Search Results (18)

Wetlands are the most important natural sources of methane. Studies on the distribution and diversity of methanotrophs, especially in tropical wetlands, are limited. The studies on wetland methanotrophs help bridge the gap in the literature for understanding the community structure of methanotrophs in tropical wetlands. Our present study documents the methanotroph diversity from various wetland habitats across Western India. Samples from various sites, such as freshwater ponds, lake sediments, mangroves, etc., located in Western India, were collected and enriched for methanotroph isolation. An established protocol for the isolation of methanotrophs from Indian rice fields, involving serial dilution and long-term incubations, was slightly modified and used. Obtaining entirely pure cultures of methanotrophs is a labor-intensive and technically challenging process. Hence, for primary level characterization, ‘methanotroph monocultures’, which have a single methanotroph culture with minimal contamination, were established. Twenty monocultures and eight pure cultures of methanotrophs were obtained in this study. The pmoA gene has been used for the phylogenetic characterization of methanotrophs for the last 25 years. Monocultures were from seven genera: the Methylomonas, Methylocystis, Methylosinus, Methylocaldum, Methylocucumis, Methylomagnum, and Methylolobus genera. Eight pure cultures were obtained, which were strains of Methylomonas koyamae, Methylosinus sporium, and Methylolobus aquaticus. A maximum number of cultures belonged to the Type I genus Methylomonas and to the Type II genus Methylocystis. Thus, the cultivation-based community studies of methanotrophs from wetland habitats in India expanded the current knowledge about the methanotroph diversity in such regions. Additionally, the cultivation approach helped us obtain new methanotrophs from this previously unexplored habitat, which can be used for further biotechnological and environmental applications. The isolated monocultures can either be used as MMCs (mixed methanotroph consortia) for environmental applications or further purified and used as pure cultures. Full article

Biogas digestive effluent (BDE) is a nutrient-enriched source that can be utilized as an organic fertilizer for rice cultivation without synthetic fertilizer (SF) application. However, a primary concern is the stimulation of methane (CH₄) emissions due to the enrichment of the labile organic carbon, a favorite substrate of methanogenic archaea. Methanotrophs potentially reduce greenhouse gas (GHG) emissions from rice fields owing to metabolizing CH₄ as a carbon source and energy. We therefore examined the effect of the application of methanotroph-inoculated BDE to the rice cultivated paddy soil on GHG emissions and rice productivity under a pot experiment. Methanotrophs (Methylosinus sp. and Methylocystis sp.), isolated from the Vietnamese Mekong Delta’s rice fields, were separately inoculated to the heated BDE, followed by a 5-day preincubation. Methanotroph-inoculated BDE was supplied to rice cultivation to substitute SF at 50% or 100% in terms of nitrogen amount. The results showed that the total CH₄ emissions increased ~34% with the application of BDE. CH₄ emissions were significantly reduced by ~17–21% and ~28–44% under the application of methanotroph-inoculated BDE at 100% and 50%, respectively. The reduction in CH₄ was commensurate with the augmentation of pmoA transcript copy number under methanotroph-inoculated BDE. In addition, methanotroph-inoculated BDE application did not increase nitrous oxide (N₂O) emissions and adversely affect rice growth and grain productivity. This study highlighted the BDE-recirculated feasibility for a lower CH₄ emission rice production based on methanotrophs where high CH₄-emitting fields were confirmed. Full article

Over the past decade, copper (Cu) has been recognized as a crucial metal in the differential expression of soluble (sMMO) and particulate (pMMO) forms of methane monooxygenase (MMO) through a mechanism referred to as the “Cu switch”. In this study, we used Methylosinus trichosporium OB3b as a model bacterium to investigate the range of Cu concentrations that trigger the expression of sMMO to pMMO and its effect on growth and methane oxidation. The Cu switch was found to be regulated within Cu concentrations from 3 to 5 µM, with a strict increase in the methane consumption rates from 3.09 to 3.85 µM occurring on the 6th day. Our findings indicate that there was a decrease in the fold changes in the expression of methanobactin (Mbn) synthesis gene (mbnA) with a higher Cu concentration, whereas the Ton-B siderophore receptor gene (mbnT) showed upregulation at all Cu concentrations. Furthermore, the upregulation of the di-heme enzyme at concentrations above 5 µM Cu may play a crucial role in the copper switch by increasing oxygen consumption; however, the role has yet not been elucidated. We developed a quantitative assay based on the naphthalene–Molisch principle to distinguish between the sMMO- and pMMO-expressing cells, which coincided with the regulation profile of the sMMO and pMMO genes. At 0 and 3 µM Cu, the naphthol concentration was higher (8.1 and 4.2 µM, respectively) and gradually decreased to 0 µM naphthol when pMMO was expressed and acted as the sole methane oxidizer at concentrations above 5 µM Cu. Using physical protein–protein interaction, we identified seven transporters, three cell wall biosynthesis or degradation proteins, Cu resistance operon proteins, and 18 hypothetical proteins that may be involved in Cu toxicity and homeostasis. These findings shed light on the key regulatory genes of the Cu switch that will have potential implications for bioremediation and biotechnology applications. Full article

The widespread use of tetracycline antibiotics in the poultry and cattle sectors endangers both human health and the terrestrial ecosystem. Chlortetracyclines (CTCs), in particular, have been proven to affect soil microorganisms in addition to plants in the terrestrial ecosystem. In order to assess the effects of CTC on soil properties, eggplant growth, and soil microorganisms, a potted experiment was carried out in this study. CTC significantly reduced the levels of ammonium nitrogen (NH₄⁺–N) and nitrite nitrogen (NO₂⁻–N) in soil. Meanwhile, the eggplant’s growth was clearly hampered. CTC dramatically and dose-dependently lowered the fluorescence parameters except the quantum yield of non-regulated energy dissipation (Φ_NO). Rhodoplanes and Cupriavidus, which were involved in N cycle, were enriched by 10 mg/kg CTC, according to results about different microorganisms at the genus level. Flavisolibacter was reduced by 10 and 50 mg/kg CTC, while Methylosinus and Actinocorallia were enriched by 250 mg/kg CTC. Redundancy analysis highlighted the profound impact of CTC on the soil microbial community, where strong correlations were observed with soil potential of hydrogen (pH), nitrate nitrogen (NO₃⁻–N), and NO₂⁻–N. These findings demonstrated the interdependence between the microbial community and soil characteristics, with CTC primarily affecting the microbes responsible for nitrogen cycling. Consequently, chlortetracycline poses potential hazards to both eggplant plants and the soil microbes in eggplant cultivation soil. Full article

Rice fields are one of the important anthropogenic sources of methane emissions. Methanotrophs dwelling near the rice roots and at the oxic–anoxic interface of paddy fields can oxidize a large fraction of the generated methane and are therefore considered to be important. Nitrogen fixation in rice root-associated methanotrophs is well known. Our aim in this study was to explore the potential of methanotrophs as bio-inoculants for rice and the studies were performed in pot experiments in monsoon. Ten indigenously isolated methanotrophs were used belonging to eight diverse genera of Type Ia, Type Ib, and Type II methanotrophs, including the newly described genera and/or species, Methylocucumis oryzae and Methylolobus aquaticus, as well as Ca. Methylobacter oryzae and Ca. Methylobacter coli. Additionally, two consortia (Methylomonas strains and Methylocystis-Methylosinus strains) were used. Nitrogen fixation pathways or nifH genes were detected in all of the used methanotrophs. Plant growth promotion (PGPR) was seen in terms of increased plant height and grain yield. Nine out of twelve (seven single strains and two consortia) showed positive effects on grain yield (6–38%). The highest increase in grain yield was seen after inoculation with Ca. Methylobacter coli (38%) followed by Methylomonas consortium (35%) and Methylocucumis oryzae (31%). Methylomagnum ishizawai inoculated plants showed the highest plant height. Methylocucumis oryzae inoculated plants showed early flowering, grain formation, and grain maturation (~17–18 days earlier). In all the pot experiments, minimal quantities of nitrogen fertilizer were used with no additional organic fertilizer inputs. The present study demonstrated the possibility of developing methanotrophs as bio-inoculants for rice agriculture, which would promote plant growth under low inputs of nitrogenous fertilizers. Although the effect of methanotrophs on methane mitigation is still under investigation, their application to reduce methane emissions from rice fields could be an added advantage. Full article

Here, a syntrophic process was developed to produce polyhydroxy-β-butyrate (PHB) from a gas stream containing CH₄ and CO₂ without an external oxygen supply using a combination of methanotrophs with the community of oxygenic photogranules (OPGs). The co-culture features of Methylomonas sp. DH-1 and Methylosinus trichosporium OB3b were evaluated under carbon-rich and carbon-lean conditions. The critical role of O₂ in the syntrophy was confirmed through the sequencing of 16S rRNA gene fragments. Based on their carbon consumption rates and the adaptation to a poor environment, M. trichosporium OB3b with OPGs was selected for methane conversion and PHB production. Nitrogen limitation stimulated PHB accumulation in the methanotroph but hindered the growth of the syntrophic consortium. At 2.9 mM of the nitrogen source, 1.13 g/L of biomass and 83.0 mg/L of PHB could be obtained from simulated biogas. These results demonstrate that syntrophy has the potential to convert greenhouse gases into valuable products efficiently. Full article

Methanotrophs are bacteria that can consume methane as their sole carbon and energy source to produce a wide variety of high-value products such as lipids, biopolymers, ectoine, and single cell proteins (SCPs). Collected samples from various sources were subjected to DNA extraction followed by 16S rRNA analysis to determine the identity and relative abundance of their microbial population. Several taxa of methanotrophs were detected in the samples including Type I (Methylobacter), Type X (Methylocaldum), Type II (Methylocystis, Methylosinus, and Beijerinckia), and Type III (Verrucomicrobium). This paper expounds the effects of environmental/cultivation conditions on the growth and population of different types of methanotrophs. The results could be used to systematically identify source(s) of natural consortia that can be enriched and developed to produce specific target product(s) under a given cultivation conditions/limitations. Full article

Particulate methane monooxygenase (pMMO), a membrane-bound enzyme having three subunits (α, β, and γ) and copper-containing centers, is found in most of the methanotrophs that selectively catalyze the oxidation of methane into methanol. Active sites in the pMMO of Methylosinus trichosporium OB3b were determined by docking the modeled structure with ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene. The docking energy between the modeled pMMO structure and ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene was −5.2, −5.7, −4.2, and −3.8 kcal/mol, respectively, suggesting the existence of more than one active site within the monomeric subunits due to the presence of multiple binding sites within the pMMO monomer. The evaluation of tunnels and cavities of the active sites and the docking results showed that each active site is specific to the radius of the substrate. To increase the catalysis rates of methane in the pMMO of M. trichosporium OB3b, selected amino acid residues interacting at the binding site of ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene were mutated. Based on screening the strain energy, docking energy, and physiochemical properties, five mutants were downselected, B:Leu31Ser, B:Phe96Gly, B:Phe92Thr, B:Trp106Ala, and B:Tyr110Phe, which showed the docking energy of −6.3, −6.7, −6.3, −6.5, and −6.5 kcal/mol, respectively, as compared to the wild type (−5.2 kcal/mol) with ethylbenzene. These results suggest that these five mutants would likely increase methane oxidation rates compared to wild-type pMMO. Full article

This paper analyzed the degradation pathways of petroleum hydrocarbon degradation bacteria, screened the main degradation pathways, and found the petroleum hydrocarbon degradation enzymes corresponding to each step of the degradation pathway. Through the Copeland method, the best inoculation program of petroleum hydrocarbon degradation bacteria in a polluted site was selected as follows: single oxygenation path was dominated by Streptomyces avermitilis, hydroxylation path was dominated by Methylosinus trichosporium OB3b, secondary oxygenation path was dominated by Pseudomonas aeruginosa, secondary hydroxylation path was dominated by Methylococcus capsulatus, double oxygenation path was dominated by Acinetobacter baylyi ADP1, hydrolysis path was dominated by Rhodococcus erythropolis, and CoA path was dominated by Geobacter metallireducens GS-15 to repair petroleum hydrocarbon contaminated sites. The Copeland method score for this solution is 22, which is the highest among the 375 solutions designed in this paper, indicating that it has the best degradation effect. Meanwhile, we verified its effect by the Cdocker method, and the Cdocker energy of this solution is −285.811 kcal/mol, which has the highest absolute value. Among the inoculation programs of the top 13 petroleum hydrocarbon degradation bacteria, the effect of the best inoculation program of petroleum hydrocarbon degradation bacteria was 18% higher than that of the 13th group, verifying that this solution has the best overall degradation effect. The inoculation program of petroleum hydrocarbon degradation bacteria designed in this paper considered the main pathways of petroleum hydrocarbon pollutant degradation, especially highlighting the degradability of petroleum hydrocarbon intermediate degradation products, and enriching the theoretical program of microbial remediation of petroleum hydrocarbon contaminated sites. Full article

Priority pollutants such as polyethylene (PE) microplastic, lead (Pb²⁺), and cadmium (Cd²⁺) have attracted the interest of environmentalists due to their ubiquitous nature and toxicity to all forms of life. In this study, periphytic biofilms (epiphyton and epixylon) were used to bioremediate heavy metals (HMs) and to biodegrade PE under high (120,000 ppm) methane (CH₄) doses. Both periphytic biofilms were actively involved in methane oxidation, HMs accumulation and PE degradation. Epiphyton and epixylon both completely removed Pb²⁺ and Cd²⁺ at concentrations of 2 mg L⁻¹ and 50 mg L⁻¹, respectively, but only partially removed these HMs at a relatively higher concentration (100 mg L⁻¹). Treatment containing 12% ¹³CH₄ proved to be most effective for biodegradation of PE. A synergistic effect of HMs and PE drastically changed microbial biota and methanotrophic communities. High-throughput 16S rRNA gene sequencing revealed that Cyanobacteria was the most abundant class, followed by Gammaproteobacteria and Alphaproteobacteria in all high-methane-dose treatments. DNA stable-isotope probing was used to label ¹³C in a methanotrophic community. A biomarker for methane-oxidizing bacteria, pmoA gene sequence of a ¹³C-labeled fraction, revealed that Methylobacter was most abundant in all high-methane-dose treatments compared to near atmospheric methane (NAM) treatment, followed by Methylococcus. Methylomonas, Methylocystis, Methylosinus, and Methylocella were also found to be increased by high doses of methane compared to NAM treatment. Overall, Cd⁺² had a more determinantal effect on methanotrophic activity than Pb²⁺. Epiphyton proved to be more effective than epixylon in HMs removal and PE biodegradation. The findings proved that both epiphyton and epixylon can be used to bioremediate HMs and biodegrade PE as an efficient ecofriendly technique under high methane concentrations. Full article

The Methylocystis and Methylosinus are two of the five genera that were included in the first taxonomic framework of methanotrophic bacteria created half a century ago. Members of both genera are widely distributed in various environments and play a key role in reducing methane fluxes from soils and wetlands. The original separation of these methanotrophs in two distinct genera was based mainly on their differences in cell morphology. Further comparative studies that explored various single-gene-based phylogenies suggested the monophyletic nature of each of these genera. Current availability of genome sequences from members of the Methylocystis/Methylosinus clade opens the possibility for in-depth comparison of the genomic potentials of these methanotrophs. Here, we report the finished genome sequence of Methylocystis heyeri H2^T and compare it to 23 currently available genomes of Methylocystis and Methylosinus species. The phylogenomic analysis confirmed that members of these genera form two separate clades. The Methylocystis/Methylosinus pan-genome core comprised 1173 genes, with the accessory genome containing 4941 and 11,192 genes in the shell and the cloud, respectively. Major differences between the genome-encoded environmental traits of these methanotrophs include a variety of enzymes for methane oxidation and dinitrogen fixation as well as genomic determinants for cell motility and photosynthesis. Full article

Cadmium (Cd), chromium (Cr) and lead (Pb) are heavy metals that have been classified as priority pollutants in aqueous environment while methane-oxidizing bacteria as a biofilter arguably consume up to 90% of the produced methane in the same aqueous environment before it escapes into the atmosphere. However, the underlying kinetics and active methane oxidizers are poorly understood for the hotspot of epipelon that provides a unique micro-ecosystem containing diversified guild of microorganisms including methane oxidizers for potential bioremediation of heavy metals. In the present study, the Pb²⁺, Cd²⁺and Cr⁶⁺ bioremediation potential of epipelon biofilm was assessed under both high (120,000 ppm) and near-atmospheric (6 ppm) methane concentrations. Epipelon biofilm demonstrated a high methane oxidation activity following microcosm incubation amended with a high concentration of methane, accompanied by the complete removal of 50 mg L⁻¹ Pb²⁺ and 50 mg L⁻¹ Cd²⁺ (14 days) and partial (20%) removal of 50 mg L⁻¹ Cr⁶⁺ after 20 days. High methane dose stimulated a faster (144 h earlier) heavy metal removal rate compared to near-atmospheric methane concentrations. DNA-based stable isotope probing (DNA-SIP) following ¹³CH₄ microcosm incubation revealed the growth and activity of different phylotypes of methanotrophs during the methane oxidation and heavy metal removal process. High throughput sequencing of ¹³C-labelled particulate methane monooxygenase gene pmoA and 16S rRNA genes revealed that the prevalent active methane oxidizers were type I affiliated methanotrophs, i.e., Methylobacter. Type II methanotrophs including Methylosinus and Methylocystis were also labeled only under high methane concentrations. These results suggest that epipelon biofilm can serve as an important micro-environment to alleviate both methane emission and the heavy metal contamination in aqueous ecosystems with constant high methane fluxes. Full article

Methylosinus trichosporium OB3b is an obligate aerobic methane-utilizing alpha-proteobacterium. Since its isolation, M. trichosporium OB3b has been established as a model organism to study methane metabolism in type II methanotrophs. M. trichosporium OB3b utilizes soluble and particulate methane monooxygenase (sMMO and pMMO respectively) for methane oxidation. While the source of electrons is known for sMMO, there is less consensus regarding electron donor to pMMO. To investigate this and other questions regarding methane metabolism, the genome-scale metabolic model for M. trichosporium OB3b (model ID: iMsOB3b) was reconstructed. The model accurately predicted oxygen: methane molar uptake ratios and specific growth rates on nitrate-supplemented medium with methane as carbon and energy source. The redox-arm mechanism which links methane oxidation with complex I of electron transport chain has been found to be the most optimal mode of electron transfer. The model was also qualitatively validated on ammonium-supplemented medium indicating its potential to accurately predict methane metabolism in different environmental conditions. Finally, in silico investigations regarding flux distribution in central carbon metabolism of M. trichosporium OB3b were performed. Overall, iMsOB3b can be used as an organism-specific knowledgebase and a platform for hypothesis-driven theoretical investigations of methane metabolism. Full article

Microorganisms play an important role in animal nutrition, as they can be used as a source of food or feed. The aim of the study was to determine the nutritional elements and fatty acids contained in the biomass of methanotrophic bacteria. Four bacterial consortia composed of Methylocystis and Methylosinus originating from Sphagnum flexuosum (Sp1), S. magellanicum (Sp2), S. fallax II (Sp3), S. magellanicum IV (Sp4), and one composed of Methylocaldum, Methylosinus, and Methylocystis that originated from coalbed rock (Sk108) were studied. Nutritional elements were determined using the flame atomic absorption spectroscopy technique after a biomass mineralization stage, whereas the fatty acid content was analyzed with the GC technique. Additionally, the growth of biomass and dynamics of methane consumption were monitored. It was found that the methanotrophic biomass contained high concentrations of K, Mg, and Fe, i.e., approx. 9.6–19.1, 2.2–7.6, and 2.4–6.6 g kg⁻¹, respectively. Consequently, the biomass can be viewed as an appropriate feed and/or feed additive for supplementation with macroelements and certain microelements. Moreover, all consortia demonstrated higher content of unsaturated acids than saturated ones. Thus, methanotrophic bacteria seem to be a good solution, in natural supplementation of animal diets. Full article

The genes encoding adenosine triphosphate (ATP)- and polyphosphate (polyP)-dependent glucokinases (Glk) were identified in the aerobic obligate methanotroph Methylomonas sp. 12. The recombinant proteins were obtained by the heterologous expression of the glk genes in Esherichia coli. ATP-Glk behaved as a multimeric protein consisting of di-, tri-, tetra-, penta- and hexamers with a subunit molecular mass of 35.5 kDa. ATP-Glk phosphorylated glucose and glucosamine using ATP (100% activity), uridine triphosphate (UTP) (85%) or guanosine triphosphate (GTP) (71%) as a phosphoryl donor and exhibited the highest activity in the presence of 5 mM Mg²⁺ at pH 7.5 and 65 °C but was fully inactivated after a short-term incubation at this temperature. According to a gel filtration in the presence of polyP, the polyP-dependent Glk was a dimeric protein (2 × 28 kDa). PolyP-Glk phosphorylated glucose, mannose, 2-deoxy-D-glucose, glucosamine and N-acetylglucosamine using polyP as the phosphoryl donor but not using nucleoside triphosphates. The K_m values of ATP-Glk for glucose and ATP were about 78 μM, and the K_m values of polyP-Glk for glucose and polyP_(n=45) were 450 and 21 μM, respectively. The genomic analysis of methanotrophs showed that ATP-dependent glucokinase is present in all sequenced methanotrophs, with the exception of the genera Methylosinus and Methylocystis, whereas polyP-Glks were found in all species of the genus Methylomonas and in Methylomarinum vadi only. This work presents the first characterization of polyphosphate specific glucokinase in a methanotrophic bacterium. Full article