Performance and microbial community of hydrogenotrophic methanogenesis under thermophilic and extreme-thermophilic conditions

Accepted Manuscript Performance and Microbial Community of Hydrogenotrophic Methanogenesis under Thermophilic and Extreme-thermophilic Conditions Nanshi Dong, Fan Bu, Qi Zhou, Samir Kumar Khanal, Li Xie PII: DOI: Reference:

S0960-8524(18)30773-9 https://doi.org/10.1016/j.biortech.2018.05.105 BITE 20011

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

8 May 2018 29 May 2018 30 May 2018

Please cite this article as: Dong, N., Bu, F., Zhou, Q., Khanal, S.K., Xie, L., Performance and Microbial Community of Hydrogenotrophic Methanogenesis under Thermophilic and Extreme-thermophilic Conditions, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.05.105

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Performance and Microbial Community of Hydrogenotrophic Methanogenesis under Thermophilic and Extreme-thermophilic Conditions Nanshi Donga, Fan Bua, Qi Zhoua, Samir Kumar Khanalc, Li Xiea,b a, State Key Laboratory of Pollution Control and Resource Reuse, Key Laboratory of Yangtze River Water Environment, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, PR China b, Shanghai Institute of Pollution Control and Ecological Security, Shanghai 200092, P.R. China c, Department of Molecular Biosciences and Bioengineering (MBBE), University of Hawai’i at Mānoa, 1955 East-West Road, Agricultural Science Building 218, Honolulu, HI 96822, USA

Abstract: In this study, hydrogenotrophic methanogenesis with respect to methanogenic activity and microbial structures under extreme-thermophilic conditions were examined, and compared with the conventional thermophilic condition. The hydrogenotrophic methanogens were successfully acclimated to the temperatures of 55, 65 and 70°C. Although acclimation was slower at 65 and 70oC, hydrogenotrophic

methanogenesis

remained

fairly

stable.

High-throughput

sequencing using 16S rRNA analysis showed that the higher temperatures resulted in single archaea community dominated by hydrogenotrophic Methanothermobacter. Moreover,

the

syntrophic

Thermodesulfovibrio

at

bacteria

55oC

to

changed

from

Coprothermobacter

Thermodesulfovibrio

at

70oC.

and

Specific

hydrogenotrophic methanogenic rate at 70°C was 98.6±4.2 Nml CH4/g VS/hr, which was over 4-folds higher than that at 55°C. The lag phase under extreme-thermophilic conditions was longer than thermophilic condition, which was probably due to the archaeal structure with low diversity. Extreme-thermophilic condition resulted in a shift

in

methanogenesis

pathway

from

acetoclastic

methanogenesis

to

hydrogenotrophic methanogenesis with the enrichment of Methanothermobacter thermautotrophicus.

Keywords: Biogas upgrading, Extreme-thermophilic condition, Hydrogenotrophic methanogenesis, Microbial community

1. Introduction Anaerobic digestion has been widely adopted both to stabilize diverse organic wastes and to produce renewable energy in the form of biogas. The produced biogas primarily contains 50-75% CH4 and 25-40% CO2, and is commonly used for heat and electricity generation in combined heat and power unit (Li and Khanal, 2016). Upgrading of biogas with CH4 content over 90% has a high volumetric calorific value (~36 MJ/Nm3), and could provide several opportunities for its utilization, such as natural gas that could be injected into a gas grid, compressed natural gas as a transportation fuel and in the synthesis of methanol among others (Deng and Hagg, 2010; Waugh, 2012). In recent years, biological upgrading via external H2-supplementationas as an alternative upgrading technology, has attracted significant attention (Bassani et al., 2015; Luo and Angelidaki, 2012). CO2 in biogas could be captured by biologically combining with H2 to produce CH4 through hydrogenotrophic methanogenesis (Rittmann, 2015). Pure culture of hydrogenotrophic methanogens, such as Methanobacterium thermautotrophicus, Methanothermobacter marburgensis and Methanococcus maripaludis had been investigated for their potential to convert CO2 and H2 into CH4 (De Poorter et al., 2007; Goyal et al., 2015; Martin et al., 2013; Seifert et al., 2013). Microbial contamination, however, was reported with pure cultures and the H2 conversion efficiency decreased from 89 to 62%, when the substrates were switched from synthetic H2/CO2 mixture to real biogas (Martin et al., 2013). Compared to pure cultures, mixed culture system with diverse microbial interactions is considered more resilient to various environmental perturbations (Angelidaki et al., 2011).

Hydrogen supplementation into mixed cultures led to

an increase in

hydrogenotrophic methanogens and syntrophic bacteria, indicating that the shift of hydrogen-mediated hydrogenotrophic pathway enhanced the biogas upgrading (Bassani et al., 2015). CH4 content as high as 95% was obtained through hydrogenotrophic methanogens enriched from digested sludge and cattle manure (Luo and Angelidaki, 2012). Both mesophilic and thermophilic anaerobic sludges had biomethanation potential from H2 and CO2, while CH4 yield under thermophilic condition (55°C) was 60% higher than that under mesophilic condition (37°C) (Luo and Angelidaki, 2012). Temperature influenced the microbial community distribution of mixed cultures. About one-third of the methanogens were hydrogenotrophic methanogens under mesophilic conditions (Fey and Conrad, 2000), while they became dominant under thermophilic conditions (Bassani et al., 2015; Pap et al., 2015). In addition, metaproteome analysis further demonstrated that methanogenic pathway in a mixed culture system shifted from acetoclastic to hydrogenotrophic methanogenesis with increasing temperature from 43 to 52oC (Kohrs et al., 2014). Recently various research studies reported the effects of temperatures on the microbial

community

Hydrogenotrophilic

under

extreme-thermophilic

methanogens

related

to

conditions

(65-80oC).

Methanothermobacter

thermautotrophicus were the dominant methanogens at 70°C. In the above studies, organic substrates, such as glucose, and waste activated sludge were used (Tang et al., 2008; Wu et al., 2016). Ahring and his co-workers found that methanogenic activity measured with glucose, acetate, propionate and butyrate reduced significantly, and only hydrogen consuming methanogens showed an enhanced activity at 65oC, which was nearly 1.5-folds higher than that at 55°C (Ahring et al., 2001). Thus, biomethanation from H2 and CO2 is assumed to be significantly enhanced under

extreme-thermophilic condition, considering the adaptation of hydrogenotrophic methanogens to higher temperature. However, high temperature may limit the solubility of gaseous substrates such as H2 and CO2 in the aqueous phase. To the best of our knowledge, studies on hydrogenotrophic methanogenesis using mixed culture under extreme-thermophilic condition and associated microbial community have not been reported. Based on above rationales, the overall goal of this study was to evaluate the performance

of

hydrogenotrophic

methanogenesis

under

thermophilic

and

extreme-thermophilic conditions using anaerobic granular sludge fed with H2 and CO2. Coenzyme F420 content and associated microbial community were examined. Gaseous substrates consumption, CH4 yield and metabolites formed at 55, 65 and 70°C were also compared, along with the discussion on specific methane activity with H2/CO2 and lag phase for methane production.

2. Materials and methods 2.1 Inoculum The granular sludge obtained from an upflow anaerobic sludge blanket (UASB) reactor treating a local paper mill wastewater, was used as an inoculum. The collected UASB granule sludge was stored at -20°C and was slowly thawed at 4°C over a period of 2 days, then washed twice with distilled water before being used as an inoculum. The total solids (TS) and VS of the inoculum were 143.5±9.5 and 101.9±6.2 g/L, respectively, with pH of 8.0±0.1. Basal anaerobic (BA) medium was used in the acclimation experiment and in the hydrogenetrophic methanogenic activity (HMA) assay was conducted as described in Angelidaki and Sanders (2004).

2.2 Experimental setup and operation 2.2.1 Mixed culture cultivation with H2/CO2 at 55, 65 and 70°C The inoculum was acclimated at temperatures of 55, 65 and 70°C (±1°C) in a series of 300-ml bottles with a working volume of 100 ml using three water-bath shakers at a speed of 120±5 rpm. The bottles were assembled with a welded stainless steel gas tube and butyl-rubber sealing gasket, and tested for gas-tightness prior to the start of experiment. Each bottle contained 80 ml BA medium and 20 ml inoculum with a VS concentration of 20 g/L. All the bottles, except controls, were flushed with high-purity H2 (>99.99%) for 5 min and sealed. The bottles were then injected with 45.8 Nml of CO2 (purity >99.99%, and corrected to standard temperature and pressure (STP) conditions hereafter denoted as N). For the control group, the bottles were flushed with N2 (purity >99.99%) for 5 min, sealed, and then injected with 45.8 Nml of N2 (purity >99.99%) to maintain the same initial pressure. Both head-space gas and liquid samples were sampled every other day, 10% of the mixed culture (10 ml) was replaced with same volume fresh BA medium, and the pH in each bottle was adjusted during sampling to 7.5±0.1 using HCl and/or NaOH. The bottles were then reinjected with H2/CO2 or N2. During the initial period (5-20 days), the H2/CO2 ratio was increased by injecting additional 55 Nml of H2 (purity >99.99%) into the bottles following CO2 injection, to allow consumption of excess CO2. From day 83 to138, the bottles were operated without pH control, and gaseous substrates (H2/CO2) were supplemented once a week. On day 139, the operation was restarted with supplementation of H2/CO2 every day. After 159 days of acclimation, the sludge samples were collected from each bottle for coenzyme F420 and microbial community analysis. Coenzyme F420 was extracted and analyzed spectrophotometrically as described in Eirich et al. (1978).

2.2.2 Specific methanogenic activity (SMA) with H2/CO2 SMA assays of the sludge acclimated at temperatures of 55, 65 and 70 oC during steady-state condition were carried out with supplementation of H2/CO2. The acclimated sample (5 ml) was transferred to a 133-ml gas-tight glass bottle containing 15 ml of BA medium. The bottle was then flushed with high-purity H2 (>99.99%) for 5 min, sealed and injected with 27.5 Nml of CO2 (purity >99.99%). Six set of bottles were prepared, sampled individually every 4 hr, and then discarded. For each temperature (55, 65 and 70°C), two of these series served as duplicates for the acclimation group and a third series, comprising of bottles flushed with N2, as the control. An inhibition experiment using 2-bromoethanesulfonate (40 mmol/L) (to suppress hydrogenotrophic methanogenesis) was conducted similar to the SMA experiment. Modified Gompertz model was used to fit the experimental data for CH4 production as given below (Lü et al., 2013; Lay et al., 1997), y  P  exp( exp(

Re  ( λ  x)  1)) P

(1)

Where P is the optimum CH4 production (Nml), R the maximum CH4 production rate (Nml CH4/hr), and λ the lag phase (hr). The independent variable x and dependent variable y are the reaction time (hr) and the CH4 production (Nml), respectively. Each three-parameter-set was estimated by nonlinear curve-fitting using Origin®, with a minimum residual sum of squared errors between the experimental data and model curves.

2.3 Analytical methods Biogas production was calculated based on the headspace volume and pressure. The latter was measured using a pressure transducer (Coates et al., 1996). Gas

composition was determined using gas chromatography equipped with a thermal conductivity detector (GC-TCD) (Agilent 6890N, CA, USA) as described previously (Xie et al., 2014). The pH of the liquid sample was measured immediately using a HQ11D pH meter (HACH, CO, USA) to avoid interference with CO2 desorption. Liquid samples were centrifuged at 10,000 rpm (15550 × g) for 10 min and the supernatant was analyzed for chemical oxygen demand (COD) and individual volatile fatty acids (VFAs). The COD was analyzed as per Standard Methods (APHA, 2005). The individual VFAs were analyzed using gas chromatography (GC) equipped with flame ionization detector (GC-FID) (Thermo Fisher Scientific, MA, USA), and analytical column CPWAX52CB (30 m × 0.25 mm × 0.25 μm; Agilent Technologies, CA, USA). The TS and VS contents of the sludge were measured according to Standard Methods (APHA, 2005).

2.4 High-throughput 16S rRNA gene sequencing and analysis 2.4.1 DNA extraction and quantification Both the inoculum and the acclimated samples were sequenced and analyzed to identify the microbial communities under different conditions. Total DNA was extracted using the E.Z.N.A. ® soil DNA kit (Omega Bio-tek, GA, USA) following the manufacturer’s instructions. The DNA concentration was measured using Qubit 2.0 fluorimeter (Life Technologies, MA, USA), and the quality of the analysis was checked using the Agilent 2100 bioanalyzer (Agilent Technologies, CA, USA) to ensure that adequate amounts of high-quality genomic DNA were extracted. 2.4.2 16S rRNA gene amplification by polymerase chain reaction (PCR) The target was the V3-V4 hypervariable region of the bacterial and archaeal 16S

rRNA genes. Polymerase chain reaction (PCR) was started immediately after DNA extraction. The 16S rRNA V3-V4 amplicon was amplified using KAPA HiFi Hot Start ready mix (TaKaRa Bio Inc., Japan). The two universal bacterial 16S rRNA gene amplicon PCR primers (PAGE purified) were the amplicon PCR forward (CCTACGGGNGGCWGCAG) and reverse (GACTACHVGGGTATCTAATCC) primers (Herlemann et al., 2011). Archaea were identified using the amplicon PCR forward

(GYGCASCAGKCGMGAAW)

and

reverse

(GGACTACVSGGGTATCTAAT) primers (Takai and Horikoshi, 2000). The reaction was set up as follows, microbial DNA (10 ng/μl) 2μl, amplicon PCR forward primer (10 μM) 1μl, amplicon PCR reverse primer (10 μM) 1μl, 2× KAPA HiFi Hot Start Ready Mix 15 μl (total 30 μl). The plate was sealed and PCR performed in Applied Biosystems 9700 (ThermoFisher Scientific, USA) using the following program, 1 cycle of denaturing at 95°C for 3 min, first 5 cycles of denaturing at 95°C for 30 s, annealing at 45°C for 30 s, elongation at 72°C for 30 s, then 20 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, elongation at 72°C for 30 s and a final extension at 72°C for 5 min. The PCR products were checked using electrophoresis in 1 % (w/v) agarose gels in TBE buffer (Tris, boric acid, EDTA) stained with ethidium bromide (EB) and visualized under UV light. 2.4.3 16S gene library construction and sequencing AMPure XP beads (Beckman Coulter, USA) were used to purify the free primers and primer dimer species in the amplicon products. A library was constructed using the universal Illumina adaptor and index. Depending on the coverage needs, all libraries were pooled for one run. Amplicons from each reaction mixture were pooled in equimolar ratios based on their concentration. The reaction products were sequenced using the Illumina MiSeq system (Illumina, CA, USA) according to the

manufacturer’s instructions. After sequencing, data were collected as follows, (1) The two short Illumina readings were assembled by PEAR (v0.9.6) software according to the overlap and fastq files were processed to generate individual fasta and qual files, which could then be analyzed by standard methods. (2) Sequences containing ambiguous bases and any longer than 480 base pairs (bp) were dislodged and those with a maximum homopolymer length of 6 bp were allowed (Köchling et al., 2015). And sequence short than 200 bp were removed. (3) All identical sequences were merged into one. (4) Sequences were aligned according to a customized reference database. (5) The completeness of the index and the adaptor was checked and removed all of the index and the adaptor sequence. (6) Noise was removed using the Pre.cluster tool. Chimeras were detected by using Chimera UCHIME. All the software was in the mothur package. 2.4.4 Data processing Effective sequences after processing were submitted to the Ribosomal Database Project (RDP) Classifier to confirm the archaeal and bacterial sequences. Species richness and diversity statistics, including the Coverage, Chao1, ACE, Simpson, and Shannon indexes, were also calculated using mothur (Schloss et al., 2009). The modified pipeline is described in the mothur website (www.mothur.org). Finally, all effective sequences without primers were submitted for downstream analysis (Kozich et al., 2013).

2.5 Statistical analysis The analysis of variance (ANOVA) was performed using Microsoft Office Excel, and p-value <0.05 was considered to be statistically significant.

3. Results and discussion 3.1 H2/CO2 bio-methanation in semi-continuous mixed cultures at 55, 65 and 70oC In this study, H2 and CO2 biomethanation using anaerobic granular sludge as inoculum was conducted under semi-continuous mode at thermophilic (55°C) and extreme-thermophilic (65 and 70°C) conditions. Profiles of CH4 production, H2/CO2 consumption, and the VFAs concentration with operation time, are shown in Fig. 1. In the first 5 days, CH4 was produced rapidly. The supplemented CO2 at a ratio of 4:1 (v/v) was gradually consumed at the three tested temperatures. A higher H2/CO2 ratio of 5 was maintained during day 5 to 20 to allow the consumption of excess CO2. It was then reduced to 4 once the accumulation of H2 was observed, and the CH4 production gradually increased and stabilized. The CH4 production stabilized within 40 days at 55oC where as it took nearly 60 days at 65 and 70°C. CH4 productions at three tested temperatures were stable and reached the theoretical value 45.8 Nml based on the amount of CO2 supplemented after enrichment. The results showed that acclimation occurred more rapidly at 55°C than those at 65 and 70°C. Although the extreme-thermophilic condition had a longer acclimation time, their CH4 production were relatively stable as shown in Fig. 1. On day 139, the three systems were restarted with pH controlled at 7.5. The CH4 productions were 43.3±3.0, 46.1±2.3, and 46.9±1.6 Nml CH4/day at 55, 65 and 70°C, respectively. The CH4 produced at the tested temperatures was higher than that in the control groups. The CH4 produced was not significantly different among three temperatures (p-value ~ 0.23), indicating that hydrogenotrophic methanogenesis was the main pathway for methane production. Initially, the VFAs concentration was relatively high, and increased with the increasing temperature. The initial total VFA concentration at 70°C was almost 3-and

2-folds higher than that at 55°C and 65°C (1,300, 2,300 and 4,400 mg/L as acetic acid equivalent (HAc) at temperatures of 55, 65 and 70°C, respectively) (Fig. 1). At all temperature conditions, the VFA levels decreased gradually, and the VFAs concentrations were in the range of 10-150 mg-HAc/L at the end of the experiment. This was mainly attributed to the dilution effect due to media replacement every other day. Moreover, the initial high VFAs production as discussed above, was contributed by the hydrolysis of anaerobic sludge, and the high temperature enhanced the sludge hydrolysis. At the end of the enrichment (day 150), the reduction of VS content decreased to 55% at 70°C due to hydrolysis. Although the same levels of VFAs were observed in the tested conditions and the controls (without H2/CO2 supplementation), CH4 production was negligible in the controls. It is therefore apparent that CH4 productions at three tested temperatures were associated with hydrogenotrophic methanogenesis utilizing CO2 and H2, and not from acetoclastic methanogenesis. In this study, hydrogenotrophic methanogens were successfully enriched under the three temperature condition, and their activities were not inhibited by the presence of high VFAs concentrations.

3.2 Relative concentration of coenzyme F420 under the three temperature conditions Coenzyme F420 plays an important role as an electron carrier in methanogenesis and facilitates the reduction of methenyl-H4MPT to methylene-H4MPT to accomplish electron transfer (Deppenmeier, 2002; Zabranska et al., 1985). The coenzyme F420 content in acetoclastic methanogens were much lower than that in hydrogenotrophic methanogens. In this study, coenzyme F420 of sludge (at day 159) and inoculum were measured to determine the effects of temperature on the relative intensity of

hydrogenotrophic methanogenesis. Relative concentration of coenzyme F420 broth with H2/CO2 were similar, 3.18±0.27, 3.48±0.56 and 3.66±0.31×10-2 mmol/g-VS at 55, 65 and 70°C, respectively, the values were significantly higher than the inoculum (2.47±0.10×10-2 mmol/g-VS) (p-value <0.01). Coenzyme F420 was abundant in mixed cultures broth with H2/CO2. Recent study demonstrated that the F420 related processes would be directly coupled to and most sensitive to H2 concentrations (Hendrickson et al., 2007). Therefore, the results further demonstrated that the hydrogenotrophic methanogens were enriched at the three tested temperatures. However, the coenzyme F420 does not reflect the differences in activity of hydrogenotrophic methanogens among the tested thermophlic and extreme-thermophilic conditions. Thus, further study was conducted to examine the microbial communities using 16S rRNA gene high-throughput sequencing technique as discussed in the following section.

3.3 Composition of microbial community at 55, 65 and 70oC In this study, a total 228,163 sequence reads were tested by 16S rRNA gene high-throughput sequencing of the V3-V4 region of the acclimated sludges at 55, 65 and 70°C. The length of most sequences were between 350 to 400 bp with an average length of 380 bp after quality-filtered process. All the sequences were clustered into operational taxonomic units (OTUs) with the identity threshold of 0.97. The rarefaction curves of archaea and bacteria showed that the number of sequencing for archaea (more than 23,000) and bacteria (more than 45,000) were sufficient, since all the curves reached plateau. The differences in the microbial communities are indicated by the alpha-diversity indices as shown in Table 1. Based on the coverage values of archaea (>99.0%) and bacteria (>97.2%), the majority of the total OTUs were detected and included in the assay. The lower Shannon values of archaea under

two extreme-thermophilic conditions (0.44 at 70°C, 0.64 at 65°C) than that at 55°C (1.34) suggested that the diversity of archaea decreased with the increasing temperature. However, the diversity of the bacterial community was more complex in contrary to the archaea. Extreme-thermophilic temperature, especially at 70°C showed the highest bacterial diversity, and it declined with decreasing temperature (Shannon and Simpson indices were 3.70 and 0.05 at 70°C, 3.21 and 0.10 at 65°C, and 3.03 and 0.14 at 55°C, respectively). The higher OTU numbers and Shannon diversity of bacteria compared to archaea further indicated that bacteria were more diverse than archaea. In addition, the higher temperature seemed to reduce the diversity of archaea; but enriched the diversity of bacteria. The effective gene sequences of each sample were assigned to different taxa levels (from genus to phylum). The identified phyla of archaea community for the samples were mainly Euryarchaeota and Thaumarchaeota. The relative abundance of Euryarchaeota (including methanogens) ranged from 82.6% to 95.5% in four samples. Fig. 2 shows the taxonomic classfication of archaea sequences by RDP classfier, and the distribution in order and genus levels for the tested samples. At the order level, the inoculum used in this study mainly consisted of Methanosarcinales (81.2% of total archaea), with Methanobacteriales accounting for only a small percentage (10.0% of total archaea). Methanosarcinales decreased to 50.5% at 55°C, and was not detectable at 65 and 70°C, whereas Methanobacteriales increased to 41.4, 82.5 and 92.9% at 55, 65 and 70°C, respectively. Compared to inoculum, enrichment of hydrogenotrophic methanogens was achieved with a major change in the archaea community. With respect to genus level, Methanosarcinales was primarily represented by strict acetoclastic Methanothrix, while Methanobacteriales comprised of two hydrogenotrophic Methanothermobacter and Methanobacterium.

Archaea community of extreme-thermophilic sludge samples were mostly homogeneous and the only methanogen detected was Methanothermobacter (82.4% at 65oC, 92.8% at 70oC). Further identification showed that the species of Methanothermobacter were Methanothermobacter thermautotrophicus in the cultures. However, 50.4% Methanothrix, 39.0% Methanothermobacter and 2.1% Methanobacterium were detected at 55°C. The microbial communities varied from acetoclastic to hydrogenotrophic when the acclimation temperature was increased from thermophilic to extreme-thermophilic. The genus Methanothermobacter, mediating hydrogenotrophic methanogenesis, was reported to be dominant in other extreme-thermophilic

biogas

reactor.

Wu

et

al.

(2016)

reported

that

Methanothermobacter was enriched with abundance of 87.3% at 70°C in anaerobic digester treating waste activated sludge. The above results further indicated that organisms related to the genus Methanothermobacter was the dominant methanogen in the culture broth at >65oC. Thus, it was probably that the acclimation temperature played the critical role, not the substrates (organics or H2/CO2) determing the methanogenic communities. Extreme-thermophlic condition should be beneficial for the enrichment of hydrogenotrophic methanogens. Bacteria struture among the three acclimated samples showed significant difference compared to the inoculum. At the phylum level, the inoculum mainly consisted of Proteobacteria (70.8% of total bacteria) and Chloroflexi (10.4% of total bacteria), while the phylum Firmucutes was identified at a lower proportion of 5.4%. However, in

samples

acclimated

with

H2/CO2

under

both

thermophilic

and

extreme-thermophilic conditions, the dominance of the phylum Firmicutes about 50% was observed. This finding was in close agreement with previous studies in which Firmicutes was the predominant phylum with 32% of the microbial community at

55oC after supplementation of H2 (Bassani et al., 2017). Wu and his co-workers also reported that phylum Firmicutes was dominant (~43.2%) in the extreme-thermophilic anaerobic digester (70oC) of waste activated sludge, and concluded that digestion at 70oC was important to increase the proportion of Firmicutes in the sludge (Wu et al., 2016). Based on the results of these studies, the phylum Firmicutes was not affected by the substrates, but governed by the temperature, especially thermophilic or extreme-thermophilic conditions ≥ 55oC. In all acclimated samples, Proteobacteria were reduced to less than 12% and Chloroflexi was nearly non-detectable. Nitrospirae was the second most abundant phylum with relative abundance of about 10%. The phylum Synergistetes accounted for 8.3% at 55oC in the acclimated samples, but it decreased continuously with increasing temperatures. The bacteria communities at order and genus levels in inoculum and the three acclimated

samples

are

presented

in

Fig.

3.

At

the

order

level,

Thermoanaerobacterales (phylum Firmicutes) was dominant in all three acclimated samples, but with a decreasing tend with increasing temperature (40.1% of total bacteria at 55°C, 35.2% at 65°C and 22.9% at 70°C). While two orders, Clostridiales and Bacillales, belonging to phylum Firmicutes, increased with increasing temperature (3.5% and 1.8% of total bacteria at 55°C, 9.5% and 4.5% at 65°C, and 14.9% and 9.9% at 70°C, respectively), Nitrospirales (phylum Nitrospirae) seemed to be independent of temperature, and was stable at 13.6% of total bacteria at 55°C, 17.7% at 65°C and 14.9% at 70°C. At the genus level, the richness and diversity of bacterial community increased with increasing temperature (Fig. 3). The unclassified Coprothermobacter, representing Thermoanaerobacterales, were dominant at 55°C (33.3%) and 65°C (21.4%), but decreased to 1.8% at 70°C. Coprothermobacter is mainly identified as thermophilic

proteolytic bacteria that could produce hydrogen from protein. These bacteria can be isolated from most of the methanogenic environments and were previously reported to be syntrophically related to hydrogenotrophic methanogens (Gagliano et al., 2015; Luo and Angelidaki, 2013; Yabu et al., 2011). Higher degradation rate of proteinaceous substrate was achieved by co-culture of Coprothermobacter and Methanothermobacter thermautotrophicus than mono-culture, and metabolite was converted from H2 to CH4 in co-culture (Sasaki et al., 2011). In previous study, genus Coprothermobacter was also dominant in a 65°C single-stage wasted activated sludge (WAS) digester, and the relative abundance at SRT of 6, 7.5 and 10 days were 54.1%, 58.4% and 66.0%, respectively (Chen et al., 2018), which were much higher than our study. This was likely due to the fact that the digester was fed with WAS at high organic loading rate continuously, and large amount of organic substrates might have benefitted the growth of Coprothermobacter. In addition, our study showed a declining abundance of Coprothermobacter strains with increasing temperature. It might due to the unclassified Coprothermobacter, which was not adapted to higher temperature. But also due to the fact that that lesser amount of organic compounds remained under extreme-thermophilic conditions due to higher endogenous degradation during acclimation period. Further research is needed to investigate this. Another interesting finding is related to identification of genus Thermodesulfovibrio (order Nitrospirales) with around 15% of total bacteria in all three acclimated samples. But different species of Thermodesulfovibrio were existed in these temperature conditions. For example, at two extreme-thermophilic conditions, Thermodesulfovibrio aggregans contributed 17.6% of total bacteria at 65°C and 14.8% at 70°C, while it only accounted 2.2% of total bacteria at 55°C. The major species at 55°C belonged to unclassified species, with nearly 11.4% of total bacteria,

which was undetectable under extreme-thermophilic conditions. Thermodesulfovibrio relatives and Methanothermobacter thermautotrophicus were found to be enriched and could be associated with syntrophic group by using anaerobic sludge obtained from a methanol-treatment bioreactor as inoculum feeding with H2/CO2 (Roest et al., 2005). Sekiguchi et al. (2008) further revealed that Thermodesulfovibrio were able to grow in co-culture with Methanothermobacter thermautotrophicus without sulfate as an electron acceptor. Apart from laboratory-scale bioreactor, the syntrophic relationship of Thermodesulfovibrio and hydrogenotrophic methanogens can also be found in natural ecosystem. Nazina et al. (2006) revealed that diverse bacterial community represented by Thermodesulfovibrio and Coprothermobacter were found in extreme-thermophilic petroleum reservoirs conducting syntrophic processes closely related to Methanothermobacter thermautotrophicus. The proportion of Exiguobacterium (Bacillales, Firmicutes) was found to increase with increasing temperature (1.4% at 55oC, 3.4% at 65oC and 8.7% at 70oC), and the majority of Exiguobacterium in this study was Exiguobacterium mexicanum. Exiguobacterium was reported to be a facultatively thermophilic bacteria, participating in carbohydrate fermentation and produce lactate as main product (Crapart et al., 2007). In this study, we observed high VFA concentration at 65 and 70oC. Therefore, the increase of Exiguobacterium was in agreement with the increased in VFA concentration. Caldanaerobacter (Thermoanaerobacterales, Firmicutes) (0.02% at 55oC, 3.0% at 65oC and 8.8% at 70oC), is hydrogenetrophic thermophilic bacterium that can grow lithotrophically on CO to produce CO2 and H2 (Fardeau et al., 2004).

3.4 SMA with H2/CO2 at 55, 65 and 70oC SMA assays with H2/CO2 were conducted to quantify and compare the hydrogenotrophic activity under thermophilic and extreme-thermophilic conditions. As shown in Fig. 4, amounts of methane obtained under thermophilic were in agreement with that under extreme-thermophilic conditions, however the production rate and lag phase were significantly different. CH4 production started after nearly 8 and 16 hr of inoculation under thermophilic and extreme-thermophilic conditions, respectively. Although higher temperature extended the lag phase, CH4 production rate increased rapidly. The experimental data confirmed well with the modified Gompertz equation (r2 >0.993, Fig. 4), and the relevant parameters are summarized in Table 2. The lag phase times (λ) were 17.1 and 15.3 hr at 65 and 70°C, respectively, which were nearly 2-times longer than that at 55°C (8.0 hr). Initially, it was thought that the low availability of H2/CO2 at higher temperature due to low solubility, could have contributed to a longer lag phase at higher temperature. However, the solubility of these gaseous substrates was not significantly different under thermophilic and extreme-thermophilic conditions. Although the lag phase was longer under extreme-thermophilic conditions, CH4 production rate was the highest. The SMA results for H2/CO2 showed the highest specific hydrogenotrophic methanogenic rate at 70°C with 98.6±4.2 Nml CH4/g VS/hr, which was over 4-folds higher than that at 55°C (23.9±2.1 Nml CH4/g VS/hr) and 1.4-folds higher than that at 65°C (68.1±6.1 Nml CH4/g VS/hr). The SMA at 65°C was nearly 3-folds higher than that at 55°C, which was in close agreement with other study (Ahring et al., 2001). The above results demonstrated that hydrogenotrophic methanogenesis was influenced by temperature, and higher temperature enhanced the SMA activity with H2/CO2. Moreover, in this study,

2-bromoethanesulfonate was added into the SMA experiment to examine whether other hydrogen consuming pathways contributed to H2 consumption. The results showed that no H2 was consumed during the experiment under extreme-thermophilic conditions, while about 17.8% of H2 was consumed under thermophilic conditions. This suggests that under extreme-thermophilic conditions, hydrogenoptrophic methanogens were the only hydrogen consumers. Fig. 5 describes the evolution of performance and microbial community of hydrogenotrophic methanogenesis from 55 to 70°C. CH4 was mainly produced by hydrogenotrophic methanogens utilizing H2/CO2, and the presence of VFAs did not contribute to methane production under thermophilic and extreme-thermophilic conditions. Dominant methanogenic community shifted from acetoclastic to hydrogenotrophic methanogens indicating the importance of temperature on their distribution. More homogenous methanogens were enriched at 70oC, and Methanothermobacter thermautotrophicus occupied 99.8% of total methanogens. The longer lag phase time (about 15 hr) at 70oC was probably due to the homogenous methanogens M. thermautotrophicus, which may need longer time to acclimatize. While more diversified bacteria structure was obtained at 70oC, along with the increasing numbers of unclassified species. The syntrophic bacteria associated with hydrogenotrophic Methanothermobacter changed from Coprothermobacter and Thermodesulfovibrio at 55oC to Thermodesulfovibrio and Exiguobacterium at 70oC. Specific hydrogenotrophic methanogenic rate at 70°C was 98.6±4.2 Nml CH4/g VS/hr, which was over 4-folds higher than that at 55°C. It was suspected that the more diversified bacteria attributed to the increased methane production rate.

4. Conclusions Hydrogenotrophic

methanogens

were

successfully

enriched

under

extreme-thermophilic condition. The diversity of archaea decreased with increasing temperature, mainly dominated by M. thermautotrophicus. More diversified bacteria structure

was

observed.

The

dominant

bacteria

Coprothermobacter

and

Thermodesulfovibrio were syntrophically related to M. thermautotrophicus at 55oC, shifting to Thermodesulfovibrio and Exiguobacterium at 70oC. Methane production rate at 70oC was over 4-folds higher than that at 55°C, indicating that activity of hydrogenotrophic methanogens was enhanced. The longer lag-phase at 65 and 70oC was probably related with the simplification in methanogens. The above results showed

a

great

potential

of

hydrogenotrophic

methanogenesis

under

extreme-thermophilic condition for industrial applications. Supplementary material for this work can be found in e-version of this paper online. Acknowledgement This study was supported by National Science Foundation of China (Grant No. 51678424 and 51378373), the Fundamental Research Funds for the Central Universities and PCRRE16105.

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Fig. 1 Variation of gas composition (CH4, CO2 and H2) and VFAs concentration at 55oC (a, b), 65oC (c, d) and 70oC (e, f)

Fig. 2 Archaeal community of inoculum and the acclimated samples at 55, 65 and 70oC (a. order level, b. genus level; Others, represents the sum of microbes besides listed and unclassified whose percentage was < 0.5% at Genus level)

Fig. 3 Bacterial community of inoculum and the acclimated samples at 55, 65 and 70oC (a. order level, b. genus level; Others, represents the sum of microbes besides listed and unclassifiedwhose percentage was < 2% at Order level and < 3% at Genus level)

Fig. 4 Modified Gompertz Fitting of hydrogenotrophic methanogenesis activity at 55, 65 and 70oC

Fig. 5 Evolution of performance and microbial community of hydrogenotrophic methanogenesis from 55°C to 70°C

30

Table 1 Comparison of the Alpha diversity indexes of the archaea communities among three acclimated sludge samples at 55, 65 and 70oC Sample

Sequence reads

OTU numbers

Shannon index

Simpson

Coverage

55oC

29954

339

1.34

0.39

0.990

65oC

23745

264

0.64

0.70

0.990

70oC

28479

268

0.44

0.86

0.992

55oC

48998

1338

3.03

0.14

0.978

65oC

45469

1285

3.21

0.10

0.977

70oC

51518

1807

3.70

0.05

0.972

Archaea

Bacteria

31

Table 2 Parameters of modified Gompertz fitting of hydrogenotrophic methanogenesis T o ( C) 55 65 70

P (Nml) 25.9 25.9 25.9

Rmax (Nml CH4/hr) 2.2±0.2 4.2±0.4 4.6±0.2

λ (hr) 8.0±0.6 17.1±0.3 15.3±0.1

r2 0.993 0.998 0.999

SMA (Nml CH4/g VS-hr) 23.9±2.1 68.1±6.1 98.6±4.2

32

33

Highlights: 

Hydrogenotrophic methanogenesis was stable under extreme-thermophilic condition.



Higher CH4 rate was achieved at 70oC, but with longer lag phase.



Homogenous archaea and diversified bacteria community were obtained at 65 and 70oC.



Methanothermobacter thermautotrophicus was dominant with 92% at 70oC.



Dominant bacteria were syntrophiclly related to M. thermautotrophicus.

34