Electrobiocorrosion by microbes without outer-surface cytochromes.

Anaerobic microbial corrosion of iron-containing metals causes extensive economic damage. Some microbes are capable of direct metal-to-microbe electron transfer (electrobiocorrosion), but the prevalence of electrobiocorrosion among diverse methanogens and acetogens is poorly understood because of a lack of tools for their genetic manipulation. Previous studies have suggested that respiration with 316L  stainless steel as the electron donor is indicative of electrobiocorrosion, because, unlike pure Fe0, 316L  stainless steel does not abiotically generate H2 as an intermediary electron carrier. Here, we report that all of the methanogens (Methanosarcina vacuolata, Methanothrix soehngenii, and Methanobacterium strain IM1) and acetogens (Sporomusa ovata and Clostridium ljungdahlii) evaluated respired with pure Fe0 as the electron donor, but only M. vacuolata, Mx. soehngenii, and S. ovata were capable of stainless steel electrobiocorrosion. The electrobiocorrosive methanogens required acetate as an additional energy source in order to produce methane from stainless steel. Cocultures of S. ovata and Mx. soehngenii demonstrated how acetogens can provide acetate to methanogens during corrosion. Not only was Methanobacterium strain IM1 not capable of electrobiocorrosion, but it also did not accept electrons from Geobacter metallireducens, an effective electron-donating partner for direct interspecies electron transfer to all methanogens that can directly accept electrons from Fe0. The finding that M. vacuolata, Mx. soehngenii, and S. ovata are capable of electrobiocorrosion, despite a lack of the outer-surface c-type cytochromes previously found to be important in other electrobiocorrosive microbes, demonstrates that there are multiple microbial strategies for making electrical contact with Fe0.

Lack of physiological evidence for cytochrome filaments functioning as conduits for extracellular electron transfer.

Extracellular cytochrome filaments are proposed to serve as conduits for long-range extracellular electron transfer. The primary functional physiological evidence has been the reported inhibition of Geobacter sulfurreducens Fe(III) oxide reduction when the gene for the filament-forming cytochrome OmcS is deleted. Here we report that the OmcS-deficient strain from that original report reduces Fe(III) oxide as well as the wild-type, as does a triple mutant in which the genes for the other known filament-forming cytochromes were also deleted. The triple cytochrome mutant displayed filaments with the same 3 nm diameter morphology and conductance as those produced by Escherichia coli heterologously expressing the G. sulfurreducens PilA pilin gene. Fe(III) oxide reduction was inhibited when the pilin gene in cytochrome-deficient mutants was modified to yield poorly conductive 3 nm diameter filaments. The results are consistent with the concept that 3 nm diameter electrically conductive pili (e-pili) are required for G. sulfurreducens long-range extracellular electron transfer. In contrast, rigorous physiological functional evidence is lacking for cytochrome filaments serving as conduits for long-range electron transport. IMPORTANCE: Unraveling microbial extracellular electron transfer mechanisms has profound implications for environmental processes and advancing biological applications. This study on Geobacter sulfurreducens challenges prevailing beliefs on cytochrome filaments as crucial components thought to facilitate long-range electron transport. The discovery of an OmcS-deficient strain's unexpected effectiveness in Fe(III) oxide reduction prompted a reevaluation of the key conduits for extracellular electron transfer. By exploring the impact of genetic modifications on G. sulfurreducens' performance, this research sheds light on the importance of 3-nm diameter electrically conductive pili in Fe(III) oxide reduction. Reassessing these mechanisms is essential for uncovering the true drivers of extracellular electron transfer in microbial systems, offering insights that could revolutionize applications across diverse fields.

Integrated CO2 capture and conversion via H2-driven CO2 biomethanation: Cyclic performance and microbial community response.

This study investigated the use of H2-driven CO2 biomethanation for integrated CO2 capture and conversion (iCCC). Anaerobic chambers containing Na2CO3-amended microbial growth medium provided with H2 were inoculated with anaerobic granular sludge. Microorganisms were enriched that could regenerate carbonate by using the bicarbonate formed from CO2 absorption to generate methane. Multiple absorption-regeneration cycles were performed and effective restoration of CO2 absorption capacity and stable carbonate recycling via CO2 biomethanation were observed for CO2 absorbents adjusted to three different pH values (9.0, 9.5, and 10.0). The pH = 10.0 group had the highest CO2 absorption capacity; 65.3 mmol/L in the 5th cycle. A slight alkaline inhibition of acetoclastic methanogenesis occurred near the end of regeneration, but had limited impact on the cyclic performance of the iCCC process. Microbial communities were dominated by H2-utilizing and alkali-tolerant species that could participate in CO2 biomethanation and survive under alternating neutral and alkaline conditions.

Detrimental impact of the Geobacter metallireducens type VI secretion system on direct interspecies electron transfer.

Direct interspecies electron transfer (DIET) is important in anaerobic communities of environmental and practical significance. Other than the need for close physical contact for electrical connections, the interactions of DIET partners are poorly understood. Type VI secretion systems (T6SSs) typically kill competitive microbes. Surprisingly, Geobacter metallireducens highly expressed T6SS genes when DIET-based co-cultures were initiated with Geobacter sulfurreducens. T6SS gene expression was lower when the electron shuttle anthraquinone-2,6-disulfonate was added to alleviate the need for interspecies contact. Disruption of hcp, the G. metallireducens gene for the main T6SS needle-tube protein subunit, and the most highly upregulated gene in DIET-grown cells eliminated the long lag periods required for the initiation of DIET. The mutation did not aid DIET in the presence of granular-activated carbon (GAC), consistent with the fact that DIET partners do not make physical contact when electrically connected through conductive materials. The hcp-deficient mutant also established DIET quicker with Methanosarcina barkeri. However, the mutant also reduced Fe(III) oxide faster than the wild-type strain, a phenotype not expected from the loss of the T6SS. Quantitative PCR revealed greater gene transcript abundance for key components of extracellular electron transfer in the hcp-deficient mutant versus the wild-type strain, potentially accounting for the faster Fe(III) oxide reduction and impact on DIET. The results highlight that interspecies interactions beyond electrical connections may influence DIET effectiveness. The unexpected increase in the expression of genes for extracellular electron transport components when hcp was deleted emphasizes the complexities in evaluating the electromicrobiology of highly adaptable Geobacter species. IMPORTANCE Direct interspecies electron transfer is an alternative to the much more intensively studied process of interspecies H2 transfer as a mechanism for microbes to share electrons during the cooperative metabolism of energy sources. DIET is an important process in anaerobic soils and sediments generating methane, a significant greenhouse gas. Facilitating DIET can accelerate and stabilize the conversion of organic wastes to methane biofuel in anaerobic digesters. Therefore, a better understanding of the factors controlling how fast DIET partnerships are established is expected to lead to new strategies for promoting this bioenergy process. The finding that when co-cultured with G. sulfurreducens, G. metallireducens initially expressed a type VI secretion system, a behavior not conducive to interspecies cooperation, illustrates the complexity of establishing syntrophic relationships.

Methane production by Methanothrix thermoacetophila via direct interspecies electron transfer with Geobacter metallireducens.

Methanothrix is widely distributed in natural and artificial anoxic environments and plays a major role in global methane emissions. It is one of only two genera that can form methane from acetate dismutation and through participation in direct interspecies electron transfer (DIET) with exoelectrogens. Although Methanothrix is a significant member of many methanogenic communities, little is known about its physiology. In this study, transcriptomics helped to identify potential routes of electron transfer during DIET between Geobacter metallireducens and Methanothrix thermoacetophila. Additions of magnetite to cultures significantly enhanced growth by acetoclastic methanogenesis and by DIET, while granular activated carbon (GAC) amendments impaired growth. Transcriptomics suggested that the OmaF-OmbF-OmcF porin complex and the octaheme outer membrane c-type cytochrome encoded by Gmet_0930, were important for electron transport across the outer membrane of G. metallireducens during DIET with Mx. thermoacetophila. Clear differences in the metabolism of Mx. thermoacetophila when grown via DIET or acetate dismutation were not apparent. However, genes coding for proteins involved in carbon fixation, the sheath fiber protein MspA, and a surface-associated quinoprotein, SqpA, were highly expressed in all conditions. Expression of gas vesicle genes was significantly lower in DIET- than acetate-grown cells, possibly to facilitate better contact between membrane-associated redox proteins during DIET. These studies reveal potential electron transfer mechanisms utilized by both Geobacter and Methanothrix during DIET and provide important insights into the physiology of Methanothrix in anoxic environments. IMPORTANCE Methanothrix is a significant methane producer in a variety of methanogenic environments including soils and sediments as well as anaerobic digesters. Its abundance in these anoxic environments has mostly been attributed to its high affinity for acetate and its ability to grow by acetoclastic methanogenesis. However, Methanothrix species can also generate methane by directly accepting electrons from exoelectrogenic bacteria through direct interspecies electron transfer (DIET). Methane production through DIET is likely to further increase their contribution to methane production in natural and artificial environments. Therefore, acquiring a better understanding of DIET with Methanothrix will help shed light on ways to (i) minimize microbial methane production in natural terrestrial environments and (ii) maximize biogas formation by anaerobic digesters treating waste.

Enhancing anaerobic digestion of food waste with granular activated carbon immobilized with riboflavin.

Previous studies have shown that anaerobic digestion of food waste can be enhanced by addition of conductive materials that stimulate direct interspecies electron transfer (DIET) between bacteria and methanogens. However, at extremely high organic loading rates (OLRs), volatile fatty acids (VFAs) still tend to accumulate even in the presence of conductive materials because of an imbalance between the formation of fermentation products and the rate of methanogenesis. In this study, granular activated carbon (GAC) immobilized with riboflavin (GAC-riboflavin) was added to an anaerobic digester treating food waste. The GAC-riboflavin reactor operated stably at OLRs as high as 11.5 kgCOD/ (m3·d) and kept VFA concentrations below 69.4 mM, COD removal efficiencies, methane production rates, and biogas methane concentrations were much higher in the GAC-riboflavin reactor than the GAC- and non-amended reactors. Transcripts associated with genes that code for proteins involved in DIET based metabolism were somewhat more highly expressed by Methanothrix in the GAC-riboflavin reactor. However, it is unlikely that riboflavin acted as an electron shuttle to stimulate DIET. Rather, it seemed to provide nutrients that enhanced the growth of microorganisms involved in the anaerobic digestion process, including those that are capable of DIET.

Enhancement effects of dissolved organic matter leached from sewage sludge on microbial reduction and immobilization of Cr(VI) by Geobacter sulfurreducens.

Sewage sludge has a high concentration of dissolved organic matter (DOM) which contains compounds that can serve as electron donors or shuttles for metal reduction by dissimilatory metal reducing bacteria (DMRB). In this study, Cr(VI) removal by G. sulfurreducens, a common DMRB present in anaerobic soils, was examined in the presence or absence of sludge DOM. Two different types of sludge DOM were tested; composted sludge DOM (C-DOM) and anaerobically digested sludge DOM (A-DOM). Both sludge DOMs enhanced Cr(VI) reduction by G. sulfurreducens, but C-DOM was more effective likely because it had higher concentrations of humic substances that served as electron shuttles. Transcriptomic studies indicated that G. sulfurreducens utilizes several different mechanisms to tolerate chromium including extracellular Cr(VI) reduction and immobilization by outer membrane c-type cytochromes and electrically conductive pili, intracellular Cr(VI) reduction by triheme cytochromes and NAD(P)H FMN reductase proteins, and chromium efflux by several P-type ATPase and RND transporter proteins. Microscopy experiments also showed that Cr(III) crystals formed on the surface of the cells, indicating that extracellular Cr(VI) reduction and adsorption was involved in the chromium removal process. These results help provide insight into the potential use of sewage sludge as an additive to enhance the bioremediation of chromium contaminated soils.

Efficient nitrogen removal from leachate by coupling Anammox and sulfur-siderite-driven denitrification.

High concentrations of nitrate can be generated during anaerobic ammonium oxidation (Anammox) wastewater treatment processes. Addition of sulfur to Anammox reactors stimulates the growth of sulfur-driven denitrifying (SADN) bacteria that can reduce nitrate to nitrogen gas. However, protons released during the SADN process lower the pH of the system and inhibit Anammox activity. The system will keep stable when pH is in the range of 7.5-8.5. This study showed that addition of siderite stabilized the reactor system and significantly improved the nitrogen removal process. In fact, even when concentrations of total nitrogen were 477.15 ± 16.84 mg/L, the sulfur/siderite reactor maintained nitrogen removal efficiencies >90%, while efficiencies in the sulfur reactor were < 80%. Anammox accounted for 31% of the bacterial sequences in the sulfur/siderite reactor compared to only 14% in the sulfur reactor with the majority of sequences clustering with Ca. Brocadia. An abundance of c-type cytochromes in anammox aggregates in the sulfur-siderite reactor also indicated that anammox activity was higher in this system.

Electromicrobiology: the ecophysiology of phylogenetically diverse electroactive microorganisms.

Electroactive microorganisms markedly affect many environments in which they establish outer-surface electrical contacts with other cells and minerals or reduce soluble extracellular redox-active molecules such as flavins and humic substances. A growing body of research emphasizes their broad phylogenetic diversity and shows that these microorganisms have key roles in multiple biogeochemical cycles, as well as the microbiome of the gut, anaerobic waste digesters and metal corrosion. Diverse bacteria and archaea have independently evolved cytochrome-based strategies for electron exchange between the outer cell surface and the cell interior, but cytochrome-free mechanisms are also prevalent. Electrically conductive protein filaments, soluble electron shuttles and non-biological conductive materials can substantially extend the electronic reach of microorganisms beyond the surface of the cell. The growing appreciation of the diversity of electroactive microorganisms and their unique electronic capabilities is leading to a broad range of applications.

Cytochrome OmcS Is Not Essential for Extracellular Electron Transport via Conductive Pili in Geobacter sulfurreducens Strain KN400.

The multi-heme c-type cytochrome OmcS is one of the central components used for extracellular electron transport in the Geobacter sulfurreducens strain DL-1, but its role in other microbes, including other strains of G. sulfurreducens, is currently a matter of debate. Therefore, we investigated the function of OmcS in the G. sulfurreducens strain KN400, which is even more effective in extracellular electron transfer than the DL-1 strain. We found that deleting omcS from strain KN400 did not negatively impact the rate of Fe(III) oxide reduction and that the cells expressed conductive filaments. Replacing the wild-type pilin gene with the aro-5 pilin gene eliminated the OmcS-deficient strain's ability to transport electrons to insoluble electron acceptors and diminished filament conductivity. These results are consistent with the concept that electrically conductive pili are the primary conduit for long-range electron transfer in G. sulfurreducens and closely related species. These findings, coupled with the lack of OmcS homologs in other microbes capable of extracellular electron transfer, suggest that OmcS is not a common critical component for extracellular electron transfer. IMPORTANCE OmcS has been widely studied and noted to be one of the key components for extracellular electron exchange by the Geobacter sulfurreducens strain DL-1. However, the true importance of OmcS warrants further investigation because it is well known that few bacteria, even within the Geobacteraceae family, contain OmcS homologs, and many bacteria that are capable of extracellular electron transfer lack an abundance of any type of outer surface c-type cytochrome. In addition, there is debate about the importance of OmcS filaments in the mechanism of extracellular electron transport to insoluble electron acceptors by G. sulfurreducens. It has been suggested that filaments comprised of OmcS rather than e-pili are the predominant conductive filaments expressed by G. sulfurreducens. However, the results presented here, along with multiple other sources of evidence, indicate that OmcS filaments cannot be the primary, conductive, protein nanowires expressed by G. sulfurreducens.

Different outer membrane c-type cytochromes are involved in direct interspecies electron transfer to Geobacter or Methanosarcina species.

Direct interspecies electron transfer (DIET) may be most important in methanogenic environments, but mechanistic studies of DIET to date have primarily focused on cocultures in which fumarate was the terminal electron acceptor. To better understand DIET with methanogens, the transcriptome of Geobacter metallireducens during DIET-based growth with G. sulfurreducens reducing fumarate was compared with G. metallireducens grown in coculture with diverse Methanosarcina. The transcriptome of G. metallireducens cocultured with G. sulfurreducens was significantly different from those with Methanosarcina. Furthermore, the transcriptome of G. metallireducens grown with Methanosarcina barkeri, which lacks outer-surface c-type cytochromes, differed from those of G. metallireducens cocultured with M. acetivorans or M. subterranea, which have an outer-surface c-type cytochrome that serves as an electrical connect for DIET. Differences in G. metallireducens expression patterns for genes involved in extracellular electron transfer were particularly notable. Cocultures with c-type cytochrome deletion mutant strains, ∆Gmet_0930, ∆Gmet_0557 and ∆Gmet_2896, never became established with G. sulfurreducens but adapted to grow with all three Methanosarcina. Two porin-cytochrome complexes, PccF and PccG, were important for DIET; however, PccG was more important for growth with Methanosarcina. Unlike cocultures with G. sulfurreducens and M. acetivorans, electrically conductive pili were not needed for growth with M. barkeri. Shewanella oneidensis, another electroactive microbe with abundant outer-surface c-type cytochromes, did not grow via DIET. The results demonstrate that the presence of outer-surface c-type cytochromes does not necessarily confer the capacity for DIET and emphasize the impact of the electron-accepting partner on the physiology of the electron-donating DIET partner.

Cytochrome-mediated direct electron uptake from metallic iron by Methanosarcina acetivorans.

Methane-producing microorganisms accelerate the corrosion of iron-containing metals. Previous studies have inferred that some methanogens might directly accept electrons from Fe(0), but when this possibility was more intensively investigated, H2 was shown to be an intermediary electron carrier between Fe(0) and methanogens. Here, we report that Methanosarcina acetivorans catalyzes direct metal-to-microbe electron transfer to support methane production. Deletion of the gene for the multiheme, outer-surface c-type cytochrome MmcA eliminated methane production from Fe(0), consistent with the key role of MmcA in other forms of extracellular electron exchange. These findings, coupled with the previous demonstration that outer-surface c-type cytochromes are also electrical contacts for electron uptake from Fe(0) by Geobacter and Shewanella species, suggest that the presence of multiheme c-type cytochromes on corrosion surfaces might be diagnostic for direct metal-to-microbe electron transfer and that interfering with cytochrome function might be a strategy to mitigate corrosion.

Extracellular Electron Exchange Capabilities of Desulfovibrio ferrophilus and Desulfopila corrodens.

Microbial extracellular electron transfer plays an important role in diverse biogeochemical cycles, metal corrosion, bioelectrochemical technologies, and anaerobic digestion. Evaluation of electron uptake from pure Fe(0) and stainless steel indicated that, in contrast to previous speculation in the literature, Desulfovibrio ferrophilus and Desulfopila corrodens are not able to directly extract electrons from solid-phase electron-donating surfaces. D. ferrophilus grew with Fe(III) as the electron acceptor, but Dp. corrodens did not. D. ferrophilus reduced Fe(III) oxide occluded within porous alginate beads, suggesting that it released a soluble electron shuttle to promote Fe(III) oxide reduction. Conductive atomic force microscopy revealed that the D. ferrophilus pili are electrically conductive and the expression of a gene encoding an aromatics-rich putative pilin was upregulated during growth on Fe(III) oxide. The expression of genes for multi-heme c-type cytochromes was not upregulated during growth with Fe(III) as the electron acceptor, and genes for a porin-cytochrome conduit across the outer membrane were not apparent in the genome. The results suggest that D. ferrophilus has adopted a novel combination of strategies to enable extracellular electron transport, which may be of biogeochemical and technological significance.

Mechanisms for Electron Uptake by Methanosarcina acetivorans during Direct Interspecies Electron Transfer.

Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here, we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in coculture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in M. acetivorans grown in coculture versus pure-culture growth on acetate revealed that transcripts for the outer-surface multiheme c-type cytochrome MmcA were higher during DIET-based growth. Deletion of mmcA inhibited DIET. The high aromatic amino acid content of M. acetivorans archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based coculture as well as the archaellum-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based cocultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outer-surface electrical contacts for DIET in a methanogen. IMPORTANCE The conversion of organic matter to methane plays an important role in the global carbon cycle and is an effective strategy for converting wastes to a useful biofuel. The reduction of carbon dioxide to methane accounts for approximately a third of the methane produced in anaerobic soils and sediments as well as waste digesters. Potential electron donors for carbon dioxide reduction are H2 or electrons derived from direct interspecies electron transfer (DIET) between bacteria and methanogens. Elucidating the relative importance of these electron donors has been difficult due to a lack of information on the electrical connections on the outer surfaces of methanogens and how they process the electrons received from DIET. Transcriptomic patterns and gene deletion phenotypes reported here provide insight into how a group of Methanosarcina organisms that play an important role in methane production in soils and sediments participate in DIET.

Identification of optimal parameters for treatment of high-strength ammonium leachate by mixed communities of heterotrophic nitrifying/aerobic denitrifying bacteria.

Heterotrophic nitrifying and aerobic denitrifying bacteria (HNADB) are important for partial nitrification treatment of high strength ammonium leachate. However, conditions for their optimal performance in mixed reactor systems have yet to be determined. In this study, optimal parameters were identified and included free ammonia (FA) concentrations below 40 mg/L, a dissolved oxygen concentration of 1.2 mg/L, a carbon to nitrogen ratio of 5 and a reflux ratio of 4. These conditions were applied to a continuous anoxic/oxic membrane moving biofilm reactor treating raw incineration leachate with high total ammonium nitrogen (TAN = 1400 mg/L). Ammonium conversion and nitrogen removal efficiencies of 99% and 86% were achieved. Autotrophic ammonia oxidizing bacteria were inhibited at FA concentrations above 25 mg/L. HNADB, particularly Paracoccus species, contributed to ammonium conversion at high FA (25-40 mg/L). These results show that leachate with high TAN and FA can be treated using parameters that support the growth of HNADB.

Stainless steel corrosion via direct iron-to-microbe electron transfer by Geobacter species.

Microbial corrosion of iron-based materials is a substantial economic problem. A mechanistic understanding is required to develop mitigation strategies, but previous mechanistic studies have been limited to investigations with relatively pure Fe(0), which is not a common structural material. We report here that the mechanism for microbial corrosion of stainless steel, the metal of choice for many actual applications, can be significantly different from that for Fe(0). Although H2 is often an intermediary electron carrier between the metal and microbes during Fe(0) corrosion, we found that H2 is not abiotically produced from stainless steel, making this corrosion mechanism unlikely. Geobacter sulfurreducens and Geobacter metallireducens, electrotrophs that are known to directly accept electrons from other microbes or electrodes, extracted electrons from stainless steel via direct iron-to-microbe electron transfer. Genetic modification to prevent H2 consumption did not negatively impact on stainless steel corrosion. Corrosion was inhibited when genes for outer-surface cytochromes that are key electrical contacts were deleted. These results indicate that a common model of microbial Fe(0) corrosion by hydrogenase-positive microbes, in which H2 serves as an intermediary electron carrier between the metal surface and the microbe, may not apply to the microbial corrosion of stainless steel. However, direct iron-to-microbe electron transfer is a feasible route for stainless steel corrosion.

Correlation of Key Physiological Properties of Methanosarcina Isolates with Environment of Origin.

It is known that the physiology of Methanosarcina species can differ significantly, but the ecological impact of these differences is unclear. We recovered two strains of Methanosarcina from two different ecosystems with a similar enrichment and isolation method. Both strains had the same ability to metabolize organic substrates and participate in direct interspecies electron transfer but also had major physiological differences. Strain DH-1, which was isolated from an anaerobic digester, used H2 as an electron donor. Genome analysis indicated that it lacks an Rnf complex and conserves energy from acetate metabolism via intracellular H2 cycling. In contrast, strain DH-2, a subsurface isolate, lacks hydrogenases required for H2 uptake and cycling and has an Rnf complex for energy conservation when growing on acetate. Further analysis of the genomes of previously described isolates, as well as phylogenetic and metagenomic data on uncultured Methanosarcina in anaerobic digesters and diverse soils and sediments, revealed a physiological dichotomy that corresponded with environment of origin. The physiology of type I Methanosarcina revolves around H2 production and consumption. In contrast, type II Methanosarcina species eschew H2 and have genes for an Rnf complex and the multiheme, membrane-bound c-type cytochrome MmcA, shown to be essential for extracellular electron transfer. The distribution of Methanosarcina species in diverse environments suggests that the type I H2-based physiology is well suited for high-energy environments, like anaerobic digesters, whereas type II Rnf/cytochrome-based physiology is an adaptation to the slower, steady-state carbon and electron fluxes common in organic-poor anaerobic soils and sediments. IMPORTANCE Biogenic methane is a significant greenhouse gas, and the conversion of organic wastes to methane is an important bioenergy process. Methanosarcina species play an important role in methane production in many methanogenic soils and sediments as well as anaerobic waste digesters. The studies reported here emphasize that the genus Methanosarcina is composed of two physiologically distinct groups. This is important to recognize when interpreting the role of Methanosarcina in methanogenic environments, especially regarding H2 metabolism. Furthermore, the finding that type I Methanosarcina species predominate in environments with high rates of carbon and electron flux and that type II Methanosarcina species predominate in lower-energy environments suggests that evaluating the relative abundance of type I and type II Methanosarcina may provide further insights into rates of carbon and electron flux in methanogenic environments.

Enhanced recovery of nitrous oxide from incineration leachate in a microbial electrolysis cell inoculated with a nosZ-deficient strain of Pseudomonas aeruginosa.

High concentrations of nitrous oxide were recovered from partial nitrification treated leachate in a microbial electrolysis cell (MEC) inoculated with a nosZ-deficient strain of Pseudomonas aeruginosa. N2O conversion efficiencies > 90% were achieved when a potential of 0.8 V was applied to the MEC. The ΔnosZ strain was enriched in the 0.8 V MEC, but Achromobacter dominated the non-current control. Nitric oxide reductase genes were highly expressed by ΔnosZ cells growing in the 0.8 V MEC, consistent with enhanced nitrous oxide production rates. Concentrations of phenazine derivatives and transcripts from phenazine biosynthesis genes were also high in the 0.8 V MEC. Phenazine derivatives are known to act as electron shuttles, enhance biofilm formation, and help ward off competitors, thereby increasing the survivability of the ΔnosZ strain in the MEC. These results show that applied current stabilized growth of the ΔnosZ strain in the reactor and allowed it to sustainably generate high concentrations of nitrous oxide.

High efficiency in-situ biogas upgrading in a bioelectrochemical system with low energy input.

Biogas produced from anaerobic digestion usually contains 30%-50% CO2, much of which must be removed, before utilization. Bioelectrochemical biogas upgrading approaches show promise, however, they have not yet been optimized for practical applications. In this study, a bioelectrochemical system with low energy input (applied cathode potential of -0.5 V vs. standard hydrogen electrode, SHE) was used for in-situ biogas upgrading. High efficiency CO2 conversion (318.5 mol/d/m2) was achieved when the system was operated with an organic load of 1.7 kgCOD/(m3 d). Methane content in the upgraded biogas was 97.0% and CO2 concentrations stayed below 3%, which is comparable to biogas upgraded with more expensive and less sustainable physiochemical approaches. The high efficiency of this approach could likely be attributed to a significant enrichment of Methanothrix (92.7%) species on the cathode surface that were expressing genes involved in both acetogenic methanogenesis and direct electron transfer (DET). Electromethanogenesis by these organisms also increased proton consumption and created a higher pH that increased the solubility of CO2 in the bioreactor. In addition, CO2 removal from the biogas was likely further enhanced by an enrichment of Actinobacillus species known to be capable of CO2 fixation. Artificial neural network (ANN) models were also used to estimate CH4 production under different loading conditions. The ANN architecture with 10 neurons at hidden layers fit best with a mean square error of 6.06 × 10-3 and R2 of 0.99.