Showing papers on "Methanosarcina barkeri published in 2020"

Carbon nanotubes accelerate acetoclastic methanogenesis: From pure cultures to anaerobic soils

Abstract: Direct interspecies electron transfer (DIET) between electricigens and methanogens has been shown to favour CO2 reduction to produce biomethane. Furthermore, DIET is accelerated by conductive materials. However, whether conductive materials can promote other methanogenic pathways is unclear due to a lack of detailed experimental data and the poor mechanistic studies. Here, we hypothesized that conductive carbon nanotubes (CNTs) stimulate acetoclastic methanogenesis independently of electricigens in pure cultures of Methanosarcina spp. and anaerobic wetland soil. We found a significant increase in the methane production rate during the growth phase, e.g. from 0.169 mM to 0.241 mM after addition of CNTs on the 3rd day. CNTs did not increase the abundance of electromicroorganisms or the electron transfer rate in anaerobic soils, using via microbial diversity and electrochemical analysis. 13C–CH3COOH labelling, stable carbon isotope fractionation and the CH3F inhibitor of acetoclastic methanogenesis were used to distinguish methanogenic pathways. CNTs mainly accelerated acetoclastic methanogenesis rather than CO2 reduction in both pure cultures and anaerobic soils. Furthermore, the presence of CNTs slightly alleviate the inhibition of CH3F on acetoclastic methanogenesis during the pure culture of Methanosarcina barkeri and Methanosarcina mazei with the production of more than 0.3 mM methane. CNTs closely attached to the cell surface were observed by transmission electron microscopy. Proteome analysis revealed a stimulation of protein synthesis with about twice the improvement involved in –COOH oxidation and electron transfer. Overall, our findings demonstrate that conducting CNTs favor methane production and that the mechanism involved is acetoclastic methanogenesis via acetate dismutation, at least partly, rather than classical CO2 reduction.

Fully Productive Cell-Free Genetic Code Expansion by Structure-Based Engineering of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase

Abstract: Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (ge...

Formation of Zerovalent Iron in Iron-Reducing Cultures of Methanosarcina barkeri

Abstract: Methanogenic archaea have been shown to reduce iron from ferric [Fe(III)] to ferrous [Fe(II)] state, but minerals that form during iron reduction by different methanogens remain to be characterized. Here, we show that zerovalent iron (ZVI) minerals, ferrite [α-Fe(0)] and austenite [γ-Fe(0)], appear in the X-ray diffraction spectra minutes after the addition of ferrihydrite to the cultures of a methanogenic archaeon, Methanosarcina barkeri (M. barkeri). M. barkeri cells and redox-active, nonenzymatic soluble organic compounds in organic-rich spent culture supernatants can promote the formation of ZVI; the latter compounds also likely stabilize ZVI. Methanogenic microbes that inhabit organic- and Fe(III)-rich anaerobic environments may similarly reduce Fe(III) to Fe(II) and ZVI, with implications for the preservation of paleomagnetic signals during sediment diagenesis and potential applications in the protection of iron metals against corrosion and in the green synthesis of ZVI.

Dual RNase and β-lactamase Activity of a Single Enzyme Encoded in Archaea.

Abstract: β-lactam antibiotics have a well-known activity which disturbs the bacterial cell wall biosynthesis and may be cleaved by β-lactamases. However, these drugs are not active on archaea microorganisms, which are naturally resistant because of the lack of β-lactam target in their cell wall. Here, we describe that annotation of genes as β-lactamases in Archaea on the basis of homologous genes is a remnant of identification of the original activities of this group of enzymes, which in fact have multiple functions, including nuclease, ribonuclease, β-lactamase, or glyoxalase, which may specialized over time. We expressed class B β-lactamase enzyme from Methanosarcina barkeri that digest penicillin G. Moreover, while weak glyoxalase activity was detected, a significant ribonuclease activity on bacterial and synthetic RNAs was demonstrated. The β-lactamase activity was inhibited by β-lactamase inhibitor (sulbactam), but its RNAse activity was not. This gene appears to have been transferred to the Flavobacteriaceae group especially the Elizabethkingia genus, in which the expressed gene shows a more specialized activity on thienamycin, but no glyoxalase activity. The expressed class C-like β-lactamase gene, from Methanosarcina sp., also shows hydrolysis activity on nitrocefin and is more closely related to DD-peptidase enzymes. Our findings highlight the need to redefine the nomenclature of β-lactamase enzymes and the specification of multipotent enzymes in different ways in Archaea and bacteria over time.

Effect of nickel, cobalt, and iron on methanogenesis from methanol and cometabolic conversion of 1,2-dichloroethene by Methanosarcina barkeri.

Abstract: Methanogens are responsible for the last step in anaerobic digestion (AD), in which methane (a biofuel) is produced. Some methanogens can cometabolize chlorinated pollutants, contributing for their removal during AD. Methanogenic cofactors involved in cometabolic reductive dechlorination, such as F430 and cobalamin, contain metal ions (nickel, cobalt, iron) in their structure. We hypothesized that the supplementation of trace metals could improve methane production and the cometabolic dechlorination of 1,2-dichloroethene (DCE) by pure cultures of Methanosarcina barkeri. Nickel, cobalt, and iron were added to cultures of M. barkeri growing on methanol and methanol plus DCE. Metal amendment improved DCE dechlorination to vinyl chloride (VC): assays with 20 µM of Fe3+ showed the highest final concentration of VC (5× higher than in controls without Fe3+ ), but also in assays with 5.5 µM of Co2+ and 5 µM of Ni2+ VC formation was improved (3.5-4× higher than in controls without the respective metals). Dosing of metals could be useful to improve anaerobic removal of chlorinated compounds, and more importantly decrease the detrimental effect of DCE on methane production in anaerobic digesters.

Substrate-dependent incorporation of carbon and hydrogen for lipid biosynthesis by Methanosarcina barkeri.

Abstract: Dual stable isotope probing has been used to infer rates of microbial biomass production and modes of carbon fixation. In order to validate this approach for assessing archaeal production, the methanogenic archaeon Methanosarcina barkeri was grown either with H2 , acetate or methanol with D2 O and 13 C-dissolved inorganic carbon (DIC). Our results revealed unexpectedly low D incorporation into lipids, with the net fraction of water-derived hydrogen amounting to 0.357±0.042, 0.226±0.003 and 0.393±0.029 for growth on H2 /CO2 , acetate and methanol, respectively. The variability in net water H assimilation into lipids during growth of M. barkeri on different substrates is possibly attributed to different Gibbs free energy yields, such that higher energy yield promoted the exchange of hydrogen between medium water and lipids. Because NADPH likely serves as the portal for H transfer, increased NADPH production and/or turnover associated with high energy yield may explain the apparent differences in net water H assimilation into lipids. The variable DIC and water H incorporation into M. barkeri lipids imply systematic, metabolic patterns of isotope incorporation and suggest that the ratio of 13 C-DIC vs D2 O assimilation in environmental samples may serve as a proxy for microbial energetics in addition to microbial production and carbon assimilation pathways. This article is protected by copyright. All rights reserved.

Kinetic and substrate complex characterization of RamA, a corrinoid protein reductive activase from Methanosarcina barkeri.

Abstract: In microbial corrinoid-dependent methyltransferase systems, adventitious Co(I)-corrinoid oxidation halts catalysis and necessitates repair by ATP-dependent reductive activases. RamA, an activase with a C-terminal ferredoxin domain with two [4Fe-4S] clusters from methanogenic archaea, has been far less studied than the bacterial activases bearing an N-terminal ferredoxin domain with one [2Fe-2S] cluster. These differences suggest RamA might prove to have other distinctive characteristics. Here, we examine RamA kinetics and the stoichiometry of the corrinoid protein:RamA complex. Like bacterial activases, K+ stimulates RamA. Potassium stimulation had been questioned due to differences in the primary structure of bacterial and methanogen activases. Unlike one bacterial activase, ATP is not inhibitory allowing the first determination of apparent kinetic parameters for any corrinoid activase. Unlike bacterial activases, a single RamA monomer complexes a single corrinoid protein monomer. Alanine replacement of a RamA serine residue corresponding to the serine of one bacterial activase which ligates the corrinoid cobalt during complex formation led to only moderate changes in the kinetics of RamA. These results reveal new differences in the two types of corrinoid activases, and provide direct evidence for the proposal that corrinoid activases act as catalytic monomers, unlike other enzymes that couple ATP hydrolysis to difficult reductions.

Dual RNase and β-lactamase activity of a single enzyme encoded in most Archaea

Abstract: β-lactams targeting the bacterial cell wall are not active on archaea. Here, we figure out that annotation of genes as β-lactamase in Archeae on the basis of homologous genes, initially annotated β-lactamases, is a remnant of the identification of the original activities of this group of enzymes, which in fact, have multiple functions including nuclease, ribonuclease, β-lactamase, or glyoxalase; which may specialized over time. We expressed a class B β-lactamase enzyme from Methanosarcina barkeri that digest penicillin G. Moreover, while a weak glyoxalase activity was detected, a significant ribonuclease activity on bacterial and synthetic RNAs was demonstrated. The β-lactamase activity was inhibited by a β-lactamase inhibitor (sulbactam), but its RNAse activity was not. This gene appears to has been transferred to the Flavobacteriaceae group including Elizabethkingia genus in which the expressed gene shows a more specialized activity toward resistance to tienanmicin but no glyoxalase activity. The expressed class C-like β-lactamase gene, also from Methanosarcina sp., shows also hydrolysis activity and was more closely related to DD-peptidase enzymes than known bacterial class C β-lactamases. Our findings highlight the requalification needness of annotated enzymes as β-lactamases and the specification overtime of multipotent enzymes in different ways in Archaea and bacteria.

Adaptation of Methanosarcina barkeri 227 as acetate scavenger for succinate fermentation by Actinobacillus succinogenes.

Abstract: Acetate is the main by-product from microbial succinate production. In this study, we performed acetate removal by Methanosarcina barkeri 227 for succinate fermentation by Actinobacillus succinogenes 130Z. The acetoclastic methanogen M. barkeri requires similar environmental factors to A. succinogenes, and the conditions required for co-cultivation were optimized in this study: gas used for anaerobicization, strain adaptation, medium composition, pH adjustment, and inoculation time points. M. barkeri 227 was adapted to acetate for 150 days, which accelerated the acetate consumption to 9-fold (from 190 to 1726 mmol gDW−1 day−1). In the acetate-adapted strain, there was a noticeable increase in transcription of genes required for acetoclastic pathway—satP (acetate transporter), ackA (acetate kinase), cdhA (carbon monoxide dehydrogenase/acetyl-CoA synthase complex), and mtrH (methyl-H4STP:CoM methyltransferase), which was not induced before the adaptation process. The activities of two energy-consuming steps in the pathway—acetate uptake and acetate kinase—increased about 3-fold. This acetate-adapted M. barkeri could be successfully applied to succinate fermentation culture of A. succinogenes, but only after pH adjustment following completion of fermentation. This study suggests the utility of M. barkeri as an acetate scavenger during fermentation for further steps towards genetic and process engineering.