Microbial Community Responses of an Anaerobic Enrichment to Temperature Shocks

MICROBIAL COMMUNITY RESPONSES OF AN ANAEROBIC ENRICHMENT TO TEMPERATURE SHOCKS

RAN MEI

THESIS

Submitted in partial fulfillment of the requirements for the degree of Master of Science in Environmental Engineering in Civil Engineering in the Graduate College of the University of Illinois at Urbana-Champaign 2015

Urbana, Illinois

Adviser:

Professor Wen-Tso Liu, Director of Research

ABSTRACT

Anaerobic digestion (AD) accomplishes degradation of complex organics in wastewater to methane and CO2 mediated by ecological cooperation of microorganisms with different metabolic functions. Previous studies have reported that temperature fluctuation could lead to

AD process instability and failure, perhaps through disruption of microbial cooperation.

Nevertheless, understanding of how AD microbiota respond to temperature shock is poor.

Specifically, we have yet to identify organisms vulnerable to heat shock or responsible for recovering post-shock AD activity. To systematically address these questions, a mesophilic benzoate-degrading methanogenic enrichment was exposed to different levels of temperature perturbation from 45°C to 70°C for 5 or 15 min. Perturbations over this temperature gradient revealed three types of post-shock methane production profiles: no inhibition (45 and 50°C), initial inhibition with gradual recovery (55 and 60°C), and complete inhibition (70°C). These responses were also reflected in the RNA- and DNA- based 16S rRNA gene analysis of the microbial community. The primary benzoate-degrading syntroph (Syntrophus-related population) was highly affected by heat shock temperature, and its abundance was crucial to the restoration of benzoate degradation after the perturbation. In contrast, two major methanogen populations were relatively stable regardless whether methane production was inhibited. Other bacteria showed different response patterns, indicating distinct physiological and ecological traits. For example, a Firmicutes-related population rose to >50% abundance post shock, perhaps surviving through spore formation and thriving on detritus degradation. Members of “Ca. Cloacimonetes”,

Spirochaetes, Bacteroidetes and Thermotogae were also stimulated while Desulfovibrio and another Spirochaetes member was inhibited. Though the exact roles of these minor microbial

ii populations are unknown, their ubiquity across AD suggests that they are functionally important.

Overall, temperature perturbation provided ecological characterization of core microbial populations in AD and can further generate knowledge on how to deal with unexpected heat shock, a common accident, thus improve the stability and performance of AD processes.

iii TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION...………………………………………………………..... 1 CHAPTER 2: EXPERIMENTAL PROCEDURES..……………………………………...... 4 CHAPTER 3: RESULTS……………….………………………………………………….... 8 CHAPTER 4: DISCUSSION…………...…………………………………………………… 12 REFERENCES…………………………………..…………………………………………... 18 FIGURES AND TABLES…………………………………………………………………… 22 APPENDIX A -- SUPPLEMENTAL FIGURES AND TABLES…………………...……… 27

iv CHAPTER 1

INTRODUCTION

Anaerobic digestion (AD) is a promising technology for wastewater treatment that capitalizes on cooperation between many microbial guilds of microorganisms to accomplish conversion of complex organics to methane and CO2. The widely accepted generic scheme of metabolic network consists of fermenters who decompose organic polymers to volatile fatty acids (VFA) and H2, syntrophs who produce acetate and H2 from VFAs and methanogens who convert acetate and H2 to methane (Schink & Stams, 2006). Recent studies applying culture- independent technologies reveal that there are still many yet-to-be-cultured microbes in AD, with significant abundance but unclear roles (Narihiro & Sekiguchi, 2007). Limited insights into these uncharacterized microbes hamper the construction of a comprehensive picture of AD system.

On the other hand, operational and environmental variations have considerable effects on

AD’s performance (Leitão, Van Haandel, Zeeman, & Lettinga, 2006). Among many types of disturbances, temperature shock receives enormous interests because temperature control is indispensable for all systems and failures of control are to some extent inevitable. Several studies have been done to compare the reactor performance at different temperature and instability was observed when the operation temperature deviated from optimal conditions (Bouallagui et al.,

2004; Choorit & Wisarnwan, 2007; Sanchez, Borja, Weiland, Travieso, & Martın, 2001).

Accidental temperature fluctuations were shown to cause instability of the performance and recovery would take a long time (C.-L. Chen et al., 2004; Zinder, Anguish, & Cardwell, 1984).

What are the responses of microbes in AD to heat shock? Are they able to recover the degradation of substrates and methanogenesis after the shock? Although these studies

1 documented the effects of temperature shock on system performance, the resistance and resilience of microbial community, which is essentially responsible to the conversion of organic substrates to methane and CO2, was not clearly reported. It is conceivable that behaviors of community members after heat shock would exhibit extraordinary heterogeneity, depending upon their physiologies and functions. In addition, different magnitude of heat shock would give rise to distinct responses, which would help to define the lethal threshold for engineering applications.

Analyzing 16S ribosome RNA and its coding gene (16S rRNA gene) is a common approach for microbial community survey. The concentration of rRNA is proportional to the number of cellular ribosomes, which roughly represents the translation potential and metabolic activity. Conversely, rRNA gene concentration is more representative of cell numbers. The 16S rRNA: rRNA gene ratio of specific taxa could serve as an indicator of the average activity of individual cells (Blazewicz, Barnard, Daly, & Firestone, 2013). This approach is especially helpful for examining species-specific evolution across time to identify the drivers of activity and abundance fluctuations (Hunt et al., 2013). Several studies showed that 16S rRNA: rRNA gene ratio and growth rate is linear and positive, so it is a species-specific measure of metabolic activity in natural communities (Campbell & Kirchman, 2012; Campbell, Yu, Heidelberg, &

Kirchman, 2011; Muttray & Mohn, 1999). This proxy of in-situ activity was further confirmed by correlating against Micro-CARD-FISH, which is a direct measure of bacterial metabolism

(Salter et al., 2014).

We have previously obtained a series of enrichment cultivations from AD sludge

(Narihiro, Nobu, Kim, Kamagata, & Liu, 2014). In this study, the benzoate-degrading enrichment culture was exposed to different levels of temperature perturbation from 45°C to

2 70°C for 5 or 15 min. A microbial community survey was performed based on 16S rRNA and rRNA gene, to unravel the microbial community response of an anaerobic enrichment culture to temperature shocks.

3 CHAPTER 2

EXPERIMENTAL PROCEDURES

2.1 Enrichment culture

Cultures used in this study were originally enriched with benzoate from anaerobic digester sludge at Urbana (Illinois, USA) as described previously together with the enrichment processes and media preparation (Narihiro et al., 2014). N2/CO2 (80:20, vol/vol) was used to degas media and exchange headspace gas at 0.2 MPa. 20 mM sodium benzoate was added as sole carbon source. All cultures were maintained at 37 °C after inoculation. Headspace gas compositions (with triplicates) and chemical compounds (formate, acetate, propionate, butyrate and benzoate) concentrations (with duplicates) were monitored as the previous study described

(Narihiro et al., 2014).

2.2 Heat shock and biomass sampling

When the methane concentration reached around mid point of maximum production, i.e.,

16 days after inoculation, all cultures except for control were exposed to heat shock using water bath with the temperature settings of 45 °C, 50 °C, 55 °C, 60 °C and 70 °C (Table. 1). Bottles were completely immersed to get efficient heat transfer and timing started when the liquid inside that bottle reached -2 °C of target temperature. For each temperature setting, heat shock lasted for 5 min and 15 min. After heat shock, all bottles were maintained back at 37 °C. At the day of heat shock (day 16), biomass sample was taken for DNA and RNA extraction from control bottles. At day 42, samples were taken from control and all treatments. At day 55 and 77, samples were taken from 55 °C (5 and 15 min) and 60 °C (5 min), respectively. At the end of the

4 cultivation, samples were taken from 70 °C (15 min) at day 86, 60 °C (15 min) and 70 °C (5 min) at day 88.

2.3 Nucleic acid extraction, PCR and sequencing

10 mL liquid containing biomass from one bottle was taken for DNA extraction using the

FastDNA SPIN Kit for Soil (MP Biomedicals, Carlsbad, CA, USA) according to the manufacturer’s instructions. DNA concentration was determined using Qubit 2.0 Fluorometer.

Approximately 100 ng of the genomic DNA was used as template for PCR amplification in a reaction volume of 25 µL. A primer set (515F/909R) targeting the V4-V5 region of 16S rRNA gene was used for amplification with a dual-indexing approach (Kozich, Westcott, Baxter,

Highlander, & Schloss, 2013). PCR was performed with Bullseye standard Taq DNA polymerase 2.0 master mix (MIDSCI, St. Louis, MO, USA) and the thermal cycling protocol consisting of initial denaturation (94°C, 3 min), 25 cycles of denaturation (94 °C, 30 s), annealing (55 °C, 45 s) and extension (72 °C, 1 min), and a final extension (72 °C, 10 min). The

PCR amplicons were purified using the Wizard SV Gel and PCR Clean-Up System (Promega,

Fichburg, WI, USA) and quantified by Qubit 2.0 Fluorometer.

Liquid containing biomass from an entire bottle (~80 mL) was taken for RNA extraction with triplicates or duplicates with the protocol described previously (Narihiro et al., 2009).

Briefly, biomass was centrifuged at 4 °C and transferred to lysing matrix tubes containing 1.4 mm ceramic spheres, 0.1 mm silica spheres and one 4 mm glass bead. 1 mL pH 5.1 buffer (10 mM EDTA, 50 mM sodium acetate) was added and the remaining volume of the tube was filled with phenol equilibrated with pH 5.1 buffer. The tubes were homogenized for 40 seconds on a bead-beating device. Acid-phenol/chloroform/isoamyl alcohol (125:24:1) and chloroform were

5 used to purify RNA molecules, which were further precipitated by ethanol at -80 °C. RNA extraction from bottles of 70 °C 15 min failed due to insufficient biomass. DNase treatments were performed with both RNase-free DNase Set (Qiagen, Valencia, CA, USA) and Turbo

DNA-free (Ambion, Austin, TX, USA). DNA amplifying PCR was carried out to detect genomic

DNA contamination in the purified RNA. Reverse transcription and 16S rRNA gene amplification were performed simultaneously with the primer described above and the

Superscript III One Step RT-PCR with Platinum Taq (Invitrogen, Carlsbad, CA, USA). The thermal cycling protocol, which was slightly modified based on the manufacture’s instruction, consists of cDNA synthesis (55°C, 30min), initial denaturation (94°C, 2 min), 30 cycles of denaturation (94 °C, 15 s), annealing (55 °C, 30 s) and extension (68 °C, 1 min), and a final extension (68 °C, 5 min). The amplicons were purified and quantified as above-mentioned.

PCR/RT-PCR products from each sample were mixed to get a pooled amplicon library with final concentration of ~10 ng/µL. Sequencing was performed on Illumina Miseq Bulk

2x300nt paired-end system at the Roy J. Carver Biotechnology Center at the University of

Illinois at Urbana-Champaign.

2.4 Operational taxonomic unit analysis

Raw sequences were assembled, screened and trimmed using Mothur (Schloss et al.,

2009) with maximum sequence length of 400 bp and quality score of 20. The output data were subsequently analyzed using QIIME 1.8.0 (Caporaso, Kuczynski, et al., 2010) for OTU picking with the open-reference strategy. UCLUST (Edgar, 2010) was used to group sequences into

OTUs based on the similarity against Greengenes database with 97% identity cut-off. For each

OTU, the most abundant sequence was selected as the representative which was further aligned

6 using PyNAST (Caporaso, Bittinger, et al., 2010) and chimeric sequences were removed using

ChimeraSlayer (Haas et al., 2011). The taxonomy was assigned by BLAST (Camacho et al.,

2009) with maximum e-value of 0.001. α-diversity indices including Chao1, Shannon and observed species were calculated by QIIME. Principal component analysis (PCA) of all the

OTUs from all samples was performed using the CANOCO 4.5 program (Braak & Šmilauer,

2002). Phylogenetic tree of major OTUs (> 0.5%) was constructed using the ARB program

(Ludwig et al., 2004) using the methods of neighbor joining and parsimony (Hugenholtz, Tyson,

Webb, Wagner, & Blackall, 2001). Relative abundance was calculated based on OTU table generated by QIIME. Nonparametric (Kendall and Spearman) regression analyses were performed in R.

2.5 Nucleotide sequences accession number

The sequence data obtained in this study have been deposited under DDBJ/EMBL/GenBank accession no. PRJDB3842.

7 CHAPTER 3

RESULTS

3.1 Methane production affected by heat shock

It took approximately 30 days for the enrichment culture to stoichiometrically convert 1.6 mmol benzoate to 4 mmol methane (Fuchs 2011) with an average CH4 production rate of 2.39

-1 mmol CH4 d during exponential phase (Fig.1 and A1). Compared with this non-disturbed control, three distinct CH4 production patterns were observed in response to heat shock: (A) no suppression in CH4 production, (B) stuntedness in CH4 production rate with later recovery of full

CH4, and (C) suspension of CH4 production. Community exposed to heat shock of 45 and 50 °C showed no suppression of CH4 production regardless of shock duration (5 and 15 min) (pattern

A; Fig. 1A and B). Heat shock at 55 °C (5 and 15 min) decreased CH4 production rate to an

-1 average rate of 0.8 mmol CH4 d (pattern B; Fig. 1C). For short (5 min) 60 °C heat shock, we

-1 observed a 30-day suspension followed by low CH4 production rate (1.06 mmol CH4 d ) until reaching max CH4 production comparable to the control (pattern C then B; Fig. 1D). Extreme heat shocks at 60 °C (15 min) and 70 °C (5 and 15 min) led to CH4 production suspension for the entire duration of the experiment (90 days) (pattern C; Fig. 1D and 1E). These observations were in correspondence with benzoate and acetate concentrations profile (Fig. A1).

3.2 Overview of microbial community responses to heat shock

To evaluate the specific microbial community response to the different heat shock levels shown above, we took samples at multiple time points (Table 1) in accordance with the observed methane production patterns: pattern A were sampled at maximum CH4; pattern B were sampled

8 during stuntedness and at eventual recovery of maximum CH4; pattern C were sampled at suspension for two times. We performed community analysis by sequencing 16S ribosomal

DNA and RNA (95,000 reads per sample) to adequate depth based on saturated rarefaction curves and high Good’s coverage (Fig. A2 and Table. A1). Principal component analysis (PCA) revealed that microbial communities at time points exhibiting similar CH4 production patterns had similar structure (Fig. 2). Based on 16S rRNA gene (Fig. 2A), communities with unsuppressed CH4 production (pattern A; open circle) were similar to the control community with no heat shock (solid circle). Interestingly, while communities with suspended CH4 production (pattern C; open triangle) were distinct from those aforementioned, communities with recovered CH4 production (open square) became quite similar to the unsuppressed communities.

Communities with stunted activity (pattern B; open diamond) were clearly distinguishable from the other two groups. On the other hand, repeating this analysis on 16S rRNA (Fig. 2B) demonstrated same results with DNA with the observation that that those communities with stunted activity were similar to the communities exhibiting normal CH4 production. This gradual shift reflected dynamics of community structure as a result of heat shock. It’s conceivable that heat shock affected key microbes that contributed to community structure as well as metabolism network. In order to further elucidate the microbial ecology, predominant microbes with over

0.5% frequencies were identified using OTU-level phylogenetic analyses (Fig. A3).

Control communities exhibited consistence between day 16 and 42, featured with the syntrophic metabolic network (Fig. 3 & Fig. A4). An OTU related to Syntrophus buswellii

(3688498, 99.73% 16S rRNA gene similarity) was identified as the predominant bacteria species that convert benzoate to acetate, hydrogen and CO2. Other bacteria clades, i.e., Spirochaetes,

Firmicutes, “Ca. Cloacimonetes” (previously known as WWE1), Synergistetes, Bacteroidetes

9 and Thermotogae were found whereas their abundance was not high (<4%), presumably maintaining and supporting the consortia. An OTU related to acetoclastic methanogen

Methanosaeta concilii (103219, 100%) dominated in archaea domain, splitting acetate to CH4 and CO2. OTUs related to hydrogenotrophic methanogens including Methanoculleus receptaculi

(4409069, 99.74%) and Methanofollis liminatans (329, 100%) were also observed (~2%), reducing CO2 to CH4 with hydrogen. The community structures based on 16S rRNA and rRNA gene are essentially similar, whereas Syntrophus and Methanosaeta populations are more dominant in the RNA pool, indicating the significant roles of these two organisms.

Microbial community structures (Fig. 3) further elucidated the responses to heat shock revealed by methane production and PCA. At day 42, unsuppressed communities resembled control communities except that an OTU related to Syntrophus aciditrophicus (32886, 100%) emerged to abundance of 6-7%. Apparent community drift was observed in stunted communities where S. buswellii was the one who suffered most from heat shock with a drastic decrease in

DNA frequency (to 10-20%). Several originally minor bacterial OTUs arose including

Soehngenia saccharolytica (228603, 100%) within Firmicutes, Mesotoga infera (77438, 100%) within Thermotogae, “Ca. Cloacimonetes” member (534698) and Bacteroidetes member

(717240). Similar trends were observed in suspended communities where S. buswellii kept decreasing especially on RNA frequency while Soehngenia and “Ca. Cloacimonetes” OTUs predominated. Although the absolute population sizes of Methanosaeta and Methanoculleus actually shrank since the total nucleic acids amount dropped drastically, they were quite stable in term of relative abundance. All the recovered communities, regardless whether they were stunted or suspended at day 42, regained a community similar to unsuppressed ones after two to five weeks. In contrast, if the communities stayed suspended, there was no change after seven weeks.

3.3 16S rRNA and rRNA gene correlation

The relationship between 16S rRNA (activity) and rRNA gene (abundance) of major

OTUs was calculated to understand their specific activity (rRNA: rRNA gene) in response to heat shock. A strongly positive correlation was revealed by statistical analysis (Fig. 4) (Kendall’s and Spearman’s P<2.2 e-16, Kendall’s tau=0.567, Spearman’s rho=0.753, n=272), which defined a region of normal function by linear regression. Most OTUs in all communities were located within the region that represented normal level of function. OTUs that significantly deviated from this region were identified as either Syntrophus buswellii or Soehngenia saccharolytica. All five OTUs counts with low RNA: DNA ratio (blue dots below the regression region) were

S.buswellii, exclusively from suspended communities. For seven OTUs counts with high RNA:

DNA ratio (above the regression region), two of them were S.buswellii in stunted communities and the other five were S.saccharolytica in suspended communities. In contrast, Methanosaeta related OTUs were all in the region of normal ratio, together with all the other major OTUs.

11 CHAPTER 4

DISCUSSION

4.1 Syntrophs

Syntrophus-related species were frequently observed as benzoate degraders in AD containing aromatic compounds (Chen, Wu et al. 2009, Narihiro, Terada et al. 2009, Perkins,

Scalfone et al. 2011). Two major OTUs in this study were identified as Syntrophus species,

3688498 (S. buswellii, 99.73%) and 32886 (S. aciditrophicus, 100%), both of which were reported to be mesophiles in pure cultures (Auburger and Winter 1995, Jackson, Bhupathiraju et al. 1999). The abundance (16S rRNA gene frequency) and activity (16S rRNA frequency) of S. buswellii was as high as 80% in control communities, implying its major role as benzoate degrader. At low strength heat shock (40 and 50 °C), its abundance, activity and specific activity

(16S rRNA: rRNA gene) were stable compared with control, indicating normal function undisturbed by heat shock. Further increasing temperature caused severe inhibition so that although DNA residue remained, specific activity in suspended communities drastically dropped to the lowest level among all major OTUs under all conditions (Fig. 4). This observation was in line with the ceased methane production. Nonetheless, it could survive heat shock as strong as 60

°C 5 min, which went far beyond the temperature range for growth when it was in pure culture without sporulation mechanism reported. When the methane production was stunted, activity restored faster than abundance, rendering specific activity very high (Fig. 4), and eventually got back to same level as control after methane production recovered. Heat shock of 60 °C 15 min or higher caused lethal and irreversible detriments, leading to complete shut down of methane production. The tightly coupled correlation between the population of this OTU and methane

12 production suggested its indispensable role in proceeding and resuming benzoate degradation in the methanogenic consortia.

The S. aciditrophicus-related OTU32886 was likely to be a minor benzoate degrader with low abundance of 0.5% in control samples but was stimulated to 5% after heat shock of 45 and

50 °C. Its abundance dropped back to less than 0.5% once S.buswellii recovered, suggesting its compensating role in the benzoate degradation when the major degrader suffered from heat shock. Its capability to syntrophically degrade certain fatty acids, e.g., butyrate and hexanoate

(Jackson, Bhupathiraju, Tanner, Woese, & McInerney, 1999), could probably lead to the increasing methane production under slight heat shock. Since there was no report about competition between two Syntrophus species, further investigations are necessary to elucidate this co-occurrence of phylogenetically close and functionally similar microorganisms.

4.2 Other bacterial OTUs

OTUs classified into bacterial groups including Spirochaetes, “Ca. Cloacimonetes”,

Firmicutes, Synergistetes, Bacteroidetes and Thermotogae were frequently observed in AD systems. Speculations of their roles were made in several studies, for example, Synergistetes might degrade amino acids (Riviere et al., 2009), Bacteroidetes were believed to be involved in decomposing of complex organics by fermentation (S. Chen & Dong, 2005; Kampmann et al.,

2012), Spirochaetes were known to ferment carbohydrates and other polymers (Singh, Kumar,

Sarbhai, & Tripathi, 2012). These OTUs were observed in this study with higher than 0.1% abundance and their responses to heat shock reflected the hypothetical roles of maintaining the microbial community by degrading biomass.

13 Some OTUs that were minor members in control samples benefited from heat shock and became dominant species, presumably owing to utilization of lysed biomass resulted from heat shock. OTU228603 had low abundance (<0.1%) in all control and unsuppressed samples, but emerged after heat shock stronger than 55°C and dominated in all suspended samples (Fig. 3). Its specific activity after heat shock was among the highest in all OTUs in all samples (Fig.4).

Along with incubation, its abundance and activity remained at high level until the end of experiments, regardless whether the methane production resumed, suggesting its irrelevance to substrate degradation and methane production. Phylogenetic analysis revealed that it was closely related to a clostridia species Soehngenia saccharolytica (100%) which was reported to perform dismutation of benzaldehyde to benzoate and benzylalcohol (Parshina et al., 2003). Considering that this reaction cannot support growth, it was proposed in several studies (Fujiwara et al., 2006;

Mayumi et al., 2013; Zhang, Dong, Jiang, Xu, & Eberl, 2006) as a sugar fermenter with acetate, hydrogen and CO2 as products but had nothing to do with aromatic ring fission or further oxidation (Trably, Batstone, Christensen, Patureau, & Schmidt, 2008). Its occurrence in high temperature, high-pressure oil reservoir environments, along with its spore-forming ability

(Parshina et al., 2003), suggested its adaptation to severe environments. Based on previous ecology studies it was likely to be a saccharide fermenter that was able to form spore to survive heat shock and compete out other potential fermenters when large amount of cell debris were available after strong heat shock.

Similar effects by heat shock was observed in OTU77438 related to Mesotoga infera

(100%), who was proved to be a heterotrophic sugar degrader (Hania et al., 2013) and two

Bacteroidales-related OTUs (717240 and 110066).

14 Not all potentially biomass degraders benefited from heat shock, even though their close relatives did. Two OTUs, 534698 and 1577238, were classified into the candidate bacterial division “Ca. Cloacimonetes” that was first discovered within a municipal anaerobic sludge digester in Evry, France (Chouari et al., 2005). “Ca. Cloacimonetes” species was frequently observed in AD systems but the function was unclear (Chouari et al., 2005; Ju & Zhang, 2014;

Limam et al., 2014). OTU1577238 was related to Candidatus Cloacimonas acidaminovorans

(100%) that had the only sequenced genome within the division who demonstrated the potential to utilize amino acids and butyrate (Pelletier et al., 2008). OTU534698 was located in another clade with no closely related isolates. In contrast with the constant but low abundance (0.1%-3%) of OTU1577238, OTU534698, potentially degrading peptides and amino acid derived from detritus biomass, was stimulated by strong heat shock to an abundance higher than 20%, inferring their different physiological properties and ecological functions. Another two OTUs

(4417515 and 576755) were identified as Spirochaetes species within the SA-8 clade with the highest abundance of 22%. Nonetheless, OTU576755 was inhibited by heat shock while

OTU4417515 was mostly abundant after heat shock higher than 55°C.

The abundance of two Synergistetes related OTUs (2566736 and 4393521) may not be significantly affected by heat shock, indicating their important roles in maintaining the function of microbial consortia, for instance, degrading small amount of cell debris that were permanently available in culture. A Desulfovibio-related OTU (4469781), constantly present in control and unsuppressed samples, was completely washed out by strong heat shock.

4.3 Methanogens

15 Methanosaeta, a specialized methanogen that only uses acetate, is widely distributed in

AD systems and regarded as a principal methane producer (Smith & Ingram-Smith, 2007).

OTU103219 (related to Methanosaeta concilii, 100%) was ubiquitously found in all samples with 5-20% DNA and RNA frequencies, which was fairly stable in contrast with Syntrophus.

However, its absolute population in suspended samples should be low since the total nucleic acids amount decreased, which could explain the ceased methane production.

Hydrogenotrophic methanogens are core methanogens that supported syntrophy in AD

(Narihiro et al., 2014). Two OTUs were identified as H2 scavengers, 4409069 (related to

Methanoculleus receptaculi, 99.74%) and 329 (related to Methanofollis liminatans, 100%) with abundance of 1-7% and 0.1-10%, respectively. Similar with Methanosaeta sp., the relative population size of Methanoculleus sp. was not significantly affected by heat shock, in consistent with the report that the optimal growth temperature for Methanoculleus receptaculi is 50-55 °C

(Cheng et al., 2008). Methanofollis liminatans, capable of using hydrogen and some alcohols, could not grow at temperature higher than 45 °C (Zellner et al., 1999), which led to its decreasing population after strong heat shock.

4.4 Conclusions

In a simplified methanogenic microbial community that was derived from anaerobic digester sludge, distinct responses to temperature perturbations of different magnitude were observed. Syntrophus-related species, the major benzoate degrader, were sensitive to heat shock but could recover activity as long as the perturbation did not exceed a lethal threshold. Their activities were crucial in recovery of substrates degradation and methane production once accidental disturbance occurred. Methane producers demonstrated higher resistance and stability.

16 Other species, presumably biomass degraders, showed different responses depending on their physiological properties and ecological niches in the methanogenic consortia. In this sense, heat shock as well as other types of disturbances may serve as an effective approach to enrich and study the generally minor groups to get a better understanding of their features and functions.

Overall, a systematic and comprehensive study about the microbial responses to environmental factors is of high value to improve the performance of anaerobic digestion systems.

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21 FIGURES AND TABLES

Fig.1. Methane gas production affected by heat shock of 45 °C (A), 50 °C (B), 55 °C (C), 60 °C (D) and 70 °C (E).

Open box, control incubated under 37 °C; open circle, heat shock for 5 min; open triangle, 15 min. Solid line, pattern A, no suppression in CH4 production; gray line, pattern B, stuntedness in CH4 production rate with later recovery of full; dotted line, pattern C, suspension of CH4 production.

Fig.2. Principal component analysis of microbial community structure based on frequencies of 16S rRNA gene (A) and 16S rRNA (B) sequences. Solid circle, control communities; open circle, unsuppressed; open square, recovered; open diamond, stunted; open triangle, suspended.

42, heat shock at 45 °C for 5 min, sample was taken 42 days after inoculation. Bubble sizes sizes Bubble inoculation. after days 42 taken was sample 5 min, for 45 °C at shock heat 42, -

5 -

e.g.,45

Solid circle,Solid 16SrRNA gene; opencircle, 16SrRNA. Label bubblesover indicates heat conditionshock and

Microbial community structure of all communities based on 16S rRNA gene and rRNA sequences for major major for sequences rRNA and gene rRNA 16S on based communities all of structure community Microbial

Fig.3. OTUs. samplingdate, (DNARNA) oracids nucleic demonstrates OTUs.Histogram of frequency RNA or sequences DNA represent (area) concentrationsfrom certain volumesof culture

Fig.4. Correlation between 16S rRNA and rRNA gene frequencies of major OTUs. Blue, Syntrophus buswellii related OTU; red, Soehngenia saccharolytica related OTU; green, Methanosaeta concilii related OTU; gray, all the other major OTUs. Solid circles, the three mentioned OTUs in suspended communities; open circles, the three mentioned OTUs in stunted communities. Slope of lines is determined by linear regression.

Table 1. Sample calendar

Treatment Sampling Date Temperature Duration 16 42 55 77 86 88 37°C Control Control

5 min Unsuppressed 45 °C 15 min Unsuppressed

5 min Unsuppressed 50 °C 15 min Unsuppressed

5 min Stuntedness Recovered 55 °C 15 min Stuntedness Recovered

5 min Suspended Recovered 60 °C 15 min Suspended Suspended

5 min Suspended Suspended 70 °C 15 min Suspended Suspended

Table 1. Sample calendar

Table S1. Sequencing results

Culture Nucl. Seq No.1 PD2 Chao1 Observed Species Good's Coverage Condition

DNA 89951 26.97 598 374 0.998 Control RNA 76592 23.96 576 371 0.998 DNA 98969 25.74 527 362 0.999 Unsuppressed RNA 63519 30.77 742 490 0.997 DNA 79260 31.16 586 437 0.998 Stunted RNA 81258 44.21 1142 802 0.996 DNA 114202 27.40 580 384 0.999 Recovered RNA 98331 33.08 818 555 0.998

DNA 84958 27.99 610 408 0.988 Suspended RNA 95992 60.47 1443 1174 0.983

1. After assembly and quality trimming 2. Phylogenetic distance

APPENDIX A

SUPPLEMENTAL FIGURES AND TABLES

Fig.A1. Acetate and benzoate concentration profiles. Four treatments were shown to represent different patterns.

Fig.A2. Rarefaction curves of all sequenced samples based on observed species.

Fig.A3. Distance matrix tree of major OTUs.

Methanosaeta, 6.0% Other Bacteria, 0.7% Other Bacteria, 6.8% Methanoculleus, 3.3% Firmicutes, 0.1% Synergistetes, 1.4% Methanosaeta, 10.4% Firmicutes, 0.5% Methanofollis, 3.0% Bacteroidetes, 0.1% Synergistetes, 0.9% Other Archaea, 0.2% “Ca. Cloacimonetes”, 0.4% Methanoculleus, 2.6% Bacteroidetes, 1.5% Spirochaetes, 0.2% Methanofollis, 2.8% “Ca. Cloacimonetes”, 3.0% Other Archaea, 0.05% Spirochaetes, 3.7%

Syntrophus, 71.5% Syntrophus, 81.2%

(A) (B)

Fig.A4. Microbial community compositions of control cultures based on frequencies of 16S rRNA gene (A) and 16S rRNA (B) sequences.

30 Table 1. Sample calendar