Showing papers on "Methanosarcina barkeri published in 2017"

Effect of humic acids on the activity of pure and mixed methanogenic cultures

Abstract: The impact of humic acid (HA) on methanogenic activity was investigated. Methanogenic crushed granular sludge and pure cultures of mesophilic methanogens were incubated in batch cultures with HA. Initial methane production rates and substrate consumption rates were quantified. In the presence of 1 kg m −3 HA, the methane production rate of all hydrogenotrophic methanogens was inhibited by more than 75%, except Methanospirillum hungatei that was not inhibited up to 5 kg m −3 HA. The acetoclastic Methanosarcina barkeri was completely inhibited by HA ≥1 kg m −3 . However, Methanosaeta concilii was only slightly affected by HA up to 3 kg m −3 . When methanogenic granular sludge was incubated with HA, the specific methanogenic activity (SMA) tests showed less inhibition, when compared to the pure cultures of methanogens. The SMA test with H 2 /CO 2 , formate and acetate showed reduced initial methane production rate of 42%, 23% and 40%, respectively. Differences in HA susceptibility were explained by differences in cell wall structure.

Streptomyces albus: A New Cell Factory for Non-Canonical Amino Acids Incorporation into Ribosomally Synthesized Natural Products

Abstract: The incorporation of noncanonical amino acids (ncAAs) with different side chains into a peptide is a promising technique for changing the functional properties of that peptide. Of particular interest is the incorporation of ncAAs into peptide-derived natural products to optimize their biophysical properties for medical and industrial applications. Here, we present the first instance of ncAA incorporation into the natural product cinnamycin in streptomycetes using the orthogonal pyrrolysyl-tRNA synthetase/tRNAPyl pair from Methanosarcina barkeri. This approach allows site-specific incorporation of ncAAs via the read-through of a stop codon by the suppressor tRNAPyl, which can carry different pyrrolysine analogues. Five new deoxycinnamycin derivatives were obtained with three distinct pyrrolysine analogues incorporated into diverse positions of the antibiotic. The combination of partial hydrolysis and MS/MS fragmentation analysis was used to verify the exact position of the incorporation events. The introdu...

Methanosarcina Spherical Virus, a Novel Archaeal Lytic Virus Targeting Methanosarcina Strains.

Abstract: A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Go1 as the host. Due to its spherical morphology, the virus was designated Methanosarcina spherical virus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction. Functions were predicted for some of these ORFs, i.e., such as DNA polymerase, ATPase, and DNA-binding protein as well as envelope (structural) protein. MetSV-derived spacers in CRISPR loci were detected in several published Methanosarcina draft genomes using bioinformatic tools, revealing a potential protospacer-adjacent motif (PAM) motif (TTA/T). Transcription and expression of several predicted viral ORFs were validated by reverse transcription-PCR (RT-PCR), PAGE analysis, and liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Analysis of core lipids by atmospheric pressure chemical ionization (APCI) mass spectrometry showed that MetSV and Methanosarcina mazei both contain archaeol and glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The MetSV host range is limited to Methanosarcina strains growing as single cells (M. mazei, Methanosarcina barkeri and Methanosarcina soligelidi). In contrast, strains growing as sarcina-like aggregates were apparently protected from infection. Heterogeneity related to morphology phases in M. mazei cultures allowed acquisition of resistance to MetSV after challenge by growing cultures as sarcina-like aggregates. CRISPR/Cas-mediated resistance was excluded since neither of the two CRISPR arrays showed MetSV-derived spacer acquisition. Based on these findings, we propose that changing the morphology from single cells to sarcina-like aggregates upon rearrangement of the envelope structure prevents infection and subsequent lysis by MetSV.IMPORTANCE Methanoarchaea are among the most abundant organisms on the planet since they are present in high numbers in major anaerobic environments. They convert various carbon sources, e.g., acetate, methylamines, or methanol, to methane and carbon dioxide; thus, they have a significant impact on the emission of major greenhouse gases. Today, very little is known about viruses specifically infecting methanoarchaea that most probably impact the abundance of methanoarchaea in microbial consortia. Here, we characterize the first identified Methanosarcina-infecting virus (MetSV) and show a mechanism for acquiring resistance against MetSV. Based on our results, we propose that growth as sarcina-like aggregates prevents infection and subsequent lysis. These findings allow new insights into the virus-host relationship in methanogenic community structures, their dynamics, and their phase heterogeneity. Moreover, the availability of a specific virus provides new possibilities to deepen our knowledge of the defense mechanisms of potential hosts and offers tools for genetic manipulation.

Inhibitory Effect of Coumarin on Syntrophic Fatty Acid-Oxidizing and Methanogenic Cultures and Biogas Reactor Microbiomes.

Abstract: Coumarins are widely found in plants as natural constituents having antimicrobial activity. When considering plants that are rich in coumarins for biogas production, adverse effects on microorganisms driving the anaerobic digestion process are expected. Furthermore, coumarin derivatives, like warfarin, which are used as anticoagulating medicines, are found in wastewater, affecting its treatment. Coumarin, the structure common to all coumarins, inhibits the anaerobic digestion process. However, the details of this inhibition are still elusive. Here, we studied the impact of coumarin on acetogenesis and methanogenesis. First, coumarin was applied at four concentrations between 0.25 and 1 g · liter-1 to pure cultures of the methanogens Methanosarcina barkeri and Methanospirillum hungatei, which resulted in up to 25% less methane production. Acetate production of syntrophic propionate- and butyrate-degrading cultures of Syntrophobacter fumaroxidans and Syntrophomonas wolfei was inhibited by 72% at a coumarin concentration of 1 g · liter-1 Coumarin also inhibited acetogenesis and acetoclastic methanogenesis in a complex biogas reactor microbiome. When a coumarin-adapted microbiome was used, acetogenesis and methanogenesis were not inhibited. According to amplicon sequencing of bacterial 16S rRNA genes and mcrA genes, the communities of the two microbiomes were similar, although Methanoculleus was more abundant and Methanobacterium less abundant in the coumarin-adapted than in the nonadapted microbiome. Our results suggest that well-dosed feeding with coumarin-rich feedstocks to full-scale biogas reactors while keeping the coumarin concentrations below 0.5 g · liter-1 will allow adaptation to coumarins by structural and functional community reorganization and coumarin degradation.IMPORTANCE Coumarins from natural and anthropogenic sources have an inhibitory impact on the anaerobic digestion process. Here, we studied in detail the adverse effects of the model compound coumarin on acetogenesis and methanogenesis, which are two important steps of the anaerobic digestion process. Coumarin concentrations lower than 0.5 g · liter-1 had only a minor impact. Even though similar inhibitory effects can be assumed for coumarin derivatives, little effects on the anaerobic treatment of wastewater are expected where concentrations of coumarin derivatives are lower than 0.5 g · liter-1 However, when full-scale reactors are fed with coumarin-rich feedstocks, the biogas processes might be inhibited. Hence, these feedstocks should be utilized in a well-dosed manner or after adaptation of the microbial community.

Characterization of the Microbial Communities in Rumen Fluid Inoculated Reactors for the Biogas Digestion of Wheat Straw

Abstract: The present study investigated the effect of rumen fluid (RF) concentration on the methane production through anaerobic digestion of wheat straw in batch mode, and compared the microbial communities in RF and RF inoculated reactors by 16S rRNA genes sequencing. Six levels of RF concentration including 1%, 5%, 10%, 15%, 20% and 25% (v/v) were used in reactors R1, R5, R10, R15, R20 and R25 respectively. The results revealed that lower than or equal to 5% RF concentrations resulted in reactor acidification and low methane production. The highest methane yield of 106 mL CH4 g VS−1 was achieved in R10, whereas higher RF concentrations than 10% could not improve the methane production significantly. Methanosarcina barkeri was abundant in the well-working reactors, and Methanobacterium was dominant in the poor-working reactors, implying the archaeal communities in reactors had changed greatly from the Methanobrevibacter-dominated RF. Although the relative abundance of Clostridium and Ruminococcus were greatly different between RF and reactors, the Bacteroidetes and Firmicutes communities were dominant in all the tested samples. The results indicated that the in vitro anaerobic conditions had altered the rumen methanogenic communities significantly and the facultative acetoclastic Methanosarcina was important for the methane production in the RF seeded reactors.

Magnetite production and transformation in the methanogenic consortia from coastal riverine sediments

Abstract: Minerals that contain ferric iron, such as amorphous Fe(III) oxides (A), can inhibit methanogenesis by competitively accepting electrons. In contrast, ferric iron reduced products, such as magnetite (M), can function as electrical conductors to stimulate methanogenesis, however, the processes and effects of magnetite production and transformation in the methanogenic consortia are not yet known. Here we compare the effects on methanogenesis of amorphous Fe (III) oxides (A) and magnetite (M) with ethanol as the electron donor. RNA-based terminal restriction fragment length polymorphism with a clone library was used to analyse both bacterial and archaeal communities. Iron (III)-reducing bacteria including Geobacteraceae and methanogens such as Methanosarcina were enriched in iron oxide-supplemented enrichment cultures for two generations with ethanol as the electron donor. The enrichment cultures with A and non-Fe (N) dominated by the active bacteria belong to Veillonellaceae, and archaea belong to Methanoregulaceae and Methanobacteriaceae, Methanosarcinaceae (Methanosarcina mazei), respectively. While the enrichment cultures with M, dominated by the archaea belong to Methanosarcinaceae (Methanosarcina barkeri). The results also showed that methanogenesis was accelerated in the transferred cultures with ethanol as the electron donor during magnetite production from A reduction. Powder X-ray diffraction analysis indicated that magnetite was generated from microbial reduction of A and M was transformed into siderite and vivianite with ethanol as the electron donor. Our data showed the processes and effects of magnetite production and transformation in the methanogenic consortia, suggesting that significantly different effects of iron minerals on microbial methanogenesis in the iron-rich coastal riverine environment were present.

Phylogenomic proximity and metabolic discrepancy of Methanosarcina mazei Go1 across methanosarcinal genomes.

Abstract: Methanosarcina mazei Go1 is a heterotrophic methanogenic archaean contributing a significant role in global methane cycling and biomethanation process. Phylogenomic relatedness and metabolic discrepancy of this genome were described herein by comparing its whole genome sequence, intergenomic distance, genome function, synteny homologs and origin of replication, and marker genes with very closely related genomes, Methanosarcina acetivorans and Methanosarcina barkeri. Phylogenomic analysis of this study revealed that genome functional feature and metabolic core of M. mazei and M. barkeri could be originated from M. acetivorans. The metabolic core of these genomes shares a common evolutionary origin to perform the metabolic activity at different environmental niches. Genome expansion, dynamics and gene collinearity were constrained and restrained the conservation of the metabolic core genes by duplication events occurring across methanosarcinal genomes. The Darwinian positive selection was an evolutionary constraint to purify the function of core metabolic genes. Using genome-wide metabolic survey, we found the existence of four novel putative metabolic pathways such as complete methanogenesis from acetate, indole-3-acetate biosynthesis V, 4-aminobutyrate degradation III, galactosamine biosynthesis I and siroheme biosynthesis. Overall, the present study would provide a stand point to revisit its phylogenomic status in order to understand the origin and evolution history of this organism.

Physiological Evidence for Isopotential Tunneling in the Electron Transport Chain of Methane-Producing Archaea.

Abstract: Many, but not all, organisms use quinones to conserve energy in their electron transport chains. Fermentative bacteria and methane-producing archaea (methanogens) do not produce quinones but have devised other ways to generate ATP. Methanophenazine (MPh) is a unique membrane electron carrier found in Methanosarcina species that plays the same role as quinones in the electron transport chain. To extend the analogy between quinones and MPh, we compared the MPh pool sizes between two well-studied Methanosarcina species, Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, to the quinone pool size in the bacterium Escherichia coli We found the quantity of MPh per cell increases as cultures transition from exponential growth to stationary phase, and absolute quantities of MPh were 3-fold higher in M. acetivorans than in M. barkeri The concentration of MPh suggests the cell membrane of M. acetivorans, but not of M. barkeri, is electrically quantized as if it were a single conductive metal sheet and near optimal for rate of electron transport. Similarly, stationary (but not exponentially growing) E. coli cells also have electrically quantized membranes on the basis of quinone content. Consistent with our hypothesis, we demonstrated that the exogenous addition of phenazine increases the growth rate of M. barkeri three times that of M. acetivorans Our work suggests electron flux through MPh is naturally higher in M. acetivorans than in M. barkeri and that hydrogen cycling is less efficient at conserving energy than scalar proton translocation using MPh.IMPORTANCE Can we grow more from less? The ability to optimize and manipulate metabolic efficiency in cells is the difference between commercially viable and nonviable renewable technologies. Much can be learned from methane-producing archaea (methanogens) which evolved a successful metabolic lifestyle under extreme thermodynamic constraints. Methanogens use highly efficient electron transport systems and supramolecular complexes to optimize electron and carbon flow to control biomass synthesis and the production of methane. Worldwide, methanogens are used to generate renewable methane for heat, electricity, and transportation. Our observations suggest Methanosarcina acetivorans, but not Methanosarcina barkeri, has electrically quantized membranes. Escherichia coli, a model facultative anaerobe, has optimal electron transport at the stationary phase but not during exponential growth. This study also suggests the metabolic efficiency of bacteria and archaea can be improved using exogenously supplied lipophilic electron carriers. The enhancement of methanogen electron transport through methanophenazine has the potential to increase renewable methane production at an industrial scale.

Physiological and molecular characterizations of the interactions in two cellulose-to-methane cocultures

Abstract: Background The interspecies interactions in a biomethanation community play a vital role in substrate degradation and methane (CH4) formation. However, the physiological and molecular mechanisms of interaction among the microbial members of this community remain poorly understood due to the lack of an experimentally tractable model system. In this study, we successfully established two coculture models combining the cellulose-degrading bacterium Clostridium cellulovorans 743B with Methanosarcina barkeri Fusaro or Methanosarcina mazei Go1 for the direct conversion of cellulose to CH4.

In Vitro Methanol Production from Methyl Coenzyme M Using the Methanosarcina Barkeri MtaABC Protein Complex

Abstract: Methanol:coenzyme M methyltransferase is an enzyme complex composed of three subunits, MtaA, MtaB, and MtaC, found in methanogenic archaea and is needed for their growth on methanol ultimately producing methane MtaABC catalyzes the energetically favorable methyl transfer from methanol to coenzyme M to form methyl coenzyme M Here we demonstrate that this important reaction for possible production of methanol from the anaerobic oxidation of methane can be reversed in vitro To this effect, we have expressed and purified the Methanosarcina barkeri MtaABC enzyme, and developed an in vitro functional assay that demonstrates MtaABC can catalyze the energetically unfavorable (ΔG° = 27 kJ/mol) reverse reaction starting from methyl coenzyme M and generating methanol as a product Demonstration of an in vitro ability of MtaABC to produce methanol may ultimately enable the anaerobic oxidation of methane to produce methanol and from methanol alternative fuel or fuel-precursor molecules © 2017 American Institute of Chemical Engineers Biotechnol Prog, 33:1243-1249, 2017

Different substrate regimes determine transcriptional profiles and gene co-expression in Methanosarcina barkeri (DSM 800).

Abstract: Methanosarcina barkeri (DSM 800) is a metabolically versatile methanogen and shows distinct metabolic status under different substrate regimes. However, the mechanisms underlying distinct transcriptional profiles under different substrate regimes remain elusive. In this study, based on transcriptional analysis, the growth performances and gene expressions of M. barkeri fed on acetate, H2 + CO2, and methanol, respectively, were investigated. M. barkeri showed higher growth performances under methanol, followed by H2 + CO2 and acetate, which corresponded well with the variations of gene expressions. The α diversity (evenness) of gene expressions was highest under the acetate regime, followed by H2 + CO2 and methanol, and significantly and negatively correlated with growth performances. The gene co-expression analysis showed that "Energy production and conversion," "Coenzyme transport and metabolism," and "Translation, ribosomal structure, and biogenesis" showed deterministic cooperation patterns of intra- and inter-functional classes. However, "Posttranslational modification, protein turnover, chaperones" showed exclusion with other functional classes. The gene expressions and especially the relationships among them potentially drove the shifts of metabolic status under different substrate regimes. Consequently, this study revealed the diversity-related ecological strategies that a high α diversity probably provided more fitness and tolerance under natural environments and oppositely a low α diversity strengthened some specific physiological functions, as well as the co-responses of gene expressions to different substrate regimes.

Pure culture, co-culture and whole ecosystem investigations of single anaerobes, partnerships and microbial communities in anaerobic digestion

Abstract: Anaerobic Digestion (AD) comprises the microbiological breakdown of complex organic materials in the absence of oxygen to produce biogas, which can be used as a renewable fuel. However, there is a knowledge gap regarding the microbiology that underpins the AD process, especially the specific synergies and competitions, between the individual species involved. Growth and substrate consumption profiles of pure cultures, and co-cultures, of three model AD organisms, Methanosarcina barkeri, Acetobacterium woodii and Methanococcus maripaludis, were investigated to determine their interactions under H2-CO2 and acetate feeding at both moderate and low temperatures. A common medium was also used for two of the strains for this study. Temporal CH4 and volatile fatty acid (VFA) concentrations in assays were determined for all combinations at each growth phase. Results showed that the substrates and temperatures strongly influenced the growth rates of each strain and co-culture. M. barkeri grew fastest in H2-CO2 at 37°C (doubling time (dt): 12.44 hours) compared to under acetate (dt: 34.34 hours) or at 15°C (dt: 28.25 hours). A. woodii grew fastest in H2-CO2 at 35°C (dt: 17.13 hours) compared to at 15°C in H2-CO2 (dt: 27.90 hours) and M. maripaludis grew at an optimum of dt = 32.30 hours in the CP medium on its preferred substrate H2-CO2 as the main energy source, at 37°C. Cocultures of M. barkeri partnered with A. woodii and M. barkeri partnered with M. maripaludis demonstrated the impact of competitions on their pure culture counterparts regarding growth and metabolisms. Future studies will include analyses of individual strains and mixed-species consortia by genomics and transcriptomics, and metagenomics and metatranscriptomics, respectively.