Methylomonas defluvii sp. nov., a type I methane-oxidizing bacterium from a secondary sedimentation tank of a wastewater treatment plant.

An aerobic methanotroph was isolated from a secondary sedimentation tank of a wastewater treatment plant and designated strain OY6T. Cells of OY6T were Gram-stain-negative, pink-pigmented, motile rods and contained an intracytoplasmic membrane structure typical of type I methanotrophs. OY6T could grow at a pH range of 4.5-7.5 (optimum pH 6.5) and at temperatures ranging from 20 °C to 37 °C (optimum 30 °C). The major cellular fatty acids were C14 : 0, C16 : 1ω7c/C16 : 1ω6c and C16 : 1ω5c; the predominant respiratory quinone was MQ-8. The genome size was 5.41 Mbp with a DNA G+C content of 51.7 mol%. OY6T represents a member of the family Methylococcaceae of the class Gammaproteobacteria and displayed 95.74-99.64 % 16S rRNA gene sequence similarity to the type strains of species of the genus Methylomonas. Whole-genome comparisons based on average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) confirmed that OY6T should be classified as representing a novel species. The most closely related type strain was Methylomonas fluvii EbBT, with 16S rRNA gene sequence similarity, ANI by blast (ANIb), ANI by MUMmer (ANIm) and dDDH values of 99.64, 90.46, 91.92 and 44.5 %, respectively. OY6T possessed genes encoding both the particulate methane monooxygenase enzyme and the soluble methane monooxygenase enzyme. It grew only on methane or methanol as carbon sources. On the basis of phenotypic, genetic and phylogenetic data, strain OY6T represents a novel species within the genus Methylomonas for which the name Methylomonas defluvii sp. nov. is proposed, with strain OY6T (=GDMCC 1.4114T=KCTC 8159T=LMG 33371T) as the type strain.

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.

Synergy effects of Methylomonas koyamae and Hyphomicrobium methylovorum under methanethiol stress.

Methanotrophs are able to metabolize volatile organic sulfur compounds (VOSCs), excrete organic carbon during CH4 oxidation, and influence microbial community structure and function of the ecosystem. In return, microbial community structure and environmental factors can affect the growth metabolism of methanotrophs. In this study, Methylomonas koyamae and Hyphomicrobium methylovorum were used for model organisms, and methanethiol (MT) was chosen for a typical VOSC to investigate the synergy effects under VOSC stress. The results showed that when Hyphomicrobium methylovorum was co-cultured with Methylomonas koyamae in the medium with CH4 used as the carbon source, the co-culture had better MT tolerance relative to Methylomonas koyamae and oxidized all CH4 within 120 h, even at the initial MT concentration of 2000 mg m-3. The optimal co-culture ratios of Methylomonas koyamae to Hyphomicrobium methylovorum were 4:1-12:1. Although MT could be converted spontaneously to dimethyl disulfide (DMDS), H2S, and CS2 in air, faster losses of MT, DMDS, H2S, and CS2 were observed in each strain mono-culture and the co-culture. Compared with Hyphomicrobium methylovorum, MT was degraded more quickly in the Methylomonas koyamae culture. During the co-culture, the CH4 oxidation process of Methylomonas koyamae could provide carbon and energy sources for the growth of Hyphomicrobium methylovorum, while Hyphomicrobium methylovorum oxidized MT to help Methylomonas koyamae detoxify. These findings are helpful to understand the synergy effects of Methylomonas koyamae and Hyphomicrobium methylovorum under MT stress and enrich the role of methanotrophs in the sulfur biogeochemical cycle. KEY POINTS: • The co-culture of Methylomonas and Hyphomicrobium has better tolerance to CH3SH. • Methylomonas can provide carbon sources for the growth of Hyphomicrobium. • The co-culture of Methylomonas and Hyphomicrobium enhances the removal of CH4 and CH3SH.

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).

Crucial Role of the Chaperonin GroES/EL for Heterologous Production of the Soluble Methane Monooxygenase from Methylomonas methanica MC09.

Methane is a widespread energy source and can serve as an attractive C1 building block for a future bioeconomy. The soluble methane monooxygenase (sMMO) is able to break the strong C-H bond of methane and convert it to methanol. The high structural complexity, multiplex cofactors, and unfamiliar folding or maturation procedures of sMMO have hampered the heterologous production and thus biotechnological applications. Here, we demonstrate the heterologous production of active sMMO from the marine Methylomonas methanica MC09 in Escherichia coli by co-synthesizing the GroES/EL chaperonin. Iron determination, electron paramagnetic resonance spectroscopy, and native gel immunoblots revealed the incorporation of the non-heme diiron centre and homodimer formation of active sMMO. The production of recombinant sMMO will enable the expansion of the possibilities of detailed studies, allowing for a variety of novel biotechnological applications.

Catalytic and Spectroscopic Properties of the Halotolerant Soluble Methane Monooxygenase Reductase from Methylomonas methanica MC09.

The soluble methane monooxygenase receives electrons from NADH via its reductase MmoC for oxidation of methane, which is itself an attractive C1 building block for a future bioeconomy. Herein, we present biochemical and spectroscopic insights into the reductase from the marine methanotroph Methylomonas methanica MC09. The presence of a flavin adenine dinucleotide (FAD) and [2Fe2S] cluster as its prosthetic group were revealed by reconstitution experiments, iron determination and electron paramagnetic resonance spectroscopy. As a true halotolerant enzyme, MmoC still showed 50 % of its specific activity at 2 M NaCl. We show that MmoC produces only trace amounts of superoxide, but mainly hydrogen peroxide during uncoupled turnover reactions. The characterization of a highly active reductase is an important step for future biotechnological applications of a halotolerant sMMO.

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.

Selection of methanotrophic platform for methanol production using methane and biogas.

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.

Crystal structure of a novel homodimeric l-ribulose 3-epimerase from Methylomonus sp.

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.

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.

Cellulose nanocrystals coated with a tannic acid-Fe3+ complex as a significant medium for efficient CH4 microbial biotransformation.

Microbial biotransformation of CH4 gas has been attractive for the production of energy and high-value chemicals. However, insufficient supply of CH4 in a culture medium needs to be overcome for the efficient utilization of CH4. Here, we utilized cellulose nanocrystals coated with a tannic acid-Fe3+ complex (TA-Fe3+CNCs) as a medium component to enhance the gas-liquid mass-transfer performance. TA-Fe3+CNCs were well suspended in water without agglomeration, stabilized gas bubbles without coalescence, and increased the gas solubility by 20 % and the kLa value at a rapid inlet gas flow rate. Remarkably, the cell growth rate of Methylomonas sp. DH-1 as model CH4-utilizing bacteria improved with TA-Fe3+CNC concentration without any cytotoxic or antibacterial properties, resulting in higher metabolite production ability such as methanol, pyruvate, formate, and succinate. These results showed that TA-Fe3+CNCs could be utilized as a significant component in the culture medium applicable as a promising nanofluid for efficient CH4 microbial biotransformation.

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.

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.

Enhancement of nitrite reduction and enrichment of Methylomonas via conductive materials in a nitrite-dependent anaerobic methane oxidation system.

Nitrite-dependent anaerobic methane-oxidizing (n-damo) process has a promising prospect in anaerobic wastewater treatment, utilizing methane as the sole electron source to remove nitrite. However, the metabolic activity of n-damo bacteria is too low for practical application. This study aimed to stimulate n-damo process by introducing conductive nano-magnetite and/or electron shuttle anthraquinone-2,6-disulfonate (AQDS), and also set a comparative treatment of adding insulated ferrihydrite. The results showed that the nitrite reduction rate was enhanced the most significantly in treatment with nano-magnetite, approximately 1.6 times higher than that of the control without any supplement. While ferrihydrite application showed an adverse effect on n-damo process. The well-known aerobic methane oxidizer Methylomonas spp. was found to be enriched under n-damo condition with the supplementation of nano-magnetite and/or AQDS, but abundance of n-damo bacteria did not exhibit significant increase. It was hypothesized that Methylomonas spp. could be survived under anaerobic n-damo condition using oxygen produced by n-damo bacteria for the self-growth, and the nitrite reduction could be promoted through the enhancement of microbial interspecies electron transfer triggered by the introduction of conductive materials. It opens a new direction for the stimulation of n-damo activity, which needs more evidences to verify the hypothetic mechanism.

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).

Combined genome-centric metagenomics and stable isotope probing unveils the microbial pathways of aerobic methane oxidation coupled to denitrification process under hypoxic conditions.

Obligate aerobic methanotrophs have been proven to oxidize methane and participate in denitrification under hypoxic conditions. However, this phenomenon and its metabolic mechanism have not been investigated in detail in aerobic methane oxidation coupled to denitrification (AME-D) process. In this study, a type of hypoxic AME-D consortium was enriched and operated for a long time in a CH4-cycling bioreactor with strict anaerobic control and the nitrite removal rate reached approximately 50 mg N/L/d. Metagenomics combined with DNA stable-isotope probing demonstrated that the genus Methylomonas, which constitutes type I aerobic methanotrophs, was the dominant member and contributed to methane oxidation and partial denitrification. Metagenomic binning recovered a near-complete (98%) draft genome affiliated with the family Methylococcaceae containing essential genes that encode nitrite reductase (nirK), nitric oxide reductase (norBC) and hydroxylamine dehydrogenase (hao). Metabolic reconstruction of the selected Methylococcaceae genomes also revealed a potential link between methanotrophy and partial denitrification.

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.

Stable-isotopic analysis and high-throughput pyrosequencing reveal the coupling process and bacteria in microaerobic and hypoxic methane oxidation coupled to denitrification.

Microaerobic and hypoxic methane oxidation coupled to denitrification (MAME-D and HYME-D) occur in stabilized landfills with leachate recirculation when biological denitrification is limited by lack of organics. To evaluate nitrate denitrification efficiency and culture MAME-D/HYME-D involved bacteria, a leach bed bioreactor semi-continuous experiment was conducted for 60 days in 5 runs, under nitrate concentrations ranging of 20 mg/L-55 mg/L, wherein 5% sterile leachate was added during runs 4 and 5. Although the HYME-D system demonstrated high denitrification efficiency (74.93%) and nitrate removal rate reached 2.62 mmol N/(L⋅d), the MAME-D system exhibited a denitrification efficiency of almost 100% and nitrate removal rate of 4.37 mmol N/(L⋅d). The addition of sterile leachate increased the nitrate removal rate in both systems, but caused the decrease of methane consumption in HYME-D. A stable isotope batch experiment was carried out to investigate the metabolic products by monitoring the 13CO2 and 15N2O production. The production of organic intermediates such as citrate, lactic acid, acetate, and propionic acid were also observed, which exhibited a higher yield in HYME-D. Variations in the microbial communities were analyzed during the semi-continuous experiment. MAME-D was mainly conducted by the association of type Ⅰ methanotroph Methylomonas and the methylotrophic denitrifier Methylotenera. Methane fermentation processed by Methylomonas under hypoxic conditions produced more complex organic intermediates and increased the diversity of related heterotrophic denitrifiers. The addition of sterile real leachate, resulting in increase of COD/N, influenced the microbial community of HYME-D system significantly.

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.