Linking methanotroph phenotypes to genotypes using a simple spatially resolved model ecosystem.

Connecting genes to phenotypic traits in bacteria is often challenging because of a lack of environmental context in laboratory settings. Laboratory-based model ecosystems offer a means to better account for environmental conditions compared with standard planktonic cultures and can help link genotypes and phenotypes. Here, we present a simple, cost-effective, laboratory-based model ecosystem to study aerobic methane-oxidizing bacteria (methanotrophs) within the methane-oxygen counter gradient typically found in the natural environment of these organisms. Culturing the methanotroph Methylomonas sp. strain LW13 in this system resulted in the formation of a distinct horizontal band at the intersection of the counter gradient, which we discovered was not due to increased numbers of bacteria at this location but instead to an increased amount of polysaccharides. We also discovered that different methanotrophic taxa form polysaccharide bands with distinct locations and morphologies when grown in the methane-oxygen counter gradient. By comparing transcriptomic data from LW13 growing within and surrounding this band, we identified genes upregulated within the band and validated their involvement in growth and band formation within the model ecosystem using knockout strains. Notably, deletion of these genes did not negatively affect growth using standard planktonic culturing methods. This work highlights the use of a laboratory-based model ecosystem that more closely mimics the natural environment to uncover bacterial phenotypes missing from standard laboratory conditions, and to link these phenotypes with their genetic determinants.

Imbalanced metabolism induced NH4+ accumulation and its effect on the central metabolism of Methylomonas sp. ZR1.

Nitrogen and carbon are the two most essential nutrient elements, and their metabolism is tightly coupled in single carbon metabolic microorganisms. However, the nitrogen metabolism and the nitrogen/carbon (N/C) metabolic balance in single-carbon metabolism is poorly studied. In this study, the nitrogen metabolism pattern of the fast growing methanotrophs Methylomonas sp. ZR1 grown in methane and methanol was studied. Effect study of different nitrogen sources on the cell growth of ZR1 indicates that nitrate salts are the best nitrogen source supporting the growth of ZR1 using methane and methanol as carbon source. However, its metabolic intermediate ammonium was found to accumulate with high N/C ratio in the medium and consequently inhibit the growth of ZR1. Studies of carbon and nitrogen metabolic kinetic under different N/C ratio conditions indicate that the accumulation of NH4+ is caused by the imbalanced nitrogen and carbon metabolism in ZR1. Feeding carbon skeleton α-ketoglutaric acid could effectively relieve the inhibition effect of NH4+ on the growth of ZR1, which further confirms this assumption. qPCR analysis of the expression level of the central metabolic key enzyme gene indicates that the nitrogen metabolic intermediate ammonium has strong regulation effect on the central nitrogen and carbon metabolism in ZR1. qPCR-combined genomic analysis confirms that a third ammonium assimilation pathway glycine synthesis system is operated in ZR1 to balance the nitrogen and carbon metabolism. Based on the qPCR result, it was also found that ZR1 employs two strategies to relieve ammonium stress in the presence of ammonium: assimilating excess ammonium or cutting off the nitrogen reduction reactions according to the available C1 substrate. Validating the connections between single-carbon and nitrogen metabolism and studying the accumulation and assimilation mechanism of ammonium will contribute to understand how nitrogen regulates cellular growth in single-carbon metabolic microorganisms.

Methylomonas rapida sp. nov., a novel species of fast-growing, carotenoid-producing obligate methanotrophs with high biotechnological potential.

The genus Methylomonas accommodates strictly aerobic, obligate methanotrophs, with their sole carbon and energy sources restricted to methane and methanol. These bacteria inhabit oxic-anoxic interfaces of various freshwater habitats and have attracted considerable attention as potential producers of a single-cell protein. Here, we characterize two fast-growing representatives of this genus, strains 12 and MP1T, which are phylogenetically distinct from the currently described Methylomonas species (94.0-97.3 % 16S rRNA gene sequence similarity). Strains 12 and MP1T were isolated from freshwater sediments collected in Moscow and Krasnodar regions, respectively. Cells of these strains are Gram-negative, red-pigmented, highly motile thick rods that contain a type I intracytoplasmic membrane system and possess a particulate methane monooxygenase (pMMO) enzyme. These bacteria grow between 8 and 45 °C (optimum 35 °C) in a relatively narrow pH range of 5.5-7.3 (optimum pH 6.6-7.2). Major carotenoids synthesized by these methanotrophs are 4,4'-diaplycopene-4,4'-dioic acid, 1,1'-dihydroxy-3,4-didehydrolycopene and 4,4'-diaplycopenoic acid. High biomass yield, of up to 3.26 g CDW/l, is obtained during continuous cultivation of MP1T on natural gas in a bioreactor at a dilution rate of 0.22 h-1. The complete genome sequence of strain MP1T is 4.59 Mb in size; the DNA G + C content is 52.8 mol%. The genome encodes four rRNA operons, one pMMO operon and 4,216 proteins. The genome sequence displays 82-85 % average nucleotide identity to those of earlier described Methylomonas species. We propose to classify these bacteria as representing a novel species of the genus Methylomonas, M. rapida sp. nov., with the type strain MP1T (=KCTC 92586T = VKM B-3663T).

Methylomonas albis sp. nov. and Methylomonas fluvii sp. nov.: Two cold-adapted methanotrophs from the river Elbe and emended description of the species Methylovulum psychrotolerans.

Three strains of methanotrophic bacteria (EbAT, EbBT and Eb1) were isolated from the River Elbe, Germany. These Gram-negative, rod-shaped or coccoid cells contain intracytoplasmic membranes perpendicular to the cell surface. Colonies and liquid cultures appeared bright-pink. The major cellular fatty acids were 12:0 and 14:0, in addition in Eb1 the FA 16:1ω5t was also dominant. Methane and methanol were utilized as sole carbon sources by EbBT and Eb1, while EbAT could not use methanol. All strains oxidize methane using the particulate methane monooxygenase. Both strains contain an additional soluble methane monooxygenase. The strains grew optimally at 15-25 °C and at pH 6 and 8. Based on 16S rRNA gene analysis recovered from the full genome, the phylogenetic position of EbAT is robustly outside any species clade with its closest relatives being Methylomonas sp. MK1 (98.24%) and Methylomonas sp. 11b (98.11%). Its closest type strain is Methylomonas methanica NCIMB11130 (97.91%). The 16S rRNA genes of EbBT are highly similar to Methylomonas methanica strains with Methylomonas methanica R-45371 as the closest relative (99.87% sequence identity). However, average nucleotide identity (ANI) and digital DNA-DNA-hybridization (dDDH) values reveal it as distinct species. The DNA G + C contents were 51.07 mol% and 51.5 mol% for EbAT and EbBT, and 50.7 mol% for Eb1, respectively. Strains EbAT and EbBT are representing two novel species within the genus Methylomonas. For strain EbAT we propose the name Methylomonas albis sp. nov (LMG 29958, JCM 32282) and for EbBT, we propose the name Methylomonas fluvii sp. nov (LMG 29959, JCM 32283). Eco-physiological descriptions for both strains are provided. Strain Eb1 (LMG 30323, JCM 32281) is a member of the species Methylovulum psychrotolerans. This genus is so far only represented by two isolates but Eb1 is the first isolate from a temperate environment; so, an emended description of the species is given.

Identification of efficient prokaryotic cell-penetrating peptides with applications in bacterial biotechnology.

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.

Genetically Engineered Methanotroph as a Platform for Bioaugmentation of Chemical Pesticide Contaminated Soil.

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.

Genome-scale revealing the central metabolic network of the fast growing methanotroph Methylomonas sp. ZR1.

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.

Methylomonas rhizoryzae sp. nov., a type I methanotroph isolated from the rhizosphere soil of rice.

A gammaproteobacterial methanotroph, strain GJ1T, was isolated from a rhizosphere soil sample of rice in Nanjing, China. The cells were Gram-negative, motile rods with a single polar flagellum, and they contained type I intracytoplasmic membranes. The cells formed pink colonies. The strain possessed both the particulate methane monooxygenase enzyme (pMMO) and the soluble methane monooxygenase enzyme (sMMO). pxmABC, encoding a divergent methane monooxygenase (pXMO), and nifH, which encodes dinitrogenase reductase, were also present. Methane and methanol were utilized as sole carbon sources, while other carbon sources, including acetate, pyruvate, succinate, citrate, malate, glucose, urea, methylamine, ethanol and formate, could not be utilized by strain GJ1T. Cell grew optimally at 25-33 °C (range 16-37 °C), pH 6.0-8.0 (range 5.5-8.5) and 0-1.2% NaCl (no growth above 1.5% NaCl). Phylogenetic analyses based on the 16S rRNA gene, pmoA and nifH showed that the isolate belongs to the genus Methylomonas of the family Methylococcaceae within the class Gammaproteobacteria. The major quinone was determined to be MQ-8, and the major fatty acids were observed to be C16:1 and C14:0. The genome size of strain GJ1T is about 4.55 Mb, and the DNA G + C content of the strain was determined to be 53.67 mol% within the range of the genus Methylomonas (47-58 mol%) reported at present. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain GJ1T and Methylomonas koyamae Fw12E-YT among the genus Methylomonas were the highest, and they were only 74.66% and 21.40%, respectively. In consequence, results of phenotypic characterization and phylogenetic analyses support strain GJ1T as a novel species within the genus Methylomonas, namely, Methylomonas rhizoryzae sp. nov.. The type strain is GJ1T (= ACCC 61706).

Isolation of a methane-oxidizing bacterium that bioremediates hexavalent chromium from a formerly industrialized Suburban River.

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. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerobic methanotrophic bacteria are known for bioremediation of an increasing range of organic and inorganic pollutants, using methane as carbon and energy source. Previously, one laboratory methanotroph strain, Methylococcus capsulatus Bath, was known to bioremediate toxic chromium (VI) by reducing it to chromium (III). Here, a newly isolated methanotroph strain, Methylomonas koyamae SHU1, has been shown able to remediate chromium (VI). This indicates that chromium (VI) bioremediation is not unique to M. capsulatus and moreover adds weight to the suggestion that methanotrophs may contribute directly to chromium (VI) detoxification in nature and in polymicrobial bioremediation fed with methane.

Anaerobic methane oxidation coupled to chromate reduction in a methane-based membrane biofilm batch reactor.

Chromate can be reduced by methanotrophs in a membrane biofilm reactor (MBfR). In this study, we cultivated a Cr(VI)-reducing biofilm in a methane (CH4)-based membrane biofilm batch reactor (MBBR) under anaerobic conditions. The Cr(VI) reduction rate increased to 0.28 mg/L day when the chromate concentration was ≤ 2.2 mg/L but declined sharply to 0.01 mg/L day when the Cr(VI) concentration increased to 6 mg/L. Isotope tracing experiments showed that part of the 13C-labeled CH4 was transformed to 13CO2, suggesting that the biofilm may reduce Cr(VI) by anaerobic methane oxidation (AnMO). Microbial community analysis showed that a methanogen, i.e., Methanobacterium, dominated in the biofilm, suggesting that this genus is probably capable of carrying out AnMO. The abundance of Methylomonas, an aerobic methanotroph, decreased significantly, while Meiothermus, a potential chromate-reducing bacterium, was enriched in the biofilm. Overall, the results showed that the anaerobic environment inhibited the activity of aerobic methanotrophs while promoting AnMO bacterial enrichment, and high Cr(VI) loading reduced Cr(VI) flux by inhibiting the methane oxidation process.

Metabolic engineering of the type I methanotroph Methylomonas sp. DH-1 for production of succinate from methane.

Methane-utilizing methanotrophs are fascinating systems for methane bioconversion. Methylomonas sp. DH-1, a novel type I methanotroph isolated from brewery sludge, has been evaluated as a promising candidate for an industrial bio-catalyst. Succinate has been considered one of the top building block chemicals for the agricultural, food, and pharmaceutical industries. In this study, Methylomonas sp. DH-1 was engineered to accumulate succinate as a desired product. The TCA cycle and enzymes diverting carbon flux to acetate or formate were modified or deleted to improve succinate productivity. By deleting succinate dehydrogenase (sdh) in the TCA cycle, succinate production increased dramatically ∼10 times compared to that of the wild type. In addition, the maximum succinate titer of ∼134 mg/L (DS-GL) was achieved by integrating glyoxylate shunt enzymes from the E. coli MG1655 strain. Pyruvate formate lyase (pfl) and acetate kinase-phosphotransacetylase (ack-pta) genes were disrupted to further concentrate carbon flux to the TCA cycle. However, these additional disruptions of competitive pathways did not affect cell growth or succinate production positively. The mutant strain DS-GL, which showed the best succinate production, was grown in a fed-batch bioreactor, and higher cell growth and succinate production (∼195 mg/L succinate with 0.0789 g-succinate/g-methane yield) were achieved. In this study, we demonstrated a novel platform for microbial conversion of methane to succinate using methanotroph.

A comparative transcriptome analysis of the novel obligate methanotroph Methylomonas sp. DH-1 reveals key differences in transcriptional responses in C1 and secondary metabolite pathways during growth on methane and methanol.

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.

Interspecies Chemical Signaling in a Methane-Oxidizing Bacterial Community.

Multiple species of bacteria oxidize methane in the environment after it is produced by anaerobic ecosystems. These organisms provide reduced carbon substrates for species that cannot oxidize methane themselves, thereby serving a key role in these niches while also sequestering this potent greenhouse gas before it enters the atmosphere. Deciphering the molecular details of how methane-oxidizing bacteria interact in the environment enables us to understand an important aspect that shapes the structures and functions of these communities. Here we show that many members of the Methylomonas genus possess a LuxR-type acyl-homoserine lactone (acyl-HSL) receptor/transcription factor that is highly homologous to MbaR from the quorum-sensing (QS) system of Methylobacter tundripaludum, another methane oxidizer that has been isolated from the same environment. We reconstitute this detection system in Escherichia coli and use mutant and transcriptomic analysis to show that the receptor/transcription factor from Methylomonas sp. strain LW13 is active and alters LW13 gene expression in response to the acyl-HSL produced by M. tundripaludum These findings provide a molecular mechanism for how two species of bacteria that may compete for resources in the environment can interact in a specific manner through a chemical signal.IMPORTANCE Methanotrophs are bacteria that sequester methane, a significant greenhouse gas, and thereby perform an important ecosystem function. Understanding the mechanisms by which these organisms interact in the environment may ultimately allow us to manipulate and to optimize this activity. Here we show that members of a genus of methane-oxidizing bacteria can be influenced by a chemical signal produced by a possibly competing species. This provides insight into how gene expression can be controlled in these bacterial communities via an exogenous chemical signal.

Physiological Effect of XoxG(4) on Lanthanide-Dependent Methanotrophy.

A recent surprising discovery of the activity of rare earth metals (lanthanides) as enzyme cofactors as well as transcriptional regulators has overturned the traditional assumption of biological inertia of these metals. However, so far, examples of such activities have been limited to alcohol dehydrogenases. Here we describe the physiological effects of a mutation in xoxG, a gene encoding a novel cytochrome, XoxG(4), and compare these to the effects of mutation in XoxF, a lanthanide-dependent methanol dehydrogenase, at the enzyme activity level and also at the community function level, using Methylomonas sp. strain LW13 as a model organism. Through comparative phenotypic characterization, we establish XoxG as the second protein directly involved in lanthanide-dependent metabolism, likely as a dedicated electron acceptor from XoxF. However, mutation in XoxG caused a phenotype that was dramatically different from the phenotype of the mutant in XoxF, suggesting a secondary function for this cytochrome, in metabolism of methane. We also purify XoxG(4) and demonstrate that this protein is a true cytochrome c, based on the typical absorption spectra, and we demonstrate that XoxG can be directly reduced by a purified XoxF, supporting one of its proposed physiological functions. Overall, our data continue to suggest the complex nature of the interplay between the calcium-dependent and lanthanide-dependent alcohol oxidation systems, while they also suggest that addressing the roles of these alternative systems is essential at the enzyme and community function level, in addition to the gene transcription level.IMPORTANCE The lanthanide-dependent biochemistry of living organisms remains a barely tapped area of knowledge. So far, only a handful of lanthanide-dependent alcohol dehydrogenases have been described, and their regulation by lanthanides has been demonstrated at the transcription level. Little information is available regarding the concentrations of lanthanides that could support sufficient enzymatic activities to support specific metabolisms, and so far, no other redox proteins involved in lanthanide-dependent methanotrophy have been demonstrated. The research presented here provides enzyme activity-level data on lanthanide-dependent methanotrophy in a model methanotroph. Additionally, we identify a second protein important for lanthanide-dependent metabolism in this organism, XoxG(4), a novel cytochrome. XoxG(4) appears to have multiple functions in methanotrophy, one function as an electron acceptor from XoxF and another function remaining unknown. On the basis of the dramatic phenotype of the XoxG(4) mutant, this function must be crucial for methanotrophy.

A novel methanotroph in the genus Methylomonas that contains a distinct clade of soluble methane monooxygenase.

Aerobic methane oxidation is a key process in the global carbon cycle that acts as a major sink of methane. In this study, we describe a novel methanotroph designated EMGL16-1 that was isolated from a freshwater lake using the floating filter culture technique. Based on a phylogenetic analysis of 16S rRNA gene sequences, the isolate was found to be closely related to the genus Methylomonas in the family Methylococcaceae of the class Gammaproteobacteria with 94.2-97.4% 16S rRNA gene similarity to Methylomonas type strains. Comparison of chemotaxonomic and physiological properties further suggested that strain EMGL16-1 was taxonomically distinct from other species in the genus Methylomonas. The isolate was versatile in utilizing nitrogen sources such as molecular nitrogen, nitrate, nitrite, urea, and ammonium. The genes coding for subunit of the particulate form methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), and methanol dehydrogenase (mxaF) were detected in strain EMGL16-1. Phylogenetic analysis of mmoX indicated that mmoX of strain EMGL16-1 is distinct from those of other strains in the genus Methylomonas. This isolate probably represents a novel species in the genus. Our study provides new insights into the diversity of species in the genus Methylomonas and their environmental adaptations.

Synthesizing value-added products from methane by a new Methylomonas.

AIMS: Methane and methanol are potential carbon sources of industrial micro-organisms in addition to crop-derived bio-carbon sources. Methanotrophs that can utilize these simple, stable and large amounts chemicals are expected to be developed into 'cell factories' for the production of specific chemicals. In this study, a methanotroph that can synthesize lycopene, C30 carotenoid and exopolysaccharides (EPS) with relative better performances from C1 substrates was isolated, and its performances were evaluated. METHODS AND RESULTS: The isolated strain was identified as Methylomonas sp. ZR1 based on 16S rRNA sequence analysis. Its maximum specific growth rate achieved 0·200 h-1 under flask culture conditions, and 0·386 h-1 in bubble column reactors. ZR1 was able to utilize 35 g l-1 of methanol and even exhibited slight growth in the presence of 40 g l-1 of methanol. Furthermore, ZR1 was proved to synthesize lycopene (C40 carotenoids) besides the C30 carotenoids through HPLC-DAD and HPLC-MS/MS analysis methods. Its carotenoid extracts exhibited excellent antioxidative activities measured by the ABTS+ method. Plenty of polysaccharides were also synthesized by ZR1, the components of the polysaccharides were identified as glucose, mannose and galactose with a proportion of 1 : 2 : 1 by GC-MS, and its yield achieved 0·13 g g-1 cell dry weight. CONCLUSIONS: The isolated strain has great potential for the production of value-added bioproducts from C1 compounds because of its excellent C1 substrate utilizing abilities and its abilities to naturally synthesize lycopene, C30 carotenoids and EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we isolated a fast-growing methanotroph, its C1 carbon substrate utilizing ability is excellent in comparison with reported methanotrophs. Furthermore, besides polysaccharides and C30 carotenoids which were commonly synthesized by methanotrophs, our findings suggested that C40 lycopene could also be naturally synthesized from methane by methanotrophs.

Selective bio-oxidation of propane to acetone using methane-oxidizing Methylomonas sp. DH-1.

Propane is the major component of liquefied petroleum gas (LPG). Nowadays, the use of LPG is decreasing, and thus utilization of propane as a chemical feedstock is in need of development. An efficient biological conversion of propane to acetone using a methanotrophic whole cell as the biocatalyst was proposed and investigated. A bio-oxidation pathway of propane to acetone in Methylomonas sp. DH-1 was analyzed by gene expression profiling via RNA sequencing. Propane was oxidized to 2-propanol by particulate methane monooxygenase and subsequently to acetone by methanol dehydrogenases. Methylomonas sp. DH-1 was deficient in acetone-converting enzymes and thus accumulated acetone in the absence of any enzyme inhibition. The maximum accumulation, average productivity and specific productivity of acetone were 16.62 mM, 0.678 mM/h and 0.141 mmol/g cell/h, respectively, under the optimized conditions. Our study demonstrates a novel method for the bioconversion of propane to acetone using methanotrophs under mild reaction condition.

Production of uracil from methane by a newly isolated Methylomonas sp. SW1.

Methane is an abundant, inexpensive one-carbon feedstock and one of the most powerful greenhouse gases. Because it does not compete with food demand, it is considered a promising carbon feedstock for the production of valuable products using methanotrophic bacteria. Here, we isolated a novel methanotrophic bacterium, Methylomonas sp. SW1, from a sewage sample obtained from Wonju City Water Supply Drainage Center, Republic of Korea. The conditions for uracil production by Methylomonas sp. SW1, such as Cu2+ concentration and temperature were investigated and optimized. As a result, Methylomonas sp. SW1 produced uracil from methane as a sole carbon source with a titer of 2.1mg/L in 84h without genetic engineering under the optimized condition. The results in this study demonstrate the feasibility of using Methylomonas sp. SW1 for the production of uracil from methane. This is the first report of uracil production from gas feedstock by methanotrophic bacteria.

Methane oxidation coupled to nitrate reduction under hypoxia by the Gammaproteobacterium Methylomonas denitrificans, sp. nov. type strain FJG1.

Obligate methanotrophs belonging to the Phyla Proteobacteria and Verrucomicrobia require oxygen for respiration and methane oxidation; nevertheless, aerobic methanotrophs are abundant and active in low oxygen environments. While genomes of some aerobic methanotrophs encode putative nitrogen oxide reductases, it is not understood whether these metabolic modules are used for NOx detoxification, denitrification or other purposes. Here we demonstrate using microsensor measurements that a gammaproteobacterial methanotroph Methylomonas denitrificans sp. nov. strain FJG1(T) couples methane oxidation to nitrate reduction under oxygen limitation, releasing nitrous oxide as a terminal product. Illumina RNA-Seq data revealed differential expression of genes encoding a denitrification pathway previously unknown to methanotrophs as well as the pxmABC operon in M. denitrificans sp. nov. strain FJG1(T) in response to hypoxia. Physiological and transcriptome data indicate that genetic inventory encoding the denitrification pathway is upregulated only upon availability of nitrate under oxygen limitation. In addition, quantitation of ATP levels demonstrates that the denitrification pathway employs inventory such as nitrate reductase NarGH serving M. denitrificans sp. nov. strain FJG1(T) to conserve energy during oxygen limitation. This study unravelled an unexpected metabolic flexibility of aerobic methanotrophs, thereby assigning these bacteria a new role at the metabolic intersection of the carbon and nitrogen cycles.

Methane-derived carbon flow through microbial communities in arctic lake sediments.

Aerobic methane (CH4 ) oxidation mitigates CH4 release and is a significant pathway for carbon and energy flow into aquatic food webs. Arctic lakes are responsible for an increasing proportion of global CH4 emissions, but CH4 assimilation into the aquatic food web in arctic lakes is poorly understood. Using stable isotope probing (SIP) based on phospholipid fatty acids (PLFA-SIP) and DNA (DNA-SIP), we tracked carbon flow quantitatively from CH4 into sediment microorganisms from an arctic lake with an active CH4 seepage. When 0.025 mmol CH4 g(-1) wet sediment was oxidized, approximately 15.8-32.8% of the CH4 -derived carbon had been incorporated into microorganisms. This CH4 -derived carbon equated to up to 5.7% of total primary production estimates for Alaskan arctic lakes. Type I methanotrophs, including Methylomonas, Methylobacter and unclassified Methylococcaceae, were most active at CH4 oxidation in this arctic lake. With increasing distance from the active CH4 seepage, a greater diversity of bacteria incorporated CH4 -derived carbon. Actinomycetes were the most quantitatively important microorganisms involved in secondary feeding on CH4 -derived carbon. These results showed that CH4 flows through methanotrophs into the broader microbial community and that type I methanotrophs, methylotrophs and actinomycetes are important organisms involved in using CH4 -derived carbon in arctic freshwater ecosystems.