Biodegradation of fluoranthene by Paenibacillus sp. strain PRNK-6: a pathway for complete mineralization.

A high-efficiency fluoranthene-degrading bacterium Paenibacillus sp. PRNK-6 was isolated from PAH-contaminated soil. The strain degrades 96% (240 mg l-1) of fluoranthene in 48 h. Various metabolic intermediates of fluoranthene catabolism were identified by gas chromatography (GC) and gas chromatography-high resolution mass spectrometry (GC-HRMS). Metabolite characterization, metabolite-feeding experiments, and appropriate enzyme activities in the cell-free extracts suggest the existence of a bifurcated pathway down the phthalic acid for complete mineralization of fluoranthene in PRNK-6. In this strain, fluoranthene catabolism begins by the attack on the fused aromatic ring portion of fluoranthene. Two terminal aromatic metabolites protocatechuate and catechol undergo ring cleavage by protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase, respectively, and enter the central metabolism.

Enhanced utilization of fluorene by Paenibacillus sp. PRNK-6: Effect of rhamnolipid biosurfactant and synthetic surfactants.

The present investigation was to study the effect of different non-ionic surfactants (Tween-80, Tween-60, Tween-40, Tween-20, Triton X-100) and a rhamnolipid biosurfactant on the degradation of fluorene by Paenibacillus sp. PRNK-6. An enhancement in the growth, as well as fluorene utilization by this strain were observed in the presence of biosurfactant and non-ionic surfactants except Tween-20 and Triton X-100. Triton X-100 and Tween-20 were toxic to this bacterium. The strain PRNK-6 utilized 75% of fluorene (280mg/L) in 24h in an unamended condition. On the other hand, the complete utilization of higher concentration fluorene (320mg/L) by this strain was noticed when the medium was amended with Tween-80 (1.5% v/v) within 24h of incubation. Whereas, 90.6%, 96.5% and 96.7% of fluorene (280mg/L) was utilized when amended with Tween-60 (3.5% v/v), Tween-40 (3% v/v) and biosurfactant (25mg/L) respectively. Biosurfactant promoted the fluorene degradation potential of PRNK-6 as 96.2% of 320mg/L fluorene was degraded within 24h. Further, the added tween series surfactants and a biosurfactant have increased the cell surface hydrophobicity of the PRNK-6. Thus correlating with the enhanced degradation of the fluorene.

The response of Paracoccus sp. SKG to acetonitrile-induced oxidative stress.

Organic solvents enhance intracellular oxidative stress and induce various physiological responses in bacteria. The study shows the morphological changes in Paracoccus sp. SKG when exposed to higher concentrations of acetonitrile, which alter the composition of the membrane fatty acid that accompanies the increase in K(+) efflux. This enhances the oxidative stress with greater activities of catalase and super oxide dismutase (SOD). The increased oxidative stress results in the generation of free radicals, which was confirmed by electron paramagnetic resonance (EPR) studies. The free radical scavenging activities were measured by ABTS and DPPH to understand the non-enzymatic defensive system during oxidative stress. The studies demonstrate the increase in free radicals in association with enzymatic and non-enzymatic defense systems under solvent stress.

Indole-3-acetic acid biosynthetic pathway and aromatic amino acid aminotransferase activities in Pantoea dispersa strain GPK.

This investigation deals with the production of IAA by a bacterial isolate Pantoea dispersa strain GPK (PDG) identified by 16S rRNA gene sequence analysis. HPLC and Mass spectral analysis of metabolites from bacterial spent medium revealed that, IAA production by PDG is Trp-dependent and follows indole-3-pyruvic acid (IPyA) pathway. Substrate specificity study of aromatic amino acid aminotransferase (AAT) showed high activities, only when tryptophan (Trp) and α-ketoglutarate (α-kg) were used as substrates. AAT is highly specific for Trp and α-kg as amino group donor and acceptor, respectively. The effect of exogenous IAA on bacterial growth was established. Low concentration of exogenous IAA induced the growth, whereas high concentration decreased the growth of bacterium. PDG treatment significantly increased the root length, shoot length and dry mass of the chickpea and pigeon pea plants.

Purification and physiochemical characterization of melanin pigment from Klebsiella sp. GSK.

The bacterium capable of producing melanin pigment in the presence of L-tyrosine was isolated from crop field soil sample and identified as Klebsiella sp. GSK based on morphological, biochemical and 16S rDNA sequencing. The polymerization of this pigment occurs outside the cell wall, which has granular structure as melanin ghosts. The chemical characterization of pigment particles showed acid resistant, alkali soluble, insoluble in most of the organic solvents and water. The pigment gets bleached when subjected to the action of oxidants as well as reductants. This pigment was precipitated with FeCl3, ammoniacal silver nitrate and potassium ferricynide. The pigment showed high absorbance in the UV region and decreased absorbance when shifted towards the visible region. The melanin pigment was further charecterized by FT-IR and EPR spectroscopy. A key enzyme 4-hydroxyphenylacetic acid hydroxylase catalyzes the formation of melanin pigment by hydroxylation of L-tyrosine was detected in this bacterium. Inhibition studies with specific inhibitor kojic acid and KCN proved that melanin is synthesized by DOPA-Melanin pathway.

Metabolism of acenaphthylene via 1,2-dihydroxynaphthalene and catechol by Stenotrophomonas sp. RMSK.

Stenotrophomonas sp. RMSK capable of degrading acenaphthylene as a sole source of carbon and energy was isolated from coal sample. Metabolites produced were analyzed and characterized by TLC, HPLC and mass spectrometry. Identification of naphthalene-1,8-dicarboxylic acid, 1-naphthoic acid, 1,2-dihydroxynaphthalene, salicylate and detection of key enzymes namely 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-1,2-dioxygenase in the cell free extract suggest that acenaphthylene metabolized via 1,2-dihydroxynaphthalene, salicylate and catechol. The terminal metabolite, catechol was then metabolized by catechol-1,2-dioxygenase to cis,cis-muconic acid, ultimately forming TCA cycle intermediates. Based on these studies, the proposed metabolic pathway in strain RMSK is, acenaphthylene --> naphthalene-1,8-dicarboxylic acid --> 1-naphthoic acid --> 1,2-dihydroxynaphthalene --> salicylic acid --> catechol --> cis,cis-muconic acid.

Purification and characterization of NAD-dependent n-butanol dehydrogenase from solvent-tolerant n-butanol-degrading Enterobacter sp. VKGH12.

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is capable of utilizing n-butanol and contains an NAD+-dependent n-butanol dehydrogenase (BDH). The BDH from n-butanol-grown Enterobacter sp. was purified from a cell-free extract (soluble fraction) to near homogeneity using a 3-step procedure. The BDH was purified 15.37-fold with a recovery of only 10.51, and the molecular mass estimated to be 38 kDa. The apparent Michaelis-Menten constant (Km) for the BDH was found to be 4 mM with respect to n-butanol. The BDH also had a broad range of substrate specificity, including primary alcohols, secondary alcohols, and aromatic alcohols, and exhibited an optimal activity at pH 9.0 and 40oC. Among the metal ions studied, Mg2+ and Mn2+ had no effect, whereas Cu2+, Zn2+, and Fe2+ at 1 mM completely inhibited the BDH activity. The BDH activity was not inhibited by PMSF, suggesting that serine is not involved in the catalytic site. The known metal ion chelator EDTA had no effect on the BDH activity. Thus, in addition to its physiological significance, some features of the enzyme, such as its activity at an alkaline pH and broad range of substrate specificity, including primary and secondary alcohols, are attractive for application to the enzymatic conversion of alcohols.

Degradation of cinnamic acid by a newly isolated bacterium Stenotrophomonas sp. TRMK2.

A bacterium Stenotrophomonas sp. TRMK2 capable of utilizing cinnamic acid was isolated from agro-industrial waste by enrichment culture technique. This strain completely utilizes 5 mM cinnamic acid within 18 h of incubation. The different metabolites formed during the degradation of cinnamic acid were characterized by GC-HRMS. The involvement of various enzymes, namely cinnamate reductase, 3-phenylpropionic acid hydroxylase, p-hydroxybenzoic acid hydroxylase and protocatechuate 3,4-dioxygenase in cinnamic acid degradation was demonstrated. A catabolic pathway for cinnamic acid in Stenotrophomonas sp. TRMK2 is as follows: Cinnamic acid; 3-Phenylpropionic acid; 3-(4-Hydroxyphenyl) propionic acid; 4-Hydroxy benzoic acid and Protocatechuic acid. Further, this strain is capable of utilizing various phenolic compounds.

A comparative study of utilization of single and mixed phenolic compounds by individual and mixed culture.

Three bacterial strains; Pseudomonas sp. TRMK1, Stenotrophomonas sp. TRMK2 and Xanthomonas sp. TRMK3 were isolated from agro-industrial waste by enrichment culture technique that are capable of utilizing phenolic acids as sole source of carbon and energy. These strains were found to utilize p-coumaric, ferulic and caffeic acid. The individual strains utilized 5 mM of mixed phenolic acids within 20 h of incubation. The bacterial consortium composing these strains was prepared and studied the efficient degradation of phenolic compounds. The bacterial consortium showed the enhanced utilization of 30 mM individual and 25 mM mixed phenolic acids within 32 and 40 h of incubation, respectively. The degradation efficiency of these strains in all the above experiments was above 90%. The prepared bacterial consortium serves as a suitable method for the in situ application of sites contaminated with wide range of phenolic compounds.

Utilization of Phenylpropanoids by Newly Isolated Bacterium Pseudomonas sp. TRMK1.

A bacterium Pseudomonas sp. TRMK1 capable of utilizing various phenylpropanoids was isolated from agro-industrial waste by enrichment culture technique. It is gram-negative, motile, aerobic, and able to utilize three different phenolic acids such as p-coumaric, ferulic, and caffeic acids at concentrations of 5, 10, and 15 mM in 18 h of incubation. The residual concentration of phenolic acids was analyzed by HPLC. The catabolic pathway of p-coumaric, ferulic, and caffeic acids is suggested based on the characterization of metabolic intermediates by GC, GC-HRMS, and different enzymatic assays. Further, Pseudomonas sp. TRMK1 utilizes a wide range of mixture of phenolic acids present in the synthetic effluent.

Biodegradation of butyronitrile and demonstration of its mineralization by Rhodococcus sp. MTB5.

A nitrile utilizing bacterium Rhodococcus sp. MTB5 was previously isolated in our laboratory by the enrichment culture technique. It is able to utilize butyronitrile as sole carbon, nitrogen, and energy source. Maximum butyronitrile degrading property of this strain has been investigated. Results reveal that 100, 98, and 88 % degradation was achieved for 2, 2.5, and 3 % butyronitrile, respectively. The strain is capable of growing in as high as 5 % butyronitrile concentration. A two-step pathway involving nitrile hydratase (NHase) and amidase was observed for the biodegradation of butyronitrile. Complete degradation (mineralization) of butyronitrile with the help of metabolite feeding experiment was reported. The significance of this investigation was the capability of the strain to completely degrade and its ability to grow on higher concentrations of butyronitrile. These potential features make it a suitable candidate for practical field application for effective in situ bioremediation of butyronitrile contaminated sites.

Catabolism of homophthalic acid by a soil bacterium.

A microorganism capable of degrading homophthalic acid as a sole source of carbon was isolated from soil. The strain was tentatively identified as Pseudomonas sp. Oxygen uptake studies were carried out with possible intermediates. Assays for several different enzymes were performed. Homophthalic acid may be metabolized by this bacterium via p-hydroxyphenyl acetic acid and homogentisic acid intermediates.

Initial degradation of dimethylphthalate by esterases from Bacillus species.

Dimethylphthalate (DMP), one of the phthalate esters, is used in the manufacture of plasticizers, insect repellents, and synthetic fibers, and contributes to environmental pollution. In the present study, we report a novel bacterium belonging to the Bacillus sp., which has the ability to utilize DMP as the sole source of carbon. The esterases from the cell-free extract of the Bacillus de-esterified DMP. Native polyacrylamide gel electrophoresis showed the presence of four isoesterases designated Et1--4. The isoesterases Et-4 and Et-1 showed a higher preference towards DMP hydrolysis as compared with Et-2 and 3. A megaplasmid of about 60 kb was detected in this bacterium. The ability of this bacterium to utilize DMP as the sole source of carbon was lost upon plasmid curing. The isoesterases Et-1--4 were absent in the cell-free extracts of the cured bacterium. The results from our studies clearly demonstrate that de-esterification is the initial step in the degradation of DMP and the genes for these esterases seem to be harbored on the plasmid in this bacterium.

Cloning and nucleotide sequence analysis of xylE gene responsible for meta-cleavage of 4-chlorocatechol from Pseudomonas sp. S-47.

Pseudomonas sp. S-47 expresses catechol 2,3-dioxygenase (C230) catalyzing the conversion of 4-chlorocatechol (4CC) as well as catechol to 5-chloro-2-hydroxymuconic semialdehyde and 2-hydroxymuconic semialdehyde, respectively, through meta-ring cleavage. The xylE gene encoding C230 for meta-cleavage was cloned from strain S-47 and its nucleotide sequence was analyzed. The pRES101 containing the xylE gene exhibited high C230 activity toward catechol and 4CC without altering the substrate specificity from natural strain. The xylE gene was composed of 924 bp and encoded polypeptide of molecular mass 35 kDa containing 307 amino acids. A deduced amino acid sequence of the C230 from strain S-47 exhibited over 80% identity with those of Pseudomonas putida mt-2, Pseudomonas putida G7, and Pseudomonas sp. CF600. However, it shows below 45% identity with those of Pseudomonas cepacia LB400 and Pseudomonas sp. KKS102. The C230 of strain S-47 was conserved in the amino acids (His150, His214, Glu261) for metal binding ligands and those (His199, His242, and Tyr251) for catalytic sites. Therefore, Pseudomonas sp. S-47 can be explained as acting by degrading catechol as well as 4CC by xylE-encoding C230 which was fused by N domain of nahH and C domain of dmpB from other Pseudomonas strains.

Metabolism of polycyclic aromatic hydrocarbons by Aspergillus niger.

Aspergillus niger metabolised polycyclic aromatic hydrocarbons such as naphthalene, anthracene and phenanthrene. The maximum weight of mycelium was obtained at 8-10 days of incubation. TLC and HPLC analysis revealed the accumulation of metabolites in resting cell culture. The metabolism of naphthalene and anthracene follows the gentisate pathway, where as phenenthrene was metabolised via protocatechuate in this organism.

Degradation of naphthalene by a Pseudomonas strain NGK1.

A Pseudomonas strain NGK1 capable of degrading naphthalene as a sole carbon source was isolated from biological waste treatment effluent. After 48 hr of incubation in 0.1% naphthalene mineral salts medium, the bacterial culture showed irregular clumped cells and salicylic acid (68 micrograms per ml) in the medium (pH 3.8). The strain degraded naphthalene through salicylate and catechol as was evidenced by metabolite characterization, oxygen uptake and enzymatic studies.

Degradation of 3-hydroxybenzoic acid by a Bacillus species.

A Bacillus sp. isolated by 4-hydroxybenzoate enrichment culture technique is capable of utilizing 3-hydroxybenzoate as the sole source of carbon and energy. The organism degraded 3-hydroxybenzoic acid through the intermediate formation of protocatechuic acid. 3-Hydroxybenzoate 4-hydroxylase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities were shown in cell-free extracts. The formed protocatechuate is metabolized through both the ortho and meta cleavage pathway.

Influence of DMF-induced oxidative stress on membrane and periplasmic proteins in Paracoccus sp. SKG.

The present study describes the N,N-dimethylformamide (DMF)-induced oxidative stress in Paracoccus sp. SKG. The oxidative stress was evaluated by analysing membrane and periplasmic proteins and K+ efflux, as well as by monitoring the activities of antioxidant enzymes like catalase, superoxide dismutase (SOD) and glutathione S-transferase (GST). The exposure of bacterial cells to a higher concentration of DMF resulted in the modification of membrane fatty acid composition which is accompanied by K+ efflux. Further, this oxidative stress resulted in increased periplasmic protein which can be attributed to the induction of GST and methionine sulphoxide reductase (Msr) enzymes under solvent stress. Paracoccus sp. SKG is tolerant to high concentrations of DMF up to 6% (v/v) and its toxic effects. DMF concentration-dependent induction of GST and Msr activities advocates the significant role of these enzymes in the bacterial defence system. The present study provides information which helps us to understand the ROS scavenging machinery in bacteria. The high tolerance of Paracoccus sp. SKG to DMF can be efficiently explored for various bioremediation and biotransformation applications.

Indole-3-acetic acid biosynthesis in Fusarium delphinoides strain GPK, a causal agent of Wilt in Chickpea.

Fusarium delphinoides (Ascomycota; Nectriaceae) is an indole-3-acetic acid (IAA) producing plant pathogen and a causal agent of wilt in chickpea. The IAA biosynthetic pathway in F. delphinoides strain GPK (FDG) was examined by analyzing metabolic intermediates and by feeding experiments. Gas chromatograph (GC) analysis of FDG culture filtrates showed the presence of metabolic intermediates of indole-3-pyruvic acid (IPyA), indole-3-acetamide (IAM), and tryptamine (TRA) pathways. The different IAA biosynthetic pathways were further confirmed by identifying the presence of different enzymes of these pathways. Substrate specificity study of aromatic amino acid aminotransferase revealed that the enzyme is highly specific for tryptophan (Trp) and α-ketoglutarate (α-kg) as amino group donor and acceptor, respectively. Furthermore, the concentration-dependent effect of exogenous IAA on fungal growth was established. Low concentration of exogenous IAA increases the fungal growth and at high concentration it decreases the growth of FDG.

Generation of continuous packed bed reactor with PVA-alginate blend immobilized Ochrobactrum sp. DGVK1 cells for effective removal of N,N-dimethylformamide from industrial effluents.

Effective removal of dimethylformamide (DMF), the organic solvent found in industrial effluents of textile and pharma industries, was demonstrated by using free and immobilized cells of Ochrobactrum sp. DGVK1, a soil isolate capable of utilizing DMF as a sole source of carbon, nitrogen. The free cells have efficiently removed DMF from culture media and effluents, only when DMF concentration was less than 1% (v/v). Entrapment of cells either in alginate or in polyvinyl alcohol (PVA) failed to increase tolerance limits. However, the cells of Ochrobactrum sp. DGVK1 entrapped in PVA-alginate mixed matrix tolerated higher concentration of DMF (2.5%, v/v) and effectively removed DMF from industrial effluents. As determined through batch fermentation, these immobilized cells have retained viability and degradability for more than 20 cycles. A continuous packed bed reactor, generated by using PVA-alginate beads, efficiently removed DMF from industrial effluents, even in the presence of certain organic solvents frequently found in effluents along with DMF.