Proteomics and Metabolomics Analyses to Elucidate the Desulfurization Pathway of Chelatococcus sp.

Desulfurization of dibenzothiophene (DBT) and alkylated DBT derivatives present in transport fuel through specific cleavage of carbon-sulfur (C-S) bonds by a newly isolated bacterium Chelatococcus sp. is reported for the first time. Gas chromatography-mass spectrometry (GC-MS) analysis of the products of DBT degradation by Chelatococcus sp. showed the transient formation of 2-hydroxybiphenyl (2-HBP) which was subsequently converted to 2-methoxybiphenyl (2-MBP) by methylation at the hydroxyl group of 2-HBP. The relative ratio of 2-HBP and 2-MBP formed after 96 h of bacterial growth was determined at 4:1 suggesting partial conversion of 2-HBP or rapid degradation of 2-MBP. Nevertheless, the enzyme involved in this conversion process remains to be identified. This production of 2-MBP rather than 2-HBP from DBT desulfurization has a significant metabolic advantage for enhancing the growth and sulfur utilization from DBT by Chelatococcus sp. and it also reduces the environmental pollution by 2-HBP. Furthermore, desulfurization of DBT derivatives such as 4-M-DBT and 4, 6-DM-DBT by Chelatococcus sp. resulted in formation of 2-hydroxy-3-methyl-biphenyl and 2-hydroxy -3, 3/- dimethyl-biphenyl, respectively as end product. The GC and X-ray fluorescence studies revealed that Chelatococcus sp. after 24 h of treatment at 37°C reduced the total sulfur content of diesel fuel by 12% by per gram resting cells, without compromising the quality of fuel. The LC-MS/MS analysis of tryptic digested intracellular proteins of Chelatococcus sp. when grown in DBT demonstrated the biosynthesis of 4S pathway desulfurizing enzymes viz. monoxygenases (DszC, DszA), desulfinase (DszB), and an NADH-dependent flavin reductase (DszD). Besides, several other intracellular proteins of Chelatococcus sp. having diverse biological functions were also identified by LC-MS/MS analysis. Many of these enzymes are directly involved with desulfurization process whereas the other enzymes/proteins support growth of bacteria at an expense of DBT. These combined results suggest that Chelatococcus sp. prefers sulfur-specific extended 4S pathway for deep-desulphurization which may have an advantage for its intended future application as a promising biodesulfurizing agent.

Mechanism of Adsorptive Removal of Methylene Blue Using Dried Biomass of Rhizopus oryzae.

Adsorption is an efficient way to remove synthetic dyes from industrial effluent. Here, we show mechanism of adsorptive removal of cationic dye methylene blue (MB) from its aqueous solution using dried biomass of Rhizopus oryzae as a biosorbent. The optimum pH and temperature for adsorption was found to be 7.0 and 28 °C, respectively. Scanning electron microscopy (SEM) of the biomass suggested distinct changes in surface topology post-MB adsorption, while Fourier transform infrared (FTIR) study indicated chemical interaction between the surface of the biomass and MB. Chemical modification of -OH and -C=O groups of biomass reduced the MB adsorption and corroborated with the FTIR analyses. Kinetics study revealed that the adsorption rate was fast initially and reached equilibrium at 4 h following a pseudo-second-order-kinetics. The adsorption isotherm followed Freundlich isotherm model with n value of 1.1615.The dried biomass of R. oryzae can be used as a potent biosorbent for the removal of MB.

Differential mode of attack on membrane phospholipids by an acidic phospholipase A2 (RVVA-PLA2-I) from Daboia russelli venom.

An acidic phospholipase A2 (RVVA-PLA2-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA2-I was non-lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA2 enzyme. RVVA-PLA2-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA2-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA2-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA2s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA2-I suggested the correlation between catalytic and membrane damaging activities of this PLA2 enzyme. Our study advocates that the presence of a large number of PLA2-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA2 enzyme.

Biodegradation of benzene, toluene, and xylene (BTX) in liquid culture and in soil by Bacillus subtilis and Pseudomonas aeruginosa strains and a formulated bacterial consortium.

PURPOSE: The major aromatic constituents of petroleum products viz. benzene, toluene, and mixture of xylenes (BTX) are responsible for environmental pollution and inflict serious public concern. Therefore, BTX biodegradation potential of individual as well as formulated bacterial consortium was evaluated. This study highlighted the role of hydrogen peroxide (H(2)O(2)), nitrate, and phosphate in stimulating the biodegradation of BTX compounds under hypoxic condition. MATERIALS AND METHODS: The individual bacterium viz. Bacillus subtilis DM-04 and Pseudomonas aeruginosa M and NM strains and a consortium comprising of the above bacteria were inoculated to BTX-containing liquid medium and in soil. The bioremediation experiment was carried out for 120 h in BTX-containing liquid culture and for 90 days in BTX-contaminated soil. The kinetics of BTX degradation either in presence or absence of H(2)O(2), nitrate, and phosphate was analyzed using biochemical and gas chromatographic (GC) technique. RESULTS: Bacterial consortium was found to be superior in degrading BTX either in soil or in liquid medium as compared to degradation of same compounds by individual strains of the consortium. The rate of BTX biodegradation was further enhanced when the liquid medium/soil was exogenously supplemented with 0.01 % (v/v) H(2)O(2), phosphate, and nitrate(.) The GC analysis of BTX biodegradation (90 days post-inoculation) in soil by bacterial consortium confirmed the preferential degradation of benzene compared to m-xylene and toluene. CONCLUSIONS: It may be concluded that the bacterial consortium in the present study can degrade BTX compounds at a significantly higher rate as compared to the degradation of the same compounds by individual members of the consortium. Further, addition of H(2)O(2) in the culture medium as an additional source of oxygen, and nitrate and phosphate as an alternative electron acceptor and macronutrient, respectively, significantly enhanced the rate of BTX biodegradation under oxygen-limited condition.

Biodegradable and biocompatible epoxidized vegetable oil modified thermostable poly(vinyl chloride): thermal and performance characteristics post biodegradation with Pseudomonas aeruginosa and Achromobacter sp.

The increased production of municipal solid waste by the disposal of plastic materials heightens the urgency to develop biodegradable materials for daily use. In vitro-biodegradation study on poly(vinyl chloride) (PVC) plasticized by epoxidized Mesua ferrea L. seed oil at three different weight percentages (PVC/ENO ratio of 75/25, 50/50 and 25/75) was conducted by using Pseudomonas aeruginosa and Achromobacter sp. bacteria. The test bacterial species were able to grow on the polymer matrix by using it as a source of energy; however the pristine PVC did not support the microbial growth. The PVC/ENO material of 25/75 ratio showed the highest percent (%) of biodegradation compared to other tested systems. The bacterial count and the dry biomass post 180 days of inoculation in 25/75 plasticized PVC suggested bacterial growth at the expense of degradation of the system. The tensile strength of 25/75 PVC/ENO system, post 180 days of inoculation by Pseudomonas aeruginosa and Achromobacter sp. decreased by about 53% and 43% respectively. Further, surface erosion phenomenon and structural change of the matrix after bacterial growth, as studied by FTIR and SEM analysis of PVC/ENO of 25/75 ratio exhibited noticeable deterioration post 180 days of inoculation.

Bioremediation and reclamation of soil contaminated with petroleum oil hydrocarbons by exogenously seeded bacterial consortium: a pilot-scale study.

PURPOSE: Spillage of petroleum hydrocarbons causes significant environmental pollution. Bioremediation is an effective process to remediate petroleum oil contaminant from the ecosystem. The aim of the present study was to reclaim a petroleum oil-contaminated soil which was unsuitable for the cultivation of crop plants by using petroleum oil hydrocarbon-degrading microbial consortium. MATERIALS AND METHODS: Bacterial consortium consisting of Bacillus subtilis DM-04 and Pseudomonas aeruginosa M and NM strains were seeded to 20% (v/w) petroleum oil-contaminated soil, and bioremediation experiment was carried out for 180 days under laboratory condition. The kinetics of hydrocarbon degradation was analyzed using biochemical and gas chromatographic (GC) techniques. The ecotoxicity of the elutriates obtained from petroleum oil-contaminated soil before and post-treatment with microbial consortium was tested on germination and growth of Bengal gram (Cicer aretinum) and green gram (Phaseolus mungo) seeds. RESULTS: Bacterial consortium showed a significant reduction in total petroleum hydrocarbon level in contaminated soil (76% degradation) as compared to the control soil (3.6% degradation) 180 days post-inoculation. The GC analysis confirmed that bacterial consortium was more effective in degrading the alkane fraction compared to aromatic fraction of crude petroleum oil hydrocarbons in soil. The nitrogen, sulfur, and oxygen compounds fraction was least degraded. The reclaimed soil supported the germination and growth of crop plants (C. aretinum and P. mungo). In contrast, seeds could not be germinated in petroleum oil-contaminated soil. CONCLUSIONS: The present study reinforces the application of bacterial consortium rather than individual bacterium for the effective bioremediation and reclamation of soil contaminated with petroleum oil.