Thermophilic Endospore-Forming Bacteria As Models for Exploring Microbial Dispersal in Time and Space

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2017 Thermophilic Endospore-Forming Bacteria as Models for Exploring Microbial Dispersal in Time and Space

Cramm, Margaret

Cramm, M. (2017). Thermophilic Endospore-Forming Bacteria as Models for Exploring Microbial Dispersal in Time and Space (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/28742 http://hdl.handle.net/11023/4275 master thesis

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UNIVERSITY OF CALGARY

Thermophilic Endospore-Forming Bacteria as Models for Exploring Microbial Dispersal

in Time and Space

Margaret Anne Cramm

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL

FULFILLMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

GRADUATE PROGRAM IN BIOLOGICAL SCIENCES

CALGARY, ALBERTA

DECEMBER, 2017

Thermophilic endospore-forming bacteria – “ thermospores ” – are particularly useful model organisms for exploring microbial biogeography because they remain viable for long geologic time periods owing to a dormant state that confers resistant to extreme conditions. Using high temperature incubation experiments and 16S rRNA gene amplicon sequencing, geographic and temporal thermospore dispersal in marine sediments was explored. Thermospores detected in surface sediments across the North Atlantic are likely to originate from multiple warm temperature habitats, and are viable in sediments buried ~15 000 years ago. These approaches also revealed thermospore viability following the extreme stress of prolonged -80°C exposure. Thermospore viability and dispersal on a scale of millions of years was explored in a 1.2 km long sediment core. Uneven thermospore germination posed a challenge for thermospore detection but their capacity for use as models of biological dispersal remains valid.

ii Preface

This thesis is the original, unpublished, independent work by the author, Margaret Cramm

iii Acknowledgements

I would like to thank the funding agencies NSERC, Genome Canada, MEOPAR, ArcticNet and, the Province of Alberta for supporting my research.

I would like to thank Anirban Chakraborty for teaching me everything I know about microbiological labwork. Anirban’s patience and dedicaton to teaching me was central to the establishment of my research foundation and the completion of this thesis.

Addionally, I would also like to thank Carmen Li for her patience and assistance with the molecular work required for this research. Carmen was integral in the collection and interpretation of DNA sequencing data that was central to this research.

Emil Ruff is a wizard of microbial community analysis. I am grateful for Emil’s dedication to teaching me that went beyond simply teaching me analysis techniques but also ensured that I understood the underlying concepts.

Finally, I would like to thank Casey Hubert, my supervisor, for mentoring and championing me throughout my graduate student research career. I am particularly grateful to Casey for allowing me to steer the direction of my research based on my curiosity, increasing the quality of my scientific writing, and supporting me in participating in unique domestic and international research opportunities that elevated my research skill set. My time as a graduate student has offered me diverse learning experiences that have advanced me as a researcher and evolved me personally. Much of what I have gained during this time is a direct result of my sense of adventure meeting Casey’s open-minded approach to graduate student learning and for this reason I know that of all the supervisors in the world Casey is the best one for me.

iv Dedication

I would like to dedicate this work to Albert Stephen Bishop Cramm and Ann Ida (Appelt) Cramm whose support made my education and participation in this research possible.

v Table of Contents

Abstract ...... ii Preface...... iii Acknowledgements ...... iv Dedication ...... v Table of Contents ...... vi List of Tables ...... ix List of Figures ...... xi List of Supplementary Tables ...... xv List of Supplementary Figures ...... xvi List of Abbreviations ...... xx Chapter 1: Introduction ...... 1 1.1 Bacterial endospores ...... 1 1.2 Endospore strategies for survival and longevity ...... 3 1.3 Thermophilic endospore-forming bacteria ...... 5 1.4 Microbial biogeography and thermospores ...... 5 1.5 Thermospores in anoxic environments ...... 8 1.6 Panspermia and the last universal common ancestor ...... 10 1.7 Thesis overview ...... 14 Chapter 2: Materials and Methods ...... 15 2.1 Sediment sample collection ...... 15 2.2 High temperature incubation ...... 17 2.3 Sulfate and organic acid measurement ...... 17 2.4 16S rRNA gene amplicon sequencing and analyses ...... 18 2.4.1 DNA extraction ...... 18 2.4.2 16S rRNA gene amplification and sequencing ...... 18 2.4.3 16S rRNA gene amplicon sequence analysis ...... 19 Chapter 3: Freezing tolerance of thermophilic endospores in Arctic marine sediment ...... 21 3.1 Abstract ...... 21 3.2 Introduction ...... 23 3.3 Materials and Methods ...... 25 3.3.1 Freezing pretreatment ...... 25 3.3.2 High temperature incubation ...... 25 3.3.3 Sulfate and organic acid measurement ...... 26 3.3.4 DNA extraction and 16S rRNA gene amplicon sequencing ...... 26 3.3.5 Gas composition measurement after freezing pretreatment ...... 27 3.3.6 Repeat freezing pretreatment and incubation to observe the effect of O2 contamination on bacterial community structure ...... 27

vi 3.4 Results ...... 29 3.4.1 Sulfate reduction during 50°C incubations ...... 29 3.4.2 Organic acid depletion ...... 30 3.4.3 16S rRNA gene amplicon library analysis and thermospore OTU identification ...... 31 3.4.4 Gas analysis after freezing pretreatment and the gas permeability of rubber stoppers after freezing ...... 40 3.4.5 Bacterial community structure after repeat pre-freezing and incubation ...... 40 3.5 Discussion ...... 47 Chapter 4: Lateral biogeography of thermophilic endospores in North Atlantic sediment ...... 52 4.1 Abstract ...... 52 4.2 Introduction ...... 53 4.3 Materials and Methods ...... 56 4.3.1 North Atlantic sediment locations ...... 56 4.3.2 High temperature incubation ...... 57 4.3.3 Sulfate and organic acid measurement ...... 58 4.3.4 16S rRNA gene amplicon sequencing and analysis ...... 59 4.4 Results ...... 60 4.4.1 Microbial activity and community structure during 50°C sediment incubation 60 4.4.1.1 Scotian Slope West ...... 64 4.4.1.2 Scotian Slope Centre ...... 65 4.4.1.3 Scotian Slope East ...... 65 4.4.1.4 Labrador Shelf ...... 66 4.4.1.5 Frobisher Bay ...... 66 4.4.1.6 Davis Strait...... 67 4.4.1.7 Pond Inlet ...... 68 4.4.1.8 Baffin Bay North ...... 69 4.4.1.9 Svalbard ...... 69 4.4.2 Shared thermospore OTU identification ...... 70 4.4.3 Thermospore OTU occurrence and site connection to North Atlantic subpolar gyre ...... 73 4.4.4 Assessing thermospore OTU absence in the largest 16S rRNA gene library 77 4.5 Discussion ...... 78 Chapter 5: Microbial temporal dispersal in the North Atlantic: tracking thermospores through time...... 82 5.1 Abstract ...... 82 5.2 Introduction ...... 83 5.3 Materials and Methods ...... 85 5.3.1 Sediment sites ...... 85 5.3.2 High temperature incubation ...... 86 5.3.3 Sulfate and organic acid measurement ...... 86 5.3.4 16S rRNA gene amplicon sequencing and analysis ...... 86 5.3.5 Representative sequence comparison ...... 87 5.4 Results ...... 88 5.4.1 Microbial activity and community structure ...... 88

vii 5.4.1.1 Scotian Slope West ...... 92 5.4.1.2 Scotian Slope Centre ...... 92 5.4.1.3 Scotian Slope East ...... 93 5.4.2 Representative sequence comparison ...... 94 5.5 Discussion ...... 96 Chapter 6: Thermophilic endospore longevity in deep sediment of the Nankai Trough over geologic timescales ...... 100 6.1 Abstract ...... 100 6.2 Introduction ...... 102 6.2.1 IODP Expedition 370 and the high temperature limit of life in sediments of the deep biosphere ...... 102 6.2.2 Endospores in very deep and very old sediments ...... 103 6.2.3 What is the maximum depth at which viable thermospores are detected as dormant endospores?...... 104 6.2.4 Are thermospores of the phylotypes found at the deepest sediment layer present in throughout the shallower horizons of the core? ...... 105 6.2.5 Do thermospores germinate in the warm horizons of the deep core? ...... 105 6.3 Materials and Methods ...... 107 6.3.1 Sediment site and recovery ...... 107 6.3.2 High temperature incubation ...... 108 6.3.3 Sulfate and organic acid measurement ...... 108 6.3.4 16S rRNA gene amplicon sequencing and analysis ...... 109 6.3.5 Estimation of cell numbers in sediment ...... 110 6.4 Results ...... 111 6.4.1. Sulfate reduction and organic acid utilization ...... 111 6.4.2. 16S rRNA gene amplicon library analysis ...... 111 6.4.3 Estimated cell numbers in microcosm sediments ...... 123 6.5 Discussion ...... 124 Chapter 7: Summary and Outlook ...... 127 7.1 Experimental summary and main conclusions ...... 127 7.2 Inconsistency among biological replicates ...... 128 7.3 Concluding remarks ...... 132 Bibliography ...... 133 Supplementary Tables ...... 150 Supplementary Figures ...... 168

viii List of Tables

Table 2.1: Details of marine ocean sediment used in this study.

Table 3.1: Thermospore OTUs detected after a 10-day pre-freezing treatment at -80°C, -20°C, or left unfrozen (positive control) after 53 days of incubation at +50°C.

Table 3.2: Thermospore OTUs detected after a 9-day pre-freezing treatment at -80°C, - 20°C, or left unfrozen (positive control) after 7 days of incubation at +50°C.

Table 4.1: Summary of the sulfate reduction, organic acid utilization, and increase in Firmicutes relative abundance after 50°C incubation. A “+” symbol represents the occurrence of sulfate reduction, organic acid decrease, or increase in Firmicutes relative abundance over the 50°C incubation over 14 days. The symbols “+” and “-“ represent a positive or negative result respectively in each replicate for the occurrence of sulfate reduction, organic acid utilization, or increase in Firmicutes relative abundance over the incubation period. The first symbol represents the result in the first replicate, the second represents the result in the second replicate, etc.

Table 4.2: Summary of the thermospore OTUs identified in each sediment after 50°C incubation. A “+” in a coloured box identifies a thermospore OTU as present in incubations of that sediment. A blue box represents a thermospore OTU that was identified in an incubation that also showed sulfate reduction and a decrease in organic acid concentration. A green box represents a thermospore OTU that was identified in an incubation that showed a reduction in organic acid concentration but no sulfate reduction. A grey box represents a thermospore OTU that was identified in an incubation that did not show either sulfate reduction or a decrease in organic acid concentration.

Table 4.4: Thermospore OTU identification based on BLAST 16S rRNA gene sequence similarity. Sites from which each thermospore OTU was identified after 50°C incubation are listed. Of forty-one thermospore OTUs identified, only the nine that are identified in sediment from more than one location are listed.

Table 5.1: Summary of the sulfate reduction, organic acid utilization, and increase in Firmicutes relative abundance after 50°C incubation. A “+” symbol represents the occurrence of sulfate reduction, organic acid decrease, or increase in Firmicutes relative abundance over the 50°C incubation. The symbols “+” and “-“ represent a positive or negative result respectively in each replicate for the occurrence of sulfate reduction, organic acid utilization, or increase in Firmicutes relative abundance over the 28-day incubation period. The first symbol represents the result in the first replicate, the second represents the result in the second replicate, etc.

ix Table 5.2: OTUs identified in 50°C incubations of Scotian Slope sediments from various depths based on BLAST searches of the NCBI database using 16S rRNA gene amplicon sequence data. The closest cultured isolates are shown.

Table 6.1: Sediment depth and corresponding in situ temperature for the sediments used in the microcosm incubations.

Table 6.2: Relative abundances (%) of dominant phyla based on 16S rRNA gene amplicon sequencing. Actinobacteria, Firmicutes, and Proteobacteria are present in every sample including the sediment-free, medium-only control incubation samples and both DNA extraction negatives.

Table 6.3: Relative abundances (%) of phyla more abundant in the incubation slurry samples than the sediment-free, medium-only control incubations and the DNA extraction negatives. Atribacteria and Chloroflexi are the only phyla found in some samples at relative abundances that are higher than those found in any of the controls.

Table 6.4: Atribacteria and Chloroflexi OTUs identified from sediment from 206 mbsf and 254 mbsf to be real constituents of the in situ microbial community. OTU relative abundances are listed in Table S6.1.

Table 6.5: Thermospore OTUs identified from incubations of different depths of sediment at 50°C, 60°C, or 70°C.

x List of Figures

Figure 1.1: Phase contrast microscopy of thermophilic sulfate-reducing Desulfotomaculum sp. spores isolated from Svalbard sediment (photo credit: Flemming Mønsted, Aarhus University). Endospores are seen as white spots within the rod-shaped cells.

Figure 1.2: Fermenting and sulfate-reducing bacteria are found in the subsurface. Examples shown are spore-forming and thermophilic bacteria. This figure is modified from Hubert et al. (2010). Examples of fermenting thermospores include Desulfotomaculum thermosubterraneum (isolated from an underground mine, Kaksonen et al., 2006), Caloranaerobacter azorensis (isolated from a Mid-Atlantic Ridge hydrothermal vent chimney; Wery et al., 2001) and Thermosipho affectus (isolated from a deep-sea hydrothermal vent; Podosokorskaya et al., 2011). Examples of sulfate- reducing thermospores include Desulfotomaculum arcticum (isolated from arctic sediment, Vandieken et al., 2006), Desulfotomaculum geothermicum (isolated from geothermal ground water; Daumas et al., 1988), and Desulfotomaculum thermocisternum (isolated from North Sea oil reservoir formation water; Nilsen et al., 1996).

Figure 3.1: Sulfate concentrations in pre-frozen microcosms over 53 days of incubation at 50°C. Individual measurements of the -80°C pre-frozen microcosms are shown in Figure S3.1). Columns represent the average across triplicates and error bars show standard error.

Figure 3.2: Organic acid concentration over 53 days of incubation at 50°C. The -80°C pretreated microcosms show less replicatable organic acid depletion (Figure S3.2). Values represent the average of triplicates and error bars show standard error.

Figure 3.3 Community structure based on 16S rRNA gene amplicon sequencing before and after 50°C incubation. a) Phylum-level community structure before and after 7 and 53 days of 50°C incubation. “Unknown” phylum refers to the collection of OTUs of which a phylum-level classification cannot be confidently made by the MetaAmp application (Dong et al., 2017). “Total >1%” refers to the collection of phyla that were present in less than 1% relative abundance. An increase in the relative abundance of Firmicutes is seen in all treatments after incubation. b) Class-level community structure within the phylum Firmicutes after 7 and 53 days of 50°C incubation. Replicates 1,2, and 3 are plotted from left to right in each set of three; the replicates for the positive control replicates were pooled for Day 0 and Day 7 were pooled during DNA extraction so their libraries are a representation of three replicates.

Figure 3.4: Relative abundances of Bacilli and Clostridia and sulfate concentrations in the microcosms pre-frozen at -80°C and -20°C for 10 days and positive control microcosms over 53 days at 50°C. Error bars show standard error.

xi Figure 3.5: Sulfate reduction and relative abundance of Desulfotomaculum spp. in pretreated microcosms at -80°C and -20°C for 10 days and positive control microcosms over 53 days at 50°C. Δ Sulfate refers to the absolute value of the sulfate concentration decrease and is plotted against the axis on the rights side. Error bars show standard error.

Figure 3.6: Non-metric multidimensional scaling plots showing the bacterial community similarities between freezing pretreatments based on 16S rRNA gene amplicon sequencing. Microcosms frozen at -80°C for 10 days are indicated by blue circles, those frozen at -20°C for 10 days are indicated by green circles, and those that remained unfrozen (4°C) are indicated by yellow circles. Circle size represents diversity based on richness and evenness with larger circles representing richer communities and is calculated using Inverse Simpson Index. Libraries generated from samples before 50°C incubation are joined and identified by label “Day 0”. a) Circles representing libraries obtained from samples after 7 days and 53 days of 50°C incubation are grouped together. b) Circles representing libraries obtained from samples from the different treatments are grouped to their freezing pretreatment (“-80°C”, “-20°C”, “Positive Control”).

Figure 3.7: O2 concentration in triplicate empty serum bottles flushed with N2:CO2 gas and pretreated at -80°, -20°C and 4°C for 2 days. Headspace was measured in triplicate serum bottles for each pretreatment.

Figure 3.8: NMDS plot comparing the bacterial community structures of microcosms subjected to the different pretreatments of the repeat experiment. Microcosms frozen at -80°C are represented by blue circles, those frozen at -20°C are represented by green circles, and those that remained unfrozen (4°C) are represented by yellow circles. Circle size represents diversity based on richness and evenness Circle size represents diversity based on richness and evenness with larger circles representing richer communities and is calculated using Inverse Simpson Index. Circles are joined according to the time of measurement. Circles visualizing the community structure at Day 0 identify the community structure after freezing treatment and pasteurization but before 50°C incubation. Circles joined and labelled “Before Pasteurization” represent the community structure after freezing but before pasteurization and 50°C incubation.

Figure 3.9: Bacterial community structure based on 16S rRNA gene amplicon sequencing after 50°C incubation in the repeat experiment in which the headspace was exchanged to ensure anoxic conditions during the incubation. Amplicon libraries prepared from sediment after freezing pretreatment but before pasteurization are shown to the right. a) Phylum-level community structure after 7 days of 50°C incubation. b) Class-level community structure within the phylum Firmicutes after 7 days of 50°C incubation.

Figure 3.10: Sulfate concentrations in microcosms of the repeat experiment over 53 days of incubation at 50°C. Error bars show standard error.

xii Figure 4.1: North Atlantic surface sediment used in 50°C thermospore incubations. Sites indicated in the map are Scotian Slope West (SSW), Scotian Slope Center (SSC), Scotian Slope East (SSE), Labrador Shelf (LS), Frobisher Bay (FB), Davis Strait (DS), Pond Inlet (PI), Baffin Bay North (BBN), and Svalbard (SV).

Figure 4.2: Phylogeny of the nine shared thermospore OTUs based on maximum likelihood phylogeny estimation. Scale bar identifies 1% sequence divergence. The tree is rooted by Geobacter metallireduces of the Proteobacteria lineage.

Figure 4.3: Average number of instances of thermospore OTU identification or instances of shared OTU identification at sediment sites connected to the North Atlantic subpolar gyre (CS) and sediment sites not connected to the North Atlantic subpolar gyre (NCS). An “incidence of thermospore OTU identification” refers to the number of thermospore OTU that are identified at a particular location. An “incidence of shared OTU” refers to the number of OTUs at a particular location that are also found at one or more other locations. The same OTU may be found in more than one sediment location and is counted as an incidence of OTU identification at each location. “CS shared OTUs” refers to the number of instances of shared OTUs counted at all CS sites. “NCS shared OTUs” refers to the number of instances of shared OTUs counted at all NCS sites. Error bars show standard error in the number of OTUs identified among the sites or the number of shared OTU instances among the sites. CS represents “Connected Sites” and refers to the sites in the North Atlantic that are connected to the North Atlantic subpolar gyre or the Norwegian Current. NCS represents “Not Connected Sites” and refers to the sites in the North Atlantic that are not connected to the North Atlantic subpolar gyre or the Norwegian Current.

Figure 4.4: The identified thermospore OTUs are classified into 2 categories: those found at only one location and those that are present in more than one location (shared). OTUs are further categorized based on their site’s connectedness to North Atlantic subpolar gyre: OTUs only found at one CS site or one NCS site, OTUs that are shared and found at CS and NCS sites, OTUs that are shared and found at CS sites only, or OTUs that are shared and found at NCS sites only. CS represents “Connected Sites” and refers to the sites in the North Atlantic that are connected to the North Atlantic subpolar gyre or the Norwegian Current. NCS represents “Not Connected Sites” and refers to the site in the North Atlantic that are not connected to the North Atlantic subpolar gyre or the Norwegian Current. All 41 thermospore OTUs detected in this study are represented in this chart.

Figure 5.1: Map of Scotian Slope sediment sites Scotian Slope West (SSW), Scotian Slope Centre (SSC), and Scotian Slope East (SSE).

Figure 6.1: Map showing the site of IODP Expedition 370 core C0023A drilling site (yellow circle).

xiii medium-only control. The group labeled “<1%” includes the sum of the relative abundances of phyla that are present in less than 1% in every library. Only phyla >1% relative abundance are shown in each legend.

Figure 6.3: Non-metric multidimensional scaling plots showing the community similarities between incubations of sediment from five different depths (206, 254, 440, 700, and 865 mbsf), sediment-free, medium-only controls, and the DNA extraction negatives. Sizes of the circles indicate the richness of the community calculated by Inverse Simpson Index with larger circles representing more diverse communities. The NMDS calculations did not reach convergence after 20 iterations. a) NMDS plot showing samples before (green) and after (yellow) incubation at 50°C, 60°C, 70°C. Circles representing communities before incubation are interspersed with circles representing communities after incubation and no grouping by incubation day (Day 0 vs. Day 21) is observed. b) NMDS plot showing samples based on inoculation sediment depth. Samples from 206 mbsf (red), 254 mbsf (yellow), 440 mbsf (green), 700 mbsf (blue), 865 mbsf (purple), and sediment-free, medium-only control (black) incubations and the DNA extraction negatives (white) do not group together based on sediment depth or controls.

xiv List of Supplementary Tables

Table S2.1: Number of replicate cores used as inoculum sediment for replicates from each site

Table S2.2: Number of reads in each amplicon library sample that passed quality control and the percent these read numbers represent of the total reads that were sequenced in each sample.

Table S3.1: Thermospore OTUs enriched after a 10 day pre-freezing treatment at - 80°C, -20°C, or unfrozen (positive control) after 53 days of incubation at 50°C. Relative abundances of each OTU after 7 days and 53 days of incubation are shown for each replicate. Where numbers are absent, the relative abundance was <1%.

Table S3.2: Thermospore OTUs enriched after a 9 day pre-freezing treatment at -80°C, -20°C, or unfrozen (positive control) in the repeat experiment after 7 days of incubation at 50°C. Relative abundances of each OTU are shown for each replicate. Where numbers are absent, the relative abundance was <1%.

Table S4.1: Taxa identification of 41 thermospore OTUs based on BLAST 16S rRNA amplicon sequence similarity.

Table S6.1: Relative abundances (%) of Atribacteria and Chloroflexi OTUs that are >1% of the total amplicon library in at least one replicate of incubations of sediment from 206 mbsf and 254 mbsf but not more than 2 reads in any control (sediment-free, medium- only control incubations and DNA extraction negatives).

xv List of Supplementary Figures

Figure S2.1: Number of reads passing quality control for each sequencing sample in Chapter 3. The first number in the sample name indicates the pretreatment temperature, the second indicates the replicate number, and the third identifies the number of days of incubation. The percent of the total reads in each sample is listed in Table S2.2.

Figure S2.2: Number of reads passing quality control in each sequencing sample used in Chapters 4 and 5. The first letters identify the sediment location, when present the second identifies the depth of the sediment in cm below seafloor, when present the third indicates the replicate number, and the fourth represents the number of days of incubation. The horizontal axis is log scale to accommodate a uniquely large sample read number of sample Fbay_R1_t14. The percent of the total reads in each sample is listed in Table S2.2.

Figure S2.3: Number of reads passing quality control in each sequencing sample used in Chapter 6. The sample name begins with the depth of the sediment and is followed by the incubation temperature, replicate number, and the number of days of incubation. The percent of the total reads in each sample is listed in Table S2.2.

Figure S3.1: Sulfate concentrations in individual -80°C pre-frozen microcosm replicates over 53 days of incubation at 50°C.

Figure S3.2: Organic acid concentration in -80°C pre-frozen microcosm replicates over 53 days of incubation at 50°C. The microcosms pretreated at -80°C for 10 days show some organic acid depletion; these same replicates also show some sulfate reduction.

Figure S3.3: Relative abundances of Bacilli and Clostridia and sulfate concentrations in the microcosms pre-frozen at -80°C. Individual replicates of sediment pre-frozen at - 80°C for 10 days are shown. Figure S4.1: Sulfate concentration (a), organic acid concentration (b), phylum-level community structure (c) and Firmicutes OTU relative abundance (d) of Scotian Slope West sediment 50°C incubations. a) Bars represent the average of 6 replicates and error bars show standard error. b) Organic acid concentrations of each replicate are plotted individually. c-d) Only those phyla and OTUs >1% relative abundance are shown in the legends.

Figure S4.2: Sulfate (a) and organic acid (b) measurement of media only control incubations for Scotian Slope West incubations. Error bars show standard error.

Figure S4.3: Sulfate concentration (a-b), organic acid concentration (c), phylum-level community structure (d) and Firmicutes OTU relative abundance (e) of Scotian Slope Centre sediment 50°C incubations. a) Bars represent the average of 3 replicates and error show standard error. b) Sulfate concentrations in each replicate are plotted individually. c) Organic acid concentrations of each replicate are plotted individually. d-e) Only those phyla and OTUs >1% relative abundance are shown in the legends.

xvi

Figure S4.4: Sulfate concentration (a), organic acid concentration (b), phylum-level community structure (c) and Firmicutes OTU relative abundance (d) of Scotian Slope East sediment 50°C incubations. a) Bars represent the average of 3 replicates and error bars show standard error. b) Organic acid concentrations of each replicate are plotted individually. c-d) Only those phyla and OTUs >1% relative abundance are shown in the legends.

Figure S4.5: Sulfate (a) and organic acid (b) measurement of media only control incubations for Scotian Slope Center and Scotian Slope East incubations. Error bars show standard error.

Figure S4.6: Sulfate concentration (a-b), organic acid concentration (c), phylum-level community structure (d) and Firmicutes OTU relative abundance (d) of Labrador Shelf sediment 50°C incubations. a) Bars represent the average of 3 replicates and error bars show standard error. b) Organic acid concentrations of each replicate are plotted individually. d-e) Only those phyla and OTUs >1% relative abundance are shown in the legends.

Figure S4.7: Sulfate concentration (a-b), organic acid concentration (c), phylum-level community structure (d) and Firmicute OTU relative abundance (e) of Frobisher Bay sediment 50°C incubations. a) Bars represent the average of 3 replicates and error bars show standard error. b) Sulfate concentrations in each replicate are plotted individually. c) Organic acid concentrations of each replicate are plotted individually. d-e) Only those phyla and OTUs >1% relative abundance are shown in the legends.

Figure S4.8: Sulfate concentration (a-b), organic acid concentration (c), phylum-level community structure (d) and Firmicutes OTU relative abundance (e) of Davis Strait sediment 50°C incubations. a) Bars represent the average of 3 replicates and error bars show standard error. b) Sulfate concentrations in each replicate are plotted individually. c) Organic acid concentrations of each replicate are plotted individually. d-e) Bars represent the combined phylum-level and OTU-level abundances of the three replicates. Only those phyla and OTUs >1% relative abundance are shown in the legends.

Figure S4.9: Sulfate concentration (a), organic acid concentration (b), phylum-level community structure (c) and Firmicutes OTU relative abundance (d) of Pond Inlet sediment 50°C incubations. a) Bars represent the average of 3 replicates and error bars show standard error. b) Organic acid concentrations of each replicate are plotted individually. c-d) Only those phyla and OTUs >1% relative abundance are shown in the legends.

Figure S4.10: Sulfate (a) and organic acid (b) measurement of media only control incubations for Labrador Shelf, Frobisher Bay, and Pond Inlet incubations. Error bars show standard error.

Figure S4.11: Sulfate concentration (a-b), organic acid concentration (c), phylum-level community structure (d) and Firmicutes OTU relative abundance (e) of Baffin Bay North

xvii sediment 50°C incubations. a) Bars represent the average of 3 replicates and error bars show standard error. b) Sulfate concentrations of each replicate are plotted individually. c) Organic acid concentrations of each replicate are plotted individually. d-e) The bars represent the combined relative abundance at the phylum level (d) or the OTU level (e) of the three replicates. Additionally, the sum of relative abundances of OTUs present less than 1% was 2.4% and is shown in (e). Only those phyla and OTUs >1% relative abundance are shown in the legends.

Figure S4.12: Sulfate (a) and organic acid (b) measurement of media only control incubations for David Strait and North Baffin Bay incubations. Error bars show standard error.

Figure S4.13: Sulfate concentration (a), organic acid concentration (b), phylum-level community structure (c) and Firmicutes OTU relative abundance (d) of Svalbard sediment 50°C incubations. These incubations are the same incubations discussed in Chapter 3 as positive controls. a) Bars represent the average of 3 replicates and error bars show standard error. b) Organic acid concentrations of each replicate are plotted individually. c-d) Bars represent the combined phylum-level and OTU-level abundances of the three replicates. Only those phyla and OTUs >1% relative abundance are shown in the legends.

Figure S5.1: Sulfate concentration (a) and organic acid concentration (b) of incubations of Scotian Slope West sediment. a) Bars represent the average of 6 replicates for incubations of sediment from 0 cm and the average of 3 replicates for incubations of sediment from 260 cm and 525 cm. Error bars show standard error. b) Histograms of the organic acid concentration of replicates are plotted individually.

Figure S5.2: Phylum-level community structure (a) and OTU-level structure within the Firmicutes community (b) of incubations of Scotian Slope West sediment. Only phyla abundant >1% in a replicate are shown in the legend. b) The OTU-level of the Firmicutes component of the 16S gene amplicon libraries is only shown for 0 cm sediment since replicates from incubations of this depth only show an increase in the relative abundance of Firmicutes. Percentages represent total relative abundance of each OTU in the complete amplicon library. Only those OTUs present >1% relative abundance of the total library are shown in the legend. The OTU’s closest neighbors based on sequence similarity are listed in Table 5.2.

Figure S5.3: Sulfate concentration (a) and organic acid concentration (b) of incubations of Scotian Slope Centre sediment. a) Bars represent the average of 3 replicates. Error bars show standard error. b) Histograms of the organic acid concentrations of replicates are individually shown.

Figure S5.4: Phylum-level community structure (a) and OTU-level structure of the Firmicutes community (b) of incubations of Scotian Slope Centre sediment. Only phyla abundant >1% in a replicate are shown in the legend. Only OTUs abundant >1% in a replicate are shown in the legend.

xviii Figure S5.5: Sulfate concentration (a) and organic acid concentration (b) of incubations of Scotian Slope East sediment. a) Bars represent the average of 3 replicates and error show standard error. b) Histograms of the organic acid concentrations in each replicate are plotted separately.

Figure S5.6: Phylum-level community structure (a) and OTU-level structure of the Firmicutes community (b) of incubations of Scotian Slope East sediment. a) Only phyla abundant >1% in a replicate are shown in the legend. b) Only OTUs present >1% relative abundance of the total library are shown in the legend.

Figure S6.1: Sulfate concentration over 21 days of incubation at 50°C, 60°C or 70°C of sediment from five different sediment depths (206, 254, 440, 700, and 865 mbsf) and a medium-only control. Sulfate reduction is not observed in any of the incubations. Bars represent the average of triplicates in incubations of sediment from 440, 700, and 865 mbsf and the medium-only control. Bars represent the average of duplicates of incubations of sediment from 206 and 254 mbsf. Error bars show standard error.

Figure S6.2: Organic acid concentration over 21 days of incubation at 50°C, 60°C, or 70°C of sediment from five different sediment depths (206, 254, 440, 700, and 865 mbsf) and a medium-only control. The concentrations of lactate, acetate, succinate, propionate, and butyrate remain constant over 21 days. Bars represent the average of triplicates in incubations of sediment from 440, 700, and 865 mbsf and the medium-only control. Bars represent the average of duplicates of incubations of sediment from 206 and 254 mbsf. Error bars show standard error.

xix List of Abbreviations

BBN – Baffin Bay North bp – base pair cmbsf – centimetres below seafloor DPA – dipicolinic acid DS – Davis Strait FB – Frobisher Bay LS – Labradore Slope LUCA – Last universal common ancestor mbsf – metres below seafloor PGM – phosphoglucerate mutase PI – Pond Inlet SASP – Small acid-soluble protein SSC – Scotian Slope Centre SSE – Scotian Slope East SSW – Scotian Slope West Sv – Svalbard 3PGA – 3-phosophoglyceric acid

xx Chapter 1: Introduction

1.1 Bacterial endospores

During the latter half of the 19th century, the exploration of bacterial endospores, or “spores”, began. Cohn (1876) wondered why some microorganisms were able to survive treatment of 100°C while others could not (Gould, 2006). At the same time Koch (1888) began observing the sporulation in spore-formers such as Bacillus anthracis and was the first to describe the sporulation process (Gould, 2006). Since then, the structure and physiology of the spore has been investigated in detail and much is known

1 about sporulation and dormancy. Knowledge of endospore physiology has become important for a wide range of topics in biology – from investigations into practical concerns such as medical sterilization and food storage to speculative inquiries such as astrobiology and the history of life on earth. Endospores are specialized structures made by some members of the bacterial phylum Firmicutes that allow the cell to survive, in a dormant yet viable state, periods of stress that would otherwise kill the vegetative cell. Endospores are known to remain viable after exposure to high pressures (>800 MPa or 7900 atm) and the vacuum of space, gamma- and UV-radiation, wet and dry heat (in some cases >150°C), freeze- drying, chemical toxins, predation, and desiccation (Fairhead et al., 1994; Setlow, 1994; Nicholson et al., 2000; Margosch et al., 2004; Klobutcher et al., 2006; O’Sullivan et al., 2015). They can survive these hostile conditions because of some unique physiological features and strategies that address the major reasons environmental stresses cause cell death – protein denaturation and DNA damage. A vegetative bacterium with the ability to sporulate produces an endospore usually in response to nutrient limitation in the spore-former’s habitat although other stressors specific to the organism such as changes in temperature (Bahl et al., 1995) or pH (Al-Hinai et al., 2015) may also trigger sporulation. Sporulation begins with the activation of spo0A, the master transcriptional regulator for sporulation in all spore- formers (Galperin et al., 2012). During endospore formation the cell asymmetrically divides into a mother cell and a forespore, which becomes the endospore. As sporulation progresses several physiological features develop, such as core dehydration and DNA stabilization, which prepare the spore for survival against many types of stress. At the completion of sporulation, the endospore is released when the mother cell lyses. Morphological features of the endospore include a dehydrated core that contains the DNA surrounded by a core membrane and a cortex of peptidoglycan (McKenney et al., 2013). The outer layer of the endospore, the spore coat, contains species-specific proteins that may contribute to environment selectivity during germination (McKenney et al., 2013). The mature endospore is metabolically inactive and lacks high-energy compounds such as ATP and NADH but it does contain substrates required for their production (e.g. 3-phosphoglyceric acid (PGA)). Enzymes acting on these substrates

2 are inactive in the spore core thus maintaining the metabolic dormancy of the spore (Setlow, 1994). When environmental conditions improve, the endospore germinates and returns to a vegetative, metabolically active, state. Signals for germination –germinants – are species specific and include low-molecular weight nutrients, ions, and high pressure; heat increases spore responsiveness to germinants (Setlow et al., 2017). Additionally, low levels of spontaneous germination occur. Products of endospore germination and growth, such as fragments of peptidoglycan, are also germinants (Setlow et al., 2017). Germination includes rehydration of the spore core, degradation of proteins bound to the DNA (e.g. SASPs), activation of enzymes for the production of ATP and NADH, and the release of the spore specific compound dipicolinic acid (DPA) (Setlow, 2007; Setlow et al., 2017). Once germinated, the vegetative cell is metabolically active and can grow and divide but it is vulnerable to stresses that it was resistant to in the endospore state.

1.2 Endospore strategies for survival and longevity

The endospore is specialized such that it can survive a variety of stresses that would be lethal to its vegetative counterpart. Around twenty percent of the dry weight of the spore is dipicolinic acid (DPA), which exists as a 1:1 chelate with Ca2+ and contributes to DNA stability during exposure to heat stress, desiccation, and hydrogen peroxide (Setlow, 2007). The core of the endospore is more acidic with a pH ~1.2 units below that of corresponding vegetative cells (Setlow and Setlow, 1980). The lower pH in the spore core inhibits the enzyme phosphoglycerate mutase (PGM) thereby regulating the catabolism of the high energy compound 3-phosophoglyceric acid (3PGA) and contributing to the maintenance of metabolic dormancy (Setlow, 1994; Setlow, 2007). The core of the spore is dehydrated; the water content of the spore core is 28% to 50% wet weight while the water content of the corresponding vegetative cell is 75% to 80% wet weight (Nicholson et al., 2000). Enzymes and ions in the spore core likely exist in a gel-like state that is difficult to permeate and may prevent small molecule toxins from damaging the contents of the core, although the permeability of the core membrane is likely also a barrier to many compounds (Black and Gerhardt, 1961; Carstensen et al.,

3 1971). While the dehydration of the core is not enough to prevent the movement of water molecules it may contribute to the maintenance of enzymes in an immobile and inactive state (Cowan et al., 2003; Sunde et al., 2009). Dehydration has also been correlated to an increase the heat tolerance of dormant endospores (Nakashio and Gerhardt, 1985; Beaman and Gerhardt, 1986). Additionally, endospores contain a family of DNA binding proteins called small acid-soluble proteins (SASPs) that protect the spore from damage due to heat, UV radiation, desiccation, and toxins (Setlow, 2007). These are highly conserved across all spore-formers and make ~50% of the protein in the spore (Setlow, 2007). SASPs are considered to be the major feature protecting spore DNA and are essential for maintaining viability after exposure to DNA damaging stresses such as UV radiation, toxins and dry heat. These specialized features of endospores mean that in this state the cell can survive stresses lethal to its vegetative neighbors. An intriguing consequence of endospore physiology and resistance mechanisms is that spores are potentially able to remain dormant yet viable for very long time periods. Endospores have been revived from 25-million year old amber and 250-million year old halite crystal (Cano and Borucki, 1995; Vreeland et al., 2000). These studies have faced some criticism (Graur and Pupko, 2001; Hebsgaard et al., 2005), while still garnering support and acceptance from many in the scientific community. An independent analysis of endospore thermal inactivation kinetics measured by their decimal reduction value, the time it takes for the viable population to decrease by a factor of 10, indicates that endospores may remain viable over millions of years (Nicholson, 2003). Much of the criticism of these studies about reviving ancient endospores comes from the observation that DNA molecules are too chemically unstable to survive over these time scales. However, most explorations into the longevity of ancient DNA study naked DNA not DNA protected in endospores which have special features that stabilize the DNA, the most significant being the SASP proteins discussed above.

1.3 Thermophilic endospore-forming bacteria

Thermospores are endospores of thermophilic endospore-forming bacteria and thus require temperatures between 45°C and 80°C to germinate, grow, and divide. Although the designation is somewhat anthropocentric, thermophiles are considered extremophiles because the high temperature environments in which they thrive are considered “extreme” for most life on Earth. Endospores of thermophilic bacteria have some advantages that may lead to increased stress tolerance and longevity. Endospores made in environments of higher temperatures achieve a greater degree of desiccation and thus are more resistant to wet heat and some chemical toxins than those generated by mesophiles at lower temperatures (Beaman and Gerhardt, 1986; Melly et al., 2002); the mechanism for achieving greater desiccation in thermophilic endospores in unknown. Additionally, in the endospore thermal inactivation study by Nicholson (2003) mentioned above, thermospores were calculated to survive periods of dormancy longer than mesophilic endospores with an estimated longevity exceeding 2 billion years. In recent years thermospores have been found in permanently cold marine sediment by high temperature incubation experiments (Hubert et al., 2009; de Rezende et al., 2013; Müller et al., 2014; Volpi et al., 2017). These misplaced thermophiles are members of the dormant microbial seed bank. Dormant thermophiles found in permanently cold sediment are immediately identified as foreign simply because they are unable to grow and divide in cold environments. Their presence in these sediments suggest that dispersal mechanisms are acting on these thermospores and transporting them from their original warm habitat to the cold seabed sediments where they are found.

1.4 Microbial biogeography and thermospores

Microbial biogeography is the study of the distribution of microbes across space and time. Hanson et al. (2012) described selection, mutation, drift, and dispersal as the

5 four main processes driving microbial biogeography. Selection encompasses the processes by which environmental features shape microbial diversity. For example, only hyperthermophilic microorganisms adapted to very high temperatures (80°C) can grow and divide in the areas adjacent to black smokers from hydrothermal fields. Drift is the changes in frequency of genotypes or species (depending on the taxonomic resolution) due to chance events and mutation is the random change in genetic sequence that may lead to altered phenotype. Selection and mutation may together cause a shift in the frequency of genotypes; for example, a random mutation in the hyperthermophile above may lead to more efficient metabolism at high temperature causing this new strain of hyperthermophile to dominate the community. Dispersal is the movement and successful colonization of a taxon at a new geographic location. For example, the new strain of hyperthermophile might be ejected from the black smoker in rising fluids, become suspended in the prevailing ocean current, and arrive at a new hydrothermal fluid environment that it is able to colonize. Movement during microbial dispersal may be directed, controlled by the movement of the bacteria by motility features such as flagella, or passive, requiring a vector for relocation. The most commonly studied microbial biogeographic pattern is the distance- decay relationship, the decrease in community similarity with increasing spatial distance, and together the four processes described above shape this pattern (Hanson et al., 2012). Selection and drift create the distance-decay pattern by creating variation with geographic distance and historical selective forces may affect present-day microbial diversity even after those selective forces are gone. Mutation adds to genetic variability and affects the pattern primarily at high taxonomic resolution, while dispersal shapes the distance-decay pattern by reducing patchiness. If dispersal is too great, this process may interfere with the microbial community structure resulting from historical selection and drift and minimize the distance-decay pattern (Hanson et al., 2012). Dispersal limitation is the confinement of an organism to a certain location or geographic region; this occurs either when an organism is unable to freely move from one location to another, or when an organism is unable to establish itself in a new location. There is some debate over whether microorganisms display dispersal limitation based on restricted movement or whether their biogeography is due to environmental

6 heterogeneity alone (Horner-Devine et al., 2004). Some studies show that microorganisms display some dispersal limitation (Whitaker et al., 2003; Abell and Bowman, 2005; Agogué et al., 2011; Hamden et al., 2013; Müller et al., 2014) while others suggest a persistent seed bank of all microorganisms at all locations with variations in microbial community structure being due to environmental selection (Caporaso et al., 2012; Gibbons et al., 2013). The debate continues partially because different taxonomic levels are used to observe microbial diversity and community structure, and thereby measure biogeography, in different studies (Horner-Devine et al., 2004; Ramette and Tiedje, 2007; Hanson et al., 2012). It is in this context that thermospores become useful tools for biogeographical studies. Because their requirement for warm temperatures identifies thermospores as foreigners to any cool environment (<45°C), their presence suggests that dispersal has occurred and that a dispersal vector is connecting this cool site with the thermospores’ original warm source habitat. Furthermore, because thermospores are dormant at cool temperatures in a protective endospore, environmental selection does not affect thermospores’ dispersal pattern; nor does drift since absence of cell division prevents changes in genotype from occurring and altering the thermospore community structure. Widespread dispersal of endospores has been implicated in the prevention of species divergence for geographically distant Bacillus sp. (Roberts and Cohan, 1995) showing that the endospore physiology reduces the effect of biogeographic processes, such as selection, mutation, and drift that could lead to endemic populations of endospore- forming bacteria. In the marine environment, thermospores are often found in cool sediment (e.g. Hubert et al., 2009; de Rezende et al., 2013; Müller et al., 2014; Volpi et al., 2017). Warm habitats connected to the marine environment include petroleum reservoirs and oceanic spreading centers and associated hydrothermal vent systems. These are likely candidate habitats of thermospores found in cool sediments and they can often connect to the water column through seeping fluids that may act as a vector passively transporting thermospores from their warm subsurface environments to the cool sediments where they are found.

7 1.5 Thermospores in anoxic environments

Candidate source habitats for thermospores include warm temperature geologic features that are anoxic such as hydrothermal vent systems, hydrocarbon reservoirs, mud volcanoes, and deep sediments and sulfate-reducing bacteria have been isolated from these environments (Muyzer and Stams, 2008). Thermophilic Desulfotomaculum spp. – spore-forming sulfate reducers – are often found in deep subsurface environments or fluids connected to these deep potential habitats (Ollivier et al., 2007; Aüllo et al., 2013) such as oil reservoirs (Gittel et al., 2009), oilfield production waters (Tardy-Jacquenod et al., 1996, Liu et al., 2008; Lan et al., 2011), and hydrothermal vent carbonate chimneys (Brazelton et al., 2006). In these anoxic environments sulfur compounds such as sulfate, thiosulfate, sulfite, and sulfur can serve as terminal electron acceptors for microorganisms. In addition to sulfate reduction, thermospore germination and anoxic growth using fermentation has been shown (Volpi et al., 2017). Examples of thermospore species are shown in Figure 1.2 alongside metabolic features. While there are thermophilic spore-formers that are aerobic, anaerobic thermospores have been targeted in the present research to investigate those that are most likely to come from these deep anoxic environments.

9 1.6 Panspermia and the last universal common ancestor

There are practical reasons to study microbial biogeography using thermospores, for example Hubert and Judd (2010) have suggested a method for using the dispersal pattern of thermospores in marine hydrocarbon seep prospecting. However, the biogeographical themes touched upon in this thesis connect to broader questions about thermospores, biogeography, and passive dispersal. Panspermia, the theory that life is dispersed throughout the universe by vectors such as meteors, was introduced in Chapter 3. This theory is sensational but there is evidence that panspermia may be possible. Panspermia involves three stages - ejection from the donor planet, travel through space, and capture by the recipient planet. Lithopanspermia is the movement of life between planetary bodies using rock-like vectors, such as comets or meteors, and is the mechanism for non-anthropogenic panspermia that is most plausible. For lithopanspermia to occur, rocky material containing endolithic life must be expelled from a donor planet at escape velocity and the encased life must survive this ejection process. Launch into space may happen when an asteroid or comet hits a planet and rocks in the area near the impact site, the spallation zone, are launched into space at velocities exceeding the planet’s escape velocity. For example, the escape velocity is 5 km/s for a rocky mid-sized planet such as Mars and the material ejected from the surface would experience acceleration up to ~3.8 × 106 m/s2 (~390 000 x g) and jerk forces (the rate of change of acceleration) of ~6 × 109 m/s3 (Nicholson, 2009). Using ultracentrifugation and ballistics tests, endospores of Bacillus subtilis were shown to remain viable (40-100%) after subjection to the velocity, acceleration, and jerk forces encountered during simulated impact-ejection from Mars (Mastrapa et al., 2001, Fajardo-Cavazos et al., 2007). Furthermore, Horneck et al. (2008) showed that Bacillus subtilis spores survive temperatures and pressures experienced by Martian meteorites that have been found on earth. The results of these studies show that the spore survival during the first step of lithopanspermia, impact-ejection from the donor planet, is possible. After ejection into space from a donor planet, life must survive the harsh conditions of space over timescales allowing for the transit between the donor planet

10 and a recipient planet. Endospores are known to survive the kinds of stressors that would be associated with exposure to space such as desiccation, vacuum pressures, radiation, and extreme hot and cold temperatures (Nicholson et al., 2000). The most damaging agent to maintained endospore viability in space is radiation, although a few centimeters of rock material is enough to shield UV, x-rays, and solar particles (Mileikowsky et al., 2000). Even without protection Bacillus subtilis endospores remained viable (1-2% survival) after 6 years of direct exposure to space conditions on the Long Duration Exposure Facility, an orbiting space laboratory operational between 1984 and 1990 (Horneck, 1993). Endopore survival increased significantly (70-90% survival) when protected in a buffer or glucose solution (Horneck, 1993, Horneck et al., 1994), or when protected by rocky meteor-like material as was shown during the SPORES experiment on the International Space Station (Panitz et al., 2015). Other extra-planetary experiments have corroborated these results showing the survival of endospores to space conditions (Horneck et al., 2010). However, surviving the harsh environment of space is not enough; endospores must remain dormant for timescales that allow for the extraterrestrial rock housing life to be received by a donor planet. It is this longevity component of the lithopanspermia endospore dispersal requirement and scenario that distinguishes thermospores from mesophilic bacterial endospores. As discussed previously, thermospores are predicted to have longer survival times than mesophilic endospores based on thermal inactivation kinetics as defined by decimal reduction times (Nicholson, 2003). Estimates by Nicholson (2003) suggest that thermophilic endospores of Bacillus stearothermophilus can remain viable for over 2 billion years. This is enough time for a meteorite originating from Mars to collide with Earth since transit times from Mars to Earth can be as short as 16 000 years (Gladman et al., 1996). Interstellar transit would be much longer, but with transit times in the tens of millions of years, thermospores could still remain viable during such transport. The last step in a successful lithopanspermia event is capture by a habitable planet and colonization. To survive re-entry, endolithic spores must survive intense heat and forces of impact as the life-bearing meteor descends towards the planet. Fortuitously, atmospheric planets provide gentler re-entry events because the atmosphere slows the meteor to velocities of 102 m/s before impact. Impact further aids

11 the life seeding process on the recipient planet by shattering the meteor and spreading pieces of it on the planet’s surface (Nicholson, 2000). Because re-entry lasts only a few minutes, only the surface millimeters of the meteor experience significant heating. Using laboratory and artificial meteorite experiments, endospores have been shown to survive the forces of re-entry both naked (Barney et al., 2016) and within a protective rock casing (Fajardo-Cavazos et al., 2005; Slobodkin et al., 2015). Slobodkin et al. (2015) report the survival of thermophilic endospores protected by just 1.4 cm of basalt after atmospheric entry of an artificial meteorite through Earth’s atmosphere at 7.6 km/s leading to surface temperatures of 1100°C. Arguments in favour of endospore survival at each stage of lithopanspermia imply that this process is possible. For panspermia to occur the following assumptions must be made. Firstly, the donor planet must bear life, similar to Bacillus sp. endospores, and this life must exhibit tolerances to the extreme stresses encountered during ejection, space transit, and capture, and must maintain viability for timescales required to be captured by a recipient planet. Secondly, an event causing rock material bearing such life to be ejected at escape velocity from the donor planet must occur. Thirdly, a recipient planet with an environment suitable for the organism’s growth and proliferation must capture the meteor harbouring this extraterrestrial life. Nicholson (2007) has described the probability of the occurrence of panspermia (PAB) as follows:

PAB = PBIZ × PEE × PSL × PSS × PSE × PSI × PREL × PSP

PBIZ = probability of an impact event in a biologically inhabited zone

PEE = probability that rocks containing endolithic microbes are ejected onto an escape trajectory

PSL = probability of the organism surviving launch

PSS = probability of the organism surviving transit through space

PSE = probability of the organism surviving entry through the recipient planet’s atmosphere

PSI = probability of the organism surviving impact with the recipient planet

PREL= probability of release from the rock to the surface of the recipient planet

12 PSP = probability of survival and proliferation on the recipient planet

One might imagine the chances of lithopanspermia occurring naturally to be very low based on the equation above. Regardless of the actual probability of panspermia, life on Earth in the form of bacterial endospores can survive all the steps required in the process. Therefore, it is possible that life on earth or elsewhere originated by this process, and that processes such as these could be occurring throughout the universe. It is estimated that there are ~ 1010 habitable planets in the Milky Way alone (Lingam and Loeb, 2017). Recently, seven planets were found in the TRAPPIST-1 system and three of these are in the habitable zone, the region where liquid water may exist (Gillon et al., 2017). Panspermia between the three habitable planets of the TRAPPIST-1 system has been explored (Lingam and Loeb, 2017) demonstrating that questions about extraterrestrial life and panspermia are not exclusively Earth-centric. Additionally, opportunities for anthropogenic panspermia have existed since the onset of the space age. The possibility of extraterrestrial contamination from returning spacecraft or of terrestrial contamination of planetary objects is taken seriously, e.g. NASA has a policy for preventing contamination of Earth and other celestial bodies during extraterrestrial missions (Zurbuchen, 2017). While it is scientifically important to avoid, contamination, natural or anthropogenic, may be evidence of biogeographical patterns of dispersal extending beyond our planet. Naturally, the question that arises after the acceptance of panspermia as a experimentally-tested possible method of biological dispersal throughout the universe is: is modern life on Earth the result of a panspermia event? There is evidence to suggest that the last universal common ancestor (LUCA), the cell from which all modern life on earth evolved, may have been a thermophilic spore-former and, as will be discussed throughout this thesis, thermophilic spore-formers may be the most able organisms to survive panspermia. Through phylogenetic analysis of 6.1 million prokaryotic genes, Weiss et al. (2016) identified 355 proteins likely possessed by LUCA. These genes point to it living in a thermophilic environment. While sporulation proteins were not part of those identified as belonging to LUCA, microorganisms related to acetogenic clostridia (spore-forming Firmicutes) as well as lineages of methanogenic archaea were identified

13 as the modern microorganisms most closely resembling LUCA. Based on the criteria used for identifying LUCA’s proteins, one of which was the requirement that the protein be present at least two phyla of Bacteria and Achaea, sporulation proteins must be excluded (Weiss et al., 2016) but Tocheva et al. (2016) argue that LUCA was a spore former. They propose that the emergence of the diderm (two membranes) phenotype arose from a sporulating monoderm (one membrane) that retained its second membrane during germination. Monoderms and diderms are found throughout the tree of life and many of the outer membrane proteins found in diderms share homology. The evolutionary tree that reduces the number of times the traits of sporulation and the presence of an outer membrane must be gained and lost identifies a spore-forming diderm as the last universal common ancestor (Tocheva et al., 2016). Weiss et al. (2016) identifies a thermophile and Tocheva et al. (2016) identifies a spore-former as possible LUCAs for life on earth and since organisms with both these microbial physiologies may also survive panspermia, it is possible that the ancestor to all life on Earth was originally a thermophilic, spore-forming extraterrestrial.

1.7 Thesis overview

This thesis investigates the use of thermospores as model organisms for studying microbial dispersal. Chapter 3 highlights the extreme survivability of thermospores by showing their continued viability even after freezing at -80°C. Chapter 4 explores thermospore passive dispersal geographically throughout North Atlantic marine surface sediment. Temporal dispersal of thermospores in the North Atlantic is analyzed in Chapter 5 by observing thermospore presence along a sediment core correlated with geologic time. Thermospore temporal dispersal over millions of years is examined in Chapter 6 in the examination of the thermophilic microbial community structure of a 1.2 km sediment core.

14 Chapter 2: Materials and Methods

The materials and methods described below are those that are common to each experiment described in this thesis. Methods specific to certain experiments will be described in the Materials and Methods section of the associated chapter.

2.1 Sediment sample collection

Marine sediment was collected from areas around the North Atlantic and Nankai Trough (Table 2.1, Figures 4.1 and 6.1). Piston coring and sediment handling of cores from the Scotian Slope was done as described in Mosher et al. (2004). Surface sediment from the Labrador Shelf and the location in Baffin Bay (Figure 4.1) were sampled using a box corer. Samples were stored at 4°C in anoxic packaging (i.e. sealed glass containers or plastic bags) until further use to prevent the germination of thermospores during storage.

15 Table 2.1: Details of marine ocean sediment used in this study.

Station Name Year Geographic Longitude Latitude Water Coring Core Length Location Depth Method1 (m) Svalbard 2007 Svalbard 79°42.006 N 11°05.199 E 212 B Surface2 Davis Strait 2013 Baffin Bay 69°36.04 N 65°48.16 W 330 B Surface2 Baffin Bay North 2014 Baffin Bay 76°18.836 N 71°06.788 W 656 B Surface2 Pond Inlet 2015 Baffin Bay 72°29.29 N 78°46.86 W 376 B Surface2 Frobisher Bay 2015 Baffin Bay 68°36.731 63°38.41 W 121 B Surface2 Labrador Shelf 2015 Labrador Shelf 58°55.609 N 62°09.321 W 141 B Surface2 Scotian Slope West 2015 Scotian Slope 42°03.1616 N 64°42.0124 W 2016 P, T 5.55 m Scotian Slope 2015 Scotian Slope 42°21.6127 N 62°27.9289 W 2208 P, T 9.02 m Centre Scotian Slope East 2015 Scotian Slope 43°00.7864 N 60°12.8241 W 2342 P, T 6.90 m C0023A 2016 Nankai Trough 32°22.00’ N 134°57.58’ E 4775.5 F, R, X 1177 m 1Coring Methods B: box core F: Short advance hydraulic piston coring system P: Piston core R: Rotary core barrel T: Extended punch coring system X: Extended shoe coring system

2Surface sediment includes sediment from ~ 0-0.05 mbsf

16 2.2 High temperature incubation

Sediment was anoxically incubated in an oven at 50°C in artificial seawater medium (Isaksen et al., 1994) amended with sulfate (20 mM) and the organic acids formate, lactate, acetate, succinate, propionate, and butyrate (each to final concentration of 1 mM). A 1 mM solution of this organic acids mixture would support the reduction of 8.75 mM sulfate to sulfide should all the organic acids be used to reduce sulfate completely (Janssen et al., 1996; Martins et al., 2015; Muyzer et al., 2008). Microcosms were prepared with sediment and the seawater medium in a 1:2 (w/v) ratio in 50 mL or 120 mL serum bottles sealed with black rubber stoppers and flushed with

9:1 mol% N2/CO2 gas for 1 min to obtain anoxic conditions. Biological triplicates were established by adding inoculum sediment directly from the storage package rather than sediment slurries. When available, replicates were established from different replicate cores from the same geographic location (Table S2.1). Pasteurization at 80°C for one hour before incubation at high temperature selected for the enrichment of thermophilic endospore-forming bacteria because pasteurization destroys many vegetative cells while incubation at 50°C selects for thermophiles. Microcosms were subsampled during the course of the experiment. The subsamples were centrifuged at 14 800 rpm into supernatant and pellet fractions and stored at -20°C. Microcosms were incubated unshaken at 50°C for up to 53 days.

2.3 Sulfate and organic acid measurement

Sulfate and organic acid concentration was measured from the supernatant fraction of a subsample from each microcosm at various time points during the incubation to monitor microbial activity. The sulfate concentration was obtained by either the turbidimetric method (Nemati et al., 2001) or ion chromatography (Dionex ICS-5000, Thermo Scientific). Organic acid concentrations were measured with ultra-high performance liquid chromatography (Ultimate 3000 RSLCnano UHPLC system, Thermo Scientific) at a 0.6 ml min-1 flow rate using either the Acclaim™ Organic Acid LC Column

17 (5μm, 4 × 250 mm, Thermo Scientific) or the Aminex HPX-87H column (5μm, 7.8 × 300 mm, Bio Rad).

2.4 16S rRNA gene amplicon sequencing and analyses

2.4.1 DNA extraction

DNA extraction was done on subsample slurries (300 mL) or pellets (0.3 g) using the in-house method described by Foght et al. (2015) or the DNeasy PowerSoil Kit (formerly the PowerSoil DNA Isolation Kit, MoBio) (Qiagen). DNA extraction negatives were established by performing the DNA extraction protocol on the buffer solution (300 mL) used in the corresponding extraction method. PCR on the DNA extraction negatives, as described in section 2.4.2, confirmed the absence of contamination introduced during the extraction process.

2.4.2 16S rRNA gene amplification and sequencing

A 444 bp fragment of the V3-V4 hypervariable region of the 16S rRNA gene was amplified using the primer pair S-D-Bact-0341-a-S-17 and S-D-Bact-0785-a-A21 (Klindworth et al., 2013). To minimize PCR bias, triplicate PCR reactions were carried out in 25 µl volumes (12.5 µl 2x KAPA HiFi Hot Start Ready Mix, 2.5 µl of each primer (1 mM), 1-5 µl template DNA, sterile water up to a total volume of 25 µl). The DNA was initially denatured at 95°C for 5 min, then 10 cycles of denaturing at 95°C for 30 sec, annealing for 60°C for 45 sec, and extension at 72°C for 1 min, followed by 20 cycles of denaturing at 95°C for 30 sec, annealing at 55°C for 45 sec, and extension at 72°C for 1 min, then a final extension at 72°C for 5 min. Amplified 16S rRNA fragments 350 bp in length were prepared for sequencing as per Dong et al. (2017) and sequenced on a MiSeq Benchtop DNA sequencer (Illumina).

18 2.4.3 16S rRNA gene amplicon sequence analysis

Community analysis was done using the MetaAmp pipeline (Dong et al., 2017). The number of reads passing the quality control step of the MetaAmp pipeline for each sample’s amplicon read library is shown in Figure 2.1. The MetaAmp pipeline established operational taxonomic units (OTUs) based on 97% sequence identity; these OTUs were used as functional units for assigning taxonomy. Paired end merging options for the MetaAmp program were 100 bp for the minimum length of overlap and 8 bp as the maximum number of mismatches in the overlap region. The quality filtering options used were the maximum number of differences to the primer sequence being 1 bp, the maximum number of expected errors was 1 bp, and length of the amplicon trimmed to 350 bp. All analyses were done using OTU tables, generated by MetaAmp (versions 1.3 and 2.0) to calculate Bray-Curtis dissimilarity matrices with the software environment R (R Core Team, 2013) and a community analysis workflow (S.E. Ruff, unpublished data). The workflow is based on custom R scripts and the R packages ‘vegan’ (Oksansen et al., 2012) and ‘cluster’ (Maechler et al., 2017). The Bray-Curtis algorithm was used because it considers OTU presence/absence and abundance, giving relatively more weight to those OTU that have higher relative abundance. This is especially important when a few populations dominate the communities. The similarity of sample communities was assesed visualized using non-metric multidimensional scaling (NMDS) based on dissimilarity matrices generated by MetaAmp (Dong et al., 2017). The significance of the NMDS ordinated groups was tested using Analysis of Similarity (ANOSIM). Bacterial community diversity was calculated using the Inverse Simpson Index. Germination experiments were required for the detection of thermospore OTUs in this study. After incubation at 50°C, viable thermospores are presumed to have germinated and grown in the microcosms and thus are assumed to be thermophilic, because they grew at 50°C, and present as endospores in the original sediment, because they survived pasteurization at 80°C for 1 hour (see section 2.2). Thermospore OTUs (defined by sharing 97% sequence identity) from amplicon libraries were identified

19 for further consideration and analysis based on the following criteria. Sequences were to be affiliated to the phylum Firmicutes, since all known endospore-formers are members of this phylum (Galperin et al., 2012). In addition, OTUs had to be present in at least one post-incubation sample in greater than 1% relative abundance. Furthermore, the number of reads for each thermospore OTU identified as such had to be present in less than two reads in the corresponding pre-incubation sample. Allowing up to two reads prevents the presence of one or two reads in a before-incubation (time 0) library from interfering with the identification of a thermospore OTU present in an after-incubation library (>1% relative abundance). In every sample explored in this thesis two reads is far below the up to 0.16% of reads in an amplicon library that may be due to carryover contamination and index misassignment (Nelson et al., 2014) (see Figure S2.1 and S2.2 for total read abundances per sample). An increase in relative abundance from no greater than 2 reads to over 1% total relative abundance may demonstrate that germination occurred since DNA is difficult to extract from spores without specialized procedures (Wunderlin et al., 2014). Significance of OTU relative abundance was confirmed using the STAMP application (Parks et al., 2014) using a two-sided Fisher’s Exact test, preferred for its accuracy with small counts (Parks and Beiko, 2010), the Bonferroni multiple test correction to prevent false positives, and a p-value of <0.050 on the subsampled amplicon libraries. Nearest neighbours to the identified OTUs based on sequence similarity were found using the BLAST application (Madden, 2002). The representative sequences for each OTU are chosen based on the UPARSE-OTU algorithm and used for nearest neighbour identification in BLAST. Phylogenetic trees for the identified OTUs were calculated in ARB (Ludwig et al., 2004) using the maximum likelihood (phyML) tree generated using >1300 bp full-length 16S rRNA reference sequences of closely related bacteria. Phylogenetic trees were visualized and prepared for publication using iTOL (Letunic and Bork, 2006).

20 Chapter 3: Freezing tolerance of thermophilic endospores in Arctic marine sediment

3.1 Abstract

Dormant endospore-forming thermophiles found in permanently cold marine sediments offer a useful model for studying microbial biogeography and passive dispersal. The dormant endospore phenotype confers resistance to unfavorable environmental conditions, allowing dispersal to occur without environmental selective pressure. Accordingly, thermophilic endospores are found in surface sediments from around the world, including the fjords of Svalbard in the high Arctic where temperatures are close to 0°C year-round. To study the resilience of Arctic thermophilic endospores to different freezing temperatures, their viability following various periods of freezing was examined. Marine sediment was frozen at -20°C or -80°C for two and ten days prior to pasteurization (one hour at 80°C) followed by incubation at 50°C for 53 days. Sulfate reduction was observed following both freezing pretreatments indicating a robust persistence of thermophilic spores of sulfate-reducing bacteria. The onset of sulfate reduction at 50°C was delayed in -80°C pre-frozen microcosms, which exhibited more variability between triplicates, compared to non-frozen and -20°C pre-frozen microcosms. Microbial communities in all microcosms were evaluated by 16S rRNA gene amplicon sequencing, revealing an increase in the relative abundance of known thermophilic spore-forming Firmicutes in all microcosms. Microbial community analyses revealed community shifts during 50°C incubation, and that unfrozen and -20°C pre- frozen microcosm communities were more similar to each other than either were to community structure of -80°C pre-frozen microcosms. The latter were more variable between replicates with apparent dominance of Bacilli that were only detected following -80°C freezing. Certain spore-forming taxa that showed germination at 50°C in non- frozen microcosms appeared unable to survive freezing at -20°C and -80°C (e.g. Sporosalibacterium spp.) while others remained viable after -80°C freezing pretreatments (e.g. Bacilli, Caloranaerobacter spp. and Clostridium halophilum). Athough the differential freezing tolerance observed among thermophilic endospores in

21 this study may have been due to O2 contamination upon -80°C pretreatment. The existence of -80°C tolerance in some thermospores of classes Clostridia and Bacilli is shownThese results are important for assessing thermospore viability after -80°C storage or other historical exposure to very cold freezing temperatures, such as in outer space.

22 3.2 Introduction

Spores of tThermophilic endospore-forming bacteria (thermospores) offer a unique model for studying the biogeography of microorganisms. By existing in a dormant state at temperatures below 50°C they can be dispersed across hostile environments without suffering adverse effects. While they must originate in warm environments such as petroleum reservoirs, mud volcanoes, geothermal ground fluids, and hydrothermal vent fluids (Aüllo et al.,2013; Daumas et al., 1988; Detmers et al., 2004; Green-Saxena et al., 2012; Hallmann et al., 2008; He et al., 2013; Müller et al. 2014, Tardy-Jacquenod et al., 1996), marine thermospores are passively dispersed through the water column and deposited in marine sediment globally contributing to a dormant microbial seed bank (de Rezende et al., 2013; Hubert et al., 2009, 2010; Hubert and Judd, 2010; Lennon and Jones, 2011; Müller et al., 2014). This study seeks to expand upon the study of endospore freezing tolerance by exploring the effect of freezing on the viability of thermophilic endospores. While thermophilic endospores are unlikely to ever encounter extremely low temperatures such as -80°C, the low temperature tolerance of thermospores is interesting for several reasons. Firstly, biological samples are often frozen for storage to preserve the integrity of the sample. Knowledge of thermospore freezing tolerance is useful in deciding the types of samples one can use to investigate thermospores, e.g. in studies of biogeography discussed in later chapters. For example, this study was initiated to determine whether sediment samples stored in a 4°C cold room that experienced a malfunction resulting in 2 days below 0°C were still suitable for use in studying the thermospores that were known to exist in the sediment. Secondly, studying the lower temperature limits of thermospores can help elucidate the diversity of thermospore phylotypes within an environment. It is likely that different thermospores have different tolerances to freezing temperatures since they also have different tolerances to extremely high temperatures (O’Sullivan et al., 2015). When a community is stressed by high temperatures, radiation, or toxic chemicals, for example, different members may be better equipped to respond to that stress than others. Thermospore phylotypes that are usually outcompeted in a particular

23 environment may have the opportunity to thrive when thermospore phylotypes that are unable to withstand exposure to the stressor perish. Based on this premise, a larger subset of the thermospore community can be studied by incubating environmental samples under different conditions, including different stressors to encourage the growth of different thermospore phylotypes. Freezing, for various durations of time and at different temperatures, may be one way to stress thermospores and effectively enrich different phylotypes. Thirdly, environments exist where temperatures fall below -80°C despite these environments being largely absent from Earth. Due to their dormancy and resistance to radiation, temperature, and pressure extremes, spore forming bacteria, are models for exploring panspermia, the idea that life evolved beyond Earth but was dispersed to it by an ancient dispersal vector, e.g. meteor (Nicholson, 2009). Interstellar particle temperature is ten degrees Kelvin (-263°C) and Bacillus subtilis spores have been used to study spore viability in the low temperatures, vacuum pressures, and high UV environment of space (Horneck, 1993; Weber and Greenberg, 1985). Nicholson (2003) explored endospore thermal inactivation kinetics based on published decimal reduction values (the time required for a particular treatment to reduce the viable spore population by a factor of 10). The results suggest that thermophilic spores are more likely than their mesophilic relatives to survive the dormancy timescales required for interstellar travel. Only recently have thermospores been studied with regards to the maintenance of viability during exposure to the conditions encountered during panspermia (Slobodkin et al., 2015); previous studies of endospore survival in space-like conditions focused exclusively on the survival of mesophilic endospores (Weber and Greenberg, 1985; Horneck, 1993; Nicholson et al., 2000). The tolerance of thermospores to stressors of outer space should be explored further in light of their potential ability to maintain viability longer than their mesophilic counterparts (Nicholson, 2003). This study does this by exploring the freezing tolerance of thermophilic endospores from marine arctic sediment. It identifies possible differential thermospore activity and bacterial community response upon incubation at high temperature following freezing.

24 3.3 Materials and Methods

3.3.1 Freezing pretreatment

Marine surface sediment from Smeerenburgfjorden (79°42.82’ N 11°05.19’ E) with a year-round in situ temperature of 2°C and known to harbour thermophilic endospore-forming sulfate-reducing bacteria (Hubert et al., 2009) was used in this study. The sediment was stored at 4°C after sampling until the experimental procedure was performed, as described in section 2.1. Microcosms were prepared with 15 g of sediment and flushed with 9:1 mol% N2/CO2 gas to ensure anoxic conditions within 120 mL serum bottles. Before the addition of medium, the sediment microcosms received one of the following freezing pretreatments: -20°C for 2 days, -20°C for 10 days, -80°C for 2 days, or -80°C for 10 days. A parallel set of microcosms was left unfrozen to serve as a positive control since thermospores have previously been enriched from this sediment after storage at 4°C (Hubert et al., 2009).

3.3.2 High temperature incubation

After freezing, 30 mL of medium containing sulfate (20 mM) and amended with 1 mM organic acids (formate, lactate, acetate, succinate, propionate, and butyrate, as described in section 2.2) was added to each of the microcosms. Additional to the organic acids, 1 mM ethanol was added to enhance thermospore germination in this sediment (recommended by CA Hanson, 2015, personal communication, February 24, 2015). Following pasteurization at 80°C for one hour, microcosms were incubated at 50°C for 53 days to enrich for thermophilic endospore-forming sulfate-reducing bacteria. Triplicate microcosms for each of the conditions described above were subsampled immediately before and after pasteurization and then daily for 7 days and a final time after 53 days. Subsampling occurred as described in section 2.2. and the subsampled aliquots were centrifuged to separate supernatant and solid fractions. Supernatant from

25 the subsamples were mixed 1:1 (v/v) with 2% zinc acetate solution to inhibit sulfate oxidization. All subsamples were stored at -20°C.

3.3.3 Sulfate and organic acid measurement

Sulfate and organic acids were measured over the time course of the experiment to monitor microbial activity. The sulfate concentration of each subsample was obtained using a turbidimetric method described by Nemati et al. (2001). Sample supernatant (0.1 mL) was added to 0.9 mL of a 1:50 dilution of conditioning agent (25 mL glycerol, 37.5 mL concentrated HCl, 37.5 mL NaCl, 50 mL 95% ethanol, and MilliQ water to a final volume of 500 mL) and mixed with excess finely ground BaCl2. After 30 minutes, the optical density was measured at 420nm. Organic acid concentrations in the amended microcosms were measured with ultra-high performance liquid chromatography (Ultimate 3000 RSLCnano UHPLC system, Thermo Scientific) using the Acclaim™ Organic Acid LC Column (Thermo Scientific) as described in section 2.3. Formate was not analyzed as the concentration was poorly resolved by the Acclaim™ Organic Acid LC Column. Acetate was not analyzed due to the addition of zinc acetate to the subsamples.

3.3.4 DNA extraction and 16S rRNA gene amplicon sequencing

DNA was extracted from subsamples taken on days 0, 7, and 53 from the unfrozen positive control incubations and those that had been frozen at -20°C or -80°C for 10 days. DNA was obtained from the sediment slurry pellets using the in-house method described by Foght et al. (2015). To amplify the bacterial 16S rRNA genes in the extracted DNA of the subsamples, PCR was performed as described in section 2.4.2. Amplified 16S rRNA gene fragments were sequenced on a MiSeq Benchtop DNA sequencer (Illumina) as described in section 2.4.2. Thermospores were identified based on the criteria outlined in section 2.4.3 in which an OTU is considered a thermospore OTU if it is of the Firmicutes phylum, increases in relative abundance >1% of the total

26 16S rRNA gene amplicon library after 50°C incubation, and the corresponding before- incubation library contains <2 reads. Thermospore OTUs were considered “viable” if detected and identified as thermospores based on the criteria outlined in section 2.4.3.

3.3.5 Gas composition measurement after freezing pretreatment

Empty microcosm bottles sealed with rubber stoppers were flushed with 9:1 mol%

N2:CO2 gas for one minute in a process identical to the headspace flushing done on the sediment incubation microcosms as described in section 2.2. Gas composition (O2, N2, and CO2) was measured by gas chromatography using an electron capture detector (7890B Gas Chromatograph System, Agilent Technologies) before and after 2 days of freezing treatment at -80°C, -20°C, or +4°C (controls).

3.3.6 Repeat freezing pretreatment and incubation to observe the effect of O2 contamination on bacterial community structure

The incubation of Arctic sediment after freezing at -80°C and -20°C was repeated to observe the effect of oxygen on the bacterial community structure during the 50°C incubation. The experiment was repeated with the following changes: marine sediment was frozen at either -80°C or -20°C for 9 days; anoxic conditions were ensured during the 53-day incubations by the addition of a headspace flush with 9:1 (v/v) N2:CO2 gas after the freezing pretreatment but before the 80°C pasteurization and 50°C incubation (the original experiment lacked this headspace flush); DNA extractions were performed using the DNeasy PowerSoil Kit (Qiagen); the Day 0 DNA extractions were pooled for sequencing such that the Day 0 replicates were combined into one amplicon library. Additionally, the organic acid concentration in the medium in the repeat experiment was 0.1 mM, a 10-fold decrease from the organic acid concentration of the medium in the original experiment (1 mM). The 10-fold decrease in organic acid concentration supplied to the microcosms was not intentional; it was caused by an erroneous organic acid stock

27 concentration. Sediment stored at 4°C was again used as the positive control. 16S rRNA gene amplicon libraries of each sample before incubation and after 7 days of 50° incubation were sequenced.

28 3.4 Results

3.4.1 Sulfate reduction during 50°C incubations

Patterns of sulfate reduction in each microcosm incubated at 50°C differed depending on the freezing pretreatment. After 7 days of 50°C incubation, the sulfate concentration in the -20°C pretreated microcosms decreased similarly to the unfrozen sediment incubated at 50°C whereas there was no decrease in sulfate in the -80°C pretreated microcosms (Figure 3.1). After 53 days, the -80°C pretreated microcosms also exhibited sulfate reduction in some but not all replicates (Figure 3.1). Sulfate reduction was inconsistent between replicates of the -80°C treatment with Replicates 2 and 3 of the -80°C freezing for 10 days treatment group showing sulfate reduction while the other four replicates pretreated at -80°C show little or no sulfate reduction over 53 days of 50°C incubation (Figure S3.1). Where sulfate reduction was observed (in four out of the six -80°C pre-frozen microcosms; Figure S3.1) the decrease ranged from 20% to 77% of the original concentration after 53 days.

16 14 12 10 8 6 4 2

Sulfate Sulfate Concentration (mM)

Day 0 Day Day0 Day7 Day7 Day0 Day7 Day0 Day7 Day0 Day7

Day 53 Day Day53 Day53 Day53 Day53 -80°C for 2 days -80°C for 10 days -20°C for 2 days -20°C for 10 days positive control

Figure 3.1: Sulfate concentrations in pre-frozen microcosms over 53 days of incubation at 50°C. Individual measurements of the -80°C pre-frozen microcosms are shown in Figure S3.1. Columns represent the average across triplicates and error bars show standard error.

29 3.4.2 Organic acid depletion

Concentrations of the organic acids lactate, succinate, propionate, and butyrate decreased in the -20°C pretreated microcosms and unfrozen positive control microcosms over 53 days (Figure 3.2). Results were consistent across all -20°C pre- frozen incubations, regardless of whether they were frozen at -20°C for two or ten days, and resembled the unfrozen positive controls. Organic acid depletion profiles in the - 80°C pretreated sediment incubations were different and were less consistent across replicates after 53 days (Figure S3.2). Despite sulfate reduction in only two of the six replicates that were pre-frozen at -80°C, propionate and butyrate concentrations reached zero after 53 days in all microcosms that were frozen for 10 days and in one replicate that was frozen for 2 days. Interestingly, lactate concentrations only decreased in -80°C pretreated microcosms that were frozen for ten days and not in microcosms frozen for two days. Succinate concentrations decreased in only one of the six -80°C pretreated microcosms and this appeared to be unconnected to sulfate reduction.

1.5

0.5

Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7

Organic Acid Organic Acid Concentration (mM) -0.5

Day 53 Day 53 Day Day53 Day53 Day53 -80°C for 2 -80°C for 10 -20°C for 2 -20°C for 10 positive control days days days days

Lactate Succinate Propionate Butyrate Figure 3.2: Organic acid concentration over 53 days of incubation at 50°C. The -80°C pretreated microcosms show less reproducible organic acid depletion (Figure S3.2). Values represent the average of triplicates and error bars show standard error.

30 3.4.3 16S rRNA gene amplicon library analysis and thermospore OTU identification

Thirty-one thermospores OTUs were identified from 16S rRNA gene libraries constructed after 7 and 53 days of incubation across the different treatments. Twenty- five of these taxa were affiliated with the class Clostridia and six to the class Bacilli (Table 3.1). On average, the number of thermospore OTUs detected in the each of the replicates of a given pretreatment was comparable (approximately 10 per replicate). However, the identities of the OTUs varied between different freezing pretreatments. Interestingly, Bacilli were only detected in the -80°C pretreated incubations (3 of 3 microcosms) whereas thermospore OTUs of class Clostridia were detected in all replicates of the two pretreatment conditions and the positive control (9 of 9 microcosms). Representative sequences for each OTU were compared to published 16S rRNA genes sequences using nucleotide BLAST (Madden, 2002) to identify the closest relatives based on 16S rRNA gene sequence similarity, which are summarized in Table 3.1.

Table 3.1: Thermospore OTUs detected after a 10-day pre-freezing treatment at -80°C, -20°C, or left unfrozen (positive control) after 53 days of incubation at +50°C. ++ indicates an OTU detected at a relative abundance of >10% in at least one triplicate + indicates an OTU detected at a relative abundance between 1% and 10% in at least one triplicate (Table is on following page.)

Closest Relative (BLAST ID) % Accession -80°C -20°C Positive ID number Control Bacilli OTU 21 Bacillus boroniphilus 96 NR_041275.1 ++ OTU 8 Bacillus hisashii 98 NR_144578.1 ++ OTU 31 Geobacillus thermantarcticus 95 NR_117156.1 ++ OTU 61 Anoxybacillus calidus 98 NR_125532.1 + OTU 88 Bacillus benzoevorans 97 NR_044828.1 + OTU 920 Bacillus hisashii 97 NR_144578.1 +

Clostridia OTU 2 Proteiniborus ethanooligenes 88 NR_125623.1 ++ ++ ++ OTU 4 Clostridilisalibacter spp. 98 KC555195 + + OTU 5 Desulfotomaculum peckii 94 NR_109724.1 ++ ++ + OTU 7 Clostridium halophilum 96 NR_125713.1 ++ + + OTU 8 Caloranaerobacter ferrireducens 97 NR_125860.1 + + + OTU 9 Lutispora thermophila 96 NR_041236.1 + + + OTU 10 Desulfotomaculum thermosapovorans 96 NR_119247.1 + + + OTU 12 Desulfonisporus sp. AAN04 97 NR_026497.1 + + OTU 15 Halothermothrix orenii 91 NR_074915.1 + ++ OTU 22 Tepidimicrobium xylanilyticum 89 NR_116042.1 + + OTU 24 Irregularibacter muris 97 NR_144613.1 + OTU 34 Desulfitibacter alkalitolerans 89 NR_042962.1 + OTU 35 Desulfonisporus sp. AAN04 94 AB436739.1 + OTU 40 Tepidimicrobium xylanilyticum 96 NR_116042.1 + OTU 45 Sporosalibacterium faouarense 95 NR_116364.1 + OTU 47 Desulfotomaculum peckii 89 NR_109724.1 ++ + OTU 51 Gracilibacter thermotolerans 91 NR_115693.1 + + OTU 64 Brassicibacter thermophilus 99 NR_137216.1 + OTU 69 Anaerovirgula multivorans 99 NR_041291.1 + OTU 87 Defluvitalea saccharophila 99 NR_117912.1 + OTU 90 Carboxydothermus siderophilus 87 NR_044272.1 + OTU 110 Halothermothrix orenii 91 NR_074915.1 + OTU 115 Gelria glutamica 91 NR_041819.1 + + OTU 121 Tindallia californiensis 91 NR_025162.1 + OTU 149 Clostridium caminithermale 96 NR_041887.1 + + +

32 An increase in the relative abundance of Firmicutes, the bacterial phylum containing all known endospore-formers, was observed after 50°C incubation in both treatment groups and the positive control (Figure 3.3a). Clostridia, the class within the phylum Firmicutes that contains all known sulfate-reducing thermospores, increased in relative abundance in both experimental treatment groups and the positive control after 50°C incubation; however, Bacilli, a class within the phylum Firmicutes that is generally, although not exclusively, associated with aerobic metabolism, increased in relative abundance only in the treatment groups pre-frozen at -80°C (Figure 3.3b). Relative abundances of Bacilli thermospores in the -80°C pretreated sediment microcosms were highest after 7 days of incubation and had decreased after 53 days (Figure 3.3b). These Bacilli OTUs are associated to the genera Bacillus, Anoxybacillus, and Geobacillus and their lower relative abundances after 53 days coincides with reduction of sulfate and an increase in Clostridia OTUs (Figures 3.4a and S3.3). Sulfate-reducing Desulfotomaculum, members of the class Clostridia, were detected in all incubations after 53 days, and their increased relative abundances were concomitant with decreases in sulfate (Figure 3.5).

33 a) Phylum-level community structure

100 90 80 70 60 50 40 30

20 Relative Abundance Relative Abundance (%) 10 0 Day 0 Day 7 Day 53 Day 0 Day 7 Day 53 Day 0 Day 7 Day 53 -80°C -20°C positive control

Acidobacteria Actinobacteria Atribacteria Bacteroidetes Chlorobi Chloroflexi Cyanobacteria Firmicutes Fusobacteria Gracilibacteria Latescibacteria Marinimicrobia- Proteobacteria Saccharibacteria Spirochaetae Unknown <1% b) Class-level community structure

100 90 80 70 60 50 40 30

Relative Abundance Relative Abundance (%) 20 10 0 Day 0 Day 7 Day 53 Day 0 Day 7 Day 53 Day 0 Day 7 Day 53 -80°C -20°C positive control Clostridia Bacilli

34 Figure 3.3 Community structure based on 16S rRNA gene amplicon sequencing before and after 50°C incubation. a) Phylum-level community structure before and after 7 and 53 days of 50°C incubation. “Unknown” refers to the collection of OTUs of which a phylum-level classification cannot be confidently made by the MetaAmp application (Dong et al., 2017). “Total >1%” refers to the collection of phyla that were present in less than 1% relative abundance. An increase in the relative abundance of Firmicutes is seen in all treatments after incubation. b) Class-level community structure within the phylum Firmicutes after 7 and 53 days of 50°C incubation. Replicates 1,2, and 3 are plotted from left to right in each set of three; the replicates for the positive control replicates were pooled for Day 0 and Day 7 were pooled during DNA extraction so their libraries are a representation of three replicates.

16 100 14 90 80 12 70 10 60 8 50 6 40 30 4 20

2 Relative Abundance (%)

Sulfate Sulfate Concentration (mM) 10

0 0

Day0 Day7 Day0 Day7 Day0 Day7

Day53 Day53 Day53 -80°C -20°C positive control Sulfate Clostridia Bacilli

35 25 9 8 20 7 6 15 5 4 10 3 5 2

Relative abundance (%) 1

0 0

Decrease Decrease sulfate in conc. (mM)

Day 7 Day Day0 Day0 Day7 Day0 Day7

Day53 Day53 Day53 -80°C -20°C positive control Desulfotomaculum Δ Sulfate

Figure 3.5: Sulfate reduction and relative abundance of Desulfotomaculum spp. in pretreated microcosms at -80°C and -20°C for 10 days and positive control microcosms over 53 days at 50°C. Δ Sulfate refers to the absolute value of the sulfate concentration decrease and is plotted against the axis on the rights side. Error bars show standard error.

Within the Clostridia, correlations between the occurrence and relative abundance patterns of OTUs and the on freezing pretreatments were evident. Several OTUs were observed in both treatment groups and the positive control after 50°C; these are affiliated with Proteiniborus ethanoligenes, Desulfotomaculum spp., Clostridium spp., Caloranaerobacter ferrireducens, and Lutispora thermophila (Table 3.1). Certain OTUs were only present in the -80°C frozen sediment after 50°C incubation; these include those closely related to Desulfitibacter alkalitolerans, Tepidimicrobium xylanilyticum, Brassicibacter thermophiles, Defluvitalea saccharophila, Anaerovirgula multivorans, and Carboxydothermus siderophilus (Table 3.1). OTUs associated to Desulfonisporus spp., Tepidimicrobium xylanilyticum, Gracilibacter thermotolerans, Clostridilisalibacter spp., and Gelria glutamica apparently remained viable (based on the criteria in 2.4.3 and 3.3.4. after -20°C freezing pretreatment but not after -80°C pretreatment. Still other OTUs were not detected after freezing at -20°C or -80°C (e.g.

36 affiliated with Sporosalibacterium faouarense and Desulfonisporus sp.) (Table 3.1). Interestingly, some different OTUs affiliated with the same species showed different pre- freezing temperature tolerances (e.g. a Desulfonisporus sp. was not detected after - 20°C pretreatment while another was, and one OTU affiliated to Tepidimicrobium xylanilyticum did was not detected after -80°C pretreatment while another related to the same bacterium was only observed in microcosms pre-frozen at -80°C). Non-metric multidimensional scaling showing the richness and diversity of the total bacterial community structure of the samples in each treatment group illustrates that bacterial community structures in microcosms that were pre-frozen at -80°C were more variable between replicates after 50°C incubation than enrichments from the -20°C treatment group and the positive control (Figures 3.6a and 3.6b). Circles representing libraries obtained from samples after 7 days and 53 days of 50°C incubation are grouped together and show that as the 50°C incubation proceeded, the communities in all pre-freezing treatment groups shifted (Figure 3.6a). ANOSIM comparing the dissimilarity between the joined groups identified an R statistic of 0.682 and a significance of p<0.001. The larger separation between circles representing -80°C pretreated replicates in Figure 3.6b suggests that these communities were more different from each other than the replicates in the -20°C and unfrozen pretreatment experiments which cluster more closely together. ANOSIM comparing the dissimilarity between the groups joined by freezing pretreatment identified an R statistic of 0.785 (P<0.001) (Figure 3.6b). The -20°C pretreated microcosm communities were more similar to those of the positive control microcosms than the -80°C pretreated microcosms. This might be due to the increase in relative abundance of Bacilli that occurred exclusively in the in the latter treatment.

37 a)

38 b)

39 3.4.4 Gas analysis after freezing pretreatment and the gas permeability of rubber stoppers after freezing

The headspace gas composition of empty serum bottles flushed with N2:CO2 gas after freezing pretreatments was measured. Although oxygen concentrations remained stable at very low concentrations (0.26 ± 0.05 mol%) in the 4°C and -20°C pretreated empty microcosms, they increased from 0.25 ± 0.06 to 10.08 ± 2.81 after 2 days of freezing at -80°C (Figure 3.7) suggesting gas permeability or the rubber stoppers offering an insufficient seal at -80°C but not at -20°C or 4°C.

14 12 10 8

6 (mol%) 4

2 Oxygen Oxygen Concentration 0 Day 0 Day 2 Day 0 Day 2 Day 0 Day 2 -80°C -20°C 4°C

3.4.5 Bacterial community structure after repeat pre-freezing and incubation

After repeating the experiment with the additional step of a headspace flush with

N2:CO2 gas to ensure anoxic conditions in the microcosms after the freezing pretreatment, the bacterial community structure of the -80°C pretreated microcosms after 7 days of 50°C incubation more closely resembled the bacterial community

40 structure of the -20°C pretreated microcosms and the unfrozen positive control microcosms after 50°C incubation (Figure 3.8). ANOSIM comparing the dissimilarity between the joined groups found an R statistic of 0.773 and a significance of 0.001 (Figure 3.8). This result differs from the original experiment where the bacterial community structures of the -80°C pretreated microcosms are more different from the bacterial community structure of the -20°C pretreated and positive control microcosms (Figure 3.6). An increase in the relative abundance of Firmicutes was seen in all treatments after incubation (Figure 3.9a.) An increase in the relative abundance of Clostridia is observed in both treatment groups and the positive control and Bacilli were present in low relative abundances after incubation in both treatment groups and the positive control (Figure 3.9b). Despite the increase in Firmicutes relative abundance after 7 days of 50°C incubation in the -80°C pretreated microcosms of the repeat experiment (Figure 3.9a), a trend that was also seen in the original experiment (Figure 3.3a), an increase in the relative abundance of Bacilli after 7 days of 50°C incubation in the -80°C pretreated microcosms, which was observed in the original experiment, is not observed in the repeat experiment (Figure 3.3b, Figure 3.9b).

Figure 3.8: NMDS plot comparing the bacterial community structures of microcosms subjected to the different pretreatments of the repeat experiment. Microcosms frozen at - 80°C are represented by blue circles, those frozen at -20°C are represented by green circles, and those that remained unfrozen (4°C) are represented by yellow circles. Circle size represents diversity based on richness and evenness Circle size represents diversity based on richness and evenness with larger circles representing richer communities and is calculated using Inverse Simpson Index. Circles are joined according to the time of measurement. Circles visualizing the community structure at Day 0 identify the community structure after freezing treatment and pasteurization but before 50°C incubation. Circles joined and labelled “Before Pasteurization” represent the community structure after freezing but before pasteurization and 50°C incubation.

42 a) Phylum-level community structure

100 90 80 70 60 50 40 30

20 Relative Abundance Relative Abundance (%) 10 0 Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 -80°C -20°C 4°C -80°C -20°C positive control Before Pasteurization

Actinobacteria Atribacteria Bacteroidetes Chloroflexi Cyanobacteria Firmicutes Proteobacteria Spirochaetae < 1% b) Class-level community structure

100

30 Relative Abundance Relative Abundance (%) 20

0 Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 -80°C -20°C 4°C -80°C -20°C positive control Before Pasteurization Clostridia Bacilli

43 Figure 3.9: Bacterial community structure based on 16S rRNA gene amplicon sequencing after 50°C incubation in the repeat experiment in which the headspace was exchanged to ensure anoxic conditions during the incubation. Amplicon libraries prepared from sediment after freezing pretreatment but before pasteurization are shown to the right. a) Phylum-level community structure after 7 days of 50°C incubation. b) Class-level community structure within the phylum Firmicutes after 7 days of 50°C incubation.

The increase in relative abundance of Firmicutes after 7 days of 50°C in the repeat experiment is less than that of the original experiment (Figures 3.3a and 3.9a). Likewise, microbial activity is less in the microcosms of the repeat experiment (Figure 3.10) than the original experiment (Figure 3.1). This result likely reflects the lower concentration (0.1 mM) of electron donors (organic acids) available to the germinating thermospore population in the repeat experiment compared to the concentration of electron donors (1 mM) in the original experiment and prevents a direct comparison between thermopsore OTUs identified in the original experiment and those identified in the repeat experiment. This repeat experiment did however establish an explanation for the enrichment of Bacilli OTUs in the original -80°C pretreated microcosms. The absence of a large enrichment of Bacilli OTUs in this repeat experiment (Figure 3.9b) where microcosms were ensured to be anoxic demonstrates that the presence of oxygen was likely the cause of the large Bacilli enrichment in the -80°C pretreated microcosms in the original experiment.

44 30

5 Sulfate Sulfate Concentration (mM)

Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7

Day53 Day53 Day53 Day53 -80°C -20°C positive control No Sediment Control

Figure 3.10: Sulfate concentrations in microcosms of the repeat experiment over 53 days of incubation at 50°C. Error bars show standard error.

Table 3.2 shows the thermospore OTUs identified in the repeat experiment. While there were Bacilli sp. identified in the -80°C and -20°C pretreated groups of the repeat experiment, their relative abundance after 7 days of incubation at 50°C was <6% in all replicates (Table S3.2) which is less than the <28% relative abundance (Table S3.1) seen for the Bacilli OTUs in the original experiment.

45 Table 3.2: Thermospore OTUs detected after a 9-day pre-freezing treatment at -80°C, - 20°C, or left unfrozen (positive control) after 7 days of incubation at +50°C. ++ indicates an OTU detected at a relative abundance of >10% in at least one triplicate + indicates an OTU detected at a relative abundance between 1% and 10% in at least one triplicate

Closest Relative (BLAST ID) % Accession -80°C -20°C positive ID number control Bacilli

OTU 19 Tepidibacillus fermentans 99 NR_125657.1 + + OTU 20 Bacillus hisashii 98 NR_144578.1 + +

Clostridia

OTU 2 Proteiniborus ethanoligenes 98 NR_044093.1 ++ ++ ++ OTU 6 Clostridium halophilum 96 NR_125713.1 ++ + ++ OTU 8 Caloranaerobacter ferrireducens 97 NR_135860.1 + + + OTU 9 Desulfotomaculum thermosapovorans 96 NR_119247.1 ++ ++ + OTU 15 Carboxydothermus siderophilus 87 NR_044272.1 ++ OTU 21 Desulfotomaculum peckii 94 NR_109724.1 + + + OTU 24 Brassicibacter thermophilus 99 NR_137216.1 + OTU 29 Halothermothrix orenii 91 NR_074915.1 + + OTU 30 Desulfonispora thiosulfatigenes 91 NR_026497.1 + + OTU 33 Desulfitispora alkaliphila 88 NR_116807.1 +

OTU 40 Tepidimicrobium xylanilyticum 96 NR_116042.1 + + OTU 75 Clostridium caminithermale 96 NR_041887.1 + + + OTU 84 Defluviitalea saccharophila 99 NR_117912.1 + + + OTU 188 Desulfotomaculum geothermicum 100 NR_042044.1 +

46 3.5 Discussion

Previous studies have shown that thermospores from permanently cold marine sediments such as the Arctic germinate upon incubation at high temperature (Volpi et al., 2017; Müller et al., 2014; de Rezende et al., 2013; Hubert et al., 2009, 2010). The utility of these misplaced and dormant thermophiles in understanding the biogeography of microbes is that they allow the processes of passive dispersal to be explored. In this study, as in previous studies (e.g. Hubert et al., 2009, de Rezende et al., 2013), the decrease in sulfate concentrations upon incubation at 50°C after pasteurization at 80°C in some microcosms is concomitant to the growth of sulfate-reducing bacteria. Sulfate reduction and organic acid depletion observed in some microcosms of each of the experimental pretreatments (-80°C, -20°C) and the positive control (+4°C) point to the activity of thermophilic organisms upon incubation at 50°C and their ability to remain viable after freezing at -20°C and -80°C. These results are in alignment with previously reported studies suggesting no loss in spore viability after -20°C storage (Freeman and Wilcox, 2003; Mah et al., 2009) and extend the lower temperature limit for maintaining viability to -80°C for some thermospores. Non-uniform sulfate reduction after 53 days in the -80°C pretreated microcosms suggests that sulfate reducers may be sporadically enriched under these conditions. The most likely reason for delayed or absent sulfate reduction in the -80°C pretreated microcosms is the presence of O2. The eventual decrease in sulfate observed after 53 days could be due to the growth of sulfate reducing organisms that only became active after O2 was depleted by aerobic growth. Two of the six -80°C pretreated sediment incubations did not show any decrease in sulfate during the incubation period; these same two incubations also did not experience a drop in organic acid concentration suggesting minimal anaerobic microbial activity. Still, some organic acid depletion in - 80°C pretreated sediment incubations prior to when sulfate reduction was observed may suggest microbial activity that is unconnected to sulfate reduction and possibly connected to aerobically respiring organisms in the sediment that use organic acids as electron donors (e.g. Bacillus boroniphilus and Geobacillus thermoantarcticus, Table 3.1)(Nicolaus et al., 1996; Ahmed et al., 2007) or, fermenting organisms that may use

47 the same organic acids. While the pretreatment freezing temperature did affect changes in the sulfate and organic acid concentration during the 53 day incubation period, the length of pre-freezing treatment (2 vs 10 days) whether at -20°C or -80°C did not greatly affect the sulfate or organic acid depletion profiles. Analysis of enriched bacterial communities following freezing pretreatments points to a possible taxonomic cause of the sulfate reduction and organic acid depletion seen in each of the treatment groups. The increase in relative abundance of Clostridia in the -20°C pretreated sediment incubations is similar to that of the positive control group. This result parallels previous studies that have shown an increase in Clostridia tied to a decrease in sulfate upon high temperature incubation of previously unfrozen marine sediment (Hubert et al., 2009, 2010; de Rezende et al., 2013; Müller et al., 2014). This result along with similar organic acid depletion and sulfate reduction profiles may suggest that freezing at -20°C does not affect the viability of most dormant thermospore phylotypes in cold sediments (Table 3.1). The -80°C pretreated sediment incubations had a large increase in relative abundance of Bacilli after 7 days of incubation. The Bacilli OTUs identified in the -80°C incubations belong to the genera Bacillus, Anoxybacillus, and Geobacillus, which contain aerobes or facultative anaerobes, but very few strict anaerobes (De Vos et al., 2009; Goh et al., 2014). The most likely reason for their increased relative abundance after 7 days of incubation is exposure to O2 which likely entered the microcosm bottles during pre-freezing at -80°C. Between 7 days and 53 days of incubation sulfate was reduced in the microcosms pretreated at -80°C and sulfate-reducing thermophiles become relatively abundant which suggests that the O2 had been depleted and the microcosms had become anoxic. It is possible that aerobic activity of Bacilli early on during the incubation depleted the O2 and created an anoxic environment that allowed the growth of sulfate reducers, which we see become abundant by 53 days. However, Bacilli OTUs were present even after anoxic conditions were ensured (in the repeat experiment) demonstrating that the Bacilli OTUs are not exclusively caused by the presence of O2 although their large relative abundance in the original experiment may have been caused by O2. This is corroborated by identification of the closest neighbor based on 16S rRNA gene similarities (Tables 3.1 and 3.2). In the original freezing experiment, two Bacilli OTUs are closely affiliated with Bacillus

48 boroniphilus and Geobacillus thermantarcticus, both aerobes (Nicholaus et al., 1996; Ahmed et al., 2007), while in the repeat experiment when conditions were ensured to be anoxic, the only Bacilli OTUs enriched were Tepidibacillus fermentans and Bacillus hisashii, an anaerobe and a facultative anaerobe respectively (Slobodkina et al., 2013; Nishida et al., 2015).

Regardless of the presence of O2 in the first experiment, in the -80°C pretreated microcosms, many of the same Clostridia thermospore OTUs identified in the -20°C and positive control microcosms were present. The detection of multiple thermospore OTUs in every pretreatment condition leads to the conclusion that some thermospores are resistant to freezing and for some the lower temperature limit for maintaining viability is below -80°C. Only two of the identified thermospore OTUs were not detected after freezing treatment yet present in the positive controls; these were affiliated with Sporosalibacterium faouarense and Desulfonisporus sp. (Table 3.1). A few OTUs were detected after freezing at -20°C but not -80°C (affiliated with Tepidimicrobium sp., Desulfonisporus sp., and Clostridilisalibacter sp., amongst others) (Table 3.1). Still others were present in all three treatments (affiliated to Proteiniborus sp. and Desulfotomaculum spp.). The presence of different thermospore OTUs in libraries of microcosms of different freezing pretreatments could suggest differential freezing tolerance among thermospores. While genes for the sporulation process are quite conserved and likely arose near the root of the Firmicutes evolutionary history since the early branching classes of Clostridia and Bacilli both contain endospore-formers (Galperin et al., 2013), there are some differences between the complement of sporulation genes possessed by different spore-formers that may confer differential low temperature tolerance. Fairhead et al. (1994) observed that the absence of small acid-soluble spore proteins (SASPs) contributed to a decrease in spore viability after freeze-drying and thus suggested that SASPs are responsible for spore tolerance to this temperature stress. SASPs bind to DNA within the spore and are known to protect it from radiation and dry heat. At the class level Bacilli and Clostridia differ notably in their SASP complement (Galperin et al., 2012) and within each class the number of SASPs varies as well. Bacilli generally contain between 11 and 22 different SASP genes while Clostridia often contain only two

49 (Galperin et al., 2012). It is possible that the increase in the number of SASP genes leads to increased tolerance to freezing in thermospore OTUs; further studies into the relationship between SASP gene complements and thermospore freezing tolerance may shed light on a genomic determinant to freezing tolerance. Notably, the number of thermospore OTUs detected in both the first experiment and the repeat experiment was larger after freezing pretreatment (-20°C and -80°C) compared to the positive control. A likely cause for this is the release of organic substrates from the sediment after the freeze-thaw event. Dead biomass is thought to be a primary source of organic substrates following a freeze-thaw cycle and the surviving microorganisms are shown to use these released substrates (Skogland et al., 1988; Schimel et al., 1996; Herrmann and Witter, 2002). Increases in organic substrates to the microcosm environment after treatment at -80°C and -20°C may have been responsible for the increased thermospore diversity in these treatment groups, although presumably necromass is supplemented to all microcosms, including the positive controls, as the mesophilic organisms die in response to pasteurization at 80°C and incubation at 50°C. Based on this study, we have yet to find the low temperature tolerance limit of thermophilic endospores for maintaining viability during dormancy if one exists at all. Some studies have explored the low temperature tolerance of mesophilic spore-formers, usually pure cultures of Bacillus (Fairhead et al., 1994; Weber and Greenberg, 1985; Miyamoto-Shinohara et al., 2008), but thermophilic spore-formers may have a unique tolerance to freezing. Based on thermal inactivation kinetics of mesophiles and thermophiles, Nicholson (2003) found that thermophilic endospore-formers have survival probabilities that are much higher than mesophilic endospore-formers. While Nicholson (2003) did not determine a cause for the difference in survival probability between mesophiles and thermophiles, damage to spore DNA (Nicholson et al., 2000, 2003; Setlow 1995, 2001), particularly spontaneous depurination (Lindahl, 1993), is thought to be responsible for reduced viability. As such, strategies that prevent DNA damage, such as SASP proteins that prevent DNA depurination (Fairhead et al., 1993; Setlow 1995) will allow the spore to remain viable for longer. These same longevity mechanisms could also maintain viability of spores after freezing (i.e. SASPs protect the spore during freezing drying (Fairhead et al., 1994)).

50 To date there are very few studies exploring the freezing tolerance of endospores. This is the first study exploring the freezing tolerance of a microbial endospore population from an environmental sample and the first study exploring the freezing tolerance of thermophilic endospores specifically. Further research into the involvement of SASPs in Firmicutes’ freezing tolerance should be investigated to understand their possible involvement in maintaining viability. The importance of understanding endospore freezing tolerance is evident when considering their function as model organisms for studying microbial dispersal and thermophilic endospores, with their potentially higher survival probabilities, are specifically important in this role. These organisms may help us understand the extent to which microorganisms may be dispersed globally and between earth and other celestial bodies in our solar system providing knowledge on possible histories for life on earth and the extent to which human exploration beyond earth will affect the dispersal of life beyond our planet.

51 Chapter 4: Lateral biogeography of thermophilic endospores in North Atlantic sediment

4.1 Abstract

Detection of dormant thermospores in permanently cold sediments can be used to study the potential vectors causing geographic dispersal of microorganisms in the marine environment. Surface sediments from nine locations in the North Atlantic were pasteurized (80°C for 1 hour) and incubated at 50°C to enrich thermospores in the sediment. Thermospore OTUs were detected in all nine sediments. Nine of the 41 thermospore OTUs detected were identified in more than one sediment and OTU 2 (affiliated with Symbiobacterium ostreiconchae) and OTU 46 (affiliated with Bacillus thermoamylovorans) appeared in incubations of the most sediments, which were three and five different sediments respectively. An analysis of the thermospores shared between different sediments demonstrated that while the North Atlantic subpolar gyre is a primary driver of North Atlantic circulation, this current is not exclusively responsible for the dispersal of the identified thermospores.

52 4.2 Introduction

Microbial biogeography is the study of microorganisms across geographic space and over geologic time. Hanson et al. (2012) identified selection, mutation, drift, and dispersal as the processes central to shaping microbial biogeography. The process of dispersal, defined by Hanson et al. (2012) as bacterial movement to and successful colonization, including activity and reproduction, at a new location, can be divided into two components, bacterial movement and successful colonization. Bacteria possessing the endospore phenotype have an increased ability to survive passive movement beyond their original location because the robust nature of the endospore phenotype allows the endospore to withstand stresses that would otherwise have destroyed the vegetative cell. Thermophilic bacteria, those with temperature optima between 45°C and 80°C, will only grow in environments with temperatures within their growth range and thus their growth is limited to these environments. Thermospores can be dispersed widely because they remain viable after exposure to environmental stress as endospores, yet their successful establishment in a new location is limited to warm environments. For these reasons, thermospores are a useful model organism for the study of the movement aspect of microbial dispersal specifically. Their movement is passive and unaffected by confounding factors of selection, mutation, and drift because the thermospores are dormant and resistant to environmental stressors during their dispersal. In this way, thermospores are similar to abiotic particles except they are biotic and are able to germinate and colonize a warm temperature environment should that environment be encountered during their passive dispersal. When a warm environment is not encountered and thermospores are deposited in an environment unsuitable for germination, they contribute to the dormant microbial seed bank and overall biodiversity of that site (Lennon and Jones, 2011). The microbial seed bank is composed of the microorganisms in a particular location that are metabolically inactive and not reproducing. These organisms do not participate in local biogeochemical cycling and so do not alter the geochemical properties of the environment nor do they impact the growing populations in that environment. They may however become active when the conditions change and become suitable for their

53 activity and growth. For thermospores lying dormant in a cool location, an increase in temperature (e.g. in an experiment) may cause germination and growth so that the active community structure and dominant microbial activities shift to reflect that of the thermophilic endospore-forming bacteria. The ability to withstand environmental stress and remain viable during passive dispersal implies that endospore-forming bacteria should be very good dispersers and could potentially be cosmopolitan (present everywhere) in the dormant microbial seed bank globally. Can endospore-forming bacteria, possibly the most equipped of all microorganisms for passive dispersal, show dispersal limitation across large geographic distances? Thermospores, with their tolerance to environmental stress and inability to germinate in cool environments characteristic to most of the earth’s surface biosphere, offer a biological mechanism and tool for addressing this question. It has long been observed that thermophilic microorganisms can be enriched from cool environments (McBee and McBee, 1955; Bartholomew and Paik, 1966; Isaksen et al., 1994; Marchant et al., 2002; Lee et al., 2005). Hubert et al. (2009) showed that thermophilic endospore-forming bacteria can be enriched from Arctic sediment and that the flux of these thermophiles is continuous to the Arctic environment. Microorganisms are dispersed thousands of kilometers through the atmosphere (Bovallius et al., 1980; Griffin et al., 2002). Likewise, ocean currents have been shown to disperse microorganisms (Wilkins et al., 2013) and thermospores have been detected and enumerated in the water column above sediments in which they are found (de Rezende et al., 2013; Volpi et al., 2017) providing a link between the dispersal vector of ocean currents, and the seabed sediment. Some microbial dispersal limitation in the marine environment has been shown. Agogué et al. (2011) demonstrated that bacterial communities cluster according to water masses and Salazar et al. (2016) showed that microorganisms attached to particles show oceanic basin specificity. Additionally, Müller et al. (2014) illustrated that thermospores found in sediments globally exhibit both cosmopolitanism and dispersal limitation, which is the uneven probability of dispersal (Hanson, et al., 2012). The North Atlantic subpolar gyre is a dominant feature of North Atlantic ocean circulation. It is composed of a branch of the North Atlantic Current that veers west after

54 crossing the Mid-Atlantic Ridge at a latitude of 48°N. It then follows the northern edges of the seas and basins of the North Atlantic south of Iceland, Greenland, and Baffin Island towards the Labrador slope where the water cools and descends, forming the deep water of the North Atlantic (Marzocchi et al., 2015). This counterclockwise current is called the North Atlantic subpolar gyre. A branch of the North Atlantic Current veers northeast after crossing the Mid-Atlantic Ridge becoming the Norwegian Current that moves into the Nordic sea and meets the western edges of Svalbard (Nøst and Isachsen, 2003; Marzocchi et al., 2015). The Mid-Atlantic Ridge systems have been considered a warm temperature source of thermospores (Hubert et al., 2009; de Rezende et al., 2013, Müller et al., 2014). These geochemically rich systems support organisms from all domains of life (Cerqueira et al., 2013) and thermophiles with optima near 50°C, including relatives to those identified in this study, may inhabit suitable hot environments in areas adjacent to hydrothermal vents. Deming and Baross (1993) have suggested that fluids emanating from hydrothermal plumes, which may rise hundreds of metres before becoming neutrally buoyant (Lupton et al., 1985), may carry thermophiles to the water column above. Other studies have suggested that these plumes may contribute to the dispersal of vent-associated organisms (Jackson et al., 2010; Dick et al., 2013). The North Atlantic Current connects hydrothermal environments, such as the Lost City hydrothermal system found near the Mid-Atlantic Ridge, to other areas of the North Atlantic and may be a dispersal vector of thermospores in the water column. In this study, the dispersal of thermospores in North Atlantic sediment was assessed using 50°C microcosm incubations of surface sediment from nine different locations in the North Atlantic. To asses the likelihood of thermospore dispersal from the Mid-Atlantic Ridge to sediment at locations connected to the North Atlantic subpolar gyre, and to assess thermospore endemism and cosmopolitanism in the North Atlantic, thermospore OTUs occurring in 50°C incubations from nine different North Atlantic sediment sites were compared.

55 4.3 Materials and Methods

4.3.1 North Atlantic sediment locations

Figure 4.1: North Atlantic surface sediment used in 50°C thermospore incubations. Sites indicated in the map are Scotian Slope West (SSW), Scotian Slope Center (SSC), Scotian Slope East (SSE), Labrador Shelf (LS), Frobisher Bay (FB), Davis Strait (DS), Pond Inlet (PI), Baffin Bay North (BBN), and Svalbard (SV).

Thermospore biogeography studies use the germination of thermospores to circumvent the difficulties associated with detecting thermophilic endospore-forming bacteria based on molecular methods alone (de Rezende et al., 2017). Nine sites in the North Atlantic were used for incubation experiments in this study. Figure 4.1 shows the locations of all nine sediments and Table 2.1 lists the geographic details of each site. Each site was designated as one of two groups: sites that were connected (Connected

56 Sites, abbreviated to CS) to the North Atlantic subpolar gyre and sites that were not connected (Not Connected Sites, abbreviated to NCS) to the North Atlantic subpolar gyre (Table 4.3). Five of the nine sediment sites were considered connected (CS) to the North Atlantic subpolar gyre (Scotian Slope West, Scotian Slope Centre, Scotian Slope East, Labrador Shelf, and Svalbard). The other four of the nine sediment sites were considered not connected (NCS) to the North Atlantic subpolar gyre (Frobisher Bay, Davis Strait, Pond Inlet, and Baffin Bay North). Svalbard was designated as connected to the North Atlantic subpolar gyre (CS) despite not being geographically connected because it is connected to the Norwegian Current, a north extending arm of the North Atlantic Current that branches after the North Atlantic Current crosses the Mid-Atlantic Ridge, thus the Norwegian Current possesses a similar history to the North Atlantic subpolar gyre current. The sediment from each site was stored anoxically at 4°C prior to 50°C incubation. Sediment from sites Baffin Bay North and Davis Strait were stored in glass containers with airtight lids. Sediments from the seven other sites were stored in anoxic plastic bags sealed with binder fasteners.

4.3.2 High temperature incubation

Anoxic 50°C sediment incubations were established as described in section 2.2. Microcosms were amended with the organic acids formate, lactate, acetate, succinate, propionate, and butyrate, to a final concentration of 1 mM each. An error in a stock solution led to microcosms of sediment from station Baffin Bay North and Davis Strait receiving organic acids at concentrations 10-fold below that of the seven other sediment incubations (0.1 mM) which was only detected after the conclusion of the incubation experiment. Svalbard sediment incubations analyzed in this study are the same incubations discussed in Chapter 3 as positive controls. These Svalbard microcosms contained 1mM of ethanol in addition to the 1 mM of the organic acids formate, lactate, acetate, succinate, propionate, and butyrate; the eight other sediment incubations in this study lacked ethanol in their medium. Differences in electron donor concentrations, as is the case with incubations of sediment from Baffin Bay North and Davis Strait, and

57 electron donor type, as is the case with incubations of sediment from Svalbard, mean that a particular OTU found in incubations of a certain electron donor mixture or concentration cannot be considered absent in incubations of the other condition even if it is not identified in that other condition. OTU identification is further discussed in section 2.4.3. Pasteurization before incubation and subsampling over the incubation period occurred as per section 2.2. Subsamples were mixed 1:1 (v/v) with a 2% solution of zinc acetate before centrifugation and separation of the supernatant and pellet fractions before storage at -20°C.

4.3.3 Sulfate and organic acid measurement

Sulfate concentration of the subsamples taken during the course of the experiment was measured using both the turbidimetric method (Nemati et al., 2001) (Svalbard samples only) and ion chromatography (Dionex ICS-5000, Thermo Scientific) as described in section 2.3. Organic acid concentrations were measured by UHPLC as described in section 2.3 using the Acclaim™ Organic Acid LC Column (5μm, 4 × 250 mm; Thermo Scientific) for the Svalbard incubation samples and the Aminex HPX-87H column (5μm, 7.8 × 300 mm, Bio Rad) for all other samples. Organic acid measurements were taken at Day 0 and Day 26, 28 or 53 in all sediment incubations, but if sulfate reduction was observed at an intermediate time point, then organic acids were also measured at that time point. The microcosms were amended with the organic acids formate, lactate, succinate, propionate, butyrate, and acetate, but acetate concentrations were not analyzed because the addition of zinc acetate to subsamples during the subsampling procedure to squelch sulfate oxidization interfered with the detection of acetate in the subsample. The Acclaim™ Organic Acid LC Column (5μm, 4 × 250 mm; Thermo Scientific) used for measurement of organic acids in the Svalbard samples poorly measured formate and thus formate was unable to be measured with confidence in the Svalbard samples.

58 4.3.4 16S rRNA gene amplicon sequencing and analysis

DNA extraction of the Svalbard incubations from days 0 and 7 were done using the in-house method described by Foght et al. (2015). All other DNA extractions were done using the DNeasy Powersoil Kit (Qiagen). 16S rRNA gene amplification and sequencing was done as described in section 2.4.2 for all samples. Community analysis and thermospore OTU identification were done as described in section 2.4.3.

59 4.4 Results

4.4.1 Microbial activity and community structure during 50°C sediment incubation

Sulfate and organic acid concentrations were monitored over the 28-, 30-, or 53- day 50°C incubation to monitor microbial activity during that time. Sulfate reduction occurred upon 50°C incubation in many of the microcosms although often not in each biological replicate. A decrease in organic acid(s) over the high temperature incubation was also observed in most microcosms. Microcosms of sediment from Davis Strait and Baffin Bay North received only a tenth of the organic acid concentration as microcosms of sediment from all other sites. As such, organic acid concentrations remained very low in the Davis Strait and Baffin Bay North microcosms over the 28-day incubation. All known thermospores belong to the bacterial phylum Firmicutes, thus an enrichment of thermospore-forming bacteria during the 50°C incubation is predicted to reveal a simultaneous increase in Firmicutes relative abundance. The relative abundance of Firmicutes was measured through a phylum-level analysis of 16S rRNA gene libraries as described in section 2.4.3. 16S rRNA gene libraries from before incubation (Day 0) and after 50°C incubation (Day 7, Day 14, and or Day 53) were made to assess the community-level response at the phylum-level and OTU-level to anoxic incubation at 50°C under the conditions described in section 2.2 and 2.3.2. A summary of sulfate reduction, organic acid utilization, and increase in Firmicutes relative abundance is shown in Table 4.1. In each microcosm that displays microbial activity as evidenced by sulfate reduction and/or a decrease in organic acid concentration, an increase in Firmicutes relative abundance is observed in the microbial community. In some cases an increase in Firmicutes relative abundance is also observed in replicates where no sulfate reduction or decrease in organic acid concentration has occurred (Table 4.1 and Figures S4.4, S4.8, S4.9, S4.11) but there is no instance of a replicate undergoing sulfate reduction or a decrease in organic acid concentration without a corresponding increase in Firmicutes relative abundance (Table 4.1). These results suggest that sulfate reduction and decreases in organic acid concentration, which are measures of microbial activity, are associated to increases in Firmicutes relative abundance in these microcosms. Notably, the increase in Firmicutes

60 relative abundance occurs in at least one replicate from incubations of sediment from each of the nine sites in this study. The results of incubations of each sediment site are discussed below. A summary of the results is shown in Table 4.1. A summary of the OTUs identified at each sediment site is shown in Table 4.2.

Table 4.1: Summary of the sulfate reduction, organic acid utilization, and increase in Firmicutes relative abundance after 50°C incubation. The symbols “+” and “-” represent a positive or negative result respectively in each replicate for the occurrence of sulfate reduction, organic acid utilization, or increase in Firmicutes relative abundance over the incubation period. The first symbol represents the result in the first replicate, the second represents the result in the second replicate, etc. * Scotian Slope West sediment incubations had six biological replicates. ** Triplicate samples were pooled for 16S rRNA sequencing. (Table is shown on the next page.)

Sediment site Measure Result Sulfate reduction ------Scotian Slope West* Organic acid utilization - - + + - + Increase in Firmicutes relative abundance - - + + - + Sulfate reduction - - + Scotian Slope Centre Organic acid utilization - - + Increase in Firmicutes relative abundance - - + Sulfate reduction - - - Scotian Slope East Organic acid utilization - - + Increase in Firmicutes relative abundance + - + Sulfate reduction + + + Svalbard Organic acid utilization + + + Increase in Firmicutes relative abundance + + + Sulfate reduction - - + Labrador Shelf Organic acid utilization + + + Increase in Firmicutes relative abundance + + + Sulfate reduction - - - Baffin Bay North** Organic acid utilization - - - Increase in Firmicutes relative abundance + Sulfate reduction - - - Pond Inlet Organic acid utilization - - - Increase in Firmicutes relative abundance + + - Sulfate reduction - - - Davis Strait** Organic acid utilization - - - Increase in Firmicutes relative abundance + Sulfate reduction - + + Frobisher Bay Organic acid utilization + + + Increase in Firmicutes relative abundance + + +

62 Table 4.2: Summary of the thermospore OTUs identified in each sediment after 50°C incubation. A “+” in a coloured box identifies a thermospore OTU as present in incubations of that sediment. A blue box represents a thermospore OTU that was identified in an incubation that also showed sulfate reduction and a decrease in organic acid concentration. A green box represents a thermospore OTU that was identified in an incubation that showed a reduction in organic acid concentration but no sulfate reduction. A grey box represents a thermospore OTU that was identified in an incubation that did not show either sulfate reduction or a decrease in organic acid concentration.

Sediment site Thermospore OTU SSW SSC SSE LS FB DS PI BBN Sv 2 + + + 4 + 5 + 12 + + 14 + 17 + 18 + 21 + 34 + + 44 + 46 + + + + + 55 + 74 + 89 + + 121 + 136 + + 145 + 168 + 196 + 212 + 216 + 219 + 220 + 224 + 244 + 339 + Table continued on next page.

63 Sediment site Thermospore OTU SSW SSC SSE LS FB DS PI BBN Sv 359 + 411 + 412 + 469 + + 514 + 522 + 532 + 562 + 610 + 642 + + 748 + 1146 + 1236 + 1535 + 2250 + 4497 +

4.4.1.1 Scotian Slope West

Sulfate reduction was not observed in incubations of Scotian Slope West (SSW) sediment, but decreases in organic acid concentration and increases in Firmicutes relative abundance were observed in 3 of 6 replicates (Table 4.1). OTUs 12, 14, 224, and 514 were identified after incubation at 50°C (Table 4.2). Figure S4.1 shows the sulfate concentration, organic acid concentration, phylum-level community structure and relative abundances of Firmicutes OTUs in sediment incubated from the Scotian Slope West site. Sulfate reduction did not occur over 11 days of incubation at 50°C, but decreases in formate and succinate concentrations in replicates 3, 4, and 6, and a decrease in butyrate concentration in replicate 4, suggest microbial activity in these replicates at 50°C. Replicates 3, 4, and 6 also show an increase in Firmicutes relative abundance after 50°C incubation (Figure S4.1c) while the phylum-level structure of replicates 1, 2 and 5 does not change suggesting that the changes in sulfate and organic acid concentration in replicates 3, 4 and 6 may be driven by Firmicutes

64 enrichment. After 50°C incubation replicates 3 and 6 are dominated by a taxon OTU 12 (Thermicanus aegyptius) while replicate 4 is dominated by OTU 14 (Vulcanibacillus modesticaldus) (Figure S4.2d; Table S4.1).

4.4.1.2 Scotian Slope Centre

OTU 12 was the only thermospore identified in Scotian Slope Centre sediment incubations (Table 4.2). Only replicate 3 from incubations (50°C) of this sediment shows sulfate reduction after 28 days (Figure S4.3b). This replicate also shows decreases in formate, lactate, succinate, propionate, and butyrate (Figure S4.3c) and its phylum-level community structure shifts to being dominated by Firmicutes after 50°C incubation (Figure S4.3d). After incubation at 50°C the 16S rRNA gene amplicon library of replicate 3 is dominated OTU 12 (Thermicanus aegyptius) (Table S4.1) which is also seen in incubations of Scotian Slope West sediment (Figure S4.1d). Conversely, replicates 1 and 2 do not display any changes and thus show no evidence of thermospore enrichment.

4.4.1.3 Scotian Slope East

Five thermospore OTUs were identified in sediment from Scotian Slope East (Table 4.2). While sulfate is not reduced in any incubations of this sediment (Figure S4.4a), replicate 3 shows evidence of microbial activity by a decrease in formate and lactate concentration over 28 days of incubation (Figure S4.4b). An increase in Firmicutes relative abundance occurs in replicates 1 and 3 of 50°C incubations from this site, but only in replicate 3 does Firmicutes represent the majority of the relative abundance (Figure S4.4c). The Firmicutes community of replicate 3 is composed of OTU 2 (Symbiobacterium ostreiconchae), OTU 5 (Desulfotomaculum tongense), and OTU 21 (Aneurinibacillus thermoaerophilus) while the Firmicutes community of replicate 1, which makes up a much smaller proportion of the relative abundance, is composed of

65 completely different thermospore OTUs, OTU 46 (Bacillus thermoamylovorans) and OTU 4497 (Bacillus hisashii) (Figure S4.4d).

4.4.1.4 Labrador Shelf

Table 4.2 shows that seven thermospore OTUs were identified in 50°C incubations of Labrador Shelf sediment, one of which, OTU 46 (Bacillus thermoamylovorans), was also identified in incubations of Scotian Slope East sediment. Figure S4.6 shows that while sulfate reduction only happens in replicate 3 of Labrador Shelf sediment incubations, decreases of organic acid concentration are observed in each of the replicates. Replicate 1 shows a decrease in lactate and butyrate concentration, and to a lesser extent, a decrease in propionate concentration over the 28-day incubation. Replicate 2 shows a decrease in propionate and butyrate over the incubation period. Replicate 3, the only replicate in which sulfate reduction was observed shows an almost complete decrease in formate, propionate, and butyrate to 0 mM during the 50°C incubation. The decrease in propionate in replicate 1 should not be considered caused by the sediment incubation because the sediment-free, media-only control for this incubation experiment showed a similar decrease in propionate in one replicate over the incubation period (Figure S4.10b). An increase in Firmicutes relative abundance is observed in all 3 replicates of Labrador Shelf incubations (Figure S4.6d). The Firmicutes community in all three replicates contained OTU 34 (Caloranaerobacter ferrireduces) after 50°C incubation. Replicate 1 also contained OTU 89 (Clostridium halophilium). Replicates 2 and 3 contained OTU 46 (Bacillus thermoamylovorans). OTU 220 (Defluvitalea saccharophila) was present in replicate 2 while OTU 469 (Clostridium caminithermale) was present in replicate 3 (Table S4.1).

4.4.1.5 Frobisher Bay

Twelve thermospore OTUs were identified in 50°C incubations of sediment from Frobisher Bay (Table 4.2). Replicates 2 and 3 of incubations of sediment from Frobisher

66 Bay showed sulfate reduction by 7 days of 50°C incubation (Figure S4.7a-b). These 2 replicates also show substantial decreases in organic acid concentration. All 5 organic acids decrease in concentration to, or nearly to, 0 mM after 28 days in replicate 2 while all organic acids except succinate follow the same trend in replicate 3 (Figure S4.7c). Sulfate reduction was not detected in replicate 1 but decreases in formate, lactate, propionate, and butyrate were detected over the 28-day incubation in this replicate. A decrease in propionate was also observed in one replicate of the medium only control incubations (Figure S4.10b), hence observations of propionate decrease warrants caution in replicates 1, 2 and 3 of Frobisher Bay incubations. Increases in Firmicutes relative abundance was observed in all three replicates of Frobisher Bay incubations (Figure S4.7d). Each of the replicates had very different Firmicutes communities. Replicate 1 was primarily dominated OTU 2 (Symbiobacterium ostreiconchae) and secondarily OTU 4 (Therminicola carboxydiphila) followed by OTU 55 (Caldibacillus debilis). The Firmicutes community of replicate 2 consists of OTU 2 (Symbiobacterium ostreiconchae), OTU18 (Desulfotomaculum geothermicum), OTU 17 (Bacillus kyonggiensis), OTU 74 (Desulfotomaculum reducens) (Day 14), OTU 1146 (Bacillus oceanisediminis), OTU 2250 (Thermoactinomyces intermedius), and OTU 55 (Caldibacillus debilis). Replicate 3 was dominated by OTU 44 (Tepidibacillus fermentans), OTU 642 (Desulfotomaculum geothermicum), OTU 216 (Clostridium thermosuccinogenes), and OTU 55 (Caldibacillus debilis) (Table S4.1). Despite thermospore enrichment in all three replicates there is not a shared OTU present amongst all three. OTU 2 (Symbiobacterium ostreiconchae), which was also present in the Scotian Slope East sediment incubations (Table 4.2), and OTU 55 (Caldibacillus debilis) are each present in two of the three replicates while all the other OTUs indicated in Table 4.2 are only present in a single replicate.

4.4.1.6 Davis Strait

Sulfate reduction and organic acid concentration decrease were not observed in incubations of sediment from Davis Strait yet a single thermospore, OTU 46 (Bacillus

67 thermoamylovorans), was identified in 50°C incubatons of sediment from this site (Table 4.2). Sulfate concentrations were unstable during the first 9 days of the 50°C incubation but after 30 days the sulfate concentration remained at levels similar to those measured at day 0 (Figure S4.8b). Given that these microcosms only received 0.1 mM of the formate, lactate, acetate, succinate, propionate, and butyrate (a 10-fold reduction in the organic acid concentration compared to the other microcosms, section 4.3.2) only 0.875 mM of sulfate could be reduced assuming that all the organic acids were utilized in sulfate reduction. This provides a possible reason to for the lack of sulfate reduction in incubations of this sediment. Despite the lack of evidence of microbial activity, there was a small increase in Firmicutes relative abundance after 14 days of incubation at 50°C (Figure S4.8c). The Firmicutes relative abundance was composed almost entirely of OTU 46 (Bacillus thermoamylovorans) (Figure S4.8d), an OTU also present in incubated sediment from the Scotian Slope East site and the Labrador Shelf (Table 4.2).

4.4.1.7 Pond Inlet

Sulfate reduction did not occur in 50°C incubations of sediment from Pond Inlet nor was there a notable decrease in organic acids (Table 4.1). A decrease in propionate concentration was observed in replicate 1 of the Pond Inlet sediment incubations but this observation must be treated with caution since a medium only control for this incubation experiment also showed a decrease in propionate during the incubation (Figure S4.10b). Firmicutes relative abundance increased slightly after 50°C incubation in replicates 1 and 2 of the Pond Inlet incubations (Figure S4.9c). This increase in Firmicutes abundance is primarily due to an increase in OTU 46 (Bacillus thermoamylovorans) relative abundance (Figure S4.9d). OTU 46 was also identified in incubated sediment from the Labrador Shelf, Davis Strait and Scotian Slope East (Table 4.2).

68 4.4.1.8 Baffin Bay North

There was no sulfate reduction or decrease in organic acids in incubations of sediment from Baffin Bay North, nevertheless eight thermospore OTUs were identified (Table 4.1 and 4.2). As observed in in Davis Strait incubations (Figure S4.8), the sulfate concentration in Baffin Bay North sediment incubations were variable over the incubation period but after 30 days of incubation at 50°C, no sulfate had been reduced in any of the replicates (Figure S4.11b). While there is a decrease in sulfate concentration after 30 days compared to the Day 0 sulfate measurement, the large variability of sulfate concentration during the first 9 days of the incubation suggest that this high sulfate measurement at Day 0 may be an artifact of the subsampling or sulfate measurement procedure (Figure S4.11a-b). As discussed in sections 4.3.2 and 4.4.1.6, sediment incubations of Baffin Bay North received 10-fold lower concentrations of the 6 organic acids thus providing lower concentrations of electron donors for sulfate reduction. Organic acids concentration was very low from the onset of the incubation and no detectable organic acid concentration decrease was observed (Figure S4.11c). There was a small increase in Firmicutes relative abundance after 14 days of incubation at 50°C even though there was no other evidence of microbial activity (Figure S4.11d). There were 8 thermospore OTUs identified in this incubation, all in <4% relative abundance (Figure 4.11e). OTU 2 (Symbiobacterium ostreiconchae) was also identified in sediment from Frobisher Bay and Scotian Slope East. OTU 642 (Desulfotomaculum geothermicum) was also found in sediment from Frobisher Bay (Table 4.2). OTU 46 (Bacillus thermoamylovorans), which was also identified in sediment from Scotian Slope East, Labrador Slope, Davis Strait, and Pond Inlet was also found in this sediment (Table 4.2).

4.4.1.9 Svalbard

Svalbard sediment incubations (described in Chapter 3) show sulfate reduction and a decrease in lactate, succinate, propionate and butyrate concentration over the 53- day incubation (Figure S4.13a-b). An increase in Firmicutes relative abundance is

69 observed in all 3 replicates of Svalbard sediment incubations after 50°C incubation and 15 thermospore OTUs were identified (Table 4.1 and 4.2). The taxa making up the Firmicutes community is very similar between the 3 replicates and contains 15 OTUs. Other than the minor differences in relative abundance of each taxa in the replicates the only other difference between the replicates is that replicate 3 contains OTU 196 (Halothermothrix orenii) while replicates 1 and 2 do not contain this OTU in their amplicon libraries (Figure S4.13e). Sediment from Svalbard shares thermospore OTU 136 (Desulfotomaculum peckii) with sediment from Baffin Bay North and shares OTU 469 (Clostridium caminithermale) with sediment from the Labrador Shelf (Table 4.2).

4.4.2 Shared thermospore OTU identification

Table 4.3: Sediment site type, connected to North Atlantic subpolar gyre (CS) or not connected to North Atlantic subpolar gyre (NCS), and the number of thermospore OTUs identified after incubation at 50°C for 14 days. Sediment site Site type Thermospore OTU count Scotian Slope West (SSW) CS 4 Scotian Slope Centre (SSC) CS 1 Scotian Slope East (SSE) CS 5 Svalbard (SV) CS 15 Labrador Shelf (LS) CS 7 Baffin Bay North (BBN) NCS 8 Pond Inlet (PI) NCS 1 Davis Strait (DS) NCS 1 Frobisher Bay (FB) NCS 12

Thermospore OTU(s) were identified in 50°C incubations of each of the nine sediment locations in the North Atlantic (Table 4.2) based on the thermospore identification criteria described in Section 2.4.3. Nine of 41 OTUs were identified in

70 incubations from more than one sediment location; these shared OTUs are listed in Table 4.4 and their phylogenetic relationship is shown in Figure 4.2. Only two shared OTUs were present in more than two sediment locations. OTU 46, which was 97% similar to Bacillus thermoamylovorans over a 350 bp region of the V3-V4 region of the 16S rRNA gene based on BLAST search (Madden, 2002) sequence similarity (as described in Section 2.4.3), was found in five of the nine locations – the most of any of the identified OTUs. OTU 2, which was 100% similar to Symbiobacterium ostreiconchae over a 350 bp region of the V3-V4 region of the 16S rRNA gene based on BLAST search (Madden, 2002) sequence similarity, was found in three of the nine locations. Seven more thermospore OTUs were found in two locations and 32 were found in only one location.

Figure 4.2: Phylogeny of the nine shared thermospore OTUs based on Maximum Likelihood phylogeny estimation. Scale bar identifies 1% sequence divergence. Deltaproteobacterial Geobacter metallireducens is included as an outgroup.

71 Table 4.4: Thermospore OTU identification based on BLAST 16S rRNA gene sequence similarity. Sites from which each thermospore OTU was identified after 50°C incubation are listed. Of forty-one thermospore OTUs identified, only the nine that are identified in sediment from more than one location are listed. Shared BLAST ID % ID Accession Sites OTU number OTU 2 Symbiobacterium ostreiconchae 100 NR_134208.1 SSE FB BBN OTU 12 Thermicanus aegyptius 100 NR_025355.1 SSC SSW OTU 34 Caloranaerobacter ferrireducens 97 NR_135860.1 LS SV OTU 46 Bacillus thermoamylovorans 97 NR_117028.1 SSE BBN PI DS LS OTU 89 Clostridium halophilum 96 NR_125713.1 LS SV OTU 136 Desulfotomaculum peckii 94 NR_109724.1 BBN SV OTU 244 Desulfotomaculum thermosapovorans 96 NR_119247.1 SSW SV OTU 469 Clostridium caminithermale 97 NR_041887.1 LS SV OTU 642 Desulfotomaculum geothermicum 95 NR_042044.1 FB BBN

72 4.4.3 Thermospore OTU occurrence and site connection to North Atlantic subpolar gyre

The average number of thermospore OTUs identified in sites connected to the North Atlantic subpolar gyre (CS) and those not connected to the North Atlantic subpolar gyre (NCS) were not notably different with averages of 6.4 and 5.5 respectively (Figure 4.4). This study suggests that a sediment site’s connectedness to the North Atlantic subpolar gyre does not increase the number of thermospores in the sediment, although the number of sites (nine) and the number of thermospore OTUs identified (forty-one) is likely too low to make this study conclusive (chi-squared statistics were unable to be done on the data owing to the high number of samples with less than five OTUs)

4 Instances of OTU Identification of OTU Instances

0 CS NCS CS shared NCS shared OTUs OTUs

74 Thermospores have been found in the water column above sediments from which they are enriched in incubation experiments (de Rezende et al., 2013; Volpi et al., 2017) therefore, a sediment site may be expected to share a thermospore OTU with another sediment site if there are water currents connecting the two sites. Most of the thermospore OTUs identified in this study are found in a single sediment location (Figure 4.5). While there are nine OTUs that are shared between different locations (indicated in Figure 4.5 as OTUs “shared between one or more sites”) the number of enriched OTUs found at one site that are also found in another location (shared OTUs) is not significantly different between sites connected to the North Atlantic subpolar gyre (CS), and those not connected to the North Atlantic subpolar gyre (NCS) with 2.8 and 2.0 incidences of shared OTUs identified respectively (Figure 4.4). (Note that an “instance of a shared OTU” differs slightly from simply a “shared OTU”; while “shared OTU” refers to the biogeography of the OTU that is detected – one that is identified at more than one site – “incidences of a shared OTU” refers to the event of a shared OTU being detected at a site, thus a single OTU that is found in two locations will be counted as two incidences of shared OTU identification. For example, in this study, there are 22 instances of a shared OTU being detected but there are only 9 shared OTUs) These data suggest that a site’s connectedness to the North Atlantic subpolar gyre does not increase the number of incidences thermospore OTUs detected in the sediment will also be found in another location. A site connected to the North Atlantic subpolar gyre (CS) appears no be more likely to hold an OTU that is also found in another location than a site that is not connected to the North Atlantic subpolar gyre (NCS).

75 18

8 Number of of OTUs Number 6

NCS

Only CS Only

Only NCS Only CS and NCS and CS Found at only one site Shared between one or more sites

76 4.4.4 Assessing thermospore OTU absence in the largest 16S rRNA gene library

Since the detection and identification of thermospore OTUs is dependent on their germination and growth, the lack of a particular OTU’s detection does not confirm its absence in situ at the sediment location. The largest amplicon library in this study (2 157 989 reads passing quality control) came from Frobisher Bay incubation replicate 1 after 14 days of incubation (Figure 2.1). Only two thermospore OTUs were identified from this sample according to the cut off criteria discussed in section 2.4.3. However, this library also contains reads in abundance between 3 and 138 of other thermospore OTUs identified in other locations. If the presence of these reads, despite being in number far below the 0.16% error threshold described by Nelson et al. (2014), could allow for these taxa to be considered identified thermospore OTUs in this sample, then only five more OTUs would be added to the number of shared OTUs, bringing the total number of shared OTUs in the dataset to fourteen. Of the forty-one thermospore OTUs detected, twenty-seven are not found in the largest library in even a single read.

77 4.5 Discussion

Incubations (50°C) of sediment from nine different locations in the North Atlantic led to the enrichment and identification of forty-one thermospore OTUs, nine of which were detected in more than one sediment location. A thermospore phylotype was present in at least one biological replicate from each sediment location implying that thermospore presence is common to North Atlantic sediments despite temperatures that are too low to allow for thermospore germination. Since temperatures are too low for growth of thermophilic endospore-forming bacteria, their presence in the sediment as dormant endospores is presumably owing to passive dispersal from warm locations (Hubert et al., 2009; Hubert and Judd, 2010). This passive dispersal would rely on water movements of which ocean currents may be a primary driver. Sediment sites connected to the North Atlantic subpolar gyre did not have a significantly higher number of thermospore OTUs nor did these sites have a significantly higher detectable number of shared thermospore OTUs. Likewise, thermospore OTUs present in 50°C incubations from sites connected to the North Atlantic subpolar gyre were not more likely to be found in other connected sites (CS) than unconnected sites (NCS). This study suggests that connectedness to the subpolar gyre, and the Mid- Atlantic Ridge hydrothermal systems that it traverses, does not affect the dormant thermospore community in a way detectable by the methods employed in this study. A larger number of sediment sites and incubation conditions that promote the germination of a more diverse population of thermophilic endospore-formers, eg. the addition of aerobic incubations (see Chapter 3) and/or media more selective for fermentative physiology (Volpi et al., 2017) that may lead to a larger number of identified thermospore phylotypes per site may provide data that could make these findings conclusive. The results of this study showing no notable difference in thermospore numbers (shared and not-shared OTUs) may suggest that thermospores deposited into marine sediment may be coming from sources other than and/or in addition to the Mid-Atlantic Ridge spreading centers. Alternatively, the dispersal of thermospores from the Mid- Atlantic Ridge spreading centers may extend far beyond those sites connected to the North Atlantic subpolar gyre and the other currents immediately connected to the North

78 Atlantic Current (Norwegian Current) to more distant sediments. Warm environments other than those of the of the hydrothermal vent systems at the Mid-Atlantic Ridge include oil production facilities (Magot, 2005) such as those found in the Barents Sea or spreading centers and hydrothermal vent systems such as those found along the Gakkel ridge in the Arctic Ocean (Edmonds et al., 2003). Similarly, cold seeps, which may be connected to deeper and warmer hydrocarbon reservoirs, are a possible source of thermophiles (Hubert and Judd, 2010). These warm sites are also connected to the water column above the sediment by seeping fluids and could similarly be a source of thermospores. Like the North Atlantic Current and its connection to the Mid-Atlantic Ridge system, the Arctic Ocean has currents connecting the Gakkel ridge to other sites within the Arctic Ocean as well as to the northern currents of the North Atlantic (Nøst and Isachsen, 2003); these warm locations are other possible source habitats of thermospores found in sediment of the North Atlantic. Thermospore OTUs of particular interest in this study were those that were present in more than one sediment location. Of these, two OTUs were present in more than two locations – OTU 46 was present in five locations and OTU 2 was present in three locations. Interestingly, OTU 46 shared a phylogenetic lineage with Bacillus thermoamylovorans, an endospore forming Bacilli. Endospores, specifically those of Bacillus subtilis, have long been used to study the possibility of panspermia, the theory that life is dispersed throughout the universe (Weber and Greenberg, 1985; Horneck, 1993; Nicholson et al., 2000; Nicholson, 2009). Radiation is a primary stress to life exposed to space conditions and spores are known to have an increased tolerance to radiation and other stressors, eg. temperature extremes, pressure extremes, and tolerance to toxins (Setlow, 1994). In addition, very slow decay rates allow endospores to remain viable for thousands to millions of years (Cano and Borucki, 1995; Vreeland et al., 2000; de Rezende et al., 2013) and it has been proposed that thermophilic endospores may remain viable for even longer (Nicholson, 2003). The tolerance to radiation and other stressors and viability after very long periods of dormancy make endospore-formers prime candidates to studying the feasibility of interplanetary/interstellar microbial dispersal. The phylogenetic proximity of OTU 46 to species known to survive the conditions of space highlights the importance of

79 understanding the dispersal capabilities of these bacteria to prevent the accidental occurrence of panspermia during human space exploration. OTU 2 shows close phylogenetic relationship to Symbiobacterium ostreiconchae of the Symbiobacterium lineage of the phylum Firmicutes, a syntrophic organism which requires commensalism with Geobacillus stearothermophilus or Ureibacillus spp. (Ohno et al., 2000, Sugihara et al., 2008; Shiratori-Takano et al., 2014). S. ostraconchae, G. stearothermophilus, and Ureibacillus sp. are moderately thermophilic and produce endospores (Fortina et al., 2001; Nazina et al., 2001; Shiratori-Takano et al., 2014). Interestingly, there were no occurrences in this study of a thermospore phylotype or a member of the family Bacillaceae that was common to all 3 locations in which OTU 2 was found. The syntrophic partner taxon with OTU 2 at its original warm source was either not equally dispersed about the North Atlantic with OTU 2 or OTU 2 is common to multiple warm temperature sources, and living commensally with different Bacillaceae sp. Alternatively, the taxon living syntrophically with OTU 2 may lack the endospore phenotype and would thus fail to be detected after the incubation conditions and subsequent implementation of the thermospore identification protocol used in this study. It is important to note that the OTUs used in this study are grouped based on sharing 97% similarity along the 16S rRNA gene amplicon. Because of this, OTU 2 and OTU 46 may be groupings of many different taxa that happen to share 97% identity along the 16S rRNA gene amplicon. Oligotyping, a technique that allows taxonomy within OTUs to be resolved (Eren et al., 2013), would provide a more robust analysis of the presence of shared OTU at different sediment locations and should be done in future biogeographic studies using thermospores. The absence of a thermospore OTU is difficult to prove since not detecting it based on the criteria in section 2.4.3 simply implies that the organism did not increase in abundance over the course of the 50°C incubation – no claim can be made about its absence in situ. Based on the identification method used in this study (section 2.4.3), the best observation of an OTU being absent is if it was missing in the largest 16S rRNA gene amplicon library. Replicate 1 of the 14 day Frobisher Bay incubation sample had over two million reads yet many of the OTUs detected by the criteria in section 2.4.3 in other samples are not present in even a single read in this library.

80 The presence of thermospore OTUs in some sediments but not in others suggests that these thermospores are not cosmopolitan to the sediment of the North Atlantic, although this cannot be claimed conclusively due to failure to detect spores that did not germinate. Müller et al. (2014) demonstrated that thermospore phylotypes are not equally distributed in the global ocean providing some evidence pointing to the endemism of some thermospores; they suggested connectivity to ocean circulation as the cause of differential thermospore presence between sediments, a theory the present biogeographical study cannot corroborate. Understanding the geophysical processes that contribute to thermospore passive dispersal leads to understanding microbial dispersal beyond thermospores specifically and the unique physiologies associated with thermophily and endospore formation make thermospores seemingly good model organisms to study these processes. Further research on thermospore dispersal may help resolve questions of microbial endemism and further understanding of the factors contributing to the dormant microbial seed bank. Additionally, better knowledge of the dispersal capabilities of endospore-forming bacteria is important to prevent the contamination of potentially sterile sites, a sensational example of this being the prevention of contamination of extraterrestrial environments during human space exploration. However, inconsistencies in OTU presence that arise among replicates cast some doubt on the effectiveness of using germination experiments to trace these dormant members of the rare biosphere and more consistent mechanisms of detection may be required before thermospores can unequivocally be considered model organisms for microbial passive dispersal.

81 Chapter 5: Microbial temporal dispersal in the North Atlantic: tracking thermospores through time.

5.1 Abstract

Previously the use of thermospores as model microorganisms has been proposed for the study of geographic microbial dispersal and biogeography, but the ability to remain dormant possibly over geologic time scales make these organisms also especially suitable for studying temporal microbial biogeography and dispersal. In the marine setting, an exploration of thermospore populations at different sediment depths may illuminate temporal patterns that can be correlated to absolute time by sediment age-dating. Sediment from three depths of three sediment cores from the Scotian Slope of Atlantic Canada were used to prepare slurries that were pasteurized (80°C for 1 hour) then incubated at 50°C for 28 days to promote the enrichment of thermospores. Evidence of thermospore germination, based on sulfate reduction, organic acid concentration decrease, and increases in Firmicutes relative abundance in 16S rRNA gene amplicon libraries after 50°C incubation was inconsistent between replicates. However, the presence of thermospores could be demonstrated in surface samples from each location and in some cases in sediments from depths of 400 cm and 655 cm below seafloor (cmbsf) corresponding to ~14 000 to ~16 000 years of sedimentation respectively. Two thermospore OTUs related to Symbiobacterium ostreiconchae and Vulcanivacillus modesticaldus were identified at multiple sediment depths and are the best candidate thermospores identified here for tracking temporal dispersal on the Scotian Slope.

82 5.2 Introduction

As discussed in previous chapters, thermospores are particularly useful models for studying microbial biogeography and dispersal in the marine environment because they exist in a dormant state in the cool (~4°C) sediments of the ocean floor and are therefore not subject to environmental selection. A defining feature of endospores is that they can remain dormant yet viable for many years. DNA degradation is one of the primary reasons for microbial loss of viability but endospores possess unique strategies for protecting their DNA. The most significant attribute that allows for long-term stability of DNA within the spore is high concentrations of small acid-soluble proteins (SASPs). These proteins are specific to spore-forming bacteria, are produced during sporulation, and bind to DNA protecting it from damage during dormancy (Setlow, 2007). There are some sensational claims about reviving endospores from samples that are millions of years old. Cano and Borucki (1995) isolated a Bacillus sp. most closely related to B. sphaericus, based on phylogenetic analysis, from 25- to 40-million-year-old amber. Vreeland et al., (2000) isolated a spore-forming bacterium phylogenetically related to a lineage containing the spore-formers Bacillus marismortui and Virgibacillus pantothenticus from a brine inclusion of 250-million-year-old halite crystal. While there has been some criticism of these studies (e.g. Hebsgaard et al., 2005), these examples imply that endospores can remain viable for millions of years, and possibly longer. The extreme longevity of viable spores is supported by theoretical estimates of spore longevity. Using decimal reduction times (the time it takes a particular treatment to reduce viable spore numbers by a factor of 10), Nicholson (2003) calculated that mesophilic spores can remain viable for nearly 2 million years and thermophilic endospores can remain viable for over 2 billion years. These viability estimations were based on dormancy at temperatures between 25°C and 40°C, while temperatures below this range would increase the longevity estimates (Nicholson, 2003). Other studies of spore longevity calculated much more conservative estimates based on the observation of thermospores displaying a half-life in marine sediments. de Rezende et al. (2013) found evidence supporting a half-life of ~350 years for thermospores of sulfate-reducing bacteria in Baltic Sea sediment; this finding was

83 supported by Volpi et al. (2017) who found a half-life of ~350 years for thermospores of fermentative bacteria in the same Baltic Sea sediment. Understanding the longevity of thermospores in sediment is important when thermospores are used as models for microbial dispersal. Thus the use of germination assays as presented in this work to detect thermospores, would confirm their presence only if they are viable. The detection of ancient thermospores in sediments can reveal temporal dispersal patterns. Hubert et al. (2009) determined, based on stable thermospore abundance in a 23 cm deep surface sediment column, that thermospores were constantly dispersed to the Arctic at a rate of 2 × 108 m-2 year-1. de Rezende et al. (2013), despite observing a thermospore half-life, were able to detect thermospores throughout a deeper sediment column (6.5 m) corresponding to 4500 years of sedimentation and thereby inferred that the original source of these thermospores is unrelated to human or industrial activity. Hubert and Judd (2010) have suggested that thermospore detection at various depths in marine sediments may reveal the presence of an intermittent supply vector, such as ephemeral hydrocarbon seepage, connecting warm subsurface environments to the surface sediments above. These studies show that the deposition of thermospores throughout time provides information about the extent and possible sources of passive dispersal. Here, sediments from three depths in three cores ranging from 5.5 m to 9 m in depth were incubated at 50°C to test for the presence of viable thermospore populations at various depths and assess patterns of thermospore temporal dispersal along the Scotian Slope.

84 5.3 Materials and Methods

5.3.1 Sediment sites

Figure 5.1: Map of Scotian Slope sediment sites Scotian Slope West (SSW), Scotian Slope Centre (SSC), and Scotian Slope East (SSE).

Piston cores from three seabed locations along the Scotian Slope were retrieved by the crew of the CCGS Hudson during expedition 2015-018 between June 28, 2015 and July 6, 2015. Details of the sampling sites are provided in Table 2.1. Cores from the three stations Scotian Slope West, Scotian Slope Centre, and Scotian Slope East were 555 cm, 902 cm, and 690 cm in length respectively. These cores were subsampled for microbiology at higher frequency along the core than most of the other cores extracted from the seabed during the expedition, thus these were the cores chosen for this study. Sediments from three different depths along each core (top, middle and bottom) were

85 sub-sampled for incubation experiments (Table 5.1). Sediments were stored at 4°C until they were used to set up high temperature sediment slurry incubations.

5.3.2 High temperature incubation

Anoxic incubations at 50°C were conducted with nine different sediments (three depths from each of the three cores) in microcosm slurries amended with organic acids as described in section 2.2. The incubations were subsampled (described in section 2.2) and mixed with a 1:1 (v/v) with 2% zinc acetate solution before separation of the supernatant and pellet fractions and storage at -20°C. Subsampling occurred daily for the first 7 days of incubation, after which time the frequency of subsampling decreased to every second day, and to once per week after 14 days. The incubations lasted 28 days.

5.3.3 Sulfate and organic acid measurement

As discussed in section 2.3, sulfate and organic acid concentrations were measured using ion chromatography (Dionex ICS-5000, Thermo Scientific) and ultra- high performance liquid chromatography (Ultimate 3000 RSLCnano UHPLC system, Thermo Scientific), respectively. The Aminex HPX-87H column (5μm, 7.8 × 300 mm, Bio Rad) was used in the UHPLC for the measurement of organic acids. Acetate concentrations were not analyzed due to the addition of zinc acetate to subsamples during the subsampling procedure that confounded the acetate concentration in the subsample.

5.3.4 16S rRNA gene amplicon sequencing and analysis

DNA extractions for all samples were performed using the DNeasy Powersoil Kit (Qiagen). Sections 2.4.2 and 2.4.3 describe the methods by which 16S rRNA genes

86 were amplified and sequenced and the method for microbial community analysis and thermospore phylotype identification, respectively.

5.3.5 Representative sequence comparison

Representative sequences of OTUs identified in multiple sediment depths along the Scotian Slope were established by analyzing each sample individually using the MetaAmp pipeline (Dong et al., 2017). The representative sequences of these OTUs were aligned and viewed using SeaView (Galtier et al., 1996).

87 5.4 Results

5.4.1 Microbial activity and community structure

Decreases in sulfate and organic acid concentrations were used as indications of microbial activity during the 28-day 50°C anoxic incubation of Scotian Slope sediment from various depths. Results of the three surface sediment incubations were previously discussed in Chapter 4 but are considered again here alongside incubations of the two deeper sediments, a mid depth and the core bottom depth, for each sediment, to evaluate thermospores as a function of sediment depth and age. Sulfate only decreased in one out of 27 Scotian Slope sediment microcosm incubations (replicate 3 of Scotian Slope Centre 0 cm sediment), whereas decreases in organic acid concentration were more common (8 out of 27) and congruent with increases in Firmicutes relative abundance upon 50°C incubation (Table 5.1). Since all known thermospores belong to the Firmicutes lineage, an increase in the relative abundance of Firmicutes upon 50°C incubation was considered a signal for thermospore presence and spore germination. Such increases in Firmicutes were observed after 7 and/or 14 days in all microcosms where organic acid(s) concentrations decreased during the 28-day incubation suggesting that these decreases were associated to the increase of Firmicutes. The organic acid concentrations of two microcosms (replicate 1 from Scotian Slope West 0 cm and replicate 1 from Scotian Slope West 655 cm) did not change over the 28-day incubation despite showing increases in Firmicutes. A summary of the sulfate reduction, organic acid utilization, and increase in Firmicutes relative abundance is listed in Table 5.1. Results of the incubations of individual sediment sites are discussed below.

88 Table 5.1: Summary of the sulfate reduction, organic acid utilization, and increase in Firmicutes relative abundance after 50°C incubation. A “+” symbol represents the occurrence of sulfate reduction, organic acid decrease, or increase in Firmicutes relative abundance over the 50°C incubation. The symbols “+” and “-“ represent a positive or negative result respectively in each replicate for the occurrence of sulfate reduction, organic acid utilization, or increase in Firmicutes relative abundance over the 28-day incubation period. The first symbol represents the result in the first replicate, the second represents the result in the second replicate, etc. * Scotian Slope West 0 cm sediment incubations had six biological replicates. (Table is shown on the following page.)

Location Depth Measure Result Sulfate reduction ------0 cm Organic acid utilization - - + + - + Increase in Firmicutes relative abundance - - + + - + Sulfate reduction - - - Scotian Slope 260 cm Organic acid utilization - - - West Increase in Firmicutes relative abundance - - - Sulfate reduction - - - 550 cm Organic acid utilization - - - Increase in Firmicutes relative abundance - - - Sulfate reduction - - + 0 cm Organic acid utilization - - + Increase in Firmicutes relative abundance - - + Sulfate reduction - - - Scotian Slope 405 cm Organic acid utilization - - - Centre Increase in Firmicutes relative abundance - - - Sulfate reduction - - - 902 cm Organic acid utilization - - - Increase in Firmicutes relative abundance - - - Sulfate reduction - - - 0 cm Organic acid utilization - - + Increase in Firmicutes relative abundance + - + Sulfate reduction - - - Scotian Slope 400 cm Organic acid utilization + + + East Increase in Firmicutes relative abundance + + + Sulfate reduction - - - 690 cm Organic acid utilization - - - Increase in Firmicutes relative abundance + - -

90 Table 5.2: OTUs identified in 50°C incubations of Scotian Slope sediments from various depths based on BLAST searches of the NCBI database using 16S rRNA gene amplicon sequence data. The closest cultured isolates are shown.

OTU # Sediment location and BLAST ID % ID Accession depth number OTU 2 Scotian Slope East 0 cm Symbiobacterium 100 NR_134208.1 Scotian Slope East 655 cm ostreiconchae OTU 5 Scotian Slope East 0 cm Desulfotomaculum 93 NR_133738.1 tongense OTU 12 Scotian Slope West 0 cm Thermicanus 100 NR_025355.1 Scotian Slope Centre 0 cm aegyptius OTU 14 Scotian Slope West 0 cm Vulcanibacillus 98 NR_042421.1 Scotian Slope East 400 cm modesticaldus OTU 21 Scotian Slope East 0 cm Aneurinibacillus 95 NR_112216.1 thermoaerophilus OTU 27 Scotian Slope East 400 cm Limnochorda pilosa 99 NR_136767.1 OTU 33 Scotian Slope East 400 cm Moorella 87 NR_125518.1 perchloratireducens OTU 46 Scotian Slope East 0 cm Bacillus 97 NR_117028.1 thermoamylovorans OTU 244 Scotian Slope West 0 cm Desulfotomaculum 96 NR_119247.1 thermosapovorans OTU 514 Scotian Slope West 0 cm Symbiobacterium 92 NR_134208.1 ostreiconchae OTU 4497 Scotian Slope East 0 cm Bacillus hisashii 97 NR_144578.1

91 5.4.1.1 Scotian Slope West

Anoxic 50°C incubations of Scotian Slope West sediment show evidence of thermospore enrichment only in surface sediment (0 cm) (Table 5.1, Figure S5.1). Sulfate was not reduced in sediment incubations of any depth of the Scotian Slope West sediment over 11 days of 50°C incubation (Figure S5.1a). Decreases in formate, succinate, and butyrate were observed in replicates 3, 4 and 6 of surface sediment incubations (Figure 5.2b). Firmicutes relative abundance increased in replicates 3, 4 and 6 of surface sediment incubations after incubations at 50°C (Figure S5.2a) and these emergent communities of Firmicutes were dominated by organisms closely related to Thermicanus aegyptius (OTU 12) and Vulcanibacillus modesticaldus (OTU 14) (Table 5.2). OTU 12 is dominant in replicates 3 and 6 while OTU 14 is dominant in replicate 4 (Figure S5.2b). The library of replicate 4 also contains OTU 244 (Desulfotomaculum sp.) and OTU 514 (Symbiobacterium sp.). Incubations of sediment from 260 cm and 525 cm did not display any evidence of sulfate reduction, decreases in organic acid concentration, or increase of Firmicutes relative abundance (Figure S5.1 and S5.2). The 16S rRNA gene amplicon libraries of these deeper sediments were dominated by Proteobacteria and Bacteroidetes and their relative abundances did not change during the 50°C incubation.

5.4.1.2 Scotian Slope Centre

Similar to incubations of Scotian Slope West, only incubations from surface sediment (0 cm) showed evidence of thermospore enrichment (Table 5.1, Figure S5.3). As discussed in section 4.4.1.2, sulfate reduction occurred in one replicate in incubations from 0 cm (Figure S4.3b). Replicate 3 of surface sediment incubations exhibited sulfate reduction, a decrease in all five organic acid concentrations, and an increase in Firmicutes that is, as was seen in two of the Scotian Slope West incubations, dominated almost completely by OTU 12, a taxon most closely related to Thermicanus aegyptius (Figure S5.4, Table 5.2). All other replicates of surface sediment incubations

92 and incubations from sediment of 405 cm and 875 cm depth showed no evidence of microbial activity or increases in Firmicutes relative abundance and therefore no thermospores were identified from the deeper sediment incubations. As was seen in the deeper sediment incubations of Scotian Slope West, the 16S rRNA gene amplicon libraries of incubations of sediment from 405 cm was dominated by Proteobacteria and Bacteroidetes and the relative abundance did not change during the 50°C incubation. Communities in 50°C incubations of sediment from 875 cm are primarily composed of Proteobacteria with low relative abundance of Atribacteria (Figure S5.4a).

5.4.1.3 Scotian Slope East

Anoxic 50°C incubations of sediment from Scotian Slope East demonstrate thermospore presence at all three depths (0 cm, 400 cm, and 655 cm) along the core (Table 5.1). Although sulfate was not reduced in any of the microcosms established from different depths in this sediment core, organic acid concentrations decreased in sediment incubations from 0 cm (replicate 3) and 400 cm depth (replicates 1, 2, and 3) (Figure S5.6b). Formate, lactate, and propionate decreased in replicate 3 of 0 cm sediment incubations over the 28-day 50°C incubation. Succinate and propionate decrease in all 3 replicates of incubations of sediment from 400 cm depth. Additionally, formate and lactate decrease in replicates 2 and 3 of incubations of sediment from 400 cm depth. Organic acid concentrations did not decrease in incubations of sediment from 655 cm depth (Figure S5.5b). In each replicate of the 0 cm and 400 cm depth incubations in which the organic acid concentration decreases over the 28-day incubation an increase in the relative abundance of Firmicutes is evident. Firmicutes relative abundance increases in replicates 1 and 3 of microcosms of sediment from 0 cm after 50°C incubation (Figure S5.6a). In replicate 1 of the surface (0 cm) incubations, the increase in relative abundance of Firmicutes is associated to increases in OTU 46 (Bacillus sp.) and OTU 4497 (Bacillus sp.) while in replicate 3 the increase in relative abundance is associated to increases in OTU 5 (Desulfotomaculum sp.) and OTU 21 (Aneurinibacillus sp.) after 7 days of 50°C incubation and OTUS 5, 21, and 2

93 (Symbiobacterium sp.) after 14 days of 50°C incubation (Figure S5.6b). The increase in Firmicutes relative abundance of incubations from 400 cm sediment is driven by a different taxon in each of the replicates with replicates 1, 2, and 3 being dominated by OTU 14 (Vulcanibacillus sp.), OTU 33 (Moorella sp.), and OTU 27 (Limnochorda sp.) respectively (Figure S5.6b, Table 5.2). A small increase in Firmicutes was observed only at Day 14 in replicate 1 of incubations of sediment from 655 cm. This increase in relative abundance of Firmicutes in replicate 1 of incubations of sediment from 655 cm can be accounted for by an increase in OTU 2, which made up the majority of the Firmicutes community. The Firmicutes relative abundance increased slightly and thermospores were identified according to the thermospore identification cut-off criteria listed in section 2.4.3 in two microcosms of sediment from Scotian Slope East despite the absence of sulfate reduction or changes in organic acid concentration that would signal microbial activity. Replicate 1 of the surface sediment incubations showed an increase in Firmicutes (to 16.6% of the Firmicutes relative abundance) that was driven by an increase in OTU 2 and OTU 4497, which are most closely related to Symbiobacterium ostreiconchae and Bacillus hisashii respectively (Figure S5.6b; Table 5.2). Similarly, replicate 1 of the 655 cm sediment incubations also showed a small increase in Firmicutes (to 3.2% relative abundance) also driven by an increase in OTU 2 (most closely related to Symbiobacterium ostreiconchae) despite sulfate reduction and organic acid utilization in this microcosm not being observed (Figure S5.5).

5.4.2 Representative sequence comparison

Comparison of the representative sequences of OTU 2 from incubations of sediment from Scotian Slope East at 0 cm and 655 cm and OTU 14 from incubations of sediment from Scotian Slope West at 0cm depth and Scotian Slope East at 400 cm depth was performed to assess the likelihood that sequences from these OTUs correspond to the same organism as defined by sharing 97% sequence similarity of the reads assigned to the taxon. Alignments of the representative sequences of OTU 2 identified at 0 cm and 655 cm of sediment from Scotian Slope East show 100%

94 sequence identity. This provides evidence supporting the suggestion that the taxon belonging to OTU 2 has the same 16S rRNA gene sequence and that it may thus be the same organism in both depths. Likewise, alignments of the representative sequence of OTU 14 at 0 cm of Scotian Slope West sediment and 400 cm depth of Scotian Slope East sediment show 100% sequence similarity and thus the OTU 14 found in two different locations may be the same organism.

95 5.5 Discussion

A sediment core into which thermospores have been continually or intermittently introduced may act as a thermospore archive for studying temporal microbial geography. In concept, since endospores can stay dormant yet viable for very long time periods after their deposition into sediment, age-dating the sediment can correlate thermospore deposition to absolute time making estimates of dispersal rates and constancy possible. However, the results described in this chapter cannot confidently support this idea and they illustrate that enriching the same OTU from different sediment depths is not always possible even though the sediment column is intact (i.e. sedimentation happened without a sediment mass transfer event). The Scotian Slope is covered by a ~1-2 m layer of sediment originating on the Nova Scotia Shelf and deposited on the slope in the last 10,000 years (the Holocene epoch) due to pelagic and hemipelagic processes involving biogenic and terrigenic sediment particles settling on the seafloor (Mosher et al., 1994). Using δ18O ratios, radiocarbon dating and micropaleontology, sediments at various near-sediment-surface depths on the Scotian Slope have been age-dated (Keigwin and Jones, 1995). Therefore, sediment depths that thermospores are found in can be roughly correlated to the time at which that sediment was deposited. In this study, two taxa were found at multiple sediment depths along the Scotian Slope, these are OTU 2 and OTU 14, which are most similar to Symbiobacterium ostreiconchae and Vulcanibacillus modesticaldus respectively. The OTU related to Symbiobacterium sp. (OTU 2) was present in surface sediment and sediment 655 cm below seafloor (cmbsf) at the Scotian Slope East site. These observations, combined with sediment age dating of nearby sites established by Keigwin and Jones (1995), suggests that OTU 2 has been dispersed to this site for ~16 000 years. Curiously, this OTU was not identified in incubations of sediment from 400 cmbsf from the same sediment core. The absence of this OTU from the middle section of the sediment core may be explained by intermittent dispersal. Fluid and gas release from hydrocarbon seeps and mud volcanoes, which are possible vectors connecting warm subsurface environments to the ocean above, are not always constant; the release may be episodic

96 and only occur to relieve geologic pressure (Roberts and Carney, 1997; MacDonald et al., 2000). Although detected ubiquitously in terrestrial and marine environments (Ueda et al., 2001; Sugihara et al., 2008), Symbiobacterium sp. have not yet been isolated from hydrocarbon seeps or mud volcanoes but the organism is enriched in anoxic, high temperature conditions similar to these environments making them possible habitats. The sporadic nature of some vectors (e.g. hydrocarbon seeps) linking warm subsurface habitats to the water column above may lead to the intermittent release of thermospores into the marine environment. An alternative dispersal mechanism could be passive movement through mass sediment transport. While the source of the brown and grey coloured sediment at the surface of the Scotian Slope East site is likely the Nova Scotia Shelf, the source of the red-brown sediment found at 655 cmbsf is likely the Gulf of the St. Lawrence (Piper and Skene, 1998). Thus far there is no published research connecting the movement of thermospores with sediment transport but since the sediment sources are different between the surface and 655 cmbsf, the dispersal of the Symbiobacterium OTU to this site is unlikely to be via sediment transport. A more plausible dispersal history may involve fluid flow that was occurring simultaneous to the sediment depositional periods. As discussed in chapter 4, the same Symbiobacterium OTU (OTU 2) was also detected in surface sediment from Frobisher Bay and Baffin Bay North 2360 km and 3740 km away respectively making evident the wide extent of dispersal experienced by this organism. The Vulcanibacillus sp. (OTU 14) was detected in surface sediment at the Scotian Slope West site and at sediment at 400 cmbsf at the Scotian Slope East site. The sediment from both of these sites is sourced from the Nova Scotia Shelf, and based on a sedimentation rate established by Keigwin and Jones (1995), endospores found 400 cmbsf were deposited ~14 000 years ago (if they were introduced into the sediment during sedimentation as is assumed here). This Vulcanibacillus OTU was not found in the surface sediment or the sediment 655 cmbsf at the Scotian Slope East site. The apparent absence of this OTU in the sediment 655 cmbsf may be explained by the organism originating in a warm source connected to a marine vector in closer proximity to the Nova Scotia Shelf sediment than the Gulf of St. Lawrence, where the sediment 655 cmbsf is derived. The absence of OTU 14 detection in the surface sediment of

97 Scotian Slope West is curious since the surface sediments of all three sites along the Scotian Slope, as well as the sediment from 400 cmbsf are in the same geographic region, all derived from the Nova Scotia Shelf, and were deposited simultanously. As discussed in Chapter 4, the apparent absence of an OTU in sediment incubations does not demonstrate certain absence in situ. Inconsistency within biological replicates is a result regularly observed throughout the experiments detailed in chapters 3 through 6 of this thesis preventing the conclusive determination of absence. A discussion about biological replicate reproducibility in section 7.2 may further contribute to an understanding about the apparent absence of OTU 14 from the surface sediment of the Scotian Slope East and the Scotian Slope Centre sites. Notably, no thermospores were identified from sediment below the surface in two of the three cores explored in this study. Despite identical incubation conditions, thermospore OTUs identified in the surface sediment of these two cores were not identified from deeper sediment. The result might be explained by thermospore half- lives. de Rezende et al. (2013) and Volpi et al. (2017), both observed a decrease in viable marine thermospore abundance with sediment depth and calculated a half-life of ~350 years. If thermospore abundances at the sites Scotian Slope West and Scotian Slope Centre are low at the surface then it is possible that no viable thermospores are present in the deeper sediments at these sites due to the thermospores’ half-lives. Results presented here suggest that spores of Symbiobacterium and Vulcanibacillus remained viable for up to ~14 000 years and ~16 000 years, respectively. The ages discussed above are based on the sediment accumulation rate of the Scotian Slope generally and not on radiocarbon dating of the specific sediment used in this study so the age dates should be viewed with some caution. The dates assume that the sediment age correlates to its depth, but this may be confounded by sediment mass transfer or mixing by benthic organisms. Direct radiocarbon dating of the sediments used in this study would improve age estimates. The ramification of spores surviving ~15 000 years is exciting for reasons beyond simply charting microbial dispersal with time. A sediment column with thermospores deposited over thousands of years may provide researchers with a “time-stamped” catalogue of viable thermospores of various ages. Protected for very long periods in the

98 spore, the genome of the same OTU found at different sediment layers could be compared to study rates of genomic change. This may reveal thermospore generation times at their source leading to a better understanding of their warm, possibly subsurface, habitats. Rates of evolution may also be calculated since the spores are viable and phenotypic traits can be observed in culture experiments. Similarly, germinated spores of different sediment depths can be competed against each other in fitness experiments. The longevity of the endospore viability unlocks the possibility of exploring many biological questions that relate to time and life’s history.

99 Chapter 6: Thermophilic endospore longevity in deep sediment of the Nankai Trough over geologic timescales

6.1 Abstract

Thermophilic endospore-forming bacteria (thermospores) are model organisms for exploring microbial temporal dispersal since they are dormant at low temperatures yet could remain viable for geologic time periods and can be experimentally revived years after deposition in marine sediments. Many questions about thermospore longevity in marine sediments remain unanswered. Do dormant thermospores experience a death decay or half-life over long time scales? Are dormant thermospores able to germinate into vegetative cells as temperatures increase during sediment burial? Are these same bacteria found again as dormant endospores in deeper sediment horizons at temperatures exceeding the growth range of corresponding vegetative cells, and if so is the spore decay rate or half-life in these warmer layers the same as in shallower cooler sediments? Is there evidence in thermospore genomes of genomic change or evolution over timescales corresponding to different sediment layers? To observe the thermospore population over geologic timescales and begin to resolve the questions above, slurries prepared with 35 g of marine sediment from 5 different depth intervals between 206 mbsf (metres below seafloor) and 865 mbsf of a 1.2 km sediment core, ranging in temperature from 30°C to 94°C, and dating back to 10 Mya were pasteurized at 80°C for one hour, and subsequently incubated at 50°C, 60°C, and 70°C for 21 days. Monitoring sulfate and organic acid concentrations revealed no microbial activity over the 21-day incubation period. Accordingly, comparing slurry sub-samples taken before and after the 21-day incubation period using a MiSeq amplicon library approach revealed microbial community structure was unchanged suggesting no enrichment of thermophilic spore-forming bacteria under these conditions. Furthermore, the microbial community structure in all samples before and after high temperature incubation resembled that obtained from sequencing and DNA extraction negatives with the exception of the phyla Atribacteria and Chloroflexi which appear to be detected only in the shallower cooler sediments of the core. This result, combined with preliminary cell

100 count data from the sediments being lower than expected, suggests the in situ thermospore numbers within the sediment intervals used in this experiment may be too low to allow for meaningful enrichment of thermophilic endospore-forming bacteria under these experimental conditions. These results highlight the difficulties faced when exploring sparse, dormant life in deep sediments.

101 6.2 Introduction

Thermospore longevity deduced by their presence in deep, old age-dated sediments, a topic that was explored in Chapter 5, can be more remarkably explored in sediments even deeper and older than those contained in the <10 m sediment cores obtained from the Scotian Slope. Cores containing marine sediment more ancient than that of the Scotian Slope must be longer to contain within it a larger sediment age range. A very long core obtained from an International Ocean Discovery Program (IODP) expedition was used to investigate thermospore longevity in very deep, and accordingly very old, sediments.

6.2.1 IODP Expedition 370 and the high temperature limit of life in sediments of the deep biosphere

The aim of IODP Expedition 370 was to explore the temperature limit of the subsurface sedimentary biosphere by measuring the microbial, biochemical, geochemical, and geophysical properties of a 1.2 km sedimentary core in the Nankai Trough off the coast of Cape Muroto south of the Island of Shikoku, Japan. This location is well suited to investigate the temperature limit of life in the subsurface since the sediment column is overlying the zone where the Philippine Sea plate and the Eurasian plate meet causing advective heating that leads to a temperature gradient three times steeper than typically seen in sediment globally (Heuer et al., 2017). Here, the temperature range between the sediment surface and 1.2 km depth is between 2°C and 120°C (Heuer et al., 2017). Currently, the known temperature limit for life is 122°C (Takai et al., 2008) and there are several microorganisms with temperature optima well above 100°C, although these organisms have been isolated from nutritionally rich hydrothermal sites (Stetter, 1999; Blöchl et al., 1997; Kashefi and Lovely, 2003; Takai et al., 2008). Microorganisms in the deep subsurface face extreme nutrient limitation and as a result these populations have much slower metabolisms and turnover rates than their surface-living counterparts (Hoehler and Jørgensen, 2013). Life in this nutrient limited zone will likely not tolerate the extreme temperatures such as those found at

102 hydrothermal sites because biomolecules are unstable at high temperatures (Bernhardt et al., 1984) and therefore energetically expensive to maintain. The energy-depleted environment of the deep biosphere may as such be inhospitable to hyperthermophiles that have larger energy requirements for survival due to increased biomolecule damage as temperatures increase (Stetter, 1999; Lever et al., 2015). In oil reservoir environments of the deep subsurface the temperature limit of life is ~80°C (Head et al., 2003; Wilhelms et al., 2001), possibly a consequence of the increased energy demand required of life living at hyperthermophilic temperatures. The temperature range (2°C to 120°C) explored in the sediment core (1.2 km) obtained on Expedition 370 likely encompassed the biotic-abiotic transition zone dictated by temperature stress in the deep biosphere (Heuer et al., 2017).

6.2.2 Endospores in very deep and very old sediments

Endospores, a specialized structure made by some members of the bacterial phylum Firmicutes, allow these cells to remain dormant yet viable for many, possibly millions, of years (Cano and Borucki, 1995; Vreeland et al., 2000; Nicholson, 2003). Endospores are found in deep subsurface marine sediments in similar numbers to vegetative cells (Langerhuus et al., 2012; Lomstein et al., 2012). Lomstein et al. (2012) found vegetative cells and endospores in abundances of 106 to 107 per cm3 in sediments off the continental shelf near the Peruvian coast that were up to 250 mbsf corresponding to 10-million-year-old sediment. Thermophilic endospore-forming bacteria (thermospores), which likely originated from warm, possibly subsuperficial, habitats, such as mud volcanoes, oil reservoirs, and hydrothermal vent systems (Hubert et al., 2009) are deposited in marine sediment during sedimentation after passive dispersal from their original warm location (Hubert and Judd, 2010; de Rezende et al., 2013; Volpi et al., 2017). These spores are buried in the sediment as sedimentation continues and each parcel of sediment moves deeper into the subsurface (Hubert and Judd, 2010). Thermospores with a temperature optima of more than 50°C have been found dormant in sediments globally (Müller et al., 2014) where the temperatures of the marine

103 sediment and the overlying water column are too cold to promote germination and growth. Germination of these thermospores can be induced from these sediments by incubating them at high temperatures (Hubert et al., 2009; de Rezende et al., 2013; Müller et al., 2014; Volpi et al., 2017). Sediment recovered on Expedition 370 is useful for exploring many unresolved questions about thermospore longevity and dispersal in the subsurface. During Expedition 370 drilling, 578 m of core material was retrieved from sediment depths between 189 and 1180 mbsf corresponding to a temperature range of 30°C to 120°C and dating back 14 Mya (Heuer et al., 2017). Therefore, this sample set allows large time and temperature ranges to be assessed in explorations of key parameters potentially affecting thermospore longevity in deep sediment.

6.2.3 What is the maximum depth at which viable thermospores are detected as dormant endospores?

While Firmicutes are not typically a substantial part of deep subsurface microbial communities (Fry et al., 2008), deep biosphere studies rely on DNA extraction for 16S rRNA gene sequencing; DNA within the protection of the endospore is difficult to extract without specialized procedures targeting their extraction specifically (e.g. Wunderlin et al., 2014). Therefore, it is likely that cells existing as endospores are missed in these standard diversity-screening assays. As discussed above, endospores have been detected in sediment 260 mbsf dating to 10 Mya (Lomstein et al., 2012). Measurements of endospores in deep sediments have relied on techniques such as measurement of the endospore biomarker dipicolinic acid (DPA) that does not require endospore germination for detection (Langerhuus et al., 2012; Lomstein et al., 2012) thus the number of viable endospores is not being assayed. How many of these endospores found in deep sediments exist as viable thermospores? Germination experiments in marine sediments <6.5 mbsf have discovered thermospore half-lives between 300-350 years (de Rezende et al., 2013; Volpi et al., 2017) suggesting a decrease in viability with depth and thus, with age. de Rezende et al. (2013) found that by 6.5 mbsf, corresponding to 4500 years of sedimentation, the number of viable thermospores was

104 1 per cubic cm. Despite this half-life, mesophilic endospores have been experimentally germinated after purportedly lying dormant for millions of years (Cano and Borucki, 1995; Vreeland et al., 2000) and thermospores are predicted to have further increased longevity (Nicholson, 2003). Core C0023A obtained on IODP Expedition 370 potentially allows for thermospores’ viability and decay rates to be assayed on much longer timescales than in previous thermospore germination studies.

6.2.4 Are thermospores of the phylotypes found at the deepest sediment layer present in throughout the shallower horizons of the core?

Once the deepest viable thermospores in Core C0023A are discovered, questions of dispersal continuity and the duration of temporal dispersion of thermospores into the sediment can be explored. Hubert et al. (2009) showed that there is a constant flux of thermospores into arctic sediment at a rate of 2 x 108 thermospores per square metre annually. Thermospore phylotypes that are continually dispersed over geological timescales allow for the investigation a third question concerning thermospore evolution by assessing mutations in their genomes over time, given that the thermospores are dormant in the sediment core and therefore not undergoing cell division and any additional mutations associated with replication. This approach would thus be capturing any genomic change happening at a population level in the thermospores’ original habitat prior to their dispersion to the C0023A site. Change in the genomes of thermospores found in the sediment column can be correlated to absolute time through age dating the sediment horizons in which they are found.

6.2.5 Do thermospores germinate in the warm horizons of the deep core?

Temperature may confound these explorations of thermospore dispersal and evolution over geological timescales. As the ambient temperature increases with depth, appropriate temperatures for thermospore germination will eventually be reached and thermospores may leave their dormant state. Will increases in temperature that happen

105 with depth allow the thermospores present in the warming sediment to germinate and grow, replenishing their numbers? In addition to temperature, key factors that would govern thermospore germination also include the availability of energy and nutrient sources. As the sediment continues heating, exceeding the temperature activity ‘window’ for a given thermospore population, will the thermospores die or sporulate to persist even deeper in the form of dormant endospores? If so, is the half-life (discussed above in section 6.2.3) of these thermospores in this deeper, hotter sediment the same as the half-life in the shallower, cooler sediments on the other side of the temperature activity window? Questions such as those described in sections 6.2.3 to 6.2.5 are part of larger questions about life in the deep biosphere. How is the deep biosphere inoculated with life and where is this life originating? How does dormancy contribute to the microbial community structure in the deep biosphere? The first steps toward answering these questions require that the thermospore populations at different depths of subsurface sediment be explored; Expedition 370 and core C0023A provides an opportunity to begin this exploration.

106 6.3 Materials and Methods

6.3.1 Sediment site and recovery

Figure 6.1: Map showing the site of IODP Expedition 370 core C0023A drilling site (yellow circle).

Sediment was obtained from site C0023A (32°22.00’N, 134°57.98’E) in the Nankai Trough 180 km south of Cape Muroto off the Japanese island of Shikoku during IODP Expedition 370 (Table 2.1). The advanced piston corer temperature tool (APCT-3) was used to measure in situ temperatures at depths up to 410.5 mbsf (Heuer et al., 2017). The temperatures of the <410.5 mbsf sediment depths were used to create a temperature model for estimating the temperature in deeper sediments (Heuer et al., 2017). Geological evidence (eg. sulfate mineralization in deeper sediments) supported the temperature model (Heuer et al., 2017). Sediment was stored at 4°C in heat sealed aluminum bags that were flushed with N2 gas to maintain anoxic conditions.

107 6.3.2 High temperature incubation

Sediment (35 g) from 5 different depths (Table 6.1) was ground into fine powder on a sterile bench that had been deinonized (to reduce static charges) using autoclaved ceramic mortars and pestles. Slurries were prepared by adding artificial seawater medium to anoxic serum bottles containing the various sediments and pasteurizing the microcosms as described in section 2.2. After pasteurization, triplicate microcosms made with sediment of each sediment depth were incubated at 50°C, 60°C, at 70°C for 21 days. Microcosms of sediment from 206 mbsf and 254 mbsf were set up in duplicate due to the limited amount of sediment from these depths available to be shared amongst expedition participants and objectives. Microcosms were subsampled daily for the first 7 days of incubation then every 2 to 4 days up to 21 days of incubation. Subsamples were stored at -20°C until being processed for sulfate and organic acid measurement and DNA extraction for 16S rRNA gene amplicon sequencing.

Table 6.1: Sediment depth and corresponding in situ temperature for the sediments used in the microcosm incubations. depth in situ sediment (mbsf) temperature 206 30°C 254 36°C 440 56°C 700 80°C 865 94°C

6.3.3 Sulfate and organic acid measurement

Sulfate and organic acid concentrations in the subsamples were measured from the supernatant fraction of centrifuged slurry subsamples. Sulfate and organic acids were measured using ion chromatography (Dionex ICS-5000, Thermo Scientific) and

108 ultra-high performance liquid chromatography (Acclaim™ Organic Acid LC Column, Ultimate 300 RSL Cnano UHPLC system, Thermo Scientific) as described in section 2.3. As was described in chapters 3 and 4, the Acclaim™ Organic Acid LC Column poorly measured formate concentration so only concentrations of lactate, acetate, succinate, propionate, and butyrate are reported.

6.3.4 16S rRNA gene amplicon sequencing and analysis

The DNeasy PowerSoil Kit (formerly the PowerSoil Isolation Kit, MoBio) (Qiagen) was used to extract DNA from all microcosms including the sediment-free, medium-only controls, before (Day 0) and after (Day 21) incubation from microcosms incubated 50°C, 60°C and 70°C. The V3-V4 region of the 16S rRNA gene was amplified using the universal primers U515F/U806R. PCR was done in 30 µl volumes consisting of 15 µl 2 × Mighty Amp Buffer (TaKaRa Bio), 0.6 µl Mighty Amp DNA Polymerase Ver. 3 (TaKaRa Bio), 0.9 µl of each primer, 2 µl DNA template, and 10.9 µl sterile water. PCR was done using a different method than was described in section 2.4.2. It began with an initial denaturation for 2 min at 98°C and was followed by 35 cycles of denaturation at 98°C for 5 sec, annealing at 55°C for 15 sec, and elongation at 68°C for 30 sec. This PCR procedure included additional denaturation, annealing and elongation cycles compared to the procedure described in section 2.4.3. in order to force the generation of DNA product from samples containing undetectable DNA concentrations (data not shown). Unfortunately, this approach also left the PCR reactions particularly vulnerable to amplifying contaminating sequences. Amplicon fragments were sequenced from all samples, including those from the sediment-free, medium-only controls and the DNA extraction negatives, on MiSeq (Illumina). Community analysis was performed as described in section 2.4.3 with the exception of ANOSIM statistics on the NMDS plot as the algorithm was unable to reach convergence after 20 iterations and no clustering of the samples could be observed. Since there was no evidence of enrichment in any of the microcosms after incubation, according to the criteria described in section 2.4.3, thermospore phylotypes were not identified with confidence.

109 The cut-off criteria for identifying thermospore OTUs in section 2.4.3 was modified to systematically determine the in situ community members of the sediment and differentiate them from contaminating sequences. To be considered present in the inoculum sediment, the OTU must be present in at least one sediment sample >1% relative abundance and the number of reads for each OTU identified must be less than 2 reads in the sediment-free, medium-only control incubation samples and the DNA extraction negatives. These criteria were used to determine OTUs of Atribacteria and Chloroflexi. A combination of the original cut-off criteria and the modified version was used to identify thermospore OTUs that could be members of the in situ community within the large signal of contaminating sequences.

6.3.5 Estimation of cell numbers in sediment

Cell numbers in the sediment of each microcosm were estimated based on global averages of sediment density (Tenzer and Gladkikh, 2013) and calculated from microscopic cell counts using the method developed by Morono et al. (2009) from sediment at various depths from site C0023A (Heuer et al., 2017).

110 6.4 Results

6.4.1. Sulfate reduction and organic acid utilization

Measurements of sulfate and organic acid concentration were used to assess microbial activity and growth in the incubated microcosms; sulfate may serve as an electron acceptor for sulfate-reducing microorganisms and organic acids may serve as an electron source for sulfate reducers and fermentative microorganisms. Sulfate concentration, measured before and after 21 days of incubation at 50°C, 60°C and 70°C for microcosms of all sediment depths, remained constant suggesting no sulfate reduction occurred during the 21-day incubation (Figure S6.1). Likewise, concentrations of lactate, acetate, succinate, propionate, and butyrate also remained constant over the 21-day incubation in all microcosms (Figure S6.2). Since sulfate reduction and organic acid utilization or production was not observed in any microcosms there is no evidence to support the existence of microbial activity or growth in incubations of the sediments at 50°C, 60°C or 70°C.

6.4.2. 16S rRNA gene amplicon library analysis

The phylum-level community structure was similar in every microcosm, including the sediment-free, medium-only controls and the DNA extraction negatives, before and after incubation at 50°C, 60°C, or 70°C. Proteobacteria was the dominant phylum in every sample making up 60% to 90% of each library (Figure 6.2). Other notable phyla included Actinobacteria (4% to 21% of each library) and Firmicutes (2% to 18% of each library) (Table 6.2). The relative abundance of Proteobacteria, Actinobacteria, and Firmicutes in each of the libraries is similar between the experimental sediment sample incubation libraries, the sediment-free, medium-only control libraries, and the DNA extraction negative libraries. This similarity prevents the discrimination of community structure as caused by the presence of the sediment and incubation treatment from the community structure resulting from amplification of contaminating sequences during the DNA extraction and amplification process.

111 a) 206 mbsf

100 90 80 70 60 50 40 30 20

Relative Abundance Relative Abundance (%) 10

Day 0 Day 0 Day Day0 Day0 Day0 Day0

Day21 Day21 Day21 Day21 Day21 Day21 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 50°C 60°C 70°C

Actinobacteria Aerophobetes Atribacteria Bacteroidetes Chlorobi Chloroflexi Deinococcus-Thermus Euryarchaeota Firmicutes Proteobacteria <1% b) 254 mbsf

100 90 80 70 60 50 40 30 20

Relative Abundance Relative Abundance (%) 10

Day0 Day0 Day0 Day0 Day0 Day0

Day 21 Day 21 Day Day21 Day21 Day21 Day21 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 50°C 60°C 70°C Actinobacteria Aerophobetes Atribacteria Bacteroidetes Chloroflexi Deinococcus-Thermus Firmicutes Parcubacteria Proteobacteria <1%

112 c) 440 mbsf

100 90 80 70 60 50 40 30 20 Relative Abundance Relative Abundance (%) 10

Day0 Day0 Day0 Day0 Day0 Day0 Day0 Day0 Day0

Day21 Day21 Day21 Day21 Day21 Day21 Day21 Day21 Day21 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 50°C 60°C 70°C Actinobacteria Bacteroidetes Chlorobi Cyanobacteria Deinococcus-Thermus Firmicutes Fusobacteria Proteobacteria d) 700 mbsf

100 90 80 70 60 50 40 30 20

Relative Abundance Relative Abundance (%) 10

Day0 Day0 Day0 Day0 Day0 Day0 Day0 Day0 Day0

Day 21 Day 21 Day Day21 Day21 Day21 Day21 Day21 Day21 Day21 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 50°C 60°C 70°C

Actinobacteria Bacteroidetes Cloacimonetes Deinococcus-Thermus Firmicutes Proteobacteria <1%

113 e) 865 mbsf

100 90 80 70 60 50 40 30

20 Relative Abundance Relative Abundance (%) 10

Day0 Day0 Day0 Day0 Day0 Day0 Day0 Day0 Day0

Day21 Day21 Day21 Day21 Day21 Day21 Day21 Day21 Day21 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 50°C 60°C 70°C Actinobacteria Bacteroidetes Cyanobacteria Deinococcus-Thermus Elusimicrobia Firmicutes Proteobacteria f) Medium-only control

100 90 80 70 60 50 40 30 20

Relative Abundance Relative Abundance (%) 10

Day0 Day0 Day0 Day0 Day0 Day0 Day0 Day0 Day0

Day 21 Day Day21 Day21 Day21 Day21 Day21 Day21 Day21 Day21 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 50°C 60°C 70°C

Actinobacteria Bacteroidetes Cloacimonetes Cyanobacteria Deinococcus-Thermus Firmicutes Proteobacteria <1%

114 g) DNA extraction negative

100 90 80 70 60 50 40 30 20

Relative Abundance Relative Abundance (%) 10 0 Day 0 Day 21 DNA extraction negative

Actinobacteria Firmicutes Proteobacteria

Figure 6.2: Phylum-level community structure based on 16S rRNA gene amplicon sequencing after 21 days of incubation at 50°C, 60°C, or 70°C of sediment from five different sediment depths (206, 254, 440, 700, and 865 mbsf) and a sediment-free, medium-only control. The group labeled “<1%” includes the sum of the relative abundances of phyla that are present in less than 1% in every library. Only phyla >1% relative abundance are shown in each legend.

115 Table 6.2: Relative abundances (%) of dominant phyla based on 16S rRNA gene amplicon sequencing. Actinobacteria, Firmicutes, and Proteobacteria are present in every sample including the sediment-free, medium-only control incubation samples and both DNA extraction negatives. *The amplicon library was composed of 2 reads and not suitable for community analysis

50°C 60°C 70°C

Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3

Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21

Actinobacteria 6.66 8.04 10.82 7.94 10.52 4.55 7.91 6.69 10.98 6.91 21.06 13.47

206 mbsf Firmicutes 10.28 12.34 3.55 8.72 7.50 7.23 12.78 5.49 14.08 6.55 6.03 6.60

Proteobacteria 81.56 79.47 84.67 59.75 77.36 66.34 78.38 66.22 66.81 80.59 72.52 78.43

Actinobacteria 7.38 13.11 4.84 6.31 5.94 7.17 7.20 9.57 8.90 12.50 9.81 8.74

254 mbsf Firmicutes 8.23 3.69 9.62 12.29 7.26 6.76 8.88 1.70 6.17 3.55 10.66 18.27

Proteobacteria 80.76 72.50 67.52 78.13 83.69 75.47 81.81 81.05 83.81 80.23 74.47 66.11

Actinobacteria 8.45 6.01 10.25 8.49 14.30 5.11 8.55 6.05 5.76 0* 9.35 7.72 6.37 10.56 7.54 11.21 9.21 10.90

440 mbsf Firmicutes 7.00 8.46 4.19 6.16 12.54 5.87 7.26 6.28 10.49 0* 9.588 10.68 10.25 9.78 7.40 6.70 8.41 6.76

Proteobacteria 83.28 80.10 83.75 82.81 71.50 83.89 83.63 85.13 82.79 100* 76.78 81.28 82.84 79.01 84.85 81.58 81.40 78.36

Actinobacteria 9.48 11.60 8.04 9.43 11.71 5.68 10.80 11.22 4.70 7.88 6.62 12.57 7.05 12.16 11.79 10.51 6.58 6.90

770 mbsf Firmicutes 9.66 7.45 5.71 9.91 11.70 16.53 7.86 9.03 6.74 10.28 11.08 4.89 11.17 8.74 5.68 11.58 9.84 7.91

Proteobacteria 78.39 80.48 85.72 77.61 76.37 73.70 80.17 78.99 87.51 80.70 81.82 81.21 80.68 78.71 82.17 76.80 82.21 84.14

Actinobacteria 9.48 8.52 9.71 10.33 5.60 10.08 4.61 11.03 10.29 4.54 10.02 9.56 5.62 12.52 3.63 7.06 9.42 12.96

865 mbsf Firmicutes 6.92 13.9 3.48 14.05 11.33 16.60 5.83 16.02 1.89 9.04 9.60 11.96 9.51 9.54 12.28 13.04 13.52 9.06

Proteobacteria 82.52 76.87 85.93 73.40 82.67 71.89 87.24 72.54 85.69 84.41 78.55 77.30 83.86 76.94 74.62 78.38 76.68 77.62

Actinobacteria 7.49 4.71 13.56 14.21 7.54 6.90 14.81 8.31 7.17 6.64 11.14 5.22 11.82 11.05 9.04 4.56 9.39 6.42

Control Firmicutes 7.63 4.92 13.53 8.22 6.70 5.83 11.18 11.47 6.27 11.43 12.19 8.27 13.96 10.29 12.97 13.17 7.43 11.36

Proteobacteria 80.88 89.96 72.46 77.38 82.88 86.59 73.29 79.43 86.40 80.57 74.17 65.31 71.43 76.17 77.00 81.47 82.02 80.71

Day 0 Day 21

DNA Actinobacteria 8.26 7.25 extraction Firmicutes 11.99 10.82 negative Proteobacteria 77.83 81.56

116 Non-metric multidimensional scaling plots show that the community structure of samples before high temperature incubation is not distinct from samples after high temperature incubation since no clustering of circles representing samples based on incubation (Day 0 vs. Day 21) is observed (Figure 6.3a). Furthermore, this same plot, when also revealing the depth of the sediment in the microcosm sediment slurry samples, sediment-free, medium-only control microcosm samples or DNA extraction negative samples (Figure 6.3b) does not reveal any clustering according to sediment depth. This evidence further supports the assumption that the community structures reflected in the 16S rRNA gene amplicon libraries are primarily a result of contaminating sequences and are thus all similar in structure.

117 a) b)

118 Despite the community structure of incubations of sediment from all depths being confounded by contaminating sequences (Figure 6.3b), some bacterial phyla are identified in the sediment incubations but not in the controls, these are Atribacteria and Chloroflexi. In a modification of the OTU cut-off criteria described in section 2.4.3, operational taxonomic units (OTUs) designated at the phylum level were considered present in the sediment if their relative abundance was above 1% in at least one microcosm and these phyla were not present >2 reads in the control groups (medium-only control incubation samples and the DNA extraction negatives). Using these criteria, Atribacteria and Chloroflexi were identified as in situ microbial constituents of sediments from 206 mbsf and 254 mbsf in relative abundances up to 11% and 5% respectively. Atribacteria and Chloroflexi were not identified in incubations of the deeper sediments from 440 mbsf, 700 mbsf, and 865 mbsf (Table 6.3). The presence and abundance of Atribacteria and Chloroflexi are not associated with incubation time (i.e. these phyla, considered here to be operational taxonomic units (OTUs), do not satisfy the thermospore cut-off criteria outlined in section 2.4.3 that requires that the OTU be present at 2 reads or less in the Day 0, before incubation, library) therefore their detection does not suggest a community shift in response to incubation temperature nor that they are thermospores. Rather their presence in sediment microcosms but not in controls suggests the possibility that they are actual sample-derived sequences and not contamination.

119 Table 6.3: Relative abundances (%) of phyla more abundant in the incubation slurry samples than the sediment-free, medium-only control incubations and the DNA extraction negatives. Atribacteria and Chloroflexi are the only phyla found in some samples at relative abundances that are higher than those found in any of the controls.

50°C 60°C 70°C

Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Day Day 0 Day 21 Day 0 Day 21 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21

206 mbsf Atribacteria 0.0034 0 0 7.5131 0 11.4086 0.0026 5.2637 2.884 4.1494 0.0049 0.0037 Chloroflexi 0 0 0.1676 11.6991 0 8.7868 0.0026 8.8016 2.833 0.9668 0 0

265 mbsf Atribacteria 3.1405 2.4662 3.5452 0 0 7.342 0.0025 0 0 2.3755 0 1.5635 Chloroflexi 0 3.6332 9.0774 2.327 0 0.9042 0.8194 4.0444 0 0 0 1.1632

440 mbsf Atribacteria 0 0 0 0 0 0 0.0057 0 0 0 0 0 0.007 0.0064 0 0.0024 0.0027 0 Chloroflexi 0.0028 0.0025 0 0.0024 0 0.0016 0 0.0023 0 0 0 0 0 0 0.0032 0.0024 0 0.0533

700 mbsf Atribacteria 0 0.003 0 0.093 0 0 0 0.0017 0.0023 0 0 0 0 0.0013 0 0.0024 0 0 Chloroflexi 0 0.003 0.0031 0.0692 0 0 0 0 0 0 0 0 0.5041 0 0 0.0016 0 0

865 mbsf Atribacteria 0.0035 0.002 0 0 0 0 0 0.0021 0.0066 0.1403 0 0 0.0011 0 0 0 0 0.0027

Chloroflexi 0 0.002 0.0031 0 0 0 0 0 0.0033 0.0455 0.0027 0 0.0018 0.0025 0 0 0 0.0027

Control Atribacteria 0 0 0 0 0 0 0 0 0.0021 0 0.0021 0 0 0 0 0 0 0.0052 Chloroflexi 0 0 0.0033 0 0 0 0 0 0 0.004 0.0021 0.0057 0 0 0.0054 0 0.0023 0

Day 0 Day 21

DNA extraction Atribacteria 0 0 negatives Chloroflexi 0.0031 0

120 Although the Atribacteria and Chloroflexi are not thermospores, the possibility of their sequences providing a signal of their existence in the sediment inoculum amongst the contaminating sequences from 206 mbsf and 254 mbsf encouraged a finer analysis of their OTUs, this time at the 97% similarity level, which more generally corresponds to genus level taxonomic resolution. Three OTUs were identified from the phylum Atribacteria, while 11 were identified from the phylum Chloroflexi (Table 6.4). These 14 OTUs were present >1% relative abundance in at least one replicate of incubations of sediment from 206 mbsf and 254 mbsf yet absent or nearly absent (present in less than 2 reads) in the sediment-free, medium-only controls and the DNA extraction negatives and thus satisfied the criteria for being considered OTUs from the in situ communities of sediment from 206 mbsf and 254 mbsf.

Atribacteria Chloroflexi OTU 36 OTU 50 OTU 41 OTU 69 OTU 158 OTU 243 206 mbsf OTU 165 OTU 135 OTU 489 OTU 277 OTU 36 OTU 50 OTU 41 OTU 69 OTU 158 OTU 195 254 mbsf OTU 183 OTU 135 OTU 273

121 Table 6.5: Thermospore OTUs identified from incubations of different depths of sediment at 50°C, 60°C, or 70°C.

OTU BLAST ID % ID Accession Relative Sediment Incubation number Abundance (%) depth temperature OTU 322 Abyssicoccus albus 99 NR_146680.1 1.55 206 mbsf 50°C

OTU 235 Clostridium vulturis 97 NR_148266.1 1.04 254 mbsf 50°C

OTU 184 Lactobacillus hominis 99 NR_125548.1 1.44 440 mbsf 50°C

OTU 223 Clostridium xylanolyticum 98 NR_037068.1 1.55 440 mbsf 70°C

OTU 152 Ethanoligenens 91 NR_074333.1 1.01 700 mbsf 70°C harbinense OTU 115 Nosocomiicoccus 99 NR_133032.1 2.63 700 mbsf 60°C massiliensis

Although the Firmicutes phylum was present as a large part of each amplicon library including the controls, an examination of Firmicutes at the 97% sequence similarity OTU level revealed a signal of thermospores from in situ sediment. Six OTUs were found as possible thermospore members of the in situ sediment – sequences of these OTUs were not found >2 reads in any of the control (sediment-free, medium-only incubation controls and DNA extraction negatives) nor were they found >2 reads in any of the Day 0 libraries. Table 6.5 lists the possible thermospore OTUs which were all present <3% relative abundance and in only one replicate microcosm of one sediment depth at one incubation temperature. While this leads to the possibility that minute amounts of thermospores may have been enriched, the absence of microbial activity indicators (sulfate reduction and changes in organic acid concentrations described section 6.4.2) combined with the large relative abundance of Firmicutes from contaminating sequences requires that the detection of these 6 “thermospore” OTUs be considered with skepticism. Consequently, the evidence is not strong enough to suggest thermospore enrichment in this study.

122 6.4.3 Estimated cell numbers in microcosm sediments

Preliminary cell counts obtained during IODP Expedition 370 are listed in Table 6.6, which also provides an associated estimate for the numbers of cells that were present in the sediment microcosms following set-up and before incubation. Estimated cell numbers are based on the sediment density and the preliminary cell counts for the sediment using the following calculation:

Ne = Na / ( ρ ✕ W)

Estimated cell number in the microcosm (Ne)

Actual cell count from Expedition 370 or 190 in the sediment (Na) Sediment density (ρ) Sediment weight (W)

Preliminary cell count data for sediments below 600 mbsf are unavailable.

Table 6.6: Estimated cell numbers in sediment microcosm incubations. Sediment density was based global averages of deep sediment (Tenzer and Gladkikh, 2013). Expected cell numbers per microcosm are based on the results of IODP Expedition 190 and the estimated cell numbers per microcosm are based on the preliminary results from IODP Expedition 370 (Heuer et al., 2017). Sediment Sediment Weight of IODP Exp 190 Expected IODP Exp 370 Estimated depth density sediment in Preliminary cell numbers Preliminary cell numbers (mbsf) (g/cc) Microcosms cell count per cell count per (g) (cells/cc) microcosm (cells/cc) microcosm 206 1.7 35 106 107 104 105 254 1.75 35 106 107 104 105 440 1.8 35 105 106 101 102 Data not Data not Data not Data not 700 1.9 35 available available available available Data not Data not Data not Data not 865 2.05 35 available available available available

123 6.5 Discussion

Incubation of sediment from five depths (206, 254, 440, 770, and 865 mbsf) from core C0023A in the Nankai Trough at 50°C, 60°C, and 70°C did not result in enrichment of thermophilic endospore-forming bacteria. The absence of sulfate reduction and organic acid utilization indicate that there was no microbial metabolic activity by sulfate- reducing or fermentative thermophiles detected using the methods listed in section 6.3.3 over 21 days of incubation. The lack of change in the community structure further suggests that thermospores were not enriched. Comparison between the community structure of the controls (sediment-free, medium-only incubations as well as DNA extraction negatives) and the sediment-inoculated microcosms suggests that the community structure identified is primarily a result of contamination. Contamination is not surprising especially in instances where DNA concentrations from experimental samples are very low (Salter et al., 2014; Weiss et al., 2014). However, even with large percentages of the libraries likely representing contamination some phyla were observed to be present only in sediment-inoculated library samples, and never in the controls. The phyla Atribacteria (formerly OP9/JS1; no cultured representatives) and Chloroflexi are commonly detected in subseafloor sediments and generally constitute a considerable proportion of marine sediment microbial diversity (Fry et al., 2008; Parkes et al., 2014). Both Atribacteria and Chloroflexi were previously found in sediments (4.15 mbsf) of the Nankai Trough during Expedition 190 (Fry et al., 2008) and the results here may also suggest their presence in situ. All endospore-formers belong to the phylum Firmicutes and while members of Firmicutes were detected in all sediment-inoculated incubations, their detection at the phylum level could not be an indication of the presence of in situ Firmicutes in the sediment used in this study because they were present in similar abundances in the controls. Despite this, a higher phylogenetic analysis of the Firmicutes revealed six thermospore OTUs that were identified as possible components of the in situ thermospore community although their detection invokes caution and all were found in only one sediment depth and therefore were unable to be used to explore questions about thermospore longevity and dispersal continuity introduced in section 6.2. Overall,

124 the germination of thermospores in the sediments is very small if it is indeed sediment derived at all. Preliminary cell count data determined cell numbers to be on the order of 104 cells/cm3 down to 350 mbsf, and deeper than this the cell numbers dropped quickly to around 101 cells/cm3 at 450 mbsf (Table 6.4; Heuer et al., 2017). The cell counts increased to 103 cells/cm3 in sediment 525 mbsf but then dropped again in the deeper sediments (Heuer et al., 2017). Cell counts below 600 mbsf from IODP Expedition 370 have yet to be published. These cell counts are much lower than previous cell abundance measurements in other deep sediments. Cell counts from sediment obtained on ODP Expedition 190 at the same site (site 1174) suggested cell abundances of >105 cells/cm3 to a depth of 500 mbsf, counts below detection limit in sediment below 500 mbsf, and then an increase to 106 cells/cm3 in sediment between 775 mbsf and 800 mbsf (Heuer et al., 2017). The incubations in this study used 35 g of sediment and based on the preliminary cell counts this equates to between 102 and 105 cells per microcosms of 206 mbsf, 254 mbsf, 440 mbsf while the cell count data reported by IODP Expedition 190 suggested that the counts would be between 106 and 107 cells per microcosm (Table 6.4). Cell count data from C0023A was not available at the time of this thesis’s publication for 700 mbsf and 865 mbsf sediments preventing estimations of cell numbers in microcosms of these sediments. While spores were not counted in this cell count, it is reasonable to assume that low cell counts could also mean low spore counts (Lomstein et al., 2012). The low spore numbers, inferred based on the low cell counts, in sediment from the depths explored in this study may have been too low to allow for a detectable thermospore enrichment over 21 days of incubation. Likewise, the sediment may completely lack thermospores leading to the same negative result. Another reason for the absence of thermospore germination may be that the incubation conditions were unsuitable for enrichment of the thermospores present in the sediment. Certainly, if endospore numbers are similar to cell numbers in deep sediment as Lomstein et al. (2012) suggest, then endospores are present in the sediments explored in this study, particularly the shallower sediments (206 and 254 mbsf). Endospore counts, using methods such as dipicolinic acid (DPA) measurement, should be done to establish endospore numbers in this sediment column. Thermospores,

125 generally considered to be passively introduced into cool sediment (see Chapters 4 and 5), may not appear in sediment layers in numbers reflective of vegetative cells like spores in general (as observed by Lomstein et al. (2012)) since they are not influenced by the in situ sediment during dormancy and, contrary to some mesophilic spores, the thermospores’ vegetative counterparts will not be present in the cool sediment and will not provide a possible origination population for the thermospores. How many of these endospores are viable thermospores is a question that can thus far only be answered using high temperature germination experiments such as the one described in this study. The minute amounts of thermospore enrichment in this study should not discourage the exploration of thermospores in this sediment column. Sediment spore counts using dipicolinic acid (DPA) measurements should be done on various depths of this sediment core to establish a spore profile with depth. Spore counts will inform future germination experiments by providing estimated spore numbers for each sediment depth. Furthermore, diverse incubation conditions might uncover more thermospores in these sediments by giving thermospores with different growth conditions than were used here the opportunity to proliferate. The incubation conditions in this study were intended to promote the growth and germination of a specific type of endospore-forming thermophiles – anaerobic sulfate-reducers. As observed in the results of Chapter 3, oxic conditions promote the enrichment of aerobic thermospores that aren’t enriched in anoxic microcosms. Additional incubations, possibly using richer media formulations, such as those that promote the growth of fermenting microorganisms or media supplemented with different electron acceptors or donors, and longer incubation times should be attempted to uncover the full scope of thermospores in these deep sediments. Together, spore counts and thermospore germination experiments will uncover the proportion of endospores in the sediment that are both thermospores and viable. Experiments such as these may lead to the identification of thermospores throughout or localized to specific depths of the sediment column and will contribute to exploring the questions addressed in the introduction of this chapter.

126 Chapter 7: Summary and Outlook

7.1 Experimental summary and main conclusions

The four sets of experiments described here in chapters 3-6 in different ways and contexts consider thermospores as models for microbial dispersal. Each chapter explores a different aspect of thermospore survival and/or biogeography in relation to factors influencing their biogeography such as stress tolerance, lateral and temporal dispersal, and thermospore longevity. The freezing treatment experiment of Chapter 3 shows that different OTUs of thermospores in a marine sediment sample can display different freezing tolerances, with many surviving freezing at -80°C. Although environments of -80°C might not be common on Earth, maintained viability after freezing at this temperature demonstrates the hardiness of thermospores in the dormant endospore state. This connects to their function as model biological propagules in biogeography studies for two reasons. The first is that samples of convenience, i.e. donated by other investigators, are sometimes the only samples available. This can be especially true for large biogeographic studies where many sediments from different locations are desirable. Occasionally sediment samples may be stored at -80°C to preserve molecular characteristics of the sediment, such as in situ community DNA. Thermospores that remain viable after exposure to -80°C will be able to germinate from these frozen sediments, and may be useful in biogeography studies. The second reason survival after -80°C freezing in sediment sample is intriguing is that thermospores, as endospores, may be useful in studying the possibility of panspermia, as explained in greater detail below (section 7.3). Microorganisms exposed to the space environment may be exposed to temperatures as low as -263°C thus exploring the lower temperature limit of thermospores is relevant to understanding their ability to survive space travel. Chapter 4 explores the utility of thermospores for studying geographic microbial dispersal in North Atlantic sediment. Detection of thermospores in every surface sediment incubated at 50°C reveals widespread passive dispersal of thermospores throughout the North Atlantic. This study was not unable to provide evidence supporting

127 the prevailing current of the North Atlantic subpolar gyre as a vector driving the observed thermospore dispersal pattern nor was it able to support cosmopolitanism of thermospores in North Atlantic marine sediment. The identification of some OTUs in more than one of the sediment locations tested does highlight that passive dispersal of thousands of kilometers may be possible but further studies including oligotyping or multi-locus sequence analysis of the same OTUs found in multiple sediment locations would be necessary to more confidently make this claim. Temporal dispersal of thermospores over geologic timescales was investigated in Chapter 5. Thermospores were not identified in every sediment that was incubated at 50°C suggesting that as depth increased the number of viable thermospores may decrease. In one instance the same thermospore OTU was identified at two different depths in a sediment column, suggesting possible sustained dispersal over ~16 000 years. Challenges to using thermospores as biological dispersal propagules come when a sediment column does not remain intact or when viable thermospores exhibit a half-life in the sediment causing a decrease in spore numbers with depth (de Rezende et al., 2013, Volpi et al., 2017). The 1.2 km long core retrieved by IODP Expedition 370, presented in Chapter 6, provided an opportunity to delve into questions concerning long-term viability of thermospores and their response to environmental changes associated with sediment warming with depth. Low cell numbers and the consequent low DNA yield from the sediments lead to 16S rRNA libraries dominated primarily by sequences arising from contaminants. However, within these contaminant-dominated libraries two phyla, Atribacteria and Chloroflexi, were identified as possible in situ members of the sediment microbial community at 206 and 254 mbsf. An obvious signal for thermospore presence could not be detected from the deep long core using the sediment methods employed in this study, and that were successful for surface sediments (Chapters 3-5).

7.2 Inconsistency among biological replicates

To investigate the in situ dormant thermospore populations in marine sediment, enrichment cultures were required. Martinus Beijerinck (1851-1931), the early

128 microbiologist who first clearly defined the enrichment culture method, identified this method as useful for microbial ecology for two reasons, understanding the conditions under which known organisms proliferate and discovering organisms that proliferate under certain conditions. In his acceptance speech upon winning the Leeuwenhoek medal from the Koninklijke Akademie van Wetenschappen in Amsterdam (1905), Beijerinck stated:

In an experimental sense the ecological approach to microbiology consists of two complementary phases which give rise to an endless number of experiments. On the one hand it leads to investigating the conditions for the development of organisms that have for some reason or other, perhaps fortuitously, come to our attention; on the other hand to the discovery of living organisms that appear under predetermined conditions, either because they alone can develop, or because they are the more fit and win out over their competitors. Especially this latter method, in reality nothing but the broadest application of the elective culture method, is fruitful and truly scientific, and it is no exaggeration to the claim that the rapid and surprising advances in general microbiology are due to this methodology (translated by Van Niel, 1949).

The enrichment culture technique was applied in the experiments of this thesis to promote the growth and division of environmentally dormant anaerobic and thermophilic spore-forming bacteria for biogeographic investigation. A primary difficulty encountered in sediment heating enrichment experiments is the inconsistent germination of specific thermospore OTUs amongst the biological replicates. There are two possible reasons for this: stochastic presence of thermospores in the sediment samples that were used, and competitive exclusion. In an attempt to incubate biological replicates as opposed to technical replicates, microcosms of a particular sediment were not inoculated from a single homogenized sediment slurry, rather each replicate microcosm was inoculated with sediment directly from the stored samples. Furthermore, when available, different replicate sediment cores from the same

129 geographic location were used (section 2.2). In microscale diversity studies it was shown that within the same soil core microbial community structures between samples of 10 g were more similar than between samples of 0.1 g -1 g (Ramette and Tiedje, 2007). This demonstrates that the size of the environmental sample analyzed may affect the reproducibility of community diversity metrics. This phenomenon will be even more pronounced when reduced diversity enrichments, such as those resulting from heating experiments of environmental samples, are being assessed. Even though the sediment core sections used in this study were small volumes (25 cm3 -1300 cm3; see Table S2.1), heterogeneity within the sediment still leads to multiple niches over a scale of centimeters for the in situ community (Vos et al., 2009; Vos et al., 2013). While dormant thermospores are passive in the niches they encounter, properties such as pore size, hydrophobicity, and particle roughness may affect the dispersal of these passive travelers to and within the sediments. Rattray et al. (2017) observed that endospores of the thermophile Desulfotomaculum geothermicum aggregate with sedimentary particles when combined with a sediment suspension. The association between thermospores and sediment particles may lead to a patchy distribution of thermospores in the seabed following sedimentation, and cause the emergence of different thermospore communities upon heating among replicates microcosms, if they are made from small sediment volumes. In addition to uneven distributions in the sediment samples, the enrichment of different thermospore OTUs in replicate incubations may be caused by competitive exclusion of one OTU by another. In other words, while two thermospores may be present, only one becomes dominant in the thermophilic community upon experimental heating. It is possible that one OTU outcompetes another under the enrichment conditions, but if absent (possible due to patchy deposition, as explained above) the formerly outcompeted OTU can thrive. Similarly, the history of the community assembly, such as the order thermospores exit dormancy, may lead to differing community structure (Fukami and Morin, 2003). If one OTU is present in higher numbers or germinates upon heating more rapidly than another, this OTU may quickly dominate the community and leave little resources for later germinators. In incubations of Scotian Slope West surface sediment (discussed in Chapters 4 and 5), out of the three

130 replicates that showed thermospore enrichment, two had nearly identical Firmicutes communities completely dominated by OTU 12 while the other was dominated by OTU 14 (Figure 4.2). It is possible that OTU 14 was present in the parcel of sediment used to inoculate the latter replicate and was able to outcompete OTU 12. Similarly, it is possible that OTU 14 was present in all the sediment parcels used to inoculate replicates of Scotian Slope West incubations but that it was outcompeted by OTU 12, which was present only in 2/3 microcosms allowing it to become the dominant OTU. As seen in Chapter 4, the metabolisms expressed amongst replicate microcosms can be similar despite containing different thermospore OTUs. For example in incubations of Frobisher Bay sediment (discussed in Chapter 4), replicates 2 and 3 show similar levels of sulfate reduction despite the thermospore community composition being very different between these replicates (Figure 4.6b and e). The resources provided may dictate how much thermospore enrichment can occur, with the taxa that can best take advantage of these resources quickly becoming dominant. The inconsistency in increases in relative abundance of thermospore OTUs between replicates decreases the confidence with which determinations of thermospore absence can be made – determinations that are already very difficult using the sediment heating methods utilized in this study. As was discussed in Chapter 4 conclusive evidence of thermospore absence cannot be obtained using these germination experiments. Inconsistent germination of OTUs due to uneven distributions of thermospores in sediment samples could be remedied by using larger sediment volumes for enrichment experiments. This approach may also solve inconsistencies due to competitive exclusion if the sediment sample volume size is such that the in situ thermospore diversity is the same in all samples. More incubation replicates of smaller sediment volumes may work with thermospore patchy distribution in the sediment to allow for the detection of thermospores that are outcompeted alongside another thermospore taxon. Increasing the number of replicates in Scotian Slope West incubations to six rather than three, led to the detection of a viable thermospore that would otherwise have not have been discovered at this sediment location.

131 7.3 Concluding remarks

While the experiments presented in this thesis consider geographic and temporal dispersal of thermophilic endospore-forming bacteria in marine sediments of the North Atlantic and Arctic, the lessons learned from these studies have wider implications. By understanding microbial dispersal in this localized area of the global ocean, processes underlying microbial biogeography more generally may be uncovered. Thermospores, owing to their unparalleled hardiness and extreme longevity, are specifically suited to be model organisms for this research. Looking backward to the beginning of life’s history or looking forward as humans begin exploring the stars, thermospores may become important biological representatives of the resilient life on Earth.

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149 Supplementary Tables

Table S2.1: Number of replicate cores used as inoculum sediment for replicates from each site

Sediment Site Number of cores Volume of sediment (cm3) Scotian Slope West 1 160 Scotian Slope Centre 1 160 Scotian Slope East 1 160 Labrador Slope 3 80 Frobisher Bay 1 80 Davis Strait 1 80 Pond Inlet 1 80 Baffin Bay North 1 80 C0023A 206 mbsf 1 25 254 mbsf 1 25 440 mbsf 1 680 700 mbsf 1 1326 875 mbsf 1 1020

Table S2.2: Number of reads in each amplicon library sample that passed quality control and the percent these read numbers represent of the total reads that were sequenced in each sample. Sample Read number Percent of total read number (%)

-80_A1_t0 6067 7.36 -80_A1_t7 1387 4.64 -80_A1_t53 1880 4.11 -80_A2_t0 2657 6.74 -80_A2_t7 1747 5.78 -80_A2_t53 2341 4.12 -80_A3_t0 4531 6.25 -80_A3_t7 2141 5.57

150 Sample Read number Percent of total read number (%)

-80_A3_t53 5642 5.59 -20_A1_t0 4900 7.94 -20_A1_t7 2631 6.41 -20_A1_t53 3932 5.92 -20_A2_t0 2273 6.89 -20_A2_t7 1729 6.44 -20_A2_t53 2886 6.1 -20_A3_t0 4013 5.91 -20_A3_t7 3149 5.76 -20_A3_t53 21806 5.49 +4_A1_t53 6426 6.41 +4_A2_t53 3353 6.68 +4_A3_t53 4588 5.85 +4_A1_A2_A3_t53 2678 6.03 +4_A1_A2_A3_t0 2040 7.25 +4_A1_A2_A3_t7 5336 5.85

-80_B1_B2_B3_t0 55487 63.5 -80_B1_t7 67675 59.28 -80_B2_t7 62460 60.71 -80_B3_t7 60900 62.26 -20_B1_B2_B3_t0 46303 62.56 -20_B1_t7 73542 60.05 -20_B2_t7 53103 60.63 -20_B3_t7 79408 60.69 +4_B1_B2_B3_t0 37589 64.12 +4_B1_t7 112709 61.34 +4_B2_t7 89827 61.43 +4_B3_t7 18915 44.77 -80_before_pasteurization 71656 63.63 -20_before_pasteurization 60650 64.15 +4_before_pasteurization 34563 64.45

SSW_0cm_R1_t0 6167 46.52 SSW_0cm_R1_t7 6505 43.84 SSW_0cm_R1_t14 1763 38.97 SSW_0cm_R2_t0 15213 46.34 SSW_0cm_R2_t7 13052 44.15

151 Sample Read number Percent of total read number (%)

SSW_0cm_R2_t14 7934 41.19 SSW_0cm_R3_t0 15242 47.32 SSW_0cm_R3_t7 6928 39.26 SSW_0cm_R3_t14 5321 35.96 SSW_0cm_R4_t0 14954 45.23 SSW_0cm_R4_t7 6209 33.94 SSW_0cm_R4_t14 3550 29.3 SSW_0cm_R5_t0 16295 47.76 SSW_0cm_R5_t7 12292 45.08 SSW_0cm_R5_t14 7690 42.84 SSW_0cm_R6_t0 9985 43.6 SSW_0cm_R6_t7 6696 37.06 SSW_0cm_R6_t14 13833 41.25 SSW_260cm_R1_t0 18995 54.11 SSW_260cm_R1_t7 16896 47.87 SSW_260cm_R1_t14 5648 47.55 SSW_260cm_R2_t0 10718 49.2 SSW_260cm_R2_t7 24416 45.72 SSW_260cm_R2_t14 10233 45.32 SSW_260cm_R3_t0 14019 51.01 SSW_260cm_R3_t7 1557 42.66 SSW_260cm_R3_t14 16361 45.49 SSW_550cm_R1_t0 9517 53.95 SSW_550cm_R1_t7 16028 50.54 SSW_550cm_R1_t14 6358 49.45 SSW_550cm_R2_t0 16870 54.28 SSW_550cm_R2_t7 9486 50.48 SSW_550cm_R2_t14 8802 49.44 SSW_550cm_R3_t0 17287 54.7 SSW_550cm_R3_t7 19245 52.02 SSW_550cm_R3_t14 6826 49.47

SSC_0cm_R1_t0 7735 38.17 SSC_0cm_R1_t7 7469 35.97 SSC_0cm_R1_t14 16583 50.1 SSC_0cm_R2_t0 15011 36.73 SSC_0cm_R2_t7 2646 35.11 SSC_0cm_R2_t14 16354 51.5 SSC_0cm_R3_t0 10794 37.3

152 Sample Read number Percent of total read number (%)

SSC_0cm_R3_t7 5183 29.09 SSC_0cm_R3_t14 13918 46.86 SSC_405cm_R1_t0 10293 39.06 SSC_405cm_R1_t7 7134 39.61 SSC_405cm_R1_t14 16581 53.93 SSC_405cm_R2_t0 14480 37.18 SSC_405cm_R2_t7 13209 40.86 SSC_405cm_R2_t14 25838 52.19 SSC_405cm_R3_t0 11497 38.39 SSC_405cm_R3_t7 4443 37.29 SSC_405cm_R3_t14 19423 50.32 SSC_902cm_R1_t0 12800 38.99 SSC_902cm_R1_t7 7152 37.6 SSC_902cm_R1_t14 44219 54.18 SSC_902cm_R2_t0 13648 37.78 SSC_902cm_R2_t7 4179 35.82 SSC_902cm_R2_t14 8620 54.12 SSC_902cm_R3_t0 112436 37.75 SSC_902cm_R3_t7 29867 37.53 SSC_902cm_R3_t14 17339 50.67

SSE_0cm_R1_t0 6636 34.3 SSE_0cm_R1_t7 25926 35.37 SSE_0cm_R1_t14 18191 46.32 SSE_0cm_R2_t0 22548 32.13 SSE_0cm_R2_t7 12268 36.53 SSE_0cm_R2_t14 13664 44.8 SSE_0cm_R3_t0 6814 33.34 SSE_0cm_R3_t7 2964 30.67 SSE_0cm_R3_t14 283773 43.07 SSE_400cm_R1_t0 10141 36.93 SSE_400cm_R1_t7 13863 26.98 SSE_400cm_R1_t14 15110 43.72 SSE_400cm_R2_t0 31309 37.64 SSE_400cm_R2_t7 14678 24.98 SSE_400cm_R2_t14 6997 38.13 SSE_400cm_R3_t0 9779 32.62 SSE_400cm_R3_t7 3258 32.71 SSE_400cm_R3_t14 29805 41.24

153 Sample Read number Percent of total read number (%)

SSE_690cm_R1_t0 9870 37.13 SSE_690cm_R1_t7 15247 38.01 SSE_690cm_R1_t14 10828 23.22 SSE_690cm_R2_t0 11796 34.21 SSE_690cm_R2_t7 9891 36.81 SSE_690cm_R2_t14 17380 46.63 SSE_690cm_R3_t0 17407 37.17 SSE_690cm_R3_t7 12461 38.29 SSE_690cm_R3_t14 10225 48.96

BBN_t0 7612 19.41 BBN_t14 6772 17.79

PI_R1_t0 7265 32.35 PI_R1_t7 8988 34.18 PI_R1_t14 19457 42.96 PI_R2_t0 11752 32.05 PI_R2_t7 5850 34.56 PI_R2_t14 46850 50.09 PI_R3_t0 7150 29.66 PI_R3_t7 10043 31.01 PI_R3_t14 13049 49.39

DS_t0 8797 19.68 DS_t14 6270 19.86

LS_R1_t0 11010 33.47 LS_R1_t7 4267 29.44 LS_R1_t14 20840 45.31 LS_R2_t0 6152 36.91 LS_R2_t7 12497 35.2 LS_R2_t14 16420 44.44 LS_R3_t0 2450 28.83 LS_R3_t7 4761 33.55 LS_R3_t14 8463 45.92

FB_R1_t0 14265 46.28 FB_R1_t7 17528 45.51 FB_R1_t14 2157989 45.31

154 Sample Read number Percent of total read number (%)

FB_R2_t0 21443 45.92 FB_R2_t7 11938 47.06 FB_R2_t14 124008 46.15 FB_R3_t0 26432 43.24 FB_R3_t7 18356 42.04 FB_R3_t14 11068 42.34

SV_t0 1942 7.25 SV_t7 5170 5.85 SV_R1_t53 6305 6.41 SV_R2_t53 3305 6.68 SV_R3_t53 4480 5.85

206mbsf_50°C_R1_t0 29607 91.25 206mbsf_50°C_R1_t21 26940 86.04 206mbsf_50°C_R2_t0 54881 92.21 206mbsf_50°C_R2_t21 23053 82.61 206mbsf_60°C_R1_t0 50535 89.92 206mbsf_60°C_R1_t21 25174 93.55 206mbsf_60°C_R2_t0 38956 73.64 206mbsf_60°C_R2_t21 30074 83.84 206mbsf_70°C_R1_t0 37135 88.88 206mbsf_70°C_R1_t21 28341 91.62 206mbsf_70°C_R3_t0 41181 88.33 206mbsf_70°C_R3_t21 27365 86.88 254mbsf_50°C_R1_t0 59544 90.29 254mbsf_50°C_R1_t21 34709 86.5 254mbsf_50°C_R2_t0 35231 93.76 254mbsf_50°C_R2_t21 33433 85.96 254mbsf_60°C_R1_t0 31974 92 254mbsf_60°C_R1_t21 42795 85.45 254mbsf_60°C_R2_t0 40644 90.02 254mbsf_60°C_R2_t21 34343 83.83 254mbsf_70°C_R1_t0 44898 78.85 254mbsf_70°C_R1_t21 36371 91.05 254mbsf_70°C_R2_t0 62249 71.32 254mbsf_70°C_R2_t21 36968 85.76 440mbsf_50°C_R1_t0 35621 90.09

155 Sample Read number Percent of total read number (%)

440mbsf_50°C_R1_t21 40293 90.41 440mbsf_50°C_R2_t0 30795 80.85 440mbsf_50°C_R2_t21 41425 89.89 440mbsf_50°C_R3_t0 109515 84.12 440mbsf_50°C_R3_t21 60966 89.32 440mbsf_60°C_R1_t0 34959 78.85 440mbsf_60°C_R1_t21 43126 89.16 440mbsf_60°C_R2_t0 26531 77.32 440mbsf_60°C_R2_t21 2 40 440mbsf_60°C_R3_t0 41303 85.52 440mbsf_60°C_R3_t21 37429 78.79 440mbsf_70°C_R1_t0 28739 88.02 440mbsf_70°C_R1_t21 46565 86.24 440mbsf_70°C_R2_t0 31547 78.79 440mbsf_70°C_R2_t21 41039 89.59 440mbsf_70°C_R3_t0 37123 75.88 440mbsf_70°C_R3_t21 35631 86.06 700mbsf_50°C_R1_t0 37597 84.56 700mbsf_50°C_R1_t21 33589 89.53 700mbsf_50°C_R2_t0 31861 90.22 700mbsf_50°C_R2_t21 37649 82.98 700mbsf_50°C_R3_t0 25655 79.32 700mbsf_50°C_R3_t21 45604 90.3 700mbsf_60°C_R1_t0 27028 92.31 700mbsf_60°C_R1_t21 60378 87.5 700mbsf_60°C_R2_t0 43057 92.49 700mbsf_60°C_R2_t21 44513 87.3 700mbsf_60°C_R3_t0 27887 85.28 700mbsf_60°C_R3_t21 38721 92.17 700mbsf_70°C_R1_t0 45027 87.95 700mbsf_70°C_R1_t21 74293 80.26 700mbsf_70°C_R2_t0 34065 92.59 700mbsf_70°C_R2_t21 121617 83.93 700mbsf_70°C_R3_t0 38943 89.44 700mbsf_70°C_R3_t21 63757 91.35 865mbsf_50°C_R1_t0 28401 87.46 865mbsf_50°C_R1_t21 49402 85.07 865mbsf_50°C_R2_t0 32431 78.27 865mbsf_50°C_R2_t21 30758 81.57

156 Sample Read number Percent of total read number (%)

865mbsf_50°C_R3_t0 31733 84.8 865mbsf_50°C_R3_t21 36527 80.46 865mbsf_60°C_R1_t0 37944 83.68 865mbsf_60°C_R1_t21 47411 83.62 865mbsf_60°C_R2_t0 30418 83.03 865mbsf_60°C_R2_t21 28510 90.54 865mbsf_60°C_R3_t0 37475 77.39 865mbsf_60°C_R3_t21 52333 92.9 865mbsf_70°C_R1_t0 179266 86.28 865mbsf_70°C_R1_t21 40484 85.17 865mbsf_70°C_R2_t0 80273 90.22 865mbsf_70°C_R2_t21 36888 89.26 865mbsf_70°C_R3_t0 42465 91.25 865mbsf_70°C_R3_t21 37365 91.08 Control_50°C_R1_t0 35068 86.91

Control_50°C_R1_t21 42397 84.24 Control_50°C_R2_t0 30346 77.93 Control_50°C_R2_t21 47442 82.61 Control_50°C_R3_t0 54176 88.79 Control_50°C_R3_t21 25345 85.28 Control_60°C_R1_t0 40881 91.5 Control_60°C_R1_t21 38973 87.93 Control_60°C_R2_t0 47989 90.79 Control_60°C_R2_t21 49471 87.57 Control_60°C_R3_t0 46718 87.43 Control_60°C_R3_t21 35276 87.35 Control_70°C_R1_t0 53521 81.91 Control_70°C_R1_t21 36801 91.11 Control_70°C_R2_t0 37629 85.6 Control_70°C_R2_t21 35213 86.2 Control_70°C_R3_t0 43526 84.27 Control_70°C_R3_t21 37900 86.96 ExNeg_t0 32769 87.92 ExNeg_t21 32870 85.86

157

158

-80°C Replicate 1 Replicate 2 Replicate 3 Closest Neighbour (BLAST ID) % ID Accession Day Day Day Day Day Day number 7 53 7 53 7 53

Bacilli OTU 21 Bacillus boroniphilus 96 NR_041275.1 12.9 1.1 9.0 1.7 27.5 3.9 OTU 28 Bacillus hisashii 98 NR_144578.1 5.3 4.3 15.4 1.5 OTU 31 Geobacillus thermantarcticus 95 NR_117156.1 20.2 6.4 OTU 61 Anoxybacillus calidus 98 NR_125532.1 3.2 3.7 2.3 1.0 OTU 88 Bacillus benzoevorans 97 NR_044828.1 7.3 1.9 OTU 920 Bacillus hisashii 97 NR_144578.1 3.8 5.0

Clostridia

OTU 2 Proteiniborus ethanooligenes 88 NR_125623.1 2.1 8.5 7.3 19.5 8.5 30.0 OTU 4 Clostridilisalibacter sp. 98 KC555195 OTU 5 Desulfotomaculum peckii 94 NR_109724.1 13.3 4.0 OTU 7 Clostridium halophilum 96 NR_125713.1 2.3 11.0 1.5 3.1 OTU 8 Caloranaerobacter ferrireducens 97 NR_125860.1 4.8 7.5 3.3 OTU 9 Lutispora thermophila 96 NR_041236.1 1.6 Desulfotomaculum OTU 10 NR_119247.1 thermosapovorans 96 6.8 2.2 3.6 OTU 12 Desulfonisporus sp. AAN04 97 NR_026497.1

OTU 15 Halothermothrix orenii 91 NR_074915.1 1.1 4.9 OTU 22 Tepidimicrobium xylanilyticum 89 NR_116042.1 OTU 24 Irregularibacter muris 97 NR_144613.1 OTU 34 Desulfitibacter alkalitolerans 89 NR_042962.1 3.4 7.5 OTU 35 Desulfonisporus sp. AAN04 94 AB436739.1 OTU 40 Tepidimicrobium xylanilyticum 96 NR_116042.1 2.3 1.3 2.5 1.7 1.2 OTU 45 Sporosalibacterium faouarense 95 NR_116364.1 OTU 47 Desulfotomaculum peckii 89 NR_109724.1 19.1 7.2 OTU 51 Gracilibacter thermotolerans 91 NR_115693.1 OTU 64 Brassicibacter thermophilus 99 NR_137216.1 1.4 OTU 69 Anaerovirgula multivorans 99 NR_041291.1 2.9 2.8 OTU 87 Defluvitalea saccharophila 99 NR_117912.1 1.0 OTU 90 Carboxydothermus siderophilus 87 NR_044272.1 1.5 OTU 110 Halothermothrix orenii 91 NR_074915.1 OTU 115 Gelria glutamica 91 NR_041819.1 OTU 121 Tindallia californiensis 91 NR_025162.1 OTU 149 Clostridium caminithermale 96 NR_041887.1 2.3 6.3 4.4 4.6 1.1

159

-20°C Replicate 1 Replicate 2 Replicate 3 Closest Neighbour (BLAST ID) % ID Accession Day Day Day Day Day Day number 7 53 7 53 7 53

Bacilli OTU 21 Bacillus boroniphilus 96 NR_041275.1 OTU 28 Bacillus hisashii 98 NR_144578.1 OTU 31 Geobacillus thermantarcticus 95 NR_117156.1 OTU 61 Anoxybacillus calidus 98 NR_125532.1 OTU 88 Bacillus benzoevorans 97 NR_044828.1

OTU 920 Bacillus hisashii 97 NR_144578.1

Clostridia

OTU 2 Proteiniborus ethanooligenes 88 NR_125623.1 9.4 11.6 11.5 11.7 9.8 8.8 OTU 4 Clostridilisalibacter sp. 98 KC555195 5.0 8.8 4.6 9.2 5.8 8.3 OTU 5 Desulfotomaculum peckii 94 NR_109724.1 13.2 8.6 9.8 7.1 12.2 6.6 OTU 7 Clostridium halophilum 96 NR_125713.1 3.0 3.7 5.6 2.2 5.2 3.5 OTU 8 Caloranaerobacter ferrireducens 97 NR_125860.1 1.8 3.2 1.7 2.6 1.6 2.2 OTU 9 Lutispora thermophila 96 NR_041236.1 3.2 6.4 2.3 Desulfotomaculum OTU 10 NR_119247.1 thermosapovorans 96 4.7 5.3 5.6 6.2 4.9 5.7 OTU 12 Desulfonisporus sp. AAN04 97 NR_026497.1 1.0 1.3 1.4 2.8 OTU 15 Halothermothrix orenii 91 NR_074915.1 OTU 22 Tepidimicrobium xylanilyticum 89 NR_116042.1 1.8 OTU 24 Irregularibacter muris 97 NR_144613.1 1.3 1.4 OTU 34 Desulfitibacter alkalitolerans 89 NR_042962.1 OTU 35 Desulfonisporus sp. AAN04 94 AB436739.1 OTU 40 Tepidimicrobium xylanilyticum 96 NR_116042.1 OTU 45 Sporosalibacterium faouarense 95 NR_116364.1 OTU 47 Desulfotomaculum peckii 89 NR_109724.1 3.6 2.4 OTU 51 Gracilibacter thermotolerans 91 NR_115693.1 1.0 OTU 64 Brassicibacter thermophilus 99 NR_137216.1 OTU 69 Anaerovirgula multivorans 99 NR_041291.1 OTU 87 Defluvitalea saccharophila 99 NR_117912.1 OTU 90 Carboxydothermus siderophilus 87 NR_044272.1 OTU 110 Halothermothrix orenii 91 NR_074915.1 1.7 OTU 115 Gelria glutamica 91 NR_041819.1 1.1 OTU 121 Tindallia californiensis 91 NR_025162.1 1.7

OTU 149 Clostridium caminithermale 96 NR_041887.1 3.7 2.4 3.3 2.4 4.6 1.3

160

positive control Day 7 Day 53 Closest Neighbour (BLAST ID) % ID Accession number Combined Rep 1 Rep 2 Rep 3

Bacilli

OTU 21 Bacillus boroniphilus 96 NR_041275.1 OTU 28 Bacillus hisashii 98 NR_144578.1 OTU 31 Geobacillus thermantarcticus 95 NR_117156.1 OTU 61 Anoxybacillus calidus 98 NR_125532.1 OTU 88 Bacillus benzoevorans 97 NR_044828.1 OTU 920 Bacillus hisashii 97 NR_144578.1

Clostridia

OTU 2 Proteiniborus ethanooligenes 88 NR_125623.1 4.3 10.1 3.8 6.9 OTU 4 Clostridilisalibacter sp. 98 KC555195 2.4 5.1 8.8 5.8 OTU 5 Desulfotomaculum peckii 94 NR_109724.1 6.0 6.9 7.8 6.2 OTU 7 Clostridium halophilum 96 NR_125713.1 1.4 1.8 3.8 OTU 8 Caloranaerobacter ferrireducens 97 NR_125860.1 1.2 4.4 2.7 OTU 9 Lutispora thermophila 96 NR_041236.1 5.4 3.5 Desulfotomaculum OTU 10 NR_119247.1 thermosapovorans 96 3.1 5.4 3.9 3.9 OTU 12 Desulfonisporus sp. AAN04 97 NR_026497.1 1.1 1.7 2.0 OTU 15 Halothermothrix orenii 91 NR_074915.1 13.0 OTU 22 Tepidimicrobium xylanilyticum 89 NR_116042.1 1.3 1.4 OTU 24 Irregularibacter muris 97 NR_144613.1 OTU 34 Desulfitibacter alkalitolerans 89 NR_042962.1 OTU 35 Desulfonisporus sp. AAN04 94 AB436739.1 2.7 OTU 40 Tepidimicrobium xylanilyticum 96 NR_116042.1 OTU 45 Sporosalibacterium faouarense 95 NR_116364.1 1.8 1.6 OTU 47 Desulfotomaculum peckii 89 NR_109724.1 OTU 51 Gracilibacter thermotolerans 91 NR_115693.1 1.9 OTU 64 Brassicibacter thermophilus 99 NR_137216.1 OTU 69 Anaerovirgula multivorans 99 NR_041291.1 OTU 87 Defluvitalea saccharophila 99 NR_117912.1 OTU 90 Carboxydothermus siderophilus 87 NR_044272.1 OTU 110 Halothermothrix orenii 91 NR_074915.1 OTU 115 Gelria glutamica 91 NR_041819.1 1.1 OTU 121 Tindallia californiensis 91 NR_025162.1 OTU 149 Clostridium caminithermale 96 NR_041887.1 1.7 1.5 1.9

161 Table S3.2: Thermospore OTUs enriched after a 9 day pre-freezing treatment at -80°C, -20°C, or unfrozen (positive control) in the repeat experiment after 7 days of incubation at 50°C. Relative abundances of each OTU are shown for each replicate. Where numbers are absent, the relative abundance was <1%.

Clostest Relative % Accession -80°C -20°C positive control (BLAST ID) ID number Rep Rep Rep Rep Rep Rep Rep Rep Rep Bacilli 1 2 3 1 2 3 1 2 3 Tepidibacillus OTU 19 99 NR_125657.1 fermentans 5.6 2.5 OTU 20 Bacillus hisashii 98 NR_144578.1 1.6 1.6 1.9 1.5

Clostridia

Proteiniborus OTU 2 98 NR_044093.1 ethanoligenes 9.0 21.1 12.4 17.9 2.1 9.7 18.2 5.4 Clostridium OTU 6 96 NR_125713.1 halophilum 10.0 6.1 5.5 2.5 8.6 4.5 2.5 10.2 Caloranaerobacter OTU 8 97 NR_135860.1 ferrireducens 8.7 3.8 3.6 4.1 2.9 2.7 3.7 3.0 4.3 Desulfotomaculum OTU 9 96 NR_119247.1 thermosapovorans 2.5 11 7.6 5.5 11.3 3.8 5.3 Carboxydothermus OTU 15 87 NR_044272.1 siderophilus 11.5 Desulfotomaculum OTU 21 94 NR_109724.1 peckii 4.7 1.7 1.9 1.1 1.1 Brassicibacter OTU 24 99 NR_137216.1 thermophiles 3.7 Halothermothrix OTU 29 91 NR_074915.1 orenii 1.6 3.0 Desulfonispora OTU 30 91 NR_026497.1 thiosulfatigenes 1.8 4.1 1.0 Desulfitispora OTU 33 88 NR_116807.1 alkaliphile 1.6 Tepidimicrobium OTU 40 96 NR_116042.1 xylanilyticum 1.2 1.1 Clostridium OTU 75 96 NR_041887.1 caminithermale 2.1 1.2 5.1 3.2 2.9 2.6 3.9 4.1 2.4 Defluviitalea OTU 84 99 NR_117912.1 Saccharophila 1.0 1.1 1.2 1.1 1.4 1.1 Desulfotomaculum OTU 188 100 NR_042044.1 geothermicum 1.4

162 Table S4.1: Taxa identification of 41 thermospore OTUs based on BLAST 16S rRNA amplicon sequence similarity. BLAST ID % ID accession Sites number OTU_2 Symbiobacterium ostreiconchae 100 NR_134208.1 SSE FB BBN OTU_4 Therminicola carboxydiphila 99 NR_043010.1 FB OTU_5 Desulfotomaculum tongense 93 NR_133738.1 SSE OTU_12 Thermicanus aegyptius 100 NR_025355.1 SSW SSC OTU_14 Vulcanibacillus modesticaldus 98 NR_042421.1 SSW OTU_17 Bacillus kyonggiensis 97 NR_132682.1 FB OTU_18 Desulfotomaculum geothermicum 95 NR_042044.1 FB OTU_21 Aneurinibacillus thermoaerophilus 95 NR_112216.1 SSE OTU_34 Caloranaerobacter ferrireducens 97 NR_135860.1 LS SV OTU_44 Tepidibacillus fermentans 99 NR_125657.1 FB OTU_46 Bacillus thermoamylovorans 97 NR_117028.1 SSE DS PI OTU_55 Caldibacillus debilis 99 NR_029016.1 FB OTU_74 Desulfotomaculum reducens 96 NR_102770.1 FB OTU_89 Clostridium halophilum 96 NR_125713.1 LS SV OTU_121 Proteiniborus ethanoligenes 98 NR_044093.1 SV OTU_136 Desulfotomaculum peckii 94 NR_109724.1 BBN SV

Table continued on the next page.

163

BLAST ID % ID accession Sites number OTU_145 Proteinivorax tanatarense 88 NR_125623.1 SV OTU_168 Desulfotomaculum thermosapovorans 96 NR_119247.1 LS OTU_196 Halothermothrix orenii 91 NR_074915.1 SV OTU_212 Alkaliphilus crotonatoxidans 91 NR_041892.1 LS OTU_216 Clostridium thermosuccinogenes 99 NR_119284.1 FB OTU_219 Lutispora thermophila 96 NR_041236.1 SV OTU_220 Defluviitalea saccharophila 99 NR_117912.1 LS OTU_244 Desulfotomaculum thermosapovorans 96 NR_119247.1 SSW OTU_339 Tepidimicrobium xylanilyticum 96 NR_116042.1 BBN OTU_359 Desulfonispora thiosulfatigenes 89 NR_026497.1 SV OTU_411 Sporosalibacterium faouarense 95 NR_116364.1 SV OTU_412 Desulfonispora thiosulfatigenes 91 NR_026497.1 SV OTU_469 Clostridium caminithermale 97 NR_041887.1 LS SV OTU_514 Symbiobacterium ostreiconchae 92 NR_134208.1 SSW OTU_522 Bacillus thermoamylovorans 99 NR_117028.1 FB OTU_532 Gracilibacter thermotolerans 91 NR_115693.1 SV OTU_562 Tepidimicrobium xylanilyticum 89 NR_116042.1 SV OTU_610 Clostridium cochlearium 100 NR_113026.1 BBN OTU_642 Desulfotomaculum geothermicum 95 NR_042044.1 FB OTU_748 Alkaliphilus crotonatoxidans 100 NR_041892.1 BBN OTU_1146 Bacillus oceanisediminis 98 NR_118440.1 FB OTU_1236 Desulfotomaculum thermosapovorans 97 NR_119247.1 BBN OTU_1535 Gelria glutamica 91 NR_041819.1 SV OTU_2250 Thermoactinomyces intermedius 96 NR_041760.1 FB OTU_4497 Bacillus hisashii 97 NR_144578.1 SSE

164 Table S6.1: Relative abundances (%) of Atribacteria and Chloroflexi OTUs that are >1% of the total amplicon library in at least one replicate of incubations of sediment from 206 mbsf and 254 mbsf but not more than 2 reads in any control (sediment-free, medium-only control incubations and DNA extraction negatives). Table is shown from page 165 to 167.

a) Atribacteria 50°C 60°C 70°C

Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Day Day Day Day Day Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 21 Day 0 21 Day 0 21 0 21

OTU_36 0 0 0 7.3049 0 10.9716 0 0.1596 2.8194 4.0471 0 0.0037

206 mbsf OTU_41 0.0034 0 0 0 0 0 0 2.3608 0 0 0 0

OTU_158 0 0 0 0 0 0 0.0026 2.7366 0 0 0.0049 0

OTU_36 0 2.4172 0.0057 0 0 0.0047 0 0 0 0 0 0

254 mbsf OTU_41 3.1036 0 1.2688 0 0 4.5473 0 0 0 2.3178 0 1.5419

OTU_158 0.0369 0 2.2707 0 0 2.79 0.0025 0 0 0.0577 0 0.0216

OTU_36 0 0 0 0 0 0 0 0 0.0021 0 0 0 0 0 0 0 0 0.0026

Control OTU_41 0 0 0 0 0 0 0 0 0 0 0.0021 0 0 0 0 0 0 0.0026

OTU_158 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Day 0 Day 21

DNA OTU_36 0 0

extraction OTU_41 0 0 negatives OTU_158 0 0

b) 50°C 60°C 70°C Bacteroidetes Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Day Day Day Day Day Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 21 Day 0 21 Day 0 21 0 21

OTU_50 0 0 0.1658 5.83 0 2.88 0.0026 1.4331 1.9766 0 0 0

OTU_69 0 0 0 0.013 0 0 0 4.4956 0 0 0 0 206 mbsf OTU_195 0 0 0 0 0 0 0 0 0 0 0 0

OTU_243 0 0 0 1.8523 0 0.8779 0 0 0 0.0035 0 0

165 50°C 60°C 70°C

OTU_165 0 0 0.0018 0.8893 0 1.8273 0 0 0 0 0 0

OTU_183 0 0 0 0 0 0 0 0 0 0 0 0

OTU_202 0 0 0 0 0 0 0 0 0 0 0 0

OTU_135 0 0 0 0 0 0 0 1.4032 0 0 0 0

OTU_489 0 0 0 0 0 1.1599 0 0.0665 0 0 0 0

OTU_273 0 0 0 0 0 0 0 0 0 0 0 0

OTU_277 0 0 0 0 0 0.004 0 1.0507 0 0 0 0

OTU_50 0 1.8958 0 0 0 0 0.8144 1.0628 0 0 0 0.0027

OTU_69 0 0 1.0162 0 0 0 0 0 0 0 0 0

OTU_195 0 0 2.3417 0.009 0 0 0 0 0 0 0 0

OTU_243 0 0 0.0312 0 0 0 0 0 0 0 0 0.046

OTU_165 0 0 0 0 0 0 0 0 0 0 0 0

254 mbsf OTU_183 0 0 1.8081 0 0 0 0 0 0 0 0 0

OTU_202 0 0 0 0.003 0 0 0 1.6772 0 0 0 0

OTU_135 0 0 0.8146 1.3729 0 0 0 0 0 0 0 0

OTU_489 0 0 0.0426 0 0 0 0 0 0 0 0 0

OTU_273 0 1.1179 0 0 0 0 0 0 0 0 0 0

OTU_277 0 0 0.3179 0 0 0 0 0.4979 0 0 0 0

OTU_50 0 0 0 0 0 0 0 0 0 0 0 0.0057 0 0 0 0 0 0

OTU_69 0 0 0 0 0 0 0 0 0 0.002 0.0021 0 0 0 0.0027 0 0 0

OTU_195 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

OTU_243 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Control OTU_165 0 0 0.0033 0 0 0 0 0 0 0.002 0 0 0 0 0 0 0 0

OTU_183 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

OTU_202 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

OTU_135 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

166 50°C 60°C 70°C

Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Day Day Day Day 0 Day 21 Day 0 Day 0 Day 21 Day 0 Day 0 Day 21 Day 0 Day 0 21 Day 0 Day 0 21 Day 0 Day 0 21 Day 0

OTU_489 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

OTU_273 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

OTU_277 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Day 0 Day 21

DNA OTU_36 0 0 extraction OTU_41 0 0 negatives OTU_158 0 0

167 Supplementary Figures

Read Number 1 10 100 1000 10000 100000 1000000

-80_A1_t0 6067 -80_A1_t7 1387 -80_A1_t53 1880 -80_A2_t0 2657 -80_A2_t7 1747 -80_A2_t53 2341 -80_A3_t0 4531 -80_A3_t7 2141 -80_A3_t53 5642 -20_A1_t0 4900 -20_A1_t7 2631 -20_A1_t53 3932 -20_A2_t0 2273 -20_A2_t7 1729 -20_A2_t53 2886 -20_A3_t0 4013 -20_A3_t7 3149 -20_A3_t53 21806 +4_A1_t53 6426 +4_A2_t53 3353 +4_A3_t53 4588 +4_A1_A2_A3_t53 2678 +4_A1_A2_A3_t0 2040 +4_A1_A2_A3_t7 5336

-80_B1_B2_B3_t0 55487 -80_B1_t7 67675 -80_B2_t7 62460 -80_B3_t7 60900 -20_B1_B2_B3_t0 46303 -20_B1_t7 73542 -20_B2_t7 53103 -20_B3_t7 79408 +4_B1_B2_B3_t0 37589 +4_B1_t7 112709 +4_B2_t7 89827 +4_B3_t7 18915 -80_before_pasteurization 71656 -20_before_pasteurization 60650 +4_before_pasteurization 34563

168 Read Number 1 10 100 1000 10000 100000 1000000

SSW_0cm_R1_t0 6167 SSW_0cm_R1_t7 6505 SSW_0cm_R1_t14 1763 SSW_0cm_R2_t0 15213 SSW_0cm_R2_t7 13052 SSW_0cm_R2_t14 7934 SSW_0cm_R3_t0 15242 SSW_0cm_R3_t7 6928 SSW_0cm_R3_t14 5321 SSW_0cm_R4_t0 14954 SSW_0cm_R4_t7 6209 SSW_0cm_R4_t14 3550 SSW_0cm_R5_t0 16295 SSW_0cm_R5_t7 12292 SSW_0cm_R5_t14 7690 SSW_0cm_R6_t0 9985 SSW_0cm_R6_t7 6696 SSW_0cm_R6_t14 13833 SSW_260cm_R1_t0 18995 SSW_260cm_R1_t7 16896 SSW_260cm_R1_t14 5648 SSW_260cm_R2_t0 10718 SSW_260cm_R2_t7 24416 SSW_260cm_R2_t14 10233 SSW_260cm_R3_t0 14019 SSW_260cm_R3_t7 1557 SSW_260cm_R3_t14 16361 SSW_550cm_R1_t0 9517 SSW_550cm_R1_t7 16028 SSW_550cm_R1_t14 6358 SSW_550cm_R2_t0 16870 SSW_550cm_R2_t7 9486 SSW_550cm_R2_t14 8802 SSW_550cm_R3_t0 17287 SSW_550cm_R3_t7 19245 SSW_550cm_R3_t14 6826

SSC_0cm_R1_t0 7735 Chapter 4 Sequecning Samples 4 Sequecning Chapter SSC_0cm_R1_t7 7469 SSC_0cm_R1_t14 16583 SSC_0cm_R2_t0 15011 SSC_0cm_R2_t7 2646 SSC_0cm_R2_t14 16354 SSC_0cm_R3_t0 10794 SSC_0cm_R3_t7 5183 SSC_0cm_R3_t14 13918 SSC_405cm_R1_t0 10293 SSC_405cm_R1_t7 7134 SSC_405cm_R1_t14 16581 SSC_405cm_R2_t0 14480 SSC_405cm_R2_t7 13209 SSC_405cm_R2_t14 25838 SSC_405cm_R3_t0 11497 SSC_405cm_R3_t7 4443 SSC_405cm_R3_t14 19423 SSC_902cm_R1_t0 12800 SSC_902cm_R1_t7 7152 SSC_902cm_R1_t14 44219 SSC_902cm_R2_t0 13648 SSC_902cm_R2_t7 4179 SSC_902cm_R2_t14 8620 SSC_902cm_R3_t0 112436 SSC_902cm_R3_t7 29867 SSC_902cm_R3_t14 17339

169 Read Number 1 10 100 1000 10000 100000 1000000

SSE_0cm_R1_t0 6636 SSE_0cm_R1_t7 25926 SSE_0cm_R1_t14 18191 SSE_0cm_R2_t0 22548 SSE_0cm_R2_t7 12268 SSE_0cm_R2_t14 13664 SSE_0cm_R3_t0 6814 SSE_0cm_R3_t7 2964 SSE_0cm_R3_t14 283773 SSE_400cm_R1_t0 10141 SSE_400cm_R1_t7 13863 SSE_400cm_R1_t14 15110 SSE_400cm_R2_t0 31309 SSE_400cm_R2_t7 14678 SSE_400cm_R2_t14 6997 SSE_400cm_R3_t0 9779 SSE_400cm_R3_t7 3258 SSE_400cm_R3_t14 29805 SSE_690cm_R1_t0 9870 SSE_690cm_R1_t7 15247 SSE_690cm_R1_t14 10828 SSE_690cm_R2_t0 11796 SSE_690cm_R2_t7 9891 SSE_690cm_R2_t14 17380 SSE_690cm_R3_t0 17407 SSE_690cm_R3_t7 12461 SSE_690cm_R3_t14 10225

BBN_t0 7612 BBN_t14 6772

PI_R1_t0 7265 Chapter 4 Sequecning Samples 4 Sequecning Chapter PI_R1_t7 8988 PI_R1_t14 19457 PI_R2_t0 11752 PI_R2_t7 5850 PI_R2_t14 46850 PI_R3_t0 7150 PI_R3_t7 10043 PI_R3_t14 13049

DS_t0 8797 DS_t14 6270

LS_R1_t0 11010 LS_R1_t7 4267 LS_R1_t14 20840 LS_R2_t0 6152 LS_R2_t7 12497 LS_R2_t14 16420 LS_R3_t0 2450 LS_R3_t7 4761 LS_R3_t14 8463

170 Read Number 1 10 100 1000 10000 100000 1000000 10000000

FB_R1_t0 14265 FB_R1_t7 17528 FB_R1_t14 2157989 FB_R2_t0 21443 FB_R2_t7 11938 FB_R2_t14 124008 FB_R3_t0 26432 FB_R3_t7 18356 FB_R3_t14 11068

SV_t0 1942 SV_t7 5170 SV_R1_t53 6305

Chapter 4 Sequecning Samples 4 Sequecning Chapter SV_R2_t53 3305 SV_R3_t53 4480

171 Read Number 0 20000 40000 60000 80000 100000 120000 206mbsf_50°C_R1_t0 29607 206mbsf_50°C_R1_t21 26940 206mbsf_50°C_R2_t0 54881 206mbsf_50°C_R2_t21 23053 206mbsf_60°C_R1_t0 50535 206mbsf_60°C_R1_t21 25174 206mbsf_60°C_R2_t0 38956 206mbsf_60°C_R2_t21 30074 206mbsf_70°C_R1_t0 37135 206mbsf_70°C_R1_t21 28341 206mbsf_70°C_R3_t0 41181 206mbsf_70°C_R3_t21 27365 254mbsf_50°C_R1_t0 59544 254mbsf_50°C_R1_t21 34709 254mbsf_50°C_R2_t0 35231 254mbsf_50°C_R2_t21 33433 254mbsf_60°C_R1_t0 31974 254mbsf_60°C_R1_t21 42795 254mbsf_60°C_R2_t0 40644 254mbsf_60°C_R2_t21 34343 254mbsf_70°C_R1_t0 44898 254mbsf_70°C_R1_t21 36371 254mbsf_70°C_R2_t0 62249 254mbsf_70°C_R2_t21 36968

440mbsf_50°C_R1_t0 35621 440mbsf_50°C_R1_t21 40293 440mbsf_50°C_R2_t0 30795 440mbsf_50°C_R2_t21 41425 440mbsf_50°C_R3_t0 109515

Chapter 6 Sequencing Samples 6 Sequencing Chapter 440mbsf_50°C_R3_t21 60966 440mbsf_60°C_R1_t0 34959 440mbsf_60°C_R1_t21 43126 440mbsf_60°C_R2_t0 26531 440mbsf_60°C_R2_t21 2 440mbsf_60°C_R3_t0 41303 440mbsf_60°C_R3_t21 37429 440mbsf_70°C_R1_t0 28739 440mbsf_70°C_R1_t21 46565 440mbsf_70°C_R2_t0 31547 440mbsf_70°C_R2_t21 41039 440mbsf_70°C_R3_t0 37123 440mbsf_70°C_R3_t21 35631

700mbsf_50°C_R1_t0 37597 700mbsf_50°C_R1_t21 33589 700mbsf_50°C_R2_t0 31861 700mbsf_50°C_R2_t21 37649 700mbsf_50°C_R3_t0 25655 700mbsf_50°C_R3_t21 45604

172 Read Number 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000 700mbsf_60°C_R1_t0 27028 700mbsf_60°C_R1_t21 60378 700mbsf_60°C_R2_t0 43057 700mbsf_60°C_R2_t21 44513 700mbsf_60°C_R3_t0 27887 700mbsf_60°C_R3_t21 38721 700mbsf_70°C_R1_t0 45027 700mbsf_70°C_R1_t21 74293 700mbsf_70°C_R2_t0 34065 700mbsf_70°C_R2_t21 121617 700mbsf_70°C_R3_t0 38943 700mbsf_70°C_R3_t21 63757

865mbsf_50°C_R1_t0 28401 865mbsf_50°C_R1_t21 49402 865mbsf_50°C_R2_t0 32431 865mbsf_50°C_R2_t21 30758 865mbsf_50°C_R3_t0 31733 865mbsf_50°C_R3_t21 36527 865mbsf_60°C_R1_t0 37944 865mbsf_60°C_R1_t21 47411 865mbsf_60°C_R2_t0 30418 865mbsf_60°C_R2_t21 28510 865mbsf_60°C_R3_t0 37475 865mbsf_60°C_R3_t21 52333 865mbsf_70°C_R1_t0 179266 865mbsf_70°C_R1_t21 40484 865mbsf_70°C_R2_t0 80273 865mbsf_70°C_R2_t21 36888 865mbsf_70°C_R3_t0 42465 865mbsf_70°C_R3_t21 37365

Chapter 6 Sequencing Samples Sequencing 6 Chapter Control_50°C_R1_t0 35068 Control_50°C_R1_t21 42397 Control_50°C_R2_t0 30346 Control_50°C_R2_t21 47442 Control_50°C_R3_t0 54176 Control_50°C_R3_t21 25345 Control_60°C_R1_t0 40881 Control_60°C_R1_t21 38973 Control_60°C_R2_t0 47989 Control_60°C_R2_t21 49471 Control_60°C_R3_t0 46718 Control_60°C_R3_t21 35276 Control_70°C_R1_t0 53521 Control_70°C_R1_t21 36801 Control_70°C_R2_t0 37629 Control_70°C_R2_t21 35213 Control_70°C_R3_t0 43526 Control_70°C_R3_t21 37900

ExNeg_t0 32769 ExNeg_t21 32870

173

16 14 12 10 8 6 4 2

Sulfate Sulfate Concentration (mM) 0

Day 0 Day Day0 Day7 Day7 Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7

Day 53 Day Day53 Day53 Day53 Day53 Day53 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 -80°C for 2 days -80°C for 10 days

Figure S3.1: Sulfate concentrations in individual -80°C pre-frozen microcosm replicates over 53 days of incubation at 50°C.

2.5

1.5

0.5

Organic Acid Organic Acid Concentration (mM)

Day 7 Day Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7 Day0

Day53 Day53 Day53 Day53 Day53 Day53 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 -80°C for 2 days -80°C for 10 days Lactate Succinate Propionate Butyrate

174

70 16

60 14

12 50 10 40 8 30 6

20 Sulfate Concentration (mM)

4 Relative Abundance Relative Abundance (%)

10 2

0 0

Day0 Day7 Day0 Day7 Day0 Day7

Day 53 Day Day53 Day53 Replicate 1 Replicate 2 Replicate 3 Bacilii Clostridia Sulfate

175 a)

5 Sulfate Sulfate Conc. (mM)

Day0 Day11 b)

0.8

0.6

0.4

0.2

Organic Acid Organic Acid Conc.(mM) 0

Day 0 Day Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Replicate 6

176 c)

100

20 Relative Abundance Relative Abundance (%)

Day 7 Day Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day0 Day7 Day0 Day7

Day 14 Day Day14 Day14 Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Replicate 6 Acidobacteria Bacteroidetes Chlorobi Chloroflexi Firmicutes Planctomycetes Proteobacteria Unknown <1% d)

100

20 Relative Abundance Relative Abundance (%)

Day 0 Day Day0 Day7 Day7 Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7

Day 14 Day Day14 Day14 Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Replicate 6 OTU_14 OTU_12 OTU_244 OTU_514

Figure S4.1: Sulfate concentration (a), organic acid concentration (b), phylum-level community structure (c) and Firmicutes OTU relative abundance (d) of Scotian Slope West sediment 50°C incubations. a) Bars represent the average of 6 replicates and error bars show standard error. b) Organic acid concentrations of each replicate are plotted individually. c-d) Only those phyla and OTUs >1% relative abundance are shown in the legends.

177 a)

Sulfate Sulfate Conc. (mM) 5

Day0 Day11

1.2

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day0 Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 1 Replicate 3

Figure S4.2: Sulfate (a) and organic acid (b) measurement of media only control incubations for Scotian Slope West incubations. Error bars show standard error.

178 a) b)

25 25

20 20

15 15

10 10 Sulfate Sulfate Conc. (mM) 5 Sulfate Conc. (mM) 5

0 0 0 10 20 30

Incubation Time (Days)

Day0 Day9

Day14 Day28 Replicate 1 Replicate 2 Replicate 3

1.2

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day0 Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

179 d)

100

Relative Abundance Relative Abundance (%) 0

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3

Acidobacteria Aminicenantes Bacteroidetes Chloroflexi Firmicutes Proteobacteria Unknown <1%

100 80 60 40 20

Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3

OTU_12 Figure S4.3: Sulfate concentration (a-b), organic acid concentration (c), phylum-level community structure (d) and Firmicutes OTU relative abundance (e) of Scotian Slope Centre sediment 50°C incubations. a) Bars represent the average of 3 replicates and error bars show standard error. b) Sulfate concentrations in each replicate are plotted individually. c) Organic acid concentrations of each replicate are plotted individually. d-e) Only those phyla and OTUs >1% relative abundance are shown in the legends.

180 a)

Sulfate Sulfate Conc. (mM) 5

Day0 Day9 Day28 Day14 b)

1.2

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day0 Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

181 c)

100

20 Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3

Acidobacteria Actinobacteria Bacteroidetes Chloroflexi Firmicutes Planctomycetes Proteobacteria Unknown <1% d)

100 80 60 40 20

Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3

OTU_2 OTU_5 OTU_46 OTU_21 OTU_4497

182

10 Sulfate Sulfate Conc. (mM) 5

Day 9 Day Day0 Day28 Day14 b)

1.2

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day0 Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate Replicate 1 Replicate 2 Replicate 3

Figure S4.5: Sulfate (a) and organic acid (b) measurement of media only control incubations for Scotian Slope Center and Scotian Slope East incubations. Error bars show standard error.

183 a) b)

25 25

20 20

15 15

10 10 Sulfate Sulfate Conc. (mM) Sulfate Sulfate Conc. (mM) 5 5

0 0 0 10 20 30

Incubation Time (Days)

Day0 Day9

Day14 Day28

Replicate 1 Replicate 2 Replicate 3 c)

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4

Organic Acid Organic Acid Conc.(mM) 0.2

Day0 Day0 Day0 Day0 Day0

Day 28 Day Day17 Day17 Day28 Day17 Day28 Day17 Day28 Day17 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

184 d)

100

Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3

Actinobacteria Bacteroidetes Cyanobacteria Firmicutes Fusobacteria Proteobacteria <1% e)

100

Relative Abundance Relative Abundance (%) 0

Day 0 Day Day0 Day7 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3

OTU_168 OTU_34 OTU_46 OTU_89 OTU_469 OTU_220 OTU_212 <1%

185 a) b)

25 25

20 20

15 15

10 10 Sulfate Sulfate Conc. (mM) Sulfate Sulfate Conc. (mM) 5 5

0 0 0 10 20 30

Incubation Time (Days)

Day0 Day9

Day14 Day28

Replicate 1 Replicate 2 Replicate 3 c)

1.4

1.2

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day 0 Day Day0 Day5 Day9 Day0 Day5 Day9 Day0 Day5 Day9 Day5 Day9 Day0 Day5 Day9

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

186 d)

100

Relative Abundance Relative Abundance (%) 0

Day 7 Day Day0 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3

Acidobacteria Actinobacteria Bacteroidetes Chlorobi Chloroflexi Cyanobacteria Firmicutes Proteobacteria e)

100

Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day 14 Day Day14 Day14 Replicate 1 Replicate 2 Replicate 3

OTU_2 OTU_44 OTU_18 OTU_17 OTU_642 OTU_74 OTU_4 OTU_216 OTU_1146 OTU_2250 OTU_522 OTU_55 <1%

187 a) b)

30 30

25 25

20 20

15 15

10 10 Sulfate Sulfate Conc. (mM) Sulfate Sulfate Cnoc. (mM) 5 5 0 0 0 10 20 30 40

Incubation Time (Days)

Day0 Day9

Day16 Day30 Replicate 1 Replicate 2 Replicate 3 c)

1.2

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day 0 Day Day0 Day0 Day0 Day0

Day 26 Day Day26 Day26 Day26 Day26 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

188 d)

100

20 Relative Abundance Relative Abundance (%)

Day0 Day14 Acidobacteria Actinobacteria Aminicenantes Bacteroidetes Chlamydiae Chlorobi Chloroflexi Cyanobacteria Firmicutes Parcubacteria Proteobacteria Unknown <1% d)

100

Relative Abundance Relative Abundance (%)

Day0 Day14

OTU_46 <1% Figure S4.8: Sulfate concentration (a-b), organic acid concentration (c), phylum-level community structure (d) and Firmicutes OTU relative abundance (e) of Davis Strait sediment 50°C incubations. a) Bars represent the average of 3 replicates and error bars show standard error. b) Sulfate concentrations in each replicate are plotted individually. c) Organic acid concentrations of each replicate are plotted individually. d-e) Bars represent the combined phylum-level and OTU-level abundances of the three replicates. Only those phyla and OTUs >1% relative abundance are shown in the legends.

189 a)

10 Sulfate Sulfate Conc. (mM) 5

Day0 Day9 Day28 Day14 b)

1.6 1.4 1.2 1 0.8 0.6 0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day 0 Day Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

190 c)

100

Relative Abundance Relative Abundance (%) 0

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Acidobacteria Actinobacteria Bacteroidetes Chloroflexi Cyanobacteria Firmicutes Planctomycetes Proteobacteria Unknown <1% d)

100

20 Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3

OTU_46

191 a)

5 Sulfate Sulfate Conc. (mM)

Day0 Day9 Day28 Day17 b)

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2

Organic Acid Organic Acid Conc.(mM)

Day0 Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate Replicate 1 Replicate 2 Replicate 3

Figure S4.10: Sulfate (a) and organic acid (b) measurement of media only control incubations for Labrador Shelf, Frobisher Bay, and Pond Inlet incubations. Error bars show standard error.

192 a) b)

30 35 30 25 25 20 20 15 15

10 10 Sulfate Sulfate Conc. (mM) Sulfate Sulfate Cnoc. (mM) 5 5

0 0 0 10 20 30 40

Inucbation Time (Days)

Day0 Day9

Day16 Day30 Replicate 1 Replicate 2 Replicate 3 c)

1.2

0.8

0.6

0.4

Organic Acid Organic Acid Conc.(mM) 0.2

Day 0 Day Day0 Day0 Day0 Day0

Day26 Day26 Day26 Day26 Day26 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

193 d)

100 80 60 40 20

Relative Abundance Relative Abundance (%)

Day0 Day14 Acidobacteria Actinobacteria Bacteroidetes Chlorobi Chloroflexi Cyanobacteria Firmicutes Planctomycetes e)

100

20 Relative Abundance Relative Abundance (%)

Day0 Day14 OTU_2 OTU_642 OTU_46 OTU_136 OTU_339 OTU_610 OTU_1236 OTU_748 <1% Figure S4.11: Sulfate concentration (a-b), organic acid concentration (c), phylum-level community structure (d) and Firmicutes OTU relative abundance (e) of Baffin Bay North sediment 50°C incubations. a) Bars represent the average of 3 replicates and error bars show standard error. b) Sulfate concentrations of each replicate are plotted individually. c) Organic acid concentrations of each replicate are plotted individually. d-e) The bars represent the combined relative abundance at the phylum level (d) or the OTU level (e) of the three replicates. Additionally, the sum of relative abundances of OTUs present less than 1% was 2.4% and is shown in (e). Only those phyla and OTUs >1% relative abundance are shown in the legends.

194 a)

Sulfate Sulfate Cnoc. (mM) 5

Day0 Day9 Day26 Day16 b)

1.2 1 0.8 0.6 0.4 0.2

Organic Acid Organic Acid Conc.(mM)

Day0 Day0 Day0 Day0 Day0

Day26 Day26 Day26 Day26 Day26 Formate Lactate Succinate Propionate Butyrate Replicate 1 Replicate 2 Replicate 3

Figure S4.12: Sulfate (a) and organic acid (b) measurement of media only control incubations for David Strait and North Baffin Bay incubations. Error bars show standard error.

195 a)

Sulfate Sulfate Conc. (mM) 5

Day0 Day7 Day53 b)

2.5

1.5

0.5 Organic Acid Organic Acid Conc.(mM)

Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7

Day53 Day53 Day53 Day53 Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

196 c)

100 80 60 40 20

Day0 Day7

Relative Abundance Relative Abundance (%)

Day53 Day53 Day53 Combined Replicates 1, 2, and 3 replicates separated

Acidobacteria Actinobacteria Atribacteria Bacteroidetes Chlorobi Chloroflexi Cyanobacteria Firmicutes Gracilibacteria Latescibacteria Marinimicrobia- Proteobacteria d)

100 80 60 40 20

Relative Abundance Relative Abundance (%)

Day0 Day7

Day53 Day53 Day53 Combined Replicates 1, 2, and replicates 3 separated

OTU_34 OTU_196 OTU_121 OTU_89 OTU_145 OTU_469 OTU_136 OTU_244 OTU_219 OTU_359 OTU_412 OTU_532 Figure S4.13: Sulfate concentration (a), organic acid concentration (b), phylum-level community structure (c) and Firmicutes OTU relative abundance (d) of Svalbard sediment 50°C incubations. These incubations are the same incubations discussed in Chapter 3 as positive controls. a) Bars represent the average of 3 replicates and error bars show standard error. b) Organic acid concentrations of each replicate are plotted individually. c-d) Bars represent the combined phylum-level and OTU-level abundances of the three replicates. Only those phyla and OTUs >1% relative abundance are shown in the legends.

197 a)

30 25 20 15 10 5

Sulfate Sulfate Concentration (mM)

Day0 Day0 Day0

Day11

Day11 Day11 0 cm 260 cm 525 cm

1.2 0 cm 1

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day 0 Day Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Replicate 6

198 1.2 260 cm 1

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day0 Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

1.2 525 cm 1

0.8

0.6

0.4

0.2 Organic Acid Organic Acid (mM) Conc.

Day 0 Day Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

199 a)

0 cm 100 80

Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7

260 cm 100 80 60 40 20

Relative Abundance Relative Abundance (%)

Day 7 Day Day0 Day7 Day0 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Bacteroidetes Proteobacteria

525 cm 100 80 60 40 20

Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Bacteroidetes Proteobacteria

200 b)

100 0 cm 80

20 Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Replicate 6

OTU 14 OTU 12

201 a)

Sulfate Sulfate Concentration (mM) 0

Day0 Day9 Day0 Day9 Day0 Day9

Day 14 Day Day14 Day28 Day28 Day14 Day28 0 cm 405 cm 875 cm

0 cm 1.2

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day0 Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

202 405 cm 1.2 1

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day0 Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

875 cm 1.2

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day 0 Day Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

203 a)

0 cm 100 80

Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day 14 Day Day14 Day14 Replicate 1 Replicate 2 Replicate 3

Acidobacteria Aminicenantes Bacteroidetes Chloroflexi Firmicutes Proteobacteria

405 cm 100 80 60 40 20

Relative Abundance Relative Abundance (%)

Day 0 Day Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Bacteroidetes Proteobacteria

875 cm 100 80 60 40 20

Relative Abundnace Relative Abundnace (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Atribacteria Chloroflexi Proteobacteria

204 b)

0 cm 100

20 Realtive Abundance Realtive Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3 OTU 12

205 a)

5 Sulfate Sulfate Concenration (mM)

Day0 Day9 Day0 Day9 Day0 Day9

Day14 Day28 Day14 Day28 Day14 Day28 0 cm 400 cm 655 cm b)

0 cm 1.2

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day0 Day0 Day0 Day0 Day0

Day28 Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

206 400 cm 1.2 1

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day 0 Day Day0 Day0 Day0 Day0

Day 28 Day Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate

Replicate 1 Replicate 2 Replicate 3

655 cm 1.2 1

0.8

0.6

0.4

0.2 Organic Acid Organic Acid Conc.(mM)

Day 0 Day Day0 Day0 Day0 Day0

Day 28 Day Day28 Day28 Day28 Day28 Formate Lactate Succinate Propionate Butyrate Replicate 1 Replicate 2 Replicate 3

Figure S5.5: Sulfate concentration (a) and organic acid concentration (b) of incubations of Scotian Slope East sediment. a) Bars represent the average of 3 replicates and error bars show standard error. b) Histograms of the organic acid concentrations in each replicate are plotted separately.

207 a) 100

0 cm 80

Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day 14 Day Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Acidobacteria Actinobacteria Bacteroidetes Chloroflexi Firmicutes Planctomycetes Proteobacteria Unknown <1%

100 400 cm 80 60 40 20

Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day 14 Day Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Atribacteria Bacteroidetes Chloroflexi Firmicutes Proteobacteria

655 cm 100 80

Relative Abundance Relative Abundance (%)

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3 Bacteroidetes Cyanobacteria Firmicutes Proteobacteria

208 b)

100 0 cm 80 60 40 20

Day0 Day7 Day0 Day7 Day0 Day7

Relative abundance (%)

Day14 Day14 Day14 Replicate 1 Replicate 2 Replicate 3

OTU 2 OTU 5 OTU 46 OTU 21 OTU 4497

400 cm 100 80 60 40 20

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Relative Abundance Relative Abundance (%) Replicate 1 Replicate 2 Replicate 3

OTU 14 OTU 33 OTU 27 8 655 cm 6 4 2

Day0 Day7 Day0 Day7 Day0 Day7

Day14 Day14 Day14 Relative Abundance Relative Abundance (%) Replicate 1 Replicate 2 Replicate 3

OTU 2 Figure S5.6: Phylum-level community structure (a) and OTU-level structure of the Firmicutes community (b) of incubations of Scotian Slope East sediment. a) Only phyla abundant >1% in a replicate are shown in the legend. b) Only OTUs present >1% relative abundance of the total library are shown in the legend.

209 a) 206 mbsf

20 15 10 5

Sulfate Sulfate 0

Day0 Day0 Day0

Concentarion (mM)

Day21 Day21 Day21 50°C 60°C 70°C b) 254 mbsf

20 15 10

5 (mM)

Sulfate Sulfate 0

Concentration

Day0 Day0 Day0

Day21 Day21 Day21 50°C 60°C 70°C

c) 440 mbsf

20 15 10 5

(mM)

Sulfate Sulfate

Concentration

Day0 Day0 Day0

Day21 Day21 Day21 50°C 60°C 70°C d) 700 mbsf

20 15 10

5 (mM)

Sulfate Sulfate 0

Concentration

Day0 Day0 Day0

Day21 Day21 Day21 50°C 60°C 70°C

210 e) 865 mbsf

20 15 10

5 (mM)

Sulfate Sulfate 0

Concentration

Day0 Day0 Day0

Day21 Day21 Day21 50°C 60°C 70°C f) Medium-only Control

20 15 10 5

(mM)

Sulfate Sulfate

Concentration

Day0 Day0 Day0

Day21 Day21 Day21 50°C 60°C 70°C

211 a) 206 mbsf

1.5

0.5(mM)

Organic Acid Organic Acid 0 Concentration Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 50°C 60°C 70°C Lactate Acetate Succinate Propionate Butyrate b) 254 mbsf

1.5 1 0.5 0

Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Organic Acid Organic Acid

50°C 60°C 70°C Concentration (mM) Lactate Acetate Succinate Propionate Butyrate c) 440 mbsf

1.5 1 0.5 0 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21

Organic Acid Organic Acid 50° 60°C 70°C

Concentration (mM) Lactate Acetate Succinate Propionate Butyrate d) 700 mbsf

2.5 2 1.5 1 0.5 0 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21

Organic Acid Organic Acid 50°C 60°C 70°C

Concentration (mM) Lactate Acetate Succinate Propionate Butyrate

212 e) 865 mbsf

2.5 2 1.5 1 0.5

(mM) 0

Day 0 Day 21 Day 0 Day 21 Day 0 Day 21

Organic Acid Organic Acid Concentration 50°C 60°C 70°C

Lactate Acetate Succinate Propionate Butyrate f) Medium-only control

2 1.5 1 0.5 0 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21

Organic Acid Organic Acid 50°C 60°C 70°C

Concentration (mM) Lactate Acetate Succinate Propionate Butyrate

213