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Communication

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

by
Panbo Deng
1,2,
Lulu Wang
3,
Xia Li
1,
Jinshan Zhang
1,* and
Haiming Jiang
2,*
1
School of Mining and Coal, Inner Mongolia University of Science and Technology, Baotou 014010, China
2
Inner Mongolia Key Laboratory of Biomass-Energy Conversion, School of Life Science and Technology, Inner Mongolia University of Science and Technology, Baotou 014010, China
3
Inner Mongolia Changsheng Pharmaceutical Co., Ltd., Hohhot 010200, China
*
Authors to whom correspondence should be addressed.
Microbiol. Res. 2023, 14(4), 1774-1787; https://doi.org/10.3390/microbiolres14040122
Submission received: 27 September 2023 / Revised: 27 October 2023 / Accepted: 31 October 2023 / Published: 2 November 2023
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Abstract

:
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 Δhyb, or Methanospirillum hungatei, as electron-acceptor microbes, were examined. Co-cultures of G. grbiciae and G. sulfurreducens Δhyb 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 Δhyb, 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 Samples were regularly withdrawn from the co-cultures to monitor methane, fumarate, and succinate via gas or high-performance liquid chromatography.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 H2/formate-utilizing methanogen M. hungatei was observed. Additionally, G. grbiciae formed functional co-cultures with H2/formate-un-utilizing G. sulfurreducens Δhyb without the GAC or magnetite supplement. These findings indicate electron transfer between G. grbiciae and M. barkeri 800/G. sulfurreducens Δhyb is via DIET rather than H2/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 microbes.

1. Introduction

Interspecies H2/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 H2/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 CO2 to methane with electrons directly from their syntrophic partners, no pure co-cultures have been obtained.
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 Δhyb, 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.

2. Materials and Methods

2.1. Strains, Media, and Culturing Conditions

G. grbiciae (ATCC BAA-46), G. sulfurreducens Δhyb (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 Δhyb 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 CO2 and N2 (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 Δhyb) to study the metabolization of ethanol and determine the type of IET (DIET or H2/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 NaHCO3, 0.25 g NaH2PO4·H2O, 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 NaH2PO4·H2O, 0.25 g NH4Cl, 2.5 g NaHCO3, 0.1 g KCl, 1 mL 1 mmol/L Na2SeO4, 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 N2 and CO2 (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.
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 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 O2 solubility and then cooled under N2/CO2 (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 Na2S·9H2O (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 Δhyb were prepared by taking 2.5 mL of G. grbiciae (later phase of logarithmic growth) and 2.5 mL of G. sulfurreducens Δhyb (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 Δhyb were boiled to reduce O2 solubility and then cooled under N2/CO2 (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 Na2S·9H2O (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.

2.2. 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 N2/CO2 (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 H2SO4 flowing at 0.6 mL/min and detection at 215 nm.

3. Results

3.1. 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 CH4 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).
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.

3.2. 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 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 H2/CO2 (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.

3.3. 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.

3.4. 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.
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.

4. 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 H2/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 H2/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 H2 or formate with ethanol and also excluded the possibility of electron exchange through H2 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 Δhyb mutant was tested to assess the role of interspecies H2 or formate transfer within the aggregates of G. grbiciae and G. sulfurreducens Δhyb. G. grbiciae formed functional co-cultures with the G. sulfurreducens Δhyb 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 Δhyb mutant. The G. sulfurreducens Δhyb 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 Δhyb is a mutant of G. sulfurreducens PCA with a gene hyb deletion, indicating that G. sulfurreducens Δhyb can also act as an electron-acceptor microbe to form functional co-cultures through DIET. These results revealed that an alternative electron-transfer mechanism between G. grbiciae and G. sulfurreducens Δhyb may involve close cell association that confers a growth advantage when interspecies H2/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 co-cultures through DIET. Co-culturing G. grbiciae and G. sulfurreducens Δhyb 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.

5. 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 Δhyb 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 H2/formate-utilizing methanogen M. hungatei under the same conditions. The findings indicate that G. grbiciae cannot produce H2 or formate with ethanol, and electron transfer between G. grbiciae and M. barkeri 800 is not through H2/formate but DIET.
(2) G. grbiciae formed functional co-cultures with G. sulfurreducens Δhyb 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 Δhyb being unable to use H2 and formate, indicated that electron transfer between G. grbiciae and G. sulfurreducens Δhyb is via DIET rather than H2/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 electron-donor 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 Δhyb does not require these materials needs further investigation.

Author Contributions

P.D., L.W. and H.J. conceived and designed the experiments; P.D. and L.W. performed the experiments; P.D., L.W., X.L., J.Z. and H.J. analyzed the data; P.D., L.W. and H.J. wrote the paper; J.Z. and H.J. reviewed and revised the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China, grant numbers 31860011 and 51864037; the Key Technology Research Program Project of Inner Mongolia Autonomous Region, grant number 2020GG0018; the Key R&D and Achievement Transformation Plan Project of Inner Mongolia Autonomous Region, grant number 2022YFHH0026; and the Natural Science Foundation of Inner Mongolia, grant number 2018LH03021.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Flowchart of the research conducted in this study.
Figure 1. Flowchart of the research conducted in this study.
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Figure 2. Syntrophic growth of G. grbiciae/M. barkeri 800 co-cultures created by metabolizing ethanol with the generation of methane. Electron donor: ethanol (1 mmol). Control: without supplement of GAC, magnetite, or polyester felt; Polyester: five pieces of polyester felt (20 × 20 × 3 mm); GAC: 20 g/L GAC (final concentration); Magnetite: 10 mmol/L magnetite (final concentration). Each data point represents the mean ± SD of triplicate cultures.
Figure 2. Syntrophic growth of G. grbiciae/M. barkeri 800 co-cultures created by metabolizing ethanol with the generation of methane. Electron donor: ethanol (1 mmol). Control: without supplement of GAC, magnetite, or polyester felt; Polyester: five pieces of polyester felt (20 × 20 × 3 mm); GAC: 20 g/L GAC (final concentration); Magnetite: 10 mmol/L magnetite (final concentration). Each data point represents the mean ± SD of triplicate cultures.
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Figure 3. Absence of methane production from ethanol when G. grbiciae was co-cultured with the H2/formate methanogenic partner M. hungatei. Electron-donor substance: ethanol (1 mmol); Control: five pieces of polyester felt (20 × 20 × 3 mm); GAC: 20 g/L GAC (final concentration); Magnetite: 10 mmol/L magnetite (final concentration). Each data point represents the mean ± SD of triplicate cultures.
Figure 3. Absence of methane production from ethanol when G. grbiciae was co-cultured with the H2/formate methanogenic partner M. hungatei. Electron-donor substance: ethanol (1 mmol); Control: five pieces of polyester felt (20 × 20 × 3 mm); GAC: 20 g/L GAC (final concentration); Magnetite: 10 mmol/L magnetite (final concentration). Each data point represents the mean ± SD of triplicate cultures.
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Figure 4. Syntrophic growth of G. grbiciae/G. sulfurreducens Δhyb co-cultures created by metabolizing ethanol with the reduction of fumarate to succinate. (A) Changes in fumarate during G. grbiciae/G. sulfurreducens Δhyb co-cultivation. (B) Changes in succinate during G. grbiciae/G. sulfurreducens Δhyb co-cultivation. Electron-donor substance: ethanol (1 mmol); electron-acceptor substance: fumarate (1.8 mmol). Control: without supplement of GAC, magnetite, or polyester felt. GAC: 20 g/L GAC (final concentration); Magnetite: 10 mmol/L magnetite (final concentration); Polyester: five pieces of polyester felt (20 × 20 × 3 mm). Each data point represents the mean ± SD of triplicate cultures.
Figure 4. Syntrophic growth of G. grbiciae/G. sulfurreducens Δhyb co-cultures created by metabolizing ethanol with the reduction of fumarate to succinate. (A) Changes in fumarate during G. grbiciae/G. sulfurreducens Δhyb co-cultivation. (B) Changes in succinate during G. grbiciae/G. sulfurreducens Δhyb co-cultivation. Electron-donor substance: ethanol (1 mmol); electron-acceptor substance: fumarate (1.8 mmol). Control: without supplement of GAC, magnetite, or polyester felt. GAC: 20 g/L GAC (final concentration); Magnetite: 10 mmol/L magnetite (final concentration); Polyester: five pieces of polyester felt (20 × 20 × 3 mm). Each data point represents the mean ± SD of triplicate cultures.
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Figure 5. Appearance of aggregates in G. grbiciae/G. sulfurreducens Δhyb co-cultures after four transfers. Electron-donor substance: ethanol (1 mmol); electron-acceptor substance: fumarate (1.8 mmol).
Figure 5. Appearance of aggregates in G. grbiciae/G. sulfurreducens Δhyb co-cultures after four transfers. Electron-donor substance: ethanol (1 mmol); electron-acceptor substance: fumarate (1.8 mmol).
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Figure 6. Production of methane by the syntrophic growth of G. grbiciae/M. barkeri 800 co-cultures metabolizing propionate (A) and butyrate (B). Electron-donor substances: propionate (0.5 mmol), butyrate (0.5 mmol); Control: without supplement of GAC, magnetite, or polyester felt; GAC: 20 g/L GAC (final concentration); Magnetite: 10 mmol/L magnetite (final concentration).
Figure 6. Production of methane by the syntrophic growth of G. grbiciae/M. barkeri 800 co-cultures metabolizing propionate (A) and butyrate (B). Electron-donor substances: propionate (0.5 mmol), butyrate (0.5 mmol); Control: without supplement of GAC, magnetite, or polyester felt; GAC: 20 g/L GAC (final concentration); Magnetite: 10 mmol/L magnetite (final concentration).
Microbiolres 14 00122 g006
Table 1. Microbes reported as electron-donor microbes in DIET.
Table 1. Microbes reported as electron-donor microbes in DIET.
MicrobesReferences
Geobacter metallireducens GS-15[14,43,44]
Geobacter hydrogenophilus[17]
Geobacter sulfurreducens PCA[15]
Anaerobic methanotrophic archaea (ANME)[16,45]
Table 2. Microbes reported as electron-acceptors microbes in DIET.
Table 2. Microbes reported as electron-acceptors microbes in DIET.
MicrobesReferences
Geobacter sulfurreducens PCA[14,43,46,47,48]
Methanosarcina barkeri 800[48,49]
Methanosaeta harundinacea 8Ac[5,14]
Methanosarcina mazei Gö1[50]
Methanosarcina mazei 633 k.o.[50]
Methanosarcina barkeri Fusaro[50]
Methanosarcina barkeri 227[50]
Methanosarcina horonobensis HB-1[50,51]
Methanothrix thermoacetophila[44]
Methanosarcina acetivorans[52]
Methanobacterium[53]
Thiobacillus denitrificans[12]
SRB HotSeep-1[16,54]
Prosthecochloris aestaurii[15]
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Deng, P.; Wang, L.; Li, X.; Zhang, J.; Jiang, H. Geobacter grbiciae—A New Electron Donor in the Formation of Co-Cultures via Direct Interspecies Electron Transfer. Microbiol. Res. 2023, 14, 1774-1787. https://doi.org/10.3390/microbiolres14040122

AMA Style

Deng P, Wang L, Li X, Zhang J, Jiang H. Geobacter grbiciae—A New Electron Donor in the Formation of Co-Cultures via Direct Interspecies Electron Transfer. Microbiology Research. 2023; 14(4):1774-1787. https://doi.org/10.3390/microbiolres14040122

Chicago/Turabian Style

Deng, Panbo, Lulu Wang, Xia Li, Jinshan Zhang, and Haiming Jiang. 2023. "Geobacter grbiciae—A New Electron Donor in the Formation of Co-Cultures via Direct Interspecies Electron Transfer" Microbiology Research 14, no. 4: 1774-1787. https://doi.org/10.3390/microbiolres14040122

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