Natesan Manickam

An integrated (nano-bio) technique for degradation of γ-HCH contaminated soil.

We have evaluated the effect of an integrated (nano-bio) technique involving the use of stabilized Pd/Fe(0) bimetallic nanoparticles (CMC-Pd/nFe(0)) and a Sphingomonas sp. strain NM05, on the degradation of γ-HCH in soil. Factors affecting degradation such as pH, incubation temperature and γ-HCH initial concentration were also studied. The results revealed that γ-HCH degradation efficiency is ~ 1.7-2.1 times greater in integrated system as compared to system containing either NM05 or CMC-Pd/nFe(0) alone. The integration showed synergistic effect on γ-HCH degradation. Further, cell growth studies indicated that NM05 gets well acclimatized to nanoparticles, showing potential growth in the presence of CMC-Pd/nFe(0) with respect to control system. This study signifies the potential efficacy of integrated technique to become an effective alternative remedial tool for γ-HCH contaminated soil. Further research in this direction could lead to the development of effective remediation strategies for other isomers of HCH and other chlorinated pesticides as well.

Isolation and functional analysis of a glycolipid producing Rhodococcus sp. strain IITR03 with potential for degradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT).

A 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) degrading bacterium strain IITR03 producing trehalolipid was isolated and characterized from a pesticides contaminated soil. The strain IITR03 was identified as a member of the genus Rhodococcus based on polyphasic studies. Under aqueous culture conditions, the strain IITR03 degraded 282 μM of DDT and could also utilize 10mM concentration each of 4-chlorobenzoic acid, 3-chlorobenzoic acid and benzoic acid as sole carbon and energy source. The catechol 1,2-dioxygenase enzyme activity resulted in conversion of catechol to form cis,cis-muconic acid. Cloning and sequencing of partial nucleotide sequence of catechol 1,2-dioxygenase gene (cat) from strain IITR03 revealed its similarity to catA gene present in Rhodococcus sp. strain Lin-2 (97% identity) and Rhodococcus strain AN22 (96% identity) degrading benzoate and aniline, respectively. The results suggest that the strain IITR03 could be useful for field bioremediation studies of DDT-residues and chlorinated aromatic compounds present in contaminated sites.

Optimization of UA-DLLME by experimental design methodologies for the simultaneous determination of endosulfan and its metabolites in soil and urine samples by GC–MS

A simple, economical, rapid and sensitive analytical method has been developed for the simultaneous determination of endosulfan (α- and β-) and its metabolites (endosulfan ether, endosulfan hydroxy ether, endosulfan lactone, endosulfan alcohol and endosulfan sulphate) in complex samples, such as soil and urine, based on ultrasound assisted dispersive liquid–liquid microextraction (UA-DLLME) followed by gas chromatography–mass spectrometric (GC–MS) analysis. The method parameters have been optimized using response surface design experiments. Trichloroethylene (TCE) and acetone were chosen as extraction and disperser solvents respectively. After UA-DLLME, the sediment phase obtained was directly analyzed by GC–MS without any further cleanup and preconcentration procedure. Several factors which can affect the UA-DLLME extraction were screened and optimized by 27–4 Plackett–Burman design (PBD) and central composite design (CCD) experiments respectively. Based on these experiments the optimized parameters for UA-DLLME extraction were as follows: extraction solvent, (TCE, 58 μL), disperser solvent (acetone, 1.27 mL) and ionic strength (Na2SO4, 7%, w/v). Intra- and inter-day precision were expressed as percent relative standard deviation (% RSD) and were found to be less than 6.33%. The limit of detection (LOD) of all the analytes in soil and urine were found to be in the range of 0.316–2.494 ng g−1 and 0.049–0.514 ng mL−1 respectively. The proposed method was successfully applied in the analysis of soil samples contaminated with endosulfan. The method may find wide application for the routine determination of endosulfan and its metabolites in environmental and biological samples.

Description of a novel indole-oxidizing bacterium Pseudomonas indoloxydans sp. nov., isolated from a pesticide-contaminated site.

A Gram-negative, deep brown-pigmented Gammaproteobacteria, strain IPL-1(T), capable of oxidizing indole was isolated from a lindane-contaminated site and subjected to a polyphasic taxonomic study. Most of the physiological and biochemical properties, major fatty acids (C(18:1)omega7c, C(16:1)omega7c/iso C(15:0) 2OH and C(16:0)), estimated DNA G+C content (67.2mol%) and 16S rRNA gene sequence analysis showed that strain IPL-1(T) belonged to the genus Pseudomonas. Strain IPL-1(T) exhibited highest 16S rRNA gene sequence similarity with Pseudomonas pseudoalcaligenes (99.0%), followed by Pseudomonas alcaliphila (98.7%), Pseudomonas oleovorans (98.3%), Pseudomonas nitroreducens (98.0%), Pseudomonas mendocina (97.6%) and Pseudomonas stutzeri (97.4%). However, the DNA-DNA relatedness values between strain IPL-1(T) and the closely related taxa were between 22% and 61%. On the basis of differential phenotypic characteristics and genotypic distinctiveness, strain IPL-1(T) should be classified within the genus Pseudomonas as a novel species, for which the name Pseudomonas indoloxydans is proposed. The type strain is IPL-1(T) (=MTCC 8062(T)=JCM 14246(T)).

Biodegradation and dissolution of polyaromatic hydrocarbons by Stenotrophomonas sp.

The aim of this work was to study the biodegradation capabilities of a locally isolated bacterium, Stenotrophomonas sp. strain IITR87 to degrade the polycyclic aromatic hydrocarbons and also check the preferential biodegradation of polycyclic aromatic hydrocarbons (PAHs). From preferential substrate degradation studies, it was found that Stenotrophomonas sp. strain IITR87 first utilized phenanthrene (three membered ring), followed by pyrene (four membered ring), then benzo[α]pyrene (five membered ring). Dissolution study of PAHs with surfactants, rhamnolipid and tritonX-100 showed that the dissolution of PAHs increased in the presence of surfactants.

Improved polycyclic aromatic hydrocarbon degradation in a crude oil by individual and a consortium of bacteria

In this study, we report the ability of Stenotrophomonas maltophilia, Ochrobactrum anthropi, Pseudomonas mendocina, Microbacterium esteraromaticum and Pseudomonas aeruginosa to degrade multiple polycyclic aromatic hydrocarbons (PAHs) present in crude oil. The PAHs in the crude oil sample obtained from Digboi oil refinery, India were estimated to be naphthalene (10.0 mg L-1), fluorene (1.9 mg L-1), phenanthrene (3.5 mg L-1) and benzo(b)fluoranthene (6.5 mg L-1). Exposure of individual bacteria to crude oil showed high rate of biodegradation of specific PAHs by M. esteraromaticum, 81.4%-naphthalene; P. aeruginosa, 67.1%-phenanthrene and 61.0%-benzo(b)fluoranthene; S. maltophilia, 47.9%-fluorene in 45 days. However, consortium of these bacteria showed enhanced biodegradation of 89.1%-naphthalene, 63.8%-fluorene, 81% of phenanthrene and 72.8% benzo(b)fluoranthene in the crude oil. The degradation was further improved up to 10% by consortium on addition of 40 μg mL-1 rhamnolipid JBR-425 biosurfactant. These results suggest that the developed bacterial consortium has significant potential in PAH remediation.

Profiling of biodegradation and bacterial 16S rRNA genes in diverse contaminated ecosystems using 60-mer oligonucleotide microarray

We have developed an oligonucleotide microarray for the detection of biodegradative genes and bacterial diversity and tested it in five contaminated ecosystems. The array has 60-mer oligonucleotide probes comprising 14,327 unique probes derived from 1,057 biodegradative genes and 880 probes representing 110 phylogenetic genes from diverse bacterial communities, and we named it as BiodegPhyloChip. The biodegradative genes are involved in the transformation of 133 chemical pollutants. Validation of the microarray for its sensitivity specificity and quantitation were performed using DNA isolated from well-characterized mixed bacterial cultures also having non-target strains, pure degrader strains, and environmental DNA. Application of the developed array using DNA extracted from five different contaminated sites led to the detection of 186 genes, including 26 genes unique to the individual sites. Hybridization of 16S rRNA probes revealed the presence of bacteria similar to well-characterized genera involved in biodegradation of various pollutants. Genes involved in complete degradation pathways for hexachlorocyclohexane (lin), 1,2,4-trichlorobenzene (tcb), naphthalene (nah), phenol (mph), biphenyl (bph), benzene (ben), toluene (tbm), xylene (xyl), phthalate (pht), Salicylate (sal), and resistance to mercury (mer) were detected with highest intensity. The most abundant genes belonged to the enzyme hydroxylases, monooxygenases, and dehydrogenases which were present in all the five samples. Thus, the array developed and validated here shall be useful in assessing not only the biodegradative potential but also the composition of environmentally useful bacteria, simultaneously, from hazardous ecosystems.

Antibacterial effect and physical properties of chitosan and chlorhexidine-cetrimide-modified glass ionomer cements.

Aims: To compare antibacterial effect and physical properties of chitosan (CH) modified glass ionomer cement (GIC) (10% v/v), chlorhexidine-cetrimide (CHX-CT) modified GIC (2.5/2.5% w/w) and conventional GIC. Materials and Methods: A total of fifty healthy children of age 7–12 years were selected and randomly assigned to class A and B for in vivo analysis. Slabs of CH modified GIC (Group II) along with slabs of conventional GIC (Group I, control) were cemented on buccal surfaces of maxillary molars (split-mouth technique) for class A children. Similarly, slabs of CHX-CT modified GIC (Group III) were cemented against control (Group I, control) in class B children. Slabs were assessed after 48 h for microbial load of Streptococcus mutans and Lactobacillus (LB) on mitis salivarius-bacitracin and Man Rogosa Sharpe agar media, respectively. Agar diffusion test was done to access the antibacterial effect of each group against Streptococcus muatns and LB. Slabs and cylinders of GICs were made for in vitro evaluation of compressive and flexure strength in each group. Results: Comparison was done by nonparametric Kruskal–Wallis analysis followed by Dunns multiple comparison test. Categorical groups were compared by Chi-square test. The increase in antibacterial activity (Group II > III > I) (P < 0.001) and marked increase in compressive and flexure strength (Group II > I > III) were observed. Conclusions: In the view of findings, it is concluded that CH modified GIC would be effective in inhibiting the bacteria associated with dental caries along with improved physical properties when compared with CHX-CT modified GIC and conventional GIC.

Draft Genome Sequence of Alcaligenes faecalis Strain IITR89, an Indole-Oxidizing Bacterium.

ABSTRACT We report the draft genome sequence of Alcaligenes faecalis strain IITR89, a bacterium able to form indigo by utilizing indole as the sole carbon source. The Alcaligenes species is increasingly reported for biodegradation of diverse toxicants and thus complete sequencing may provide insight into biodegradation capabilities and other phenotypes.

The significance of gtf genes in caries expression: A rapid identification of Streptococcus mutans from dental plaque of child patients

Aim: Rapid phylogenetic and functional gene (gtfB) identification of S. mutans from the dental plaque derived from children. Material and Methods: Dental plaque collected from fifteen patients of age group 7-12 underwent centrifugation followed by genomic DNA extraction for S. mutans. Genomic DNA was processed with S. mutans specific primers in suitable PCR condtions for phylogenetic and functional gene (gtfB) identification. The yield and results were confirmed by agarose gel electrophoresis. Results: 1% agarose gel electrophoresis depicts the positive PCR amplification at 1,485 bp when compared with standard 1 kbp indicating the presence of S. mutans in the test sample. Another PCR reaction was set using gtfB primers specific for S. mutans for functional gene identification. 1.2% agarose gel electrophoresis was done and a positive amplication was observed at 192 bp when compared to 100 bp standards. Conclusion: With the advancement in molecular biology techniques, PCR based identification and quantification of the bacterial load can be done within hours using species-specific primers and DNA probes. Thus, this technique may reduce the laboratory time spend in conventional culture methods, reduces the possibility of colony identification errors and is more sensitive to culture techniques.