Aerobic degradation of fenvalerate by a Gram-positive bacterium, Bacillus flexus strain XJU-4.

Synthetic pyrethroid-fenvalerate-is one of the most widespread toxic pollutants and has adverse effect on living systems. However, little is known about its biotransformation mechanism in different microorganisms. To elucidate the pathway that might be involved in the catabolism of fenvalerate, we used Bacillus flexus strain XJU-4 (3-nitrobenzoate degrading organism) as an ideal fenvalerate degrading bacterium. Thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry analysis results revealed that 3-phenoxybenzoate, protocatechuate, and catechol are the three main by-products of fenvalerate metabolism. Additionally, the bacterial cell-free enzymes showed the activities of fenvalerate hydrolyzing esterase, 3-phenoxybenzaldehyde dehydrogenase, 3-phenoxybenzoate dioxygenase, phenol hydroxylase, protocatechuate 2,3-dioxygenase and catechol-2,3-dioxygenase. Thus, in strain XJU-4, protocatechuate and catechol were further metabolized through meta-cleavage pathway. Moreover, laboratory-scale soil experiments results suggest that B. flexus strain XJU-4 is a suitable contender for bioremediation of pyrethroid fenvalerate-contaminated sites.

Purification and immobilization of laccase from Trichoderma harzianum strain HZN10 and its application in dye decolorization.

In this study we report the purification of laccase produced by Trichoderma harzianum strain HZN10 (using wheat bran under solid state fermentation) and its application in decolorization of synthetic dyes. Extracellular laccase was purified to homogeneity by DEAE-Sepharose and Sephadex G-100 chromatography with specific activity of 162.5 U/mg and 25-fold purification. Purified laccase was immobilized in various entrapments like calcium alginate, copper alginate, calcium alginate-chitosan beads and sol-gel matrix. Optimization results revealed that the laccase immobilized in sol-gel was optimally active in wide pH range (4.0-7.0) and thermo-stable (50-70 °C) than free enzyme which was optimum at 50 °C and pH 6.0. Kinetic analysis showed K m of 0.5 mM and 2.0 mM and V max of 285 U/mg and 500 U/mg by free laccase and sol-gel immobilized laccase respectively with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS] substrate. Free and immobilized laccase was employed for decolorization of three different synthetic dyes (malachite green, methylene blue and congo red). High performance liquid chromatography (HPLC) analysis results revealed that approximately 100% of malachite green, 90% of methylene blue and 60% of congo red dyes at initial concentration of 200 mg/L were decolorized within 16, 18 and 20 h, respectively by laccase immobilized in sol-gel matrix in the presence of 1-hydroxybenzotriazole (HBT) mediator. During the decolorization all three synthetic dyes showed various peaks on HPLC chromatogram indicating different by-products formation. Finally, phytotoxicity analysis results revealed that the by-products of synthetic dyes (formed during decolorization) showed less toxicity against Phaseolus mungo compared to untreated synthetic dyes.

Characterization of antibiotic resistant and enzyme producing bacterial strains isolated from the Arabian Sea.

Marine bacteria are known to produce many bioactive molecules and extracellular enzymes of commercial importance. We have investigated the bacterial diversity of the coastal area of Karwar, Karnataka State, India. Among these bacterial isolates, five bacterial strains were selected and identified by their morphological, biochemical characteristics and phylogenetic analysis based on 16S rRNA gene sequences. The identified bacterial isolates, Bacillus toyonensis PNTB1, Lysinibacillus sphaericus PTB, Vibrio vulnificus PMD, Shewanella MPTDBS, and Pseudomonas chlororaphis PNTB were characterized for their tolerance to salt and antibiotics. Vibrio vulnificus PMD showed maximum tolerance at higher concentration of salt than other bacteria. These bacterial strains were screened for the production of extracellular enzymes such as lipase, cellulase, pectinase, tannase, chitinase, and L-glutaminase. Vibrio vulnificus showed maximum production of L-glutaminase enzyme. Bacillus toyonensis PNTB1 shows lipase, CM-cellulase and chitinase activities. These isolated bacterial cultures were also utilized most of the aromatic compounds at 7 mM. These findings indicate the organisms present in this zone may have more potential applications in bioremediation, agricultural, industrial, and therapeutics.

Purification and characterization of endo ß-1,4-D-glucanase from Trichoderma harzianum strain HZN11 and its application in production of bioethanol from sweet sorghum bagasse.

An acidophilic-solvent-thermostable endo ß-1,4-D-glucanase produced from a potential Trichoderma harzianum strain HZN11 was purified to homogeneity by DEAE-Sepharose and Sephadex G-100 chromatography with 33.12 fold purification with specific activity of 66.25 U/mg and molecular mass of ~55 kDa. The optimum temperature and pH were 60 °C and 5.5 retaining 76 and 85 % of activity after 3 h, respectively. It showed stability between pH 4.5-6.0 and temperature between 50-70 °C indicating thermostability. Endo ß-1,4-D-glucanase was activated by Ca2+ and Mg2+ but inhibited by Hg2+, Pb2+ and Cd2+. The effect of thiol reagents, metal chelators, oxidizing agents and surfactants on enzyme activity has been studied. Purified endo ß-1,4-D-glucanase exhibited highest specificity towards carboxymethyl cellulose. Kinetic analysis showed the K m, V max and K i (cellobiose inhibitor) of 2.5 mg/mL, 83.75 U/mg and 0.066 M, respectively. The storage stability of purified endo ß-1,4-D-glucanase showed a loss of mere 13 % over a period of 60 days. The hydrolysis efficiency of purified endo ß-1,4-D-glucanase mixed with cocktail was demonstrated over commercial enzyme. Optimized enzymatic hydrolysis of sweet sorghum and sugarcane bagasse released 5.2 g/g (36 h) and 6.8 g/g (48 h) of reducing sugars, respectively. Separate hydrolysis and fermentation of sweet sorghum bagasse yielded 4.3 g/L bioethanol (16 h) confirmed by gas chromatography-mass spectrometry (GC-MS). Morphological and structural changes were assessed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy. Elemental analysis was carried out by SEM equipped with energy dispersive X-ray technique. These unique properties prove the potentiality of enzyme for biomass conversion to biofuel and other industrial applications.

Xylanase production from Penicillium citrinum isolate HZN13 using response surface methodology and characterization of immobilized xylanase on glutaraldehyde-activated calcium-alginate beads.

The present study reports the production of high-level cellulase-free xylanase from Penicillium citrinum isolate HZN13. The variability in xylanase titers was assessed under both solid-state (SSF) and submerged (SmF) fermentation. SSF was initially optimized with different agro-waste residues, among them sweet sorghum bagasse was found to be the best substrate that favored maximum xylanase production (9643 U/g). Plackett-Burman and response surface methodology employing central composite design were used to optimize the process parameters for the production of xylanase under SSF. A second-order quadratic model and response surface method revealed the optimum conditions for xylanase production (sweet sorghum bagasse 25 g/50 ml; ammonium sulphate 0.36 %; yeast extract 0.6 %; pH 4; temperature 40 °C) yielding 30,144 U/g. Analysis of variance (ANOVA) showed a high correlation coefficient (R 2 = 97.63 %). Glutaraldehyde-activated calcium-alginate-immobilized purified xylanase showed recycling stability (87 %) up to seven cycles. Immobilized purified xylanase showed enhanced thermo-stability in comparison to immobilized crude xylanase. Immobilization kinetics of crude and purified xylanase revealed an increase in K m (12.5 and 11.11 mg/ml) and V max (12,500 and 10,000 U/mg), respectively. Immobilized (crude) enzymatic hydrolysis of sweet sorghum bagasse released 8.1 g/g (48 h) of reducing sugars. Xylose and other oligosaccharides produced during hydrolysis were detected by High-Performance Liquid Chromatography. The biomass was characterized by Scanning Electron Microscopy, Energy Dispersive X-ray and Fourier Transformation Infrared Spectroscopy. However, this is one of the few reports on high-level cellulase-free xylanase from P. citrinum isolate using sweet sorghum bagasse.

Purification, characterization, gene cloning and expression of GH-10 xylanase (Penicillium citrinum isolate HZN13).

An extracellular thermostable xylanase (Xyl-IIb) produced by Penicillium citrinum isolate HZN13 was purified to homogeneity using DEAE-Sepharose, Sephadex G-100 and Bio-Gel P-60 chromatography with specific activity of 6272.7 U/mg and 19.6-fold purification. The purification revealed the occurrence of multiple forms of xylanases (Xyl-I, Xyl-IIa, Xyl-IIb and Xyl-III). The molecular mass of highly purified Xyl-IIb was ~31 kDa with SDS-PAGE. The enzyme was cellulase-free, thermostable (55-75 °C) and acidophilic (3.5-5.0). It was activated by Ca2+, Ba2+, DTT and ß-mercaptoethanol, whereas inhibited by Hg2+, Pb2+, Ni2+ and p-CMB. Purified Xyl-IIb exhibited highest specificity toward birchwood and oat spelts xylan. Kinetics of Xyl-IIb revealed a K m of 10 mg/ml and 16.7 mg/ml and V max of 9523g and 15,873 U/mg with birchwood and oat spelts xylan, respectively, indicating high affinity toward birchwood xylan. The xylanase (Xyl-IIb) belongs to glycosyl hydrolase (GH) family 10 based on conserved regions. Xylanase-encoding gene (xynB) consists of 1501 bp with an open reading frame of 264 bp which was predicted to encode a protein having 87 amino acids and shared homology with endo-1,4-beta-xylanase (xynB) gene from Penicillium citrinum. Cloned xynB gene was expressed in E. coli BL21 (DE3) with xylanase activity (80 U/mg) and confirmed to be GH-10 Xyl-IIa based on molecular mass (~40 kDa). These properties of xylanase make it promising for their applications in biofuel industries.

Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1.

Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1

Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

Biodegradation of pesticide profenofos by the free and immobilized cells of Pseudoxanthomonas suwonensis strain HNM.

Profenofos is an organophosphate pesticide used extensively in agriculture to control pests. A bacterium capable of degrading profenofos was isolated from pesticide-contaminated soil samples and identified as Pseudoxanthomonas suwonensis strain HNM based on its morphological and biochemical characteristics and phylogenetic analysis of 16S rRNA gene sequences. 4-Bromo-2-chlorophenol was identified as a metabolite of profenofos degradation by HPLC and GC-MS analysis. The organism degraded profenofos by hydrolysis to yield 4-bromo-2-chlorophenol which was further utilized as carbon source for growth. The organism utilized various organophosphate pesticides such as temephos, quinalphos, and chloropyrifos as carbon sources. The optimum conditions for degradation of profenofos by P. suwonensis strain HMN were found to be at pH 7 and 30 °C. We have investigated the rate of degradation of profenofos by the free and immobilized cells of P. suwonensis strain HNM in various matrices such as sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA), and SA-bentonite clay. The rate of degradation of 3 and 6 mM profenofos by the freely suspended cells were compared with that by immobilized cells in batches and semi-continuous with shaken cultures. The SA-bentonite clay-immobilized cells showed higher rate of degradation of 3 and 6 mM profenofos then freely suspended cells and cells immobilized in SA and SA-PVA. The SA-bentonite clay-immobilized cells of P. suwonensis strain HNM could be reused for more than 32 cycles without losing their degradation capacity. Thus, the immobilized cells are more efficient than freely suspended cells for the degradation of organophosphate pesticide contaminated water.

Enhanced degradation of 2-nitrotoluene by immobilized cells of Micrococcus sp. strain SMN-1.

Nitrotoluenes are the toxic pollutants of the environment because of their large scale use in the production of explosives. Biodegradation of such chemicals by microorganisms may provide an effective method for their detoxification. We have studied the degradation of 2-nitrotoluene by cells of Micrococcus sp. strain SMN-1 immobilized in various matrices such as polyurethane foam (PUF), sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA), agar and polyacrylamide. The rate of degradation of 15 and 30 mM 2-nitrotoluene by freely suspended cells and immobilized cells in batches and fed-batch with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 15 and 30 mM 2-nitrotoluene than freely suspended cells and the cells immobilized in SA-PVA, polyacrylamide, SA and agar. The PUF-immobilized cells could be reused more than 24 cycles without loosing their degradation capacity and showed more tolerance to pH and temperature changes than freely suspended cells. These results revealed the enhanced rate of degradation of 2-nitrotoluene by PUF-immobilized cells of Micrococcus sp. strain SMN-1.

Biodegradation of 4-chlorobenzoic acid by Pseudomonas aeruginosa PA01 NC.

A bacterial consortium capable of degrading chloroaromatic compounds was isolated from pulp and paper mill effluents by selective enrichment on 4-chlorobenzoic acid as sole source of carbon and energy. The four different bacterial isolates obtained from bacterial consortium were identified as Pseudomonas aeruginosa AY792969 (A), P. aeruginosa PA01 NC (B), Pseudomonas sp. ZZ5 DQ113452 (C) and Pseudomonas sp. AY762360 (D) based on their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. These bacterial isolates were found to be versatile in degrading a variety of chloroaromatic compounds including fluoro- and iodobenzoic acids. P. aeruginosa PA01 NC utilized 4-chlorobenzoic acid at 2 g/l as growth substrate. Biodegradation studies have revealed that this organism degraded 4-chlorobenzoic acid through 4-chlorocatechol which was further metabolized by ortho-cleavage pathway and the dechlorination occurred after the ring-cleavage.

Biodegradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1.

A bacterial consortium capable of degrading nitroaromatic compounds was isolated from pesticide-contaminated soil samples by selective enrichment on 2-nitrotoluene as a sole source of carbon and energy. The three different bacterial isolates obtained from bacterial consortium were identified as Bacillus sp. (A and C), Bacillus flexus (B) and Micrococcus sp. (D) on the basis of their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. The pathway for the degradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1 was elucidated by the isolation and identification of metabolites, growth and enzymatic studies. The organism degraded 2-nitrotoluene through 3-methylcatechol by a meta-cleavage pathway, with release of nitrite.

Bacterial degradation of fungicide captan.

The phthalimide fungicide captan has been widely used to control plant pathogenic fungi. A strain of Bacillus circulans utilized the fungicide captan as sole source of carbon and energy. The organism degraded captan by a pathway involving its initial hydrolysis to yield cis-1,2,3,6-tetrahydrophthalimide, a compound without fungicidal activity. The formation of this compound was confirmed by HPLC, IR, NMR, and mass spectral analysis. The results also revealed that cis-1,2,3,6-tetrahydrophthalimide was further degraded to o-phthalic acid by a protocatechuate pathway. These findings indicated that there was a complete mineralization of fungicide captan by B. circulans.

Biodegradation of cypermethrin by Micrococcus sp. strain CPN 1.

A bacterium capable of utilizing pyrethroid pesticide cypermethrin as sole source of carbon was isolated from soil and identified as a Micrococcus sp. The organism also utilized fenvalerate, deltamethrin, perimethrin, 3-phenoxybenzoate, phenol, protocatechuate and catechol as growth substrates. The organism degraded cypermethrin by hydrolysis of ester linkage to yield 3-phenoxybenzoate, leading to loss of its insecticidal activity. 3-Phenoxybenzoate was further metabolized by diphenyl ether cleavage to yield protocatechuate and phenol as evidenced by isolation and identification of metabolites and enzyme activities in the cell-free extracts. Protocatechuate and phenol were oxidized by ortho-cleavage pathway. Thus, the organism was versatile in detoxification and complete mineralization of pyrethroid cypermethrin.

Biodegradation of DDT by a Pseudomonas species.

A bacterial strain capable of degrading 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) was isolated from insecticide-contaminated soil by biphenyl enrichment culture and identified as a Pseudomonas species. The organism degraded DDT through the intermediate formation of 2,3-dihydroxy-DDT, which undergoes meta-ring cleavage, ultimately yielding 4-chlorobenzoic acid as a stable metabolite.