Biodegradation of Petroleum Sludge by Methylobacterium sp. Strain ZASH.

A bacterium was isolated from sludge-contaminated soil in a petroleum refinery and tested for its ability to degrade aliphatic hydrocarbon compounds present in petroleum sludge. The isolate was grown on minimal salt media agar supplemented with 1% (w/v) petroleum sludge. The isolate was tentatively identified as Methylobacterium s p. s t rain ZASH based on the partial 16s rDNA molecular phylogeny. The bacterium grew optimally between the temperatures of 30°C and 35°C, pH 7 and 7.5, 0.5% and 1.5% (v/v) Tween 80 as the surfactant, and between 1% and 2% (w/v) peptone as the nitrogen source. The constants derived from the Haldane equation were µmax = 0.039 hr-1, Ks = 0.385% (w/v) total petroleum hydrocarbons (TPH) or 3,850 mg/L TPH, and Ki =1.12% (w/v) TPH or 11,200 mg/L. The maximum biodegradation rate exhibited by this strain was 19 mg/L/hr at an initial TPH concentration of 10,000 mg/L. Gas chromatography analysis revealed that after 15 days the strain was able to degrade all aliphatic n-alkanes investigated with different efficiencies. Shorter n-alkanes were generally degraded more rapidly than longer n-alkanes with 90% removal for C-12 compared to only 30% removal for C-36. The addition of sawdust did not improve bacterial degradation of petroleum hydrocarbons, but it assisted in the removal of remaining undegraded hydrocarbons through adsorption.

Genomic analysis, simultaneous production, and process optimization of extracellular polymeric substances and polyhydroxyalkanoates by Methylobacterium sp. ISTM1 by utilizing molasses.

In the current study, the isolated Methylobacterium sp. ISTM1 simultaneously produced both extracellular polymeric substances (EPS) and polyhydroxyalkanoates (PHA) in a single-step process. The yield of biopolymers (EPS and PHA) was enhanced by optimizing the process parameters of EPS and PHA production. Methylobacterium sp. ISTM1 was able to produce 7.18 ± 0.04 g L-1 EPS and 1.41 ± 0.04 g L-1 PHA simultaneously at optimized culture conditions i.e., 9% molasses and pH 7. The genomic analysis of the strain has identified the involved genes and pathways in the production of EPS and PHA. Both the biopolymers were found non-toxic according to the cytotoxicity analysis. The results of the current study present the potential of the bacterium Methylobacterium sp. ISTM1 produces non-toxic biopolymers by utilizing agro-industrial waste (molasses) that can be harnessed sustainably for various applications.

Effect of Treating Acid Sulfate Soils with Phosphate Solubilizing Bacteria on Germination and Growth of Tomato (Lycopersicon esculentum L.).

Acid sulfate soils contain sulfide minerals that have adverse environmental effects because they can lead to acidic drainage and prevent the establishment of vegetation. The current study examined the effect of a novel method for the restoration of these soils and the promotion of germination and plant growth. Thus, we isolated two strains of phosphate solubilizing bacteria, Methylobacterium sp. PS and Caballeronia sp. EK, characterized their properties, and examined their effects in promoting the growth of tomato plants (Lycopersicon esculentum L.) in acid sulfate soil. Compared with untreated control soil, treatment of acid sulfate soils with these bacterial strains led to increased seed germination, growth of plants with more leaves, and plants with greater levels of total-adenosine tri-phosphate (tATP). Relative to the untreated control soil, the addition of Caballeronia sp. EK led to a 60% increase in seed germination after 52 days, growth of plants with more than 3 times as many leaves, and a 45.2% increase in tATP after 50 days. This strain has potential for use as a plant biofertilizer that promotes vegetation growth in acid sulfate soils by improving the absorption of phosphorous.

Purification and characterization of formaldehyde dismutases of Methylobacterium sp. FD1.

In the present study, we purified and characterized three formaldehyde dismutases (Fdms) (EC 1.2.98.1) (Fdm1, Fdm2, and Fdm3) of Methylobacterium sp. FD1. These Fdms (with His-tag) were produced in the recombinant E. coli and purified by immobilized metal affinity chromatography from the E. coli extracts. In each of the three Fdms, the enzyme-bound coenzyme was nicotinamide adenine dinucleotide (NAD(H)) and the enzyme-bound metal was zinc. The quaternary structures of these Fdms were estimated as homotetrameric. The optimal pHs and temperatures of Fdm1, Fdm2, and Fdm3 were approximately 6.5, 6.0, and 6.0, and 35°C, 25°C, and 30°C, respectively. The Km values of Fdm1, Fdm2, and Fdm3 were 621, 865, and 414 mM, respectively. These results were similar to the properties of already-known Fdms. However, each of the Fdms of FD1 had methanol:p-nitroso-N,N-dimethylaniline oxidoreductase activity that is not found in already-known Fdms.

Two-stage fermentation optimization for poly-3-hydroxybutyrate production from methanol by a new Methylobacterium isolate from oil fields.

AIMS: We aimed to explore a new Methylobacterium isolate to produce polyhydroxybutyrate (PHB) by using methanol as a sole carbon resource and improve PHB production. METHODS AND RESULTS: A new PHB-producing isolate (Methylobacterium sp. 1805) was obtained from oil fields by using methanol as a sole carbon source. The fermentation situation of PHB production was further optimized by using Box-Behnken response surface methodology (RSM). Before optimization, the cell biomass was 0·6 g l-1 after 3-day culture and 0·3 g l-1 PHB was produced after 5-day methanol-inducing stage. The RSM growth medium was optimized as 15 g l-1 glycerol, 10 g l-1 beef extract and 0·65 g l-1 MgSO4 ·7H2 O. The RSM methanol-inducing medium was optimized as 0·65 g l-1 MgSO4 (metal ions), 20 mmol l-1 PBS pH 6·5 and final 2% methanol (v/v). The biomass and PHB production reached 1·0 and 0·55 g l-1 after 3-day culture, respectively. The PHB yield increased by about 80% when compared with before optimization. CONCLUSIONS: The optimization of a two-stage fermentation process improved PHB production from methanol by using Methylobacterium sp. 1805. SIGNIFICANCE AND IMPACT OF THE STUDY: A new Methylobacterium isolate was isolated and produced high-level PHB by using methanol as a sole carbon resource. The bacteria will provide a potential tool for C1 resource in producing PHB.

Gaseous Formaldehyde Degrading by Methylobacterium sp. XJLW.

Formaldehyde is harmful to human beings. It is widely used in chemical industry, medicine, and agriculture and is frequently discharged into the sewage. Microbial metabolism of formaldehyde has attracted increasing attention for its potential application in formaldehyde removal, especially for indoor gaseous formaldehyde degradation. Methylobacterium sp. XJLW capable of degrading formaldehyde was isolated and exhibited a strong activity for liquid formaldehyde degradation. In the present study, the survival rate of XJLW was evaluated under drought, 30 °C, 4 °C, 15 °C, 35 °C, and 40 °C. After 4 days, the average survival rate under 30°C is the greatest (83.97%) among the five temperatures. Whether the temperature was above or below 30°C, the average survival rate decreased significantly. However, the resistance of XJLW to reduced temperatures seemed better than that to increased temperatures. The average survival rate under 15°C and 4°C was 71.1% and 58.67%, while that under 35 °C and 40 °C was 49.47% and 0.1%. Two batches of gaseous formaldehyde treatments were carried out in an analog device with super absorbent polymer (SAP) as the carrier materials of XJLW. The results showed that XJLW could effectively degrade gaseous formaldehyde in the analog device for a long period.

Microbial production of uracil by an isolated Methylobacterium sp. WJ4 using methanol.

In this study, we report the production of uracil from methanol by an isolated methylotrophic bacterium, Methylobacterium sp. WJ4. The use of methanol as alternative carbon feedstock is attractive option in biotechnology. As a feedstock of biotechnological processes, methanol has distinct advantages over methane. This is not only due to physical and chemical considerations, but also to the properties of the pertinent organisms. Besides, with a wide array of biological activities and synthetic accessibility, uracil is considered as privileged structures in drug discovery. Uracil analogues have been applied to treatments of patients with cancer or viral infections. In this respect, it is meaningful to produce uracil using methanol. The effect of process parameters and methanol concentration for uracil production were investigated and optimized. Uracil production was remarkably increased to 5.76mgg cell dry weight-1 in optimized condition. The results were significant for further understanding of methylotrophic bacteria on uracil production.

Biodegradation of high concentrations of formaldehyde using Escherichia coli expressing the formaldehyde dismutase gene of Methylobacterium sp. FD1.

In the present study, formaldehyde dismutase from Methylobacterium sp. FD1 was partially purified and analyzed by nanoLC-MS/MS; it was then cloned from the genomic DNA of FD1 by PCR. The open reading frame of the formaldehyde dismutase gene of FD1 was estimated to be 1203 bp in length. The molecular weight and pI of formaldehyde dismutase (401 aa), as deduced from the FD1 gene, were calculated at 42,877.32 and 6.56, respectively. NAD(H)-binding residues and zinc-binding residues were found in the amino acid sequence of the deduced formaldehyde dismutase of FD1 by BLAST search. The resting Escherichia coli cells that were transformed with the FD1 formaldehyde dismutase gene degraded high concentrations of formaldehyde and produced formic acid and methanol that were molar equivalents of one-half of the degraded formaldehyde. The lyophilized cells of the recombinant E. coli also degraded high concentrations of formaldehyde.

Screening and the degradation conditions of DBP-degrading bacterium / 预防医学

Objective To identify a strain screen which utilize dibutyl phthalate (DBP) as the sole carbon source and to explore the optimal conditions for the degradation of DBP. Methods The solid leachate was inoculated in minimal salt medium (MSM) supplemented with DBP as the sole carbon and energy source to isolate the targeted strain. The strain was identified through colony phenotype, transmission electron microscope and 16SrDNA gene sequence analysis. The targeted strain was inoculated to the above medium with different pH and temperature. The optimal temperature and pH of the microbial degradation of DBP were studied with determination of the DBP residue and bacterial biomass. Results One bacterial strain named L6 was isolated from the solid with adding DBP as the sole carbon source. Based on its morphology, physiochemical characteristics, and 16SrDNA sequence, the strain was identified as Methylobacterium sp. The optimal pH and temperature for its biodegradation activities were 7 and 30℃, respectively. The targeted strain could degrade 85% of 800 mg/L DBP within 120 hours. Conclusion Based on the high removal rate, the isolated Methylobacterium sp. L6 has a potential for bioremediation technology of DBP pollution.

Cytotoxic (A549) and antimicrobial effects of Methylobacterium sp. isolate (ERI-135) from Nilgiris forest soil, India.

OBJECTIVE: To assess the antimicrobial and cytotoxic effects of Methylobacterium sp. isolated from soil sample of Doddabetta forest, Nilgiris, Western Ghats of Tamil Nadu. METHODS: Isolation of Methylobacterium was performed from soils by serial dilution plate technique. The strain was grown in modified nutrient gulucose agar (MNGA) medium to study the morphology and biochemical characteristics. Methylobacterium sp. was screened for its antimicrobial activity against pathogenic bacteria and fungi. The strain was subjected to 16S rRNA analysis and was identified as Methylobacterium sp. The nucleotide sequence of the 16S rRNA gene of the isolate exhibited close similarity with other Methylobacterium sp. and has been submitted to Genbank. The antibacterial substances were extracted using chloroform and ethyl acetate from MNGA medium in which ERI-135 had grown for 5 d at 30 °C. Cytotoxic effect was also studied. GC-MS analysis was carried out. The antimicrobial activity was assessed using broth micro dilution technique. RESULTS: Ethyl acetate extract showed activity against bacteria such as Bacillus subtilis, Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, Enterobacter aerogenes, Staphylococcus aureu and Staphylococcus epidermidis (S. epidermidis) and fungi such as, Candida albicans and Trichophyton rubrum. The lowest minimum inhibitory concentrations were: 250 µg/mL against S. epidermidis and 250µg/mL against K. pneumonia. The isolate had the ability to produce enzymes such as protease. The exyract showed cytotoxic effect in human adenocarcinoma cancer cell line (A549). GC-MS analysis showed the presence of isovaleric acid (3.64%), 2-Methylbutanoic acid (5.03%), isobutyramide (5.05%), N,N-oimethylformamide-di-t-butylacetal (9.79%), benzeneacetamide (15.56%), octyl butyl phthalate (3.59%) and diisooctyl phthalate (5.79) in the extract. CONCLUSIONS: Methylobacterium sp. (ERI-135) showed promising antibacterial and cytotoxic activity. This is the first report in the antimicrobial and cytotoxic effect of Methylobacterium sp.