- In situelectrochemical H(2)production for efficient and stable power-to-gas electromethanogenesis GREEN CHEMISTRY 2020; 22 (18): 6194–6203
Cultivating electroactive microbes - from field to bench.
Electromicrobiology is an emerging field investigating and exploiting the interaction of microorganisms with insoluble electron donors or acceptors. Some of the most recently categorized electroactive microorganisms became of interest to sustainable bioengineering practices. However, laboratories worldwide typically maintain electroactive microorganisms on soluble substrates, which often leads to a decrease/loss of the ability to effectively exchange electrons with solid electrode surfaces. In order to develop future sustainable technologies, we cannot rely solely on existing lab-isolates and must, therefore, develop isolation strategies for environmental strains with electroactive properties superior to strains in culture collections. In this article, we provide an overview of the studies that isolated or enriched electroactive microorganisms from the environment using an anode as the sole electron acceptor (electricity-generating / electrogens) or a cathode as the sole electron donor (electricity - consuming /electrotrophs). Next, we recommend a selective strategy for the isolation of electroactive microorganisms. Furthermore, we provide a practical guide for setting up electrochemical reactors and highlight crucial electrochemical techniques to determine electroactivity and the mode of electron transfer in novel organisms.
View details for DOI 10.1088/1361-6528/ab6ab5
View details for PubMedID 31931483
- Microbial Battery Powered Enzymatic Electrosynthesis for Carbon Capture and Generation of Hydrogen and Formate from Dilute Organics ACS ENERGY LETTERS 2019; 4 (12): 2929–36
Methanococcus maripaludis Employs Three Functional Heterodisulfide Reductase Complexes for Flavin-Based Electron Bifurcation Using Hydrogen and Formate.
Hydrogenotrophic methanogens oxidize molecular hydrogen to reduce carbon dioxide to methane. In methanogens without cytochromes, the initial endergonic reduction of CO2 to formylmethanofuran with H2-derived electrons is coupled to the exergonic reduction of a heterodisulfide of coenzymes B and M by flavin-based electron bifurcation (FBEB). In Methanococcus maripaludis, FBEB is performed by a heterodisulfide reductase (Hdr) enzyme complex that involves hydrogenase (Vhu), although formate dehydrogenase (Fdh) has been proposed as an alternative to Vhu. We have identified and purified three Hdr complexes of M. maripaludis, where homodimeric Hdr complexes containing (Vhu)2 or (Fdh)2 were found, in addition to a heterocomplex that contains both Vhu and Fdh. Formate was found in in vitro assays using the purified Hdr complex to act directly as the electron donor for FBEB via the associated Fdh. Furthermore, while ferredoxin was slowly reduced to 30% [-360 mV vs the standard hydrogen electrode (SHE)] by H2 and formate (0.8 atm and 30 mM, according to thermodynamics), the addition of CoB-S-S-CoM as the high-potential electron acceptor ( E°' = -140 mV vs SHE; to induce FBEB) resulted in the rapid and more complete reduction of Fd to 94% (-455 mV vs SHE).
View details for PubMedID 30010323
Mediator-free enzymatic electrosynthesis of formate by the Methanococcus maripaludis heterodisulfide reductase supercomplex.
2018; 254: 278–83
Electrosynthesis of formate is a promising technology to convert CO2and electricity from renewable sources into a biocompatible, soluble, non-flammable, and easily storable compound. In the model methanogen Methanococcus maripaludis, uptake of cathodic electrons was shown to proceed indirectly via formation of formate or H2by undefined, cell-derived enzymes. Here, we identified that the multi-enzyme heterodisulfide reductase supercomplex (Hdr-SC) of M. maripaludis is capable of direct electron uptake and catalyzes rapid H2and formate formation in electrochemical reactors (-800 mV vs Ag/AgCl) and in Fe(0) corrosion assays. In Fe(0) corrosion assays and electrochemical reactors, purified Hdr-SC primarily catalyzed CO2reduction to formate with a coulombic efficiency of 90% in the electrochemical cells for 5 days. Thus, this report identified the first enzyme that stably catalyzes the mediator-free electrochemical reduction of CO2to formate, which can serve as the basis of an enzyme electrode for sustained electrochemical production of formate.
View details for PubMedID 29413934
Enhanced microbial electrosynthesis by using defined co-cultures
2017; 11 (3): 704-714
Microbial uptake of free cathodic electrons presents a poorly understood aspect of microbial physiology. Uptake of cathodic electrons is particularly important in microbial electrosynthesis of sustainable fuel and chemical precursors using only CO2 and electricity as carbon, electron and energy source. Typically, large overpotentials (200 to 400 mV) were reported to be required for cathodic electron uptake during electrosynthesis of, for example, methane and acetate, or low electrosynthesis rates were observed. To address these limitations and to explore conceptual alternatives, we studied defined co-cultures metabolizing cathodic electrons. The Fe(0)-corroding strain IS4 was used to catalyze the electron uptake reaction from the cathode forming molecular hydrogen as intermediate, and Methanococcus maripaludis and Acetobacterium woodii were used as model microorganisms for hydrogenotrophic synthesis of methane and acetate, respectively. The IS4-M. maripaludis co-cultures achieved electromethanogenesis rates of 0.1-0.14 μmol cm(-2) h(-1) at -400 mV vs standard hydrogen electrode and 0.6-0.9 μmol cm(-2) h(-1) at -500 mV. Co-cultures of strain IS4 and A. woodii formed acetate at rates of 0.21-0.23 μmol cm(-2) h(-1) at -400 mV and 0.57-0.74 μmol cm(-2) h(-1) at -500 mV. These data show that defined co-cultures coupling cathodic electron uptake with synthesis reactions via interspecies hydrogen transfer may lay the foundation for an engineering strategy for microbial electrosynthesis.The ISME Journal advance online publication, 1 November 2016; doi:10.1038/ismej.2016.149.
View details for DOI 10.1038/ismej.2016.149
View details for Web of Science ID 000394542000010
Methane release from sediment seeps to the atmosphere is counteracted by highly active Methylococcaceae in the water column of deep oligotrophic Lake Constance.
FEMS microbiology ecology
2016; 92 (8)
Methane emissions from freshwater environments contribute substantially to global warming but are under strong control of aerobic methane-oxidizing bacteria. Recently discovered methane seeps (pockmarks) in freshwater lake sediments have the potential to bypass this control by their strong outgassing activity. Whether this is counteracted by pelagic methanotrophs is not well understood yet. We used a (3)H-CH4-radiotracer technique and pmoA-based molecular approaches to assess the activity, abundance and community structure of pelagic methanotrophs above active pockmarks in deep oligotrophic Lake Constance. Above profundal pockmarks, methane oxidation rates (up to 458 nmol CH4 l(-1) d(-1)) exceeded those of the surrounding water column by two orders of magnitude and coincided with maximum methanotroph abundances of 0.6% of the microbial community. Phylogenetic analysis indicated a dominance of members of the Methylococcaceae in the water column of both, pockmark and reference sites, with most of the retrieved sequences being associated with a water-column specific clade. Communities at pockmark and reference locations also differed in parts, which was likely caused by entrainment of sediment-hosted methanotrophs at pockmark sites. Our results show that the release of seep-derived methane to the atmosphere is counteracted by a distinct methanotrophic community with a pronounced activity throughout bottom waters.
View details for DOI 10.1093/femsec/fiw123
View details for PubMedID 27267930
Extracellular Enzymes Facilitate Electron Uptake in Biocorrosion and Bioelectrosynthesis
2015; 6 (2)
Direct, mediator-free transfer of electrons between a microbial cell and a solid phase in its surrounding environment has been suggested to be a widespread and ecologically significant process. The high rates of microbial electron uptake observed during microbially influenced corrosion of iron [Fe(0)] and during microbial electrosynthesis have been considered support for a direct electron uptake in these microbial processes. However, the underlying molecular mechanisms of direct electron uptake are unknown. We investigated the electron uptake characteristics of the Fe(0)-corroding and electromethanogenic archaeon Methanococcus maripaludis and discovered that free, surface-associated redox enzymes, such as hydrogenases and presumably formate dehydrogenases, are sufficient to mediate an apparent direct electron uptake. In genetic and biochemical experiments, we showed that these enzymes, which are released from cells during routine culturing, catalyze the formation of H2 or formate when sorbed to an appropriate redox-active surface. These low-molecular-weight products are rapidly consumed by M. maripaludis cells when present, thereby preventing their accumulation to any appreciable or even detectable level. Rates of H2 and formate formation by cell-free spent culture medium were sufficient to explain the observed rates of methane formation from Fe(0) and cathode-derived electrons by wild-type M. maripaludis as well as by a mutant strain carrying deletions in all catabolic hydrogenases. Our data collectively show that cell-derived free enzymes can mimic direct extracellular electron transfer during Fe(0) corrosion and microbial electrosynthesis and may represent an ecologically important but so far overlooked mechanism in biological electron transfer.The intriguing trait of some microbial organisms to engage in direct electron transfer is thought to be widespread in nature. Consequently, direct uptake of electrons into microbial cells from solid surfaces is assumed to have a significant impact not only on fundamental microbial and biogeochemical processes but also on applied bioelectrochemical systems, such as microbial electrosynthesis and biocorrosion. This study provides a simple mechanistic explanation for frequently observed fast electron uptake kinetics in microbiological systems without a direct transfer: free, cell-derived enzymes can interact with cathodic surfaces and catalyze the formation of intermediates that are rapidly consumed by microbial cells. This electron transfer mechanism likely plays a significant role in various microbial electron transfer reactions in the environment.
View details for DOI 10.1128/mBio.00496-15
View details for Web of Science ID 000355312400022
View details for PubMedID 25900658
View details for PubMedCentralID PMC4453541
Anaerobic methane oxidation coupled to denitrification is the dominant methane sink in a deep lake
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (51): 18273-18278
Anaerobic methane oxidation coupled to denitrification, also known as "nitrate/nitrite-dependent anaerobic methane oxidation" (n-damo), was discovered in 2006. Since then, only a few studies have identified this process and the associated microorganisms in natural environments. In aquatic sediments, the close proximity of oxygen- and nitrate-consumption zones can mask n-damo as aerobic methane oxidation. We therefore investigated the vertical distribution and the abundance of denitrifying methanotrophs related to Candidatus Methylomirabilis oxyfera with cultivation-independent molecular techniques in the sediments of Lake Constance. Additionally, the vertical distribution of methane oxidation and nitrate consumption zones was inferred from high-resolution microsensor profiles in undisturbed sediment cores. M. oxyfera-like bacteria were virtually absent at shallow-water sites (littoral sediment) and were very abundant at deep-water sites (profundal sediment). In profundal sediment, the vertical distribution of M. oxyfera-like bacteria showed a distinct peak in anoxic layers that coincided with the zone of methane oxidation and nitrate consumption, a strong indication for n-damo carried out by M. oxyfera-like bacteria. Both potential n-damo rates calculated from cell densities (660-4,890 µmol CH4⋅m(-2)⋅d(-1)) and actual rates calculated from microsensor profiles (31-437 µmol CH4⋅m(-2)⋅d(-1)) were sufficiently high to prevent methane release from profundal sediment solely by this process. Additionally, when nitrate was added to sediment cores exposed to anoxic conditions, the n-damo zone reestablished well below the sediment surface, completely preventing methane release from the sediment. We conclude that the previously overlooked n-damo process can be the major methane sink in stable freshwater environments if nitrate is available in anoxic zones.
View details for DOI 10.1073/pnas.1411617111
View details for Web of Science ID 000346767200054
View details for PubMedID 25472842
Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis.
2014; 8 (8): 1673-1681
Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<-414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism.
View details for DOI 10.1038/ismej.2014.82
View details for PubMedID 24844759
Characterization and phylogeny of a novel methanotroph, Methyloglobulus morosus gen. nov., spec. nov
SYSTEMATIC AND APPLIED MICROBIOLOGY
2014; 37 (3): 165-169
A novel methanotrophic gammaproteobacterium, strain KoM1, was isolated from the profundal sediment of Lake Constance after initial enrichment in opposing gradients of methane and oxygen. Strain KoM1 grows on methane or methanol as its sole source of carbon and energy. It is a Gram-negative methanotroph, often expressing red pigmentation. Cells are short rods and occur sometimes in pairs or short chains. Strain KoM1 grows preferably at reduced oxygen concentrations (pO2=0.05-0.1bar). It can fix nitrogen, and grows at neutral pH and at temperatures between 4 and 30°C. Phylogenetically, the closest relatives are Methylovulum miyakonense and Methylosoma difficile showing 91% 16S rRNA gene sequence identity. The only respiratory quinone is ubiquinone Q8; the main polar lipids are phosphatidyl ethanolamine and phosphatidyl glycerol. The major cellular fatty acids are summed feature 3 (presumably C16:1ω7c) and C16:1ω5c, and the G+C content of the DNA is 47.7mol%. Strain KoM1 is described as the type strain of a novel species within a new genus, Methyloglobulus morosus gen. nov., sp. nov.
View details for Web of Science ID 000335618900002
View details for PubMedID 24685906
Elstera litoralis gen. nov., sp nov., isolated from stone biofilms of Lake Constance, Germany
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY
2012; 62: 1750-1754
An alphaproteobacterium, strain Dia-1(T), was isolated from algae-dominated biofilms on stones from the littoral zone of Lake Constance, Germany. This bacterium was isolated after initial enrichment in spent medium obtained after growth of a diatom culture. Numerous sugars and some organic acids and alcohols served as growth substrates. The bacterium grew slowly, was strictly aerobic but microaerophilic, and did not grow in cultures shaken under air. 16S rRNA gene sequence analysis indicated that strain Dia-1(T) was distantly related to representatives of the genera Azospirillum (90-91% sequence similarity), Skermanella (88-89%), Rhodocista (87-88%) and Dongia (88-89% sequence similarity). Based on this sequence comparison, on phenotypic characterization including substrate utilization patterns, and comparison of cellular fatty acids, quinones, polar lipids and polyamines, this isolate was found to be substantially different from the genera mentioned above. On the basis of these results, a novel genus and species is proposed for this strain. The name Elstera litoralis gen. nov., sp. nov. is suggested, with strain Dia-1(T) ( = DSM 19532(T) = LMG 24234(T)) as the type strain of the type species.
View details for DOI 10.1099/ijs.0.026609-0
View details for Web of Science ID 000308849000005
View details for PubMedID 21948090
Anaerobic Oxidation of Methane in Sediments of Lake Constance, an Oligotrophic Freshwater Lake
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
2011; 77 (13): 4429-4436
Anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor has been reported for various environments, including freshwater habitats, and also, nitrate and nitrite were recently shown to act as electron acceptors for methane oxidation in eutrophic freshwater habitats. Radiotracer experiments with sediment material of Lake Constance, an oligotrophic freshwater lake, were performed to follow 14CO2 formation from 14CH4 in sediment incubations in the presence of different electron acceptors, namely, nitrate, nitrite, sulfate, or oxygen. Whereas 14CO2 formation without and with sulfate addition was negligible, addition of nitrate increased 14CO2 formation significantly, suggesting that AOM could be coupled to denitrification. Nonetheless, denitrification-dependent AOM rates remained at least 1 order of magnitude lower than rates of aerobic methane oxidation. Using molecular techniques, putative denitrifying methanotrophs belonging to the NC10 phylum were detected on the basis of the pmoA and 16S rRNA gene sequences. These findings show that sulfate-dependent AOM was insignificant in Lake constant sediments. However, AOM can also be coupled to denitrification in this oligotrophic freshwater habitat, providing first indications that this might be a widespread process that plays an important role in mitigating methane emissions.
View details for DOI 10.1128/AEM.00340-11
View details for Web of Science ID 000291985700020
View details for PubMedID 21551281
Activity and diversity of methanotrophic bacteria at methane seeps in eastern Lake Constance sediments.
Applied and environmental microbiology
2011; 77 (8): 2573-2581
The activity and community structure of aerobic methanotrophic communities were investigated at methane seeps (pockmarks) in the littoral and profundal zones of an oligotrophic freshwater lake (Lake Constance, Germany). Measurements of potential methane oxidation rates showed that sediments inside littoral pockmarks are hot spots of methane oxidation. Potential methane oxidation rates at littoral pockmark sites exceeded the rates of the surrounding sediment by 2 orders of magnitude. Terminal restriction fragment length polymorphism (T-RFLP) analysis of the pmoA gene revealed major differences in the methanotrophic community composition between littoral pockmarks and the surrounding sediments. Clone library analysis confirmed that one distinct Methylobacter-related group dominates the community at littoral pockmarks. In profundal sediments, the differences between pockmarks and surrounding sediments were found to be less pronounced.
View details for DOI 10.1128/AEM.02776-10
View details for PubMedID 21335392
- Activity and Diversity of Methanotrophic Bacteria at Methane Seeps in Eastern Lake Constance Sediments APPLIED AND ENVIRONMENTAL MICROBIOLOGY 2011; 77 (8): 2573-2581
Abundance and Activity of Methanotrophic Bacteria in Littoral and Profundal Sediments of Lake Constance (Germany)
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
2009; 75 (1): 119-126
The abundances and activities of aerobic methane-oxidizing bacteria (MOB) were compared in depth profiles of littoral and profundal sediments of Lake Constance, Germany. Abundances were determined by quantitative PCR (qPCR) targeting the pmoA gene and by fluorescence in situ hybridization (FISH), and data were compared to methane oxidation rates calculated from high-resolution concentration profiles. qPCR using type I MOB-specific pmoA primers indicated that type I MOB represented a major proportion in both sediments at all depths. FISH indicated that in both sediments, type I MOB outnumbered type II MOB at least fourfold. Results obtained with both techniques indicated that in the littoral sediment, the highest numbers of methanotrophs were found at a depth of 2 to 3 cm, corresponding to the zone of highest methane oxidation activity, although no oxygen could be detected in this zone. In the profundal sediment, highest methane oxidation activities were found at a depth of 1 to 2 cm, while MOB abundance decreased gradually with sediment depth. In both sediments, MOB were also present at high numbers in deeper sediment layers where no methane oxidation activity could be observed.
View details for DOI 10.1128/AEM.01350-08
View details for Web of Science ID 000262084800014
View details for PubMedID 18997033