Pravin Malla Shrestha

A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane

Anaerobic conversion of organic wastes and biomass to methane is an important bioenergy strategy, which depends on poorly understood mechanisms of interspecies electron transfer to methanogenic microorganisms. Metatranscriptomic analysis of methanogenic aggregates from a brewery wastewater digester, coupled with fluorescence in situ hybridization with specific 16S rRNA probes, revealed that Methanosaeta species were the most abundant and metabolically active methanogens. Methanogens known to reduce carbon dioxide with H2 or formate as the electron donor were rare. Although Methanosaeta have previously been thought to be restricted to acetate as a substrate for methane production, Methanosaeta in the aggregates had a complete complement of genes for the enzymes necessary for the reduction of carbon to methane, and transcript abundance for these genes was high. Furthermore, Geobacter species, the most abundant bacteria in the aggregates, highly expressed genes for ethanol metabolism and for extracellular electron transfer via electrically conductive pili, suggesting that Geobacter and Methanosaeta species were exchanging electrons via direct interspecies electron transfer (DIET). This possibility was further investigated in defined co-cultures of Geobacter metallireducens and Methanosaeta harundinacea which stoichiometrically converted ethanol to methane. Transcriptomic, radiotracer, and genetic analysis demonstrated that M. harundinacea accepted electrons via DIET for the reduction of carbon dioxide to methane. The discovery that Methanosaeta species, which are abundant in a wide diversity of methanogenic environments, are capable of DIET has important implications not only for the functioning of anaerobic digesters, but also for global methane production.

Promoting direct interspecies electron transfer with activated carbon

Granular activated carbon (GAC) is added to methanogenic digesters to enhance conversion of wastes to methane, but the mechanism(s) for GACs stimulatory effect are poorly understood. GAC has high electrical conductivity and thus it was hypothesized that one mechanism for GAC stimulation of methanogenesis might be to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens. Metabolism was substantially accelerated when GAC was added to co-cultures of Geobacter metallireducens and Geobacter sulfurreducens grown under conditions previously shown to require DIET. Cells were attached to GAC, but did not aggregate as they do when making biological electrical connections between cells. Studies with a series of gene deletion mutants eliminated the possibility that GAC promoted electron exchange via interspecies hydrogen or formate transfer and demonstrated that DIET in the presence of GAC did not require the electrically conductive pili and associated c-type cytochrome involved in biological interspecies electrical connections. GAC also greatly stimulated ethanol metabolism and methane production in co-cultures of G. metallireducens and Methanosarcina barkeri. Cells were attached to GAC, but not closely aggregated, suggesting little opportunity for biological electrical contacts between the species. GAC also enhanced methane production in samples from a methanogenic digester in which Methanosaeta were the predominant methanogens. The results demonstrate that GAC can promote DIET and suggest that stimulation of metabolism in methanogenic digesters can be attributed, at least in part, to the high conductivity of GAC providing better interspecies electrical connections than those that can be forged biologically.

Direct Interspecies Electron Transfer between Geobacter metallireducens and Methanosarcina barkeri

ABSTRACT Direct interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability of Methanosarcina barkeri to participate in DIET was evaluated in coculture with Geobacter metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficient G. metallireducens strain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficient G. metallireducens isolates to share electrons with M. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. When M. barkeri was grown in coculture with the H2-producing Pelobacter carbinolicus, incapable of DIET, M. barkeri utilized H2 as an electron donor but metabolized little of the acetate that P. carbinolicus produced. This suggested that H2, but not electrons derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri cocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H2 transfer. M. barkeri is the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H2 or electrons derived from DIET for CO2 reduction. Furthermore, M. barkeri is genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.

Promoting Interspecies Electron Transfer with Biochar

Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than GAC, they stimulated DIET in co-cultures of Geobacter metallireducens with Geobacter sulfurreducens or Methanosarcina barkeri in which ethanol was the electron donor. Cells were attached to the biochar, yet not in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological electrical connections. The finding that biochar can stimulate DIET may be an important consideration when amending soils with biochar and can help explain why biochar may enhance methane production from organic wastes under anaerobic conditions.

Magnetite compensates for the lack of a pilin-associated c-type cytochrome in extracellular electron exchange.

Nanoscale magnetite can facilitate microbial extracellular electron transfer that plays an important role in biogeochemical cycles, bioremediation and several bioenergy strategies, but the mechanisms for the stimulation of extracellular electron transfer are poorly understood. Further investigation revealed that magnetite attached to the electrically conductive pili of Geobacter species in a manner reminiscent of the association of the multi-heme c-type cytochrome OmcS with the pili of Geobacter sulfurreducens. Magnetite conferred extracellular electron capabilities on an OmcS-deficient strain unable to participate in interspecies electron transfer or Fe(III) oxide reduction. In the presence of magnetite wild-type cells repressed expression of the OmcS gene, suggesting that cells might need to produce less OmcS when magnetite was available. The finding that magnetite can compensate for the lack of the electron transfer functions of a multi-heme c-type cytochrome has implications not only for the function of modern microbes, but also for the early evolution of microbial electron transport mechanisms.

Interspecies Electron Transfer via Hydrogen and Formate Rather than Direct Electrical Connections in Cocultures of Pelobacter carbinolicus and Geobacter sulfurreducens

ABSTRACT Direct interspecies electron transfer (DIET) is an alternative to interspecies H2/formate transfer as a mechanism for microbial species to cooperatively exchange electrons during syntrophic metabolism. To understand what specific properties contribute to DIET, studies were conducted with Pelobacter carbinolicus, a close relative of Geobacter metallireducens, which is capable of DIET. P. carbinolicus grew in coculture with Geobacter sulfurreducens with ethanol as the electron donor and fumarate as the electron acceptor, conditions under which G. sulfurreducens formed direct electrical connections with G. metallireducens. In contrast to the cell aggregation associated with DIET, P. carbinolicus and G. sulfurreducens did not aggregate. Attempts to initiate cocultures with a genetically modified strain of G. sulfurreducens incapable of both H2 and formate utilization were unsuccessful, whereas cocultures readily grew with mutant strains capable of formate but not H2 uptake or vice versa. The hydrogenase mutant of G. sulfurreducens compensated, in cocultures, with significantly increased formate dehydrogenase gene expression. In contrast, the transcript abundance of a hydrogenase gene was comparable in cocultures with that for the formate dehydrogenase mutant of G. sulfurreducens or the wild type, suggesting that H2 was the primary electron carrier in the wild-type cocultures. Cocultures were also initiated with strains of G. sulfurreducens that could not produce pili or OmcS, two essential components for DIET. The finding that P. carbinolicus exchanged electrons with G. sulfurreducens via interspecies transfer of H2/formate rather than DIET demonstrates that not all microorganisms that can grow syntrophically are capable of DIET and that closely related microorganisms may use significantly different strategies for interspecies electron exchange.

Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH(4) into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH(4) flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using (13)C-labelled CH(4) directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to (13)CH(4) for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.

Plugging in or going wireless: strategies for interspecies electron transfer.

Interspecies exchange of electrons enables a diversity of microbial communities to gain energy from reactions that no one microbe can catalyze. The first recognized strategies for interspecies electron transfer were those that relied on chemical intermediates that are recycled through oxidized and reduced forms. Well-studied examples are interspecies H2 transfer and the cycling of sulfur intermediates in anaerobic photosynthetic communities. Direct interspecies electron transfer (DIET) in which two species establish electrical contact is an alternative. Electrical contacts documented to date include electrically conductive pili, as well as conductive iron minerals and conductive carbon moieties such as activated carbon and biochar. Interspecies electron transfer is central to the functioning of methane-producing microbial communities. The importance of interspecies H2 transfer in many methanogenic communities is clear, but under some circumstances DIET predominates. It is expected that further mechanistic studies and broadening investigations to a wider range of environments will help elucidate the factors that favor specific forms of interspecies electron exchange under different environmental conditions.

Transcriptional activity of paddy soil bacterial communities

Bulk mRNA was used to explore the transcriptional activity of bacterial communities in oxic versus anoxic paddy soil. Two microbial cDNA libraries were constructed from composite samples using semi-randomly primed RT-PCR. cDNAs averaged 500-600 bp in length and were treated as expressed sequence tags (ESTs). Clustering analysis of 805 random cDNAs resulted in 179 and 155 different ESTs for the oxic and anoxic zones respectively. Using an E-value threshold of e(-10), a total of 218 different ESTs could be assigned by blastx, while 116 ESTs were predicted novel. Both the proportion and significance of the EST assignments increased with cDNA length. Taxonomic assignment was more powerful in discriminating between the aerobic and anaerobic bacterial communities than functional inference, as most ESTs in both oxygen zones were putative indicators of similar housekeeping functions, in particular ABC-type transporters. A few ESTs were putative indicators for community function in a biogeochemical context, such as beta-oxidation of long-chain fatty acids specifically in the oxic zone. Expressed sequence tags assigned to Alpha- and Betaproteobacteria were predominantly found in the oxic zone, while those affiliated with Deltaproteobacteria were more frequently detected in the anoxic zone. At the genus level, multiple assignments to Bradyrhizobium and Geobacter were unique to the oxic and anoxic zones respectively. The phylum-level affiliations of 93 16S rRNA sequences corresponded well with two taxonomically distinct EST patterns. Expressed sequence tags affiliated with Acidobacteria and Chloroflexi were frequently detected in both oxygen zones. In summary, the soil metatranscriptome is accessible for global analysis and such studies have great potential in elucidating the taxonomic and functional status of soil bacterial communities, but study significance depends on the number and length of cDNAs being randomly analysed.

Transcriptomic and Genetic Analysis of Direct Interspecies Electron Transfer

ABSTRACT The possibility that metatranscriptomic analysis could distinguish between direct interspecies electron transfer (DIET) and H2 interspecies transfer (HIT) in anaerobic communities was investigated by comparing gene transcript abundance in cocultures in which Geobacter sulfurreducens was the electron-accepting partner for either Geobacter metallireducens, which performs DIET, or Pelobacter carbinolicus, which relies on HIT. Transcript abundance for G. sulfurreducens uptake hydrogenase genes was 7-fold lower in cocultures with G. metallireducens than in cocultures with P. carbinolicus, consistent with DIET and HIT, respectively, in the two cocultures. Transcript abundance for the pilus-associated cytochrome OmcS, which is essential for DIET but not for HIT, was 240-fold higher in the cocultures with G. metallireducens than in cocultures with P. carbinolicus. The pilin gene pilA was moderately expressed despite a mutation that might be expected to repress pilA expression. Lower transcript abundance for G. sulfurreducens genes associated with acetate metabolism in the cocultures with P. carbinolicus was consistent with the repression of these genes by H2 during HIT. Genes for the biogenesis of pili and flagella and several c-type cytochrome genes were among the most highly expressed in G. metallireducens. Mutant strains that lacked the ability to produce pili, flagella, or the outer surface c-type cytochrome encoded by Gmet_2896 were not able to form cocultures with G. sulfurreducens. These results demonstrate that there are unique gene expression patterns that distinguish DIET from HIT and suggest that metatranscriptomics may be a promising route to investigate interspecies electron transfer pathways in more-complex environments.