Carbon cloth stimulates direct interspecies electron transfer in syntrophic co-cultures
Introduction
Materials that have the potential to support biofilm growth can enhance anaerobic digestion of organic wastes to methane (Adu-Gyamfi et al., 2012). One of the materials that has shown promise is carbon cloth (Sasaki et al., 2007, Sasaki et al., 2009, Sasaki et al., 2010, Tatara et al., 2008, Zhang et al., 2012, Zhao et al., 2013). In these studies the enhanced methane production in the presence of carbon cloth was attributed to its ability to promote microbial attachment. However, another possibility is that the conductive properties of carbon cloth will impact electron exchange between microorganisms similar to other conductive materials which were used to mediate electron transfer between cells and electrodes or other cells (Chen et al., 2014, Cruz Viggi et al., 2014, Kato et al., 2012, Liu et al., 2012, Liu et al., 2014, Rotaru et al., 2014a). It has been recently discovered that some methanogens can receive electrons from an electron-generating microorganism either directly – using molecular electric connections (Chen et al., 2014, Liu et al., 2012, Rotaru et al., 2014a, Rotaru et al., 2014b), or indirectly – using conductive minerals (Chen et al., 2014, Kato et al., 2012, Liu et al., 2012, Liu et al., 2014).
DIET is an alternative to interspecies H2/formate transfer for syntrophic electron exchange between microbial species (Rotaru et al., 2014a, Rotaru et al., 2014b, Summers et al., 2010). DIET was initially described in co-cultures of Geobacter metallireducens and Geobacter sulfurreducens growing in medium in which ethanol was the electron donor and fumarate was the electron acceptor (Summers et al., 2010). G. metallireducens can metabolize ethanol, but cannot use fumarate as an electron acceptor (Lovley et al., 1993), whereas G. sulfurreducens cannot metabolize ethanol, but can respire fumarate, which is then reduced to succinate (Caccavo et al., 1994). The co-culture adapted to metabolize ethanol with the reduction of fumarate (Summers et al., 2010). Multiple lines of evidence (Rotaru et al., 2012, Shrestha et al., 2013a, Shrestha et al., 2013b, Summers et al., 2010) suggested that the electron transfer between the species was via the Geobacter pili that have metallic-like conductivity (Malvankar et al., 2011, Reguera et al., 2005). The possibility of interspecies H2/formate transfer was ruled out by the fact that G. metallireducens is unable to metabolize ethanol with the production of H2 or formate (Rotaru et al., 2012, Shrestha et al., 2013a, Shrestha et al., 2013b), and the fact that interspecies electron exchange remained effective when the co-cultures were initiated with a G. sulfurreducens strain incapable of H2 and formate uptake, because the genes encoding formate dehydrogenase and an uptake hydrogenase were deleted (Rotaru et al., 2012).
Methanosaeta and Methanosarcina species, which are often abundant in anaerobic digesters (Angenent et al., 2004, De Vrieze et al., 2012, McMahon et al., 2004, Morita et al., 2011, Steinhaus et al., 2007), are also capable of receiving electrons via DIET (Rotaru et al., 2014a, Rotaru et al., 2014b). Methanosaeta harundinacea or Methanosarcina barkeri grew in defined co-cultures with ethanol-metabolizing G. metallireducens (Rotaru et al., 2014a, Rotaru et al., 2014b), but only with strains of G. metallireducens that could produce pili which are electrically conductive (Malvankar et al., 2011, Reguera et al., 2005). Metatranscriptomic analysis, as well as an assessment of metabolic potential and granule conductivity suggested that Methanosaeta species in a digester treating simulated brewery wastes also reduced carbon dioxide to methane with electrons derived from DIET (Morita et al., 2011, Rotaru et al., 2014b).
Although the biological electrical connections necessary for DIET are sufficient for effective syntrophic metabolism, studies with granular activated carbon (GAC), biochar, or nano-magnetite minerals demonstrated that DIET could be promoted via the conductive materials (Chen et al., 2014, Liu et al., 2012, Liu et al., 2014). For example, amending G. metallireducens–G. sulfurreducens or G. metallireducens–M. barkeri co-cultures with GAC greatly accelerated the initial rate of interspecies electron exchange (Liu et al., 2012). In the presence of GAC, digester granules in which Methanosaeta species were the predominant methanogens produced methane 2.5-fold faster than in GAC-free controls (Liu et al., 2012). GAC is 3000-fold more conductive than the Geobacter pili and in the presence of GAC even pili-deficient strains can participate in DIET (Liu et al., 2012, Rotaru et al., 2014a). Electron-donating and accepting cells attached onto GAC, which served as a conduit for electron transfer between species.
This study aimed to reveal if carbon cloth, often used in rector design, presumably because of its biomass retention properties (Sasaki et al., 2007, Sasaki et al., 2009, Sasaki et al., 2010, Tatara et al., 2008, Zhang et al., 2012, Zhao et al., 2013) would rather serve as an electrical conduit to promote DIET. As control we tested non-conductive cotton cloth with similar biomass retention properties. Additionally, we examined if the conductivity of carbon cloth affected interspecies H2 transfer. Learning about the impact of carbon cloth on electron transfer mechanism will assist future reactor designs and improve methane production during anaerobic digestion.
Section snippets
Microorganisms, media and growth conditions
All pure cultures and co-cultures were incubated in 27 mL pressure tubes with 10 mL medium under anoxic conditions with a gas phase of 80:20 of N2:CO2. G. sulfurreducens strain DL1 (ATCC 51573) and various mutant strains were transferred routinely on NBF medium with 10 mM acetate as the electron donor and 40 mM fumarate as the electron acceptor (Coppi et al., 2004). G. metallireducens strain GS-15 (ATCC 53774) and mutant strains were transferred routinely on FC medium with 10 mM ethanol as electron
Carbon cloth stimulation of DIET in Geobacter co-cultures
The ability of carbon cloth to promote DIET was first examined with co-cultures of G. metallireducens and G. sulfurreducens, because the availability of a diversity of gene-deletion mutants facilitates analysis of electron transfer mechanisms in these co-cultures. Addition of carbon cloth to co-cultures of G. metallireducens and G. sulfurreducens in medium with ethanol as the electron donor and fumarate as the electron acceptor stimulated syntrophic metabolism of ethanol (Fig. 1A) with the
Conclusions
These results demonstrate that carbon cloth can promote DIET. Carbon cloth may be particularly effective in accelerating the initial electron exchange between species, eliminating the need for biological connections via structures such as conductive pili. The selective attachment of Methanosarcina and Methanosaeta species to carbon cloth in methanogenic digesters is consistent with the finding that methanogens within this family are capable of DIET. Thus, the conductivity of carbon cloth should
Acknowledgements
We thank Louis Raboin for facilitating SEM analysis, Joy Ward and Henry Xu for laboratory assistance. The first author thanks the Oversea Study Program of Guangzhou Elite Project, and the Innovative Doctorial Candidates Training Project of Sun Yat-sen University. This research was supported by the Office of Science (BER), U. S. Department of Energy Award No. DESC0004485.
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