Caroline M. Plugge

Electron transfer in syntrophic communities of anaerobic bacteria and archaea

Interspecies electron transfer is a key process in methanogenic and sulphate-reducing environments. Bacteria and archaea that live in syntrophic communities take advantage of the metabolic abilities of their syntrophic partner to overcome energy barriers and break down compounds that they cannot digest by themselves. Here, we review the transfer of hydrogen and formate between bacteria and archaea that helps to sustain growth in syntrophic methanogenic communities. We also describe the process of reverse electron transfer, which is a key requirement in obligately syntrophic interactions. Anaerobic methane oxidation coupled to sulphate reduction is also carried out by syntrophic communities of bacteria and archaea but, as we discuss, the exact mechanism of this syntrophic interaction is not yet understood.

Reversible interconversion of carbon dioxide and formate by an electroactive enzyme

Carbon dioxide (CO2) is a kinetically and thermodynamically stable molecule. It is easily formed by the oxidation of organic molecules, during combustion or respiration, but is difficult to reduce. The production of reduced carbon compounds from CO2 is an attractive proposition, because carbon-neutral energy sources could be used to generate fuel resources and sequester CO2 from the atmosphere. However, available methods for the electrochemical reduction of CO2 require excessive overpotentials (are energetically wasteful) and produce mixtures of products. Here, we show that a tungsten-containing formate dehydrogenase enzyme (FDH1) adsorbed to an electrode surface catalyzes the efficient electrochemical reduction of CO2 to formate. Electrocatalysis by FDH1 is thermodynamically reversible—only small overpotentials are required, and the point of zero net catalytic current defines the reduction potential. It occurs under thoroughly mild conditions, and formate is the only product. Both as a homogeneous catalyst and on the electrode, FDH1 catalyzes CO2 reduction with a rate more than two orders of magnitude faster than that of any known catalyst for the same reaction. Formate oxidation is more than five times faster than CO2 reduction. Thermodynamically, formate and hydrogen are oxidized at similar potentials, so formate is a viable energy source in its own right as well as an industrially important feedstock and a stable intermediate in the conversion of CO2 to methanol and methane. FDH1 demonstrates the feasibility of interconverting CO2 and formate electrochemically, and it is a template for the development of robust synthetic catalysts suitable for practical applications.

The genome of Akkermansia muciniphila, a dedicated intestinal mucin degrader, and its use in exploring intestinal metagenomes.

Background The human gastrointestinal tract contains a complex community of microbes, fulfilling important health-promoting functions. However, this vast complexity of species hampers the assignment of responsible organisms to these functions. Recently, Akkermansia muciniphila, a new species from the deeply branched phylum Verrucomicrobia, was isolated from the human intestinal tract based on its capacity to efficiently use mucus as a carbon and nitrogen source. This anaerobic resident is associated with the protective mucus lining of the intestines. Methodology/Principal Findings In order to uncover the functional potential of A. muciniphila, its genome was sequenced and annotated. It was found to contain numerous candidate mucinase-encoding genes, but lacking genes encoding canonical mucus-binding domains. Numerous phage-associated sequences found throughout the genome indicate that viruses have played an important part in the evolution of this species. Furthermore, we mined 37 GI tract metagenomes for the presence, and genetic diversity of Akkermansia sequences. Out of 37, eleven contained 16S ribosomal RNA gene sequences that are >95% identical to that of A. muciniphila. In addition, these libraries were found to contain large amounts of Akkermansia DNA based on average nucleotide identity scores, which indicated in one subject co-colonization by different Akkermansia phylotypes. An additional 12 libraries also contained Akkermansia sequences, making a total of ∼16 Mbp of new Akkermansia pangenomic DNA. The relative abundance of Akkermansia DNA varied between <0.01% to nearly 4% of the assembled metagenomic reads. Finally, by testing a large collection of full length 16S sequences, we find at least eight different representative species in the genus Akkermansia. Conclusions/Significance These large repositories allow us to further mine for genetic heterogeneity and species diversity in the genus Akkermansia, providing novel insight towards the functionality of this abundant inhabitant of the human intestinal tract.

Syntrophobacter fumaroxidans sp. nov.: a syntrophic propionate-degrading sulfate-reducing bacterium.

A syntrophic propionate-oxidizing bacterium, strain MPOBT, was isolated from a culture enriched from anaerobic granular sludge. It oxidized propionate syntrophically in co-culture with the hydrogen- and formate-utilizing Methanospirillum hungateii, and was able to oxidize propionate and other organic compounds in pure culture with sulfate or fumarate as the electron acceptor. Additionally, it fermented fumarate. 16S rRNA sequence analysis revealed a relationship with Syntrophobacter wolinii and Syntrophobacter pfennigii. The G + C content of its DNA was 60.6 mol%, which is in the same range as that of other Syntrophobacter species. DNA-DNA hybridization studies showed less than 26% hybridization among the different genomes of Syntrophobacter species and strain MPOBT. This justifies the assignment of strain MPOBT to the genus Syntrophobacter as a new species. The name Syntrophobacter fumaroxidans is proposed; strain MPOBT (= DSM 10017T) is the type strain.

Biological formation of caproate and caprylate from acetate: fuel and chemical production from low grade biomass

This research introduces an alternative mixed culture fermentation technology for anaerobic digestion to recover valuable products from low grade biomass. In this mixed culture fermentation, organic waste streams are converted to caproate and caprylate as precursors for biodiesel or chemicals. It was found that acetate, as the main intermediate of anaerobic digestion, can be elongated to medium chain fatty acids with six and eight carbon atoms. Mixed microbial communities were able to produce 8.17 g l−1 caproate and 0.32 g l−1 caprylate under methanogenesis-suppressed conditions in a stable batch reactor run. The highest production rate was 25.6 mM C caproate per day with a product yield of 0.6 mol C per mol C. This elongation process occurred with both ethanol and hydrogen as electron donors, demonstrating the flexibility of the process. Microbial characterization revealed that the microbial populations were stable and dominated by relatives of Clostridium kluyveri.

Biomethanation and its potential

Biomethanation is a process by which organic material is microbiologically converted under anaerobic conditions to biogas. Three main physiological groups of microorganisms are involved: fermenting bacteria, organic acid oxidizing bacteria, and methanogenic archaea. Microorganisms degrade organic matter via cascades of biochemical conversions to methane and carbon dioxide. Syntrophic relationships between hydrogen producers (acetogens) and hydrogen scavengers (homoacetogens, hydrogenotrophic methanogens, etc.) are critical to the process. Determination of practical and theoretical methane potential is very important for design for optimal process design, configuration, and effective evaluation of economic feasibility. A wide variety of process applications for biomethanation of wastewaters, slurries, and solid waste have been developed. They utilize different reactor types (fully mixed, plug-flow, biofilm, UASB, etc.) and process conditions (retention times, loading rates, temperatures, etc.) in order to maximize the energy output from the waste and also to decrease retention time and enhance process stability. Biomethanation has strong potential for the production of energy from organic residues and wastes. It will help to reduce the use of fossil fuels and thus reduce CO(2) emission.

Metabolic flexibility of sulfate-reducing bacteria

Dissimilatory sulfate-reducing prokaryotes (SRB) are a very diverse group of anaerobic bacteria that are omnipresent in nature and play an imperative role in the global cycling of carbon and sulfur. In anoxic marine sediments sulfate reduction accounts for up to 50% of the entire organic mineralization in coastal and shelf ecosystems where sulfate diffuses several meters deep into the sediment. As a consequence, SRB would be expected in the sulfate-containing upper sediment layers, whereas methanogenic archaea would be expected to succeed in the deeper sulfate-depleted layers of the sediment. Where sediments are high in organic matter, sulfate is depleted at shallow sediment depths, and biogenic methane production will occur. In the absence of sulfate, many SRB ferment organic acids and alcohols, producing hydrogen, acetate, and carbon dioxide, and may even rely on hydrogen- and acetate-scavenging methanogens to convert organic compounds to methane. SRB can establish two different life styles, and these can be termed as sulfidogenic and acetogenic, hydrogenogenic metabolism. The advantage of having different metabolic capabilities is that it raises the chance of survival in environments when electron acceptors become depleted. In marine sediments, SRB and methanogens do not compete but rather complement each other in the degradation of organic matter. Also in freshwater ecosystems with sulfate concentrations of only 10–200 μM, sulfate is consumed efficiently within the top several cm of the sediments. Here, many of the δ-Proteobacteria present have the genetic machinery to perform dissimilatory sulfate reduction, yet they have an acetogenic, hydrogenogenic way of life. In this review we evaluate the physiology and metabolic mode of SRB in relation with their environment.

Syntrophic butyrate and propionate oxidation processes: from genomes to reaction mechanisms

In anoxic environments such as swamps, rice fields and sludge digestors, syntrophic microbial communities are important for decomposition of organic matter to CO2 and CH4 . The most difficult step is the fermentative degradation of short-chain fatty acids such as propionate and butyrate. Conversion of these metabolites to acetate, CO2 , formate and hydrogen is endergonic under standard conditions and occurs only if methanogens keep the concentrations of these intermediate products low. Butyrate and propionate degradation pathways include oxidation steps of comparably high redox potential, i.e. oxidation of butyryl-CoA to crotonyl-CoA and of succinate to fumarate, respectively, that require investment of energy to release the electrons as hydrogen or formate. Although investigated for several decades, the biochemistry of these reactions is still not completely understood. Genome analysis of the butyrate-oxidizing Syntrophomonas wolfei and Syntrophus aciditrophicus and of the propionate-oxidizing Syntrophobacter fumaroxidans and Pelotomaculum thermopropionicum reveals the presence of energy-transforming protein complexes. Recent studies indicated that S. wolfei uses electron-transferring flavoproteins coupled to a menaquinone loop to drive butyryl-CoA oxidation, and that S. fumaroxidans uses a periplasmic formate dehydrogenase, cytochrome b:quinone oxidoreductases, a menaquinone loop and a cytoplasmic fumarate reductase to drive energy-dependent succinate oxidation. Furthermore, we propose that homologues of the Thermotoga maritima bifurcating [FeFe]-hydrogenase are involved in NADH oxidation by S. wolfei and S. fumaroxidans to form hydrogen.

Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum subsp. nov., a thermophilic, syntrophic, propionate-oxidizing, spore-forming bacterium.

From granular sludge from a laboratory-scale upflow anaerobic sludge bed reactor operated at 55 degrees C with a mixture of volatile fatty acids as feed, a novel anaerobic, moderately thermophilic, syntrophic, spore-forming bacterium, strain TPO, was enriched on propionate in co-culture with Methanobacterium thermoautotrophicum Z245. The axenic culture was obtained by using pyruvate as the sole source of carbon and energy. The cells were straight rods with pointed ends and became lens-shaped when sporulation started. The cells were slightly motile. The optimum growth temperature was 55 degrees C and growth was possible between 45 and 62 degrees C. The pH range for growth of strain TPO was 6-8, with an optimum at pH 7-7.5. Propionate was converted to acetate, CO2 and CH4 by a co-culture of strain TPO with Methanobacterium thermoautotrophicum Z245. In pure culture, strain TPO could grow fermentatively on benzoate, fumarate, H2/CO2, pyruvate and lactate. Sulphate could serve as inorganic electron acceptor when strain TPO was grown on propionate, lactate, pyruvate and H2/CO2. The G+C content was 53.7 mol%. Comparison of 16S rDNA sequences revealed that strain TPO is related to Desulfotomaculum thermobenzoicum (98%) and Desulfotomaculum thermoacetoxidans (98%). DNA-DNA hybridization revealed 88.2% reassociation between strain TPO and D. thermobenzoicum and 83.8% between strain TPO and D. thermoacetoxidans. However, both organisms differ physiologically from strain TPO and are not capable of syntrophic propionate oxidation. It is proposed that strain TPO should be classified as new subspecies of D. thermobenzoicum as D. thermobenzoicum subsp. thermosyntrophicum.

ROLE OF SUBSTRATE CONCENTRATION IN PARTICLE SIZE DISTRIBUTION OF METHANOGENIC GRANULAR SLUDGE IN UASB REACTORS

Abstract The effect of influent substrate concentration on particle size distribution of methanogenic granular sludge was studied in laboratory-scale UASB reactors fed with propionate as sole carbon and energy source. A mean biomass increase of 60.9 mg VSS/d and 0.95 ml sludge/d was measured in five UASB reactors operated in parallel mode, resulting in a mean SVI of 7.8 ml sludge/g dry wt. Particle size distributions were measured by a gravimetric method and by direct image analysis. Median granule diameter increased with increasing influent substrate concentration and decreased with decreasing concentration. Increasing the influent concentration led to an increase of the methanogenic activity of the granules with propionate or acetate as test substrate.