cross-section, thinly-
bedded chert beds with desiccation cracks have abundant intraclasts (Fig. 5E).

67

4) Vuggy Chert
Vuggy chert beds are less prevalent and are generally thinner than stromatolitic chert in
respective sections. Beds are 10 to 50 cm thick in southern sections, and form resistant cliffs 2 to
3 m thick in eastern exposures (Fig. 2). Vuggy chert is light or dark gray fresh, weathering
orange or tan (Fig. 5C- D). Vuggy lithologies are most commonly observed above stromatolitic
chert, and are equally resistant. Vuggy chert contains mm- to cm-scale irregular voids that do not
follow bedding (Fig, 5C), as opposed to the laminae forming laterally-linked domes in the
stromatolitic facies. Contacts between vuggy and stromatolitic chert are often difficult to
distinguish, with isolated areas of vugs (~100 cm
2
) showing cuspate void patterns similar to
those seen in stromatolites, while stromatolites are often obscured by patches of massive chert. In
one southern section (Fig. 2, column VV), vuggy chert grades vertically into laminated
dolostones over 20 cm. The vuggy chert conformably overlies ~0.5 m of stromatolitic chert, but
vugs do not have stromatolite-like textures (Fig. 5D). Vugs immediately overlying stromatolites
are empty, while vugs below dolostones are themselves filled with dolomite (Fig. 5D). No vug
infill was observed anywhere else in stratigraphic sections (Fig. 5D).
5) Thinly-Bedded Dolostone
Thinly-bedded dolostone is observed in two southern sections of the Hartbeesfontein
Basin (Fig. 2, sections N1-SC1 and VV). Dolostone form beds 10 to 30 cm thick, and are less
resistant than surrounding chert. The dolostone is gray, tan and olive colored when fresh and
weathers to light gray (Fig. 5D). In both sections, thinly-bedded dolostones overlie stromatolitic
and vuggy chert. In section VV, vuggy chert transitions into dolostone beds, with chert vugs
bearing higher degrees of dolomite infill up-section (Fig. 5D, see previous section). Polished
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slabs of VV dolostones reveal abundant chert rip-up clasts from underlying vuggy cherts (Fig.
5F). Dolostones in N1-SC1 also overlie stromatolitic and vuggy cherts, although the exact
contact is obscured by cover. N1-SC1 dolostone has more clearly defined laminae than in section
VV, with no rip-up clasts observed.
Stromatolite Petrography
1) Stromatolitic Chert
Laterally-linked stromatolites in Hartbeesfontein cherts consist of alternating dark and
light laminae (Fig. 6). Dark laminae are between 50 and 100 µm thick, are composed of
microcrystalline quartz ~10 to 15 µm in diameter, and contain abundant detrital material (Fig.
6B, C). Opaque minerals associated with detrital material appear as yellow iron oxy-hydroxides
in reflected light (Fig. 6A). The intermittent presence of detrital material in dark laminae is
associated with lateral pinching and swelling of laminae (Fig. 6C). Detrital material is present
across all stromatolite growth angles, thicker at dome or cone apices and thinner on steeper
stromatolite flanks (Fig. 6C, E). The lateral variability and apical thickening of dark laminae
gives stromatolites within chert relatively low to moderate degrees of inheritance, with many
structures vertically changing from domal to conical morphologies (Fig. 6B). Light laminae are
composed of microcrystalline quartz averaging ~20 µm in diameter (Fig. 6C, E). Individual light
laminae are between 20 and 50 µm thick and have relatively uniform thickness. Opaque minerals
are rarely present in light laminae.
Non-laminated mesostructure also occurs within stromatolitic chert (Fig. 6B, D).
Grumelous textures (Cayeux, 1935; Cayeux, 1970) are defined by areas of microcrystalline
69

quartz (“grumeaux”) forming irregular, interconnected patches between 50 and 100 µm in
diameter (Fig. 6D). Interstitial spaces between grumeaux are filled in with quartz crystals at least
20 µm in diameter. Boundaries between stromatolitic and grumelous fabrics are often gradational
over hundreds of microns, and are typically associated with the presence of larger, mm-scale
fenestrae (Fig. 6B).
Fenestrae are pervasive in Hartbeesfontein stromatolitic chert, ranging from 0.5 mm to 1
cm in diameter (Fig. 7, 8, 11). Two types of fenestrae—rounded and ragged—are defined based
on their relationship with surrounding stromatolite laminae. Rounded fenestrae are bounded by
rings of dark filaments that conform to the fenestral margins (Figs. 6B, 7A, B, 8B, C, 11A, C).
Rounded fenestrae less than 5 mm in diameter are typically sub-circular to ovoid in shape (Fig.
7A, 8B, 11A, C). Larger rounded fenestrae have more irregular morphologies, but are bounded
by the same filamentous textures observed in smaller, more circular fenestrae (Fig. 7B). Sub-
circular fenestrae 0.5 to 1 mm in diameter are occasionally filled with an anastomosing fabric of
filaments, and are commonly surrounded by rings of filamentous bundles between 200 and 400
µm thick (Fig. 8B). In contrast to rounded fenestrae, ragged fenestrae have margins that truncate
dark and light stromatolite laminae (Fig. 7C-D). In non-laminated grumelous textures, ragged
fenestrae are distinguished by a lack of bounding filamentous bundles. Despite different
relationships with stromatolite textures, both types of fenestrae have similar patterns of
cementation, where a layer of anhedral microcrystalline quartz (< 20 µm) surrounds fenestrae
margins, and subhedral macrocrystalline quartz (with crystals between 20 and 2000 µm in
diameter) fills the remaining pore space (Figs. 7, 11A, C). Fenestrae affect the shape of
immediately adjacent stromatolite textures, but do not disrupt patterns of the underlying
stromatolite laminae (Fig. 7, 11).
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Filaments that define the edges of rounded fenestrae are distinct from the dark laminae
that form the majority of stromatolite fabrics (Figs. 7A, B, 8B, C, 11A, C). Filaments 10 µm in
diameter surround fenestrae in bundles between 250 and 400 µm thick (Figs. 7A, B, 8B).
Individual filaments are straight to curved, and do not follow crystal boundaries. Filamentous
textures occur within microcrystalline quartz, and are almost always associated with circular and
sub-circular fenestrae. In several samples, several sub-circular rounded fenestrae are separated
from each other by filamentous pillars up to 300 µm thick (Fig. 7A, 8B, 11A, C). Filamentous
pillars are often hourglass-shaped, with thicker, flared bases and tops as pillars converge with
surrounding stromatolite fabrics. In select samples, filamentous pendants extend into rounded
fenestrae but do not completely separate fenestrae from each other, often resembling broken
pillar structures (Fig. 7A).
Opaque minerals that appear yellow under reflected light are observed along the base of
several fenestrae (Figs. 7A, B, 9A). Opaque crystals typically occur between filamentous
textures and the microcrystalline quartz lining the fenestral boundaries, but also appear within
filamentous bundles or along individual filaments of anastomosing fabrics (Fig. 8A, C). While
the current mineral composition may be secondary, the persistent presence of opaque minerals as
geopetal features along fenestral bottoms and within filamentous bundles suggests a primary
origin for the presence of the opaque minerals. Micro-XRF analysis of stromatolitic chert
indicates the presence of iron and the absence of sulfur in opaque minerals associated with
fenestrae. Raman spectra of the same minerals indicate the presence of the iron oxy-hydroxide
goethite, as well as an absence of pyrite (Fig. 9). The yellow coloration of fenestrally-associated
opaque minerals under reflected light supports Raman analysis indicating the presence of
goethite.
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2) Stromatolitic Dolomite
Dolomitic stromatolites consist of alternating light and dark laminae of variable thickness
(Fig. 10). Light and dark laminae are predominantly comprised of dolomite, but also contain
opaque areas of organic carbon associated with microcrystalline quartz. Light laminae are 2.5 to
9 mm thick, and have grumelous textures. Individual dolomite rhombs occasionally reach 400
µm in diameter within light laminae (Fig. 10B). Areas of spar between grumeaux are 0.1 to 1
mm in diameter, have irregularly branching morphologies, and show no preferential orientation
either parallel or normal to laminae (Fig. 10A). Sparry zones between grumeaux exhibit drusy
cementation patterns, and exhibit no geopetal accumulations of detritus (Fig. 10B). Areas of
organic carbon 200 to 700 µm wide occasionally occur in light laminae, as determined by Raman
spectroscopy. Organic carbon is consistently associated with microcrystalline quartz, either
surrounding or within carbon-rich areas (Fig. 10C). Dark laminae vary between 0.2 and 2 mm
thick, and are composed of dolomitic microspar and microcrystalline quartz interspersed with
opaque organic carbon (Fig. 10A). The irregular thickness and undulating nature of dark laminae
form mm-scale cones and domes on the surfaces of macroscale cones defined by thicker light
laminae. Silica occurs in association with organic carbon between dolomite crystal boundaries,
with some areas featuring isolated dolomite rhombs suspended in a “matrix” of organic-rich
silica (Fig. 10A).

72

DISCUSSION
Depositional Setting
The transition from stromatolitic to thinly-bedded dolostone or chert indicates
shallowing-upward cycles within the Hartbeesfontein paleo-lake (Fig. 2). While the prevalence
of detrital material defining laminae in stromatolitic chert implies a regular source of
sedimentation, stromatolites show no evidence of current influence in plan view (Fig. 3), nor of
wave or current reworking observed in thin sections (Figs. 6, 10). Stromatolites are therefore
conservatively interpreted as forming below wave base. Vuggy chert frequently occurs between
stromatolites and thinly-bedded sediments (Fig. 2), and occasionally bears textures closely
resembling stromatolitic laminae in small, cm-scale areas. Rip-up clasts of vuggy chert within
overlying thinly-bedded dolostone indicate early silicification of sediments, followed by
disruption from wave or current activity and subsequent deposition in dolomitic sediments. (Fig.
5D, F). Vuggy chert is therefore interpreted as rapidly silicified sediments in wave-agitated
environments between deeper stromatolite facies and thinly-bedded mudflat facies, with
occasional stromatolitic textures potentially representing reworked stromatolites. The presence
of mud cracks, rip-up clasts, and clastic microbial textures in thinly-bedded cherts indicate
deposition in shallow, periodically desiccated mud-flat settings around the edges of the
Hartbeesfontein lake (Fig. 5A, B, E, F). The interpretation of a shallow mudflat environment is
further supported by previous descriptions of wave ripples and evaporites alongside mud cracks
and rip-up clasts in thinly-bedded cherts (Karpeta, 1989; 1993), though neither ripples nor
evaporites were observed in this study.
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Vuggy and massive cherts associated with stromatolites have been previously interpreted
as former magadiite beds (Karpeta, 1989; 1993). Magadiite is a hydrous sodium silicate mineral
(NaSi7O13(OH)3·4(H2O)), most commonly observed as an evaporite in alkaline lacustrine
environments (Eugster, 1967; 1969). Previous lines of evidence for magadiite precursors to
Hartbeesfontein cherts included soft-sediment deformation, reticulate cracking patterns distinct
from mud cracks, and extensive vuggy textures as magadiite converts into chert (Karpeta, 1989),
a process which involves at least 25% volume reduction (Surdam et al., 1972; Eugster, 1986).
While this study did not observe reticulate cracks, the prevalent irregular patterns of void spaces
and lack of laterally-consistent bedding structures within vuggy cherts (Fig. 5C, D) supports
previous hypotheses of a magadiitic precursor. In contrast, no observations of magadiitic textures
were observed in stromatolite outcrops or in thin sections of this study. In contrast, the detailed
preservation of filaments and associated fenestrae in Hartbeesfontein stromatolites (Figs. 7, 8)
suggest primary silicification of microbial mats. The volume reduction as magadiite converts
into chert is highly unlikely to preserve the level of detail observed within stromatolites.
A lacustrine setting for Hartbeesfontein sediments is supported by high lateral facies
variability over sub-kilometer distances, as well as frequent sub-meter facies shifts within
southern stratigraphic columns (Fig. 2). Similar scales of stratigraphic variability have supported
lacustrine interpretations of stromatolitic facies within the ~2.7 Ga Tumbiana Formation of
Western Australia (Walter, 1985, Awramik & Buchheim, 2008). The frequent facies changes are
interpreted as shifts in relative lake level over time. Stromatolite facies likely formed on flooding
surfaces caused by lake transgression (Buchheim & Awramik, 2000). As the lake regressed,
shallower, more agitated environments resulted in rip-up textures observed within vuggy cherts
and overlying thinly-bedded cherts. Shallowing culminated in periodically desiccated mudflat
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facies before subsequent transgression formed new flooding surfaces. Areas where shallowing-
upward cycles are interrupted potentially represent more rapid changes in lake level. For
example, a progression from vuggy to stromatolitic chert with no thin beds in-between could
signify limited lake regression before the subsequent flooding surface, as seen in column LP
(Fig. 2). Conversely, an immediate transition from stromatolites to thin beds is interpreted as an
accelerated lake regression, as exhibited in column BR (Fig. 2). Cyclic patterns of deeper
stromatolitic facies and shallower, more agitated environments have been noted in other
lacustrine basins (Surdam & Stanley, 1979), including the Tumbiana Formation (Awramik &
Buchheim, 2009). The confinement of laterally and vertically variable sedimentary facies within
a single intracratonic half-graben in the central Kaapvaal Craton reinforces the lacustrine
interpretation of the Hartbeesfontein Basin, as opposed to extensive, laterally consistent marine
deposits (Fig. 1B).
Stromatolite Biogenicity
Petrographic textures in both chert and dolomite stromatolites in the Hartbeesfontein
Basin suggest biogenic origins. Laminae in stromatolitic chert are defined by detrital material
that pinches and swells laterally (Fig. 6C, E). Detrital material is observed at all stromatolitic
growth angles (Fig. 6C, E). Stromatolites formed by abiogenic mineral precipitation alone are
more likely to have isopachous laminae, and would not be predicted to entrain detrital grains at
the high growth angles seen in Hartbeesfontein stromatolites (e.g., Frantz et al., 2015). The
presence of detrital material on such steep faces, beyond the angle of repose, is best explained by
the presence of microbial mats, either via filamentous trapping and binding of grains, adhesion to
75

extracellular polymeric substances (EPS), or both (Gebelein et al., 1969; Hofmann et al., 1999;
Frantz et al., 2015).
Dolomitic stromatolites do not exhibit the same detailed preservation of fenestrae or
high-angle detrital material seen in stromatolitic chert (Fig. 10), but carbonate textures still
suggest a biogenic origin. Abiogenic precipitation of stromatolites is often defined by regular
isopachous laminae comprised of fibrous carbonate fans or botryoids. In contrast, layers within
Hartbeesfontein stromatolitic dolostones contain irregular grumelous microspar textures between
laminae (Fig. 10), suggestive of microbial involvement (Monty, 1976; Grotzinger & Knoll, 1999;
Turner et al., 2000). The abundance of organic carbon within dark layers and occasionally in
light layers also suggests at least the previous presence of microbial mats if not their direct
involvement in stromatolite morphogenesis (Fig. 10).
Origin and Significance of Rounded Fenestrae in the Hartbeesfontein Stromatolites
Many fenestrae within stromatolitic chert have smooth, rounded morphologies. Rounded
fenestrae appear to be a primary component of stromatolite fabrics, as the walls of the fenestrae
appear to deform stromatolitic fabrics without truncating laminae (Figs. 7A, B, 8B, C).
Filamentous textures are particularly abundant surrounding and within the walls of rounded
fenestrae (Fig. 8). The morphology of individual filaments is independent of crystal boundaries,
indicating a primary origin as opposed to secondary mobilization of opaque materials between
quartz crystals. The most parsimonious explanation for circular fenestrae in stromatolitic chert is
the entrainment of gas within microbial mat layers. Gas can be trapped within mats either from
metabolic production within laminae (Bosak et al., 2009; 2010; Mata et al., 2012), or potentially
by entraining gas produced from subjacent mats. Gas produced within mats will hypothetically
76

only deform immediately surrounding laminae, while gas traveling up through microbial mats
will deform successive layers prior to preservation in the rock record. Fenestrae in
Hartbeesfontein cherts do not deform underlying or overlying layers, and only impact
immediately surrounding laminae (Figs. 7, 8). Therefore, the gas that caused the fenestrae to
form likely originated from microbial metabolisms within the same laminae at the time of
preservation.
Preservation of gas fenestrae has been noted within several ancient and modern microbial
structures (Bosak et al., 2009; 2010; Mata et al., 2012; Wilmeth et al., 2015; Sallstedt et al.,
2018). Textures where filamentous bundles separate rounded fenestrae are reminiscent of
“hourglass structures” (Fig. 11A-D) described within recent stromatolites from Yellowstone
National Park (Mata et al., 2012). The Yellowstone hourglass structures form when oxygen
produced by photosynthesis is entrained within mats by filamentous cyanobacteria (Mata et al.,
2012). Hourglass structures form when filaments are oriented into flared pillar structures
between oxygen bubbles, occasionally breaking apart with continued gas production (Fig. 11 B,
D). The fabric of cyanobacterial filaments surrounding rounded voids is subsequently preserved
by rapid silicification, with new laminae forming daily timeframes (Berelson et al., 2011; Mata
et al., 2012). Rapid silicification is also inferred for Hartbeesfontein stromatolites, particularly
given the evidence for shallow, agitated environments with the presence of rip-up clasts
indicating currents strong enough to transport cm-scale clasts (Fig. 5E, F). If silicification rates
were comparable in the two locations, the order of magnitude size difference in rounded
fenestrae (Fig. 11A-D) implies faster rates of gas production in Hartbeesfontein than
Yellowstone stromatolites.
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While not conclusive, oxygenic photosynthesis would be a parsimonious candidate for
the gas-producing metabolism within Hartbeesfontein stromatolites. Support for this hypothesis
comes from the abundance of filamentous microfossils in shallow aqueous environments, and the
similarity to modern stromatolite textures formed by cyanobacteria, (Mata et al., 2012). While
methane produced from methanogenic communities is a hypothetical gas candidate, methane
typically forms deeper within the sediment (Boudreau et al., 2005; Bosak et al., 2009), requiring
upward motion to become entrained within mats. The fact that the rounded pore fenestrae are
isolated to particular layers and do not appear to intrude laminae from below argues against
methane as the primary gas within Hartbeesfontein stromatolites (Figs. 7, 11). Finally, absence
of sulfur in geopetal minerals within fenestrae indicates the absence of hydrogen sulfide as a
candidate fenestral gas. In contrast, the presence of iron oxy-hydroxides within geopetal fabrics
supports the presence of an oxidizing gas such as oxygen produced by photosynthesis.
More poorly preserved fenestral fabrics occur in lacustrine stromatolites of the ~2.7 Ga
Tumbiana Formation within the Fortescue Group in Australia (Fig. 11E, F), approximately
coeval with Ventersdorp Supergroup deposits. Rounded fenestrae within Tumbiana stromatolites
have only been reported from the apices of conical morphologies (Bosak et al., 2009), and have
been compared with oxygen bubbles formed by cyanobacteria in modern mats (Bosak et al.,
2009; 2010). Flannery and Walter, 2012 also described fenestrae within conical Tumbiana
stromatolites, but argued against gas production based on fenestral and stromatolite
morphologies. In contrast, the majority of fenestrae within Hartbeesfontein stromatolites are
rounded, surrounded by filamentous microfossils, and are not concentrated along dome or cone
apices (Figs. 6, 7). The differences between Hartbeesfontein and Tumbiana fenestral textures
could be due to different rates of mat mineralization, patterns of microbial metabolisms, or a
78

combination of both factors. The similarities between various rounded fenestral fabrics in
Archean stromatolites and modern oxygen-influenced mat textures potentially provides
petrographic support for the hypothesis of oxygenic photosynthesis prior to the Great Oxidation
Event, and increases the possibility for Archean lacustrine environments to represent “oxygen
oases” (Fischer, 1965) versus the reducing conditions present in the ocean at large at the time.
CONCLUSIONS
The Hartbeesfontein stromatolites are significant among Archean microbial deposits,
both in their abundance and their detailed preservation of petrographic microstructure. In
conjunction with stromatolites from the Fortescue Group of Western Australia, the extent of
Hartbeesfontein stromatolites show that life was abundant in lacustrine settings on multiple
cratons prior to the Great Oxidation Event. The preservation of silicified and carbonate
stromatolites in lacustrine deposits as well as clastic microbialites in mudflats indicates
diversification of microbial communities into multiple non-marine environmental regimes.
Finally, the detailed preservation of filaments, fenestrae, trapped detrital grains, and abundant
organic carbon within Hartbeesfontein stromatolites provides pervasive evidence for biogenicity.
The presence of rounded fenestrae and associated filamentous textures within silicified
stromatolites indicates the presence of gas-producing metabolisms within microbial mats. The
size of fenestrae coupled with the relatively rapid silicification processes required to preserve
delicate gas-related textures indicate rapid rates of gas production. Hartbeesfontein fenestral
textures most closely resemble oxygen-influenced fabrics within modern, rapidly-lithifying
cyanobacterial stromatolites. Further examination of gas-related textures in microbialites from
Hartbeesfontein and elsewhere in the Archean can potentially provide petrographic support for
79

the hypothesis of oxygenic photosynthesis prior to the Great Oxidation Event and the presence of
oxygen oases in non-marine environments.

ACKNOWLEDGMENTS
This research was supported by the National Science Foundation Graduate Research Fellowship,
the Geological Society of America Student Research Grant, the Lewis and Clark Fund for
Exploration and Field Research, and the University of Southern California Earth Science
Department Research Fund. NJB would like to acknowledge funding support from the National
Research Foundation of South Africa in Pretoria. We thank Dr. Alyssa Bell (LA Natural History
Museum) for logistical support associated with the fieldwork of summer 2014.
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FIGURES: CHAPTER 2

FIGURE 1: A: Generalized stratigraphy of the Ventersdorp Supergroup (modified from van der
Westhuizen, 1991). B: Generalized geological map of the Hartbeesfontein Basin, modified from
Wilkinson & Coetsee, 1986. The shaded box represents the area shown in Fig. 1C. C: Locations
of stratigraphic sections in the southern Hartbeesfontein Basin.

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FIGURE 2: Measured stratigraphic sections within the Hartbeesfontein Basin (see Fig. 1 for
locations). N1-SC1, N1-SC2, and VV represent a south-to-north transect within the southern
portion of the basin, while BR and LP represent a south-to-north transect within the eastern
portion of the basin.

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FIGURE 3: Stromatolitic cherts. Hammer is 30 cm, pen is 15 cm for scale. A-C: Macroscale
domal structures. D: Vertically extensive stromatolitic laminae obscured by cm-scale patches of
massive and vuggy chert. E: Plane view of stromatolites at 2 meters in section VV.

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FIGURE 4: Stromatolitic dolomites. Hammer is 30 cm for scale. A: Stromatolites at 18 m in
section BR. Note the transition between conical and domal forms up successive stromatolite
laminae. B: Stromatolites at 20 m in section BR.

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FIGURE 5: Non-stromatolitic sedimentary features. Hammer head and pen are 15 cm for scale.
A: Mud cracks at 24.5 m in section BR. B: Structures resembling MISS in float of section N1-
SC1. C: Vuggy chert from 0.8 m in section VV. D: Vuggy chert grading upward into thinly-
bedded dolomite at 1 m in section VV. E: Thin section of rip-up clasts below mud cracks in Fig.
5A. F: Scan of rip-up clasts within Fig. 5D.

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FIGURE 6: Petrographic textures in stromatolitic cherts. A: Polished slab of silicified
stromatolite. Note yellow iron oxy-hydroxides which correspond to opaque laminae in Fig. 6B.
B: Thin section of the same stromatolite in Fig. 6A (250 µm thick). The top and bottom thirds of
the sample are representative of light and dark stromatolitic laminae patterns. The middle third of
the sample is representative of grumelous textures, and contains multiple large rounded and
irregular fenestrae. C: Stromatolitic laminae in Fig. 6B. D: Grumelous textures in Fig. 6B. E:
Cross-polarized view of detrital material along stromatolite laminae.
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FIGURE 7: Fenestral textures within stromatolitic cherts. A: Four rounded fenestrae separated
by fenestral pillars. B: A single rounded fenestrae of irregular shape. Note the presence of
opaque minerals lining the bottoms of fenestrae in Figs. 7A and 7B. C, D: Ragged fenestrae.
Filaments and laminae extend into these fenestrae from surrounding stromatolitic fabrics, as
opposed to surrounding them in rounded fenestrae.

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FIGURE 8: Filamentous textures in stromatolitic cherts. A: Filamentous bundles lining the edge
of a rounded fenestrae. B: A rounded fenestrae filled with anastamosing filaments. C: Detail of a
filamentous pillar which separates two rounded laminae (See Fig. 7A). Note the presence of
opaque minerals along certain filaments in Figs. 5A and 5C.

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FIGURE 9: A: Opaque minerals forming geopetal structures along the bottom of a Hartbeesfontein
fenestra. B: Raman spectrum of A, compared with known Raman spectra for quartz and goethite.

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FIGURE 10: Petrographic textures of stromatolitic dolomites. A: A dark-light laminae couplet.
B: Isolated dolomite rhombs within a light lamina. C: A pocket of organic matter within a light
lamina.

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FIGURE 11: Gas-related textures within stromatolites. A, C: Rounded fenestrae separated by
filamentous pillars from the Hartbeesfontein Basin. B, D: “Hourglass structures” of
cyanobacterial filaments separating fenestrae within a recent stromatolite from Yellowstone
National Park. E: A conical stromatolite from the ~2.7 Ga Tumbiana Formation of Western
Australia. F: Insert of Fig. 10C, featuring multiple rounded fenestrae within the conical apex.

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SUPPLEMENTARY APPENDIX: STROMATOLITE PHOTOMOSAICS
Rietgat Formation:
Location: Hartbeesfontein, South Africa
Age: Neoarchean (~2.7 Ga)
Paleoenvironment: Lacustrine, approximately fair-weather wave base
Stromatolitic Cherts:
p. Thin Section
Label
μm Morphology Notes
103 N1-SC1-1 100 Conical Filamentous “cobweb” textures,
coalesced fenestrae
104 N1-SC1-1 (detail) Filaments surrounding rounded
fenestrae
105 N1-SC1-1 14a 270 Domal and
conical
Hourglass structures, coalesced
fenestrae, geopetal opaques
106 N1-SC1-1 14a
(detail)
Hourglass structure
107 N1-SC1-1a 30 Domal Low inheritance from laminar to domal
structures
108 N1-SC1-1c 40 Domal and
conical
Cm-scale fenestrae, geopetal opaques,
detrital material at steep angles
109 N1-SC1-1c
(detail)
Geopetal opaques. “Floating” opaques
interpreted as hourglass structure cross-
sections.
110 N1-SC1-1c 2018
C
250 Domal Hourglass structures

111 N1-SC1-1c 2018
C (detail)
Hourglass structure.

112 N1-SC1-1c 2018
D
250 Domal and
conical
Hourglass structures, coalesced
fenestrae
113 N1-SC1-1c 2018
D (detail)
Coalesced fenestrae
114 N1-SC1-1e 40 Domal Low inheritance, detrital material at
steep angles
115 N1-SC1-1e
(detail)
Detrital material at steep angles
116 N1-SC1-1e 2018
A
250 Domal Extensive fenestral textures, coalesced
fenestrae
117 N1-SC1-1e 2018
B
250 Domal Extensive fenestral textures, coalesced
fenestrae
118 N1-SC1-1e 2018
B (detail)
Coalesced fenestrae
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119 N1-SC1-1e 2018
C
250 Domal Extensive fenestral textures, coalesced
fenestrae
120 N1-SC1-1e 2018
C (detail)
Hourglass fenestrae

Stromatolitic Dolostones:
p. Thin Section
Label
μm Morphology Notes
121 BR-7.3-i 100 Domal Alternating light and dark carbonate layers
122 BR-7.3-i (detail) Grumeux fabric in a light layer
123 BR-7.3-ii 60 Domal Alternating light and dark carbonate layers
124 BR-7.3-ii (detail) Alternating light and dark layers

Notes: All samples are in cross-sectional view. Arrows adjacent to scale bars indicate up
directions.

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

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CHAPTER 3:
RAPID RATES OF OXYGENIC PHOTOSYNTHESIS WITHIN NEOARCHEAN
STROMATOLITES
ABSTRACT
Oxygenic photosynthesis fundamentally altered biological, geological, and atmospheric
processes on early Earth, but determining the timing of this metabolism’s origins remains a
continuing endeavor. Fenestrae (former voids) preserved in Archean stromatolites provide
evidence of microbial gas production, though determining which microbial metabolisms formed
ancient stromatolite fenestrae is challenging. 2.7 Ga lacustrine stromatolites from the
Hartbeesfontein Basin of South Africa contain abundant rounded fenestrae associated with
filamentous fabrics and iron oxides ~300 million years before the Great Oxidation Event (ca. 2.4
Ga). Fenestrae associated with iron oxides in modern stromatolites from springs in Yellowstone
National Park serve as an analogue for ancient fenestral textures, where bubbles form, become
entrained by microbial filaments, and are lithified as fenestrae on a quasi-daily time frame.
Modern rates of gas production within microbial mats, including oxygen, hydrogen sulfide,
hydrogen, and methane, were examined to constrain which metabolisms could produce
Hartbeesfontein fenestrae during Yellowstone preservational timeframes. Of the metabolisms
investigated, oxygenic photosynthesis is most likely to have formed observed fenestral textures
within Hartbeesfontein stromatolites, with gas production rates orders of magnitude faster than
any other metabolism. Furthermore, hydrogen sulfide production via sulfate reduction is limited
by low Archean sulfate concentrations, the fastest fermentation rates are documented within
cyanobacterial mats, comprising only 10% of gas production, and feasible rates of
methanogenesis are restricted to organic-rich wetlands (a poor analogue for the Hartbeesfontein
paleo-environment). The presence of abundant gas fenestrae within 2.7 Ga lacustrine
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stromatolites would suggest the evolution of oxygenic photosynthesis well before the Great
Oxidation Event, and may support hypotheses for a non-marine origin of cyanobacteria/oxygen
production.
INTRODUCTION
The evolution of oxygenic photosynthesis has shaped interactions between earth-life
systems for billions of years, yet the metabolism’s origins remains contested, with estimates
spanning approximately 1.4 billion years from the Archean to the Paleoproterozoic. The
youngest bound of estimates (2.4 Ga) is defined by the Great Oxidation Event (GOE), when the
presence of an oxidizing atmosphere is marked by the appearance of widespread BIFs and
oxidized soils, the disappearance of redox-sensitive conglomerates, and the switch from mass-
independent to mass-dependent sulfur isotope fractionation (Grandstaff, 1980; Yang & Holland,
2003; Farquhar et al., 2000; Holland et al., 2002). Two prevailing hypotheses exist regarding the
relationship between the origin of oxygenic photosynthesis and the GOE. 1: Oxygenic
photosynthesis evolved hundreds of millions of years before the GOE, but most oxygen
produced was buffered by high concentrations of compounds that react with oxygen, preventing
accumulation in the atmosphere and oceans (Kump & Barley, 2007; Gaillard et al., 2011; Lyons
et al., 2014). The hypothesis suggests that local regions where oxygen production overpowered
redox buffers exhibited transient periods, or “whiffs”, of oxidation observable in the rock record
(Anbar et al., 2007; Kendall et al., 2010; Crowe et al., 2013; Riding et al., 2014), with 2.4 Ga
representing a global decrease in Earth’s redox buffering capacity (Kump & Barley, 2007;
Gaillard et al., 2011). 2: Alternately, the GOE itself represents the nearly-instantaneous aftermath
of oxygenic photosynthesis evolving ~2.4 Ga. The hypothesis suggests that oxygen production
rates would have overcome redox buffers within hundreds of thousands of years after the
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evolution of photosynthesis, given the prodigious rates of oxygen production by cyanobacteria
(Fischer et al., 2016; Ward et al., 2016; Shih et al., 2017; Soo et al., 2017). Both hypotheses have
used geochemical proxies from various sedimentary environments for evidence of oxidation
(Rye & Holland, 1998; Johnson et al., 2013), and molecular phylogenetic analyses for evidence
of ancient biological processes (Shih et al., 2017; Soo et al., 2017; Schirrmeister et al., 2013;
Magnabosco et al., 2018).
Ancient microbial mats preserved in the rock record form clear targets for the
investigation of the antiquity of oxygenic photosynthesis (Walter et al., 1983; Hofmann, 2000;
Schopf, 2006). Physical and chemical properties of stromatolites, commonly attributed to the
growth of microbial mats, have the potential to preserve evidence of specific microbial
metabolisms in ancient environments, constraining the early diversification patterns of life on
Earth (Buick, 1992; Bontognali et al., 2012). For example, the presence of primary fenestral
textures within stromatolite laminae provides evidence for gas-producing metabolisms within
ancient microbial mats (Bosak et al., 2009; Mata et al., 2012). Certain fenestral textures within
ancient microbialites resemble bubbles produced within modern microbial mats (Bosak et al.,
2009; Mata et al., 2012; Homann et al., 2015; Wilmeth et al., 2015; Sallstedt al., 2018). Gas
production within modern microbial mats forms distinctive morphologies, including cones with
bubbles along central axes (e.g., Bosak et al., 2009) and spherical fenestrae along microbial
lamina separated by filamentous pillars (hourglass-associated fenestrae) (Mata et al., 2012).
The preservation of microbial mat bubbles as fenestrae within stromatolites is dependent
on rates of gas production, bubble degeneration, and lithification within microbial mats. Slower
metabolic rates can produce large, cm-scale bubbles within mats over days to weeks, while faster
metabolisms can form similar quantities of gas within hours. Bubble degeneration occurs either
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by mechanical processes such as wave activity in agitated environments, microbial metabolisms
incorporating gases from bubbles as reactants, or by dissolution across bubble surfaces. In either
case, faster rates of gas production are more likely to produce larger bubbles. In highly-agitated
environments where large bubbles are less likely to be sustained, lithification rates must increase
to preserve observable fenestrae within microbialites. Therefore, known rates of bubble
preservation in modern stromatolites (Berelson et al., 2011) can be used in conjunction with
fenestral volumes from microbialites to provide estimates for metabolic rates within ancient
microbial mats. Here we examine the potential of various gas-producing metabolisms to form the
abundant fenestral textures observed in Neoarchean lacustrine stromatolites, constrained using
lithification rates from analogous modern stromatolites from a Yellowstone hot spring.
Yellowstone Stromatolites: Modern Analogues for Bubble Formation and Lithification
While multiple examples of bubbles and associated mat textures have been observed
within modern microbial mats (Hoehler et al., 2001; Bosak et al., 2009; 2010; Mackey et al.,
2015), siliceous stromatolites forming in Obsidian Pool Prime (OPP), Yellowstone National
Park, are currently the only modern examples known that provide evidence for both the
production and preservation of microbially-produced bubbles (Mata et al., 2012). The OPP
stromatolites provide useful analogues for gas-related fenestrae in ancient stromatolites (Figs. 1,
2, 3). Like many ancient stromatolites, laminae in OPP examples are fine (30-330 microns in
thickness) and consist of light-dark couplets (Berelson et al., 2011; Mata et al., 2012; Pepe-
Ranney et al., 2012). Laminae are composed almost exclusively of filamentous cyanobacteria
(Mata et al., 2012; Pepe-Ranney et al., 2012), where filaments are largely surface normal in light
laminae and surface parallel in dark laminae (Fig. 1A). Using novel dating techniques, Berelson
129

et al. 2011 found that light-dark couplets are formed and preserved within stromatolites on a
quasi-daily basis.
Many light laminae in the stromatolites from OPP contain “hourglass-associated
fenestrae” between 100 µm and 5 mm in diameter (Fig. 2, 3), textures where closely-associated
rounded pores are separated from each other by thin, hourglass-shaped bundles of microbial
filaments (Mata et al., 2012). Hourglass-associated fenestrae form when oxygen bubbles
produced during photosynthesis expand and distort surrounding mat textures, resulting in bubble
entrainment within laminae by filamentous cyanobacteria. In select cases, copious gas
production results in smaller bubbles coalescing into larger void spaces, splitting hourglass-
shaped filament bundles into cuspate pendants along fenestral margins (Fig. 3).
Gas is produced within laminae, as OPP stromatolite textures below hourglass-associated
fenestrae show no evidence of gas upwelling from underlying sediments. Some hourglass-
associated fenestrae in OPP stromatolites are coated with opaque oxides, providing evidence of
oxidizing conditions within bubbles (Fig. 2E). Micro-XRF analysis of the opaque minerals
indicates abundant iron with no sulfur present, with reflected light microscopy suggesting the
presence of goethite. The agitated nature of hot spring environments and the presence of aerobic
respiration within mats would typically disrupt or metabolize such bubbles before preservation.
However, in OPP, lithification occurs on the timescale of bubble formation as silica precipitates
from the spring within the microbial mat.
Thus, hourglass-associated fenestrae in OPP stromatolites indicate the following criteria
should be satisfied for bubble fenestrae preservation in modern or ancient stromatolites:
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1. A microbial community capable of producing gas. In OPP stromatolites, the gas-
producing metabolism is oxygenic photosynthesis, but other metabolisms are viable
in different communities.
2. A microbial mat with physical characteristics that allow the trapping of gas. In OPP,
the predominantly filamentous cyanobacterial community is adept at entraining gas
bubbles.
3. A rate of gas production fast enough to become trapped in microbial mat layers
without immediate physical disruption or metabolic consumption.
4. A rate of mat lithification on the timescale of the existence of a gas bubble. In
agitated environments, the timescales of bubble duration would be short, whereas in
very quiet water environments, the timescale could be longer. In OPP stromatolites,
the rate of bubble lithification is approximately daily.
Preservation of Bubble Fenestrae Within 2.7 Ga Stromatolites, South Africa
Lacustrine stromatolites from the ~2.7 Ga Rietgat Formation near Hartbeesfontein, South
Africa (Karpeta et al., 1989; Wilmeth et al., in prep) contain textures which strongly resemble
fabrics within OPP stromatolites (Figs. 1, 2, 3), indicating the rapid production of gas in
Neoarchean microbial mats. As in OPP samples, Hartbeesfontein stromatolites are composed of
light-dark laminae couplets between 70 and 150 microns thick preserved in silica (Fig. 1B).
Light laminae contain rounded fenestrae between 500 µm and 1 cm in diameter (Fig. 2, 3).
Hartbeesfontein rounded fenestrae are separated from each other by filament bundles up to 200
µm thick, with individual filaments 10 µm wide. Larger fenestrae (> 5 mm in diameter) often
bear cuspate filamentous pendants similar to Yellowstone textures formed when adjacent
bubbles coalesce and split apart bounding filaments (Fig. 3).
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Large fenestrae also bear geopetal structures of opaque minerals resembling oxides
observed in OPP stromatolites (Fig. 2F). Micro-XRF of Hartbeesfontein opaque minerals
indicates the presence of iron and the absence of sulfur, while Raman spectra indicate the
presence of goethite and the absence of pyrite (S4, Wilmeth et al., in prep.), which is further
supported by reflected light microscopy. The paragenetic sequence of diagenesis demonstrates
that oxides were present first, at the bottom of the fenestrae, followed by two generations of
pore-filing silica cement. The remarkable similarities between rounded fenestrae in
Hartbeesfontein stromatolites and hourglass-associated fenestrae in Yellowstone samples
increase confidence in similar modes of preservation for gas-related textures in each (Walter et
al., 1976; Berelson et al., 2011).
The relative rates of gas production between Hartbeesfontein and Yellowstone
stromatolites can be further constrained using observed differences in fenestral size and
depositional environment. Given the assumption of similar preservational timeframes in the two
locations, stromatolites with larger fenestrae indicate more rapid rates of gas production.
Hourglass-associated fenestrae are typically less than 200 µm wide in Yellowstone stromatolites
(Mata et al., 2012), while many Hartbeesfontein fenestrae exceed 1 mm in diameter (Fig. 2, 3).
Minimum and maximum fenestral widths are also larger in Hartbeesfontein (500 µm and 1 cm)
than OPP stromatolites (100 µm and 5 mm), indicating consistently higher volumes of gas
produced. Similarly, agitated depositional environments will disrupt bubble formation more
frequently than within calmer settings. Therefore, bubbles preserved in agitated environments
require faster gas production rates to reach sizes observed in calm environments. Evidence for
higher-energy depositional environments within Hartbeesfontein stromatolites is derived from
abundant detrital material present within dark laminae (Fig. 1B), indicating sediment transport
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onto microbial mats via wave or current activity (Wilmeth et al., in prep.). Detrital material is
occasionally present within Yellowstone dark laminae (Fig 1A), but is less abundant than in
Hartbeesfontein samples. The coupled evidence of abundant mm-scale fenestrae within agitated
depositional environments indicates relatively higher rates of gas production within
Hartbeesfontein microbial mats than in Yellowstone hot springs.
METHODS
Estimating Gas Production Within Archean Microbial Mats
Fenestral volumes within Hartbeesfontein stromatolites were estimated through
petrographic examination of thin sections. Fenestrae within thin sections are observed as two-
dimensional cross-sections of three-dimensional spaces. To estimate the volumes of each
fenestra, the cross-sectional area was measured and treated as a circle. The circle’s radius was
then used to calculate a hypothetical spherical volume. The likelihood of intersecting the largest
cross-section of a fenestral sphere or irregular void within a thin section is low, therefore our
estimates of gas volume within Hartbeesfontein stromatolites are conservative. If fenestral
volumes within ancient mats were higher than calculated estimates, faster metabolic rates would
be required to produce observed fenestrae. Given a set time of 24 hours for the preservational
window of bubbles within OPP stromatolites, the number of modern metabolic rates that can
produce observed fenestrae will decrease as fenestral volume increases.
Estimates of mat volumes preserving observed fenestrae were obtained by multiplying
thin section height and width by the diameter of the largest fenestrae (1 cm). The estimates
conservatively assume that all fenestrae observed within thin sections have diameters equal or
smaller than 1 cm, hence all fenestral volumes should be accounted for in the hypothetical mat
133

space (6.3 cm
3
for 3.3 x 1.9 x 1 cm sections, and 25 cm
3
for 5.9 x 4.2 x 1 cm sections). The
amount of gas present within a given volume of mat is expressed as mL gas/cm
3
mat, measured
by dividing fenestral volumes by mat volumes. The highest volume of gas per mat volume was
calculated to be 0.043 mL gas/cm
3
mat, which was used in subsequent comparisons with gas
production rates in modern microbial mats.
Gas Production Rates in Modern Microbial Mats
A literature survey of modern gas production rates in microbial mats was conducted to
determine the rates at which different metabolisms produce gas via the following reactions:
Oxygenic Photosynthesis: CO2 + H2O   CH2O + O2
Sulfate Reduction: SO4
2-
+ 2CH2O   H2S + 2HCO3
-

Fermentation: CH2O + H2O   2H2 + CO2
Methanogenesis: CO2 + 4H2   CH4 + 2H2O (hydrogenotrophic)
CH3COOH   CH4 + CO2 (acetoclastic)
Metabolic rates that can produce at least 0.043 mL gas/cm
3
mat within 24 hours are
capable of producing observed fenestrae in Hartbeesfontein stromatolites. A table of gas
production rates within modern microbial mats, as well as the time required for each rate to
produce Hartbeesfontein fenestrae, is provided in Supplementary Table 1. As Hartbeesfontein
fenestrae are preserved within stromatolite laminae, only studies examining in-situ gas
production within microbial mats were considered, as opposed to gas released from microbial
communities in benthic sediments or from thick organic-rich peats. When possible, net oxygen
134

production rates were used from literature sources to account for the removal of oxygen by
aerobic respiration.
Of the four metabolisms examined, fermentation and acetoclastic methanogenesis
produces carbon dioxide in addition to hydrogen and methane, respectively. However, at typical
lacustrine pH values between 6 and 8, at least 50% of carbon dioxide produced will be converted
to bicarbonate (HCO3
-
), with only trace amounts of gas remaining at pH 8. Evidence for
magadiite formation within the Hartbeesfontein Basin (Karpeta, 1989; Wilmeth et al., in prep.)
indicate alkaline conditions around pH ~10, based on modern lake chemistry (Eugster, 1967). At
such high pH values, carbon dioxide is converted to bicarbonate and carbonate ions, with only
hydrogen remaining as a gaseous metabolic product to produce bubbles. Therefore, this study
presents rates of fermentation solely in terms of hydrogen production.
Time Required To Produce Hartbeesfontein Fenestrae
The amount of time for a metabolic rate to produce a given bubble volume is the sum of:
1) the time for a gas to reach saturation within solution, and 2) once saturated, the time to
subsequently form appropriate bubble volumes. The saturation concentration of a gas in solution
is defined by Henry’s Law:
1) aqueous concentration of gas at saturation = Hcp * partial pressure of gas
Here, aqueous concentration is expressed in moles gas*liter solution
-1
, Hcp represents Henry’s
constant in units of (moles gas)*(liter solution)
-1
*(atm)
-1
, and partial pressure of gas is expressed
in atm. A gas is at saturation in a solution when the aqueous concentration is in equilibrium with
the overlying partial pressure of the same gas. In undersaturated solutions, microbial gas
production must first bring solutions to saturation before bubble production can begin. Therefore,
135

the time required to bring a solution to saturation must be accounted for when calculating the
total time of bubble formation. Since the initial aqueous concentration of gas in Archean lakes is
unknown, our calculations conservatively use initial aqueous concentrations of 0 (moles gas)*
(liter solution)
-1
. Actual lake environments would most likely have some amount of dissolved gas
present, which would decrease the amount of time necessary to saturate solutions. Changes in the
temperature and hydrostatic pressure of a solution alter Henry’s constant (Hcp), which in turn
affects the time required to bring a solution to saturation. A sensitivity analysis of times required
for metabolic rates to saturate Archean lakes with gas due to changes in temperature and
hydrostatic pressure is provided in Supplemental Tables 2 and 3. Once saturation is reached,
determining the time for a metabolism to produce a specific bubble volume involves dividing the
observed bubble volume by the rate of gas production (S1).
Geochemical Analysis of Stromatolites
Investigating the geochemistry and mineralogy of fenestral textures in stromatolites can
further constrain the identity of specific gases present within ancient microbial mats. In both
Hartbeesfontein and Yellowstone stromatolites. opaque minerals are specifically associated
along the edges and bottoms of gas-related fenestrae. Identification of fenestrally-associated
minerals can help determine the initial chemical properties of fenestral bubbles, providing further
constraints on candidate gas-producing metabolisms. Geochemical analysis of opaque minerals
within Hartbeesfontein and Yellowstone fenestrae was performed on a Horiba XploRa+ micro-
Raman spectrometer and a Horiba XGT-7200 micro-XRF analyzer at the Natural History
Museum in Los Angeles, California.

136

RESULTS
Measured rates of oxygenic photosynthesis could produce Hartbeesfontein fenestral
textures between 6 minutes and 10 hours (Fig. 4, S1). In contrast, the fastest rates of sulfate
reduction and fermentative hydrogen production could produce the same volume of gas in 6 hrs,
with maximum production times of 190 and 1600 hours respectively. Finally, measured rates of
methanogenesis could produce appropriate volumes of gas between 14 and 280,000 hours (Fig.
4, S1). The mean times for oxygenic photosynthesis, sulfate reduction, fermentative hydrogen
production, and methanogenesis to produce Hartbeesfontein fenestrae are 1.2, 45, 412, and
31,006 hours respectively (Fig. 4, S1). While all metabolisms investigated could generate gases
at a rate consistent with Yellowstone preservational windows (Table 1, S1), the ratio of
metabolic rates that fell within preservational windows vs. total measured metabolic rates was
different for each metabolism. For example, all surveyed rate measurements (27/27) of oxygen
production could produce Hartbeesfontein fenestrae in under one day, with 22 rate measurements
producing appropriate amounts of gas in under an hour (Table 1). In contrast, only 39% of
surveyed sulfate reduction rates could produce observed textures within 24 hours, with
corresponding values for fermentative hydrogen production and methanogenesis at 20% and
10%, respectively (Table 1). At conservative Yellowstone preservational windows of 48 hours,
ratios of sulfate reduction, fermentative hydrogen production and methanogenesis that can
produce Hartbeesfontein fenestrae increase to 70%, 33%, and 19%, respectively. While the same
volume of gas can hypothetically form within longer timeframes, the likelihood of bubble
degeneration becomes higher as time increases, particularly in agitated environments.
The times required for metabolic gas production to saturate Archean lakes are orders of
magnitude lower than times necessary for the same rates to subsequently produce 0.043 ml/cm3
137

of gas bubbles. For example, metabolic rates that saturate solutions in 9.4E-8 hours subsequently
produce appropriate bubble volumes in 0.072 hours, and rates that saturate solutions in 220 hours
require 320,000 hours to produce the same bubble volumes afterwards (S2). Ratios of saturation
time to bubble production time vary between 8.6E-4 and 1.1E-6 (S3). The amount of time
required to saturate solutions will be lower still in solutions where some amount of dissolved gas
is present. Sensitivity analyses of solution saturation times were calculated for each gas between
15 and 35 °C, at depths up to 10 m (S2, S3). Saturation times at 15 °C were up to 1.6 times
slower than at 35 °C, and were 1.001 times slower at 0 m depth than at 10 m depth. Therefore, at
various temperatures and depths, the majority of time required to produce fenestral textures in
Archean stromatolites is spent producing bubbles once solutions reach saturation.
DISCUSSION
Potential for Modern Metabolisms to Produce Hartbeesfontein Textures
Rapid rates of gas production are critical to producing the large fenestrae observed in
Hartbeesfontein stromatolites, given the timeframe of bubble lithification compounded with an
agitated depositional environment. Oxygenic photosynthesis has the fastest average and
maximum gas production rates of any microbial metabolism, with all measurements falling well
within the daily lithification rates of Yellowstone stromatolites (Fig 4, Table 1). Even when
accounting for synchronous aerobic respiration, modern processes of oxygenic photosynthesis
could produce observed Hartbeesfontein fenestral fabrics within 1.2 hours on average, and at a
minimum of 6 minutes (Fig. 4, S1). No other metabolism could produce Hartbeesfontein
fenestrae in under an hour, even when accounting for changes in temperature and water depth.
138

The mineralogy associated with bubble fenestrae can be used as supporting evidence to
distinguish oxygen from other gases within microbial mats. In Yellowstone stromatolites, iron
oxides form around and within hourglass-associated fenestrae when spring waters react with
oxygen bubbles (Fig. 2G). Similarly, the bases of many Hartbeesfontein fenestrae contain
geopetal iron oxy-hydroxides such as goethite (Fig. 2H, S4, Wilmeth et al., in prep).
Furthermore, no sulfur or sulfide minerals were detected within Hartbeesfontein fenestral
mineralogy via XRF or Raman spectroscopy, supporting the absence of hydrogen sulfide within
fenestrae. The close association of oxidized iron with fenestral textures is consistent with the
hypothesis that Hartbeesfontein fenestrae were initially oxygen bubbles.
While the fastest observed rates of other metabolisms (e.g., sulfate reduction,
fermentative hydrogen production, methanogenesis) fall within the Yellowstone preservational
window (24-48 hours), other evidence preserved in the Hartbeesfontein stromatolites would
argue against non-oxygenic metabolisms. For example, 70% of observed sulfate reduction rates
could produce Hartbeesfontein fenestrae within a conservative 48-hour preservational window
based solely on modern rate measurements, (Table 1, S1). However, modeled marine Archean
sulfate concentrations range between 0.2 and 0.002 mM and were likely lower in non-marine
settings, orders of magnitude lower than 28 mM within modern oceans (Habicht et al., 2002;
Crowe et al., 2014). Lower sulfate concentrations limit sulfate reduction rates (Boudreau &
Westrich, 1984; Ingvorsen & Jorgenson, 1984; Nielsen, 1987), and thus restrict the plausibility
of sulfate reduction rapidly forming large hydrogen sulfide bubbles in Archean environments.
Finally, sedimentary and petrographic evidence from Hartbeesfontein argues against sulfate
reduction as a candidate metabolism. The absence of sulfate minerals or pseudomorphs within
Hartbeesfontein subaerial and evaporite facies indicates low sulfate concentrations, and no sulfur
139

was observed in XRF and Raman analysis of opaque fenestrally-associated minerals.
Additionally, vuggy cherts present above Hartbeesfontein stromatolites have previously been
interpreted as former magadiite deposits (Karpeta, 1989). Magadiite formation occurs at
conditions around ~10 pH within alkaline lakes (Eugster, 1967), suggesting that the
Hartbeesfontein lake was rather alkaline. At pH values >8.0, the dominant sulfide species is HS
-
,
with little hydrogen sulfide present to form bubbles.
Hydrogen can be produced through a variety of metabolic pathways, predominantly via
cyanobacterial fermentation (Burow et al., 2012; Lee et al., 2014; Nielsen et al., 2015). The
fastest rates of hydrogen production occur within bubble-rich cyanobacterial mats from Guerrero
Negro, Mexico and Elkhorn Slough, USA (Hoehler et al., 2001; Burow et al., 2012). While
hydrogen production from Guerrero Negro can hypothetically produce Hartbeesfontein
structures in 6 hours, those quantities of hydrogen only comprise 10% of observed bubble
volume, with the other 90% comprised of oxygen produced by photosynthesis (Hoehler et al.,
2001). Likewise, bubble production in Elkhorn Slough ceases when mats are treated with 3-(3,4-
dichlorophenyl)-1,1-dimethylurea (DCMU), which inhibits Photosystem II in cyanobacteria
(Burow et al., 2012). Therefore, observations from modern microbial mats indicate that even the
fastest known hydrogen production rates within appropriate preservational windows are not
sufficient to produce observed Hartbeesfontein fenestrae. The same microbial communities with
high fermentation rates are more likely to produce higher quantities of oxygen during the same
preservational windows, outpacing hydrogen production (Fig.3, S1). In the agitated
environments of Hartbeesfontein stromatolite deposition, oxygen bubbles are more likely to
reach observable sizes between disruptions than hydrogen bubbles.
140

The only observed rates of methanogenesis within microbial mats that could produce
Hartbeesfontein fenestrae within a conservative 48-hour window occur in organic-rich wetlands,
with all other rates requiring thousands of hours (Fig. 4, S1) (King, 1990). However, modern
marshes and wetlands are not suitable analogues for Archean environments due to the relative
abundance of organic material present to convert to methane. Hydrothermal vents can also
plausibly provide a constant supply of hydrogen for hydrogenotrophic methanogenesis, but no
evidence for vents was observed within Hartbeesfontein sediments. Finally, while hydrogen-
producing metabolisms within mats have been observe to stimulate methanogenesis within
microbial mats, resultant methane production is still an order of magnitude slower than
appropriate preservational windows (Hoehler et al., 2001).
Based on metabolic, petrographic and sedimentary evidence, oxygenic photosynthesis is
the most likely candidate metabolism for fenestral fabrics observed within 2.7 Ga
Hartbeesfontein stromatolites. The direct investigation of an ancient microbial mat supports
geochemical and molecular clock evidence for oxygenic photosynthesis evolving well before the
Great Oxidation Event (Anbar et al., 2007; Kump & Barley, 2007; Kendall et al., 2010; Gaillard
et al., 2011; Crowe et al., 2013; Riding et al., 2014). The presence of rapid oxygen production
rates within lacustrine deposits ~300 million years before the GOE supports previous hypotheses
regarding the delay between early oxygenic photosynthesis and the eventual oxidation of Earth’s
atmosphere. One model suggests that microbial mats in terrestrial and freshwater environments
could have fueled oxidative weathering of the continents for millions of years in isolated
“oxygen oases” without impacting atmospheric oxygen levels (Lalonde & Konhauser, 2015).
Additional evidence for non-marine oxygen production comes from phylogenomic studies of
cyanobacteria, which indicate preference for freshwater environments as an ancestral trait of the
141

clade (Sanchez-Barcaldo et al., 2005; Blank & Sanchez-Baracaldo, 2010). Therefore,
Hartbeesfontein stromatolites not only corroborate evidence for pre-GOE oxygenic
photosynthesis, but also support geochemical and molecular phylogenetic models for the non-
marine evolution of cyanobacteria. While the Ventersdorp Supergroup and the roughly
equivalent Fortescue Group of Western Australia are the oldest known lacustrine deposits on
Earth, the investigation of fenestral textures within older and younger stromatolites has the
potential to extend a visual record of oxygen production and cyanobacterial expansion across
different environments over time.

ACKNOWLEDGEMENTS
This research was supported by the National Science Foundation Graduate Research Fellowship,
the Geological Society of America Student Research Grant, the Lewis and Clark Fund for
Exploration and Field Research, and the University of Southern California Earth Science
Department Research Fund. We thank the University of Johannesburg and Dr. Alyssa Bell (LA
Natural History Museum) for logistical support associated with the fieldwork of summer 2014.
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150

FIGURES: CHAPTER 3

FIGURE 1: Laminae and filaments in Yellowstone and Hartbeesfontein stromatolites. A, C, E
are from Obsidian Pool Prime, Yellowstone National Park. Blue coloration comes from epoxy
impregnation. B, D, F are from Hartbeesfontein, South Africa. A, B: Light and dark laminae
patterns within stromatolites. Dark laminae in Yellowstone stromatolites are comprised of
cyanobacterial filaments, while dark laminae in Hartbeesfontein stromatolite are defined by
detrital material. C, D: Separation of filaments along laminae. E, F: Filamentous textures within
“hourglass structures” separating fenestrae.
151

FIGURE 2: Fenestral textures within Yellowstone and Hartbeesfontein stromatolites. A, C, E
are from Obsidian Pool Prime, Yellowstone National Park. B, D, F are from Hartbeesfontein,
South Africa. A, B: Circular and sub-circular fenestrae separated by “hourglass structures”
comprised of filaments. C, D: Larger, less circular fenestrae, concentrically surrounded by
filaments. E, F: Opaque iron oxides associated with fenestrae.

152

FIGURE 3: Morphological evidence of coalescing gas bubbles within microbial mats. A:
Simplified illustration of mat cross-section with expanding gas bubbles entrained between
filamentous “hourglass structures”. B: Illustration representing a period of time after A when
bubble expansion has severed hourglass structures and formed a larger, less spherical bubble. C,
D: Fenestral examples of A and B from Yellowstone National Park. E, F: Fenestral examples
from Hartbeesfontein, South Africa.

153

FIGURE 4: A survey of gas production rates within modern microbial mats, expressed as the
time required for metabolic rates to produce fenestral textures within Hartbeesfontein
stromatolites (0.043 m mL gas/cm
3
mat). Time is represented in log(hours required to produce
fenestrae) (see Supplemental Table 1). Dashed line represents the 24-hour maximum
preservation window observed within Yellowstone stromatolites (see Table 1 for specific
number of metabolisms within the preservation window). Light green represents net oxygen
production (photosynthesis-respiration), dark green represents gross oxygen production, light
brown represents methanogenesis within wetland mats (excluding peats), and dark brown
represents methanogenesis in all other environments.
154

TABLES AND SUPPLEMENTARY INFORMATION
TABLE 1:
Comparison of surveyed metabolic rates and limiting reactants for different gas-producing
metabolisms. 24 hours represents the maximum preservational window for fenestrae in
Yellowstone stromatolites.
Metabolism
Total # of
Modern
Measurements
# of Measurements
That Can Produce
Hartbeesfontein
Fenestrae Within:
Limiting Reactants
in Archean
Environments 1 hr 12 hrs 24 hrs
Oxygenic Photosynthesis 27 22 27 27 None
Sulfate Reduction 23 0 3 9 SO4
2-
Fermentation 15 0 1 3 None
Methanogenesis 21 0 0 2 H2

SUPPLEMENTARY TABLE 1:
Compiled data for gas production rates measured in situ within microbial mats.
Ref Mat Location & Environment
Gas Production
Rate
Time to make observed
HBF fenestrae

(µmoles/cm
3
*hr) Hours Days Years

Oxygenic Photosynthesis

1 Guerrero Negro, Mexico, saltern 1.75E+00 1.10

1 Guerrero Negro, Mexico, saltern 2.30E+00 0.84

1 Guerrero Negro, Mexico, saltern 3.60E+00 0.53

2 Guerrero Negro, Mexico, saltern 3.00E+00 0.64

2 Guerrero Negro, Mexico, saltern 2.00E+01 0.10

3 Guerrero Negro, Mexico, saltern 3.00E-01 6.41

3 Guerrero Negro, Mexico, saltern 2.76E+01 0.07

4 Guerrero Negro, Mexico, saltern,
net oxygen production
3.40E+00 0.57

4 Guerrero Negro, Mexico, saltern,
net oxygen production
9.18E+00 0.21

5 Wismar Bight, Germany, intertidal,
net oxygen production
8.60E+00 0.22

5 Wismar Bight, Germany, intertidal,
net oxygen production
1.98E+01 0.10

6 La Salada de Chiprana, Spain,
hypersaline lake
1.76E+00 1.09

155

6 La Salada de Chiprana, Spain,
hypersaline lake
2.49E+00 0.77

6 La Salada de Chiprana, Spain,
hypersaline lake
3.91E+00 0.49

6 La Salada de Chiprana, Spain,
hypersaline lake
4.27E+00 0.45

7 M'Bo Islet, New Caledonia, lagoon,
net oxygen production
2.00E-01 9.62

7 M'Bo Islet, New Caledonia, lagoon,
net oxygen production
2.10E+00 0.92

7 Poe Beach, New Caledonia,
shallow back-reef, net oxygen
production
2.60E+00 0.74

7 Tabu Reef, New Caledonia, lagoon,
net oxygen production
3.40E+00 0.57

8 Solar Lake, Egypt, hypersaline lake 5.00E-01 3.85

8 Solar Lake, Egypt, hypersaline lake 2.75E+00 0.70

8 Solar Lake, Egypt, hypersaline lake 9.00E+00 0.21

8 Solar Lake, Egypt, hypersaline lake 1.20E+01 0.16

9 Solar Lake, Egypt, hypersaline
lake, net oxygen production
4.32E+00 0.45

9 Solar Lake, Egypt, hypersaline
lake, net oxygen production
8.80E+00 0.22

9 Solar Lake, Egypt, hypersaline
lake, net oxygen production
1.04E+01 0.18

9 Solar Lake, Egypt, hypersaline
lake, net oxygen production
1.30E+01 0.15

Sulfate Reduction
3 Guerrero Negro, Mexico, saltern 1.91E-01 9.0

8 Solar Lake, Egypt, hypersaline lake 2.15E-01 8.0

10 Elkhorn Slough, USA, estuary 6.43E-02 26.7 1.11

11 Guerrero Negro, Mexico, saltern 1.26E-01 13.6

11 Guerrero Negro, Mexico, saltern 2.70E-01 6.4

12 Eilat, Israel, saltern 2.29E-02 75.0 3.12

12 Eilat, Israel, saltern 2.92E-02 58.9 2.45

12 Eilat, Israel, saltern 5.00E-02 34.4 1.43

12 Eilat, Israel, saltern 5.42E-02 31.7 1.32

12 Eilat, Israel, saltern 5.42E-02 31.7 1.32

13 Lotgen Lagoon, Denmark 1.00E-01 17.2

13 Lotgen Lagoon, Denmark 1.20E-01 14.3

14 Guerrero Negro, Mexico, saltern 3.33E-02 51.6 2.15

14 Guerrero Negro, Mexico, saltern 8.33E-02 20.6

156

15 Bonaire, Netherlands Antilles,
hypersaline lake
3.33E-02 51.6 2.15

15 Bonaire, Netherlands Antilles,
hypersaline lake
3.75E-02 45.8 1.91

16 Shark Bay, Australia, intertidal 7.83E-02 21.9

17 Shark Bay, Australia, intertidal 4.10E-02 42.0 1.75

17 Shark Bay, Australia, intertidal 4.26E-02 40.4 1.68

18 Fisherman Bay, Australia, tidal 1.75E-02 98.2 4.09

18 Spencer Gulf, Australia, tidal 8.67E-02 19.8

19 Texel, Netherlands, supralittoral 9.17E-03 187.5 7.81

19 Texel, Netherlands, supralittoral 1.29E-02 133.0 5.54

Fermentation -- Hydrogen

10 Elkhorn Slough, USA, estuary 4.00E-02 48.4 2.02

14 Guerrero Negro, Mexico, saltern 2.00E-03 967.6 40.32
14 Guerrero Negro, Mexico, saltern 2.50E-03 774.1 32.25
14 Guerrero Negro, Mexico, saltern 1.17E-02 165.9 6.91
20 Elkhorn Slough, USA, estuary 8.00E-03 241.9 10.08
20 Elkhorn Slough, USA, estuary 8.33E-02 23.2
20 Elkhorn Slough, USA, estuary 1.13E-01 17.1
21 Guerrero Negro, Mexico, saltern 1.21E-03 1601.6 66.73
21 Guerrero Negro, Mexico, saltern 1.73E-03 1119.2 46.63
21 Guerrero Negro, Mexico, saltern 2.00E-03 967.6 40.32
21 Guerrero Negro, Mexico, saltern 3.15E-01 6.2
22 Guerrero Negro, Mexico, saltern 1.55E-02 124.9 5.20
22 Guerrero Negro, Mexico, saltern 3.10E-02 62.4 2.60
23 Limfjorden, Denmark, supralittoral 5.43E-02 35.6 1.48
23 Saline de Giraud, France, saltern 7.93E-02 24.4 1.02

Methanogenesis
1 Guerrero Negro, Mexico, saltern 1.07E-04 16459.3 685.8 1.9
1 Guerrero Negro, Mexico, saltern 1.91E-04 9244.9 385.2 1.1
10 Elkhorn Slough, USA, estuary 5.00E-05 35222.9 1467.6 4.0
21 Guerrero Negro, Mexico, saltern 4.06E-04 4335.1 180.6 0.5
21 Guerrero Negro, Mexico, saltern 3.60E-04 4886.4 203.6 0.6
21 Guerrero Negro, Mexico, saltern 6.25E-06 281783.6 11741.0 32.2
21 Guerrero Negro, Mexico, saltern 1.25E-05 140891.8 5870.5 16.1
24 Sippewissett Salt Marsh, USA,
intertidal wetlands
1.92E-04 9188.6 382.9 1.0
24 Sippewissett Salt Marsh, USA,
intertidal wetlands
2.50E-05 70445.9 2935.2 8.0
157

24 Sippewissett Salt Marsh, USA,
intertidal wetlands
3.25E-04 5418.9 225.8 0.6
25 Solar Lake, Egypt, hypersaline lake 2.00E-04 8805.7 366.9 1.0
25 Solar Lake, Egypt, hypersaline lake 2.67E-04 6604.3 275.2 0.8
25 Solar Lake, Egypt, hypersaline lake 4.76E-05 36984.1 1541.0 4.2
25 Solar Lake, Egypt, hypersaline lake 1.44E-04 12192.6 508.0 1.4
26 Bretagne, France, saltern 2.13E-04 8287.8 345.3 0.9
27 Denmark, wetlands 1.20E-01 14.7
27 Denmark, wetlands 1.05E-01 16.8
27 Denmark, wetlands 6.00E-02 29.4 1.2
27 Denmark, wetlands 2.40E-02 73.4 3.1
27 Denmark, wetlands 5.00E-02 35.2 1.5
27 Denmark, wetlands 8.44E-03 208.7 8.7

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162

SUPPLEMENTARY TABLE 2:
Calculated aqueous gas concentrations at saturation in Archean environments. The sensitivity of
Henry’s Constants to temperature and pressure were calculated using equations within
Supplementary Table 2 Citations 1 and 2, respectively, using data from Citations 1, 3, and 4.
Saturation concentrations of gas were calculated by multiplying calculated Henry’s constants
with estimated Archean partial pressures for each gas. Maximum estimates for Archean partial
pressure were used to provide estimates of saturation concentrations and conservative times for
metabolisms to saturate solutions (see Supplementary Table 3).

Gas Temp Depth
Henry’s
Constant
Archean
Partial
Pressure
Partial
Pressure
Citation
Saturation
Gas Conc.

°C m moles/L*atm atm moles/L
O 2 15 0 1.6E-3 2E-6 3 3.1E-09
O 2 25 0 1.3E-3 2E-6 3 2.6E-09
O 2 35 10 1.1E-3 2E-6 3 2.2E-09

H 2S 15 0 1.26E-1 1E-5 4 1.3E-06
H 2S 25 0 9.9E-2 1E-5 4 9.9E-07
H 2S 35 10 7.9E-2 1E-5 4 7.9E-07

H 2 15 0 8.2E-4 1E-3 5 8.2E-07
H 2 25 0 7.7E-4 1E-3 5 7.7E-07
H 2 35 10 7.3E-4 1E-3 5 7.3E-07

CH 4 15 0 1.7E-3 1E-3 6 1.7E-06
CH 4 25 0 1.4E-3 1E-3 6 1.4E-06
CH 4 35 10 1.2E-3 1E-3 6 1.2E-06

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164

SUPPLEMENTARY TABLE 3:
Calculated times for maximum and minimum microbial gas production rates (see Supplementary
Table 1) to saturate aqueous solutions in Archean environments, compared with times for the
same rates to subsequently form observed bubble fenestrae in Hartbeesfontein stromatolites.
Calculations for saturation gas concentrations in Archean environments were calculated in
Supplementary Table 2.
Gas T Depth
Saturation
Gas Conc.
Bubble
Gas Conc.
Gas
Production
Rate
Max/
Min
Rate
Time to
Aqueous
Saturation
Time
To
Form
Bubbles
Saturation
Time
/Bubble
Time

°C m moles/L moles/L
moles gas/
(L)(hr) hr hr

O 2 15 0 3.1E-09 2.0E-03 2.8E-02 Max 1.1E-07 0.072 1.5E-06
O 2 15 0 3.1E-09 2.0E-03 2.0E-04 Min 1.5E-05 10 1.5E-06
O 2 25 0 2.6E-09 2.0E-03 2.8E-02 Max 9.4E-08 0.072 1.3E-06
O 2 25 0 2.6E-09 2.0E-03 2.0E-04 Min 1.3E-05 10 1.3E-06
O 2 35 10 2.2E-09 2.0E-03 2.8E-02 Max 8.0E-08 0.072 1.1E-06
O 2 35 10 2.2E-09 2.0E-03 2.0E-04 Min 1.1E-05 10 1.1E-06

H 2S 15 0 1.3E-06 2.0E-03 2.7E-04 Max 4.7E-03 7.4 6.3E-04
H 2S 15 0 1.3E-06 2.0E-03 9.2E-06 Min 1.4E-01 220 6.3E-04
H 2S 25 0 9.9E-07 2.0E-03 2.7E-04 Max 3.7E-03 7.4 4.9E-04
H 2S 25 0 9.9E-07 2.0E-03 9.2E-06 Min 1.1E-01 220 4.9E-04
H 2S 35 10 7.9E-07 2.0E-03 2.7E-04 Max 2.9E-03 7.4 3.9E-04
H 2S 35 10 7.9E-07 2.0E-03 9.2E-06 Min 8.6E-02 220 3.9E-04

H 2 15 0 8.2E-07 2.0E-03 3.2E-04 Max 2.6E-03 6.3 4.1E-04
H 2 15 0 8.2E-07 2.0E-03 1.2E-06 Min 6.8E-01 1700 4.1E-04
H 2 25 0 7.7E-07 2.0E-03 3.2E-04 Max 2.4E-03 6.3 3.9E-04
H 2 25 0 7.7E-07 2.0E-03 1.2E-06 Min 6.4E-01 1700 3.9E-04
H 2 35 10 7.3E-07 2.0E-03 3.2E-04 Max 2.3E-03 6.3 3.6E-04
H 2 35 10 7.3E-07 2.0E-03 1.2E-06 Min 6.0E-01 1700 3.6E-04

CH 4 15 0 1.7E-06 2.0E-03 1.2E-04 Max 1.4E-02 17 8.6E-04
CH 4 15 0 1.7E-06 2.0E-03 6.3E-09 Min 2.7E+02 320000 8.6E-04
CH 4 25 0 1.4E-06 2.0E-03 1.2E-04 Max 1.2E-02 17 7.0E-04
CH 4 25 0 1.4E-06 2.0E-03 6.3E-09 Min 2.2E+02 320000 7.0E-04
CH 4 35 10 1.2E-06 2.0E-03 1.2E-04 Max 9.6E-03 17 5.8E-04
CH 4 35 10 1.2E-06 2.0E-03 6.3E-09 Min 1.8E+02 320000 5.8E-04

165

SUPPLEMENTARY 4:

A: Opaque minerals forming geopetal structures along the bottom of a Hartbeesfontein fenestra.
B: Raman spectrum of A, compared with known Raman spectra for quartz and goethite.

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CHAPTER 4:
ENVIRONMENTAL AND BIOLOGICAL INFLUENCES ON CARBONATE
PRECIPITATION WITHIN HOT SPRING MICROBIAL MATS IN LITTLE HOT CREEK,
CA
ABSTRACT
Microbial mats are found in a variety of modern environments, with evidence for their
presence as old as the Archean. There is much debate about the rates and conditions of processes
that eventually lithify and preserve mats as microbialites. Here we apply novel tracer
experiments to quantify both mat biomass addition and the formation of CaCO3. Microbial mats
from Little Hot Creek (LHC), California, contain calcium carbonate that may have formed
within mat layers, and thus constitute a good test case to investigate the relationship between
rates of mat growth and carbonate precipitation. The LHC mats were divided into four layers via
color and fabric and waters within and above the mat were collected to determine carbonate
saturation state. Mat samples were also collected for 16S rRNA analysis of microbial
communities in each layer. Rates of carbonate precipitation and carbon fixation were measured
in the laboratory by incubating samples from each mat layer with δ
13
C-labeled HCO3
-
for 24
hours. Comparing these rates with those from experimental controls, poisoned with NaN3 and
HgCl2, allowed for differences in biogenic and abiogenic precipitation to be determined. Carbon
fixation rates were highest in the top layer of the mat (0.17% new organic carbon/day), which
also contained the most phototrophs. Isotope-labeled carbonate was precipitated in all four layers
of living and poisoned mat samples. In the top layer, the precipitation rate in living mat samples
was negligible although abiotic precipitation occurred. In contrast, the bottom three layers
exhibited biologically enhanced carbonate precipitation. The lack of correlation between rates of
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carbon fixation and biogenic carbonate precipitation suggests more significant roles for
processes other than autotrophy to preserve mats as microbialites.
INTRODUCTION
Microbial mats have been preserved within the rock record over 3.5 billion years (e.g.,
Walter et al., 1980; Noffke et al., 2006; Schopf, 2006; Bosak et al., 2009). Structures attributed
to microbial precipitation of carbonate minerals, commonly termed microbialites, are particularly
common in the geologic past (e.g, Awramik and Sprinkle, 1999; Grotzinger and Knoll, 1999;
Riding, 2000; Bosak et al., 2013). Despite the ubiquity of microbial mats in a variety of modern
aqueous environments, few appear to be mineralized in a way that would lead to the preservation
of a microbialite. When identified, modern microbial mats that precipitate minerals provide
potential analogues for the formation of ancient microbialites (Reid et al., 2000; Visscher et al.,
2000; Dupraz et al., 2004; Dupraz & Visscher, 2005; Vasconcelos et al., 2006; Dupraz et al.,
2009; Mata et al., 2012).
The metabolic activity of microbial communities, physical mat properties, and the
physicochemical changes in the surrounding environment can influence mineral saturation states
and induce mineral precipitation (Pentecost & Riding, 1986; Arp et al., 1999a; Riding, 2000;
Visscher et al., 2000; Dupraz et al., 2009). This study defines mineralization that occurs when
the activities of living microbes act to enhance precipitation as biogenic precipitation (see also
“organomineralization sensu stricto” in Trichet & Defarge, 1995, “microbially-induced
precipitation” in Dupraz et al., 2009). Mineralization within mats that is independent of living
microbial activity is defined as abiogenic precipitation (see also “biologically-influenced
precipitation” in Frankel & Bazylinski, 2003; Dupraz et al., 2009). Three factors can influence
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the precipitation of calcium carbonate within microbial mats, whether biogenic or abiogenic in
nature: the concentrations of CO3
2-
ions, Ca
2+
ions, and surface chemistry or nucleation centers
(Dupraz & Visscher et al., 2009). The first two factors relate to the saturation state of calcium
carbonate, defined as omega ( Ω), the product of calcium and carbonate ion concentrations
divided by the solubility constant Ksp’ for the appropriate mineral (where the symbol ’ accounts
for the activities of Ca
2+
and CO3
2-
). When calcium carbonate is at equilibrium in solution, Ω =
1, with under- and over-saturated solutions bearing lower and higher values respectively. The
third factor relates to the potential for locations within microbial mats to serve as nuclei for
carbonate minerals to form (Arp et al., 1999a; Dupraz et al., 2009).
Certain metabolisms have been studied as candidates for inducing biogenic carbonate
precipitation, including oxygenic photosynthesis (Pentecost & Riding, 1986; Merz-Preiß &
Riding, 1999; Riding, 2000), anoxygenic photosynthesis (Bundeleva et al., 2012), sulfate
reduction (Visscher et al., 2000; Dupraz et al., 2004; Gallagher et al., 2012), and anaerobic
oxidation of methane (Michaelis et al., 2002). For example, cyanobacteria increase saturation
through carbon fixation by raising pH and removing CO2. Cyanobacteria can also produce
abundant exopolymeric substances which can serve as nucleation centers for carbonate
precipitation (De Philippis et al., 1998; Obst et al., 2009). However, precipitation induced via
cyanobacterial photosynthesis occurs primarily in freshwater environments with low dissolved
inorganic carbon and high calcium concentrations, and is not correlated with areas of increased
carbon fixation (Merz-Preiß & Riding, 1999; Arp et al., 1999a, 1999b, 2001). In lakes with high
dissolved inorganic carbon, aragonite precipitation is only associated with cyanobacteria which
contain an external fibrous layer on cell walls (Couradeau et al., 2013; Gerard et al., 2013).
Finally, internal accumulation of calcium has been observed in multiple cyanobacterial lineages
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from lacustrine environments and hot springs (Ragon et al., 2014; Cam et al., 2017). Therefore,
external environmental factors, cellular morphologies, and intracellular chemistry can affect
precipitation in microbial mats even when metabolisms that increase carbonate saturation are
abundant.
One method used to examine biologically-induced precipitation is the comparison of
metabolic rates with patterns of carbonate mineralization within microbial mats. However,
studies directly comparing rates of precipitation and metabolic activity remain limited. Within
modern Bahamian stromatolites, laminae with high sulfate reduction rates appear to coincide
with zones of carbonate precipitation (Visscher et al., 2000; Dupraz et al., 2004). Rates of sulfate
reduction were measured using incubations with
35
S-labeled SO4
2-
, while carbonate precipitation
was assessed through petrographic analysis (Visscher et al., 2000). In contrast, numerical models
of metabolic impact on carbonate saturation hypothesize that sulfate reduction coupled with H2S
oxidation decreases Ω (Aloisi, 2008; Meister, 2013). Bundeleva et al., (2012) found a correlation
between carbonate precipitation and biomass production through anoxygenic
photosheterotrophic growth in pure cultures of the Proteobacterium Rhodovulum in
supersaturated solutions ( Ω = 10-120).
Measuring carbonate precipitation rates within microbial mats complements constraints
on microbialite growth rates determined in studies of stromatolite accretion. Previous hypotheses
have suggested that stromatolite laminae form daily via the diurnal activity of cyanobacteria
(e.g., Doemel & Brock, 1974; Golubic & Focke, 1978; Pepe-Ranney et al., 2012). In contrast,
studies of Holocene stromatolites using
14
C dating suggest seasonal or even multiyear lamination
rates (Chivas et al., 1990; Berelson et al., 2011; Petryshyn et al, 2012). The variability in
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stromatolite accretion rates is likely due to the variety of biogenic and abiogenic precipitation
mechanisms within microbial mats. Comparing precipitation rates in relation to specific
metabolisms or physicochemical processes in turn provides constraints for modern and ancient
microbialite growth.
This study examines the effects of microbial metabolism and environmental factors on
nascent carbonate precipitation within microbial mats from Little Hot Creek, California by
focusing on the rate of carbon fixation into biomass and concomitant carbonate precipitation.
Isotope labeling experiments were used to compare rates of carbonate precipitation and
autotrophic production of organic carbon during incubations with
13
C enriched bicarbonate.
Rates of precipitation were compared between active and poisoned microbial mats to discern
biogenic from abiogenic carbonate production.
SITE DESCRIPTION AND PREVIOUS WORK
Little Hot Creek (LHC) is a stream sourced from hydrothermal springs within the Long
Valley Caldera of eastern California (Fig. 1A,B). The source of LHC is on the eastern flank of a
resurgent dome formed by a rising magma chamber beneath the caldera (Sorey et al., 2003).
Thermal waters emerge at ~80 °C from several vents (Vick et al., 2010). The vents discharge
into LHC, where the waters cool to ~50 °C over the course of approximately 30 meters. Several
recent studies have characterized the microbiology and geochemistry of parts of the site. Vick et
al. (2010) examined the microbial complement of the LHC source vents, and determined that
groups related to the Aquificae and Thermodesulfobacteria, among others, were present in the
hottest portions of the system, where the thermal waters discharge onto the surface. Source vent
environments had circumneutral pH (6.75) and calcium concentrations ~0.55 mM (Vick et al.,
171

2010). Breitbart et al., (2004) examined phage-microbial interactions in LHC streams from
several vent sources. While specific taxa were not described, the microbial communities at one
location several meters downstream (74 °C, pH 7.7) had a turnover rate of under one day based
on labeled thymidine incubations (Breitbart et al., 2004). Dendrolitic cone structures in microbial
mats from a cooler, adjacent pool (45 °C) have also been studied as a potential analogue for
ancient microbialites (Bradley et al., 2017).
Extensive microbial mats occur 25 m downstream from source waters, where flow
velocity decreases as the creek bed widens from < 1 m to 8 m across (Fig. 1C). Textures and mat
thickness differ between wholly subaqueous mats and those at the creek surface. Subaqueous
mats occur towards the center of the creek and are predominantly green with small orange
patches (Fig. 1C, 1D). Portions of the subaqueous mats several centimeters wide are partially
detached from the creek bed, forming “rollover structures” (Hagadorn, 2012) and exposing
patches of underlying sediment. The resulting voids indicate a maximum mat thickness of 1 to 2
cm. In contrast, surficial mats are orange to tan, and usually extend from creek margins up to 1 m
into LHC (Fig. 1C,1D), with smaller patches in the middle of the stream. Surficial mats rise up to
10 cm above the LHC creek bed, often reaching the creek’s surface. Surficial mat textures do not
exhibit rollover features seen in subaqueous mats.
METHODS
Field Geochemical Analysis
Microbial mats and creek waters were sampled ~30 m downstream from LHC headwaters
(Fig. 1B). Temperature, pH, calcium concentrations, TCO2 (H2CO3 + HCO3
-
+ CO3
2-
), and δ
13
C
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of the TCO2 were measured in pore waters 1 and 5 cm deep within the sampled mat.
Measurements were also made in stream waters 20 cm upstream, and 80 cm downstream from
the mat. All field geochemical measurements were performed at the same time (June 2015),
except for pH values within mat pore water, which were measured in August 2015. Temperature,
TCO2, δ
13
C, calcium concentrations and stream water pH values were virtually identical in June
and August, increasing the confidence in using August pore water pH values to calculate
saturation states. Saturation states of calcium carbonate within and around the LHC mat were
calculated in CO2SYS (Pierrot & Wallace, 2006) using pH data from a SevenGo Duo pH meter
(Mettler Toledo) and TCO2 data obtained using a cavity ringdown spectrometer (Picarro).
Samples for calcium analysis were collected from the stream using a clean 20ml syringe, filtered
onsite with a 0.45 micron filter, and preserved with 2 drops of HCl. Calcium concentration was
measured on the acidified sample using a microwave induced plasma-optical emission
epectrometer (Agiilent).
Mat Extraction and Description
A piece of surficial microbial mat was selected for removal from LHC (Fig. 2A). After
extraction, the sampled mat was placed in a container lined with sterile aluminum foil, along
with LHC water from around the mat to prevent desiccation, and was stored and transported on
ice before laboratory analyses. The sampled LHC mat was divided into four layers based on
texture and color (Fig. 2B). Layer A was 1 cm thick and graded from bright orange at the mat-
water interface to olive green at the boundary with Layer B. Layer A was extremely cohesive,
with isolated pieces retaining shape when separated from the mat. Layer B was 3 cm thick, tan,
and lacked the cohesiveness of Layer A. Layer C was 1 cm thick, light pink, and was more
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cohesive than Layers B or D, but less than Layer A. Layer D was 5 cm thick and gray, with
similar consistency to Layer B. Minerals from all layers were examined using an environmental
scanning electron microscope (SEM) (Fig. 3). No preparation of sample was required for SEM
examination.
Incubation Experiments
The rates of biogenic carbonate precipitation and autotrophic production of organic
carbon in each layer were determined using incubation experiments with
13
C-labeled HCO3
-
.
Prior to incubation, each layer was homogenized and separated into two sample groups- one for
organic δ
13
C analysis, the other for inorganic δ
13
C analysis. In both groups, three sets of triplicate
samples ~2-3 mg each, (9 total) were prepared from each layer: 1) “Negative Control”: δ
13
C
values of mat carbonates and organic carbon were obtained without incubation or addition of
δ
13
C label; 2) “Poisoned Control”: Mat samples were placed into 2 mL glass vials containing 10
µL of 300 mM NaN3 and saturated HgCl2 solution to poison the mat, filled and capped without
headspace with LHC water amended with 5 mM HCO3
-
and a δ
13
C label of +2000 ‰; 3)
“Living”: Mat samples were placed into 2 mL vials and filled and capped without headspace
with LHC water amended with 5 mM HCO3
-
, and a δ
13
C label of +2000 ‰. Table 1 gives the
initial carbonate parameters for the stream and microbial mat pore water.
For both carbonate precipitation and organic carbon production experiments, Poisoned
Controls and Living samples were incubated for 24 hours at 40 °C. To account for changing
metabolism over a diel cycle, the incubator was set on a 12-hour light/dark cycle. After
incubation, samples for organic δ
13
C analyses were treated with 1 M HCl until effervescence
ceased. All incubations were washed with phosphate-buffered saline (PBS) three times by
174

centrifugation, decanting of the supernatant, and suspension of the pellet, in order to remove
labeled HCO3
-
that was not incorporated into carbonates or organic material. After HCl treatment
and the PBS wash, all samples were oven-dried at 60 °C.
Dried samples were ground, weighed (1-3 mg) and measured for % C and δ
13
C on a
Picarro cavity ringdown spectrometer (CRDS; Santa Clara, California) (Supp. Table 2). Samples
analyzed for carbonates were acidified with phosphoric acid in a closed vessel, with evolved CO2
passing into the Picarro CRDS. Organic carbon was analyzed on de-carbonated samples by
oxidizing dried material at 1000 °C in a Costech Elemental Analyzer and passing CO2 into the
Picarro. The averages of triplicate δ
13
C measurements for Living and Poisoned Control
incubations were compared with averages of Negative Control samples from respective layers
(Figs. 6, 7). Heavier δ
13
C values in Living and Poisoned Control incubated samples compared to
Negative Control samples indicated incorporation of labeled HCO3
-
by carbonate precipitation
(in the case of carbonate analysis, Fig. 6) or production of autotrophic organic carbon (in
samples combusted via EA, Fig. 5).
An analysis of the significance of different isotope values for different treatments was
determined by comparing δ
13
C triplicate averages (Δavg) with distributions of potential Δavg
values using bootstrap analyses (Efron, 1979; Efron & Tibshirani, 1986). After calculating the
experimental Δavg between two triplicate averages (Δavg
exp
), δ
13
C values from all six samples
were resampled at random to create two novel triplicate sets. The difference in these two
resampled triplicate sets produced a new Δavg value (Δavg
res
). δ
13
C values were resampled with
replacement, which indicates that a single δ
13
C value can be selected multiple times. For
example, resampling with replacement can produce two triplicates where all six δ
13
C values are
175

the same, producing a Δavg
res
of 0. Repeating the resampling process 1000 times produced
distributions of Δavg
res
values which were then compared with the measured Δavg
exp
. Values of
Δavg
exp
greater than 95% of Δavg
res
distributions (p < 0.05) were evaluated as non-random
representations of δ
13
C that increased during incubation experiments, supporting the hypothesis
that carbonate precipitation or carbon fixation occurred. Values of Δavg
exp
were also compared
with the Δavg that could be produced by machine variability alone on two identical triplicates
(Δavg
mac
). The standard deviation (σ) for multiple CRDS δ
13
C measurements is conservatively +
0.1 ‰ (Subhas et al. 2015) σ for a triplicate average is 0.058 ‰ (0.1x3
-0.5
), and σ for Δavg is
0.082 ‰ ((0.058
2
+0.058
2
)
0.5
). The Δavg
exp
values which were larger than 2σ (0.164 ‰) have less
than 5% probability of machine variability as an origin, while Δavg
exp
values less than σ have a
14% or higher probability.
The amount of new inorganic or organic carbon produced during incubations was
calculated using an isotope mass balance approach. Our primary assumption was that new
carbonate or autotrophic organic carbon would incorporate the heavier δ
13
C value of the HCO3
-
label with no significant fractionation. In making our isotope mass balance, we use the fractional
abundance of
13
C (
13
C/(
12
C +
13
C)) in Living and Poisoned Control samples with the bicarbonate
label before and after incubation as per Hayes (1983).
Percent new growth = (1 − ( 𝐹𝐹 𝑙𝑙𝑙𝑙 𝑙𝑙 𝑙𝑙 𝑙𝑙 𝑙𝑙 − 𝐹𝐹 𝐻𝐻𝐻𝐻𝐻𝐻 3 −
)/( 𝐹𝐹 𝑝𝑝 𝑝𝑝 𝑙𝑙 𝑝𝑝 𝑝𝑝 𝑙𝑙 𝑝𝑝 𝑝𝑝 𝑐𝑐 𝑝𝑝 𝑙𝑙 𝑐𝑐 𝑐𝑐 𝑝𝑝 𝑙𝑙 − 𝐹𝐹 𝐻𝐻𝐻𝐻𝐻𝐻 3 −
)) ∗ 100 (1)
F represents the
13
C/
12
C ratio in each component measured, and HCO3- denotes the bicarbonate
spike value. Abiogenic carbonate production rates were calculated by replacing the fractional
abundances for Living and Poisoned Control with Poisoned Control and Negative Control,
176

respectively. The solution to Eq. 1 was turned into a rate by dividing percent new carbon
production by the incubation time (24 hours), and were expressed as % new carbon/day.
DNA and Community Composition Analysis
The bacterial and archaeal composition of LHC mats was determined by sequencing
amplified libraries of small subunit ribosomal RNA (16S rRNA) genes. Field samples of all
layers were amplified and sequenced in triplicate, with subsequent single analyses of mat
samples taken from each layer immediately before and after incubation (see Fig, 8).
Extraction of DNA from each sample was performed using an Xpedition™ Soil/Fecal
DNA MiniPrep kit according to manufacturer’s instructions (Zymo Research Corp., Irvine, CA,
USA). Extracted DNA was amplified using primers that spanned the V4 region of the 16S rRNA
gene between positions 519 and 802 (Escherichia coli numbering), producing a product of
approximately 266 bp. The primer pair amplifies a broad distribution of both the Bacteria and
Archaea (Klindworth et al., 2013). The forward primer (M13L-519F: 5′- GTA AAA CGA CGG
CCA GCA CMG CCG CGG TAA -3′) contained the M13 forward primer (in bold), followed by
the 16S rRNA gene-specific sequence (underlined) to allow for barcoding of each sample in a
separate reaction (Stamps et al., 2016). The reverse primer (785R: 5′-TAC NVG GGT ATC
TAA TCC-3′) was taken directly from the “S-D-Bact07850b-A-18” reverse primer in
Klindworth et al. (2013).
Each 50 µL PCR reaction consisted of: 1X 5 PRIME HOT master mix (5 PRIME Inc.,
Gaithersburg, MD), 0.2 µM of each primer, molecular grade water, and 4 µL of extracted
template DNA. The thermal cycling used for PCR was the same as described in Stamps et al.,
177

(2016). Positive (E. coli) and negative (no template) controls were also amplified along with
sample template reactions. The amplified DNA molecules were then purified using AmpureXP
paramagnetic beads (Beckman Coulter Inc., Indianapolis, IN, USA) at a final concentration of
0.8 x v/v. A second, six cycle PCR was used to add a unique 12 bp barcode to each previously
amplified sample using a forward primer containing the unique barcode+M13 forward sequence
(5′-3′) and the original 785R primer (A mapping file is available at 10.5281/zenodo.1067761).
The final barcoded PCR products were again cleaned and concentrated using AmpureXP
paramagnetic beads at a final concentration of 0.8X (v/v), quantified using the QuBit dsDNA HS
assay (Life Technologies, Carlsbad, CA, USA), pooled in equimolar amounts, and concentrated
to a final volume of 80 μL using two Amicon® Ultra-0.5 mL 30K Centrifugal Filters (EMD
Millipore, Billerica, MA, USA).
The final pooled library was sequenced using the Illumina MiSeq platform (Illumina, San
Diego, CA, USA) and the PE250 V2 chemistry. After sequencing, reads were merged and de-
multiplexed using QIIME (Caporaso et al., 2010), filtered at a minimum quality score of 20
before being clustered into sub-operational taxonomic units (sOTUs) using Deblur (Amir et al.,
2017). Raw reads were deposited into the NCBI sequencing read archive (SRA) under the
accession number SRX2830741. An R markdown notebook and all required data to recreate the
16S rRNA gene analyses presented here are available at 10.5281/zenodo.1067761. Taxonomy
was assigned using mothur (Schloss et al., 2009) against the SILVA database (Release 128)
(Pruesse et al. 2007).

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RESULTS
Environmental Characterization
The LHC mat sampled for biomass and precipitation experiments was a surficial mat ~30
m from source waters, extending 50 cm from the shoreline into creek flow (Fig. 1D). The total
mat thickness was 10 cm with a gradational contact between the deepest layer (D) and the
underlying sediment. Unconsolidated mineral crystals were noted within the mat as granulated
textures during extraction, but no lithified structures such as carbonate crusts or continuous
nodules or layers were observed. Temperature, pH, TCO2, and δ
13
C data from upstream,
downstream, and pore waters within the mat collected prior to extraction are shown in Table 1.
The temperature of surface waters decreased from 52.4 °C upstream to 38.1 °C downstream of
the sampled mat. The temperature at the mat surface was 34.1 °C, increasing to 48.4 °C at 3 cm
mat depth. The pH of stream waters above the mat in June 2015 decreased from 8.34 upstream to
8.29 downstream. The pH within mats in August 2015 increased from 8.10 to 8.30 with depth.
TCO2 of creek waters decreased downstream, while δ
13
C values of TCO2 became slightly
heavier. With increasing mat depth, TCO2 increased and δ
13
C values of TCO2 decreased (see
Table 1). Mat pore waters were supersaturated with respect to calcium carbonate, with Ω
increasing from 3.23 at 0.5 cm depth to 4.27 at 10 cm.
A Layered Mat with Extensive Carbonate Precipitation
The microbial mat contained four layers that were visually defined, labeled A through D
from top to bottom (Fig. 2B). Weight percentages of organic and inorganic carbon were
inversely correlated within these mat layers (Fig. 4, Supp. Table 1). Layer A had the highest
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amount of organic carbon (5.1 wt%) and the lowest amount of carbonate (79.7 wt%), while
Layers B, C, and D varied between 0.6 to 1.7 wt% organic carbon and 93.1 to 96.9 wt%
carbonate. Despite the high wt% carbonate, the mat was not lithified and was easily
disarticulated with a spatula.
Upon removal of the organic matter, carbonate crystals were visible in all layers via SEM
(Fig. 3). Many carbonates formed individual rhombs up to 200 µm in diameter, with an average
width of 50 µm. Carbonate crystals within LHC mats are euhedral and lack evidence for erosion
such as rounding. Euhedral crystal morphologies indicate in situ precipitation, as opposed to
trapping and binding of detrital carbonates. Crystal abundance broadly corresponded with
measured carbonate weight percentage, with Layer A exhibiting less carbonate than Layers B
through D (Fig, 4). While the weight percentage of carbonate was high in all layers, the
carbonate crystals were not interlinked and did not form a solid framework.
Production Rates of Organic and Inorganic Carbon
Carbon fixation will produce more enriched δ
13
Corg values in mat samples incubated with
labeled bicarbonate than un-incubated samples. In all layers, δ
13
Corg values were not heavier (p >
0.05, see Supp. Table 3, Fig. 5) in poisoned than un-incubated samples, indicating little to no
microbial activity in the poisoned incubations after addition of NaN3 and HgCl2 and also
indicating that the addition of bicarbonate spike did not reside on the sample surface. In contrast,
organic δ
13
C values were heavier in living than poisoned samples in each layer, implying uptake
of labeled bicarbonate through autotrophic production of organic carbon (Fig. 5). Layers A and B
exhibited the highest rates of autotrophic carbon production (both 0.17% new organic
carbon/day, p = 0.01, 0.003 respectively), followed by Layer D (0.07% new organic carbon/day,
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p = 0.01, see Fig. 7). Production rates in Layer C were an order of magnitude lower than the
other three layers (0.011% new organic carbon/day, p = 0.036).
Analogous to the carbon fixation experiment, carbonate precipitation will also produce heavier
δ
13
Cinorg values in mat samples incubated with labeled bicarbonate than in un-incubated samples.
In Layers A, C and D, δ
13
Cinorg values were higher in poisoned than un-incubated samples (p =
0.021, 0.010, and 0.013, respectively, see Fig. 6, Supp. Table 3). The incorporation of labeled
13
C into carbonates in poisoned samples provides evidence for abiogenic precipitation in the
absence of active metabolisms. Poisoned samples from Layer B did not have heavier δ
13
Cinorg
values than un-incubated samples (Fig. 7, p = 0.071). This result shows that the incorporation of
spike into carbonate is not an artifact of experiment design, but that abiotic precipitation did not
occur in Layer B.
The lack of a difference between living and poisoned carbonate production in Layer A
(Fig. 6, p = 0.38) indicates that microbial activity/presence did not foster additional carbonate
production at the mat’s surface. While the Δavg
exp
measured in Layer B was slightly lower than
the 95% confidence interval in bootstrap analysis (p = 0.058), a comparison with Δ avg
mac
shows
only a 12% probability of machine variability explaining the observed data, similar to the
probability of machine variability in Layer C (10%), which has Δavg
exp
higher than the 95%
confidence interval (p = 0.034). The similarity in machine error probability between Layer C,
which passes the bootstrap confidence interval test for precipitation, and Layer B, which falls
just short of the confidence interval, increases confidence that some biogenic carbonate
precipitation occurred in Layer B. Inorganic δ
13
C values in Layer D were heavier in living than
in poisoned samples (p = 0.038). The incorporation of additional labeled
13
C into carbonates in
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living relative to poisoned samples from Layers B, C, and D is evidence that microbial activity
plays a role in increasing the carbonate content of these layers.
Mat Communities with Depth
Cyanobacteria and Bacterioidetes were the most abundant phyla in the surface Layer A
(see Fig. 8). Members of the phylum Cyanobacteria composed ~37-40% of the community,
including the genera Calothrix, Leptolyngbya, Synechococcus, and Phormidium. The phylum
Bacteroidetes (~18-23%) held the most abundant taxa in Layer A, an uncultured member of the
Saprospiraceae (17-22%). The rest of the community was predominantly composed of members
of the phyla Chloroflexi (~6-23%), Acidobacteria (~5-9%), Verrucomicrobia (3-9%), and
Planctomycetes (~3-6%). Compared to samples preserved immediately in the field, the
Chloroflexi from Layer A seemed to respond favorably during the 24 h experimental incubation,
more than doubling in relative abundance (from ~9% to 18%). The microbial community in
Layer B may have contained abundant novel taxa, as no taxonomic classification was given to
10-12% of these sequences, and while of quality sequence, they remain unclassified.
Ignavibacteria responded favorably during transportation prior to incubation, more than doubling
from ~7 to 16%. Additionally, Aminicenantes (Candidate Phylum OP8) increased from 5.9% to
15.1% during incubations. The other most abundant classified taxa in Layer B were members of
the phyla Proteobacteria (3-9%), Chlorobi (4-7%), Planctomycetes (3-12%), and Chloroflexi (1-
4%).
Layers C and D had very similar communities based on 16S rRNA gene analysis.
Aminicenantes was the most abundant phylum in Layers C and D, at 15-73% and 46-67%,
respectively. The phylum did not respond favorably during transportation in Layer C, decreasing
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from ~70% from samples taken immediately in the field to 14.9% and 30.1% immediately prior
to and after laboratory incubation, respectively. Unclassified taxa also formed a major fraction of
the community (~10-13% in C and ~8-25% in D). Although the Ignavibacteriae were a relatively
minor fraction of the microbial community in samples from Layer C preserved in the field
(~2%), they increased in relative abundance over five-fold during incubations (5.5 - 27%). The
rest of the sequences in Layers C and D were members of the phyla Chloroflexi, Nitrospirae,
Planctomycetes, and Bathyarcheota, present as 2 to 5% of the community.
DISCUSSION
Correlating Carbon Fixation with Carbonate Precipitation within Mat Layers
Autotrophic production of organic carbon occurred in all layers of non-poisoned mats
during the incubation experiments (Fig. 7). The rate of this carbon fixation in Layers A and B at
the top of the mat (approx. 0.2 % new organic carbon/day) was more than twice as fast as that
within Layer D (0.07 % new organic carbon/day), and more than an order of magnitude greater
than within Layer C (0.01 % new organic carbon/day). Studies of other microbial mats have also
measured higher production rates of organic carbon in layers closer to mat surfaces (Buhring et
al., 2009). Van der Meer et al., (2005) examined specific biomarkers after labeled bicarbonate
incubations, and provided evidence for autotrophic organic production by members of the phyla
Cyanobacteria and Chloroflexi. Members of the Cyanobacteria phylum were the most abundant
in Layer A (35-39%), but members of the phylum Chloroflexi also represented a major fraction
of the community (8-24%). Although rates of carbon fixation in Layer B were almost equal to
Layer A, cyanobacteria were far less abundant (~0.5-1.5%). Instead, members of the phylum
Chloroflexi were more abundant than cyanobacteria in Layer B (~3-8%).
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Conversely, while Layers A and B had similar rates of carbon fixation with different
microbial communities, Layers C and D had similar communities but Layer D had higher rates of
carbon fixation (Fig. 7). These lower layers are more likely to be light-limited and contain higher
abundances of anaerobic microbes. Since incubation was performed under potentially aerobic
conditions, carbon fixation rates in Layers C and D could represent increased production from
oxygen-tolerant autotrophs than what naturally occurs in deeper mat layers in LHC. However,
the microbial community in Layer D remained stable during incubations, with no change in
percent abundance greater than 5% across all taxonomic ranks (Fig. 8). This stability within the
deepest mat community during incubations increases confidence that oxygen levels did not
impact carbon fixation rates. Chloroflexi are relatively common in both layers, but decreased
from 14.2% to 2.3% during incubation in Layer C, potentially decreasing net carbon fixation.
Finally, it is possible that future examination and identification of the ~14-26% of unclassified
taxa in Layers B, C, and D will yield further information on carbon-fixing organisms within the
LHC mats.
Biogenic carbonate precipitation within the LHC mat occurred during incubation of
Layers B, C, and D. In contrast, the highest rates of carbon fixation were in Layers A and B.
Therefore, although biogenic carbonate precipitation and carbon fixation both occur within LHC
microbial mat layers, there is no apparent correlation between the two processes within the mat
as a whole. There also appears to be no correlation between biogenic and abiogenic precipitation
in LHC mats. Both styles produce carbonate in Layers C and D, while only biogenic
precipitation is prevalent in Layer B. Layer A had the highest rate of abiogenic precipitation with
no major biogenic influence. Finally, abiogenic carbonate precipitation has no clear correlation
with carbonate saturation states within LHC mats. Calcium carbonate was supersaturated at all
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points above and within mats, with Ω increasing with depth, from 3.23 at 0.5 cm (Layer A) to
4.27 at 10 cm (Layer D). However, abiogenic precipitation rates were highest in Layer A at the
top of the mat, and were marginal within Layer B immediately below, with intermediate values
in Layers C and D. This is in contrast with previous experiments in abiotic systems, which
observe a positive correlation between precipitation rate and saturation (Shiraki & Brantley,
1995).
Layer B had the highest weight percentage of carbonate (96.9%, Fig. 4, Supp. Table 1),
but only biogenic precipitation occurred during incubation. Conversely, Layer A has the highest
abiogenic precipitation rates, and total precipitation rates (biogenic + abiogenic) equivalent to
those of Layers C and D, but the lowest carbonate percentage (79.7%). One explanation for this
discrepancy is spatial differentiation of communities as LHC mats grow over time. Taxa with
higher motility have the potential to move to more habitable environments when conditions
become less habitable (Stal, 1995; Nadeau et al., 1999). Cyanobacteria have been shown to
exhibit motile behavior in reaction to light availability, ultraviolet radiation, and chemical
gradients within mats (Richardson & Castenholtz, 1987; Bebout & Garcia-Pichel, 1995;
Ramsing et al., 1997). Carbonate precipitation has the potential to limit light availability in LHC
mats, forcing motile photoautotrophs to move upwards, differentiating a thin, relatively organic-
rich and carbonate-poor layer (Layer A) on top of carbonate-rich, organic-poor layers below
(Layers B, C, and D).
Precipitation Rates Compared with Microbialite Formation
Precipitation rates from LHC mats not only provide valuable information about the
dynamics of modern mat growth, but can also help constrain conditions for microbialite
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formation and preservation in deep time. Averaging all four layers, LHC mats are 91 weight %
carbonate, with a precipitation rate of 0.012 % new carbonate per day. The average dry sample
mass is 0.002 mg. Therefore, a typical incubation sample had 0.0018 grams of carbonate (0.002
grams total x 0.91 grams carbonate/grams total), and 2.2 E-7 grams of carbonate are precipitated
every day (0.0018 grams carbonate x 0.00012 grams new carbonate/grams carbonate). At
measured rates, a lithified microbialite with 100 weight % carbonate would take two more years
to produce. This hypothesis will be easily testable with future observations of LHC mats.
There are three potential solutions to the discrepancy between the rates of precipitation
observed within LHC mats and the lack of lithified microbialites in the same location. 1) The
incubation experiments represent a “snapshot” of microbial mat processes instead of long-term,
steady state conditions. While the short-term nature of the experiments explains the differences
between laboratory and field observations, the explanation does not include, perhaps, a specific
process that inhibits microbialite formation at LHC. While a few clades changed in abundance
during transportation or incubation, the majority remained relatively stable, indicating that major
changes in mat community composition during experiments are unlikely to explain the
discrepancy. 2) Metabolisms within LHC mats in the field inhibit precipitation or promote
dissolution which may not have been recognized in the lab experiments. Although the
communities of each layer during incubations are relatively representative of field abundances,
we introduced more light and aerobic conditions to lower layers such as C and D. Such changes
could either promote metabolisms that are not normally active in field communities, either
processes that promote carbonate precipitation, or inhibit carbonate dissolution. 3) Organic
carbon production dilutes carbonate mineralization. In this scenario, carbonate precipitation in
LHC mats could be equal or potentially higher than observed in the incubation experiments.
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However, if production rates of organic carbon are consistently higher than carbonate
precipitation rates, then the microbial mat will never completely lithify into a microbialite (see
Supplemental Data). While all three explanations merit further investigation on mats from LHC
and other environments, dilution of carbonate mineralization through organic carbon production
provides the best explanation with the current set of observations.
Hypotheses for ancient microbial growth rates vary between short-term diurnal cycles
based on modern cyanobacterial motility (Doemel & Brock, 1974; Golubic & Focke, 1978), and
slower growth over many months or years (Chivas et al., 1990; Petryshyn et al., 2012; Frantz et
al., 2014). Evidence from LHC suggests that elements of both ideas are at work in modern mats.
Patterns of LHC mat communities and carbonate growth suggest that cyanobacterial motility
does separate modern mats into upper organic-rich and lower carbonate-rich zones, but on slower
timescales than thought by previous hypotheses. At observed precipitation rates, a minimum of
23 years is required for a hypothetical LHC mat starting with no carbonate present to fully
lithify, all things being equal. However, if production rates of organic carbon consistently
outpace carbonate precipitation rates in this mat, it is possible that the mats may never become
fully lithified. It is possible to speculate that the paucity of microbialites vs. the ubiquity of
microbial mats in modern environments may reside in the balance between rates of organic
carbon production versus carbonate precipitation, where times of abundant microbialite
formation in the past may represent conditions where carbonate precipitation rates were equal to
or greater than microbial growth rates.
Our study of one-day growth rate experiments shows 1) Rates of autotrophic carbon
fixation are not correlated with rates of biogenic carbonate precipitation within certain microbial
187

mats. The results corroborate previous work demonstrating that carbon fixation only precipitates
calcium carbonate in specific chemical environments (Merz-Preiß & Riding, 1999; Arp et al.,
2001). The uppermost layers of LHC mats have the highest carbon fixation rates as well as
abundant autotrophic communities, but biogenic precipitation only occurred in Layers B, C and
D. 2) Mats with consistently higher rates of organic carbon production than carbonate
precipitation are unlikely to produce lithified microbialites. This hypothesis is supported by
recent research indicating higher rates of primary productivity in unlithified microbial mats than
within recently formed stromatolites (Schuler et al., 2017). 3) Individual microbial mat layers
can host both biogenic and abiogenic carbonate precipitation. Abiogenic precipitation occurred
in the uppermost LHC layer, and in concert with biogenic precipitation in the lower two layers.
4) Layers with the highest carbonate production rates do not always correspond with areas of
high carbonate percentage. The uppermost LHC layer had the lowest percentage of carbonate
(79.7%), but the highest rate of abiogenic carbonate precipitation, while the immediately
adjacent layer was 96.9% carbonate, but had the lowest rates of total carbonate precipitation
during incubations. Comparing differences in carbonate percentage and production rate can
potentially provide growth histories of microbial mats.

ACKNOWLEDGMENTS
This work was supported in part through funding for the 2015 International Geobiology Course
from the Agouron Institute. A permit was granted to J.R.S. from the U.S. Forest Service (Permit
#MLD15053) to conduct fieldwork and sample LHC. We acknowledge all members of the 2015
International Geobiology Course in their assistance, including Caitlin Bojanowski, James
188

Bradley, Joy Buongiorno, Luoth Chou, Leslie Daille, Gabriela Libanori, Katie Rempfert,
Danielle Santiago, Chris Trivedi, Lennart van Maldegem, Feifei Zhang, Laura Zinke, Emma
Bertran, Jonah Duckles, Sean Loyd, Victoria Petryshyn, and Russell Shapiro. We thank Nick
Rollins for analyzing and reducing data, and Julien Emile-Geay and Jeffrey Thompson for their
assistance on statistical analyses.
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Leeuw, J.W., Damsté, J.S.S., and Ward, D.M., 2005, Diel variations in carbon
metabolism by green nonsulfur-like bacteria in alkaline siliceous hot spring microbial
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Lith, Y., 2006, Lithifying microbial mats in Lagoa Vermelha, Brazil: Modern
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10.1016/j.sedgeo.2005.12.022.
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199

FIGURES: CHAPTER 4

FIGURE 1: Location of LHC microbial mats. (A) Map of California. (B) View of mat location
area looking east (downstream). Mat sampling and field geochemistry was performed on left-
hand creek margin. Green areas towards the center of creek flow represent subaqueous mats,
orange areas on creek margins represent mats at creek surfaces. (C) Aerial overview of LHC
source and mat location. (D) Location of mat sampling and field geochemistry. Creek flow is
from right to left. The yellow flag represents the Upstream location for field geochemistry, while
the left blue flag represents the Downstream location (see Table 1). The orange surficial mat was
sampled between the middle blue flag and the yellow flag (see Fig. 2A).

200

FIGURE 2: Structure of LHC microbial mats. (A) Microbial mat sample immediately after
extraction. Note the distinct textural difference between the mat surface (Layer A) and the layers
below (Layers B, C, and D). Scale bar is in cm. (B) Microbial mat after transport from field to
laboratory. Layer A represents the mat surface, Layer D represents the mat bottom.
201

FIGURE 3: SEM images of carbonate precipitates. Scale bar is 500 µm. (A) Layer A. (B) Layer
B. (C) Layer C. (D) Layer D. Note the presence of euhedral crystals, particularly in Layer B, an
indication of precipitation within mat layers as opposed to trapping and binding of detrital
carbonates.

202

FIGURE 4: Percent organic and inorganic carbon of mat layers. Points represent triplicate
averages, error bars represent one standard deviation.

203

FIGURE 5: δ
13
C of organic carbon of mat layers after incubation experiments. Points represent
triplicate averages, error bars represent one standard deviation.

204

FIGURE 6: δ
13
C of inorganic carbon of mat layers after incubation experiments. Points
represent triplicate averages (except for Layer B Living Label, which is the average of two
samples), error bars represent one standard deviation.

205

FIGURE 7: Rates of calcium carbonate precipitation and autotrophic organic carbon production.
(A) Carbonate precipitation rates. Gray bars represent the amount of new carbonate precipitated
in poisoned incubation samples as a percentage of the carbonate present prior to incubation.
Green bars represent the additional amount of new carbonate precipitated in living incubation
samples compared with poisoned incubation samples. (B) Carbon fixation rates. Bars represent
the amount of organic carbon produced through carbon fixation in living incubation samples as a
percentage of the organic carbon present prior to incubation.

206

FIGURE 8: Major orders in LHC mat layers. (A) Field triplicates. (B) Laboratory samples prior
to and after incubation experiments.

207

TABLES AND SUPPLEMENTARY INFORMATION: CHAPTER 4
TABLE 1: Field measurements of stream water and pore water at LHC taken in June 2015 (*
Indicates measurements taken in August 2015). Upstream and Downstream locations are relative
to the sampled mat.

Location
Upstream
20 cm
Downstream
80 cm
Mat Porewater: 1 cm
(A/B Boundary)
Mat Porewater: 5 cm
(Layer B)
T (°C) 52.4 38.1 38.8 48.4
TCO2 (mM) 11.91 11.88 11.52 11.59
δ
13
C (‰) -2.58 -2.49 -2.06 -2.23
pH 8.34 8.29 8.10* 8.30*
Ω 4.62 3.50 3.23 4.27
208

SUPPLEMENTARY INFORMATION:
Production Rates of Carbonate and Organic Carbon by Volume
Rates of carbonate precipitation and carbon fixation were calculated as % carbon/day.
Respective volumes of carbonate and organic carbon produced per day can be estimated using
weight percents, densities, and production rates of carbonate and organic matter. The average dry
mass of samples was 0.002 g. Averaging all four layers, carbonate was 91 weight % of LHC
mats. The composition of 0.002 g dry mat mass would therefore be 0.0018 g carbonate, and
0.0002 g organic carbon. At a precipitation rate of 0.012% new carbonate/day, 2.16 E-7 grams
carbonate would be added daily. Carbon fixation rates of 0.1% new carbon/day would result in
daily organic matter production of 2.08 E-5 grams. Dividing these rates by the densities of
calcium carbonate (2.7 g/cm3) and organic matter (~1.5 g/cm3) results in 8 E-8 cm3 carbonate
produced/day and 1.4 E-7 cm3 organic carbon produced/day. Therefore, carbon fixation
produces ~1.7 times more mat volume on average than carbonate precipitation.

209

SUPPLEMENTARY TABLE 1:
Percent organic and inorganic carbon in all mat layers. SD signifies one standard deviation for
the triplicate average.
Layer
Organic
Replicate
wt%
Corg
Average
wt% Corg SD
Inorganic
Replicate
wt%
CaCO3
Average
wt%CaCO3 SD
A 1 4.8 5.1 0.43 1 84.2 79.7 7.48
A 2 5.7

2 69.2

A 3 4.7

3 85.8

B 1 0.7 0.6 0.12 1 96.7 96.9 0.39
B 2 0.7

2 97.5

B 3 0.5

3 96.7

C 1 1.7 1.7 0.018 1 93.3 93.1 1.71
C 2 1.6

2 95.0

C 3 1.7

3 90.8

D 1 1.1 1.1 0.072 1 95.0 94.2 1.18
D 2 1.0

2 92.5

D 3 1.1

3 95.0

210

SUPPLEMENTARY TABLE 2: δ
13
C values for inorganic and organic carbon during
incubation experiments. SD signifies one standard deviation for the triplicate average.

Layer Org/Inorg
δ
13
C
Living/Poisoned/Un-
Incubated
Trip. δ
13
C Avg.
δ13C
SD
A Inorg Living Label 1 -1.14 -1.2 0.05
A Inorg Living Label 2 -1.22

A Inorg Living Label 3 -1.25

A Inorg Poisoned Label 1 -1.22 -1.22 0.08
A Inorg Poisoned Label 2 -1.13

A Inorg Poisoned Label 3 -1.32

A Inorg Un-Incubated 1 -1.43 -1.49 0.06
A Inorg Un-Incubated 2 -1.58

A Inorg Un-Incubated 3 -1.47

A Org Living Label 1 -15.87 -16.67 0.85
A Org Living Label 2 -17.85

A Org Living Label 3 -16.28

A Org Poisoned Label 1 -20.41 -20.37 0.04
A Org Poisoned Label 2 -20.32

A Org Poisoned Label 3

A Org Un-Incubated 1 -20.32 -20.42 0.12
A Org Un-Incubated 2 -20.59

A Org Un-Incubated 3 -20.36

211

Layer Org/Inorg
δ
13
C
Living/Poisoned/Un-
Incubated
Triplicate δ
13
C Avg.
δ13C
SD
B Inorg Living Label 1

-1.5 0.06
B Inorg Living Label 2 -1.55

B Inorg Living Label 3 -1.44

B Inorg Poisoned Label 1 -1.62 -1.6 0.04
B Inorg Poisoned Label 2 -1.54

B Inorg Poisoned Label 3 -1.63

B Inorg Un-Incubated 1 -1.61 -1.67 0.05
B Inorg Un-Incubated 2 -1.74

B Inorg Un-Incubated 3 -1.65

B Org Living Label 1 -18.35 -18.22 0.26
B Org Living Label 2 -18.45

B Org Living Label 3 -17.86

B Org Poisoned Label 1 -21.73 -21.78 0.08
B Org Poisoned Label 2 -21.73

B Org Poisoned Label 3 -21.89

B Org Un-Incubated 1 -21.85 -21.93 0.12
B Org Un-Incubated 2 -21.85

B Org Un-Incubated 3 -22.1

212

Layer Org/Inorg
δ
13
C
Living/Poisoned/Un-
Incubated
Triplicate δ
13
C Avg.
δ13C
SD
C Inorg Living Label 1 -0.84 -0.86 0.02
C Inorg Living Label 2 -0.85

C Inorg Living Label 3 -0.89

C Inorg Poisoned Label 1 -0.96 -0.96 0.06
C Inorg Poisoned Label 2 -1.04

C Inorg Poisoned Label 3 -0.89

C Inorg Un-Incubated 1 -1.14 -1.16 0.02
C Inorg Un-Incubated 2 -1.19

C Inorg Un-Incubated 3 -1.14

C Org Living Label 1 -18.9 -18.77 0.18
C Org Living Label 2 -18.89

C Org Living Label 3 -18.51

C Org Poisoned Label 1 -18.99 -19.11 0.12
C Org Poisoned Label 2 -19.07

C Org Poisoned Label 3 -19.28

C Org Un-Incubated 1 -18.97 -18.97 0.13
C Org Un-Incubated 2 -18.8

C Org Un-Incubated 3 -19.13

213

Layer Org/Inorg
δ
13
C
Living/Poisoned/Un-
Incubated
Triplicate δ
13
C Avg.
δ13C
SD
D Inorg Living Label 1 -1.16 -1.21 0.1
D Inorg Living Label 2 -1.25

D Inorg Living Label 3 -1.01

D Inorg Poisoned Label 1 -1.26 -1.29 0.02
D Inorg Poisoned Label 2 -1.29

D Inorg Poisoned Label 3 -1.32

D Inorg Un-Incubated 1 -1.44 -1.42 0.04
D Inorg Un-Incubated 2 -1.37

D Inorg Un-Incubated 3 -1.45

D Org Living Label 1 -19.4 -19.63 0.16
D Org Living Label 2 -19.74

D Org Living Label 3 -19.75

D Org Poisoned Label 1 -20.87 -21.15 0.34
D Org Poisoned Label 2 -20.95

D Org Poisoned Label 3 -21.63

D Org Un-Incubated 1 -20.88 -21.03 0.1
D Org Un-Incubated 2 -21.11

D Org Un-Incubated 3 -21.09

214

SUPPLEMENTARY TABLE 3: Differences in organic and inorganic carbon triplicate
averages (Δavg). L = Living Label, P = Poisoned Label, N = Unincubated. Δavg values with
bootstrap p-values < 0.05 and probability of machine variability below 5% represent carbon
fixation and carbonate precipitation in organic and inorganic carbon samples, respectively.
Exceptions include: *) In Layer B, the Δavg was calculated between 3 Living Label and 2
Poisoned Label samples, instead of two triplicates like every other comparison. The Δavg,
bootstrap p-value, and probability of machine variability are close enough to Layer C, which
meets the bootstrap and machine variability tests, to merit an interpretation of biogenic
precipitation.
ORGANIC CARBON
Layer A B C D
Sample
Groups
Compared
P - N L - P P - N L - P P - N L - P P - N L - P
Difference in
Average
δ
13
C (Δ avg)
0.058 3.70 0.15 3.56 -0.15 0.35 -0.12 1.5
% New
Organic
Carbon/Day
0.0027 0.17 0.0032 0.17 -0.0023 0.012 -0.0083 0.073
Bootstrap p-
value (one-
tailed T-test)
0.29 0.010 0.076 0.0030 0.12 0.036 0.28 0.010
Probability
of machine
variability
25% <0.01% 4% <0.01% 4% <0.01% 7% <0.01%
Autotrophy Present Present Present Present
INORGANIC CARBON
Layer A B C D
Sample
Groups
Compared
P - N L - P P - N L - P P - N L - P P - N L - P
Difference in
Average
δ
13
C (Δ avg)
0.23 0.02 0.07 0.10 0.19 0.10 0.13 0.15
% New
Inorganic
Carbon/Day
0.013 0.00093 0.0033 0.0047 0.0090 0.0048 0.0061 0.0070
Bootstrap p-
value (one-
tailed T-test)
0.021 0.38 0.071 0.058 0.010 0.034 0.013 0.038
Probability
of machine
variability
0.3% 43% 19% 12% 1% 10% 5% 1%
Carbonate
Precipitation
Not
biogenic
Biogenic
*
Not
biogenic
Biogenic Not
biogenic
Biogenic

Abstract (if available)

Abstract Distinguishing the specific processes and rates of processes that shape modern and ancient microbialites remains an enduring challenge within the field of geobiology. Microbialites are defined as “organosedimentary deposits that accrete as a result of a benthic microbial community trapping and binding detrital sediment and/or forming the locus of mineral precipitation” (Burne and Moore, 1987). This definition encompasses a diverse variety of structures observed in modern and ancient environments, including laminated stromatolites, oncoid grains, and clastic wrinkle structures, among many others (Kalkowsky, 1908

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Asset Metadata

Creator Wilmeth, Dylan Thomas (author)

Core Title Preservation of gas-related textures in microbialites: Evidence for ancient metabolisms and environments

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Geological Sciences

Publication Date 07/26/2018

Defense Date 04/20/2018

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

Tag Archean,bubbles,Great Oxidation Event,microbial metabolisms,microbialites,OAI-PMH Harvest,oxygenic photosynthesis,stromatolites

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Corsetti, Frank (committee chair), Berelson, William (committee member), Bottjer, David (committee member), Levine, Naomi (committee member)

Creator Email dwilmeth@usc.edu,dylan.wilmeth@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-32977

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