Exploit Carbon Materials to Accelerate Initiation and Enhance Process

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Exploit Carbon Materials to Accelerate Initiation and Enhance Process Stability of CO Anaerobic Open-Culture Fermentation Fan Lü, Ke-jian Guo, Hao-wen Duan, Li-ming Shao, and Pin-Jing He ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04589 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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ACS Sustainable Chemistry & Engineering

Fan Lü

E-mail:

[email protected]

Ke-jian Guo

E-mail:

[email protected]

Hao-wen Duan

E-mail:

[email protected]

Li-ming Shao

E-mail:

[email protected]

Pin-jing He

E-mail:

[email protected]

1

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Exploit Carbon Materials to Accelerate Initiation and Enhance Process Stability of CO Anaerobic Open-Culture Fermentation Fan Lü,a,b,c Ke-jian Guo, a,b,c Hao-wen Duan, a,b,c Li-ming Shao,b,c,d Pin-jing He,*,a,b,c,d a

State Key Laboratory of Pollution Control and Resource Reuse, Tongji University,

Shanghai 200092, China b

Shanghai Institute of Pollution Control and Ecological Security, Shanghai200092,

China c

Institute of Waste Treatment and Reclamation, Tongji University, Shanghai 200092,

China d

Centre for the Technology Research and Training on Household Waste in Small

Towns & Rural Area, Ministry of Housing Urban-Rural Development, Shanghai 200092, China * Corresponding to: [email protected]

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Abstract The aim of this study was to investigate the possibility of improving anaerobic fermentation of carbon monoxide (CO), a dominant component in syngas, through the assistance of different carbon materials (activated carbon, biochar, graphene, carbon cloth, and carbon fibre). Results showed that only biochar and activated carbon could promote CO utilization when the pCO increased from 0.07 to 0.21 atm and promoted CO methanization but was delayed until the pCO further increased from 0.21 to 0.41 atm. Compared to the control without carbon material addition, the CO conversion rate increased by up to 149% and 193% and the methane (CH4) production rate was up to 238% and 186%, respectively, for the biochar and activated carbon scenarios. Graphene and carbon cloth did not accelerate CO utilization but could stabilize the bio-reaction at a high pCO. Natural stable carbon isotope signals revealed that CH4 production from CO was mainly via acetate at low pCO and via hydrogen at high pCO. Result from high-throughput sequencing showed that biochar and activated carbon boosted the growth of Rikenellaceae and Thiobacillus in the solid phase. Synergistaceae which can be in co-culture with Methanobacterium was boosted in the liquid phase in biochar group and activated carbon group. Key words: syngas fermentation; carboxydotrophic methanogenesis; syngas methanization, direct interspecies electron transfer; syntrophy; mixed culture

3



Introduction

The demand for biomass-based energy is increasing because renewable biomass can be converted to valuable products (e.g. biogas or liquid biofuels). However, a significant proportion of biomass (e.g. wood, straw, bark) and majority of synthetic waste (e.g. plastics, rubber) is difficult to be biodegraded due to the refractory and polymeric characteristics.1 Thermal gasification or pyrolysis of these materials can serve as a pretreatment method to produce syngas which is mainly composed of carbon monoxide (CO), hydrogen (H2), carbon dioxide (CO2), and other trace compounds.2 Syngas can be used as a combustion fuel directly, but in some applications low volumetric energy density (usually less than 13 MJ/m3, less than 40% of natural gas) limit its utilization.3 Further conversion of waste-derived syngas to bulk chemicals and fuels is preferable for energy storage, compression, long distance transportation and subsequent liquefaction of the gas4, or to be compatible with existing gas distribution infrastructure, such as pipelines and the various well-established and efficient end use technologies. This conversion can be achieved through chemical or biotechnological processes. Chemical processes are relatively mature but normally involve high pressures and/or temperatures, catalysts, require a constant ratio of CO to H2, and tend to have a low product specificity when impurities are present in the syngas.5 On the contrary, biotechnological processes can circumvent these disadvantages and are highlighted by high yields and low energy costs.6 Open-culture 4


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fermentation using mixed cultures is preferable for waste-derived syngas owing to the enrichment of carboxydotrophic microorganisms (CO-utilizing) and robustness of the microbial system.7 The availability and low cost of these organisms makes it high in practical value. The bioconversion of CO is the core process for syngas bio-utilization, since CO is used as both carbon and energy sources by specific carboxydotrophic microorganisms. CO is also potentially lethal to these microorganisms and other accompanying microorganisms due to their high affinity to metal-containing enzymes 8

. The bio-oxidation of CO can be coupled with several respiratory processes, such as

hydrogenogenesis, sulfate-reduction, acetogenesis, and methanogenesis.8 Table 1 summarizes the metabolic routes from CO and the Gibbs free energy of each. Although many valuable substances could be generated during these processes,9-10 bioconversion of CO-rich gases using mixed cultures is more popular for the production of methane11-12 for the sake of minimizing the requirement of downstream processing. Nevertheless, a few studies that use mixed cultures to transform syngas into liquid bio-chemicals have been identified13-14. In theory, as long as there is no limiting, metabolic route from CO to CH4 is thermodynamically more favorable than others (Table 1) and should prevail. In addition, CH4 is more easily separated from the liquid phase. To be noticed, in mixed cultures, the bioconversion of CO into specific metabolites may involve a variety of metabolic pathways and different species working cooperatively or antagonistically. For example, Navarro et al.12 found that in their reactor CH4 was produced from acetic acid synthesized from CO but not 5



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directly from carboxydotrophic methanogenesis. Unfortunately, the potential toxicity of CO may affect the metabolism of CO-utilizing functional microorganisms, leading to a longer growth doubling time and affecting the metabolic cooperation among functional ones and their neighbors.9 Most methanogens cannot tolerate CO. Daniels et al.15 reported that methane production by Methanothermobacter thermautotrophicus was inhibited by a CO partial pressure (pCO) above 0.3 atm. Severe inhibition to methanization even occurred at a pCO above 0.25 atm in a mixed culture.11 Once the functional microorganisms cannot consume enough CO in a timely manner, the excess CO can further exacerbate the inhibition process, leading to the instability of the entire process.9 Furthermore, sludge usually requires long adaptation time to achieve a higher CO conversion rate to methane.12, 16 Therefore, in order to reduce the long adaptation time and to maintain system stability by alleviating CO inhibition, new ways need to be explored to augment the metabolic capability and resistibility of microorganisms (i.e. to boost the growth of functional microorganisms and to facilitate mutual cooperation between different micro-organisms). Considerable research efforts have focused on strengthening the amount and activity

of

microbes

in

anaerobic

microsystem.

The

addition

of

environmentally-friendly carbon materials has been shown to be effective methods for the anaerobic digestion of organic matter.17-20 Activated carbon with a rich pore structure and high specific surface area has been reported to aggregate functional microorganisms leading to the acceleration of methanogenesis during digester start-up 6


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and high resistant to heavy loads.17 Biochar whose specific surface area is one tenth to one percent of activated carbon also has been reported to selectively enrich functional microbes tightly attached to it and also protects them from high substrate loadings.18 Carbon cloth and graphene were also reported to promote methanogenesis in the anaerobic digestion of complex organic wastes or glucose.19-20 In addition, these materials are all conductive and might promote direct interspecies electron transfer (DIET) then lead to the faster conversion of substrates.19, 21 However, to date there is no research focusing on enhancing CO conversion by adding carbon materials in mixed culture. Nevertheless, the beneficial roles of carbon materials in the anaerobic process and their practical application value (e.g., low cost and no environmental risk of biochar, reusable features of carbon cloth, utilizing waste to produce graphene-like structure material), implies that carbon materials might serve as beneficial additives for CO fermentation. Based on the above considerations, the objective of this work was to assess whether or not five different carbon materials (i.e., activated carbon, biochar, graphene, carbon cloth, carbon fibre) could improve the reactors’ performance under different pCO in mixed cultures. Stable carbon isotope signatures and high-throughput sequencing were used to reveal the methanogenic pathway and distribution of microbial communities in association with carbon materials or being planktonic.

Materials and Methods

Preparation of inoculum 7



The sludge seed was collected from a mesophilic reactor treating paper mill wastewater. The granular sludge seed was collected under an anaerobic atmosphere, sieved through a 2-mm mesh then adapted to a CO-fed anaerobic digester for 45 days as an inoculum. The inoculum utilized in this study had a total solids (TS) content of 12% by wt and a volatile solids (VS) content of 73% dry wt.

Set up of semi-continuous CO-fermentation experiments Six identical closed-loop gas-lift reactors with a working volume of 700 mL and a headspace volume of 450 mL were used. Each reactor had an influent and effluent port, a gas inlet port with a glass pipe, and a gas outlet port connected to a 1-L gas bag using a gastight tubing. The gas inlet pipe equipped with a bubble diffuser was extended to the bottom of the reactor. CO was conveyed using a peristaltic pump and passed through the bubble diffuser from the same gas bag to form a closed loop. To avoid O2 contamination and leakage amplified by the long-time feeding and high flow rate, gas circulation was run for 3 hours once a day (every 2 hours for a period of 15 minutes) with a timer switch control. Gas flow rates were controlled at a rate of 75 mL/min. Gas in the gas bags was replaced every two days. Meanwhile, the reactor was purged with high purity (99.999%) nitrogen for a total of seven minutes to exhaust any gaseous residues in the headspace to ensure that the initial CO content of each reactor was the same when connected to the air bag. Each reactor received 60-g of sludge inoculum (equal to a VS content of 5.1 g) at the start of the experiment. After a start-up period of six days, 5 g of carbon material 8


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was added to each reactor as follows: (1) activated carbon (75–150 µm), (2) biochar (75–150 µm), (3) graphene (3.4–8 nm thickness and 3–10 layers), (4) carbon cloth (8×8×0.036 cm3), and (5) carbon fibre (8×7×0.032 cm3). The remaining reactor received no materials and was considered as the control. Basic nutrient solution (Text S1 of Supporting Information) were supplied to sustain microbial growth. All reactors temperature and pH were controlled at 35±1 ºC and 6.5 to 7.8, respectively. pH values during the experiment are shown in Figure S1 (Supporting Information). According to the pCO, the experiment was divided into four stages: I (0–62 d), II (63–92 d), III (93–120 d), and IV (121–156 d), corresponding to an initial pCO of 0.07 atm, 0.21 atm, 0.41 atm and 0.69 atm, respectively. The CO content in the fed gas was adjusted using nitrogen (N2) gas. At the end of each stage, 30 mL liquid-solid mixtures were collected, frozen in liquid nitrogen, and stored at -80 ºC.

Physio-chemical analysis of gas and liquids The stable carbon isotopic signatures of CH4 (δ13CH4) and CO2 (δ13CO2) were periodically monitored using an isotope ratio mass spectrometry (Delta V Advantage, Thermo Finniga, USA) linked to a gas chromatography (Trace Ultra, Thermo Finniga, USA). Gas components (H2, CH4, CO, CO2, O2, N2,) and alcohols (including methanol, ethanol, propanol, iso-butanol, and butanol) were measured by gas chromatography. Volatile fatty acids (VFAs, including formate, acetate, propionate, n-butyrate, iso-butyrate, n-valerate, and iso-valerate) were measured by a high-performance liquid chromatography. Dissolved organic carbon (DOC) and 9



inorganic carbon were measured by a Total Carbon/Total Nitrogen analyzer (TOC-VCPN, TNM-1, Shimadzu, Kyoto, Japan). Details of the methods are given in the Supporting Information.

DNA extraction and high-throughput sequencing of bacterial and archaeal community The liquid-solid mixtures were vortex-mixed for 1 min and centrifuged at 700×g for 5 min. The microorganisms in the supernatant were considered suspended and identified by the label “l”. The microorganisms in the sediment were considered precipitated and identified by the label “s”. The supernatant was further centrifuged at 16000 rpm for 10 min to recover any precipitants. The total DNA in each stratified fraction of the six reactors were extracted using a PowerSoil® DNA isolation kit (Mo-Bio Laboratories Inc., CA). Samples were denoted with marks consisting of three parts. “BL” represented for blank without additives, “AC” for addition with carbon material activated carbon, BC for biochar, “GR” for graphene, “CC” for carbon cloth, and “CF” for carbon fibre. “21” (0.21 atm pCO), “41” (0.41 atm pCO), and “69” (0.69 atm pCO) refer to different stages based on pCO. “s” and “l” were for the fractions of either sediment and liquid. The variable regions V4-V5 of the microbial 16S ribosomal RNA gene were amplified by PCR using the primers 515F (5'–GTGCCAGCMGCCGCGGTAA–3') and 806R 5'–GGACTACHVGGGTWTCTAAT–3'), which were selected as the sequencing primer set to simultaneously obtain bacterial and archaeal information. 10


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The pretreatment and sequencing procedure followed the method used by Amato et al. 22

. The individual sample reads were de-multiplexed based on the barcode tag

sequence and processed as described by Lü et al. 23.

Characterization of carbon materials and evaluation of CO-liquid mass transfer efficiency The surface area of the carbon materials was characterized by N2 adsorption– desorption isotherms at 77 K using a specific surface area analyzer (ASAP 2020M, Micromeritics Instrument, USA) and calculated using the Brunauer-Emmett-Teller (BET) equations. The oxygen-containing acidic and alkaline surface functional groups of the carbon materials were determined using Boehm’s titration method24. The pH, DOC, and electrical conductivity (EC) value in the water-extractable fraction of the carbon materials (shaken with deionized water at 1:10, w/v) were determined as described by Zhang et al.25. Experiment to evaluate CO-liquid mass transfer efficiency was operated in six additional bottles with different carbon materials. Details can be found in the Supporting Information.

Results and Discussion

Results

Variations of CO conversion and biogas production During stage I (0–62 d) where the initial pCO was 0.07 atm, all of the reactors 11



performed in a similar manner as indicated by an almost identical CO conversion (Figure 1a) and CH4 production rates (Figure 1b). At the end of this stage, the CO conversion rate in the BC group (i.e., the reactor with biochar) started to increase. When the pCO increased to 0.21 atm in stage II (63–92 d), the CO conversion rate in the BC group was almost two times higher than the other groups. At day 76, the CO conversion rate of the AC group (i.e., the reactor with activated carbon) began to increase, while no significant improvements were observed in the GR, CC and CF groups (i.e., the reactors with graphene, carbon cloth, carbon fiber). The CH4 production rates in the BC and AC groups remained unchanged although their CO conversion rates increased. In stage III (93–120 d, pCO 0.41 atm), H2 began to be detected (Figure 2), while the AC and BC groups still performed better than the other groups based on the CO conversion and CH4 production rates. Compared to the blank group, the CO conversion rate was increased by up to 193% and 149% and the CH4 production rate increased up to 186% and 238%, respectively. The pCO increased further to 0.69 atm in stage IV leading to the decreasing CO conversion rate in the blank group. At day 134, the CO conversion rate of the BC group reached a peak (17.6 mmol/d) then declined to a low level (< 12 mmol/d) similar to the blank group. Nevertheless, the CC and GR groups’ performance was similar relative to the blank group in the first three stages but stood out with a higher stability performance similar to the AC group in stage IV. CH4 production seemed to lag behind CO conversion. During the four stages, the yield of CH4 from CO varied with pCO (i.e. 6.7%–10.5% at 0.07 atm pCO, 2.6%–6.5% 12


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at 0.21 atm pCO, 2.9%–4.6% at 0.41 atm pCO, and 5.4%–6.9% at 0.69 atm pCO; Figure 2), while the theoretical value of CH4 yield from CO was 25% according to [4CO+2H2OCH4+3CO2]. It seemed that the yield of CH4 was negatively correlated with pCO during the first three stages except for the AC and BC groups, suggesting that the CO was toxic for methanization when unacclimated. However, all groups increased the yield of CH4 in stage IV. Furthermore, the total gas production and the methane production in all of the groups with the added carbon materials were enhancement compared with the blank group.

Variations of DOC, VFAs and alcohols Variations in DOC, VFAs, and alcohols are showed in Figure 3. In the first two stages (Day 1–92), DOC slowly increased and only methanol and ethanol were detected occasionally. VFAs, mainly acetate > 90%, began to be detected when the pCO increased to 0.41 atm and the accumulation of VFAs was observed when the pCO further increased from 0.41 to 0.69 atm. Compared to the blank group, the addition of activated carbon lead to a faster accumulation and degradation of VFAs while the addition of biochar and graphene promoted a higher yield of acetate and DOC.

Methanogenic pathway analysis via stable carbon isotope signatures The

pathways

of

acetoclastic,

hydrogenotrophic,

and

methylotrophic

methanogenesis was distinguished according to the value and the changing trend of isotope values δCH4, δCO2, and apparent fractionation factor αc (αc = 13



(δCO2+1000)/(δCH4+1000)). Previous results have suggested that αc > 1.065, αc < 1.027 and αc around 1.045 are characteristic for environments dominated by hydrogenotrophic, acetoclastic and a combination of hydrogenotrophic and acetoclastic, respectively.26-27 Unfortunately, there is limited isotopic fractionation data on the carboxydotrophic pathway. Nevertheless, the present study still endeavored to find whether the pathway shift in CO fermentation could be observed using these tools. During stage I and stage II (day 1–92), the methane production rate was maintained at low levels (< 0.2 mmol/d), and the δCH4 of the six experiments had no significant changes and were maintained at -38.5‰ – -49.0‰. There was a decline of δCH4 in the BC and AC groups during the period of stage II which corresponded to their methanogenesis initiation. The δCO2 was initially -2‰ – -11.9‰ and decreased sharply to -17.4‰ – -23.1‰. In stage III, for the BL, AC, BC, GR, CC, and CF groups, the δCH4 decrease from -45‰, -59.5‰, -60.2‰, -42.5‰, -52.4‰, and -56.6‰ to -60.8‰, -61.7‰, -64.5‰, -62.8‰, -61.3‰, and -63.0‰, respectively. However, αc had an opposite trend for changes in δCH4. αc gradually decreased in the first two stages from 1.021 – 1.047 to 1.021 – 1.029, then sharply increased in stage III, and finally stabilized at 1.047 – 1.054 in stage IV. This behavior suggests that acetoclastic methanogenesis occurred at a low pCO and hydrogenotrophic methanogenesis occurred at a high pCO. Noticeably, the time when the αc value began to increase was consistent with the time when methane production rate of each group increased, indicating the initiation of methanogenesis.

14


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Microbial community revealed by high-throughput sequencing A total of 797 operational taxonomic units (OTUs) were detected in all samples, and the number of OTUs per sample ranged from 175 to 433. Other parameters related to the microbial community diversity are shown in Table S1 (Supporting Information). Generally, the community richness in the solid fractions of the six reactors were higher than that in the liquid fractions, as reflected by a higher value of OTUs and Chao index. Moreover, the solid fractions of the groups with carbon materials had more OTUs, a higher value of Chao, and a lower Simpson value relative to the BL group. These results indicate that the addition of carbon materials enriched the community richness and diversity. The phylogenetic classification of the sequences from the 37 samples at the phylum level is summarized in Figure S2 (Supporting Information). Microorganisms in the solid fractions showed significant differences compared with those in the liquid fractions, where the former should be directly affected by the addition of carbon materials, while the latter was susceptible to the pCO. Archaeal taxonomic distribution The relative proportion of Archaea in the solid and liquid fractions accounted for 50% ± 10% and 13% ± 10.9% of the total sequences, respectively. A total of 45 OTUs were detected for Archaea (Figure 5a). The genus level identification showed that Archaea mainly consisted of Methanosaeta (36.6%–65.7%), Methanolinea (8.2%–26.8%), Methanobacterium (3.9%–25.6%), Miscellaneous Crenarchaeotic Group (MCG) (2.2%–11.7%), and uncultured archaeon WCHA1-57 (1.6%–7.7%) in 15



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the solid fraction, and Methanobacterium (15.4%–81.7%), Methanosaeta (12.7%– 69.2%), MCG (1.6%–14.4%), WCHA1-57 (1.3%–14.9%), and Methanospirillum (0.1%–26.4%) in the liquid fractions. When the pCO increased from 0.21 to 0.41 atm in stage III and 0.69 atm in stage IV, the proportion of fixed Methanobacterium was enhanced from 7% ± 4.2% to 16% ± 2.9% and 15% ± 6.0%, and significantly in the AC group from 8.4% at AC30s to 21.3% at AC60s and 19.0% at AC100s. Additionally, the predominant Methanosaeta was replaced by Methanobacterium in the AC and BC groups in the liquid fractions. Methanospirillum was also boosted in BL, AC, BC, and GR groups in the liquid fractions when the pCO further increased from 0.41 to 0.69 atm (i.e., from no more than 1% to more than 22%). Bacterial taxonomic distribution The relative proportion of Bacteria in the solid and liquid fractions accounted for 40% ± 10% and 80% ± 10% of the total sequences. A total of 742 OTUs were detected for Bacteria. Sixteen families covering 60%–80% of the total bacterial sequences were selected for further discussion (Figure 5b). Rikenellaceae (8.6%– 36.1%),

Thiobacillaceae

(1.6%-21%),

Atribacteria

spp.

(3.1%–25.8%),

Hydrogenophilaceae (1.1%–9.9%) and Anaerolineaceae (2.6%–16.0%) were the main families in the solid fractions which accounted for approximately 70%–80% in average for the 16 families. The major suspended bacteria were significantly different from the fixed ones and the bacterial composition also presented a large difference under different pCO in the different reactors. WCHB1-69, Hydrogenophilaceae, and 16


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Rikenellaceae were the dominant families when the pCO was 0.21 atm. When the pCO increased from 0.21 to 0.41 atm, Rhodocyclaceae turned out to be dominant. Synergistaceae was also enhanced and then became the predominant bacteria when the pCO further increased from 0.41 atm to 0.69 atm. In addition, Synergistaceae were boosted in AC60l/100l and BC60l/100l (AC60l 25.6%, AC100l 47.2%; BC60l 16.2%, BC100l 43.6%). Anaerolineaceae was also enriched in the solid fractions of the AC group (AC30s 15.2%, AC60s 10.4%, AC100s 14.0%, higher than others). Rikenellaceae, maintained a high abundance especially in the AC (AC30s 36.1%, AC60s 22.3%, AC100s 24.0%) and BC groups (BC30s 37.5% BC100s 28.7%) together with Atribacteria spp., there were active fermenters involved in the degradation of carbohydrates and proteins and the production of VFA and ethanol.28-30 Another abundant family in the solid fractions but not detected in the inoculum was Hydrogenophilaceae. This family was also dominant in the liquid fractions but decreased when the pCO increased. The main genus Thiobacillus in family Thiobacillaceae was reported to have the ability of CO2-fixation.31 Its percentage was higher in AC and BC groups in the solid fractions and decreased in the liquid fractions when the pCO increased, confirming that activated carbon and biochar sheltered Thiobacillus to resist the high pCO loading. Principal component analysis of microbial diversity Figure S4 shows the bio-plots from principal component analysis (PCA) of microbial diversity, respectively, based on the sequences of the total community, bacteria, and archaea. For the total microbiomes (Figure S4a), the samples could be 17



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clustered into three groups: 1) the samples from the solid fractions, relating to higher abundance of Methanosaeta; 2) the samples from the liquid fractions in stage II or stage III (pCO = 0.21 or 0.41 atm), relating to WCHB1-69, Rhodocyclaceae, and Hydrogenophilaceae; 3) the samples from the liquid fractions in the final stage when pCO was 0.69 atm, relating to Synergistaceae. These data clearly show that the microbial distribution in the solid fractions was relatively stable and less affected by pCO. Meanwhile, under the same pCO, the addition of carbon materials especially for active carbon and biochar varied the microbial contribution resulting in higher loading values. Figure S4b and Figure S4c delivered similar results but provided more details. For example, the group named “Blvii wastewater sludge bacteria” belonging to family Rikenellaceae were stable in the solid fractions. Methanobacterium was enriched in the liquid fractions of the AC group.

Discussion

CO partial pressure played a key factor in CO conversion routes In our system, the major products of CO conversion are CH4, acetate, H2, and methanol, and their yield varied with pCO (Figure 2, 3). CH4 was the main end product that could be produced from four potential pathways: direct carboxydotrophic methanogenesis, tandem reaction of CO-utilizing acetogenesis and acetoclastic methanogenesis, hydrogenogenesis

tandem

reaction

methanogenesis,

of and

CO-utilizing tandem

18


hydrogenogenesis

reaction

of

and

CO-utilizing

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methylogenesis and methylotrophic methanogenesis. When the pCO was 0.07 atm in stage I and 0.21 atm in stage II (day 1–92), the αc value (Figure 4) gradually got close to the value of the pure culture experiment with Methanosaeta (1.007–1.010)32, implying that the acetoclastic pathway became predominant pathway. In this period, acetate was not detected and the concentration of methanol and ethanol was low (

Exploit Carbon Materials to Accelerate Initiation and Enhance Process

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