Johannes C. M. Scholten

Modeling methanogenesis with a genome‐scale metabolic reconstruction of Methanosarcina barkeri

We present a genome‐scale metabolic model for the archaeal methanogen Methanosarcina barkeri. We characterize the metabolic network and compare it to reconstructions from the prokaryotic, eukaryotic and archaeal domains. Using the model in conjunction with constraint‐based methods, we simulate the metabolic fluxes and resulting phenotypes induced by different environmental and genetic conditions. This represents the first large‐scale simulation of either a methanogen or an archaeal species. Model predictions are validated by comparison to experimental growth measurements and phenotypes of M. barkeri on different substrates. The predicted growth phenotypes for wild type and mutants of the methanogenic pathway have a high level of agreement with experimental findings. We further examine the efficiency of the energy‐conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase reaction, and determine a stoichiometry for the nitrogenase reaction. This work demonstrates that a reconstructed metabolic network can serve as an analysis platform to predict cellular phenotypes, characterize methanogenic growth, improve the genome annotation and further uncover the metabolic characteristics of methanogenesis.

Integrative Analysis of Transcriptomic and Proteomic Data: Challenges, Solutions and Applications

ABSTRACT Recent advances in high-throughput technologies enable quantitative monitoring of the abundance of various biological molecules and allow determination of their variation between biological states on a genomic scale. Two popular platforms are DNA microarrays that measure messenger RNA transcript levels, and gel-free proteomic analyses that quantify protein abundance. Obviously, no single approach can fully unravel the complexities of fundamental biology and it is equally clear that integrative analysis of multiple levels of gene expression would be valuable in this endeavor. However, most integrative transcriptomic and proteomic studies have thus far either failed to find a correlation or only observed a weak correlation. In addition to various biological factors, it is suggested that the poor correlation could be quite possibly due to the inadequacy of available statistical tools to compensate for biases in the data collection methodologies. To address this issue, attempts have recently been made to systematically investigate the correlation patterns between transcriptomic and proteomic datasets, and to develop sophisticated statistical tools to improve the chances of capturing a relationship. The goal of these efforts is to enhance understanding of the relationship between transcriptomes and proteomes so that integrative analyses may be utilized to reveal new biological insights that are not accessible through one-dimensional datasets. In this review, we outline some of the challenges associated with integrative analyses and present some preliminary statistical solutions. In addition, some new applications of integrated transcriptomic and proteomic analysis to the investigation of post-transcriptional regulation are also discussed.

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.

Direct inhibition of methanogenesis by ferric iron

Observed inhibition of methanogenesis under Fe(III)-reducing conditions is usually explained by competition of methanogens and Fe(III)-reducing bacteria for the common substrates acetate and hydrogen. However, substrate competition alone cannot explain the strong inhibition of methanogenesis during Fe(III)-reduction. We demonstrate direct inhibition of methanogenesis by amorphous Fe(OH)3 at concentrations between 0 and 10 mM in experiments with pure cultures of methanogens. The sensitivity toward Fe(III) was higher for Methanospirillum hungatei and Methanosarcina barkeri grown with H2/CO2 than for Methanosaeta concilii and Methanosarcina barkeri grown with acetate. Cultures of Methanosarcina barkeri grown with H2/CO2 and methanol demonstrated a capacity for Fe(III) reduction, which suggests that Fe(III)-reduction by methanogens may also contribute to Fe(III) inhibition of methanogenesis. Our results have important implications for kinetic modelling of microbial redox processes in anoxic soils and sediments.

The effect of sulfate and nitrate on methane formation in a freshwater sediment.

A freshwater sediment from a ditch of a peat grassland near Zegveld (Province of Utrecht, The Netherlands) was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Methanogenesis started after a 10 days lagphase. After 35–40 days aceticlastic methanogens were sufficiently enriched to cause a net decrease of acetate. In the presence of sulfate methane formation was only slightly affected. The addition of nitrate led to an outcompetion of aceticlastic methanogens by nitrate reducers. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. Addition of inorganic electron acceptors resulted in an outcompetition of the syntrophic propionate and butyrate degrading consortia by the sulfate and nitrate reducers.

Global transcriptomic analysis of Desulfovibrio vulgaris on different electron donors.

Whole-genome microarrays of Desulfovibrio vulgaris were used to determine relative transcript levels in cells grown to exponential or stationary phase on a medium containing either lactate or formate as electron donor. The results showed that 158 and 477 genes were differentially expressed when comparing exponential to stationary phase in lactate- or formate-based media, respectively; and 505 and 355 genes were responsive to the electron donor used at exponential or stationary phase, respectively. Functional analyses suggested that the differentially regulated genes were involved in almost every aspect of cellular metabolism, with genes involved in protein synthesis, carbon, and energy metabolism being the most regulated. The results suggested that HynBA-1 might function as a primary periplasmic hydrogenase responsible for oxidation of H2 linked to the proton gradient in lactate-based medium, while several periplasmic hydrogenases including HynBA-1 and Hyd might carry out this role in formate-based medium. The results also indicated that the alcohol dehydrogenase and heterodisulfide reductase catalyzed pathway for proton gradient formation might be actively functioning for ATP synthesis in D. vulgaris. In addition, hierarchical clustering analysis using expression data across different electron donors and growth phases allowed the identification of the common electron donor independent changes in gene expression specifically associated with the exponential to stationary phase transition, and those specifically associated with the different electron donors independent of growth phase. The study provides the first global description and functional interpretation of transcriptomic response to growth phase and electron donor in D. vulgaris.

Effect of sulfate and nitrate on acetate conversion by anaerobic microorganisms in a freshwater sediment.

Acetate is quantitatively the most important substrate for methane production in a freshwater sediment in The Netherlands. In the presence of alternative electron acceptors the conversion of acetate by methanogens was strongly inhibited. By modelling the results, obtained in experiments with and without (13)C-labelled acetate, we could show that the competition for acetate between methanogens and sulfate reducers is the main cause of inhibition of methanogenesis in the sediment. Although nitrate led to a complete inhibition of methanogenesis, acetate-utilising nitrate-reducing bacteria hardly competed with methanogens for the available acetate in the presence of nitrate. Most-probable-number enumerations showed that methanogens (2x10(8) cells cm(-3) sediment) and sulfate reducers (2x10(8) cells cm(-3) sediment) were the dominant acetate-utilising organisms in the sediment, while numbers of acetate-utilising nitrate reducers were very low (5x10(5) cells cm(-3) sediment). However, high numbers of sulfide-oxidising nitrate reducers were detected. Denitrification might result in the formation of toxic products. We speculate that the accumulation of low concentrations of NO (<0.2 mM) may result in an inhibition of methanogenesis.

Global transcriptomics analysis of the Desulfovibrio vulgaris change from syntrophic growth with Methanosarcina barkeri to sulfidogenic metabolism

Desulfovibrio vulgaris is a metabolically flexible micro-organism. It can use sulfate as an electron acceptor to catabolize a variety of substrates, or in the absence of sulfate can utilize organic acids and alcohols by forming a syntrophic association with a hydrogen-scavenging partner to relieve inhibition by hydrogen. These alternative metabolic types increase the chance of survival for D. vulgaris in environments where one of the potential external electron acceptors becomes depleted. In this work, whole-genome D. vulgaris microarrays were used to determine relative transcript levels as D. vulgaris shifted its metabolism from syntrophic in a lactate-oxidizing dual-culture with Methanosarcina barkeri to a sulfidogenic metabolism. Syntrophic dual-cultures were grown in two independent chemostats and perturbation was introduced after six volume changes with the addition of sulfate. The results showed that 132 genes were differentially expressed in D. vulgaris 2 h after addition of sulfate. Functional analyses suggested that genes involved in cell envelope and energy metabolism were the most regulated when comparing syntrophic and sulfidogenic metabolism. Upregulation was observed for genes encoding ATPase and the membrane-integrated energy-conserving hydrogenase (Ech) when cells shifted to a sulfidogenic metabolism. A five-gene cluster encoding several lipoproteins and membrane-bound proteins was downregulated when cells were shifted to a sulfidogenic metabolism. Interestingly, this gene cluster has orthologues found only in another syntrophic bacterium, Syntrophobacter fumaroxidans, and four recently sequenced Desulfovibrio strains. This study also identified several novel c-type cytochrome-encoding genes, which may be involved in the sulfidogenic metabolism.

Complete genome sequence of Syntrophobacter fumaroxidans strain (MPOBT)

Syntrophobacter fumaroxidans strain MPOBT is the best-studied species of the genus Syntrophobacter. The species is of interest because of its anaerobic syntrophic lifestyle, its involvement in the conversion of propionate to acetate, H2 and CO2 during the overall degradation of organic matter, and its release of products that serve as substrates for other microorganisms. The strain is able to ferment fumarate in pure culture to CO2 and succinate, and is also able to grow as a sulfate reducer with propionate as an electron donor. This is the first complete genome sequence of a member of the genus Syntrophobacter and a member genus in the family Syntrophobacteraceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,990,251 bp long genome with its 4,098 protein-coding and 81 RNA genes is a part of the Microbial Genome Program (MGP) and the Genomes to Life (GTL) Program project.

Microbial dynamics in upflow anaerobic sludge blanket (UASB) bioreactor granules in response to short-term changes in substrate feed

The upflow anaerobic sludge blanket (UASB) reactor is a microcosm for the methanogenic degradation of organic matter in anaerobic environments, and depends on the auto-formation of dense 3D biofilms of 1-3 mm in diameter, referred to as granular sludge (biogranules). Past research has shown that UASB and other methanogenic reactors are extremely stable functionally, but the underlying basis of the functional stability is not well understood. In this study, microbial dynamics in the communities residing in UASB biogranules were analysed to determine responses to short-term perturbations (change in reactor feed). The reactor was fed with simulated brewery wastewater (SBWW) for 1.5 months (phase 1), acetate/sulfate for 2 months (phase 2), acetate alone for 3 months (phase 3) and then a return to SBWW for 2 months (phase 4). Analysis of 16S rRNA, methanogen-associated mcrA and sulfate reducer-associated dsrAB gene-based-clone libraries showed a relatively simple community composed mainly of the methanogenic archaea (Methanobacterium and Methanosaeta), members of the green non-sulfur (Chloroflexi) group of bacteria and Syntrophobacter, Spirochaeta, Acidobacteria and Cytophaga-related bacterial sequences. The mcrA clone libraries were dominated throughout by Methanobacterium- and Methanospirillum-related sequences. Although the reactor performance remained relatively stable throughout the experiment, community diversity levels generally decreased for all libraries in response to a change from SBWW to acetate alone feed. There was a large transitory increase noted in 16S diversity at the 2 month sampling on acetate alone, entirely related to an increase in bacterial diversity. Upon return to SBWW conditions in phase 4, all diversity measures returned to near phase 1 levels. Our results demonstrated that microbial communities, even highly structured ones such as in UASB biogranules, are very capable of responding to rapid and major changes in their environment.