Unveiling the Biochar-Respiratory Growth of Methanosarcina acetivorans Involving Extracellular Polymeric Substances

Biochar can be applied to diverse natural and engineered anaerobic systems. Biochar plays biogeochemical roles during its production, storage, and environmental dynamics, one of which is related to the global methane flux governed by methanotrophs and methanogens. Our understanding of relevant mechanisms is currently limited to the roles of biochar in methanotrophic growth, but less is known about the roles of biochar in methanogenic growth. Here, we demonstrated that biochar enhanced the methanogenic growth of a model methanogen, Methanosarcina acetivorans, and the role of biochar as an electron acceptor during methanogenic growth was confirmed, which is referred to as biochar-respiratory growth. The biochar-respiratory growth of M. acetivorans promoted the secretion of extracellular polymeric substances (EPS) with augmented electron transfer capabilities, and the removal of EPS significantly attenuated extracellular electron transfer. Identification and quantification of prosthetic cofactors for EPS suggest an important role of flavin and F420 in extracellular electron transfer. Transcriptomic analysis provided additional insights into the biochar-respiratory growth of M. acetivorans, showing that there was a positive response in transcriptional regulation to the favorable growth environment provided by biochar, which stimulated global methanogenesis. Our results shed more light on the in situ roles of biochar in the ecophysiology of methanogens in diverse anaerobic environments.


Introduction
Biochar is a carbonaceous material produced by the pyrolysis of biomass wastes at 300 to 900 °C under oxygen-limited conditions.Biochar contains high specific surface areas and internal porosity, multiple functional groups, and good electron transfer properties and has consequently been widely applied in agricultural and environmental engineering for soil improvement and pollution remediation, respectively [1][2][3].Moreover, combustion during wildfires and natural fossil fuel use usually generates temperatures between 340 and 500 °C and sometimes up to 1000 °C [4] and is estimated to produce approximately 128 ± 84 Tg of black carbon each year [5], of which biochar is an important component [6,7].Hence, biochar is already ubiquitous in diverse soils and sedimentary environments.
Biochar plays biogeochemical roles during its production, storage, and environmental dynamics, one of which is related to the emission of the atmospheric greenhouse gas methane [8].Amendment with increased amounts of biochar has been reported to decrease methane emissions from landfill and paddy soils, and mechanisms in which biochar assists in methane mitigation have been proposed [9][10][11].Biochar was discovered to facilitate microbial methane oxidation, which is a major sink for atmospheric methane, by the following two mechanisms: (1) biochar was proposed to serve as a favorable habitat for the colonization of aerobic methanotrophic bacteria in a laboratory column study due to its high specific surface area and porosity [12]; (2) biocharrespiratory growth of anaerobic methanotrophic archaea was documented in a laboratory reactor study, in which biochar Unveiling the Biochar-Respiratory Growth of Methanosarcina acetivorans Involving… 1 3 could serve as the sole extracellular electron acceptor for the anaerobic oxidation of methane [13].Nevertheless, inconsistent effects of biochar amendment on soils are usually observed, and under some conditions, biochar can promote the emission of methane [14][15][16].Although this discrepancy is recognized to be caused by different biochar physicochemical properties and soil conditions [9,10], the potential effect of biochar on the ecophysiology of methanogenic archaea, namely methanogens, must also be considered.
As an essential methane producer in the global carbon cycle, methanogens inhabit a wide range of anaerobic environments, including soils and diverse sediments [17].Methanogens are metabolically restricted both in their range of electron donors, which include H 2 , C1-methylated compounds and acetate, and electron acceptors, which consist of CO 2 and endogenous methyl groups, which all lead to methane production and energy conservation [18].Recently, certain redox-active substances, such as ferric iron (Fe 3+ ), were found to be able to act as extracellular electron acceptors for the methanogenic growth of Methanosarcina spp. that use C1-methylated compounds or acetate as electron donors [19,20].Intriguingly, the diversion of electrons from methanogenesis to extracellular Fe 3+ did not weaken methanogenic growth as commonly thought but enhanced methanogenic growth, which was referred to as Fe 3+ -respiratory methanogenic growth.Therefore, it has yet to be revealed whether methanogens are capable of biochar-respiratory growth, thereby affecting global methane dynamics, as they are capable of Fe 3+ respiration, when redox-active biochar and methanogens inevitably coexist in soil and sedimentary environments.
Therefore, we conducted this study in which a model methylotrophic and acetotrophic methanogen, Methanosarcina acetivorans, was selected to investigate the role of biochar in the methanogenic growth of this archaea.We reveal here that biochar is able to serve as an electron acceptor for M. acetivorans, enhancing its methanogenic growth.Biochemical and electrochemical approaches were employed to explore the role of extracellular polymeric substances in extracellular electron transfer.Transcriptomic analyses were conducted to investigate the biochar-assisted enhancement in methanogenic growth.This work sheds more light on the roles of biochar in the ecophysiology of methanogens.

Preparations of Biochar
Corn stover collected from farmlands of Qingdao (Shandong, China) was cleaned and then dried at 110 °C for 24 h.The dried stover was wrapped with aluminum foil and pyrolyzed at 300 °C, 500 °C, and 900 °C with a heating rate of 10 °C min −1 for 6 h in a tube furnace under N 2 .Biochar was ground in a mortar to pass through a 100-mesh sieve, then washed with milli-Q water three times to remove organic matter and other impurities, and dried at 80 °C for later use.The Brunauer-Emmett-Teller (BET) surface area of biochar was measured using an automated surface area and porosity analyzer (Micromeritics, ASAP2460) with N 2 as an adsorption gas and degassed at 120 °C for 8 h.Biochar pyrolyzed at 300 °C that has the most redox functional groups was prepared for oxidative modification as previously reported [13,21].Biochar pyrolyzed at 300 °C was treated with 20% H 2 O 2 solution at 6.25 g/L with stirring overnight, then washed with milli-Q water three times to remove excess H 2 O 2 solution, and dried at 80 °C.The properties (productivity and elemental percentage) of biochar described above were shown in Table S1.

Test for the Effect of Biochar with Different Properties on the Methanogenic Growth of M. acetivorans
M. acetivorans C2A (JCM, Japan) was cultured in a high salt (HS) medium with methanol (125 mM) as a carbon source [22].The medium was purged with N 2 /CO 2 (v/v = 8:2) to obtain anoxic conditions.Na 2 S (0.3 g L −1 ) and cysteine (0.3 g L −1 ) were added to eliminate oxygen.Afterwards, biochar with different properties were added to the medium at indicated concentrations in the "Result" section.Then, precultured M. acetivorans was inoculated into the medium at 1:100 with an initial optical density (OD 600 ) at ~ 0.06.The culture was incubated in the dark at 37 °C.Biological triplication was prepared for each culture condition.The amounts of produced methane, total cellular proteins, and ATP were measured as described below.

Test for the Effect of Biochar on the Secretion of Extracellular Polymeric Substances During the Methanogenic Growth of M. acetivorans
M. acetivorans was cultured with or without biochar (pyrolyzed at 300 °C) in biological triplication.Extracellular polymeric substances (EPS) were extracted via a modified heat extraction method [23], which could be found in the Supporting text.The concentrations of each component in EPS and the electrochemical characteristics of EPS were measured as described below.

Biochemical and Physicochemical Analysis
The production of methane in the headspace was determined using a gas chromatograph (GC-2014, Shimadzu) equipped with a GXD-502 chromatographic column.N 2 was used as the carrier gas.Gas samples (1 mL) were taken from the headspace of the cultures with a micro-injector every 1 day.Cellular protein concentrations and ATP content were measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology) and an ATP content assay kit (Beyotime Biotechnology), respectively, every 2 days.
The reduction of biochar was measured via a modified method for measuring the reduction of humic substances during microbial growth [24].The mixture of cells with biochar was collected from the culture of M. acetivorans that grew with biochar (pyrolyzed at 300 °C) on days 2, 4, 6 and 8 by centrifuging at 4500 g for 15 min.The cell pellets with biochar were resuspended in 10 mL HS medium following with the addition of ferric citrate (Fe 3+ ) at the final concentration of 2 mM.After a 5-min reaction, a 50-µL sample was taken for measuring amounts of Fe 2+ by a ferrozine assay as previously described [24].The reduction of Fe 3+ caused by reduced biochar during growth was calculated by subtracting those caused by equal amounts of cells alone collected from the culture minus the amended biochar.Biochar alone in the culture without inoculation with M. acetivorans was also measured as a negative control.
The concentration of polysaccharide in EPS was determined according to the anthrone method with glucose as the standard, which was described in detail in the Supporting texts.Humus-like compounds in EPS were determined by a three-dimensional excitation emission matrix (3-DEEM) using a fluorescence spectrophotometer (Hitachi, F7000) at an excitation wavelength range from 200 to 420 nm and an emission wavelength range from 400 to 530 nm [25].The spectrum was recorded at a scanning speed of 2400 nm min −1 .The cofactor F 420 in EPS was extracted using the protocol reported before [26] and measured by 3-DEEM at 425 nm of excitation wavelength and 472 nm of emission wavelength.

Electrochemical Analysis
All measurements were performed in a three-electrode system using an electrochemical workstation (CHI660E, CH Instruments) at room temperature with 0.2 M Na 2 SO 4 (pH 7.0) as the electrolyte.An Ag/AgCl electrode and platinum sheet electrode were used as reference electrode and counter electrode, respectively, and a glassy carbon or biochar electrode was used as the working electrodes.The biochar electrode was prepared according to a previously reported protocol [27].
Cyclic voltammetry (CV) was used to measure the electrochemical capacitance of biochar, and the electron accepting capacity (EAC) was determined via chronoamperometry in an anaerobic chamber [28]; methods were described in detail in the Supporting text.
The electrochemical impedance spectroscopy (EIS) of EPS was measured with a glassy carbon electrode as the working electrode, and the electroactive substances in EPS were measured via differential pulse voltammetry (DPV).The EIS was determined in the range of 100 kHz to 0.01 Hz, and the range of DPV was from − 0.6 to 0 V, with potential increment of 10 mV, amplitude of 50 mV, and pulse width of 0.3 s at 0.05 s sampling intervals.

Morphology Analysis
The morphology of M. acetivorans and biochar was analyzed using a field emission scanning electron microscope (SEM) (Quanta 250 FEG, FEI).Specific methods could be found in the Supporting text.

Transcriptomic Analysis
Total RNA was extracted from M. acetivorans that grew with or without biochar (pyrolyzed at 300 °C) at day 4 using TRIzol (Invitrogen).Biological triplication was performed.RNA integrity was detected according to agarose gel electrophoresis, and the RNA integrity number was measured by a bioanalyzer (Agilent2100).Ribo-Zero magnetic kit (EpiCentre) was used to remove rRNA.The fragmentation buffer was added to randomly break mRNA into small fragments of approximately 200 bp.cDNA was synthesized with reverse transcriptase and random primers using the mRNA as the template.The second strand of cDNA was digested with uracil-DNA glycosylase enzyme before PCR; therefore, only the first strand of cDNA was contained in the library.Finally, the Truseq SBS kit (300 cycles) (Illumina) was used for sequencing, and the obtained data was analyzed.

Statistical Analyses
All statistical significances for different biochar-amended groups were determined by one-way ANOVA followed by a least significant difference (LSD) test, with a p-value < 0.05 indicating statistical significance and a p-value < 0.01 indicating extreme statistical significance.The comparative factors and the statistical test p-values are indicated in each figure legend.

Role of Biochar in the Methanogenic Growth of M. acetivorans
Biochar was first produced via the pyrolysis of corn stover at 300 °C (referred to as BC-300).The addition of 50-500 mg L −1 BC-300 increased methane production (Fig. 1a) and significantly enhanced the methanogenic growth of M. acetivorans from days 2 to 6, as determined by the monitoring of total cellular proteins (p < 0.05 or 0.01) (Fig. 1b).SEM imaging showed coccoid-shaped cells of M. acetivorans growing on the external surfaces and internal pores of biochar (Fig. 1c).The reduction of BC-300 was observed during the monitoring of methanogenic growth coupled with the reduction of Fe 3+ (Fig. 1d).
To further verify the role of biochar as an electron acceptor during the methanogenic growth of M. acetivorans, an oxidized form of BC-300 (BC-300-Ox) was prepared.The abundances of oxygen-containing functional groups such as C-O at 1440 cm −1 and C = O at 1620 cm −1 in BC-300-Ox as detected by FTIR increased by 5.51% and 6.80%, respectively (Fig. S1).BC-300-Ox increased methane production to the same extent as BC-300 (Fig. 2a) and significantly enhanced total cellular proteins (Fig. 2b) and the energy source ATP for growth (Fig. 2c) by 20.55% (p < 0.01) and 14.84% (p < 0.05), respectively, compared to those for BC-300 during the methanogenic growth of M. acetivorans on day 6.Because pyrolysis temperature can affect the electrochemical properties of biochar, thereby affecting its electron accepting capacity [27], we prepared biochar by pyrolyzing the corn stover at 500 and 900 °C (referred to as BC-500 and BC-900, respectively).The increase in pyrolysis temperature from 300 to 900 °C increased the BET surface area (Fig. S2a and b), electrochemical capacitance, and EAC (Fig. S2c, d and e) of the biochar (Table 1).Both BC-500 and BC-900 increased methane production to the extent that BC-300 did (Fig. 2d), and BC-900 significantly enhanced cellular protein (Fig. 2e) and ATP (Fig. 2f) by 13.40% (p < 0.01) and 18.92% (p < 0.05), respectively, compared to BC-300 during the methanogenic growth of M. acetivorans on day 6.Therefore, all these results indicate the electronaccepting role of biochar, which enhanced the methanogenic growth of M. acetivorans.

Biochar Promoted the Secretion of EPS During the Methanogenic Growth of M. acetivorans, Which Augmented Extracellular Electron Transfer
Loosely-bound EPS (LB-EPS) and tightly-bound EPS (TB-EPS) were isolated from M. acetivorans on day 4 from 500 mg L −1 BC-300-amended and unamended (control) groups.Figure 3a and b show that the concentrations of three major components of EPS, including polysaccharides, proteins, and humus-like compounds, in both LB-EPS and TB-EPS isolated from the BC-300 group were higher than those isolated from the control group.We then speculated that the increase in the amount of EPS may improve electron transfer.Analysis of redox functional groups contained in EPS, such as quinone and carboxylic acid groups, showed that the abundances of these groups were higher in the EPS isolated from the BC-300 group than in those isolated from the control group (Fig. 3c).Furthermore, EIS analysis showed that the impedance values of EPS isolated from the BC-300 group were lower than those isolated from the control group (Fig. 3d).These results demonstrate that amendment with biochar coupled with the reduction of biochar amended at 500 mg/L during the methanogenic growth promoted the secretion of EPS during the methanogenic growth of M. acetivorans and that the EPS exhibited a higher capacity for electron transfer.
To verify the role of EPS in extracellular electron transfer, LB-EPS and TB-EPS were successively removed from the cells of the BC-300 and control groups following a previously reported method [23]; whole cells, cells minus LB-EPS, and cells minus LB&TB-EPS were acquired (Fig. 4a).An extracellular electron transfer assay was performed by monitoring the reduction of Fe 3+ .Figure 4b shows that the reduction of Fe 3+ after the removal of LB-EPS was reduced by 5.6% (p < 0.01) and 5.5% (p < 0.05) in the presence and absence of BC-300, respectively.The reduction of Fe 3+ after the removal of LB&TB-EPS was reduced by 40.3% and 23.1% in the presence and absence of BC-300, respectively (p < 0.01) (Fig. 4b).
To explore the mechanism of EPS-mediated extracellular electron transfer, redox prosthetic cofactors involved in EPS were identified and quantified.The EPS exhibited two apparent peaks at − 0.15 and − 0.48 V in the DPV measurement, which were attributed to c-type cytochrome and flavin, respectively [29,30].Quantification of the area of the two peaks indicated that the abundance of flavin was twofold higher in the EPS isolated from the BC-300 group than in the control group (p < 0.01), whereas the abundance of c-type cytochrome did not change significantly (Fig. 5a).3-DEEM was employed to quantify the cofactor F 420 of EPS, and the F 420 fluorescence of EPS isolated from the BC-300 group was fourfold higher than that isolated from the control group (p < 0.01) (Fig. 5b).

Transcriptomic Analyses Reveal Global Upregulation of Genes Involved in Methanogenesis-related Pathways in the Biochar-amended Group
Transcriptomic analyses were conducted to further investigate the enhancing effect of biochar on the methanogenic growth of M. acetivorans.The differentially expressed genes (DEGs) were screened with univariate statistical significance (log 2 (FC) > 1.0 or < − 1.0, and p < 0.05).Among the 4462 genes expressed in M. acetivorans, a total of 1534 and 221 DEGs were significantly upregulated and downregulated in  the cultures amended with biochar, respectively (Fig. S3).GO (Gene Ontology) enrichment analysis indicated that these DEGs could be classified into three categories: cellular component (CC), biological translation process (BP), and molecular function (MF) (Fig. S4).The DEGs enriched in BP, such as those involved in nitrogen compound metallic and organic substance biosynthetic processes, were significantly upregulated.The KEGG database was used to analyze the enrichment in DEGs in metabolic pathways (Fig. 6a).A total of 79 genes were enriched in methanogenesis-related pathways, among which the expression of the genes involved in the biosynthesis of coenzyme F 420 (Cof), methylfuran (Mfn), riboflavin (Rib, Arf, Rfk), phenazine (Phz, Trp), and coenzyme F 430 (Cfb) was significantly upregulated (Fig. 6b).These cofactors are essential for the enzymatic activities of key enzymes and electron transfer involved in methanogenesis.The expression of key genes directly involved in methanogenesis and energy conservation, including methyl coenzyme M reductase (Mcr), methyltetrahydrosarcinapterin:coenzyme M methyltransferase (Mtr), methanol methyltransferase (Mta), methenyl-tetrahydrosarcinapterin cyclohydrolase (Mch), F 420 -dependent N 5 N 10 -methylene-tetrahydrosarcinapterin reductase (Mer), ATP synthase and Na + /H + antiporter (Mrp), ATP synthase (ATPase), and F 420 dehydrogenase (Fpo), was significantly upregulated (Fig. 6b).Furthermore, a total of 35 and 34 genes were enriched in cellular processes and environmental information processing, respectively, including the quorum sensing and microbial secretion systems.A total of 125 genes were found to be enriched in genetic processing information, among which 28 genes were annotated as transcriptional factors or regulators and RNA polymerase involved in the process of transcription, and 88 genes annotated as ribosomal proteins, tRNA ligase, and translation initiation factors involved in the process of translation were significantly upregulated (Table S2).

Discussion
The superior electrochemical properties of biochar allow it to act as an electron acceptor or donor for electroactive bacteria, such as Geobacter species [6,31,32].Biochar interacts with electroactive bacteria by accepting electrons derived from the oxidation of organic matter or donating electrons of its own to nonoxygen terminal electron acceptors.In addition, biochar was found to stimulate interspecies electron transfer from G. metallireducens to another species of Methanosarcina, M. barkeri, by serving as an electron shuttle [33].Thus, the ability of biochar to act as an electron donor for methanogens in anaerobic environments has been proposed [33][34][35].However, for fermentative methanogenesis, electrons derived from the oxidation of organic matter must be transferred to endogenous methyl groups for methane production.Our findings have addressed an interesting question on the availability of biochar to methanogens as an electron acceptor.Similar to previously reported observations from Fe 3+ -respiratory growth [19,20], M. acetivorans used biochar as an electron acceptor to enhance methanogenetic growth.We thus tentatively designated for what we found here as biochar-respiratory growth.EPS from electroactive bacteria was suggested to play a crucial role in extracellular electron transfer [29,36,37].Biochemical and electrochemical analyses of the EPS secreted by M. acetivorans also suggest that EPS is involved in extracellular electron transfer (Figs. 3 and 5), which was further supported by an extracellular electron transfer assay using whole cells and EPS-removed cells (Fig. 4).As one of the EPS components, c-type cytochromes have been well recognized to mediate extracellular electron transfer for electroactive bacteria [38].Although c-type cytochromes were also identified from the EPS secreted by M. acetivorans, their abundances were not significantly regulated in biochar-respiratory cells compared to non-respiratory cells.In contrast, the abundances of two other redox components, flavin and F 420 , identified from EPS secreted by M. acetivorans were observed to be significantly upregulated in biochar-respiratory cells (Fig. 5a and b).In fact, the cofactor F 420 is a derivative of riboflavin, which universally functions as an electron carrier for methanogens, and multiple F 420 -dependent oxidoreductases are involved in methanogenesis and energy conservation in methanogens [39].Transcriptomic analysis was used to identify the upregulated expression of several genes encoding subunits of the membrane-bound Fpo complex, which is F 420 -dependent oxidoreductase, in the biochar-respiratory cells.In particular, MA3732 encoding the FpoF subunit, which is an intracellular electron input module for the membrane-bound Fpo  **) indicate significant differences between the BC-300 group and the control group at p < 0.01 complex, was observed to be significantly upregulated in biochar-respiratory cells [40].Fpo complex-mediated electron transfer is recognized to couple with a transmembrane H + gradient that is required for energy conservation [41].Thus, it is reasonable to speculate that the enhancement of methanogenic growth by biochar is caused by the energyconserving Fpo complex.However, we cannot exclude the possibility that other potential extracellular electron mediators may contribute to energy conservation in biochar-assisted growth.For example, humus-like compounds secreted by M. barkeri have been proposed to facilitate extracellular electron transfer [42], and higher abundances of humus-like compounds were found in the EPS of cultures amended with biochar in this study (Fig. 3b).Extracellular electron transfer via membrane-bound Rhodobacter nitrogen fixation has been known to couple with Na + gradients across membranes, which could enhance energy conservation [19].
Biochar can provide a favorable surface for the growth of aerobic methanotrophs [11,12].This favorable environment is considered to occur due to the higher specific surface area and internal porosity of biochar, which lead to increased water-retention capacity and improved gas diffusion conditions for microbial colonization.Similarly, we demonstrated that the higher the specific surface area of biochar was, the more conspicuous the effect of biochar on the methanogenic growth of M. acetivorans (Fig. 2d, e, and f).Notably, growth on the favorable surface provided by biochar promoted the secretion of EPS with augmented redox properties (Fig. 3).Transcriptomic analyses revealed the upregulation of two key genes, MA1059 and MA4667, annotated as SecY and TatC, respectively, which are involved in the extracellular protein secretion pathway [43], providing a potential explanation for the biochar-promoted secretion of EPS.Moreover, the growth surface provided by biochar is favorable to methanogenesis.The expression of many key genes directly involved in methanogenesis, including Mta, Mcr, Mtr, Mer, and Mch, was upregulated in the biochar-respiratory cells (Fig. 6b).Genes involved in methanogenesis are considered to be transcriptionally regulated in response to both extracellular and intracellular signals [44].The regulation of Mta by methanol-specific regulators (Msr) is one of the few regulators recognized thus far [45].The expression of several Msr genes, including MA0459, MA0460, MA4397, and MA4398, was upregulated in biochar-respiratory cells (Fig. 6b), suggesting that the favorable environment that biochar provides may be an extracellular signal that can stimulate methanogenesis.However, the transcriptional regulation of other key genes involved in methanogenesis with biochar as a signal remains to be revealed.

Ecological Implications
Although biochar and methanogens inevitably encounter in a variety of anaerobic environments, the role of biochar in methanogenic growth is not well understood.The M. acetivorans investigated in this work belongs to the genus Methanosarcina, which is enriched in diverse natural and engineered anaerobic environments such as freshwater and marine sediments, soil, anaerobic sludge, and compost [17,18].M. acetivorans has the largest genome among all isolated Methanosarcina strains thus far [46] and is regarded as a model that provides a mechanistic understanding of methanogenesis.Thus, M. acetivorans was selected in this work to investigate the in situ roles of biochar in the ecophysiology of methanogens in anaerobic environments.
Biochar-respiratory methanotrophic growth has been proposed as a process that assists in atmospheric methane mitigation.The biochar-respiratory methanogenic growth reported here further links the biogeochemical roles of biochar to the anaerobic methane cycle.An investigation of the Tai Lake plain showed that total soil methane emissions increased by 34% in soils amended with biochar at 40 t ha −1 [15].An investigation of paddy soil showed that biochar prepared at a low pyrolysis temperature increased the methane yield and methane production rate by 0.2 mL g −1 soil and 38%, respectively [47].It was found that fermentative bacteria such as Clostridium and Janibacter and methanogens such as Methanosarcina were significantly enriched via amendment with biochar.However, the authors of a study on landfill cover soil concluded that biochar amendment is effective in increasing methanotroph populations, which assists in reducing methane emissions [48].A consensus has emerged that the ecological consequences of amendment with biochar are highly dependent on the microbial community enriched in situ.Thus, the finding of this work extends the current understanding of the biogeochemical roles of biochar in the context of global methane dynamics.
In addition to biochar application as a soil amendment, biochar addition to anaerobic digestion systems has been recognized as a promising technology to enhance the anaerobic remediation of organic contaminants and the production of renewable biogas energy [49,50].The addition of biochar was found to reduce the lag phase of the methanogenic stage and improve methane-rich biogas production.The advantages of biochar usually include its role as an electron shuttle between fermentative bacteria and methanogens [50].The finding of this work, that is, the biochar-respiratory growth of a methanogen, provides an alternative explanation for the positive effect of biochar on anaerobic digestion systems.

Fig. 1
Fig. 1 Growth parameters and morphology for the methanogenic growth of M. acetivorans with different amounts of biochar.a Methane production.b Total cellular protein concentrations.Single asterisk (*) and double asterisks (**) indicate significant differences between the biochar-amended groups and the control group at p < 0.05 and 0.01, respectively.c SEM image.d Reduction of Fe 3+ coupled with the reduction of biochar amended at 500 mg/L during the methanogenic growth

Fig. 2
Fig. 2 Growth parameters for the methanogenic growth of M. acetivorans with different properties of biochar.a, d Methane production.b, e Total cellular protein concentrations.c, f Total cellular ATP

Fig. 3
Fig. 3 Characterizations of EPS isolated from M. Acetivorans grown with or without biochar.a The concentrations of protein and polysaccharide.PN and PS stand for protein and polysaccharide, respectively.

Fig. 4 Fig. 5
Fig. 4 Role of EPS in the extracellular reduction of Fe 3+ in BC-300 and control groups.a Schematic diagram of EPS removal.b Reduction of Fe 3+ catalyzed by the whole cells, cells minus LB-EPS, and cells minus LB&TB-EPS.Single asterisk (*) and double asterisks (**) indicate significant differences between cells minus EPS groups and the whole cells group at p < 0.05 and 0.01, respectively