Arctic Tundra Soil Bacterial Communities Active at Subzero Temperatures Detected by Stable Isotope Probing

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Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing

Gadkari, Preshita S.; McGuinness, Lora R.; Männistö, Minna K.; et.al. https://scholarship.libraries.rutgers.edu/discovery/delivery/01RUT_INST:ResearchRepository/12643411810004646?l#13643523450004646

Gadkari, P. S., McGuinness, L. R., Männistö, M. K., Kerkhof, L. J., & Häggblom, M. M. (2019). Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing. In FEMS Microbiology Ecology. Rutgers University. https://doi.org/10.7282/t3-9xqb-6e24

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Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing

Preshita S. Gadkari 1, Lora R. McGuinness 2, Minna K. Männistö 3, Lee J, Kerkhof 2, and Max M. Häggblom 1*

1 Department of Biochemistry and Microbiology and 2 Department of Marine and Coastal Sciences School of Environmental and Biological Sciences Rutgers, The State University of New Jersey New Brunswick, NJ, 08901, USA 3 Natural Resources Institute Finland, P.O. Box 16, FI-96301 Rovaniemi, Finland

One sentence summary: Cellobiose stable isotope probing reveals a subzero-active bacterial community in Arctic tundra soils

Keywords: Arctic tundra soil, subzero temperature, stable isotope probing, cellobiose, Candidatus Saccharibacteria, Melioribacteraceae, Verrucomicrobiaceae

*Corresponding author: Department of Biochemistry and Microbiology, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901-8525, USA. E-mail: [email protected]

ABSTRACT Arctic soils store vast amounts of carbon and are subject to intense climate change. While effects of thaw on the composition and activities of Arctic tundra microorganisms has been examined extensively, little is known about the consequences of temperature fluctuations within the subzero range in seasonally frozen or permafrost soils. This study identified tundra soil bacteria active at subzero temperatures using stable isotope probing (SIP). Soils from Kilpisjärvi, Finland were amended with 13C-cellobiose and incubated at 0, -4, and -16°C for up to 40 weeks. 16S rRNA gene sequence analysis of 13C-labelled DNA revealed distinct subzero-active bacterial taxa. The SIP experiments demonstrated that diverse bacteria, including members of Candidatus Saccharibacteria, Melioribacteraceae, Verrucomicrobiaceae, Burkholderiaceae, Acetobacteraceae, Armatimonadaceae, and Planctomycetaceae were capable of synthesizing 13C-DNA at subzero temperatures. Differences in subzero temperature optima were observed, for example with members of Oxalobacteraceae and Rhizobiaceae found to be more active at 0°C than at -4°C or -16°C, whereas Melioribacteriaceae were active at all subzero temperatures tested. Phylogeny of 13C-labelled 16S rRNA genes from the Melioribacteriaceae, Verrucomicrobiaceae, and Candidatus Saccharibacteria suggested that these taxa formed subzero-active clusters closely related to members from other cryo-environments. This study demonstrates that subzero temperatures impact active bacterial community composition and activity which may influence biogeochemical cycles.

INTRODUCTION The Arctic is currently exhibiting dramatic ecosystem changes. Many of these changes will have widespread implications, including alterations in Earth’s carbon budget and climate. Primarily, this is because Arctic soil environments harbor vast stores of organic carbon (C), and decomposition of these C stores is expected to be a significant contributor to atmospheric chemistry and climate (Tarnocai et al. 2009; Natali et al. 2011; Biasi et al. 2014; Jansson and Tas 2014; Schuur et al. 2015). As a consequence of a changing climate, there is a risk that these vulnerable polar environments are shifting from historic carbon sinks to contemporary carbon sources (Oechel et al. 1993; Belshe, Schuur and Bolker 2013; Natali et al. 2015). The shift from a soil carbon sink to source is largely due to microbial responses to increasing temperatures in the tundra (Biasi et al. 2014; McCalley et al. 2014; Voigt et al. 2016, 2017). While there has been extensive research on tundra soil microbial communities active at temperatures above 0°C (Sjögersten et al. 2003; Rinnan et al. 2007; Graham et al. 2012; Natali et al. 2015: 201; Schuur et al. 2015; Stark et al. 2015; Stackhouse et al. 2017), little is known about effects of temperature changes within subzero ranges on active microbial community members. Though understudied, biological activities are well established to occur below 0 °C, as long as cellular membrane fluidity is maintained (Arthur and Watson 1976; Clein and Schimel 1995; Bakermans et al. 2003; McMahon, Wallenstein and Schimel 2009; Öquist et al. 2009; Mykytczuk et al. 2013, 2016; Goordial et al. 2016; Schaefer and Jafarov 2016). Approaches to study life at subzero temperatures have included respirometry, enzymatic capabilities, microbial biomass measurements, RNA studies, as well as (meta)genome and (meta)transcriptome analyses (for reviews see Jansson and Tas 2014; Nikrad, Kerkhof and Häggblom 2016). These techniques have provided evidence that microbes are active at subzero temperatures and that their interactions are complex. However, ascertaining identities of active microorganisms is difficult with these methods because of the caveat of frozen environments—DNA or RNA may remain preserved, despite the lack of metabolic activity, while respirometry demonstrates activity but does not identify the responsible organism. To overcome this challenge, methods such as stable isotope probing (SIP) have been employed to identify and track

3 active community members relevant to understanding overall subzero microbial activities (Tuorto et al. 2014; Nikrad, Kerkhof and Häggblom 2016). In this study, we used SIP methodology to identify the active subzero bacterial community in Arctic tundra soils. We carried out SIP and 16S rRNA gene bacterial sequencing of seasonally frozen Arctic tundra soils from Kilpisjärvi in northwestern Finland using 13C-cellobiose as the carbon substrate at temperatures of -16, -4, and 0°C. Cellobiose is representative of the plant-derived C in the tundra, an important C source in the global C cycle. Active microorganisms at subzero temperatures will assimilate the 13C into their DNA when replicating their genomes, allowing for separation and sequencing of the newly synthesized 13C-DNA. Our hypothesis was that different members of the soil bacterial community would be active at the different subzero temperatures. Climate change is predicted to increase winter temperatures even more than summer temperatures in northern ecosystems (Mikkonen et al. 2015). Precipitation is predicted to increase in Arctic regions but with more in the form of rain instead of snow (Bintanja and Andry 2017). Decrease in the duration, thickness and insulation properties of snow may thus lead to colder soils in a warmer climate (Groffman et al. 2001). This study not only identified bacterial members that were active in processing cellobiose at subzero temperatures but also examined how subzero temperature ranges may structure bacterial community composition.

MATERIALS AND METHODS Study site and tundra soil collection Tundra soils for the laboratory incubations were collected from Malla Nature Preserve, Kilpisjärvi, in northwestern Finland in July of 2012. The sampling site is located at 69° 3’ 48.229” N, 20° 44' 38.791" E, within the subalpine Scandinavian mountain range, with vegetation consisting mainly of shrubs and lichens, and a soil pH of ~5 (Männistö, Tiirola and Häggblom 2007; Männistö, Tiirola and Häggblom 2009; Männistö et al. 2013). The mean annual air temperature is -2.2°C, with the soil temperature fluctuating from 0°C to -25°C in the winter (Männistö, Tiirola and Häggblom 2007; Männistö et al. 2013; Kumar et al. 2017). The growing season is only 100 days long and the soils are subject to subzero temperatures for the majority of the year

(Männistö et al. 2013). For this study, the soils were collected with a steel corer (rinsed between samples in the field) to approximately 5 cm depth in the organic layer and then placed in a plastic bag. After sampling, soils were sieved and stored in the laboratory at 4°C for 22 months to deplete them of labile carbon prior to initiating the SIP experiments.

Subzero SIP Incubation Setup A set of triplicate tundra soil incubations (0.3 g) were established in 2 mL microcentrifuge tubes with cellobiose as the carbon source to represent plant-derived organic matter. Individual soil incubations were amended with either 13C- or 12C- cellobiose (1 mg C/per gram soil C added in a total volume of 100 µL water per replicate) and incubated at temperatures of 0°C, -4°C, and -16°C. All incubations were monitored with an thermometer or temperature logger placed in a similar microcentrifuge tube stored next to the soil incubations, where daily temperature logs were maintained. The temperatures varied briefly, maintaining averages of -15.8, -4.0, and -0.4°C over the course of up to 40 weeks. We screened for the earliest time points for detectable incorporation of the 13C substrate into DNA at 0, 2, 5, 8, and 40 weeks, after which the triplicate incubations for each time point were sacrificed for DNA extraction and downstream analysis.

Soil DNA Extraction and Separation DNA was extracted using a hexadecyltrimethylammonium bromide (CTAB) — phenol-chloroform extraction method (Griffiths et al. 2000; Männistö et al. 2013). Briefly, soils were extracted with equal volumes of 5% CTAB and phenol-chloroform isoamyl alcohol (25:24:1) by bead-beating with two 3 mm glass beads on a bench-top vortexer for 3 minutes. The soil was pelleted by centrifugation and the aqueous phase was then extracted with an equal part chloroform isoamyl alcohol (24:1). The DNA was precipitated by adding 2X volume of 30% w/v polyethylene glycol (8000 MW) and incubating at 4°C for 45 minutes. This DNA pellet was rinsed with cold 100% ethanol at 4°C, followed by a 70% ethanol wash. The DNA was quantified on a 2% agarose gel and ~100 ng of this DNA sample was amended with 100 ng of archaeal 13C-labeled

5 carrier DNA from Halobacterium salinarium and 10 µg of ethidium bromide to allow for visualization of DNA bands under UV light (Gallagher et al. 2005; Tuorto et al. 2014). 12C-(“light”) and 13C-(“heavy”) DNA bands were separated by cesium chloride (CsCl) isopycnal ultracentrifugation at 225,000 x g for 48 hours and the DNA bands were collected by pipette.

Initial pre-screening for bacterial cellobiose assimilation Verification of bacterial 13C-incorporation involved amplifying 16S rRNA genes from the 13C-DNA bands using the bacterial specific 16S rRNA primers 27Forward (5'- AGAGTTTGATCCTGGC TCAG-3') and 1100Reverse (5'-AGGGTTGCGCTCGTTG-3'). PCR amplification was done as follows: initial denaturation for 2 minutes at 94 °C; followed by 27 cycles of 30 seconds at 94 °C, 40 seconds at 53 °C, and 1 minute at 72 °C; and a final elongation for 5 minutes at 72 °C. Unambiguous 13C-DNA synthesis was assessed by demonstrating positive rRNA gene amplification in the 13C-DNA bands of 13C-cellobiose amended incubations while observing no rRNA gene amplicons from the 13C-DNA carrier bands of 12C-cellobiose amended incubations. This critical quality assurance step assures that there was no contamination or shearing from the top band (12C-DNA) into the 13C-DNA carrier band. The 16S rRNA gene amplicons from both the 12C-DNA and 13C-DNA carrier bands were then digested with MspI and screened by terminal restriction fragment length polymorphism (TRFLP) for bacterial community analysis (Tuorto et al. 2014). Additionally, we screened for the earliest time points for detectable incorporation of the 13 C substrate into DNA at T0, 2, 5, 8, and 40 weeks. The incubations at 0 and -4˚C only showed 13C incorporation after 5-weeks, while the -16˚C incubation had no 13C assimilation at 5 or 8 weeks and only demonstrated unambiguous 13C incorporation after 40 weeks of incubation (Supplemental Figures S1-3).

16S rRNA Gene Amplicon Sequencing The soil incubations demonstrating 13C-DNA synthesis were subsequently selected for MiSeq sequencing by MRDNA (Shallowater, Texas, USA) using the 16S rRNA gene V4 region. 16S rRNA gene amplicons generated with 27F/1100R were re-

6 amplified using barcoded 341F (5'-CCTACGGGNGGCWGCAG-3') - 785R (5'- GACTACHVGGGTATCTAATCC-3') primers (Klindworth et al. 2013) using the following conditions: initial denaturation for 3 minutes at 94°C; followed by 28 cycles of 30 seconds at 94°C C, 40 seconds at 53°C, and 1 minute at 72°C; and a final elongation for 5 minutes at 72°C. PCR products were checked by 2% agarose gel electrophoresis, purified using Ampure XP beads (Beckman Coulter), and sequenced on the Illumina amplicon sequencing platform. Data analysis utilized MRDNA’s proprietary analysis pipeline (MRDNA, Shallowater, Texas, USA). The number of reads passing QA/QC (sequences less than 150 bp discarded along with ambiguous base calls, chimeric sequences removed) are listed in Table S1. Operational taxonomic units (OTUs) were taxonomically classified using BLASTn against the GreenGenes, RDPII, and NCBI 16S rRNA gene databases. Similarity criteria for classification at different phylogenetic levels were as follows: >97% species, 95-97% Genus, 90-95% Family, 80-85% Class, and 77- 80% Phylum. The sequence data are included in Supplemental Data.

Statistical and Phylogenetic Analyses Data were imported into ClustVis using R packages including pcaMethods (R package version 0.7.7) for Principal Component Analysis (PCA) with unit variance scaling, Singular Value Decomposition (SVD), and imputation to calculate principal components (Metsalu and Vilo 2015). Heat maps to compare relative abundances of bacterial populations were also generated with ClustVis using ggplot2 heatmap (R package version 0.7.7), unit variance scaling, hierarchical clustering, correlation distance, and average linkage (Metsalu and Vilo 2015). Representative OTUs were used for the phylogenetic analyzes. Alignments for the 16S rRNA genes were built using Molecular Evolutionary Genetics Analysis version 7.0 (Kumar, Stecher and Tamura 2016) with representative closest matches identified by Blast and with 16S rRNA genes from similar polar environments (e.g., Arctic soil, lake water, etc.). Sequences were unambiguously aligned using ClustalW and evolutionary history was inferred in MEGA7 by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei 1993). All positions containing gaps and missing data were eliminated. Initial tree(s) for the heuristic search were obtained

7 automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The bootstrap consensus tree inferred from 500 replicates represents the evolutionary history of the taxa analyzed, and branches corresponding to partitions reproduced in greater than 50% bootstrap replicates are listed (Felsenstein 1985; Kumar, Stecher and Tamura 2016). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches.

RESULTS Resident and Subzero-Active Tundra Soil Bacterial Community The dominant bacterial community of the tundra heath soil (Initial community determined from T0 samples) was comprised largely of members of the phyla Proteobacteria, Actinobacteria, Acidobacteria, Verrucomicrobia, Bacteroidetes, and Planctomycetes (Figure 1; in order of abundance). Members of Chloroflexi, Fusobacteria, Firmicutes, Cyanobacteria, Chlamydia, Ignavibacteria, Candidatus Saccharibacteria, and Armatimonadetes were also detected at lower abundance (Figure

1). The most abundant bacterial families at T0 were mostly comprised of Nitrosomonadaceae, Acidobacteriaceae, Thermomonosporaceae, Sinobacteraceae, Acidimicrobiaceae, Hyphomicrobiaceae, Holophagaceae, and Chitinophagaceae (in order of abundance). The soil at this site is frozen for approximately 8 months of the year (Männistö et al. 2013) and it is thus of interest to elucidate which members of this soil bacterial community are potentially active at temperatures of 0 °C and below. We therefore used a SIP approach to determine which members of the soil bacterial community would assimilate 13C-cellobiose at temperatures of -16, -4, and 0°C during incubation of 5 to 40 weeks. Overall, diverse members of the bacterial community were active and replicated their genomes at the different subzero temperatures. However, the subzero active community was distinctly different from that of the resident community (Figure 1). Bacteria active at subzero temperatures included members of the phyla Ignavibacteriae, Proteobacteria, Armatimonadetes, Actinobacteria, Verrucomicrobia, Candidatus

Saccharibacteria, Chloroflexi, Cyanobacteria, Firmicutes, Deinoccocus Thermus, Candidatus Brocadiaceae, and Planctomycetes. In contrast, members of the

Bacteroidetes, Chlamydia, and Fusobacteria that we detected in the T0 community were not detected in the 13C-DNA fractions at 5 or 40 weeks and were determined to be inactive on 13C-cellobiose. Furthermore, although abundant in the initial soil, members of the Actinobacteria, Acidobacteria, Bacteroidetes, and Chloroflexi were not detected at high abundance in the community that assimilated the 13C substrate. At a family level analysis, bacteria active at 0°C included members of Candidatus Saccharibacteria, Melioribacteriaceae, Armatimonadaceae, Thermacae, Desulfobacteraceae, Nocardiaeae, Oxalobacteraceae, and Prochlorococceae (Figure 1). Bacteria active at -4°C included members of the Rubrobacteraceae, Candidatus Saccharibacteria, and Oxalobacteraceae. Of particular interest was the detection of bacteria assimilating 13C-cellobiose at -16°C, including members of the Candidatus Saccharibacteria, Melioribacteriaceae, Candidatus Brocadiaceae, Pelobacteraaceae, Acetobacteraceae, Armatimonadaceae, Desulfobacteraceae, Anaerolineae, Verrucomicrobiaceae, and Planctomycetaceae. PCA demonstrated clustering of the active bacterial communities based on temperature, with the soil communities incubated at 0 and -4°C clustering closer to each other than those incubated at -16°C (Figure 2), and all clearly separated from the initial community. Several members of the resident bacterial families that were detected in the initial soil sample were apparently unable or limited in their ability to assimilate cellobiose at subzero temperatures. These included members of the Holophagaceae, Acidobacteriaceae, Chitinophagaceae, Polyangiaceae, Thermoanaerobacteraecae, Bradyrhizobiaceae, Fusobacteriaceae, Connexibacteriaceae, Ktedonobacteraceae, Hyphomicrobiaceae, Thermomonosporaceae, Solirubrobacteraceae, Sphingobacteraceae, Spartobacteria, Sinobacteraceae, Rhabdochlamydiaceae, Chlamydiaceae, Verrucomicrobia subdivison 3, and Bacteroidaceae (Figure 1). A heat map displaying the relative abundance of each bacterial family within each incubation is presented in Figure 3. From this analysis, it can be discerned that there is a strikingly high abundance of bacterial members detected in the T0 community that were not found to be active on 13C-cellobiose at <0 oC. In the 13C-cellobiose

9 amended soil incubated at 0°C, members of Candidatus Saccharibacteria were detected, along with members of Armatimonadetes, Oxalobacteraceae, Chromobacteraceae, Melioribacteraceae, Planctomycetaceae, and Verrucomicrobiaceae. Interestingly, Candidatus Brocadiaceae were most abundant at 0°C, while members of the Planctomycetaceae were dominant at 0°C and -16°C, but not as abundant at -4°C. At -4°C, members of Rhizobiaceae, were more abundant than at any other subzero temperature. In the -16°C soil incubations, members of Verrucomicrobiacaeae, Acetobacteraceae, Candidatus Saccharibacteria, Melioribacteraceae, Planctomycetaceae, and Burkholderiaceae were dominant. In the - 4°C and 0°C incubations, members of Candidatus Saccharibacteria were also detected at high relative abundances. Similarly, members of Phycisphaeraceae, were more prevalent in these -4°C and 0°C incubations. To further compare ecological patterns of bacterial families that were detected to be metabolically active in frozen soils, the diversity and species evenness was calculated for all soil cold incubations and SIP experiments. These indices were compared to that of the initial soil bacterial community in Table S2. Both diversity and evenness of the bacterial communities that were able to assimilate 13C-cellobiose at subzero temperatures was lower than that of the initial soil bacterial community.

Novel Cryo-active Bacterial Clades Phylogenetic analysis of subzero active community members, especially those of the phyla Candidatus Saccharibacteria, Verrucomicrobia and Ignavibacteria were carried out to investigate the relationships of the cryo-active OTUs of these phyla with those of isolates and uncultured bacterial phylotypes (Figures 4-6; Table S3). Within the Candidatus Saccharibacteria, the four representative subzero-active OTUs were most closely related to sequences detected in other polar or cold environments, including Svalbard ice cores, Antarctic soils, Arctic soils, thawing Canadian permafrost soils, and Chilean desert soils (Figure 4). While representatives of Candidatus Saccharibacteria have been detected in diverse environments, ranging from human oral cavities to marine sponges, the OTUs representing subzero active members of Candidatus Saccharibacteria were most closely related to representatives from cold environments.

Within the Verrucomicrobia, four representative subzero active OTUs were assessed (Figure 5). Three of the OTUs clustered closely together and were most closely related to Opitutus clones from limnopolar and meromictic lakes in Antarctica and France, respectively. The fourth OTU of the subzero active Verrucomicrobia was more divergent with no close relatives within the phylum. All subzero active OTUs clustered uniquely and represent newly distinguished cryo-active tundra soil bacteria members. An unexpected observation was the detection of subzero-active Ignavibacteria (Figure 6). Members of the Ignavibacteria have mainly been found in thermophilic environments, such as hot springs, volcanic lakes, wastewater or deep aquatic sediments. However, the six representative subzero active OTUs within the Ignavibacteria formed a distinct group divergent from the known isolates and curated sequences of the phylum. The closest sequence to these active members was from a rhizosphere soil from India.

DISCUSSION Subzero metabolism in seasonally frozen or permafrost soils has been detected by production of gases such as CO2, and CH4 (Flanagan and Veum 1974; Fahnestock, Jones and Welker 1999; Panikov and Dedysh 2000; Mikan, Schimel and Doyle 2002; Monson et al. 2006; Panikov et al. 2006; Schuur et al. 2009, 2015; Öquist et al. 2009; Drotz et al. 2010; Sistla et al. 2013), but less has been done to distinguish specific members that are metabolically active. No particular bacterial indicator species utilizing cellobiose across tundra, temperate forest, and agricultural soils was identified in a SIP study, suggesting that cellobiose assimilation is ubiquitous to most bacterial communities (Verastegui et al. 2014). This study set out to investigate how different subzero temperatures shape the active bacterial community composition in Arctic tundra soils. One the most intriguing discoveries from this study was the identification of a new group of cryo-active Ignavibacteria. The Ignavibacteria have been characterized as mesophiles to thermophiles; they have been detected in hot springs and lakes, and the two known isolates of this phylum are thermophiles (Lino et al. 2010; Podosokorskaya

11 et al. 2013; Juottonen et al. 2017). Two cultured Ignavibacteria isolates have the ability to degrade cellobiose (Lino et al. 2010; Podosokorskaya et al. 2013). The cryo-active Ignavibacteria OTUs form a distinct cluster separate from the previously described phylotypes (Figure 6). This suggests that subzero cellobiose degradation and C cycling in the cold tundra soil is part of the lifestyle of some Ignavibacteria. Other studies using metagenomics suggest that tundra soil Ignavibacteria may be active in fermentative pathways, anaerobic methanotrophy, or sulfur reduction by matching to a bin representing Coenzyme B-Coenzyme M heterodisulfide reductase genes (Johnston et al. 2016). Another interesting finding is the possibility of small pockets of anaerobiosis in our SIP incubations, hence giving rise to active anaerobic bacteria, such as Desulfobacteraceae, Anaerolinaceae, and Pelobacteraceae that were also detected in the heavy DNA fraction of 13C-cellobiose fed soils. Several OTUs belonging to members of Candidatus Saccharibacteria were detected in this study, and generally clustered with other representative phylotypes from cold soils, e.g. polar and desert environments (Figure 4) (Connon et al. 2007; Wilhelm et al. 2011; Chong et al. 2012; Zeng et al. 2013). While phylotypes from colds soils and the OTUs from this study clustered together, taxa from cold-environments appear dispersed within the phylum. This finding suggests that cold and temperate representatives of Candidatus Saccharibacteria can adapt to a wide range of temperate-cold environments. However, it is difficult to ascertain additional features about these phylotypes as there is yet no cultivated representative of this candidate phylum. The cryo-active Verrucomicrobia members identified in this study formed a relatively tight cluster, and were not closely related to other representatives from cryo- environments (Figure 5). Furthermore, members from cold environments (Gangwar et al. 2009; Borin et al. 2010; Lipson et al. 2010; Shivaji et al. 2011; Kim et al. 2015) were dispersed widely throughout the phylum, suggesting that cold tolerance or cryo-activity is a ubiquitous adaptation across the Verrucomicrobia. One of the closest relatives to the cluster of Verrucomicrobia OTUs is reported to utilize C from leaves (Yoon et al. 2010). The other close relatives are from Arctic peat soil and paddy soils, as well as soil-nematode associated members (Vandekerckhove et al. 2000; Lipson et al. 2010;

Kim et al. 2015) (Figure 5). Members of Verrucomicrobia are ubiquitous to the planet’s soil environments (Bergmann et al. 2011; Navarrete et al. 2015). Overall, many members of the Verrucomicrobia have been found in polar environments using both culture-dependent and -independent techniques (Neufeld and Mohn 2005; Fierer et al. 2012; Kim et al. 2014; Deng et al. 2015). Interestingly, known cellobiose degraders and prevalent members of the resident community of these tundra soils such as the Acidobacteriaceae (Pankratov et al. 2011; Männistö et al. 2012; Pankratov 2012; Rawat et al. 2012; Eichorst et al. 2018) were not detected in the heavy 13C-DNA fraction of the subzero incubations (Figure 1). This suggests a seasonal dynamic in the community members active in C-degradation, and may imply that the Acidobacteria are seasonally active in processing carbon or are not able to compete for the substrate with other cellobiose degraders at the subzero temperatures. The members of the Ignavibacteriae, Candidatus Saccharibacteria, and Verrucomicrobiaceae that were active at subzero temperatures were, unsurprisingly, related to families in these phyla known to degrade cellulose, cellobiose, or are suggested to have saccharolytic activities (Lino et al. 2010; Dai et al. 2016; Juottonen et al. 2017). Several members of the Melioribacteraceae, Desulfobacteraceae, Anaerolinaceae and Pelobacteraceae that are thought to be cold-adapted or have been detected in polar regions (Gittel et al. 2014; Lee et al. 2014; Goordial et al. 2016; Reyes et al. 2016) were shown to be active at various subzero temperatures in this study. Regarding the usage of cellulose/cellobiose, this study is consistent with a 13C-cellulose SIP study of soils from coastal regions, grasslands, and desert, reporting that members of Burkholderiales, Rhizobiales, and Sphingobacteriales were metabolically active (Eichorst and Kuske 2012). Cellulose degrading bacteria are ubiquitously found in soils, and appear to be adapted to the temperature of that habitat (Eichorst and Kuske 2012). There are also known diazotrophs, such as the Oxalobacteraceae, that were active at both -4 and 0 °C, suggesting key roles in the relatively C-rich, N-limited environments such as the Arctic tundra soils (Yang et al. 2014). It was hypothesized that the bacterial community active on cellobiose would differ at different subzero temperatures, as has been demonstrated in a permafrost study on

13 acetate (Tuorto et al. 2014). This effect of temperature on active bacterial community composition was demonstrated by the clustering of the temperature treated biological replicates by PCA analysis (Figure 2). The active bacterial communities clustered distinctly by temperature and separated from the initial resident tundra soil community. A tighter clustering at temperatures of 0°C and -4°C was observed, while the -16°C incubation community was more distinct. While we aimed to detect the earliest assimilation of the 13C-labeled cellobiose, it is possible that there was cross-feeding during our longest incubation time (40 weeks). Furthermore, our subzero activity at -16°C was similar to the time frame of a previous SIP study of Alaska permafrost soil (Tuorto et al. 2014) and discerns the active bacterial communities at subzero temperatures whether they assimilated 13C-cellobiose or the 13C-labelled by-products of degradation. Finally, the active bacterial community distinguished by this SIP approach is different from that detected by rRNA-profiles during the growing season in tundra soils of the same Kilpisjärvi site. Using rRNA as a measure of activity, it was determined that the Acidobacteria and Gammaproteobacteria dominated at the site in the summer (Männistö et al. 2013). Although they were not the most abundant in the resident bacterial community, members of the Planctomycetes, Deinococcus-Thermus, Actinobacteria, and Bacteroidetes were active at subzero temperatures and also detected at the summer season sampling (Männistö et al. 2013). Understanding which members of the tundra soil microbial community are active at subzero temperatures is critical for developing a better understanding of soil C cycling as well as for assessing how climate change may impact vulnerable polar environments. Precipitation patterns (from snow to rain) are predicted to change in northern ecosystems (Bintanja and Andry 2017) with a decrease in the duration, thickness and insulation properties of snow which can subsequently lead to colder soils in a warmer climate (Groffman et al. 2001). Here it was demonstrated that temperature has an impact on cryo-active bacteria through the assimilation of 13C-cellobiose into bacterial DNA. Diverse bacteria, including members of Candidatus Saccharibacteria, Melioribacteraceae, Verrucomicrobiaceae, Burkholderiaceae, Acetobacteraceae, Armatimonadaceae, and Planctomycetaceae, were capable of synthesizing 13C-DNA at subzero temperatures with differences in their subzero temperature optima. This effect

14 is further supported by differential responses in respiration and C turnover in soils at similar subzero ranges, and this study identifies the active bacterial family compositions within subzero ranges (Lloyd and Taylor 1994; Mikan, Schimel and Doyle 2002; Michaelson and Ping 2003; Bore et al. 2017). It is thus important to understand the selective pressures that drive the dynamics and activity of the bacterial community in Arctic tundra soils and determine how the dominant guilds differ in their utilization of carbon and nitrogen and their responses to fluctuations in temperature and associated environmental conditions. Ultimately, these studies will identify the mechanisms of competition leading to dominance between various microbial guilds and delineate the various drivers of microbial diversity and activity in Arctic tundra soil environments.

Acknowledgements This work was funded in part by the New Jersey Agricultural Experiment Station and the Academy of Finland.

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Figure 1. Relative abundance of bacterial phyla and families in triplicate tundra soil incubations at initial and subzero temperatures. The bars represent the bacterial community members that assimilated 13C-cellobiose at temperatures of -16, -4, and 0°C after incubation of 5 to 40 weeks compared to the T0 community. Other represents low abundance (<1%) families.

Figure 2. Principal component analysis of the bacterial family-level SIP data for each temperature compared to the T0 community. Principal component analysis was carried out using unit variance scaling was applied to rows; singular value decomposition with imputation was used to calculate principal components.

Figure 3. Heat map displaying the relative abundances (color key indicates fold difference) of tundra soil bacterial family members compared across incubations. Rows and columns were clustered using correlation distance and average linkage.

Figure 4. 16S rRNA gene phylogeny of the Candidatus Saccharibacteria comparing representative OTUs from 13C-DNA fractions from the -16, -4, and 0°C incubations with representative uncultured phylotypes. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model with a total of 398 positions in the final dataset with 500 bootstraps iterations. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap values above 50 are listed. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. OTUs from this study are in blue font, taxa from cold environments are in blue background. All taxa are listed with sample source.

Figure 5. 16S rRNA gene phylogeny of the Verrucomicrobia comparing representative OTUs from 13C-DNA fractions from the -16, -4, and 0°C incubations with representative cultivated strains and uncultured phylotypes. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model with a total of 356 positions in the final dataset with 500 bootstraps iterations. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap values above 50 are listed. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. OTUs from this study are in blue font, taxa from cold environments are in blue background. All taxa are listed with sample source.

Figure 6. 16S rRNA gene phylogeny of Ignavibacteria comparing representative OTUs from 13C-DNA fractions from the -16, -4, and 0°C incubations with representative cultivated strains and uncultured phylotypes. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model with a total of 336 positions in the final dataset with 500 bootstraps iterations. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap values above 50 are listed. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. OTUs from this study are in blue font, taxa from cold environments are in blue background. All taxa are listed with sample source.

Arctic tundra soil bacterial communities active at subzero

temperatures detected by stable isotope probing

SUPPLEMENTAL INFORMATION

Preshita S. Gadkari 1, Lora R. McGuinness 2, Minna K. Männistö 3, Lee J, Kerkhof 2,

and Max M. Häggblom 1*

1 Department of Biochemistry and Microbiology and

2 Department of Marine and Coastal Sciences

School of Environmental and Biological Sciences

Rutgers, The State University of New Jersey

New Brunswick, NJ, 08901, USA

3 Natural Resources Institute Finland, P.O. Box 16, FI-96301 Rovaniemi, Finland

Figure S1. 16S rRNA gene TRFLP profiles of light 12C-DNA and heavy 13C-DNA bands from triplicate 12C-cellobiose control (left) and 13C-cellobiose experimental (right) stable isotope probing of incubations at -16˚C after 40 week incubation. The 13C-DNA band of the 12C-cellobiose fed control cultures yielded no 16S rRNA gene amplicon and produced a flat TRFLP profile. The 13C-DNA bands of the 13C-cellobiose fed cultures were subsequently analyzed by Illumina MiSeq sequencing.

Figure S2. 16S rRNA gene TRFLP profiles of light 12C-DNA and heavy 13C-DNA bands from triplicate 12C-cellobiose control (left) and 13C-cellobiose experimental (right) stable isotope probing of incubations at -4˚C after 5 week incubation. The 13C-DNA band of the 12C-cellobiose fed control cultures yielded no 16S rRNA gene amplicon and produced a flat TRFLP profile. The 13C-DNA bands of the 13C-cellobiose fed cultures were subsequently analyzed by Illmina MiSeq sequencing.

Figure S3. 16S rRNA gene TRFLP profiles of light 12C-DNA and heavy 13C-DNA bands from triplicate 12C-cellobiose control (left) and 13C-cellobiose experimental (right) stable isotope probing of incubations at 0˚C after 5 week incubation. The 13C- DNA band of the 12C-cellobiose fed control cultures yielded no 16S rRNA gene amplicon and produced a flat TRFLP profile. The 13C-DNA bands of the 13C-cellobiose fed cultures were subsequently analyzed by Illumina MiSeq sequencing.

Table S1: Number of reads from Illumina MiSeq sequencing per biological replicate of initial resident community and subzero incubations (13C-cellobiose amended bottom bands).

Table S2. Shannon diversity index (H) and evenness (E) for OTUs assigned at the family level of replicate of initial resident community and subzero incubations (13C- cellbiose amended bottom bands).

Table S3. Partial 16S rRNA sequences of subzero-active OTUs of the Candidatus Saccharibacteria, Verrucomicrobia and Ignavibacteria depicted in Figures 4, 5 and 6, respectively.

Candidatus Saccharibacteria

>SeqRunC_OTU35 [Active 0°C -4°C -16°C] TAGGGAATTTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGCAGGATGAAGGCCTTAGGGTTGTAA ACTGCTTTTATGTATGACGATTATGACGGTAGTACATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTC ATACGGAGGATCCAAGCGTTATCCGGAATTACTGGGCGTAAAGAGTTGCGTAGGTGGCAGAGTAAGCAGAGCAT GAAAGCGTGTGGCTCAACCATACATACATGTTCTGAACTGCTCAGCTTGAGGATGAGAGAGGTAATTGGAATTCC CAGTGTAGGAGTGAAATCCGTAGATATTGGGAGGAACACCGATGGCGTAGGCAGATTACTAGCTCACTCCTGACA CTCAGGCACGAAAGCGTGGGGAGCAAACGGGATTAGATACCCCGAGTAGTC

>SeqRunB_OTU37 [Active 0°C -4°C] TAGGGAATTTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGCAGGACGAAGGCCCTCGGGTCGTAA ACTGCTTTTATCTGTGAAGATTATGACGGTAGCAGATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGT CATACGGAGGATCCAAGCGTTATCCGGAATTACTGGGCGTAAAGAGTTGCGTAGGTGGCATTGTAAGCGAGTAGT GAAAGCCTGGGGCTCAACCCCTTACCCATTACTTGAACTGCAAAGCTAGAGGATGAGAGAGGTTATTGGAATTCC TAGTGTAGGAGTGAAATCCGTAGATATTAGGAGGAACACCGATGGCGTAGGCAGATAACTGGCTCATTCCTGACA CTAAGGCACGAAAGCGTGGGTAGCAAACGGGATTAGATACCCG-GGTAGTC

> SeqRunB_OTU69 [Active 0°C -4°C -16°C] TAGGGAATTTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGCAGGATGAATGCCTTCGGGTTGTAAA CTGCTTTTATATGTGACGATTATGACAGTAGCATATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTCA TACGGAGGATCCAAGCGTTATCCGGAATTACTGGGCGTAAAGAGTTGCGTAGGTGGCAGAGTAAGTTAGTAGTG AAAGCGTTCGGCTCAACCGAATATCCATTACTAAAACTGCTCAGCTAGAGGACGAGAGAGGTTATTGGAATTCCTA GTGTAGGAGTGAAATCCGTAGATATTAGGAGGAACACCGATGGCGTAGGCAGATAACTGGCTCGTTCCTGACACT AAGGCACGAAAGCGTGGGTAGCAAACGGGATTAGATACCCCTGGTAGTC

> SeqRunB_OTU135 [Active 0°C -4°C] TAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGCAGGATGAAGGCCCTTGGGTCGTAA ACTGCTTTTCTCTGTGAGGAATATGACAGTAGCAGAGGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGT CATACGGAGGATCCAAGCGTTATCCGGAATTACTGGGCGTAAAGAGTTGCGTAGGCGGCAGAGTAAGCAGAATG TGAAATCGTGTGGCTCAACCATACACCCATATTTTGAACTGCTCAGCTAGAGGATGAGAGAGGTGGCTGGAATTC CCAGTGTAGGAGTGAAATCCGTAGATATTGGGAGGAACACCGATGGCGTAGGCAGGCCACTGGCTCATTCCTGAC GCTCAGGCACGAAAGCGTGGGGAGCGACCGGGATTAGATACCCCTGGTAGTC

Verrucomicrobia

> SeqRunC_OTU11 [Active -16°C] TTTCGAATCATTCACAATGGGGGAAACCCTGATGGTGCGACGCCGCGTGGGGGATGAAGGTCTTCGGATTGTAAA CCCCTGTCACCTGGGACTAAACCTCGGCGAAGAGCCGAGCTGAATTAACCAGGAGAGGAAGCAGTGGCTAACTCT GTGCCAGCAGCCGCGGTGATACAGAGACTGCAAGCGTTACTCGGATTCACTGGGCGTAAAGGGAGCGCAGGTGG GCAGGTGTGTCGGGCGTGAAATCCCGGGGCTTAACCCCGGAATGGCGCCCGAAACTATCTGTCTAGAGGATTGG AGAGGCGGGTGGAATTCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGAGGAACACCGACGGCGAAGGCAGC CCGCTGGACAAATCCTGACACTCAGGCTCGAAAGTATGGGG-AGCAAAAGGGATTAGATACCCTGGTAGTC

> SeqRunB_OTU16 [Active 0°C -16°C] TTTCGAATCATTCACAATGGGGGCAACCCTGATGGTGCGACGCCGCGTGGGGGATGAAGGTCTTCGGATTGTAAA CCCCTGTCACCTGGGACAAAACCCCGGCGAAGAGCCGGGCTGATTCAACCAGGAGAGGAAGCAGTGGCTAACTC TGTGCCAGCAGCCGCGGTAATACAGAGACTGCAAGCGTTACTCGGATTCACTGGGCGTAAAGGGAGCGCAGGTG GGCAGGTGTGTCGGGTGTGAAATCCCGGGGCTTAACTCCGGAACTGCACCCGAAACTACTTGCCTGGAGGATTGG AGAGGCGGGCGGAATTCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGAGGAACACCGACGGCGAAGGCAAC CCGCTGGACAAATCCTGACACTCAGGCTCGAAAGTATGGGG-AGCAAAAGGGATTAGATACCCCGGTAGTC

> SeqRunB_OTU12 [Active 0°C -4°C -16°C] TCGAGGATTTTTCTCAATGGGGGAAACCCTGAAGGAGCGACGCCGCGTGAGGGATGAAGGTCTTCGGATTGTAA ACCTCTGTCATCTGGGAACAATGTGATCTACCTAACACGTGGATTATTGATAGTACCGGAAGAGGAAGCAGTGGC TAACTCTGTGCCAGCAGCCGCGGTAATACAGAGACTGCAAGCGTTGTTCGGATTCATTGGGCGTAAAGGGTGCGC AGGCGGTTCGTTAAGTCGGATGTGAAATCTCACAGCCTAACTGTGATAGGTCATTCGAAACTGGCGGACTCGAGG GCTGGAGAGGAGACTGGAATAGTCGGTGTAGCGGTGAAATGCGTAGAGATCGACTAGAACACCGGTGGCGAAG GCGAGTCTCTGGACAGTTCCTGACGCTCAGGCACGAAAGCCAGGGGAGCAAACGGGATTAGATACCCCGGTAGT C

> SeqRunC_OTU14 [Active -16°C] TTTCGAATCATTCACAATGGGGGCAACCCTGATGGTGCGACGCCGCGTGGGGGATGAAGGTCTTCGGATTGTAAA CCCCTGTCACCTGGGACAAAACCCCGGCGAAGAGCCGGGCTGATTCAACCAGGAGAGGAAGCAGTGGCTAACTC TGTGCCAGCAGCCGCGGTAATACAGAGACTGCAAGCGTTACTCGGATTCACTGGGCGTAAAGGGAGCGCAGGTG GGCAGGTGTGTCGGGTGTGAAATCCCGGGGCTTAACTCCGGAACTGCACCCGAAACTACTTGCCTGGAGGATTGG AGAGGCGGGCGGAATTCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGAGGAACACCGACGGCGAAGGCAAC CCGCTGGACAAATCCTGACACTCAGGCTCGAAAGTATGGGG-AGCAAAAGGGATTAGATACCCGGGTAGTC

Ignavibacteria

> SeqRunB_OTU4 [Active – 0°C -4°C -16°C] TGAGGAATATTGCTCAATGGACGAAAGTCTGAAGCAGCAACGCCGCGTGAGGGATGAAGGTGCTCTGCATCGTA AACCTCTGTAGTCGGGGACAAATGTGGGGTTACTACCCCATTGATGGTACCCGAAAAGTAAGGATCGGCTAACTA CGTGCCAGCAGCCGCGGTAATACGTAGGATCCAAGCGTTGTCCGGATTTACTGGGTGTAAAGGGTGCGCAGGCG GACTGGTAAGTCAGAAGTGAAATCTCGGCGCTCAACGCCGAAACGTCTTTTGATACTGTCAGTCTTGAATCGGGG AGAGATCCGTGGAATTCCGAGTGTAGCAGTGAAATGTGTAGATATTCGGAAGAACACCAGAGGCGAAGGCAGCG GATTGGCCCTTGATTGACGCTCAGGCACGAAAGCATGGGGATCAAACAGGATTAGATACCCTGGTAGTC

> SeqRunC_OTU4 [Active 0°C -4°C -16°C] TGAGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAACGCCGCGTAAAGGATGAAGGCGCTTTGCGTTGTA AACTTTTGTAGTCGGGGACAAATTGCGGGTTATTACCCGCTTGATGGTACCCGAAAAGTAAGGATCGGCTAACTAC GTGCCAGCAGCCGCGGTAATACGTAGGATCCGAGCGTTGTCCGGATTTACTGGGTGTAAAGGGTGCGCAGGCGG ACTGGTAAGTCAGTGGTGAAATCTCGTCGCTCAACGACGAAACTGCCTTTGATACTATCAGTCTTGAATCCGGTAG AGATCTGTGGAATTCCGAGTGTAGCAGTGAAATGTGTAGATATTCGGAAGAACACCAGTGGCGAAGGCAGCAGA TTGGGCCGGTATTGACGCTCAGGCACGAAAGCATGGGGATCAAACAGGATTAGATACCCGG-TAGTC

> SeqRunB_OTU10 [Active 0°C] TTTCGAATCATTCACAATGGGCGAAAGCCTGATGGTGCGACGCCGCGTGAGGGATGAAGGTCTTCGGATTGTAAA CCTCTGTCACTGGGGAAGAAACGCTTCAATTTAATAGATTGAAGCCTGACTTAACCCGGAGAGGAAGCAGTGGCT AACTCTGTGCCAGCAGCCGCGGTAATACAGAGACTGCAAGCGTTATTCGGATTCACTGGGCGTAAAGGGTGCGCA GGTGGCCAAGTGTGTGAGGCGTGAAAGCCCGTTGCTCAACAGCGGAATTGCACCTCAAACTACATGGCTAGAGCA TTGGAGAGGGGAGCAGAATTCACGGTGTAGCAGTGAAATGCGTAGATATCGTGAGGAATACCAGAGGCGAAGG CGGCTCCCTGGACAATTGCTGACACTCAGGCACGAAAGCGTGGGGAGCAAAAGGGATTAGATACCCGGGTAGTC

> SeqRunB_OTU14 [Active 0°C] TGAGGAATATTGCTCAATGGACGAAAGTCTGAAGCAGCAACGCCGCGTGAGGGATGAAGGTGCTCTGCATCGTA AACCTCTGTAGAGGGAGACAAACTTCGAGTTACTACTCGATTGATGGTACCCCTAAAGTAAGGATCGGCTAACTAC GTGCCAGCAGCCGCGGTAATACGTAGGATCCAAGCGTTGTCCGGATTTACTGGGTGTAAAGGGTGCGCAGGCGG ACCAATAAGTCAGGAGTGAAATCTCGGCGCTCAACGCCGAAACGTCTTTTGATACTGTTGGTCTTGAATAAGCGAG AGATTCGTGGAATTCCGAGTGTAGCAGTGAAATGTGTAGATATTCGGAAGAACACCAGAGGCGAAGGCAGCGAA TTGGCGCTTTATTGACGCTCAGGCACGAAAGCATGGGGATCAAACAGGATTAGATACCCTGGTAGTC

> SeqRunC_OTU39 [Active 16°C] TGAGGAATATTGCTCAATGGACGAAAGTCTGAAGCAGCAACGCCGCGTGAGGGATGAAGGTGCTCTGCATCGTA AACCTCTGTAGTCGGGGACAAATGTGGGGTTACTACCCCATTGATGGTACCCGAAAAGTAAGGATCGGCTAACTA CGTGCCAGCAGCCGCGGTAATACGTAGGATCCAAGCGTTGTCCGGATTTACTGGGTGTAAAGGGTGCGCAGGCG GACTGGTAAGTCAGAAGTGAAATCTCGGCGCTCAACGCCGAAACGTCTTTTGATACTGTCAGTCTTGAATCGGGG AGAGATCCGTGGAATTCCGAGTGTAGCAGTGAAATGTGTAGATATTCGGAAGAACACCAGAGGCGAAGGCAGCG GATTGGCCCTTGATTGACGCTCAGGCACGAAAGCATGGGGATCAAACAGGATTAGATACCCGGGTAGTC

> SeqRunB_OTU65 [Active 0°C] TGAGGAATATTGCTCAATGGACGAAAGTCTGAAGCAGCAACGCCGCGTGAGGGATGAAGGTGCTCTGCATCGTA AACCTCTGTAGCCGGGGACAAATGTGGGATTACTATCCCATTGATGGTACCCGGAAAGTAAGGATCGGCTAACTA CGTGCCAGCAGCCGCGGTAATACGTAGGATCCAAGCGTTGTCCGGATTTACTGGGTGTAAAGGGTGCGCAGGCG GACCAATAAGTCAGGAGTGAAATCTCGGCGCTCAACGCCGAAACGTCTTTTGATACTGTTGGTCTTGAATAAGCGA GAGATTCGTGGAATTCCGAGTGTAGCAGTGAAATGTGTAGATATTCGGAAGAACACCAGAGGCGAAGGCAGCGA ATTGGCGCTTTATTGACGCTCAGGCACGAAAGCATGGGGATCAAACAGGATTAGATACCCGGGTAGTC

> SeqRunB_OTU166 [Active 0°C] TGAGGAATATTGCTCAATGGACGAAAGTCTGAAGCAGCAACGCCGCGTGAGGGATGAAGGTGCTCTGCATCGTA AACCTCTGTAGTCGGGGACAAATGTGGGGTTACTACCCCATTGATGGTACCCGAAAAGTAAGGATCGGCTAACTA CGTGCCAGCAGCCGCGGTAATACGTAGGATCCGAGCGTTGTCCGGATTTACTGGGTGTAAAGGGTGCGCAGGCG GACTGATAAGTCAGTGGTGAAATCTCGTCGCTTAACGACGAAACTGCCTTTGATACTATCAGTCTTGAATCCGGTA GAGATCTGTGGAATTCCGAGTGTAGCAGTGAAATGTGTAGATATTCGGAAGAACACCAGTGGCGAAGGCAGCAG ATTGGGCCGGTATTGACGCTCAGGCACGAAAGCATGGGGATCAAACAGGATTAGATACCCGGGTAGTC