Reduction of Gibbs free energy and enhancement of Methanosaeta by bicarbonate to promote anaerobic syntrophic butyrate oxidation

Accepted Manuscript Reduction of Gibbs free energy and enhancement of Methanosaeta by bicarbonate to promote anaerobic syntrophic butyrate oxidation Yupeng Zhang, Jianzheng Li, Fengqin Liu, Han Yan, Jiuling Li, Xue Zhang PII: DOI: Reference:

S0960-8524(18)30877-0 https://doi.org/10.1016/j.biortech.2018.06.098 BITE 20115

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

21 March 2018 24 June 2018 28 June 2018

Please cite this article as: Zhang, Y., Li, J., Liu, F., Yan, H., Li, J., Zhang, X., Reduction of Gibbs free energy and enhancement of Methanosaeta by bicarbonate to promote anaerobic syntrophic butyrate oxidation, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.06.098

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Reduction of Gibbs free energy and enhancement of Methanosaeta by bicarbonate to promote anaerobic syntrophic butyrate oxidation Yupeng Zhang1, Jianzheng Li*,1, Fengqin Liu1, Han Yan1, Jiuling Li2, Xue Zhang 1 1

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, 73 Huanghe

Road, Harbin 150090, P.R. China 2

Advanced Water Management Centre, The University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia

*Corresponding Authors. Phone: +86 (451)-86283761; Fax: +86 (451)-86283761; E-mail: [email protected] (J. Li)

1

Abstract: Bicarbonate (HCO3–) has been extensively researched as a buffer in anaerobic digestion. The effect of HCO3– concentration on syntrophic butyrate oxidation process was evaluated by batch culturing of anaerobic activated sludge, and the mechanism was further revealed by the changes of Gibbs free energy (ΔG) and the interspecies transfers of electron and proton. The results showed that butyrate degradation rate was enhanced by 32.07% when the supplement of HCO3– increased from 0 to 0.20 mol/L. However, methane production and acetate degradation were strongly inhibited by HCO3– more than 0.10 mol/L. More function of HCO3– was found as 1) decreasing the ΔG of syntrophic methanogenesis of butyrate while increasing the ΔG of methanogenesis of acetate, 2) enriching M. harundinacea and M. concilii, 3) increasing the diffusion rate of protons between the syntrophic consortia. This work would increase the anaerobic digestion efficiency by enhancing the interaction of the syntrophic consortia. Key words: Gibbs free energy; methanogens; bicarbonate; anaerobic digestion; syntrophic

1 Introduction Anaerobic digestion is known as a cost-effective process for treating high density organic wastewater and producing sustainable energy such as hydrogen (H2) and methane (CH4) (Luo et al., 2015). However, the efficiency of anaerobic digestion remains unsatisfied. In the anaerobic digestion, three trophic groups of microorganisms are mainly involved, i.e. hydrolytic-fermentative bacteria, syntrophic acetogens and methanogens (De Bok et al., 2004). As the intermediates produced by hydrolytic-fermentative bacteria, volatile fatty acids (VFAs) such as propionate and butyrate were then oxidized into a mixture of acetate and H2/CO2 or formate by syntrophic acetogens, and the mixture was converted to CH4 by methanogens (Liu et al., 2016). Due to syntrophic acetogenic reaction is endergonic under standard condition (25°C, 100 kPa), the reaction is feasible only when the produced 2

H2 or formate is consumed by methanogens (Dolfing, 2013). Besides, the syntrophic acetogenesis is regarded as one of the rate-limiting steps in anaerobic digestion (Amani et al., 2011). The accumulation of butyrate and propionate were often found in anaerobic digestion processes, which resulted in acidification of anaerobic digestion systems and deterioration of digestion performance. Therefore, it is crucial to enhance the syntrophic acetogenesis in anaerobic digestion processes. In the syntrophic methanogenesis of VFAs, methanogens act as electron sinks, and the electron transfer from syntrophic acetogens to methanogens is termed interspecies electron transfer (IET) (Rotaru et al. 2012). Traditionally, it is thought that electron carriers such as H2 or formate are diffused from acetogens to methanogens (De Bok et al., 2004). In other words, diffusion rate of the electron carriers would limit the methanogenesis of VFAs. However, recent researches show that electrons can be directly transferred from acetogens to methanogens (Lee et al., 2016; Zhao et al., 2016b). Rotaru and coworkers found that species of Geobacter and Methanosaeta could exchange electrons via direct interspecies electron transfer (DIET), which stoichiometrically converted ethanol to methane (Rotaru et al., 2014b). Their further research revealed G. metallireducens and Methanosarcina barkeri also exchanged electrons via DIET (Rotaru et al. 2014a). DIET between bacteria and methanogen is also found in natural and artificial anaerobic environment. For example, DIET between syntrophic consortia was enhanced by the conductive magnetite particles in the methanogenesis of propionate (Cruz Viggi et al., 2014). Magnetite was also identified to simulate DIET between the syntrophic consortia in the methanogenesis of butyrate (Li et al., 2015; Yamada et al., 2015). Active carbon or biochar are also demonstrated in enhancing DIET to improve VFAs degradation (Lee et al., 2016; Zhao et al., 2015; Zhao et al., 2016b). In fact, without the mediation of conductors, bacteria also transferred electron by electrically conductive pili and outer surface c-type cytochromes (Rotaru et al., 3

2014b; Summers et al., 2010). These researches indicated that DIET is a novel interspecies electron transfer mechanism in syntrophic consortia, by which VFAs can be degraded effectively (Zhao et al., 2016b). There are three pairs of reactions to syntrophic CH4 formation and an aceticlastic CH4 formation included in the methanogenesis of butyrate, i.e. H2-mediated IET [Reaction (1) and (2)], formate-mediated IET [Reaction (3) and (4)], DIET [Reaction (5) and (6)] and aceticlastic methanogenic reaction or syntrophic acetate oxidation combined with hydrogenotrophic methanogenic reaction [Reaction (7)] (Thauer et al., 1977). The total reactions of the syntrophic methanogenesis by IET or DIET are the same as expressed as Reaction (8). ΔG0′ is the Gibbs free energy change under standard conditions at pH 7. CH3CH2CH2COO– + 2H2O → 2CH3COO– + H+ + 2H2 4H2 + H+ + HCO3– → CH4 + 3H2O

ΔG0′ = +48.32 kJ/mol

ΔG0′ = -135.58 kJ/mol

CH3CH2CH2COO– + 2HCO3– → 2CH3COO– + H+ + 2HCOO– 4HCOO– + H2O + H+ → CH4 + 3HCO3–

(1) (2)

ΔG0′ = +45.60 kJ/mol

ΔG0′ = -111.00 kJ/mol

(3) (4)

CH3CH2CH2COO– + 2H2O → 2CH3COO– + 5H+ + 4e–

(5)

HCO3− + 9H+ + 8e– → CH4 + 3H2O

(6)

CH3COO– + H2O → HCO3– + CH4

ΔG0′ = -31.02 kJ/mol

2CH3CH2CH2COO– + H2O + HCO3– → 4CH3COO– + H+ + CH4

(7) ΔG0′ = -38.94 kJ/mol

(8)

Obviously, the methanogenesis via IET and DIET all employed bicarbonate (HCO3–) as electron acceptor. And HCO3– is the product of the aceticlastic methanogenesis. Though the influence of HCO3– on the anaerobic digestion have been extensively investigated, research about HCO3– was mainly focus on its buffer capacity (Kasali et al., 1989). Some phenomena were also observed that 4

HCO3– concentration altered the bacterial activity. For example, methane production with glucose as substrate was inhibited by high density of HCO3– (0.15–0.20 mol/L), and the fraction of CH4 generated via syntrophic acetate oxidation pathway generally increased with the increment of HCO3– concentration (Lin et al., 2013). In the anaerobic digestion of methanol process, methanogenesis with HCO3– buffer was faster than with phosphate buffer (Loureiro et al., 2003). The influence of HCO3– on bacterial activity suggesting that HCO3– participated in the methanogenic metabolism. In methanogenic environment, the density of electron carriers such as partial pressure of H2 (pH2) and concentration of formate is extremely low (Stams and Plugge, 2009). Thus, the actual Gibbs free energy change (ΔG) of the methanogenic reaction will be significantly influenced by substrate concentration (Leng et al., 2017). In a steady anaerobic digestion process, the VFAs concentration is usually a constant. Thereby, HCO3– concentration ([HCO3–]) becomes a crucial factor in changing the ΔG of syntrophic and methanogenic reactions. On the other hand, based on the reactions involved in syntrophic butyrate oxidation in methanogenic environment, protons are generated by syntrophic acetogenic reactions [Reaction (1), (3) and (5)] and then are consumed by methanogenic reactions [Reaction (2), (4) and (6)]. However, the proton concentration (<10-7 mol/L) in the methanogenic environment is also extremely low due to the neutral pH, indicating a diffusion flux very low (Fan et al., 2007). Though HCO3– has been reported as a carrier to enhance proton transport (Fan et al., 2007), its effect on anaerobic syntrophic oxidation of VFAs has not been well understood. As one of the dominant VFAs in anaerobic digestion, butyrate was selected as a special substrate for methane fermentation in the present work. A series of batch culturing of anaerobic activated sludge were conducted to evaluate the influence of [HCO3–] on the anaerobic syntrophic oxidation and methane production of butyrate. To understand the mechanism in the syntrophic butyrate degradation 5

consortia, 1) microbial communities in the activated sludge were investigated by clone library and terminal restriction fragment length polymorphism (T-RFLP), 2) thermodynamic constraints on the acetogenic and methanogenic reactions were evaluated, and then 3) the effect of [HCO3–] on proton transfer was discussed. This work would provide not only an insight into the effect of HCO3– on anaerobic syntrophic oxidation of VFAs, but also an approach to the enhancement of anaerobic digestion by enhancing and balancing the interaction between the syntrophic consortia.

2 Materials and Methods 2.1 Inoculum The inoculum for batch fermentation was anaerobic activated sludge that was taken from the third compartment of a 28 L laboratory-scale anaerobic baffled reactor (ABR) in the State Key Laboratory of Urben Water Resource and Environment, China. The ABR was treating sugar refinery wastewater under mesophilic condition (35°C), and the methane production rate in the third compartment was about 13.8 L/day. Mixed liquor volatile suspended solids (MLVSS) and mixed liquor suspended solids (MLSS) of the sampled anaerobic activated sludge were 4.50 g/L and 8.66 g/L, respectively.

2.2 Batch fermentation

All the batch fermentations for methanogenesis of butyrate were performed in serum bottles with the same working volume of 250 mL. Each of the bottles was filled with 100 mL basal medium and inoculated with 5 mL of the anaerobic activated sludge. The basal medium contains (mg/L): 530 Na2HPO4·2H2O, 410 KH2PO4, 300 NH4Cl, 110 CaCl2·2H2O, 100 MgCl2·6H2O, 300 NaCl, 4.5 FeCl2·4H2O, 1.65 EDTA·Na2, 0.05 H3BO3, 0.05 ZnCl2, 0.038 CuCl2·H2O, 0.05 MnCl2·4H2O, 0.05 CoCl2·6H2O, 0.092 NiCl2·6H2O, 0.026 Na2SeO3·5H2O, 0.033 Na2WO4·2H2O, 0.024 Na2MoO4·2H2O, 0.004 biotin, 0.04 niacin acid, 0.1 pyridoxin HCl, 0.02 riboflavin, 0.04 thiamine HCl, 0.02 6

cyanocobalamin, 0.02 folic acid, 0.04 lipoic acid, 0.02 aminobenzoic acid, 500 L-cysteine HCl. 1 mL/L 0.10% resazurin was added into the medium as a redox indicator. As the sole carbon source, 20 mmol/L butyrate was loaded into the basal medium. To remove oxygen, the media were boiled for 15 min and cooled to room temperature with N2 sparging. Then the media were dispensed to serum bottles with the same gas (N2) atmosphere. After loaded with the basal medium and butyrate, the serum bottles were divided into five groups according to the [HCO3–] of 0, 0.05, 0.10, 0.15, and 0.20 mol/L, respectively, adjusted by adding NaHCO3. The five groups of fermentation were named B0, B0.05, B0.1, B0.15 and B0.20, respectively. NaCl was added into each of the fermentation to give the identical Na+ concentration of 0.20 mol/L. The blank fermentations without butyrate and NaHCO3 were set to access the endogenous methanogenesis from the anaerobic digestion with activated sludge. Each of the serum bottles was inoculated with 5 mL anaerobic activated sludge. The initial pH in the five fermentations were the same 7.50 adjusted by 1 mol/L NaOH or HCl. The serum bottles were then sealed with butyl rubber stopper. Each of the five fermentations were conducted in triplicate. All fermentations were performed in a constant temperature air bath shaker (HZQ-C, Harbin Donglian Electronic Technology Development Co. Ltd., China) at 37°C and 120 rpm.

2.3 Chemical analyses During the batch fermentation processes, 0.50 mL supernatant in each serum bottle was siphoned off for analysis. pH was measured by a pH meter (DELTA 320, Mettler Toledo, USA). Volume of the produced biogas in serum bottles was measured by releasing the gas pressure with 50 mL glass syringes. Then the volume of biogas was corrected to standard temperature (0°C) and pressure (101 kPa). CH4 and CO2 in the produced biogas were analyzed by a gas chromatograph (SP-6800A, Shandong Lunan Instrument Factory, China) equipped with a 2 m stainless column packed with 7

Porapak Q (100 to 180 mesh) and a thermal conductivity detector (TCD). Temperatures of the oven, injection port, and detector were the same 80°C. N2 was used as the carrier gas at a flow rate of 40 mL/min. The injection volume was 0.50 mL. VFAs in the media were analyzed by another gas chromatograph (SP6890, Shandong Lunan Instrument Factory, China) equipped with RTX-Stabilwax glass column (30 m×0.32 mm×1 um) and a flame ionization detector (FID). Temperatures of the injection port, oven and detector were 210°C, 190°C, and 210°C, respectively. N2 was used as carrier gas with a 0.75 MPa column head pressure. The split ratio was 1:50. The injection volume was 1 µL. MLSS and MLVSS were determined according to the procedures described in the Standard Methods (APHA, 1995).

2.4 Microbial community analyses Quantitative polymerase chain reaction (qPCR), T-RFLP and clone library were introduced to investigate the microbial communities in the batch fermentations. At the end of the batch fermentations, sludge samples were collected for DNA extraction, respectively. The genomic DNA was extracted using the DNeasy PowerSoil Kit (Qiagen®, Hilden, Germany) according to the manufacturer's protocol. For qPCR, the bacterial 16S rRNA gene was amplified using ABI 7500 Real-Time PCR System and FastSYBR green master mixture (Sangon Biotech Co., Ltd, China). The primers to conduct qPCR were Bac519f (5'-CAGCMGCCGCGGTAANWC-3') and Bac907r (5'-CCGTCAATTCMTTTRAGTT-3') (Aydin et al., 2015). Each of the reaction for qPCR contained 12.50 μL 2×SGExcel FastSYBR mixture (with ROX), 1 μL each of the two primers (10 mmol/L) and 2 μL template DNA, and then to get a final volume of 25 μL with ddH 2O. For bacterial 16S rRNA gene, the PCR procedure was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 53°C for 45 s and extension at 72°C for 30 s. 8

For clone analysis, the bacterial 16S rRNA gene sequences were amplified using universal bacterial

primers,

EUB8F

(5′-AGAGTTTGATCCTGGCTCAG-3′)

and

EUB1492R

(5′-GGYTACCTTGTTA SGACTT-3′). The archaeal 16S rRNA gene sequences were amplified using the universal archaeal primers ARC8F, (5′-YCYGKTTGATCCYGSCRG-3′) and ARC931R, (5′-CCCGCCAATTC-CTTTHAG-3′). The PCR products were purified using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa, China) and ligated into the pMD19-T vector (TaKaRa, China). Plasmids were transformed into competent cells of Escherichia coli. The clones were randomly selected and sequenced by Sangon Biotech Co., Ltd (China). For 16S rRNA gene T-RFLP analysis of bacteria and archaea, PCR was amplified using the primer pairs EUB8F/EUB1492R and ARC8F/ARC931R, respectively. Both EUB8F and ARC8F were labeled with 6-carboxyfluorescein (FAM). The PCR products were purified using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa, China). The purified DNA was then digested with HaeIII for 2 h at 37°C. The digestion products were size separated by Sangon Biotech Co., Ltd (China).

2.5 Data analysis Each of the five fermentations was carried out in triplicate (n=3). The standard error for each variable was calculated. Multiple comparison tests were conducted to compare the cumulative methane production during exposure to various [HCO3–] with SPSS software. The bacterial and archaeal 16S rRNA gene sequences were clustered into operational taxonomic units (OTUs) with 97% similarity using Mothur program ver. 1.19 (Schloss, 2009). The OTUs were then compared with the 16S rRNA gene sequences database in the National Center for the Biotechnology Information (NCBI) website using BLASTN (Altschul, 1990).

9

3 Result and Discussion 3.1 Methanogenic conversion of butyrate To evaluate the effect of HCO3– on methanogenesis of butyrate, batch culturing of anaerobic activated sludge was conducted with various [HCO3–] and the performance of each fermentation was illustrated in Fig. 1. As shown in Fig. 1A, a lag phase was observed in all the fermentations. With the [HCO3–] of 0 and 0.05 mol/L in B0 and B0.05, respectively, both the lag phases lasted up to 19 d. When [HCO3–] was increased to 0.10, 0.15 and 0.20 mol/L in B0.10, B0.15 and B0.20, the lag phase was decreased to 12, 10 and 8 d, respectively. Obviously, the anaerobic syntrophic butyrate oxidation had been stimulated by the increased [HCO3–]. After the lag phase, butyrate degradation in the five fermentations all came up to a logarithmic phase. Within the logarithmic phase, the maximum butyrate degradation rate in B0, B0.05, B0.10, B0.15 and B0.20 was calculated to be about 176.86, 183.59, 185.14, 219.25 and 233.58 mg/(L∙d), respectively. The results indicated that the supplement of HCO 3– not only accelerated the butyrate degradation, but also enhanced the maximum degradation rate. Following the butyrate degradation, acetate was produced and then converted to methane via aceticlastic methanogenesis. As shown in Fig. 1B, the accumulation of acetate in the fermentation processes synchronously increased with the butyrate degradation (Fig. 1A), but then decreased due to the metabolism of aceticlastic methanogens. The maximal acetate concentration found in B0, B0.05, B0.10, B0.15 and B0.20 was 975.96, 2911.78, 2652.09, 2786.47 and 3065.54 mg/L, which was completely degraded within 37, 46, 50, 70 and 80 days, respectively (Fig. 1B). The results indicated that the methanogenesis was inhibited by the increased [HCO3–], though the syntrophic butyrate oxidation had been enhanced (Fig. 1A). The inhibition of [HCO3–] to methanogenesis was also confirmed by the decreasing specific methane yield and the maximal specific methane production rate. 10

The specific methane yield of butyrate in B0, B0.05, B0.10, B0.15 and B0.20 was about 636.68, 615.22, 609.39, 552.58 and 468.56 mL/g-butyrate, respectively (Fig. 1C). And the maximal specific methane production rate in logarithmic phase was calculated to be about 60.89, 34.66, 24.96, 8.51 and 6.37 mL/(g-butyrate∙d), respectively. The methane yield was as low as 6.07 mL in the blank fermentations (data not shown), suggested that endogenous methanogenesis of the anaerobic digestion with activated sludge was negligible. Though the supplement of HCO3– more than 0.10 mol/L inhibited the aceticlastic methanogenesis, H2 released within all the fermentation was below detection limit (data not shown), indicating the activity of hydrogenotrophic methanogenesis of the inoculated activated sludge was not inhibited. In B0.10, B0.15 and B0.20, H2 produced by the syntrophic acetogenesis of butyrate was almost consumed by hydrogenotrophic methanogens and resulted in the first logarithmic phase from the 6th day to the 15th day (Fig. 1C). And the average methane yield of B0.10, B0.15 and B0.20 after first logarithmic phase reached 139.56, 156.79 and 158.49 mL/g-butyrate, respectively. In second logarithmic phase since the 30th day, the cumulative methane was produced by both hydrogenotrophic and aceticlastic methanogens. Due to the inhibition of aceticlastic methanogenesis by the increased [HCO3–], the specific methane yield of butyrate in B0.10, B0.15 and B0.20 all decreased gradually. Because no observable inhibition of aceticlastic methanogenesis was found in both B0 and B0.05 with the [HCO3–] less than 0.05 mg/L, only one logarithmic phase was observed, in which syntrophic acetogenesis and aceticlastic methanogenesis were also involved. During methanogenesis of butyrate, CO2 was mainly produced in the aceticlastic methanogenesis [Reaction (7)]. The cumulative CO2 increased along with the cumulative CH4 in B0 (Fig. 1D), confirmed that the aceticlastic methanogenesis in B0 followed the butyrate degradation. A lag phase 11

was observed in the fermentations with [HCO3–] more than 0.05 mol/L. With the [HCO3–] of 0.05 and 0.10 mol/L in B0.05 and B0.10, the lag phases lasted up to 30 and 23 d, respectively. When [HCO3–] was increased to 0.15 and 0.20 mol/L in B0.15 and B0.20, both the lag phase was decreased to 12 d. After lag phase, cumulative CO2 increased sharply with the degradation of acetate (Fig. 1B) in B0.05 and B0.10. While CO2 production in fermentation B0.15 and B0.20 came up to the first logarithmic phase from the 10th day to the 19th days. It was interesting to find that acetate was not degraded in this period. So CO2 in the first logarithmic phase was mainly produced from the decomposing of HCO3– with pH dropped (Fig. 1E). In second logarithmic phase since the 30th day, the cumulative CO2 increased following the acetate degradation. Compared with B0, the initial pH in B0.05, B0.10, B0.15 and B0.20 was observably increased by the supplement of HCO3– (Fig. 1E). The pH in the five fermentation processes decreased firstly due to the accumulation of acetate produced by syntrophic acetogenesis of butyrate and then increased due to the consumption by aceticlastic methanogenesis. The lowest pH found in B0, B0.05, B0.10, B0.15 and B0.20 was 7.00, 7.84, 7.96, 8.21 and 8.23, respectively. And the average pH found in the B0, B0.05, B0.10, B0.15 and B0.20 reached 7.59, 8.16, 8.21, 8.43 and 8.47, respectively. The high pH would inhibit the acetate degradation because most aceticlastic methanogen prefer a neutral pH (Ma et al., 2006). It was also reported that the optimum pH for butyrate-oxidizing bacteria neutral (Jackson et al., 1999). However, with the pH ranged from 7.01-7.79, the maximum butyrate degradation rate in treatment B0 was only 176.86 mg/(L∙d) which was the lowest among the 5 treatments (Fig. 1A). The results indicated that 1) the methane fermentation process of butyrate was in stages, i.e. syntrophic acetogenesis coupled with hydrogenotrophic methanogenesis then aceticlastic methanogenesis; 2) the

12

supplied HCO3– had played as a buffer against VFAs produced; 3) the enhanced pH by additional HCO3– inhibited acetate degradation rather than butyrate degradation.

3.2 Microbial community structure To understand the performance of anaerobic syntrophic butyrate oxidation in methane fermentation with various [HCO3–], anaerobic activated sludge in the five fermentations was separately sampled at the end of culturing, and then the clone libraries were constructed based on the bacterial and archaeal 16S rRNA gene sequences, respectively. The bacterial clone library (Table 1) revealed that a lot of the OTUs were related to syntrophic degradation of VFAs. Among the identified bacteria, Syntrophus aciditrophicus can syntrophically degrade butyrate into H2 and acetate (Jackson et al., 1999). Desulfotomaculum salinum is known as a sulfate-reducing bacteria (SRB) which utilized butyrate and acetate as the electron donor to reduce sulfate (Nazina et al., 2005). Desulfomicrobium baculatum is also an SRB, but its syntrophic capability has not been proved (Zhang and Fang, 2001). Smithella propionica is also found to degrade butyrate when co-cultured with Methanospirillurn hungateii (Liu et al., 1999). Some OTUs were also found to be the related bacteria that could promote anaerobic butyrate oxidation by utilizing the intermediate and end products. Thermanaerovibrio velox is reported to promote syntrophic butyrate degradation with H2 as electron donor to reduce Sulphur (Zavarzina et al., 2000). Treponema caldarium can syntrophically oxidize acetate to H 2/CO2 in anaerobic environment with low pH2 (Lee et al., 2015). More than 45% of the bacterial 16S rRNA gene sequences were affiliated to genus Mesotoga which may syntrophically oxidize acetate through an uncharacterized acetate-oxidizing pathway (Nobu et al., 2015). Together with the hydrogenotrophic methanogens, homoacetogen Romboutsia lituseburensis should contribute to the low pH2 by converting H2/CO2 to 13

acetate (Xu et al., 2015). There were other anaerobic syntrophic bacteria were also identified from the anaerobic activated sludge, such as Syntrophorhabdus aromaticivorans (Qiu et al., 2008) and T. acidaminovorans (Sieber et al., 2012). Though syntrophic with hydrogenotrophic methanogens, these bacteria are unable to metabolize butyrate or acetate (Qiu et al., 2008; Sieber et al., 2012). Archaeal 16S rRNA gene sequences showed that methanogens in the clone library were classified

into aceticlastic methanogens (Methanosaeta), hydrogenotrophic (Methanolinea and Methanoculleus) and mixotrophic (Methanosarcina) (Table 2). It is known that hydrogenotrophic methanogens in the clone library also utilized formate as substrate to the formation of methane (Cheng et al., 2008, Imachi et al., 2008). To find out the abundance of the identified functional microorganism in each of the batch fermentation, 16S rRNA gene T-RFLP analysis of bacteria and archaea were carried out, respectively. In silico analysis of clone sequences indicated that there were 2 T-RFs (77 bp and 186 bp) were similar to the bacteria involved in syntrophic butyrate degradation (Fig. 2A). The 77 bp T-RF was related to S. aciditrophicus (Jackson et al., 1999), while 186 bp T-RF was related to D. salinum (Nazina et al., 2005). The abundance of syntrophic butyrate-oxidizing bacteria in fermentation B0, B0.05, B0.10, B0.15 and B0.20 were 22.61%, 11.12%, 4.65%, 6.99% and 6.30%, respectively. According to the abundance of acetogens, the butyrate degradation rate in B0 should be the highest among the five fermentations. However, the fact was that the butyrate degradation rate in B0 was the lowest (Fig. 1A). It was implied that the supplement of HCO3– had played as electron and proton carrier and enhanced the syntrophic butyrate degradation in the other four batch fermentations. It was found that T-RF 203 bp in the five fermentations increased following the increase of [HCO3–]. The T-RF related to Mesotoga infera was chartered by the function of anaerobic syntrophic oxidation of acetate (Nobu et 14

al., 2015). The absolute dominance of 203 bp T-RF in all the five fermentations (Fig. 2A) suggested that acetate was mainly degraded by the syntrophic acetate oxidation bacteria and hydrogenotrophic methanogens. There were 7 T-RFs of archaea were detected by the T-RFLP analysis (Fig. 2B). Among the 7 T-RFs were 21 bp, 182 bp, 213 bp and 216 bp T-RFs which were all identified as methanogens in the clone library (Table 2). The 21 bp T-RF was similar to hydrogenotrophic methanogen Methanolinea tarda (Imachi et al., 2008). It is noteworthy that M. tarda was the most outstanding methanogen in all the five fermentations. The relative abundance of M. tarda in the B0, B0.05, B0.10, B0.15 and B0.20 reached 66.23%, 63.29%, 50.41%, 53.74% and 70.13, respectively. The result confirmed that hydrogenotrophic methanogenesis was the primary pathway for methanogenesis of butyrate. While 182 bp, 213 bp and 216 bp T-RFs were related to Methanosaeta harundinacea, Methanosaeta concilii and Methanosarcina mazei, respectively. Methanosaeta was identified as aceticlastic (Ma et al., 2006; Patel and Sprott, 1990), while Methanosarcina was mixotrophic which utilized both H2 and acetate (Maestrojuan and Boone, 1991). The relative abundance of Methanosaeta harundinacea and Methanosaeta concilii was calculated 0%, 6.60%, 10.26%, 11.15% and 19.02%, respectively, indicating that Methanosaeta was stimulated by the additional HCO3–. Methanosaeta was aceticlastic which utilized acetate to produce CH4 (Jetten et al., 1992; Smith and Ingram-Smith, 2007). It was interesting to find that the increment of [HCO3–] promoted the abundance of aceticlastic methanogens, viz., M. harundinacea and M. concilii (Fig. 2B), but inhibited the aceticlastic methanogenesis (Fig. 1B). It then raised the question why HCO3– could promote their proliferation and what role did these aceticlastic methanogens act. In this study, the butyrate degradation accelerated along with the abundance of Methanosaeta and Methanosarcina increased, 15

suggesting that Methanosaeta and Methanosarcina took part in the syntrophic butyrate degradation. Usually, hydrogenotrophic methanogens act as the electron sinks to cooperate with syntrophic acetogens (Rotaru et al. 2012). However, it was the Methanosaeta and Methanosarcina rather than hydrogenotrophic Methanogens that was promoted by additional HCO3– (Fig. 2B). As a vicarious mechanism, DIET between Methanosaeta or Methanosarcina and syntrophic acetogens stood out, as both Methanosaeta and Methanosarcina have the ability to reduce HCO3– to CH4 with electron (Rotaru et al., 2014a; Rotaru et al., 2014b). Furthermore, the increment of the abundance of Methanosaeta and Methanosarcina in syntrophic VFAs degradation process can be an indicator of DIET (Lü et al., 2016). So, M. harundinacea and M. concilii might participate in the DIET rather than utilized acetate in the fermentation with [HCO3–] more than 0.05 mol/L (Fig. 3). A similar phenomenon that DIET stimulated syntrophic butyrate degradation and enriched Methanosaeta was observed by Zhao and colleagues (Zhao et al.,2016a; Zhao et al., 2016b).

3.3 Thermodynamic analysis of methanogenesis with different [HCO3–]

As illustrated in Fig. 1, the anaerobic syntrophic butyrate oxidation had been progressively enhanced (Fig. 1A), while the methanogenesis of acetate had been inhibited (Fig. 1B and C) by the increased supplement of HCO3– from 0 to 0.20 mol/L. S. aciditrophicus, D. salinum, S. propionica and T. velox were the dominant syntrophic butyrate-oxidizing bacteria in the batch fermentations (Table 1), and their optimal pH was near to neutral as that of the most methanogens (Liu et al., 1999; Nazina et al., 2005; Zavarzina et al., 2000). Without supplement of HCO3–, the pH ranged from 7.00 to 7.79 in fermentation B0, which was more suitable than the other four fermentations (B0.05, B0.10, B0.15 and B0.20) for both syntrophic butyrate-oxidizing bacteria and methanogens, so the butyrate degradation

16

in fermentation B0 should be efficient as same as acetate degradation. However, among the five fermentations, the lag phase and maximum butyrate degradation rate in fermentation B0 was the longest and the lowest, respectively (Fig. 1A). The results indicated that pH was not the only factor affecting the anaerobic syntrophic butyrate oxidation. To understand the mechanism of the enhancement of HCO3– to anaerobic syntrophic butyrate, and the inhibition of HCO3– on acetate degradation, the actual ΔG of Reaction (7) and (8) with different [HCO3–] were calculated. As shown in Reaction (7) and (8), HCO3– as the reactant or the product could affect the ΔG in the two reactions by changing the thermodynamic condition. To reveal the influence of HCO3– on the reactions, ΔG in Reaction (7) and (8) were calculated, respectively, based on Eq. (1). For reaction a[A] + b[B] = c[C] + d[D] there is

[C ]c [ D]d ΔG  ΔG  RTln [ A]a [ B]b 0

Eq.(1)

where ΔG (kJ/mol) is actual energy yield for the reaction under methanogenic conditions (298 K, pH2 10–4 atm, pCH4 0.01 atm), R is the universal gas constant [8.31 J/(mol·K)], and T is Kelvin temperature. pH with calculated to be 8.31 in the fermentation with various [HCO3–] (detail in Supplementary). The initial ionic strength in each fermentation was adjusted to the same level by adding NaCl. The calculation (Fig. 4) showed that the ΔG of Reaction (8) decreased from -118.32 to -137.17 kJ/mol when [HCO3–] increased from 0 to 0.20 mol/L, while the ΔG of Reaction (7) increased from -55.58 to -36.73 kJ/mol. The results confirmed that the anaerobic syntrophic butyrate oxidation had been enhanced by the increased [HCO3–], with the methanogenesis of acetate being inhibited. This result was consistent with the previous studies (Lin et al., 2013; Loureiro et al., 2003). 17

3.4 Proton Transfer with different [HCO3–]

As known, H2-mediated IET [Reaction (1) and (2)], formate-mediated IET [Reaction (3) and (4)] and DIET [Reaction (5) and (6)] are involved in syntrophic methanogenesis of butyrate. As showed in [Reaction (1)-(6)], each of the three IETs was depended upon proton transfer. Proton can directly transfer from syntrophic butyrate-oxidizing bacteria to methanogens or by proton carriers such as HCO3– (Fan et al., 2007). Diffusion rate of proton and proton carriers is driven by their concentration gradient and diffusion coefficient. The interspecies distance of 12.40 μm and 2.00-4.00 μm (averaged 3.00 μm) have been reported in suspended syntrophic consortia (Stams and Dong, 1995) and granular sludge or aggregates (Ishii et al., 2005; Schmidt and Ahring, 1995), respectively. Thus, the diffusion rate (W) of protons and HCO3– can be calculated by Fick's diffusion law [Eq. (2)] which was described by Fan et al. (2007) and Felchner-Zwirello et al. (2012). W=-D∙A∙ΔC/δm

Eq. (2)

where D is the diffusion coefficient (cm2/s), δm is the interspecies distance between syntrophic butyrate oxidation bacteria and methanogens (about 3.00 μm), ∆C is the maximum concentration gradient (mol/L). A is the cross-sectional area (μm2/mL) of the syntrophic consortia, which is the product of single cell surface (1.58 μm2) and copy number per milliliter of syntrophic acetogenic bacterial 16S rRNA gene (Felchner-Zwirello et al., 2012). A limitation in this type of quantitative evaluation exited because that extraction efficiency is uncertain. The calculations based on the bacterial 16S rRNA gene copies (copies/mL) and the ratio of syntrophic acetogenic bacteria (%) (Fig.2 A) were illustrated in Table 3. The diffusion coefficient of proton and HCO3– is 9.30×10-5 and 1.34×10-5 cm2/s, respectively (Fan et al., 2007). According to Eq. (2), the maximum diffusion rate of proton and HCO3– in the tested five 18

fermentations was calculated, respectively. The actual proton diffusion rate via various IETs was also calculated based on the average degradation rate of butyrate in the fermentation processes. As shown in Table 4, the diffusion of proton was very ineffective because of the extremely low proton concentration difference between syntrophic consortia (ΔC <10-7 mol/L at pH 7), though the proton diffusion coefficient (9.30×10-5 cm2/s) was much higher than that of HCO3– (1.34×10-5 cm2/s) (Fan et al., 2007). The actual proton diffusion rate via each of the three IETs were all remarkable higher than the maximum possible diffusion rates, indicating the diffusion of proton was not the main transfer pathway in the anaerobic syntrophic butyrate oxidation. On the contrary, the maximum diffusion rate of HCO3– as proton carrier was very effective and increased generally with the increase of [HCO3–]. Therefore, HCO3– was revealed as an effective proton carrier to enhance the anaerobic syntrophic butyrate oxidation.

4 Conclusions Additional HCO3– accelerated the butyrate degradation rate and enriched M. harundinacea and M. concilii. However, the additional HCO3– more than 0.10 mol/L significantly inhibited aceticlastic methanogenesis. Three functions of the HCO3– had been identified to promote the methanogenesis of butyrate: 1) decreasing the ΔG of syntrophic methanogenesis of butyrate and increasing the ΔG of methanogenesis of acetate, 2) enriching M. harundinacea and M. concilii, 3) increasing the diffusion rate of protons between the syntrophic consortia. The results revealed a novel role of HCO3– in anaerobic butyrate degradation and provided a strategy to enhance butyrate degradation when anaerobic digestion acidified.

19

Acknowledgements This research was supported and funded by the National Natural Science Foundation of China (Grant No. 51478141) and Harbin Institute of Technology Environment and Ecology Innovation Special Funds (Grant No. HSCJ201614). The authors sincerely express their profound gratitude to these institutions and all individuals who contributed towards the success of this work.

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27

Figure captions Fig. 1. Temporal change of the butyrate (A), acetate (B), cumulative methane production (C) and pH (D) Fig. 2 T-RFLP profiles of the bacteria (A) and archaea (B) 16S rRNA genes Fig. 3 Pathway of methanogenesis of butyrate Fig. 4 Effect of [HCO3–] on Gibbs free energy of methanogenesis of butyrate and methanogenesis of acetate (ΔG was calculated under 298 K, pH 8.31, pH2 10–4 atm, pCH4 0.01 atm, 20 mmol/L butyrate and acetate as reactant, 1mmol/L acetate as product)

28

Table 1 Clone library of bacteria OTU ID (GenBank

Numbers

Taxonomy

Clostridiales

Thermoanaerobacterales

Identity T-RF (bp)

Most similar sequence

accession number)

of clones

(%)

F24 (MG674677.1)

2

186

Desulfotomaculum salinum (NR115338.1)

86

F17 (MG674675.1)

1

259

Ruminococcus albus (NR115230.1)

90

F1 (MG674671.1)

1

290

Romboutsia lituseburensis (NR118728.1)

97

F30 (MG674679.1)

1

310

Proteiniclasticum ruminis (NR115875.1)

99

F31(MG674680.1)

1

231

Thermoanaerobacter mathranii

81

(NR074681.1)

Kosmotogales

F8 (MG674688.1)

6

203

Mesotoga infera (NR118572.1)

99

F33 (MG674681.1)

4

203

Mesotoga infera (NR118572.1)

99

F15 (MG674674.1)

1

203

Mesotoga infera (NR118572.1)

98

F2 (MG674676.1)

1

203

Mesotoga infera (NR118572.1)

98

F4 (MG674685.1)

1

203

Mesotoga infera (NR118572.1)

95

Desulfovibrionales

F14 (MG674673.1)

1

76

Desulfomicrobium baculatum (NR074900.1)

99

Syntrophobacterales

F12 (MG674672.1)

1

77

Syntrophus aciditrophicus (NR102776.1)

91

F7 (MG674687.1)

1

77

Smithella propionica (NR024989.1)

96

F34 (MG674682.1)

1

244

Syntrophorhabdus aromaticivorans

93

(NR041306.1)

Synergistales

F5 (MG674686.1)

1

77

Thermanaerovibrio acidaminovorans

82

(NR074520.1)

F35 (MG674683.1)

1

251

29

Thermanaerovibrio velox (NR104765.1)

85

Bacteroidales

F9 (MG674689.1)

1

411

Prolixibacter bellariivorans (NR113041.1)

88

Spirochaetales

F3 (MG674678.1)

1

310

Treponema caldarium (NR074757.1)

87

Ignavibacteriales

F36 (MG674684.1)

1

231

Ignavibacterium album (NR074698.1)

92

30

Table 2 Clone library of archaea OTU ID (GenBank Numbers T-RF

Identity

Taxonomy

Most similar sequence accession number)

Methanosarcinales

FA7 (MG603743.1)

of clones (bp)

9

Substrate (%)

182 Methanosaeta harundinacea

99

Acetate

99

Acetate

99

H2/CO2, Methylamines,

(AY970347.1)

FA33 (MG603747.1)

12

213

Methanosaeta concilii (NR028242.1)

FA2 (MG603741.1)

7

216

Methanosarcina mazei (NR074221.1)

Methanomicrobiales

FA4 (MG603742.1)

12

21

Methanolinea tarda

Acetate, Methanol

98

H2/CO2, Formate

99

H2/CO2, Formate

(NR028163.1)

FA20 (MG603745.1)

3

69

Methanoculleus receptaculi (NR043961.1)

31

Table 3 Syntrophic acetogenic bacterial 16S rRNA gene copy number Bacterial 16S rRNA

Ratio of syntrophic

Syntrophic acetogenic bacterial 16S

gene copy numbers

acetogenic bacteria

rRNA gene copy numbers

(copies/mL)

(%)

(copies/mL)

B0

9.25×107±0.07×107

22.61±0.77

2.09×107±0.09×107

B0.05

9.75×107±0.14×107

11.12±0.50

1.08×107±0.06×107

B0.10

10.22×107±0.11×107

4.65±0.21

0.48×107±0.03×107

B0.15

8.00×107±0.28×107

6.99±0.57

0.56×107±0.06×107

B0.20

9.50×107±0.14×107

6.30±0.21

0.60×107±0.03×107

Tests

32

Table 4 Maximum and actual diffusion rate of protons in the anaerobic syntrophic butyrate oxidation

Tests

Maximum diffusion rate of proton and supplied

Actual proton diffusion rate via various IETs

carrier HCO3– [μmol/(mL∙d)]

[μmol/(mL∙d)]

Proton

HCO3–

DIET

IET via H2

IET via formate

B0

0.24

0

3.35

0.67

0.67

B0.05

0.03

32926.69

3.41

0.68

0.68

B0.10

0.01

28902.32

3.71

0.74

0.74

B0.15

0.01

51127.84

4.82

0.96

0.96

B0.20

0.01

73048.48

5.16

1.03

1.03

33

Acetate concentration (mg/L)

2400

B0 B0.05 B0.10 B0.15 B0.20

A

2000 1600 1200 800 400 0 0

10

20

30

40

50

60

70

Cumulative methane production (mL/g-butyrate)

700

C 600 500 400 B0 B0.05 B0.10 B0.15 B0.20

200 100 0 0

10

20

30

40

50

60

70

80

Time (d) 9.0

E 8.5 8.0

pH

B0 B0.05 B0.10 B0.15 B0.20

7.5 7.0 6.5 0

10

20

30

40

50

60

70

80

Time (d)

34

B0 B0.05 B0.10 B0.15 B0.20

B 3000 2500 2000 1500 1000

80

Time (d)

300

3500

Cumulative conbon dioxide production (mL/g-butyrate)

Butyrate concentration (mg/L)

Fig. 1:

500 0 0

10

20

30

40

50

60

70

80

Time (d) 400

D

300 B0 B0.05 B0.10 B0.15 B0.20

200 100 0 0

10

20

30

40

50

Time (d)

60

70

80

A

100 54 bp

90

Relative abundance (%)

Smithella propionica (77 bp)

80

85 bp

70

Desulfotomaculum salinum (186 bp)

60

Mesotoga infera (203 bp) Syntrophorhabdus aromaticivorans (244 bp)

50

Thermanaerovibrio velox (251 bp)

40

271 bp

30

276 bp

20

279 bp

290 bp

10

Prolixibacter bellariivorans (411 bp)

0 Inoculum

B

B0

B0.05

B0.10

B0.15

B0.20

100

Methanolinea tarda (21bp)

Relative abundance (%)

90

58 bp

80

80 bp

70

Methanosaeta harundinacea (182bp)

60

198 bp

50

Methanosaeta concilii (213bp)

40

Methanosarcina mazei (216bp)

30 20 10

0 Inoculum

B0

B0.05

B0.10

B0.15

Fig. 2:

35

B0.20

Fig. 3:

Butyrate

Process Syntrophic Methanogenic

Syntrophus aciditrophicus

Intermediate products

Smithella propionica

Mesotoga infera

H2/CO2

Methanoculleus receptaculi

Treponema caldarium

Acetate

Methanosarcina mazei

Methanosaeta harundinacea

Methanosaeta harundinacea

Methanosaeta concilii

Methanosaeta concilii

Methanolinea tarda

CO2+CH4

36

e-

Fig. 4:

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Butyrate

Process Syntrophic Methanogenic

Syntrophus aciditrophicus

Intermediate products

Smithella propionica

Mesotoga infera

H2/CO2

Methanoculleus receptaculi

Treponema caldarium

Acetate

Methanosarcina mazei

e-

Methanosaeta harundinacea

Methanosaeta harundinacea

Methanosaeta concilii

Methanosaeta concilii

Methanolinea tarda

CO2+CH4 Pathway of methanogenesis of butyrate

38

Highlight:

1 Novel function of HCO3– in anaerobic digestion were revealed. 2 ΔG of syntrophic butyrate methanogenesis could be decreased by additional HCO3–. 3 Proton transfer among syntrophic consortia was enhanced by additional HCO3–. 4 Additional HCO3– enhanced Methanosaeta abundance but decreased acetate degradation. 5 Methanogenesis of butyrate was enhanced by additional HCO3– as a result.

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