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To develop biotechnological process for methane to methanol conversion, selection of a suitable methanotrophic platform is an important aspect. Systematic approach based on literature and public databases was developed to select representative methanotrophs Methylotuvimicrobium alcaliphilum, Methylomonas methanica, Methylosinus trichosporium and Methylocella silvestris. Selected methanotrophs were further investigated for methanol tolerance and methanol production on pure methane as well as biogas along with key enzyme activities involved in methane utilization. Among selected methanotrophs M. alcaliphilum showed maximum methanol tolerance of 6% v/v along with maximum methanol production of 307.90 mg/L and 247.37 mg/L on pure methane and biogas respectively. Activity of methane monooxygenase and formate dehydrogenase enzymes in M. alcaliphilum was significantly higher up to 98.40 nmol/min/mg cells and 0.87 U/mg protein, respectively. Biotransformation trials in 14 L fermentor resulted in increased methanol production up to 418 and 331.20 mg/L, with yield coefficient 0.83 and 0.71 mg methanol/mg of pure methane and biogas respectively. The systematic selection resulted in haloalkaliphilic strain M. alcaliphilum as one of the potential methanotroph for bio-methanol production.

The technology of single-cell protein production from natural gas is based on using thermotolerant methanotrophic bacteria with high growth rates on methane. So far, the spectrum of strains used for industrial purposes was restricted to members of the genus Methylococcus. This poses limitations to further development of this technology and fuels the search for new cultures of fast-growing methanotrophs. The later task was addressed in the present work by analyzing the sediment samples of the Chernaya River, Crimea. Molecular analysis of the microbial community composition in the sediment revealed Gammaproteobacteria as the predominant group (33‒42% of all retrieved 16S rRNA gene fragments), as well as Chloroflexi, Actinobacteria, Alphaproteobacteria, Bacteroidota, and Acidobacteria as other numerically significant community members. The methanotrophic enrichment culture obtained from the sediment contained bacteria of the genus Methylomonas as the major component, with the relative abundance of up to 60% of all 16S rRNA gene fragments. The use of various strategies for methanotroph isolation resulted in obtaining three isolates of target bacteria of the genera Methylomonas, Methylomagnum and Methylocystis. The optimal growth temperatures of these isolates were 25, 35, and 40°С, respectively. The highest specific growth rate in batch culture, 0.21 h−1, was determined for Methylocystis sp. Kr9, which displayed 99.22 and 99.13% 16S rRNA gene sequence similarity to the type strains of two Methylocystis species, Methylocystis echinoides IMET 10491T and Methylocystis parvus OBBPT, respectively.

Methane oxidation rates and diversity of methane-oxidizing microorganisms were studied in the upper sediment layers of the Laptev Sea (methane seep area, Lena fore-delta, shelf, and upper slope not affected by seeps and river flow). The highest methane concentrations and methane oxidation rates (up to 1.16 µmol СН4 dm−3 day−1) were revealed at the seep stations. Carbon dioxide was the main product of methane oxidation at all stations. Sequencing of the 16S rRNA gene fragments revealed microorganisms of the class Gammaproteobacteria, order Methylococcales. Since the share of methanotrophs was high at the sites of constant gas seepage (up to 1.93% of the total read number), they could be detected by sequencing of the native samples. Phylogenetic diversity of methane-oxidizing bacteria was not high, and all detected microorganisms belonged to the Deep-Sea 1 cluster. On the phylogenetic tree, the sequences formed a cluster together with the genus Methyloprofundus. Members of the genera Methylomonas and Methylobacter related to methanotrophs inhabiting freshwater ecosystems were revealed in enrichments from the coastal stations sediment samples.

d‐Allulose has potential as a low‐calorie sweetener which can suppress fat accumulation. Several enzymes capable of d‐allulose production have been isolated, including d‐tagatose 3‐epimerases. Here, we report the isolation of a novel protein from Methylomonas sp. expected to be a putative enzyme based on sequence similarity to ketose 3‐epimerase. The synthesized gene encoding the deduced ketose 3‐epimerase was expressed as a recombinant enzyme in Escherichia coli, and it exhibited the highest enzymatic activity toward l‐ribulose, followed by d‐ribulose and d‐allulose. The X‐ray structure analysis of l‐ribulose 3‐epimerase from Methylomonas sp. (MetLRE) revealed a homodimeric enzyme, the first reported structure of dimeric l‐ribulose 3‐epimerase. The monomeric structure of MetLRE is similar to that of homotetrameric l‐ribulose 3‐epimerases, but the short C‐terminal α‐helix of MetLRE is unique and different from those of known l‐ribulose 3 epimerases. The length of the C‐terminal α‐helix was thought to be involved in tetramerization and increasing stability; however, the addition of residues to MetLRE at the C terminus did not lead to tetramer formation. MetLRE is the first dimeric l‐ribulose 3‐epimerase identified to exhibit high relative activity toward d‐allulose., d‐Allulose has potential as a low‐calorie sweetener which can suppress fat accumulation. The enzymes capable of d‐allulose production have attracted attention. Here, we report the X‐ray structure of a novel l‐ribulose 3‐epimerase from Methylomonas sp. (MetLRE) which also has enzymatic activity towards d‐allulose, showing that MetLRE is the first dimeric l‐ribulose 3‐epimerase identified to exhibit high relative activity toward d‐allulose. ​

Methanotrophic bacteria can use methane as sole carbon and energy source. Its importance in the environment is related to the mitigation of methane emissions from soil and water to the atmosphere. Brazilian mangroves are highly productive, have potential to methane production, and it is inferred that methanotrophic community is of great importance for this ecosystem. The scope of this study was to investigate the functional and taxonomic diversity of methanotrophic bacteria present in the anthropogenic impacted sediments from Bertioga’s mangrove (SP, Brazil). Sediment sample was cultivated with methane and the microbiota actively involved in methane oxidation was identified by DNA-based stable isotope probing (DNA-SIP) using methane as a labeled substrate. After 4 days of incubation and consumption of 0.7 mmol of methane, the most active microorganisms were related to methanotrophs Methylomonas and Methylobacter as well as to methylotrophic Methylotenera, indicating a possible association of these bacterial groups within a methane derived food chain in the Bertioga mangrove. The abundance of genera Methylomonas, able to couple methane oxidation to nitrate reduction, may indicate that under low dissolved oxygen tensions some aerobic methanotrophs could shift to intraerobic methane oxidation to avoid oxygen starvation.

Biotrans 2021, Graz, Austria, 19 Jul 2021 - 22 Jul 2021; ChemBioChem e202200195 (2022). doi:10.1002/cbic.202200195 special issue: "BioTrans 2021 / Guest Editors: Mélanie Hall, Silvia M. Glück, Karl Gruber, Wolfgang Kroutil", Published by Wiley-VCH, Weinheim

Methane-oxidizing bacteria (MOB) possess the metabolic potential to assimilate the highly potent greenhouse gas, CH4, and can also synthesize valuable products. Depending on their distinct and fastidious metabolic pathways, MOB are mainly divided into Type I and Type II; the latter are known as producers of polyhydroxyalkanoate (PHA). Despite the metabolic potential of MOB to synthesize PHA, the ecophysiology of MOB, especially under high CH4 flux conditions, is yet to be understood. Therefore, in this study, a rice paddy soil receiving a high CH4 flux from underground was used as an inoculum to enrich MOB using fed-batch operation, then the enriched Type II MOB were characterized. The transitions in the microbial community composition and CH4 oxidation rates were monitored by 16S rRNA gene amplicon sequencing and degree of CH4 consumption. With increasing incubation time, the initially dominant Methylomonas sp., affiliated with Type I MOB, was gradually replaced with Methylocystis sp., Type II MOB, resulting in a maximum CH4 oxidation rate of 1.40 g-CH4/g-biomass/day. The quantification of functional genes encoding methane monooxygenase, pmoA and PHA synthase, phaC, by quantitative PCR revealed concomitant increases in accordance with the Type II MOB enrichment. These increases in the functional genes underscore the significance of Type II MOB to mitigate greenhouse gas emission and produce PHA.

Sediment samples were taken from sediment adjacent to a suburban river in Sheffield in Northern England that had suffered heavy metal pollution due to previous activity of the steel industry (between the 17th and 19th centuries). The most abundant heavy metals found in the samples were lead, chromium, nickel, arsenic and cobalt, with maximum concentrations of 412·80, 25·232, 25·196, 8·123 and 7·66 mg kg-1 , respectively. Enrichment cultures were set up using methane as carbon and energy source, as a result of which a strain of methanotroph was isolated that was shown via 16S rRNA gene sequencing to be a strain Methylomonas koyamae and given the designation SHU1. M. koyamae SHU1 removed hexavalent chromium from an initial concentration of 10 ppm, which was inhibited by the metabolic inhibitor sodium azide or the methane monooxygenase inhibitor phenylacetylene. To the authors' knowledge, this is the first description of a strain of the widely environmentally distributed genus Methylomonas that is capable of remediating hexavalent chromium.

Aerobic CH4 oxidation coupled to denitrification (AME-D) can not only mitigate the emission of greenhouse gas (e.g., CH4) to the atmosphere, but also reduce NO3− and/or NO2− and alleviate nitrogen pollution. The effects of O2 tension on the community and functional gene expression of methanotrophs and denitrifiers were investigated in this study. Although higher CH4 oxidation occurred in the AME-D system with an initial O2 concentration of 21% (i.e., the O2-sufficient condition), more NO3−-N was removed at the initial O2 concentration of 10% (i.e., the O2-limited environment). Type I methanotrophs, including Methylocaldum, Methylobacter, Methylococcus, Methylomonas, and Methylomicrobium, and type II methanotrophs, including Methylocystis and Methylosinus, dominated in the AME-D systems. Compared with type II methanotrophs, type I methanotrophs were more abundant in the AME-D systems. Proteobacteria and Actinobacteria were the main denitrifiers in the AME-D systems, and their compositions varied with the O2 tension. Quantitative PCR of the pmoA, nirS, and 16S rRNA genes showed that methanotrophs and denitrifiers were the main microorganisms in the AME-D systems, accounting for 46.4% and 24.1% in the O2-limited environment, respectively. However, the relative transcripts of the functional genes including pmoA, mmoX, nirK, nirS, and norZ were all less than 1%, especially the functional genes involved in denitrification under the O2-sufficient condition, likely due to the majority of the denitrifiers being dormant or even nonviable. These findings indicated that an optimal O2 concentration should be used to optimize the activity and functional gene expression of aerobic methanotrophs and denitrifiers in AME-D systems.

ChemBioChem (2022). doi:10.1002/cbic.202100592, Published by Wiley-VCH, Weinheim

Methanotrophs are aerobic to micro-aerophilic bacteria, which oxidize and utilize methane, the second most important greenhouse gas. The community structure of the methanotrophs in rice fields worldwide has been studied mainly using culture-independent methods. Very few studies have focused on culturing methanotrophs from rice fields. We developed a unique method for the cultivation of methanotrophs from rice field samples. Here, we used a modified dilute nitrate mineral salts (dNMS) medium, with two cycles of dilution till extinction series cultivation with prolonged incubation time, and used agarose in the solid medium. The cultivation approach resulted in the isolation of methanotrophs from seven genera from the three major groups: Type Ia (Methylomonas, Methylomicrobium, and Methylocucumis), Type Ib (Methylocaldum and Methylomagnum), and Type II (Methylocystis and Methylosinus). Growth was obtained till 10–6–10–8 dilutions in the first dilution series, indicating the culturing of dominant methanotrophs. Our study was supported by 16S rRNA gene-based next-generation sequencing (NGS) of three of the rice samples. Our analyses and comparison with the global scenario suggested that the cultured members represented the major detected taxa. Strain RS1, representing a putative novel species of Methylomicrobium, was cultured; and the draft genome sequence was obtained. Genome analysis indicated that RS1 represented a new putative Methylomicrobium species. Methylomicrobium has been detected globally in rice fields as a dominant genus, although no Methylomicrobium strains have been isolated from rice fields worldwide. Ours is one of the first extensive studies on cultured methanotrophs from Indian rice fields focusing on the tropical region, and a unique method was developed. A total of 29 strains were obtained, which could be used as models for studying methane mitigation from rice fields and for environmental and biotechnological applications.

The presence of organic micropollutant (OMP) in groundwater threatens drinking water quality and public health. Rapid sand filter (RSF) rely on biofilms with nitrifying and methanotrophic bacteria to remove ammonia and methane during drinking water production. Previous research observed the partial removal of OMPs with active nitrification and methane oxidation due to co-metabolic conversion of OMPs. However, the contribution of indigenous nitrifying and methanotrophic communities from RSF has yet to be fully explored. Accordingly, experiments were carried out with biofilm-covered sand collected from field-scale RSF, to assess the removal of nine OMPs by nitrifying and methanotrophic bacteria. Results indicated that stimulating nitrification resulted in significantly more removal of caffeine, 2,4-dichlorophenoxyacetic acid and bentazone. Stimulating methanotrophic conditions enhanced the removal of caffeine, benzotriazole, 2,4-dichlorophenoxyacetic acid and bentazone. Microbial community analysis based on 16 S rRNA gene sequencing revealed Nitrosomonas and Nitrospira are the dominant genus in the community under nitrifying conditions. The three genera Methylobacter, Methylomonas and Methylotenera were enriched under methanotrophic conditions. This study highlights that nitrifying and methanotrophic bacteria play important roles during OMP removal in field RSF. Furthermore, results suggest that bioaugmentation with an enriched nitrifying and methanotrophic culture is a promising approach to improve OMP removal in RSF.

The shrinkage of vast inland lakes affects microbially mediated soil biogeochemical processes, which are critical for maintaining ecosystem sustainability, such as microbial diversity and a balanced CH4 budget. Here we aimed to elucidate shifts in the bacterial community and methanotrophy during the shrinkage of a saline lake. Sediments and soils along a gradient transecting a saline lake, saline riparian land, and grassland were collected. The succession of microbial communities was characterized by high-throughput sequencing of the V4-V5 region of 16S rRNA genes coupled to non-metric multidimensional scaling (NMDS), linear discriminant effect size (LEfSe), community assembly, and co-occurrence network analyses. We further incubated these samples under a 10% CH4 (v/v) atmospheric condition to determine the response of methane oxidation potentials and of methanotrophs to lake shrinkage by using pmoA-based qPCR and amplicon sequencing. LEfSe and NMDS analyses showed significant differences in bacterial communities among 3 stages of lake shrinkage. The microbial taxa with the highest increase were phylogenetically affiliated with unclassified Rhizobiales, Panacagrimonas, and Pseudomonas in saline and grassland soils when compared with sediments. Microbial community assembly was largely determined by deterministic rather than stochastic processes (NTI > 2). The drastic increase of Methylocystis-like (type II) methanotrophs was observed during lake shrinkage, while type I methanotrophs showed a decreasing trend. However, upon consuming high-concentration methane of about 10%, type I methanotrophs dominated methane-oxidizing communities in lake sediment (Methylomonas), riparian saline soil (Methylomicrobium), and grassland soil (Methylobacter). Structural equation model identified soil pH, C/N ratio, and soil texture as key factors affecting methane oxidation rates and the methanotrophic community. Lake shrinkage showed profound impacts on the overall bacterial communities and methane oxidizers. Soil physico-chemical properties likely shaped the bacterial community and phylogenetically distinct methanotrophs during lake shrinkage.

Methanotrophs are the only biological sink of the greenhouse gas methane. To understand the ecological features of methanotrophs in association with plants in the methane emitting environments, we investigated the community composition and methane oxidation of methanotrophs associated with duckweeds in a fresh water lake. Duckweeds collected from Lake Biwa, Japan over three summers showed methane consumption activity between 0.0067 and 0.89 μmol h⁻¹ g⁻¹ (wet weight), with the highest values occurring from the end of July to August. The methanotrophic community on duckweeds consisted primarily of γ-proteobacterial groups including the genera Methylomonas and Methylocaldum. Further analysis of co-cultures of a methanotroph isolate with sterilized duckweed revealed that the duckweed plant as well as the duckweed spent culture supernatant exerted an enhancing effect on methane oxidation. These results indicate that duckweeds not only provide a habitat for methanotrophs but also stimulate methanotrophic growth.

Propane is the main component of liquefied petroleum gas and is derived from crude oil processing. Methanotrophic bacteria can convert various alkanes using methane monooxygenase enzyme to primary alcohols. These are further oxidized to various aldehydes by alcohol dehydrogenases or methanol dehydrogenases. In this study, 2-propanol was produced from propane using the whole cells of Methylosinus trichosporium OB3b, Methylomicrobium alcaliphilum 20Z, and Methylomonas sp. DH-1 as the biocatalysts. The biocatalytic process of converting propane to 2-propanol was optimized by the use of several inhibitors and additives, such as EDTA, sodium phosphate, and sodium formate to prevent oxidation of 2-propanol to acetone and to enhance conversion of propane to propanol. The maximum titer of 2-propanol was 0.424 g/L, 0.311 g/L, and 0.610 g/L for Methylomonas sp. DH-1, M. alcaliphilum 20Z, and M. trichosporium OB3b whole cells, respectively. These results showed that type I and type II methanotrophs could be used as the potent biocatalyst for conversion of propane to propanol.

As a homologous gene encoding microbial alkaline phosphomonoesterase, the expression of phoD is critically controlled by P availability and thus contributes to the mineralization of soil organic P under P-depleted condition. However, its role in the regulation of soil P turnover is largely unknown due to the complex coupling of physiochemical and biological processes in the P cycle, especially in paddy field. We hypothesized that 1) P fertilization would decrease the abundance of phoD gene and change the composition of phoD-harboring microbial community and 2) the high abundance of phoD-harboring microorganisms in P-poor soil would stimulate the synthesis of alkaline phosphomonoesterase, thus mitigating P limitation via the mineralization of organic P. After 42 days of rice growth, the phoD abundance negatively correlated with soil P availability, and it was significantly higher in non-fertilized treatments than in P-fertilized treatments for both rhizosphere and bulk soils. A stronger competition among phoD-harboring microorganisms was detected in non-fertilized soil than in P-fertilized soil, with Bradyrhizobium, Methylobacterium, and Methylomonas being the dominant taxa in all samples. However, the high phoD gene abundance under P-poor condition was mainly due to the growth of rare operational taxonomic units (OTUs) affiliated to Actinobacteria and Cyanobacteria (relative abundance

Anaerobic processes are widely used for treating high-strength organic wastewater. Understanding the ecological patterns of the microorganisms involved and the effect of environmental factors on microbial community are important to manage the performance of anaerobic processes. Microbial communities of 12 anaerobic sludge samples acclimated under different environmental conditions were investigated. Genera detected from these anaerobic sludge samples generally presented three distribution patterns: frequently detected with high abundance, frequently detected with low abundance and occasionally detected with permanently low abundance. The type of feed stock was one of the most important process parameters affecting the shape of microbial community (e.g., Syntrophus, Methylomonas and Methylobacillus). Dye wastewater (Bacteroides) and the supplement of conductive materials (genus T78) were also found to shape the microbial community. Some syntrophic bacteria and methanogens were rare in many anaerobic samples. However, correlation analysis suggested that rare genera are potential syntrophic partners and are responsible for syntrophic methanogenesis.

Background Methanotrophs play an important role in biotechnological applications, with their ability to utilize single carbon (C1) feedstock such as methane and methanol to produce a range of high-value compounds. A newly isolated obligate methanotroph strain, Methylomonas sp. DH-1, became a platform strain for biotechnological applications because it has proven capable of producing chemicals, fuels, and secondary metabolites from methane and methanol. In this study, transcriptome analysis with RNA-seq was used to investigate the transcriptional change of Methylomonas sp. DH-1 on methane and methanol. This was done to improve knowledge about C1 assimilation and secondary metabolite pathways in this promising, but under-characterized, methane-bioconversion strain. Results We integrated genomic and transcriptomic analysis of the newly isolated Methylomonas sp. DH-1 grown on methane and methanol. Detailed transcriptomic analysis indicated that (i) Methylomonas sp. DH-1 possesses the ribulose monophosphate (RuMP) cycle and the Embden–Meyerhof–Parnas (EMP) pathway, which can serve as main pathways for C1 assimilation, (ii) the existence and the expression of a complete serine cycle and a complete tricarboxylic acid (TCA) cycle might contribute to methane conversion and energy production, and (iii) the highly active endogenous plasmid pDH1 may code for essential metabolic processes. Comparative transcriptomic analysis on methane and methanol as a sole carbon source revealed different transcriptional responses of Methylomonas sp. DH-1, especially in C1 assimilation, secondary metabolite pathways, and oxidative stress. Especially, these results suggest a shift of central metabolism when substrate changed from methane to methanol in which formaldehyde oxidation pathway and serine cycle carried more flux to produce acetyl-coA and NADH. Meanwhile, downregulation of TCA cycle when grown on methanol may suggest a shift of its main function is to provide de novo biosynthesis, but not produce NADH. Conclusions This study provides insights into the transcriptomic profile of Methylomonas sp. DH-1 grown on major carbon sources for C1 assimilation, providing in-depth knowledge on the metabolic pathways of this strain. These observations and analyses can contribute to future metabolic engineering with the newly isolated, yet under-characterized, Methylomonas sp. DH-1 to enhance its biochemical application in relevant industries. Electronic supplementary material The online version of this article (10.1186/s12864-019-5487-6) contains supplementary material, which is available to authorized users.

Methylotrophic bacterial community is very important group of bacteria utilizing reduced carbon compounds and plays significant role in plant growth promotion (PGP), crop yield and soil fertility for sustainable agriculture. Abiotic and biotic stresses are very important factors affecting PGP in agriculture. A vast number of microbial communities play an important role in abiotic stress tolerance. The PGP methylotrophic microbes have been reported well enough to mitigate different types of biotic and abiotic stresses. The abiotic stress tolerance was well documented by several methylotrophic bacterial communities such as Hyphomicrobium, Methylarcula, Methylobacillus, Methylobacterium, Methylocapsa, Methylocella, Methyloferula, Methylohalomonas, Methylomonas, Methylophilus, Methylopila, Methylosinus, Methylotenera, Methylovirgula and Methylovorus. The abiotic stress tolerance ability of different methylotrophs and their colonization in different parts of plants under severe low temperature, high temperature, drought and salt stress conditions have been investigated in various studies. The methylotrophic communities help in proliferation of plant directly through solubilization of phosphorus, potassium and zinc, production of phytohormones viz., auxins and cytokinins; production of Fe-chelating compound, biological nitrogen fixation and ACC-deaminase activities or indirectly through productions of ammonia, siderophores and secondary metabolites. The auxin and cytokinin secreted by methylotrophs influence seed germination and plant root growth and help plants to endure water stress. On the plant surface, the abundant methylotrophs exude osmo-protectants such as sugars and alcohols which ultimately help to protect the plants from desiccation and excessive radiations. The utilisation of these potent methylotrophic strains may facilitate proper crop production, PGP by ameliorating abiotic stresses.

This work is aiming at achieving antimonate (Sb(V)) bio-reduction in a methane (CH4) based membrane biofilm reactor (MBfR), and elucidating the effect of oxygen (O2) on the performance of the biofilm. Scanning electron microscope (SEM), energy dispersive X-ray (EDS) and X-ray photoelectron spectroscopy (XPS) confirm Sb2O3 precipitates were the main product formed from Sb(V) reduction in the CH4-fed biofilm. Illumina sequencing shows Thermomonas may be responsible for Sb(V) reduction. Moreover, we found 8 mg/L of O2 in the influent irreversibly inhibited Sb(V) reduction. Metagenomic prediction by Reconstruction of Unobserved State (PICRUSt) shows that the biofilm lacked efficient defense system to the oxidative stress, leading to the great suppress of key biological metabolisms such as TCA cycle, glycolysis and DNA replication, as well as potential Sb(V) reductases, by O2. However, methanotrophs Methylomonas and Methylosinus were enriched in the biofilm with O2 intrusion, in accordance with the enhanced abundance of genes encoding aerobic CH4 oxidation. These insights evoke the theoretical guidance of microbial remediation using CH4 as the electron donor towards Sb(V) contamination, and will give us a strong reference with regard to wastewater disposal.

Methylomonas is one of the most important and frequently detected genera in aquatic habitats and rice fields. A strain of Methylomonas, Kb3, an aerobic methanotroph, was isolated from a rice field from the Western parts of India. Kb3 cells were composed of short, motile rods and formed pink colored colonies and pink pellicles in liquid. On the basis of 16S rRNA gene, the closest valid species was Methylomonas methanica strain S1 (97.9% sequence similarity). Kb3 also showed close affiliation to “Methylomonas denitrificans” FJG1, a recently described, but not yet valid species (99.1% sequence similarity) and to strain LW13, a freshwater lake Washington isolate (99.5% sequence similarity). To understand the correct taxonomical position and metabolic properties of Kb3, the whole genome was sequenced. Kb3 genome showed a complete methane oxidation pathway, the presence of pMMO and sMMO genes, genes for nitrogen fixation and potential to convert nitrite to nitric oxide and nitric oxide to nitrous oxide. Numerous genes for chemotaxis and motility were detected. Genome comparison of Kb3 with the genomes of Methylomonas methanica S1 and “Methylomonas denitrificans” FJG1 showed ortho-ANI values of 86–87%, much less than 95%, the current cut-off for species. Digital DNA-DNA hybridization also showed values of 41–42%, much lesser than the 70% cut-off, both indicating that Kb3 did not belong to any of these species. Methylomonas LW13 and Methylomonas Kb3 showed close genome level similarities, i.e. OrthoANI value of 97.9% and a DDH of 87.3% and close branching in the phylogenomic tree confirming that Kb3 and LW13 can together form a new species.

Estuary and coastal environments have essential ecosystem functions in greenhouse gas sinks and removal of nitrogen pollution. Methane-oxidizing bacteria (MOB) and ammonia-oxidizing bacteria (AOB) communities play critical functions in the estuary's tidal flat sediments. Therefore, the effects of ammonium on MOB communities and methane on AOB communities need to be further explained. In this study, microcosm incubations with different contents of ammonium or methane were conducted for a relatively short (24 h) or long (28 days) period with tidal flat sediments from the Yangtze River estuary. Subsequently, the tagged highly degenerate primer PCR and DNA-based stable isotope probing method were employed to demonstrate the effects on MOB and AOB populations. The results indicated that the methane consumption was enhanced with ammonium supplements within 24 h of incubation. Supplement of 2 μmol/g d.w.s (μmol per gram dry weight soil) NH4+ increased the amount of MOB and its proportion to the total bacteria (p < 0.05) for 28 days incubation. The ammonium supplement increased the proportion of Methylomonas and Methylobacter based on the 16S rRNA gene. According to the functional gene analysis, the MOB primarily engaged in methane oxidation include Methylomonas, Methylobacter, Methylomicrobium, and Methylosarcina, which were associated with Type Ia MOB. It suggested that ammonium supplement may promote methane oxidation by stimulating the Type Ia MOB in tidal flat sediments of the Yangtze River estuary. The current research helps understand the effect of ammonium on methane consumption in the estuary and coastal environments.

Bioaugmentation is a promising alternative in soil remediation. One challenge of bioaugmentation is that exogenous pollutant-degrading microbes added to soil cannot establish enough biomass to eliminate pollutants. Considering that methanotrophs have a growth advantage in the presence of methane, we hypothesize that genetically engineered methanotrophs could degrade contaminants efficiently in soil with methane. Here, methanotroph Methylomonas sp. LW13, herbicide bensulfuron-methyl (BSM), and two kinds of soil were chosen to confirm this hypothesis. The unmarked gene knock-in method was first developed for strain LW13. Then, BSM hydrolase encoding gene sulE was inserted into the chromosome of strain LW13, conferring it BSM-degrading ability. After inoculation, the cell amount of strain LW13-sulE in soil raised considerably (over 100 fold in 9 days) with methane provision; meanwhile, >90% of BSM in soil was degraded. This study provides a proof of the concept that genetically engineered methanotroph is a potential platform for soil remediation.

In bacterial biotechnology, instead of producing functional proteins from plasmids, it is often necessary to deliver functional proteins directly into live cells for genetic manipulation or physiological modification. We constructed a library of cell-penetrating peptides (CPPs) capable of delivering protein cargo into bacteria and developed an efficient delivery method for CPP-conjugated proteins. We screened the library for highly efficient CPPs with no significant cytotoxicity in Escherichia coli and developed a model for predicting the penetration efficiency of a query peptide, enabling the design of new and efficient CPPs. As a proof-of-concept, we used the CPPs for plasmid curing in E. coli and marker gene excision in Methylomonas sp. DH-1. In summary, we demonstrated the utility of CPPs in bacterial engineering. The use of CPPs would facilitate bacterial biotechnology such as genetic engineering, synthetic biology, metabolic engineering, and physiology studies., Lee et al. construct a cell-penetrating peptides (CPP) library and identify CPPs that can penetrate bacterial cells with minimum or no impact on cell viability. For the identified top CPP candidates, their abilities to deliver macromolecules such as I-SceI and Cre recombinase proteins to bacteria are evaluated as proof-of-concept studies for potential applications.

The uptake of extracellular electrons from cathodes by microbes is a recently discovered phenomenon. However, current knowledge on the diversity of microbes accepting extracellular electrons and populating biocathodes is scarce. Research is required to explore the distribution of extracellular electron uptake metabolism in the tree of life across microbial guilds. Here we characterize the electron uptake ability of microbial guilds enriched on H2, S2O32-, or CH4 and NH4+ from the same inoculum taken from a groundwater treatment sand-filter. We hypothesized that functional microbes having dense outer membrane cytochromes in their native pathway, such as pathways of NH4+, CH4, S2O32- and H2, can perform extracellular electron uptake. We aimed of addressing the following questions: (1) Are there any known microbial member of anticipated function performing extracellular electron transfer? (2) How does electron uptake efficiency vary between the different functional guilds? (3) How is the dissipated electron energy distributed across metabolisms? We developed and applied a novel pipeline to identify taxa utilizing direct electron energy and utilizing secondary microbial products. We report the putative direct electron uptake metabolism of types belonging to Methylomonas, UBA6140, and Nitrosomonas. Furthermore, members of Streptococcaceae, Rhizobiaceae, Streptococcus, Brevundimonas, Chryseobacterium, and Pseudomonas are detected as electroactive taxa. Our results reveal novel insights into the diversity, electrochemical activity, and metabolism of taxa performing direct electron uptake.

Methylomonas sp. ZR1 was an isolated new methanotrophs that could utilize methane and methanol growing fast and synthesizing value added compounds such as lycopene. In this study, the genomic study integrated with the comparative transcriptome analysis were taken to understanding the metabolic characteristic of ZR1 grown on methane and methanol at normal and high temperature regime. Complete Embden-Meyerhof-Parnas pathway (EMP), Entner–Doudoroff pathway (ED), Pentose Phosphate Pathway (PP) and Tricarboxy Acid Cycle (TCA) were found to be operated in ZR1. In addition, the energy saving ppi-dependent EMP enzyme, coupled with the complete and efficient central carbon metabolic network might be responsible for its fast growing nature. Transcript level analysis of the central carbon metabolism indicated that formaldehyde metabolism was a key nod that may be in charge of the carbon conversion efficiency (CCE) divergent of ZR1 grown on methanol and methane. Flexible nitrogen and carotene metabolism pattern were also investigated in ZR1. Nitrogenase genes in ZR1 were found to be highly expressed with methane even in the presence of sufficient nitrate. It appears that, higher lycopene production in ZR1 grown on methane might be attributed to the higher proportion of transcript level of C40 to C30 metabolic gene. Higher transcript level of exopolysaccharides metabolic gene and stress responding proteins indicated that ZR1 was confronted with severer growth stress with methanol than with methane. Additionally, lower transcript level of the TCA cycle, the dramatic high expression level of the nitric oxide reductase and stress responding protein, revealed the imbalance of the central carbon and nitrogen metabolic status, which would result in the worse growth of ZR1 with methanol at 30 °C.