Geobacter grbiciae —A New Electron Donor in the Formation of Co-Cultures via Direct Interspecies Electron Transfer

: Geobacter grbiciae can grow via coupling oxidation of ethanol to the reduction of various forms of soluble Fe(III) and poorly crystalline Fe(III) oxide, suggesting that G. grbiciae can act as an electron-donor microbe for forming co-cultures through direct interspecies electron transfer (DIET). In this report, potential co-cultures through DIET of G. grbiciae and Methanosarcina barkeri 800, G. sulfurreducens ∆ hyb, or Methanospirillum hungatei , as electron-acceptor microbes, were examined. Co-cultures of G. grbiciae and G. sulfurreducens ∆ hyb were performed with ethanol as the sole electron-donor substance and fumarate as the electron-acceptor substance in the presence of granular activated carbon (GAC), magnetite, or polyester felt. The conditions for co-culturing G. grbiciae and M. barkeri 800 (or M. hungatei ) were the same as those for G. grbiciae and G. sulfurreducens ∆ hyb , except fumarate was absent and different cultivation temperatures were used. All co-cultures were anaerobically cultivated. Samples were regularly withdrawn from the co-cultures to monitor methane, fumarate, and succinate via gas or high-performance liquid chromatography. G. grbiciae formed functional co-cultures with M. barkeri 800 in the presence of GAC or magnetite. No co-culture of G. grbiciae with the H 2 /formate-utilizing methanogen M. hungatei was observed. Additionally, G. grbiciae formed functional co-cultures with H 2 /formate-un-utilizing G. sulfurreducens ∆ hyb without the GAC or magnetite supplement. These ﬁndings indicate electron transfer between G. grbiciae and M. barkeri 800/ G. sulfurreducens ∆ hyb is via DIET rather than H 2 /formate, conﬁrming that G. grbiciae acts as an electron-donor microbe. Although the co-cultures of G. grbiciae and M. barkeri 800 syntrophically converted ethanol to methane through DIET, the conversion of propionate or butyrate to methane was not observed. These ﬁndings expand the range of microbes that can act as electron donors for interaction with other microbes through DIET. However, propionate and butyrate metabolism through DIET in mixed microbial communities with methane as a product requires further analysis. This study provides a framework for ﬁnding new electron-donor


Introduction
Interspecies H 2 /formate transfer has been considered for almost half a century as the main pathway for electron transfer between species [1][2][3].Direct interspecies electron transfer (DIET) has recently been proposed as a potential alternative to interspecies H 2 /formate transfer for electron transfer between species [4][5][6][7][8][9][10].DIET is a syntrophic metabolism in which electrons directly flow from one microbe to another using conductive materials (i.e., pili or cytochrome) rather than being shuttled by reduced molecules, including molecular hydrogen or formate.DIET was first found in the laboratory as a combination of Geobacter metallireducens GS-15 and Geobacter sulfurreducens PCA, which grew in a medium with ethanol as an electron-donor substance and fumarate as an electron-acceptor substance [11].Subsequently, various co-cultures that exchange electrons through DIET have been reported [5,[12][13][14][15][16][17][18] and primarily involve Geobacter species and methanogens.Moreover, the effects of various conductive materials on DIET have also been reported [7,10,[18][19][20][21][22][23][24][25][26][27].The continuing increase in the number of microbes identified to carry out electron exchange through DIET indicates that DIET is a syntrophic metabolism that is more prevalent in nature than previously thought.
Although our understanding of DIET has grown since its discovery, there remain unresolved features, including identifying the various microbes that can interact with each other through DIET.For example, Geobacter species with methanogens [5,[12][13][14][28][29][30][31] are the main co-cultures documented to exchange electrons through DIET.Only a few microbes have been documented to act as the electron-donor microbes in DIET (Table 1).Most of the reported electron-donor microbes are Geobacter species.Syntrophomonas spp.[26,[32][33][34][35], Clostridium spp.[18,36], and Defluviitoga spp.[37] have been identified to possibly act as electron-donor microbes in co-cultures with methanogens through DIET; however, these co-cultures have not been experimentally confirmed with pure cultures.In addition to a lack of sufficient understanding of the types of microbes acting as electron donors, little is known about electron-acceptor microbes.Only a few microbes have been shown to act as electron-acceptor microbes in DIET (Table 2).Among all reported electron-acceptor microbes, the majority are methanogens.Although Methanospirillum spp.[38,39], Methanobacterium spp.[40,41], and Methanolinea spp.[42] have been documented to reduce CO 2 to methane with electrons directly from their syntrophic partners, no pure co-cultures have been obtained.
Table 1.Microbes reported as electron-donor microbes in DIET.

Microbes References
Geobacter sulfurreducens PCA [14,43,[46][47][48] [12] SRB HotSeep-1 [16,54] Prosthecochloris aestaurii [15] Currently, in-depth research characterizing the types of microbes that form co-cultures through DIET is missing.Therefore, discovering more electron-donor/acceptor microbes and studying their common features will facilitate our understanding of DIET.Geobacter grbiciae can obtain energy for growth by coupling the oxidation of ethanol to the reduction of various forms of soluble Fe(III) and poorly soluble crystalline Fe(III) oxide [55], which implies that G. grbiciae acts as an electron-donor microbe to form co-cultures via DIET.In this study, the possibility of G. grbiciae, as the electron-donor microbe, in forming co-cultures through DIET with Methanosarcina barkeri 800, Methanospirillum hungatei, and Geobacter sulfurreducens ∆hyb, as electron-acceptor microbes, was investigated.This study aimed to identify new microbes that act as electron-donor microbes in forming co-cultures through DIET.We also investigated whether microbes that grow by coupling the oxidation of ethanol to the reduction of ferric (III) citrate and/or poorly crystalline ferric (III) oxide can act as electron-donor microbes in the formation of co-cultures through DIET.The possibility of co-cultures of G. grbiciae and M. barkeri 800 converting propionate and butyrate to methane was also evaluated.

Strains, Media, and Culturing Conditions
G. grbiciae (ATCC BAA-46), G. sulfurreducens ∆hyb (Hyb hydrogenase-deficient strain of G. sulfurreducens PCA (ATCC 51573)), M. barkeri 800 (DSM 800), and M. hungatei (DSM 13809) were acquired from frozen (−80 • C) stocks deposited in a laboratory culture collection.G. sulfurreducens ∆hyb is a generous gift from D R Lovley laboratory (University of Massachusetts-Amherst). Cultures were grown under strict anaerobic conditions in anaerobic pressure tubes (27 mL) or serum bottles (156 mL) sealed with thick butyl rubber stoppers and crimped with aluminum covers.A mixture of CO 2 and N 2 (20:80, v/v) was provided in the headspace.
A flowchart of the research conducted is presented in Figure 1.Initially, interspecies electron transfer (IET) between a co-culture of G. grbiciae and M. barkeri 800 was determined by examining whether a G. grbiciae/M.barkeri 800 co-culture stoichiometrically converted ethanol to methane.Observation of IET between a G. grbiciae and M. barkeri 800 co-culture would then instigate the co-culturing of G. grbiciae and M. hungatei (G.sulfurreducens ∆hyb) to study the metabolization of ethanol and determine the type of IET (DIET or H 2 /formate) between the G. grbiciae/M.barkeri 800 co-culture.Finally, co-culturing G. grbiciae and M. barkeri 800 to convert propionate and butyrate to methane was evaluated after DIET was confirmed in G. grbiciae and M. barkeri 800 co-cultures.
G. grbiciae was grown on 10 mmol/L acetate in a freshwater (FWNN) medium with 4 mmol/L disodium anthraquinone-2, 6-disulfonate (AQDS) as the electron acceptor, and incubated at 30 • C. The FWNN medium composition per liter of deionized water was 2.5 g NaHCO 3 , 0.25 g NaH 2 PO 4 •H 2 O, 0.1 g KCl, 10.0 mL vitamin solution [56], and 10.0 mL trace mineral solution [56].The influence of AQDS as an electron shuttle was eliminated by routinely growing G. grbiciae in a Fe (III)-citrate (FC) medium with 10 mmol/L acetate as an electron donor at 30 • C, as described by Tremblay et al. [57].The composition of FC medium per liter of deionized water was 13.7 g ferric citrate (FC), 0.60 g NaH 2 PO 4 •H 2 O, 0.25 g NH 4 Cl, 2.5 g NaHCO 3 , 0.1 g KCl, 1 mL 1 mmol/L Na 2 SeO 4 , 10.0 mL vitamin solution [56], and 10.0 mL trace mineral solution [56].Before co-cultivation, the G. grbiciae strain was adapted to grow in FC medium with 10 mmol/L ethanol as the electron donor substance for greater than three transfers.The preparation process of the FC medium is as follows.Initially, 13.7 g of ferric citrate was added to 800 mL of deionized water.Then, NaOH solution (10 mol/L) was added slowly to the ferric citrate solution under heating and stirring conditions until fumaric acid was dissolved completely (the pH about 7).Other components (excluding sodium acetate and vitamin solutions) were added and dissolved.Deionized water was added to the abovementioned solution until the volume was 1 L. Fifty milliliters of the prepared solution was placed into a serum bottle (156 mL) and aerated with a mixture of N 2 and CO 2 (80:20, v/v) for 30 min to remove oxygen from the solution.The serum bottle with the deoxygenated solution was sealed with a thick butyl rubber stopper and crimped with an aluminum cover.The sealed serum bottle was sterilized at 121 • C for 30 min.After cooling the solution, sodium acetate (deoxygenated and heat-sterilized) and vitamin (deoxygenated and filter-sterilized) solutions were added proportionally.The preparation process of the FWNN medium is the same as that of the FC medium, except ferric citrate and NaOH were not used.After sterilization, sodium acetate and AQDS solutions were added to the FWNN medium.
23 , 14, 4 heat-sterilized) and vitamin (deoxygenated and filter-sterilized) solutions were added proportionally.The preparation process of the FWNN medium is the same as that of the FC medium, except ferric citrate and NaOH were not used.After sterilization, sodium acetate and AQDS solutions were added to the FWNN medium.G. sulfurreducens Δhyb was grown under strict anaerobic conditions at 30 °C in pressure tubes that had 10 mL NBAF medium, which is a defined medium with acetate (a final concentration of 10 mmol/L) as the electron-donor substance and fumarate (a final concentration of 40 mmol/L) as the electron-acceptor substance [56].The composition of NBAF medium per liter of deionized water was 0.42 g KH2PO4, 0.22 g K2HPO4, 0.2 g NH4Cl, 0.38 g KCl, 0.36 g NaCl, 0.04 g CaCl2•2H2O, 0.1 g MgSO4•7H2O, 1.8 g NaHCO3, 0.5 g Na2CO3, 4.64 g fumaric acid, 0.5 mL 0.1% resazurin, 1.0 mL 100 mmol/L Na2SeO4, 10.0 mL vitamin solution [56], and 10.0 mL trace mineral solution [56].The preparation process for the NBAF medium is as follows.Initially, 4.64 g of fumaric acid was added to 800 mL of deionized water.Then, NaOH solution (10 mol/L) was added slowly to this fumaric acid solution under heating and stirring conditions until the fumaric acid was dissolved completely (the pH was ~7).Other components (excluding sodium acetate and vitamin solutions) were added and dissolved.Deionized water was added to the medium until the volume was 1 L.Ten milliliters of the prepared solution was placed into an anaerobic pressure tube (27 mL) and aerated with a mixture of N2 and CO2 (80:20, v/v) for 10 min to remove oxygen from the solution.The anaerobic pressure tube with the deoxygenated solution was sealed with a thick butyl rubber stopper and crimped with an aluminum cover.The sealed anaerobic pressure tube was sterilized at 121 °C for 30 min.After the G. sulfurreducens ∆hyb was grown under strict anaerobic conditions at 30 • C in pressure tubes that had 10 mL NBAF medium, which is a defined medium with acetate (a final concentration of 10 mmol/L) as the electron-donor substance and fumarate (a final concentration of 40 mmol/L) as the electron-acceptor substance [56].The composition of NBAF medium per liter of deionized water was 0.42 g KH 2 PO 4 , 0.22 g K 2 HPO 4 , 0.2 g NH 4 Cl, 0.38 g KCl, 0.36 g NaCl, 0.04 g CaCl 2 •2H 2 O, 0.1 g MgSO 4 •7H 2 O, 1.8 g NaHCO 3 , 0.5 g Na 2 CO 3 , 4.64 g fumaric acid, 0.5 mL 0.1% resazurin, 1.0 mL 100 mmol/L Na 2 SeO 4 , 10.0 mL vitamin solution [56], and 10.0 mL trace mineral solution [56].The preparation process for the NBAF medium is as follows.Initially, 4.64 g of fumaric acid was added to 800 mL of deionized water.Then, NaOH solution (10 mol/L) was added slowly to this fumaric acid solution under heating and stirring conditions until the fumaric acid was dissolved completely (the pH was ~7).Other components (excluding sodium acetate and vitamin solutions) were added and dissolved.Deionized water was added to the medium until the volume was 1 L.Ten milliliters of the prepared solution was placed into an anaerobic pressure tube (27 mL) and aerated with a mixture of N 2 and CO 2 (80:20, v/v) for 10 min to remove oxygen from the solution.The anaerobic pressure tube with the deoxygenated solution was sealed with a thick butyl rubber stopper and crimped with an aluminum cover.The sealed anaerobic pressure tube was sterilized at 121 • C for 30 min.After the solution cooled, sodium acetate (deoxygenated and heat-sterilized) and vitamin (deoxygenated and filter-sterilized) solutions were added.
M. barkeri 800 was grown on acetate (a final concentration of 20 mmol/L) in 50 mL DSMZ medium 120 (https://www.dsmz.de/,accessed on 5 May 2016) (removed methanol and sodium acetate) and incubated at 37 • C, as described previously [13].M. hungatei was grown under the conditions specified by the culture collections.Medium for the cultivation of M. barkeri 800 and M. hungatei was boiled to reduce O 2 solubility and then cooled under N 2 /CO 2 (80:20, v/v) for increased gas exchange.Before the addition of the inoculum, the medium was modified with 0.5 mL anaerobic sterile stocks of vitamins [56] and a 0.5 mL premix of cysteine (100 mmol/L) and Na 2 S•9H 2 O (50 mmol/L).
The co-cultures of G. grbiciae and M. barkeri 800 were prepared by mixing 2.5 mL G. grbiciae (later phase of logarithmic growth) and 2.5 mL M. barkeri 800 (later phase of logarithmic growth) cultures and inoculating into 45 mL NBM medium (modified NBAF medium created by removing fumaric acid from the NBAF medium) with ethanol (2 mol/L, 0.5 mL) as the sole electron-donor substance.Cells were grown at 37 • C. NBM medium was determined to support the growth of G. grbiciae.Experiments using G. grbiciae and M. hungatei were initiated by taking 2.5 mL G. grbiciae (later phase of logarithmic growth) and 2.5 mL M. hungatei (later phase of logarithmic growth) cultures and inoculating into 45 mL NBM medium with ethanol (2 mol/L, 0.5 mL) as the sole electron-donor substance.Cells were grown at 37 • C. Co-cultures of G. grbiciae and G. sulfurreducens ∆hyb were prepared by taking 2.5 mL of G. grbiciae (later phase of logarithmic growth) and 2.5 mL of G. sulfurreducens ∆hyb (later phase of logarithmic growth) cultures and inoculating into 45 mL NBAF medium with ethanol (2 mol/L, 0.5 mL) as the sole electron-donor substance.Cells were grown at 30 • C. The NBAF medium was determined to support the growth of G. grbiciae.All media that cultivated mixtures of G. grbiciae and methanogens/G.sulfurreducens ∆hyb were boiled to reduce O 2 solubility and then cooled under N 2 /CO 2 (80:20) for increased gas exchange.Before the addition of inoculum, all the media were modified with 0.5 mL of anaerobic filter-sterile stocks of vitamins [56] and a 0.5 mL premix of cysteine (100 mmol/L) and Na 2 S•9H 2 O (50 mmol/L).All electron-donor substances were added from anaerobic heat-sterilized stocks.GAC (8-20 mesh, 80.7 Ω −1 •m −1 , Sigma-Aldrich, St Louis, MO, USA) (20 g/L) or magnetite nanoparticles (34.0 Ω −1 •m −1 , a final concentration 10 mmol/L) were added to a suitable medium before heat-sterilization of the medium, as described previously [58,59].The increase in the methane production rate after adding activated carbon and magnetite to the co-culture system may be attributed to two reasons: (i) activated carbon and magnetite act as attachment carriers for microbial growth, which increases the chances of contact between the two microbes; (ii) activated carbon and magnetite promote electron transfer between two microbes because of their conductivity.To eliminate the influence of (i) as much as possible, five pieces of polyester felt (Heavy Duty Fabric) (20 × 20 × 3 mm) were added to the control anaerobic serum bottle.

Analytical Techniques
The stoichiometry of ethanol metabolism was measured by growing co-cultures in 50 mL of the medium in 156 mL serum bottles.Samples were withdrawn regularly with N 2 /CO 2 (80:20) degassed hypodermic syringes to monitor methane, fumarate, and succinate via gas chromatography (GC) or high-performance liquid chromatography (HLPC), as described previously [13,60].Headspace methane (0.5 mL) was sampled and injected into a GC-2014 equipped with an Rtx ® -1(30 m) column heated at 110 • C. The injector port and flame ionization detector (FID) were set at 200 • C. Samples for fumarate/succinate analysis were filtered with a syringe needle filter (0.22 µm pore diameter) and then separated via HPLC (Aminex HPX-87H column, 300 × 7.8 mm) with a mobile phase of 10.0 mmol/L H 2 SO 4 flowing at 0.6 mL/min and detection at 215 nm.

Experiments with G. grbiciae and M. barkeri 800
Methane production was monitored in the anaerobic serum bottles supplemented with and without conductive materials to assess whether G. grbiciae can co-culture with M. barkeri 800 through DIET and ethanol as the electron-donor substance.Methane was generated solely in the anaerobic serum bottle supplemented with GAC or magnetite (Figure 2).In contrast, negligible methane was generated in the control anaerobic serum bottle without added conductive particles and the anaerobic serum bottle supplemented with non-conductive polyester felt, indicating that conductive materials facilitated methane production in this system.Additionally, the anaerobic serum bottle supplemented with GAC exhibited faster and greater methane production than those with magnetite during the early growth phase.The amount of methane generated in the anaerobic serum bottle supplemented with GAC was 88.67% of the methane production expected to convert ethanol to methane (i.e., 1.5 mmol CH 4 in theory) within 44 days.This value is much higher than methane generated in the anaerobic serum bottle supplemented with magnetite (41.73%) within 44 days.However, similar levels of methane production were observed for co-cultures in anaerobic serum bottles supplemented with either magnetite or GAC after 90 days of culturing (Figure 2).

Experiments with G. grbiciae and M. barkeri 800
Methane production was monitored in the anaerobic serum bo les supplemented with and without conductive materials to assess whether G. grbiciae can co-culture with M. barkeri 800 through DIET and ethanol as the electron-donor substance.Methane was generated solely in the anaerobic serum bo le supplemented with GAC or magnetite (Figure 2).In contrast, negligible methane was generated in the control anaerobic serum bo le without added conductive particles and the anaerobic serum bo le supplemented with non-conductive polyester felt, indicating that conductive materials facilitated methane production in this system.Additionally, the anaerobic serum bo le supplemented with GAC exhibited faster and greater methane production than those with magnetite during the early growth phase.The amount of methane generated in the anaerobic serum bo le supplemented with GAC was 88.67% of the methane production expected to convert ethanol to methane (i.e., 1.5 mmol CH4 in theory) within 44 days.This value is much higher than methane generated in the anaerobic serum bo le supplemented with magnetite (41.73%) within 44 days.However, similar levels of methane production were observed for co-cultures in anaerobic serum bo les supplemented with either magnetite or GAC after 90 days of culturing (Figure 2).The results showed IET between G. grbiciae and M. barkeri 800.Co-culturing of G. grbiciae with the strict H2/formate-utilizing methanogen M. hungatei was carried out to confirm that G. grbiciae and M. barkeri 800 interact through DIET rather than H2/formate.

Experiments with G. grbiciae and M. hungatei
G. grbiciae was co-cultured with M. hungatei using ethanol as the sole electron-donor substance to assess H2/formate transfer via G. grbiciae with a H2/formate-utilizing partner M. hungatei.M. hungatei generates methane only with H2 or formate but not with ethanol.Thus, ethanol cannot be converted to methane by G. grbiciae or M. hungatei alone.As shown in Figure 3, negligible methane was generated in the anaerobic serum bo les supplemented with and without conductive materials for 133 days.As a control, however, M. hungatei generates 0.185 ± 0.002 mmol methane when grown alone in a modified DSMZ medium 120 (modified from DSMZ_Medium 120 by removing yeast extract, casitone, resazurin, Na-acetate, and methanol) with H2/CO2 (130 kPa, v/v = 80:20) within nine days.The results showed IET between G. grbiciae and M. barkeri 800.Co-culturing of G. grbiciae with the strict H 2 /formate-utilizing methanogen M. hungatei was carried out to confirm that G. grbiciae and M. barkeri 800 interact through DIET rather than H 2 /formate.

Experiments with G. grbiciae and M. hungatei
G. grbiciae was co-cultured with M. hungatei using ethanol as the sole electron-donor substance to assess H 2 /formate transfer via G. grbiciae with a H 2 /formate-utilizing partner M. hungatei.M. hungatei generates methane only with H 2 or formate but not with ethanol.Thus, ethanol cannot be converted to methane by G. grbiciae or M. hungatei alone.As shown in Figure 3, negligible methane was generated in the anaerobic serum bottles supplemented with and without conductive materials for 133 days.As a control, however, M. hungatei generates 0.185 ± 0.002 mmol methane when grown alone in a modified DSMZ medium 120 (modified from DSMZ_Medium 120 by removing yeast extract, casitone, resazurin, Na-acetate, and methanol) with H 2 /CO 2 (130 kPa, v/v = 80:20) within nine days.The result showed no IET between G. grbiciae and M. hungatei.This finding is similar to previous reports showing that M. hungatei cannot interact with G. metallireducens GS-15 [5] and G. hydrogenophilus [17] through IET.
The result showed no IET between G. grbiciae and M. hungatei.This finding is similar to previous reports showing that M. hungatei cannot interact with G. metallireducens GS-15 [5] and G. hydrogenophilus [17] through IET.

Co-Culturing G. grbiciae with the G. sulfurreducens Δhyb Mutant
In addition, we tested the co-cultivation of G. grbiciae and the G. sulfurreducens Δhyb mutant to assess the role of interspecies H2/formate transfer between G. grbiciae and G. sulfurreducens Δhyb.G. sulfurreducens Δhyb is a mutant of G. sulfurreducens PCA in which the hyb gene is deleted.This gene encodes a hydrogenase subunit, and earlier studies have shown that this strain cannot consume hydrogen [61] or formate [62].The co-cultures of G. grbiciae and the G. sulfurreducens Δhyb mutant metabolized ethanol with fumarate as the electron-acceptor substance with or without conductive materials (Figure 4).Thus, a conductive material was not required, which differs from the co-culturing of G. grbiciae and M. barkeri 800.Further research will investigate why the co-culturing of G. grbiciae and G. sulfurreducens Δhyb did not require the conducive material to metabolize ethanol.The co-culture formed large aggregates following four transfers (Figure 5), which suggested that the two microbes interact with each other.

Co-Culturing G. grbiciae with the G. sulfurreducens ∆hyb Mutant
In addition, we tested the co-cultivation of G. grbiciae and the G. sulfurreducens ∆hyb mutant to assess the role of interspecies H 2 /formate transfer between G. grbiciae and G. sulfurreducens ∆hyb.G. sulfurreducens ∆hyb is a mutant of G. sulfurreducens PCA in which the hyb gene is deleted.This gene encodes a hydrogenase subunit, and earlier studies have shown that this strain cannot consume hydrogen [61] or formate [62].The co-cultures of G. grbiciae and the G. sulfurreducens ∆hyb mutant metabolized ethanol with fumarate as the electron-acceptor substance with or without conductive materials (Figure 4).Thus, a conductive material was not required, which differs from the co-culturing of G. grbiciae and M. barkeri 800.Further research will investigate why the co-culturing of G. grbiciae and G. sulfurreducens ∆hyb did not require the conducive material to metabolize ethanol.The co-culture formed large aggregates following four transfers (Figure 5), which suggested that the two microbes interact with each other.

Syntrophic Metabolism of Propionate and Butyrate in Co-Cultures of G. grbiciae and M. barkeri 800
G. grbiciae metabolizes propionate and butyrate with AQDS, poorly crystalline iron oxide, or ferric citrate as electron-acceptor substances [55].G. grbiciae grows on ethanol by conductive particle-mediated DIET with M. barkeri 800.The use of volatile fatty acids, butyrate, and propionate as electron-donor substances to generate methane via DIET was investigated by the co-cultured G. grbiciae and M. barkeri 800.
G. grbiciae and the G. sulfurreducens Δhyb mutant metabolized ethanol with fumarate as the electron-acceptor substance with or without conductive materials (Figure 4).Thus, a conductive material was not required, which differs from the co-culturing of G. grbiciae and M. barkeri 800.Further research will investigate why the co-culturing of G. grbiciae and G. sulfurreducens Δhyb did not require the conducive material to metabolize ethanol.The co-culture formed large aggregates following four transfers (Figure 5), which suggested that the two microbes interact with each other.G. grbiciae metabolizes propionate and butyrate with AQDS, poorly cry oxide, or ferric citrate as electron-acceptor substances [55].G. grbiciae grows on conductive particle-mediated DIET with M. barkeri 800.The use of volatile fa tyrate, and propionate as electron-donor substances to generate methane vi investigated by the co-cultured G. grbiciae and M. barkeri 800.
Negligible methane was produced in the control anaerobic serum bo le aerobic serum bo le supplemented with conductive materials over 67 days w nate and butyrate were used as electron-donor substances (Figure 6).In theor propionate and 0.5 mmol butyrate should generate approximately 0.875 mm mmol methane, respectively.No greater than 0.24 µmol methane was prod both conditions, indicating that conversion of propionate or butyrate to meth observed by the co-cultured G. grbiciae and M. barkeri 800.Negligible methane was produced in the control anaerobic serum bottle and the anaerobic serum bottle supplemented with conductive materials over 67 days when propionate and butyrate were used as electron-donor substances (Figure 6).In theory, 0.5 mmol propionate and 0.5 mmol butyrate should generate approximately 0.875 mmol and 1.25 mmol methane, respectively.No greater than 0.24 µmol methane was produced under both conditions, indicating that conversion of propionate or butyrate to methane was not observed by the co-cultured G. grbiciae and M. barkeri 800.
Negligible methane was produced in the control anaerobic serum bo le and the anaerobic serum bo le supplemented with conductive materials over 67 days when propionate and butyrate were used as electron-donor substances (Figure 6).In theory, 0.5 mmol propionate and 0.5 mmol butyrate should generate approximately 0.875 mmol and 1.25 mmol methane, respectively.No greater than 0.24 µmol methane was produced under both conditions, indicating that conversion of propionate or butyrate to methane was not observed by the co-cultured G. grbiciae and M. barkeri 800.

Discussion
Methane was generated by co-culturing G. grbiciae and M. barkeri 800 with ethanol as the electron-donor substance in the presence of conductive materials: GAC or magnetite (Figure 2).In contrast, negligible methane was generated by co-culturing G. grbiciae and

Discussion
Methane was generated by co-culturing G. grbiciae and M. barkeri 800 with ethanol as the electron-donor substance in the presence of conductive materials: GAC or magnetite (Figure 2).In contrast, negligible methane was generated by co-culturing G. grbiciae and M. barkeri 800 with ethanol as the electron-donor substance in the presence of non-conductive polyester felt and in the absence of conductive materials.These results indicated IET between G. grbiciae and M. barkeri 800 in the presence of conductive materials GAC or magnetite.In addition, the conductive materials facilitate methane production in this system.This stimulatory effect may be ascribed to the establishment of DIET between G. grbiciae and M. barkeri 800 with conductive particle materials acting as electron conduits.Previous studies on DIET have indicated that some (semi) conductive materials (including magnetite, carbon cloth, GAC, and biochar) can accelerate the direct electron transfer between microbes [7,10,13,[18][19][20][21][22][23][24][25][26][27]34,35].Methane was generated at a faster rate and higher levels in co-cultures supplemented with GAC than in co-cultures supplemented with magnetite during the early growth phase.This observation indicates that GAC may afford better interspecies electrical connections than magnetite because of the high specific surface area of GAC.Moreover, this result also indicates that GAC may provide a better environmental niche for the growth of microbes and provide better access to the substrate because GAC is a strong absorbent.
The electron transfer between G. grbiciae and M. barkeri 800 may occur through H 2 /formate or DIET.M. hungatei has been used as a potential electron-acceptor microbe to form co-cultures with G. metallireducens GS-15 [5] and G. hydrogenophilus [17] with ethanol as the electron-donor substance.The study of these co-cultures revealed whether G. metallireducens GS-15 and G. hydrogenophilus can act as electron-donor microbes through DIET with ethanol as the electron-donor substance.Therefore, G. grbiciae was co-cultured with the strict H 2 /formate-utilizing methanogen M. hungatei to determine the electron-transfer pathway between G. grbiciae and M. barkeri 800.G. grbiciae failed to co-culture with M. hungatei, and negligible methane was generated (Figure 3).This finding is similar to earlier reports that M. hungatei cannot interact with G. metallireducens GS-15 [5] and G. hydrogenophilus [17] through DIET, respectively.The results showed that G. grbiciae cannot produce H 2 or formate with ethanol and also excluded the possibility of electron exchange through H 2 or formate between G. grbiciae and M. barkeri 800.This result, in turn, validated the hypothesis that G. grbiciae interacts with M. barkeri 800 through DIET.
In addition, to further confirm that G. grbiciae can serve as an electron-donor microbe in forming co-cultures through DIET, the co-culturing of G. grbiciae and the G. sulfurreducens ∆hyb mutant was tested to assess the role of interspecies H 2 or formate transfer within the aggregates of G. grbiciae and G. sulfurreducens ∆hyb.G. grbiciae formed functional co-cultures with the G. sulfurreducens ∆hyb mutant because this co-culture was observed to metabolize ethanol with fumarate as the electron-acceptor substance supplemented with or without conductive materials (Figure 4).Moreover, the co-culture formed large aggregates following four transfers (Figure 5), suggesting that the two microbes interact.These findings indicated an IET between G. grbiciae and the G. sulfurreducens ∆hyb mutant.The G. sulfurreducens ∆hyb mutant has been shown to not use hydrogen [61] and formate [62].In addition, G. sulfurreducens PCA can act as an electron-acceptor microbe to form functional co-cultures through DIET [43,47,48].G. sulfurreducens ∆hyb is a mutant of G. sulfurreducens PCA with a gene hyb deletion, indicating that G. sulfurreducens ∆hyb can also act as an electronacceptor microbe to form functional co-cultures through DIET.These results revealed that an alternative electron-transfer mechanism between G. grbiciae and G. sulfurreducens ∆hyb may involve close cell association that confers a growth advantage when interspecies H 2 /formate transfer is no longer possible.This observation, in turn, further indirectly validated the hypothesis that G. grbiciae can act as the electron-donor microbe in forming cocultures through DIET.Co-culturing G. grbiciae and G. sulfurreducens ∆hyb did not require a conductive material, and why this co-culturing was successful without a conductive material is a future study.
G. grbiciae can metabolize propionate and butyrate with AQDS, poorly crystalline iron oxide, or ferric citrate as electron acceptors [55].In addition, it has also been reported that propionate [33,[63][64][65][66] and butyrate [67][68][69] can be converted to methane through DIET by some enrichments from anaerobic digester systems.The possibility of propionate and butyrate acting as electron-donor substances for the successfully constructed co-culture of G. grbiciae and M. barkeri 800 was examined.Negligible methane was generated in the anaerobic serum bottle supplemented with or without conductive materials when propionate and butyrate were used as the electron-donor substances of G. grbiciae/M.barkeri 800 co-cultures (Figure 6).This observation indicated that the G. grbiciae and M. barkeri 800 co-culture was unable to convert propionate or butyrate to methane.Wang et al. have also shown that the conversion of propionate and butyrate to methane through DIET by co-culturing G. metallireducens GS-15 and M. barkeri 800 and G. metallireducens GS-15 and M. harundinacea 8Ac was unsuccessful, even though these co-cultures converted propanol and butanol to their respective fatty acids (propionate and butyrate) and methane through DIET [14], and G. metallireducens GS-15 can metabolize propionate and butyrate with poorly crystalline iron oxide or ferric citrate as electron acceptors [70].Why co-cultures of G. grbiciae and M. barkeri 800 cannot convert propionate or butyrate to methane through DIET requires further investigation.Although some enrichments that might interact through DIET with propionate [33,[63][64][65][66] or butyrate [67][68][69] as the sole electron-donor substance have been described, none of the enrichments was isolated with the purpose of DIET.Additionally, no pure co-culture formed through DIET with propionate or butyrate as the sole electron-donor substance has been acquired.Although the co-cultures of G. grbiciae and M. barkeri 800 syntrophically converted ethanol to methane through DIET, they did not convert propionate or butyrate to methane.However, this does preclude the possibility that other strains can use butyrate or propionate to produce methane through DIET.Thus, the conversion of propionate or butyrate to methane through DIET should be further examined using other pure co-cultures.

Conclusions
The possibility of G. grbiciae serving as the electron-donor microbe to form co-culture through DIET has been assessed with M. barkeri 800, M. hungatei, and G. sulfurreducens ∆hyb as the electron-acceptor microbes.Additionally, propionate and butyrate acting as electron-donor substances in co-cultures formed through DIET for methane production have been evaluated using the co-culture of G. grbiciae and M. barkeri 800.The following results were obtained.
(1) G. grbiciae formed co-cultures with M. barkeri 800 in the presence of GAC or magnetite when ethanol served as the electron-donor substance.However, G. grbiciae failed to form functional co-cultures with the strict H 2 /formate-utilizing methanogen M. hungatei under the same conditions.The findings indicate that G. grbiciae cannot produce H 2 or formate with ethanol, and electron transfer between G. grbiciae and M. barkeri 800 is not through H 2 /formate but DIET.
(2) G. grbiciae formed functional co-cultures with G. sulfurreducens ∆hyb with or without supplementation with GAC or magnetite when ethanol served as the electron-donor substance and fumarate as the electron-acceptor substance.This finding, combined with G. sulfurreducens ∆hyb being unable to use H 2 and formate, indicated that electron transfer between G. grbiciae and G. sulfurreducens ∆hyb is via DIET rather than H 2 /formate.
(3) Although co-cultures of G. grbiciae and M. barkeri 800 syntrophically converted ethanol to methane through DIET, the conversion of propionate or butyrate to methane was not observed.
In summary, G. grbiciae was discovered as a new electron-donor microbe in DIET.This discovery widens the range of microbes that serve as electron-donor microbes in DIET syntrophic systems.Moreover, the results confirmed that a microbe can be an electrondonor microbe in co-cultures through DIET if the microbe can grow by coupling the oxidation of ethanol to the reduction of ferric (III) citrate and/or poorly crystalline ferric (III) oxide.This result provides impetus to find new potential electron-donor microbes.Although co-culturing G. grbiciae and M. barkeri 800 failed to convert propionate and butyrate to methane, it is plausible that other strains can use butyrate or propionate to produce methane through DIET.For example, propionate and butyrate metabolism through DIET in mixed microbial communities with methane production have been reported, and further efforts with pure co-cultures are needed to confirm these previous observations.Furthermore, the rationale on why forming a co-culture of G. grbiciae and M. barkeri 800 requires the supplement of conductive materials while forming a co-culture of G. grbiciae and G. sulfurreducens ∆hyb does not require these materials needs further investigation.

Figure 1 .
Figure 1.Flowchart of the research conducted in this study.

Figure 1 .
Figure 1.Flowchart of the research conducted in this study.