Thermodynamic analysis of direct interspecies electron transfer in syntrophic methanogenesis based on the optimized energy distribution

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Please cite this article as: Liu, Y., Gu, M., Yin, Q., Du, J., Wu, G., Thermodynamic analysis of direct interspecies electron transfer in syntrophic methanogenesis based on the optimized energy distribution, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122345

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Thermodynamic analysis of direct interspecies electron transfer in syntrophic methanogenesis based on the optimized energy distribution

Yu Liu, Mengqi Gu, Qidong Yin, Jin Du, Guangxue Wu*

Guangdong Province Engineering Research Center for Urban Water Recycling and Environmental Safety, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong, China

*Corresponding author E-mail: [email protected]

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Abstract The aim of this study was to investigate the syntrophic methanogenesis from the perspective of energy transfer and competition. Effects of redox materials and redox potential on direct interspecies electron transfer (DIET) were analyzed through thermodynamic analysis based on the energy distribution principle. Types of redox materials could affect the efficiency of DIET via changing the total energy supply of the syntrophic methanogenesis. Decreasing system redox potential could facilitate DIET through increasing the total available energy. The competition between hydrogenotrophic methanogens and DIET methanogens might be the reason for the low proportion of the DIET pathway in the syntrophic methanogenesis. A mechanism of facilitation of DIET was proposed based on the energy distribution. Providing sufficient electrons, inhibiting hydrogenotrophic methanogens and adding more competitive redox couples to avoid hydrogen generation might be beneficial for the facilitation of DIET.

Keywords: Direct interspecies electron transfer; Thermodynamic analysis; Redox materials; Energy distribution

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1. Introduction Anaerobic biological treatment is a core technology for sustainable wastewater treatment. It can convert complex organic carbon to bioenergy methane (CH4) with the cooperation of different microorganisms. During the syntrophic methanogenesis, Gibbs free energy of the acetogenic reaction for producing acetate and hydrogen (H2) is usually positive (Thauer et al., 1977), indicating that this reaction is thermodynamically unfavorable. Interspecies hydrogen transfer (IHT) is an effective pathway for methanogens to utilize H2 and decrease the H2 partial pressure in the system so as to make the acetogenic reaction thermodynamically favorable. The suitable partial pressure of H2 for acetogenesis is below 10-4 atm (Logan et al., 2002), while H2 needs to be maintained at a relatively high partial pressure for hydrogenotrophic methanogenesis. Besides, the slow H2 transfer rate usually results in the low efficiency of methanogenesis. Compared with IHT, direct interspecies electron transfer (DIET) is an electron transfer pathway through pili or c-type cytochrome OmcS instead of the diffusion of soluble substances (Lovley, 2011), which could avoid the accumulation of H2 and result in the faster electron transfer rate (Cruz Viggi et al., 2014). DIET showed a slight thermodynamic advantage (0.7 KJ/mol) compared with formate-mediated interspecies electron transfer, and DIET could improve the metabolic efficiency to compensate for the external limitations (Storck et al., 2016), which makes DIET a more effective electron transfer pathway to promote methanogenesis.

Some abiotic redox materials (RMs), such as granular activated carbon, carbon cloth and hematite (Fe2O3), can also function as redox mediators in the DIET pathway (Chen et al., 3

2014a; Zhu et al., 2015; Barua et al., 2019). DIET has been confirmed in co-cultures of Geobacter and Methanosaeta or Methanosarcina through pili and c-type cytochrome (Rotaru et al., 2014a and 2014b). Some RMs could function as a substitution of c-type cytochrome or pili to transfer electrons. For example, activated carbon could replace pili and c-type cytochrome to transfer electrons in co-cultures of Geobacter metallireducens and Geobacter sulfurreducens (Liu et al., 2012). The addition of hematite could also facilitate the generation of c-type cytochrome or humic substances to improve the redox property of extracellular polymeric substances (EPS) and thus facilitate DIET (Ye et al., 2018). In addition, the promotion of DIET with the dosage of RMs might be related with the characteristics of RMs (Zhu et al., 2015; Zhang et al., 2018b). For example, the specific surface area of hematite was negatively related with the promotion of DIET (Zhu et al., 2015). However, Zhang et al. (2018b) found that the surface functional group and surface charge of biochar might determine the facilitating effect on DIET. Yuan et al. (2018) pointed out that the functional group and surface charge of biochar was related with the redox-active properties, resulting in different efficiencies of DIET. However, Wang et al. (2018) found that the addition of biochar could facilitate DIET by decreasing the redox potential of system. All these results demonstrated that RMs showed different abilities to promote DIET, but the facilitation mechanism still requires further investigation.

Thermodynamic analysis is a useful method to connect substance conversion with energy generation and utilization. Through thermodynamic analysis, Gu et al. (2019) found that RMs with relatively low redox potentials could promote the DIET pathway for methanogenesis. 4

However, previous thermodynamic studies only focused on the promotion of the reaction rate of DIET from the aspect of substance transformation. Thermodynamic analysis of DIET associated with energy is less conducted. It has been found that 90% of energy loss in DIET is used as the activation energy required for cofactors to transfer electrons (Storck et al., 2016), and DIET might provide more energy for syntrophic microorganisms than IHT (Cheng and Call, 2016). When ethanol was oxidized to generate electrons, it could provide 30 kJ/mol more energy than producing hydrogen (Mei et al., 2018), indicating the priority of DIET from the perspective of energy. This result reveals that energy supplying and utilization might be important for investigating DIET.

Energy supply, distribution and utilization have been investigated intensively. Schink (1997) pointed out that the syntrophic relationship in the anaerobic system is related to not only substance transformation, but also energy transfer. Syntrophic microorganisms could share the total energy generated from the methanogenic process unequally for growth and movement (Oh et al., 2018; Seitz et al., 1990). Walker et al. (2012) found that the total energy provided from the fermentation of lactic acid by the co-cultures of Desulfovibrio and Methanococcus was -82.2 kJ/mol, but the energy assigned to Desulfovibrio and Methanococcus was -60 kJ/mol and -20 kJ/mol, respectively. Another example is that energy assigned to butyric acid oxidizing bacteria, hydrogenotrophic methanogens and acetoclastic methanogens was 17%, 9-10% and 73-74%, respectively (Junicke et al., 2016). These results demonstrated that energy was not distributed equally and syntrophic partners may compete for energy. However, a comprehensive thermodynamic analysis associated with energy 5

supply and distribution in syntrophic methanogenesis via DIET has not been reported yet.

This study aimed to investigate the energy distribution of syntrophic methanogenesis with DIET and effects of RMs through thermodynamic analysis. Effect of proportions of the DIET pathway were examined to reveal the energy competition between syntrophic microorganisms. Moreover, a mechanism of DIET facilitation was proposed.

2. Materials and methods Catabolism reaction (CAT) and anabolism reaction (AN) were used to represent the metabolism of microorganisms. The reactions relating to the conversion of ethanol to methane with/without the DIET pathway and the calculation of Gibbs free energy (ΔG) are shown in Table 1. The reactions in Table 1 all satisfy the balance of atom and valence. The electron donor half reactions are the oxidation of organic carbon, and the electron accept half reactions are the reduction of H+ to H2 or the generation of CH4. The catabolism reactions were the sum of electron donor and acceptor half reactions based on the balance of electrons. The DIET reactions were adopted from previous studies (Rotaru et al., 2014a and 2014b). The anabolism reactions were according to Heijnen (2002). The biomass yield coefficient (Y), maximum specific growth rate (μmax), decay coefficient (kd) and all other thermodynamic parameters were calculated according to Heijnen (2002) and Kleerebezem and van Loosdrecht (2010). The Gibbs free energy of the DIET reaction was calculated with the consideration of the redox potential of RM (ERM) (Moscoviz et al., 2017). The reference electrode for all the redox potential used in this study was the standard hydrogen electrode. 6

The ΔG of the reactions in metabolism of microorganisms were calculated as following: a1R1 + a2R2 → b1P1 + b2P2 ΔGθ = [biΔGθ (Pi) − aiΔGθ (Ri)] Where ΔGθ represents the Gibbs free energy of reactants Ri and products Pi in the reaction under standard condition. ai and bi are the stoichiometric parameters in theoretical reactions. CAT reaction is the degradation of organic carbon (ethanol in this study) to generate acetate or carbon dioxide (CO2) and AN reaction is the biomass synthesis reaction.

All the reactions in this study were modified with 308 K and pH=7. The modification of Gibbs free energy is according to the following equation: ∏c(Pi)bi

ΔG = ΔGθ + RTln∏c(Ri)ai Where R is the gas constant with the value of 8.314 J/(mol·K). T is the temperature of reaction (K). c(Pi) and c(Ri) represent the concentration of reactants and products, respectively.

The ΔG of the DIET reaction is calculated as the following equation: a1R1 + a2R2 + nRM-→ b1P1 + b2P2 + nRM ΔG = ΔGθ + nFERM/103 Where RM/RM- represents the redox couple transferring electrons between acidogenic bacteria and methanogens. F refers to faraday constant with the value of 96485 C/mol. ERM is the redox potential of RM used in this study. 7

The heterotrophic growth/autotrophic growth dissipation energy ΔGdis (kJ/mol) is calculated according to the following equation: Gdis = 200 + 18(6 – No.C)1.8 + exp {[(3.8 − γ)2]0.16 (3.6 + 0.4No.C)} Where No.C is the number of carbon atom in organic carbon (e.g. for ethanol, No.C = 2). γ represents the reduction degree of substance. For example, for ethanol, γ = 6.

The biomass yield coefficient Y (C-mol biomass/mol substance) is calculated as the following equation: λ = (−Gdis − GAN)/GCAT Y = 1/(λYSCAT + YSAN) Where YSCAT and YSAN refer to the stoichiometric coefficient of ethanol in CAT and AN reactions, respectively. In this study, YSCAT has a fixed value of 1 to represent the catabolism reaction with 1 mol substance. YSAN is the stoichiometric coefficient of ethanol in AN reaction with 1 mol biomass produced. For example, YSAN is 2 in reaction 2 (Table 1).

The calculation of maintenance coefficient mG (kJ/(C-mol biomass·h)) is according to the following equation: mG = 4.5exp[

―69000 1 1 ( ― )] T 298 R

The decay coefficient kd (h-1) is calculated according to the following equation: kd =

mG Gdis + (γX/γD)( ― GCAT) 8

Where γD represents the reduction degree of electron donor, and γX is the reduction degree of biomass with the value of 4.2.

The maximal production rate of Gibbs energy by catabolism qmax (KJ/h) is calculated G according to the following equation: = 3( ― ∆GCAT/γD)exp[ qmax G

―69000 1 1 )] ( ― R T 298

The calculation of maximum specific growth rate μmax (h-1) is according to the following equation: G dis μmax = (qmax G ― m )/∆G

To calculate the thermodynamic parameters of syntrophic methanogenesis, some principles were set to ensure the accuracy of calculations. The principles are as follows: (1) All the reactions were modified with T = 308 K and pH = 7. The concentrations of all the liquid state reactants and products were 1 mol/L and the partial pressure of gas was 1 atm. (2) The energy supplying reaction was identified as the reaction that had a negative Gibbs free energy without consideration of ATP generation. There was no energy loss in the energy distribution and transfer process and the energy utilization efficiency was 100%. The net available energy was the sum of Gibbs free energy of all the reactions involved in the system. The total available energy was the sum of Gibbs free energy of energy supplying reactions and the energy distribution had no priority within all the reactions in the system. The minimum energy for each reaction had to satisfy μmax > kd > 0 to maintain 9

the growth of microorganisms. (3) All the reactants in methanogenic reactions, such as acetate, H2 and electrons, were all from the oxidation of organic carbon such as ethanol. The ratio of different methanogenic reactions was dependent on the ratio of acetate, H2 and electrons produced from 1 mol organic carbon. CO2 was set as the non-limiting factor due to its adequate existence in most systems. (4) The reaction rate was proportional to the growth rate of microorganisms, so the growth rate was used to represent the reaction rate. When the generation rate of acetate, H2 and electrons was equal to the consumption rate, the distribution coefficient was the best for the balance of the system (referred to as the optimum point).

In the syntrophic methanogenesis without DIET, the energy supplying reactions included reaction 7 (ΔGA) and reaction 9 (ΔGH). The net available Gibbs free energy was ∆G = ∆GE + ∆GA + 2/4∆GH = -91.44 kJ/mol. The energy distribution coefficient for reaction 1, reaction 7 and reaction 9 was a1, a2 and (1 – a1 – a2), respectively. When the generation rates of acetate and H2 were equal to their consumption rates, i.e., μmax_E = μmax_A and 2μmax_E = 4μmax_H, the system reached a balance with no acetate and H2 accumulation.

In the system with DIET pathway, the syntrophic methanogenesis included reaction 3, reaction 7 and reaction 11 when ethanol was used as the organic carbon. The net available energy was the same as the syntrophic methanogensis without DIET (-91.44 kJ/mol). However, whether reaction 3 was an energy supplying reaction relied on the value of ERM. 10

When the value of ERM was above -0.258 V, the energy supplying reactions included reaction 3 and reaction 7. When ERM was between -0.406 V and -0.258 V, reaction 3, 7 and 11 were all energy supplying reactions and the total available energy was -91.44 kJ/mol. When ERM was below -0.406 V, reaction 7 and reaction 11 were the energy supplying reactions.

In the real methanogenic system, acetoclastic methanogenesis, hydrogenotrophic methanogenesis and DIET pathway may co-exist. The effects of ratios of the DIET pathway was investigated. The ratio of the DIET pathway α (0 < α < 1) represented the ratio of electrons which could be used for CH4 generation directly through DIET instead of H2 generation. Since the competition for electrons only existed between DIET and hydrogenotrophic pathways, the ratio of hydrogenotrophic pathway was set as (1-α). The actual ratio of DIET pathway should be calculated as 0.8α. The net available energy was -91.44 kJ/mol. The energy distribution coefficients for four reactions (reaction 5, 7, 9 and 11) were a1, a2, a3 and (1 – a1 – a2 – a3), respectively. When μmax_E = μmax_A, 2(1 – α)μmax_E = 4μmax_H and 4αμmax_E = 8μmax_D, the system reached a balance without accumulation of any intermediates.

3. Results and discussion 3.1 Thermodynamic analysis of syntrophic methanogenesis Ethanol was one of the most suitable organic carbon for methanogenesis, and the degradation rate of ethanol was much faster than that of acetate or propionate (Liu et al., 2019). The energy distribution of syntrophic methanogenesis of ethanol without DIET is shown in the 11

supplemental materials. Zone I represented that there was no acetate and H2 accumulation in the system and all acetate and H2 were used for methane production. It was the best zone for methanogenesis. Zone II and IV represented H2 accumulation and acetate accumulation, respectively. Zone III represented the accumulation of both acetate and H2, which was the worst for methanogenesis. The growth rate of methanogen is much slower than that of acetogen (Haarstrick et al., 2004), resulting in easy accumulation of acetate and H2 during the anaerobic methanogenesis (Zone III). To promote the degradation of ethanol and avoid the accumulation of acetate and H2, the distribution of energy should be controlled in Zone I. It indicated that most of available energy should be distributed to acetoclastic methanogenesis and hydrogenotrophic methanogenesis. Increasing energy distribution coefficient of acetoclastic methanogenesis could promote the efficiency of methanogenesis distinctly.

DIET, which has been identified between Geobacter and Methanosaeta or Methanosarcina (Rotaru et al., 2014a and 2014b), is a methanogenic pathway with high efficiency. Geobacter could be enriched when ethanol was used as the organic carbon (Zhao et al., 2016; Xing et al., 2017), which made ethanol the best organic carbon for DIET. c-type cytochrome (ERM = -0.29 V) was an important redox material in the electron transport chain in the DIET pathway (Ueki et al., 2018; Baek et al., 2018). Thermodynamic analysis of ethanol syntrophic methanogenesis with c-type cytochrome as RM and the four Zones representing different conditions are shown in Fig. 1. The optimum point had the same distribution coefficient as syntrophic methanogenesis without DIET. When hydrogenotrophic methanogenesis does not exist, all the electrons would be utilized by the DIET pathway. The Gibbs free energy of 12

hydrogenotrophic methanogenesis reaction and DIET reaction with c-type cytochrome as RM was almost the same. Therefore, the energy distribution was also similar under these two conditions, and the total reaction rate of methanogenesis had no priority over syntrophic methanogenesis without DIET. The total reaction rate of methanogenesis through the DIET pathway is determined by acetogenic reaction rate, electron transfer rate and methanogenic reaction rate. Therefore, in the DIET pathway, the facilitation of the acetogenic and methanogenic reaction rates might not be responsible for the facilitation of methanogenesis. Although many studies have pointed out that DIET could facilitate the total methanogenesis rate and CH4 production, it might be due to the faster electron transfer rate instead of the reaction rate (Cruz Viggi et al., 2014), which could not be revealed through the thermodynamic analysis.

In the DIET pathway, ethanol was firstly oxidized to acetate and electrons. Then acetate was used to generate CH4 directly and methanogens could utilize CO2 and electrons through the DIET pathway to generate CH4 in the co-cultures of Geobacter metallireducens and Methanosaeta harundinacea (Rotaru et al., 2014a). With thermodynamic analysis and the experimental data shown in Rotaru et al. (2014a), the reaction rate of ethanol oxidation was slow during the initial stage. Since DIET was dependent on the generation of electrons from the ethanol oxidation, DIET showed no priority. During this stage, the distribution of energy was in Zones I and II (Fig. 1). When the rate of ethanol oxidation increased, energy distribution was changed to Zones III and IV, and energy distributed to ethanol oxidation and DIET increased. Therefore, the DIET pathway was promoted and the total rate of 13

methanogenesis increased. However, the reaction rate of the ethanol oxidation was higher than that of the acetoclastic methanogenesis, resulting in the accumulation of acetate. At the end of the reaction, ethanol was consumed completely and electrons were not sufficient for the DIET pathway. Acetoclastic methanogenesis was the dominant pathway for CH4 production with the consumption of accumulated acetate. It demonstrated that the energy could be transferred from Zones I and II to III and IV and then back to I and II. Similarly, energy distribution showed the same transferring tendency in the co-cultures of Geobacter metallireducens and Methanosarcina barkeri when ethanol was used as the organic carbon (Rotaru et al., 2014b). Acetate accumulation also appeared in this research, indicating that energy was transferred from Zones III and IV to I and II. In the Zone I, methanogenesis was dominant in the system and showed the priority of DIET to facilitate methanogenesis. But it seemed unlikely to maintain the energy distribution of methanogenic system in Zone I because when the energy distribution was in Zone I, the total reaction rate was always slow due to the relatively low substance or electron concentration.

Therefore, energy distribution might be one of the most important factors affecting efficiency of syntrophic methanogenesis through DIET, and transfer of energy might be related with the microbial community.

3.2 Effect of redox potential on DIET For methanogenesis systems with different redox materials, the net available energy was the same. It is hard to understand that the addition of different redox materials did not affect the 14

net available energy, resulting in no differences on the energy distribution. When ERM was in different ranges, the energy supplying reactions in the methanogenesis system changed. For example, When ERM was above -0.258 V, reaction 3 and reaction 7 were energy supplying reactions. But when ERM was between -0.406 V and -0.258 V, reaction 3, 7 and 11 were energy supplying reactions. It indicated that the energy supplying mode might be affected with the addition of redox materials. To further investigate the effect of redox materials on the methanogenesis system, the total available energy, the sum of Gibbs free energy of all energy supplying reactions, was adopted for analysis. Redox potential of RMs could affect the Gibbs free energy of reactions in the DIET pathway (Table 1). ERM above -0.406 V could facilitate ethanol oxidation to generate electrons instead of H2 (reaction 3). When ERM was below -0.258 V, the DIET methanogenesis pathway could be promoted (reaction 11). All these indicated that ERM could affect the energy supplying in the system and the addition of RMs could change the total available energy of the system.

When ERM was above -0.258 V, increasing ERM could improve energy supplying through promoting ethanol oxidation to facilitate methanogenesis (Table 2). When ERM ranged from -0.406 V to -0.258 V, the total available energy remained -91.44 kJ/mol. When ERM was below -0.406 V, decreasing ERM could increase the total available energy. These results demonstrated that a suitable ERM could improve the total available energy to facilitate methanogenesis. Since ERM could hardly be below -0.406 V, carbon materials with a wide range of redox potential from +0.195 V to +0.576 V (Goldin et al., 2008) were used as an example to discuss as follows. Compared with c-type cytochrome, the relatively higher total 15

energy with carbon materials resulted in the higher reaction rate of methanogenesis. Chen et al. (2014a) found that carbon cloth could accelerate metabolism of DIET co-cultures but did not promote metabolism of co-cultures involved in interspecies H2 transfer. The enrichment of Geobacter and Methanosaeta species suggested that interspecies electron transfer shifted from IHT to DIET with the addition of conductive carbon cloth (Zhao et al., 2017). Biochar could accelerate DIET as an electron shuttle in the co-cultures of Geobacter and Methanosarcina (Yuan et al., 2018). The reasons for carbon materials promoting DIET were mostly divided into two types. The first one is the enrichment of syntrophic microorganisms (Zhao et al., 2017). The other one is that carbon materials can serve as the electron shuttle and the functional groups (e.g. quinone) on the surface of carbon materials can accept and donate electrons reversibly (Yuan et al., 2018). In this study, a new explanation from the perspective of energy was proposed. ERM of carbon materials could provide more energy to facilitate DIET methanogenesis and increase reaction rate of methanogenesis.

In the calculation with ERM between -0.406 V and -0.258 V, the total available energy remained the same, indicating the similar condition for c-type cytochrome and Fe2O3 as RMs (Table 2). In previous studies, the addition of different RMs showed different facilitation abilities (Chen et al., 2014a and 2014b; Wang et al., 2019). It seemed unreasonable that there was no difference between c-type cytochrome and Fe2O3. In reality, the system energy is dynamic and the yield coefficient of methanogens could not be realized under the optimized condition. Therefore, other factors (i.e., the redox potential of system) affecting the performance of DIET should be existed. For example, Wang et al. (2019) found that the 16

addition of 10 g/L magnetite could decrease the redox potential of system by 25% and increase CH4 production by 34.4%. Zhang et al. (2018a) added granular activated carbon/nano zero-valent iron in the methanogenesis system and found that redox potential of system decreased from -490 mV to -500 mV. These results showed that redox potential of system might have more important effects than redox potential of RMs on methanogenesis.

The system redox potential should be calculated with the sum of all the redox reactions involved in the anaerobic process. Usually, in a complex system, the redox potential should be varied. Generally, the best redox potential range for methanogenesis is from -400 mV to -200 mV (Hirano et al., 2013). To investigate the effect of system redox potential on DIET, -0.6 V, -0.4 V and -0.2 V were set as the system redox potential to compare the performance of DIET (Table 3). Since the confirmed DIET pathway did not co-exist with the hydrogenotrophic pathway, only reactions 3, 7 and 11 were included in the methanogenic system for comparison. Under the optimized condition with different system redox potentials, decreasing system redox potential could facilitate DIET distinctly. Decreasing redox potential from -0.2 V to -0.4 V, the reaction rate of DIET increased by 164%, which might further increase the CH4 production rate. Decreasing system redox potential could increase the total available energy and then promote DIET. Hirano et al. (2013) used the electro-chemical method to investigate methanogenesis and found that when the voltage was -0.8 V, CH4 production increased by 1.6 times. In addition, voltage might have a more obvious effect on microbial community (Dou et al., 2018). It demonstrated that the system redox potential might be the key factor affecting methanogenesis and the relatively low redox potential could 17

facilitate methanogenesis and affect microbial community to promote DIET.

3.3 Effect of proportions of the DIET pathway on methanogenesis Despite that a low redox potential could facilitate syntrophic methanogenesis, the distribution coefficient of DIET decreased even when the total energy increased (Table 3). It indicated that improving total available energy could increase methanogenesis reaction rate and more energy was assigned to the ethanol oxidation. Although it is well-known that DIET outcompeted interspecies H2/formate transfer due to its low energy consumption (Lovley, 2017), the energy used for the DIET pathway to produce CH4 might be different. Lin et al. (2017) found that electron-consuming methanogens utilizing CO2 and electrons to produce CH4 was thermodynamically unfavorable, demonstrating that this reaction might need more extra energy generated from other reactions, which was opposite to acetoclastic and hydrogenotrophic methanogenesis (Table 1). It indicated that the priority of DIET might not be related with low energy consumption. The electron transfer rate of DIET is much faster than that of interspecies H2/formate transfer (Cruz Viggi et al., 2014), and this might be the most important advantage of DIET rather than energy consumption. Therefore, the proportion of DIET might be the key factor for facilitating methanogenesis.

To investigate the effect of the proportion of the DIET pathway on methanogenesis, ethanol oxidation, acetoclastic methanogenesis, hydrogenotrophic methanogenesis and DIET were combined in a system. Considering that c-type cytochrome was used as the RM (Fig. 2), when the proportion of the DIET pathway was below 12.8%, reaction 5 could not supply energy for 18

the system. Due to the priority of hydrogenotrophic methanogenesis, the DIET pathway could not facilitate methanogenesis with a high efficiency. When the proportion of the DIET pathway increased above 12.8%, the total energy remained the same (-91.44 kJ/mol). When the proportion of the DIET pathway was increased to 32%, there was no accumulation of H2 or electrons, hydrogenotrophic methanogenesis and DIET could perform well. If the proportion was above 48%, which should be considered as the priority of the DIET pathway, the electrons would be accumulated.

The same tendency was found when biochar (ERM = -0.0174 V) and carbon materials (ERM = +0.195 V) were used as RM and the proportion for electron accumulation was changed to 18.8% and 14.8%, respectively. Electron accumulation indicated that there would be other redox reactions consuming these electrons. The competition for electrons between hydrogenotrophic methanogenesis and DIET existed in the methanogenesis system. Since the system only contained four reactions, H+/H2 might be the only redox couple to consume these electrons, resulting in the enhancement of hydrogenotrophic methanogenesis. Therefore, the DIET pathway might not be prior to hydrogenotrophic methanogenesis in a mixed system and the proportion of DIET pathway could not reach a high level. It might be important to inhibit hydrogenotrophic methanogenesis to facilitate DIET for high-efficiency methanogenesis.

The effects of redox materials on hydrogentrophic and DIET pathways might be diverse. For example, Zhao et al. (2018) found that addition of ferroferric oxide (Fe3O4) could decrease the relative abundance of hydrogenotrophic methanogens and enrich the microorganisms possibly 19

involved in the DIET to facilitate CH4 production. However, the addition of magnetite might not change the reaction rate of hydrogenotrophic methanogenesis (Jing et al., 2017). Although the activity of hydrogenotrophic methanogens might not be affected obviously with the addition of all kinds of RMs, inhibition of H2 generation might be another choice to facilitate the DIET pathway. The DIET pathway could not consume all the electrons even when the proportion of DIET was high. Therefore, another redox couple that had higher redox potential than H+/H2 could outcompete H+/H2 to utilize the electrons and benefit the DIET pathway.

3.4 Proposed mechanism of facilitation of DIET based on energy distribution Assuming that energy distribution generally existed in the methanogenesis system, a mechanism of methanogenesis was proposed based on the energy distribution (Fig. 3). From the perspective of energy distribution, higher energy distributed to DIET through increasing the total available energy could contribute to the promotion of DIET. Patrick et al. (1973) presented a redox potential control system to obtain a wide range of redox potential (from approximately -250 mV to +600 mV) with hydrogen-nitrogen mixtures, which could be used to obtain a relatively low redox potential to benefit DIET. The addition of RMs could not only work as electron shuttles for speeding up the electron transfer rate, but also enrich functional microorganisms for DIET, such as Geobacter (Xu et al., 2013). In addition, the dosage of RMs could also decrease the redox potential of system (Wang et al., 2019), which would further increase the available energy for methanogenesis.

Based on the above analysis, the hydrogenotrophic methanogenesis might compete with DIET 20

for both electrons and energy. As a result, the proportion of DIET could not reach a relatively high level. It indicated that reducing the function of hydrogenotrophic methanogens might be a strategy to promote DIET. For example, the addition of Fe3O4 could decrease the abundance of hydrogenotrophic methanogens (Zhao et al., 2018). However, although hydrogenotrophic methanogenesis did not exist in co-cultures of Geobacter metallireducens and Methanosaeta harundinacea when ethanol was the organic carbon, the proportion of DIET was only 33% (Rotaru et al., 2014a). It demonstrated that hydrogenotrophic methanogenesis might not be the only reason for the relatively low proportion of DIET in methanogenesis, electrons supply limitation should also be taken into consideration when CO2 was not a limiting factor. When electron supply was limited in the methanogenesis system, extra electron donor might be added to facilitate DIET. For example, zero-valent iron could serve as electron donor to facilitate the reduction from CO2 to CH4 (Feng et al., 2014). Therefore, sufficient electrons and inhibition of hydrogenotrophic methanogenesis might be crucial to the promotion of DIET.

Increasing the proportion of DIET through the addition of RMs to a certain level could result in the accumulation of electrons. Under this condition, a more positive redox couple than H+/H2 utilizing electrons preferentially should be applied to avoid H2 generation and competition between hydrogenotrophic methanogens and DIET methanogens. For example, Fe3+ had a much more positive oxidability than H+, which might be more competitive than H+ and could utilize electrons preferentially. If Fe3+ would be added into the methanogenesis system, DIET could be promoted while electrons were consumed by Fe3+ instead of H+ to 21

inhibit hydrogenotrophic methanogenesis. It might be another strategy for DIET facilitation.

4. Conclusions Energy distribution might be one of the most important factors affecting efficiency of syntrophic methanogenesis through DIET. The enhancement of DIET was related to types of RMs. Low redox potential of system could facilitate DIET through increasing the total available energy. The proportion of DIET could not reach a certain high level due to the competition for electrons and energy between hydrogenotrophic methanogenesis and DIET. Furthermore, sufficient electrons, inhibition of hydrogenotrophic methanogens and addition of competitive redox couple to avoid H2 generation, might be all crucial to the facilitation efficiency of DIET.

Acknowledgements This research was supported by the National Natural Science Foundation of China (51878371) and the Shenzhen Science and Technology Innovation Committee (JCYJ20170817161106801).

Supplementary Materials E-supplementary data for this work can be found in e-version of this paper online.

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28

ess

Figures and Tables Table 1 Catabolism and anabolism reactions of the syntrophic methanogenesis. Pathway 1 Conventional

2 3

DIET

4

xidation

genesis

5 DIET: Conventional =α:(1-α)

6

7 Acetoclastic

8

9 Hydrogenotrophic

10 11

DIET

12

Reactions CAT: CH3CH2OH + H2O → CH3COO- + 2H2 + H+ AN: 0.5CH3CH2OH + 0.2NH4+ → CH1.8O0.5N0.2 + 0.9H2 + 0.2H+ CAT: CH3CH2OH + H2O + 4RM → CH3COO- + 5H+ + 4RMAN: 0.5CH3CH2OH + 0.2NH4+ + 1.8RM → CH1.8O0.5N0.2 + 2H+ + 1.8RMCAT: CH3CH2OH + H2O + 4αRM → CH3COO- + 2(1-α)H2 + (1+4α)H+ + 4αRMAN: 0.5CH3CH2OH + 0.2NH4+ + 1.8αRM → CH1.8O0.5N0.2 + 0.9(1-α)H2 + (0.2+1.8α)H+ + 1.8αRMCAT: CH3COO- + H+ → CH4 + CO2 AN: 0.5CH3COO- + 0.2NH4+ + 0.3H+ + 0.1H2 → CH1.8O0.5N0.2 + 0.5H2O CAT: CO2 + 4H2 → CH4 + 2H2O AN: CO2 + 2.1H2 + 0.2NH4+ → CH1.8O0.5N0.2 + 1.5H2O + 0.2H+ CAT: CO2 + 8H+ + 8RM- → CH4 + 2H2O + 8RM AN: CO2 + 4H+ + 0.2NH4+ + 4.2RM- → CH1.8O0.5N0.2 + 1.5H2O + 4.2RM

Note: T=35℃, the partial pressure of gas was 1 atm, and the concentrations of substances were 1 mol/L.

29

ΔG (kJ/mol) +8.37 +31.53

-156.54-385.94ERM

-42.675-173.67ERM

8.37-164.91α-385.

31.53-74.205α-173

-34.47 +27.35

-130.7 -20.76

199.12+771.88ERM

152.39+405.2384E

ge

Table 2 Effects of redox potential of RMs on energy distribution. RMs

E (V)

ΔG (kJ/mol)

Fe3+/Fe2+

+0.772

-488.95

Carbon material

+0.195

-266.27

Biochar

-0.0174

-184.29

AQDS/ AHQDS

-0.184

-119.99

Fe2O3/ Fe2+

-0.287

-91.44

c-type cytochrome

-0.290

-91.44

Unknown material1

-0.500

-127.87

Unknown material2

-0.600

-166.46

58 V

8V

6V

06 V

*

Parameters Distribution Coefficient* Y (C-mol/mol) kd (h-1) μmax (h-1) Distribution Coefficient Y (C-mol/mol) kd (h-1) μmax (h-1) Distribution Coefficient Y (C-mol/mol) kd (h-1) μmax (h-1) Distribution Coefficient Y (C-mol/mol) kd (h-1) μmax (h-1) Distribution Coefficient Y (C-mol/mol) kd (h-1) μmax (h-1) Distribution Coefficient Y (C-mol/mol) kd (h-1) μmax (h-1) Distribution Coefficient Y (C-mol/mol) kd (h-1) μmax (h-1) Distribution Coefficient Y (C-mol/mol) kd (h-1) μmax (h-1)

Ethanol oxidation 0.2715 0.2228 0.0131 0.3325 0.2676 0.1071 0.0142 0.1712 0.2638 0.0704 0.0147 0.1118 0.2572 0.0434 0.0150 0.0652 0.2512 0.0315 0.0152 0.0445 0.2512 0.0315 0.0152 0.0445 0.2583 0.0431 0.0150 0.0709 0.2625 0.0553 0.0148 0.0989

Acetoclastic methanogenesis 0.3418 0.3078 0.0214 0.3325 0.3450 0.1818 0.0231 0.1712 0.3481 0.1305 0.0239 0.1118 0.3535 0.0882 0.0245 0.0652 0.3583 0.0689 0.0247 0.0445 0.3583 0.0689 0.0247 0.0445 0.3525 0.0935 0.0244 0.0709 0.3492 0.1190 0.0240 0.0989

Representing the energy distribution coefficient at the optimal point. T=35 ℃ ; the partial

pressure of gas was 1 atm; the concentrations of substances were 1 mol/L.

30

DIET methanog 0.386 0.115 0.010 0.166 0.387 0.078 0.010 0.085 0.388 0.059 0.010 0.055 0.389 0.042 0.011 0.032 0.390 0.034 0.011 0.022 0.390 0.034 0.011 0.022 0.389 0.050 0.011 0.035 0.388 0.067 0.010 0.049

V)

.2

.4

.6

Table 3 Effect of the redox potential of system on energy distribution. ΔG (kJ/mol)

-77.19

-154.38

-231.56

*

Parameters

Ethanol oxidation

Acetoclastic methanogenesis

Distribution Coefficient*

0.2466

0.3621

0.3913

Y (C-mol/mol)

0.0263

0.0590

0.0287

0.0153

0.0249

0.0111

μmax (h-1)

0.0342

0.0342

0.0171

Distribution Coefficient

0.2614

0.3501

0.3886

Y (C-mol/mol)

0.0550

0.1111

0.0555

kd (h-1)

0.0148

0.0241

0.0109

μmax (h-1)

0.0901

0.0901

0.0451

Distribution Coefficient

0.2663

0.3460

0.3876

Y (C-mol/mol)

0.0828

0.1604

0.0808

kd (h-1)

0.0144

0.0234

0.0108

μmax (h-1)

0.1460

0.1460

0.0730

kd

(h-1)

Representing the energy distribution coefficient at the optimal point. T=35℃; the partial

pressure of gas was 1 atm; the concentrations of substances were 1 mol/L.

31

DIET methanogen

Figures I: μmax_E < μmax_A, 4μmax_E < 8μmax_D

II: μmax_E < μmax_A, 4μmax_E > 8μmax_D

III: μmax_E > μmax_A, 4μmax_E > 8μmax_D

IV: μmax_E > μmax_A, 4μmax_E < 8μmax_D

The optimal point: a1=0.2512, a2=0.3583, a3=0.3905 (b)

(a) 4μmax_E = 8μmax_D μmax_E = μmax_A

I

II

IV

μmax_E = kd_E

III

μmax_A = kd_A

μmax_D = kd_D

Fig. 1 (a) Energy distribution of syntrophic methanogenesis of ethanol with c-type cytochromes as RMs through DIET pathway, and (b) parameters of microorganisms involved in different processes at the optimal point.

32

μmax_E < μmax_A, 2μmax_E < 4μmax_H, 4μmax_E < 8μmax_D

μmax_E < μmax_A, 2μmax_E > 4μmax_H , 4μmax_E < 8μmax_D

μmax_E > μmax_A, 2μmax_E > 4μmax_H, 4μmax_E < 8μmax_D

μmax_E > μmax_A, 2μmax_E < 4μmax_H, 4μmax_E < 8μmax_D

μmax_E < μmax_A, 2μmax_E < 4μmax_H, 4μmax_E > 8μmax_D

μmax_E > μmax_A, 2μmax_E < 4μmax_H, 4μmax_E > 8μmax_D

α = 0.1

I

IV

I

III

II

α = 0.3

IV III

II

III

α = 0.8

Fig. 2 Effect of the proportion of DIET pathway with c-type cytochromes as RMs on methanogenesis. 33

Fig. 3 The proposed mechanism of syntrophic methanogenesis of ethanol based on energy distribution principle.

34

Graphical Abstract

35

Highlights 

Redox materials changed energy supplying mode in syntrophic methanogenesis.



A low system redox potential could facilitate the DIET methanogenic pathway.



Promoted methanogenesis was related with the increased available energy.



Energy competition existed between hydrogenotrophic and DIET methanogenesis.

47.

36