Tobacco-associated with Methylophilus sp. FP-6 enhances phytoremediation of benzophenone-3 through regulating soil microbial community, increasing photosynthetic capacity and maintaining redox homeostasis of plant.

Benzophenone-3 (BP-3) has attracted widespread attention due to its large accumulation in the environment and its potential toxicity effects to human. This study aimed to investigate the effects of the combined application of tobacco and Methylophilus sp. strain FP-6 with both plant growth promoting (PGP) traits and BP-3 degradation function on BP-3 remediation in soil. The results showed that about 79.18% of BP-3 was removed from the soil after 30 days of plant culture inoculated with the FP-6 strain, which was significantly higher than the plant-alone treatment. Simultaneously, inoculation with strain FP-6 significantly improved growth performance, biomass production, antioxidant levels, osmoregulation substance, photosynthetic capacity and chlorophyll accumulation in tobacco. Moreover, the application of FP-6 shifted the bacterial community, and enhanced the abundance of BP-3-degrading or soil nutrient cycling-affecting bacteria (e.g., Chloroflexi, Bryobacter, MND1 and Myxococcales), which might be valuable for the promotion of plant growth and degradation of BP-3 in the soil. The results from this study gave first insights into the enhancement of BP-3 removal efficiency from soil by phytoremediation assisted with bacteria possessing both PGP properties and BP-3 degradation function. The role of soil bacterial community in this remediation process was also discussed.

Comparison of the Wild-Type Obligate Methylotrophic Bacterium Methylophilus quaylei and its Isogenic Streptomycin-Resistant Mutant via Metal Nanoparticle Generation.

Metal nanoparticles synthesized by green methods with the use of microorganisms are currently one of the most closely studied types of nanomaterials. It has accurately been shown that the characteristics of metal nanoparticles generated in the presence of different bacteria vary. For the two isogenic strains of obligate methylotrophic bacteria of the wild type (M. quaylei MTT) and its streptomycin-resistant mutant (M. quaylei SMR), the pleiotropic character of streptomycin resistance mutation in the SMR cells has been revealed. It has been shown that both cultures can generate silver nanoparticles. There is a dramatic difference in the formation of palladium nanoparticles, which are formed only in the presence of cells of the streptomycin-resistant mutant M. quaylei SMR. This study shows that closely related isogenic strains of obligate methylotrophic bacteria can be distinguished by the spectra of biogenic nanoparticles of two noble metals. While palladium nanoparticles are only generated by the cells of the streptomycin-resistant mutant M. quaylei SMR, biogenic silver nanoparticles can be generated from both cultures. Thus, the assessment of the ability of microorganisms to form biogenic nanoparticles of different metals allows the revelation of subtle metabolic differences of even close cultures.

Isolation and characterization of a novel benzophenone-3-degrading bacterium Methylophilus sp. strain FP-6.

Benzophenone-3 (BP-3) is extensively applied in sunscreens and some other related cosmetic products. It is necessary to efficiently and safely remove BP-3 from environments by application of various treatment technologies. However, to the authors' knowledge, BP-3 biodegradation by a single bacterial strain has not been reported before. In this study, a Gram-negative aerobic bacterium capable of degrading BP-3 as a sole carbon source was isolated from a municipal wastewater treatment plant and classified as Methylophilus sp. FP-6 according to BIOLOG GEN III and 16S rDNA analysis. Methanol was chosen for further experiments as a co-metabolic carbon source to enhance the microbial degradation efficiency of BP-3. Orthogonal and one-way experiments were all performed to investigate the optimal culture conditions for degradation of BP-3 by Methylophilus sp. FP-6. The degradation rate of BP-3 reached about 65% after 8 days of incubation with strain FP-6 under optimal culture conditions. The half-life (t1/2) of BP-3 biodegradation by strain FP-6 was estimated as 2.95 days according to the BP-3 degradation curve. The metabolite intermediates generated during the BP-3 degradation process were analyzed by LC-MS/MS and three metabolite products were identified. According to the analysis of metabolic intermediates, three pathways for degradation of BP-3 by strain FP-6 were proposed. The results from this study gave first insights into the potential of BP-3 biodegradation by a single bacterial strain.

The Draft Genome Sequence of Methylophilus sp. D22, Capable of Growing Under High Concentration of Methanol.

In this study, a wild type Methylophilus sp. strain D22 belonging to the family Methylophilus was isolated and characterized, which shows high tolerance towards methanol, as it can grow under 50 g/L of methanol. Methylophilus sp. strain D22 was isolated from the lake sludge in Nanjing Tech University, China. The assembled draft genome contains one circular chromosome with 3,004,398 bp, 49.7% of GC content, and 2107 predicted encoding proteins. Sequence-based genomic analysis demonstrates that the assimilation pathway of ribulose monophosphate (RuMP) pathway and dissimilation pathway of tetrahydromethanopterin (H4MPT) pathway are co-existing and contribute to the high methanol utilization efficiency.

Evidence for photolytic and microbial degradation processes in the dissipation of leptospermone, a natural ß-triketone herbicide.

Bioherbicides appear as an ecofriendly alternative to synthetic herbicides, generally used for weed management, because they are supposed to have low side on human health and ecosystems. In this context, our work aims to study abiotic (i.e., photolysis) and biotic (i.e,. biodegradation) processes involved in the fate of leptospermone, a natural ß-triketone herbicide, by combining chemical and microbiological approaches. Under controlled conditions, the photolysis of leptospermone was sensitive to pH. Leptospermone has a half-life of 72 h under simulated solar light irradiations. Several transformation products, including hydroxy-leptospermone, were identified. For the first time, a bacterial strain able to degrade leptospermone was isolated from an arable soil. Based on its 16S ribosomal RNA (rRNA) gene sequence, it was affiliated to the Methylophilus group and was accordingly named as Methylophilus sp. LS1. Interestingly, we report that the abundance of OTUs, similar to the 16S rRNA gene sequence of Methylophilus sp. LS1, was strongly increased in soil treated with leptospermone. The leptospermone was completely dissipated by this bacteria, with a half-life time of 6 days, allowing concomitantly its growth. Hydroxy-leptospermone was identified in the bacterial culture as a major transformation product, allowing us to propose a pathway of transformation of leptospermone including both abiotic and biotic processes.

Improved TNT detoxification by starch addition in a nitrogen-fixing Methylophilus-dominant aerobic microbial consortium.

In this study, a novel aerobic microbial consortium for the complete detoxification of 2,4,6-trinitrotoluene (TNT) was developed using starch as a slow-releasing carbon source under nitrogen-fixing conditions. Aerobic TNT biodegradation coupled with microbial growth was effectively stimulated by the co-addition of starch and TNT under nitrogen-fixing conditions. The addition of starch with TNT led to TNT mineralization via ring cleavage without accumulation of any toxic by-products, indicating improved TNT detoxification by the co-addition of starch and TNT. Pyrosequencing targeting the bacterial 16S rRNA gene suggested that Methylophilus and Pseudoxanthomonas population were significantly stimulated by the co-addition of starch and TNT and that the Methylophilus population became predominant in the consortium. Together with our previous study regarding starch-stimulated RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) degradation (Khan et al., J. Hazard. Mater. 287 (2015) 243-251), this work suggests that the co-addition of starch with a target explosive is an effective way to stimulate aerobic explosive degradation under nitrogen-fixing conditions for enhancing explosive detoxification.

[Phosphate-solubilizing activity of aerobic methylobacteria].

Phosphate-solubilizing activity was found in 14 strains of plant-associated aerobic methylobacteria belonging to the genera Methylophilus, Methylobacillus, Methylovorus, Methylopila, Methylobacterium, Delftia, and Ancyclobacter. The growth of methylobacteria on medium with methanol as the carbon and energy source and insoluble tricalcium phosphate as the phosphorus source was accompanied by a decrease in pH due to the accumulation of up to 7 mM formic acid as a methanol oxidation intermediate and by release of 120-280 µM phosphate ions, which can be used by both bacteria and plants. Phosphate-solubilizing activity is a newly revealed role of methylobacteria in phytosymbiosis.

Salt stress tolerance of methylotrophic bacteria Methylophilus sp. and Methylobacterium sp. isolated from coal mine spoils.

Two methylotrophic strains of Bina coalmine spoil BNV7b and BRV25 were identified based on physiological traits and 16S rDNA sequence as Methylophilus and Methylobacterium species.' The strains exhibited similar carbon utilization but differed in N utilization and their response to the metabolic inhibitors. Methylophilus sp. was less tolerant to salt stress and it viability declined to one tenth within 4 h of incubation in 2M NaCI due to membrane damage and leakage of the intracellular electrolytes as evident from malondiaaldehyde (MDA) assay. In 200 mM NaCI, they exhibited increased superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activity while in 500 mM NaCI, enzyme activities declined in Methylophilus sp. and increased in Methylobacterium sp. Among exogenously applied osmoprotectants proline was most efficient; however, polyols (mannitol, sorbitol and glycerol) also supported growth under lethal NaCI concentration.

[Major results of research and development of heterogenous biocatalysts for regenerated water clearing from harmful admixture].

The biological method of clearing atmospheric condensate in pressurized habitats exploits filters with a heterogenic biocatalyst produced by way of immobilizing harmless for human, animal and plant microoganisms on water-insoluble solid carrier--foam polyvinyl-formal (FPVF), and a hydrogen peroxide biofilter containing triacetate cellulose-immobilized catalase. Experience of forming an immobilized bacterial association as a polyenzyme system is particularly promising for development of advanced biotechnologies. Biocatalysts with expanded applicability can be manufactured using a FPVF-immobilized associative bacterial culture composed of Paracoccus denitrificans, Pseudomonas esterophilus and Methilopila capsulata. In aerobic condition at room temperature the heterogenic biocatalyst is capable to transform harmful organics in atmospheric condensate, e.g. methyl amine, ethyl acetate, acetic acid, ethanol and acetone into the end-products, i.e. carbon dioxide and water. Ammonia is consumed by 3 cultures as a source of nitrogen.

[Effect of exogenous fatty acids on the growth and production of exopolysaccharides of obligately methylotrophic bacterium Methylophilus quaylei].

Accelerating growth and increasing exopolysaccharide production in obligate methylotrophic bacterium Methylophilus quaylei were observed in the presence of C12-C18 fatty acids added to the growth media. Sodium oleate was the best growth factor. Based on data on the composition of the free fatty acids fraction in the cells and the values of the zeta-potential and fluorescence anisotropy of whole cells, we suggested that fatty acids were incorporated in the outer membrane of M. quaylei.

Changes in bacterial community of anthracene bioremediation in municipal solid waste composting soil.

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in a municipal solid waste (MSW) composting site. Knowledge of changes in microbial structure is useful to identify particular PAH degraders. However, the microbial community in the MSW composting soil and its change associated with prolonged exposure to PAHs and subsequent biodegradation remain largely unknown. In this study, anthracene was selected as a model compound. The bacterial community structure was investigated using terminal restriction fragment length polymorphism (TRFLP) and 16S rRNA gene clone library analysis. The two bimolecular tools revealed a large shift of bacterial community structure after anthracene amendment and subsequent biodegradation. Genera Methylophilus, Mesorhizobium, and Terrimonas had potential links to anthracene biodegradation, suggesting a consortium playing an active role.

[Stearic acid methyl ether: a new extracellular metabolite of the obligate methylotrophic bacterium Methylophilus quaylei].

Methyl esters of fatty acids, free fatty acids, and hydrocarbons were found in the culture liquid and in the cellular lipids of the obligate methylotrophic bacterium Methylophilus quaylei under optimal growth conditions and osmotic stress. The main extracellular hydrophobic metabolite was methyl stearate. Exogenous free fatty acids C16-C18 and their methyl esters stimulated the M. quaylei growth and survivability, as well as production of exopolysaccharide under osmotic and oxidative stress, playing the role of growth factors and adaptogens. The order of hydrophobic supplements according to the ability to stimulate bacterial growth is C18 : 1 > C18 : 0 > C16 : 0 > methyl oleate > methyl stearate > no supplements > C14: 0 > C12 : 0. The mechanism underlying the protective action of fatty acids and their methyl esters is discussed.

Methylophilus flavus sp. nov. and Methylophilus luteus sp. nov., aerobic, methylotrophic bacteria associated with plants.

Novel yellow, obligately methylotrophic and restricted facultatively methylotrophic bacteria, respectively designated strains Ship(T) and Mim(T), with the ribulose monophosphate pathway of C(1) assimilation are described. Cells were strictly aerobic, Gram-negative, asporogenous, non-motile rods that multiply by binary fission, were mesophilic and neutrophilic and synthesized indole-3-acetic acid and exopolysaccharide. The predominant cellular fatty acids were C(16 : 0) and C(16 : 1). The major ubiquinone was Q-8. The predominant phospholipids were phosphatidylethanolamine and phosphatidylglycerol; diphosphatidylglycerol was absent. The two strains lacked α-ketoglutarate dehydrogenase and glutamate dehydrogenase. They assimilated ammonium via the glutamate cycle enzymes glutamine synthetase and glutamate synthase. The DNA G+C contents of strains Ship(T) and Mim(T) were 50.7 and 54.5 mol% (T(m)), respectively. The level of 16S rRNA gene sequence similarity between these strains was very high (99.8 %) but they shared a low level of DNA-DNA relatedness (44 %). Based on 16S rRNA gene sequence analysis and low levels of DNA-DNA relatedness with the type strains of recognized species of the genus Methylophilus (31-36 %), strains Ship(T) and Mim(T) are considered to represent novel species of the genus Methylophilus, for which the names Methylophilus flavus sp. nov. (type strain Ship(T) =DSM 23073(T) =VKM B-2547(T) =CCUG 58411(T)) and Methylophilus luteus sp. nov. (type strain Mim(T) =DSM 22949(T) =VKM B-2548(T) =CCUG 58412(T)) are proposed.

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.

[Deuterium oxide as a stress factor to the methylotrophic bacterium Methylophilus sp]. / Oksid deiteriia kak stressovyi faktor u metilotrofnykh bakterii Methylophilus sp.

The adaptation of the methylotrophic bacterium Methylophilus sp. B-7741 to growth in highly deuterated media was studied. For the first time, we showed the cross adaptation of bacterial cells to deuterated media and oxidative and osmotic stresses. The activity at catalase in deuterated cells was higher than in the control cells. Deuterated cell-free culture liquids showed protective effects on the growth of Methylophilus sp. B-7741 in deuterated media, which was manifested as an increase in the deuterated biomass yield. These data and the data available in the literature suggest that the mechanisms of bacterial cell adaptation to heavy water and to oxidative and osmotic stresses are similar.

[Effect of deuteration on methanol dehydrogenase activity in Methylophilus sp. B-7741]. / Vliianie deiterirovaniia na aktivnost' metanoldegidrogenazy Methylophilus sp. B-7741.

We studied the effect of deuterium oxide present in the medium on the activity of methanol dehydrogenase (EC 1.1.99.8) from methylotrophic bacteria Methylophilus sp. B-7741. Methanol dehydrogenase activity in extracts of the biomass obtained in a highly deuterated medium (2H-enzyme) was 34-47% of enzyme activity in the control biomass, which depended on reaction conditions. The isotopic effects of substrate deuterium (methanol) for 1H-enzyme and 2H-enzyme were 1.37 +/- 0.05 and 1.38 +/- 0.01, respectively. We revealed for the first time the reverse isotopic effect of solvent deuterium in the reaction catalyzed by methanol dehydrogenase (0.80 +/- 0.02 and 0.60 +/- 0.01 for 1H-enzyme and 2H-enzyme, respectively).

[Phylogenetic analysis of aerobic methylotrophic bacteria, using dichloromethane]. / Filogeneticheskii analiz aérobnykh metilotrofnykh bakterii, ispol'zuiushchikh dikhlormetan.

The phylogenetic relationships of 12 aerobic dichloromethane-degrading bacteria that implement different C1-assimilation pathways was determined based on 16S ribosomal RNA sequences and DNA-DNA hybridization data. The restricted facultative methylotroph "Methylophilus leisingerii" DM11 with the ribulose monophosphate pathway was found to belong to the genus Methylophilus cluster of the beta subdivision of the phylogenetic kingdom Proteobacteria. The facultative methylotroph Methylorhabdus multivorans DM13 was assigned to a separate branch of the alpha-2 group of Proteobacteria. Paracoccus methylutens DM12, which utilizes C1-compounds via the Calvin cycle was found to belong to the alpha-3 group of the Proteobacteria (more precisely, to the genus Paracoccus cluster). Thus, phylogenetic analysis confirmed the taxonomic status of these recently characterized bacteria. According to the degree of DNA homology, several novel strains of methylotrophic bacteria were divided into three genotypic groups within the alpha-2 group of the Proteobacteria. Genotypic group 1, comprising strains DM1, DM3, and DM5 through DM9, and genotypic group 3, comprising strain DM10, were phylogenetically close to the methylotrophic bacteria of the genus Methylopila, whereas genotypic group 2 (strain DM4) was close to bacteria of the genus Methylobacterium. The genotypic groups obviously represent distinct taxa of methylotrophic bacteria, whose status should be confirmed by phenotypic analysis.