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Molecular Characterization of camphor utilizing bacterial isolates from refinery sludge and detection of target loci-Cytochrome P-450 cam mono oxygenase (cam C gene) by PCR and gene probe.

ABSTRACT: This study presents the isolation and molecular characterization of bacterial strains utilizing 1, 7, 7-Trimethylbicyclo (2.2.1) heptane-2-one (camphor) as a sole source of carbon, isolated from the biomass sludge sample collected from an effluent treatment plant of Mathura Refinery Limited (MRL), India. Initial screening was carried out where the 16S rDNA PCR was performed using reported eubacterial primer set followed by Amplified Ribosomal DNA Restriction Analysis (ARDRA). About 47% of the isolates have shown unique ARDRA pattern based on which, 15 distinct isolates were selected and tested for the presence of cam C gene that was successfully demonstrated by PCR using gene specific primers. A Dot-Blot experiment was designed to detect the cam C loci in the plasmid DNA of all camphor isolates based on non-radioactive "Biotin-Streptavidin" detection system. The bacterial identity with respect to partial 16S rDNA gene sequences of all camphor isolates placed them in 9 major genera viz., Pseudomonas sp., Staphylococcus sp., Alcaligenes sp., Agromyces sp., Stenotrophomonas sp., Reichenowia sp., Achromobacter sp., Brevibacterium sp. and Pseudaminobacter sp. A detailed phylogentic tree was also constructed to establish their evolutionary status from the gene sequence data.

SUBMITTER: Bhuvaneswari G

PROVIDER: S-EPMC3647104 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Molecular Characterization of camphor utilizing bacterial isolates from refinery sludge and detection of target loci-Cytochrome P-450 cam mono oxygenase (cam C gene) by PCR and gene probe.

Bhuvaneswari Ganesan G

SpringerPlus 20130417 1

This study presents the isolation and molecular characterization of bacterial strains utilizing 1, 7, 7-Trimethylbicyclo (2.2.1) heptane-2-one (camphor) as a sole source of carbon, isolated from the biomass sludge sample collected from an effluent treatment plant of Mathura Refinery Limited (MRL), India. Initial screening was carried out where the 16S rDNA PCR was performed using reported eubacterial primer set followed by Amplified Ribosomal DNA Restriction Analysis (ARDRA). About 47% of the is ...[more]

PMID: 23667817

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Project description:BackgroundSustainable management of voluminous and hazardous oily sludge produced by petroleum refineries remains a challenging problem worldwide. Characterization of microbial communities of petroleum contaminated sites has been considered as the essential prerequisite for implementation of suitable bioremediation strategies. Three petroleum refinery sludge samples from North Eastern India were analyzed using next-generation sequencing technology to explore the diversity and functional potential of inhabitant microorganisms and scope for their on-site bioremediation.ResultsAll sludge samples were hydrocarbon rich, anaerobic and reduced with sulfate as major anion and several heavy metals. High throughput sequencing of V3-16S rRNA genes from sludge metagenomes revealed dominance of strictly anaerobic, fermentative, thermophilic, sulfate-reducing bacteria affiliated to Coprothermobacter, Fervidobacterium, Treponema, Syntrophus, Thermodesulfovibrio, Anaerolinea, Syntrophobacter, Anaerostipes, Anaerobaculum, etc., which have been well known for hydrocarbon degradation. Relatively higher proportions of archaea were detected by qPCR. Archaeal 16S rRNA gene sequences showed presence of methanogenic Methanobacterium, Methanosaeta, Thermoplasmatales, etc. Detection of known hydrocarbon utilizing aerobic/facultative anaerobic (Mycobacterium, Pseudomonas, Longilinea, Geobacter, etc.), nitrate reducing (Gordonia, Novosphigobium, etc.) and nitrogen fixing (Azovibrio, Rhodobacter, etc.) bacteria suggested niche specific guilds with aerobic, facultative anaerobic and strict anaerobic populations. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) predicted putative genetic repertoire of sludge microbiomes and their potential for hydrocarbon degradation; lipid-, nitrogen-, sulfur- and methane- metabolism. Methyl coenzyme M reductase A (mcrA) and dissimilatory sulfite reductase beta-subunit (dsrB) genes phylogeny confirmed methanogenic and sulfate-reducing activities within sludge environment endowed by hydrogenotrophic methanogens and sulfate-reducing Deltaproteobacteria and Firmicutes members.ConclusionRefinery sludge microbiomes were comprised of hydrocarbon degrading, fermentative, sulfate-reducing, syntrophic, nitrogen fixing and methanogenic microorganisms, which were in accordance with the prevailing physicochemical nature of the samples. Analysis of functional biomarker genes ascertained the activities of methanogenic and sulfate-reducing organisms within sludge environment. Overall data provided better insights on microbial diversity and activity in oil contaminated environment, which could be exploited suitably for in situ bioremediation of refinery sludge.

Dataset Information

Molecular Characterization of camphor utilizing bacterial isolates from refinery sludge and detection of target loci-Cytochrome P-450 cam mono oxygenase (cam C gene) by PCR and gene probe.

Publications

Molecular Characterization of camphor utilizing bacterial isolates from refinery sludge and detection of target loci-Cytochrome P-450 cam mono oxygenase (cam C gene) by PCR and gene probe.

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