Methylotrophic Bacterial Strains Research Articles

Metal uptake potential of four methylotrophic bacterial strains from coal mine spoil, exploring a new possible agent for bioremediation

Abstract The abundance of total metal tolerant culturable bacteria and methylotrophic bacteria was numerated in the barren mine spoil. Metal resistant methylotrophic bacteria were isolated and identified through biochemical testing and 16SrRNA gene sequence analysis. The metal uptake potential of the isolates was determined. The size of the methylotrophic bacteria was significantly low (104) compared to total bacterial population (108). The methylotrophic population was less tolerant to the tested metals viz., Cd, Cu, Cr, Ni and Zn. The metal resistant isolates showed close similarity with Pseudomonas, Methylophilus and Methylobacterium sp. Lower concentration (5–10 μ M) of al the tested metals was growth stimulatory. MIC values were lowest for Cd and highest for Zn. At the same time, MIC value was highest for Pseudomonas strain and lowest for Methylobacterium. Highest metal uptake was observed for Cd and lowest for Cr. Metal uptake potential varied with strain as well as metal species. There was no relation between the intracellular metal accumulation and metal adsorbed to the bacterial cell wall. Lowest adsorption capacity was exhibited by the Pseudomonas strain. Highest adsorption was for Cu and lowest for Ni. Metal uptake was rapid during first 4 h, and then after rate of uptake declined. It suggests that methylotrophic bacteria can be used as potential accumulator of Cd.

Methylobacterium frigidaeris sp. nov., isolated from an air conditioning system.

A reddish pink-pigmented, Gram-stain-negative, aerobic and methylotrophic bacterial strain, designated strain IER25-16T, was isolated from a laboratory air conditioning system in the Republic of Korea. Cells were motile rods showing catalase- and oxidase-positive reactions. Strain IER25-16T grew at 10-40 °C (optimum, 30 °C), at pH 4.0-7.0 (optimum, pH 5.0-7.0) and in the presence of 0-1.0 % (w/v) NaCl (optimum, 0 %). The major respiratory quinone was ubiquinone-10 and ubiquinone-9 was also detected as the minor respiratory quinone. Summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c) was detected as the predominant fatty acids. The genomic DNA G+C content of strain IER25-16T was 70.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain IER25-16T belonged to the genus Methylobacterium of the class Alphaproteobacteria. Strain IER25-16T was most closely related to Methylobacterium platani PMB02T (97.9 %), Methylobacterium aquaticum GR16T (97.9 %) and Methylobacterium tarhaniae N4211T (97.5 %). The average nucleotide identity and in silico DNA-DNA hybridization values between strain IER25-16T and M. platani, M. aquaticum and M. tarhaniae were 88.3, 88.8 and 89.6 % and 36.2, 37.3 and 39.3 %, respectively. The phenotypic and chemotaxonomic features and the phylogenetic inference clearly suggested that strain IER25-16T represents a novel species of the genus Methylobacterium, for which the name Methylobacteriumfrigidaeris sp. nov. is proposed. The type strain is strain IER25-16T (=KACC 19280T=JCM 32048T).

Metagenomic survey of methanesulfonic acid (MSA) catabolic genes in an Atlantic Ocean surface water sample and in a partial enrichment.

Methanesulfonic acid (MSA) is a relevant intermediate of the biogeochemical cycle of sulfur and environmental microorganisms assume an important role in the mineralization of this compound. Several methylotrophic bacterial strains able to grow on MSA have been isolated from soil or marine water and two conserved operons, msmABCD coding for MSA monooxygenase and msmEFGH coding for a transport system, have been repeatedly encountered in most of these strains. Homologous sequences have also been amplified directly from the environment or observed in marine metagenomic data, but these showed a base composition (G + C content) very different from their counterparts from cultivated bacteria. The aim of this study was to understand which microorganisms within the coastal surface oceanic microflora responded to MSA as a nutrient and how the community evolved in the early phases of an enrichment by means of metagenome and gene-targeted amplicon sequencing. From the phylogenetic point of view, the community shifted significantly with the disappearance of all signals related to the Archaea, the Pelagibacteraceae and phylum SAR406, and the increase in methylotroph-harboring taxa, accompanied by other groups so far not known to comprise methylotrophs such as the Hyphomonadaceae. At the functional level, the abundance of several genes related to sulfur metabolism and methylotrophy increased during the enrichment and the allelic distribution of gene msmA diagnostic for MSA monooxygenase altered considerably. Even more dramatic was the disappearance of MSA import-related gene msmE, which suggests that alternative transporters must be present in the enriched community and illustrate the inadequacy of msmE as an ecofunctional marker for MSA degradation at sea.

Hydrogen and carbon isotope fractionation during degradation of chloromethane by methylotrophic bacteria

Chloromethane (CH3Cl) is a widely studied volatile halocarbon involved in the destruction of ozone in the stratosphere. Nevertheless, its global budget still remains debated. Stable isotope analysis is a powerful tool to constrain fluxes of chloromethane between various environmental compartments which involve a multiplicity of sources and sinks, and both biotic and abiotic processes. In this study, we measured hydrogen and carbon isotope fractionation of the remaining untransformed chloromethane following its degradation by methylotrophic bacterial strains Methylobacterium extorquens CM4 and Hyphomicrobium sp. MC1, which belong to different genera but both use the cmu pathway, the only pathway for bacterial degradation of chloromethane characterized so far. Hydrogen isotope fractionation for degradation of chloromethane was determined for the first time, and yielded enrichment factors (ε) of −29‰ and −27‰ for strains CM4 and MC1, respectively. In agreement with previous studies, enrichment in 13C of untransformed CH3Cl was also observed, and similar isotope enrichment factors (ε) of −41‰ and −38‰ were obtained for degradation of chloromethane by strains CM4 and MC1, respectively. These combined hydrogen and carbon isotopic data for bacterial degradation of chloromethane will contribute to refine models of the global atmospheric budget of chloromethane.

Methylobacterium bullatum sp. nov., a methylotrophic bacterium isolated from Funaria hygrometrica

A novel, pink-pigmented aerobic, facultatively methylotrophic bacterial strain (F3.2 T) isolated from the phyllosphere of Funaria hygrometrica, was analyzed using a polyphasic approach. Cells were Gram-negative, motile rods, strictly aerobic and non-spore-forming and exhibited surface structures varying in quantity, distribution and morphology. The isolate grew at 10–33 °C over a pH range of 5.5–8.0 and in the presence of less than 1.0% NaCl. Strain F3.2 T shared less than 70% DNA–DNA binding to the next type strain of the genus Methylobacterium ( M. adhaesivum DSM 17169 T). In addition to the major cellular fatty acid C 18:1 ω7c (81.7%), present in all Methylobacterium species (and also members of the genus Alphaproteobacteria), a high value (11.7%) of the fatty acids (summed feature) C 16:1 ω7c and/or iso-C 15:02OH was determined. Phylogenetic analyses based on 16S rDNA and methanol dehydrogenase gene sequences, DNA–DNA hybridization values, chemotaxonomic and phenotypic characteristics indicate that the strain F3.2 T represents a novel species within the genus Methylobacterium. We propose the name Methylobacterium bullatum sp. nov. for this species. The type strain is the strain F3.2 T (DSM 21893 T = LMG 24788 T).

Methylophilus rhizosphaerae sp. nov., a restricted facultative methylotroph isolated from rice rhizosphere soil

Three facultative methylotrophic bacterial strains, designated CBMB127(T), CBMB145 and CBMB147, were isolated from the rhizosphere soil of rice and characterized. The strains produced indole-3-acetic acid and siderophores, had 1-aminocyclopropane-1-carboxylate deaminase activity and sulfur oxidation property and also methanol dehydrogenase. Phylogenetic analysis based on the 16S rRNA and methanol dehydrogenase (mxaF) gene sequences showed that Methylophilus methylotrophus was their close relative. The results of the phenotypic, phylogenetic and genotypic analyses showed that strains CBMB127(T) and CBMB145, with 99.4 % 16S rRNA gene sequence similarity and 99 % DNA-DNA hybridization, could be distinguished from recognized species of Methylophilus. Therefore strain CBMB127(T) and CBMB145 are considered to represent a novel species of Methylophilus, for which the name Methylophilus rhizosphaerae sp. nov. is proposed, with CBMB127(T) (=KACC 13099(T)=NCCB 100233(T)) as the type strain. Strain CBMB147 represents a novel strain of the species Methylophilus methylotrophus.

Methylobacterium phyllosphaerae sp. nov., a pink-pigmented, facultative methylotroph from the phyllosphere of rice

A pink-pigmented, aerobic, facultatively methylotrophic bacterial strain, CBMB27T, isolated from leaf tissues of rice (Oryza sativa L. 'Dong-Jin'), was analysed using a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Methylobacterium oryzae, Methylobacterium fujisawaense and Methylobacterium mesophilicum; strain CBMB27T showed sequence similarities of 98.3, 98.5 and 97.3 %, respectively, to the type strains of these three species. DNA-DNA hybridization experiments revealed low levels (<38 %) of DNA-DNA relatedness between strain CBMB27T and its closest relatives. The sequence of the 1-aminocyclopropane-1-carboxylate deaminase gene (acdS) in strain CBMB27T differed from those of close relatives. The major fatty acid of the isolate was C(18 : 1)omega7c and the G+C content of the genomic DNA was 66.8 mol%. Based on the results of 16S rRNA gene sequence analysis, DNA-DNA hybridization, and physiological and biochemical characterization, which enabled the isolate to be differentiated from all recognized species of the genus Methylobacterium, it was concluded that strain CBMB27T represents a novel species in the genus Methylobacterium for which the name Methylobacterium phyllosphaerae sp. nov. is proposed (type strain CBMB27T =LMG 24361T =KACC 11716T =DSM 19779T).

Novel pollutant-resistant methylotrophic bacteria for use in bioremediation

Thirty one novel methylotrophic bacterial strains were isolated from a range of soil and sediment sources (both pristine and polluted) under different enrichment regimes. They were characterised physiologically and classified by their 16S rRNA gene sequence. A great taxonomical and phenotypical variety was recovered. Some of the isolates display interesting features of resistance to heavy metals, arsenate or organic pollution and four can be considered real ‘super-bugs’ for their ability to withstand extremely high concentrations of a variety of pollutants. A description of the 31 strains is presented in this work.

Novel pollutant-resistant methylotrophic bacteria for use in bioremediation

Thirty one novel methylotrophic bacterial strains were isolated from a range of soil and sediment sources (both pristine and polluted) under different enrichment regimes. They were characterised physiologically and classified by their 16S rRNA gene sequence. A great taxonomical and phenotypical variety was recovered. Some of the isolates display interesting features of resistance to heavy metals, arsenate or organic pollution and four can be considered real `super-bugs' for their ability to withstand extremely high concentrations of a variety of pollutants. A description of the 31 strains is presented in this work.

Carbon isotope fractionation of methyl bromide during agricultural soil fumigations

The isotopic composition ofmethyl bromide (CH3Br) has been suggestedto be a potentially useful tracer forconstraining the global CH3Br budget. Inorder to determine the carbon isotopiccomposition of CH3Br emitted from the mostsignificant anthropogenic application(pre-plant fumigation) we directly measured theδ13C of CH3Br released duringcommercial fumigation. We also measured theisotopic fractionation associated withdegradation in agricultural soil under typicalfield fumigation conditions. The isotopiccomposition of CH3Br collected in soilseveral hours after injection of the fumigantwas −44.5‰ and this value increased to −20.7‰over the following three days. The mean kineticisotope effect (KIE) associated withdegradation of CH3Br in agricultural soil(12‰) was smaller than the reported value formethylotrophic bacterial strain IMB-1, isolatedfrom previously fumigated agricultural soil,but was similar to methylotrophic bacterialstrain CC495, isolated from a pristine forestlitter zone. Using this fractionationassociated with the degradation of CH3Brin agricultural soil and the meanδ13C of the industriallymanufactured CH3Br (−54.4‰), we calculatethat the agricultural soil fumigation sourcehas a carbon isotope signature that ranges from−52.8‰ to −42.0‰. Roughly 65% ofindustrially manufactured CH3Br is usedfor field fumigations. The remaining 35% isused for structural and post-harvestfumigations with a minor amount used duringindustrial chemical manufacturing. Assumingthat the structural and post-harvest fumigationsources of CH3Br are emitted withoutsubstantial fractionation, we calculate thatthe δ13C of anthropogenicallyemitted CH3Br ranges from −53.2‰ to −47.5‰.

Isolation and characterization of methanesulfonic Acid-degrading bacteria from the marine environment

Two methylotrophic bacterial strains, TR3 and PSCH4, capable of growth on methanesulfonic acid as the sole carbon source were isolated from the marine environment. Methanesulfonic acid metabolism in these strains was initiated by an inducible NADH-dependent monooxygenase, which cleaved methanesulfonic acid into formaldehyde and sulfite. The presence of hydroxypyruvate reductase and the absence of ribulose monophosphate-dependent hexulose monophosphate synthase indicated the presence of the serine pathway for formaldehyde assimilation. Cell suspensions of bacteria grown on methanesulfonic acid completely oxidized methanesulfonic acid to carbon dioxide and sulfite with a methanesulfonic acid/oxygen stoichiometry of 1.0:2.0. Oxygen electrode-substrate studies indicated the dissimilation of formaldehyde to formate and carbon dioxide for energy generation. Carbon dioxide was not fixed by ribulose bisphosphate carboxylase. It was shown that methanol is not an intermediate in methanesulfonic acid metabolism, although these strains grew on methanol and other one-carbon compounds, as well as a variety of heterotrophic carbon sources. These two novel marine facultative methylotrophs have the ability to mineralize methanesulfonic acid and may play a role in the cycling of global organic sulfur.

Isolation and characterization of four methylotrophic bacterial strains

AbstractFour pure methylotrophic bacterial strains isolated from Kuwaiti soil were designated KISRI‐5, KISRI‐6.1, KISRI‐512 and KISRI‐5112, and deposited in NCIMB under NCIB numbers 12135, 12136, 12137 and 12138, respectively. Characterization tests showed that they grew well on methanol and slowly on glucose, at temperatures up to 44 °C and pH 7.0. However, they could not grow on numerous other organic carbon sources. The four strains were very close taxonomically, with minor differences in colony characteristics, lipids pattern, mole G + C% and resistance to certain antibiotics. All strains utilized monomethylamine, but not methane or trimethylamine; all contained ubiquinones with eight isoprene units (Q8), and had C16 as their major fatty acid component. Their characteristics indicate that they are different strains of restricted type M facultative methylotrophs, and could be considered new members of the genus Methylophilus.

Selection of Methylotrophic Microorganisms for the Formation of Single Cell Protein

Methanol-assimilating microorganisms were isolated from 27 soil samples. Thirty-one (26 bacteria and 5 yeasts) out of 385 isolates were found to be methylotrophs. Fungal and actinomycetes isolates did not grow on methanol as a sole carbon source. The growth parameters of yeast and bacterial isolates in shake flasks cultures widely varied from one isolate to another. The methylotrophic bacterial strains 148-16, 118-11,018-01 and 108-11 showed the highest specific growth rates, being 0.083,0.059,0.058 and 0.057 h-1 respectively. Pichia sp. 2y-17 gave a short exponential phase as compared with bacterial isolates. The methylotrophic bacterial strain 148-16 gave the highest biomass and protein productivity (113.30 and 73.88 mg/100 ml culture respectively). Effective yield of tested yeast was found to be of a low value. Microbial growth was highly affected by the high concent- ration of formaldehyde.

Copper requirement and its influence on the growth of the obligate methylotrophic bacterial strain 4025

Requirement of copper for growth of the obligate methanol-utilizing bacterium, strain 4025 was investigated. Strong demand for copper compounds was determined in the surface growth on methanol and methylamine. A pronounced requirement for copper and iron was evident during submerged growth of the strain on methanol; both elements affected growth and methanol utilization of the cultures. The optimal concentration of several copper compounds was 1.2 μmoles per liter. The copper effect was more pronounced with ammonium than with nitrate as the nitrogen source.