Banwari Lal

EVALUATION OF INOCULUM ADDITION TO STIMULATE IN SITU BIOREMEDIATION OF OILY-SLUDGE-CONTAMINATED SOIL

ABSTRACT A full-scale study evaluating an inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil was conducted at an oil refinery where the indigenous population of hydrocarbon-degrading bacteria in the soil was very low (103 to 104 CFU/g of soil). A feasibility study was conducted prior to the full-scale bioremediation study. In this feasibility study, out of six treatments, the application of a bacterial consortium and nutrients resulted in maximum biodegradation of total petroleum hydrocarbon (TPH) in 120 days. Therefore, this treatment was selected for the full-scale study. In the full-scale study, plots A and B were treated with a bacterial consortium and nutrients, which resulted in 92.0 and 89.7% removal of TPH, respectively, in 1 year, compared to 14.0% removal of TPH in the control plot C. In plot A, the alkane fraction of TPH was reduced by 94.2%, the aromatic fraction of TPH was reduced by 91.9%, and NSO (nitrogen-, sulfur-, and oxygen-containing compound) and asphaltene fractions of TPH were reduced by 85.2% in 1 year. Similarly, in plot B the degradation of alkane, aromatic, and NSO plus asphaltene fractions of TPH was 95.1, 94.8, and 63.5%, respectively, in 345 days. However, in plot C, removal of alkane (17.3%), aromatic (12.9%), and NSO plus asphaltene (5.8%) fractions was much less. The population of introduced Acinetobacter baumannii strains in plots A and B was stable even after 1 year. Physical and chemical properties of the soil at the bioremediation site improved significantly in 1 year.

In Situ Bioremediation Potential of an Oily Sludge-Degrading Bacterial Consortium

A field-scale study was conducted in a 4000 m2 plot of land contaminated with an oily sludge by use of a carrier-based hydrocarbon-degrading bacterial consortium for bioremediation. The land belonged to an oil refinery. Prior to this study, a feasibility study was conducted to assess the capacity of the bacterial consortium to degrade oily sludge. The site selected for bioremediation contained approximately 300 tons of oily sludge. The plot was divided into four blocks, based on the extent of contamination. Blocks A, B, and C were treated with the bacterial consortium, whereas Block D was maintained as an untreated control. In Block A, at time zero, i.e., at the beginning of the experiment, the soil contained as much as 99.2 g/kg of total petroleum hydrocarbon (TPH). The application of a bacterial consortium (1 kg carrier-based bacterial consortium/10 m2 area) and nutrients degraded 90.2% of the TPH in 120 days, whereas in block D only 16.8% of the TPH was degraded. This study validates the large-scale use of a carrier-based bacterial consortium and nutrients for the treatment of land contaminated with oily sludge, a hazardous hydrocarbon waste generated by petroleum industry.

Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites.

ABSTRACT The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.

Degradation of Polycyclic Aromatic Hydrocarbons by a Newly Discovered Enteric Bacterium, Leclercia adecarboxylata

ABSTRACT A bacterial strain, PS4040, capable of degrading polycyclic aromatic hydrocarbons for use as the sole carbon source was isolated from oily-sludge-contaminated soil. The 16S rRNA gene showed 98.8% homology to that of Leclercia adecarboxylata. Comparative molecular typing with the clinical strain of L. adecarboxylata revealed that there were few comigrating and few distinct amplimers among them.

Prevalence of Duodenal Ulcer‐Promoting Gene (dupA) of Helicobacter pylori in Patients with Duodenal Ulcer in North Indian Population

Background:   The duodenal ulcer (DU)‐promoting gene (dupA) of Helicobacter pylori has been identified as a novel virulent marker associated with an increased risk for DU. The presence or absence of dupA gene of H. pylori present in patients with DU and functional dyspepsia in North Indian population was studied by polymerase chain reaction (PCR) and hybridization analysis.

Rapid detection and quantification of bisulfite reductase genes in oil field samples using real-time PCR

Sulfate-reducing bacteria (SRB) pose a serious problem to offshore oil industries by producing sulfide, which is highly reactive, corrosive and toxic. The dissimilatory sulfite reductase (dsr) gene encodes for enzyme dissimilatory sulfite reductase and catalyzes the conversion of sulfite to sulfide. Because this gene is required by all sulfate reducers, it is a potential candidate as a functional marker. Denaturing gradient gel electrophoresis fingerprints revealed the presence of considerable genetic diversity in the DNA extracts achieved from production water collected from various oil fields. A quantitative PCR (qPCR) assay was developed for rapid and accurate detection of dsrB in oil field samples. A standard curve was prepared based on a plasmid containing the appropriate dsrB fragment from Desulfomicrobium norvegicum. The quantification range of this assay was six orders of magnitude, from 4.5 x 10(7) to 4.5 x 10(2) copies per reaction. The assay was not influenced by the presence of foreign DNA. This assay was tested against several DNA samples isolated from formation water samples collected from geographically diverse locations of India. The results indicate that this qPCR approach can provide valuable information related to the abundance of the bisulfite reductase gene in harsh environmental samples.

Diversity of culturable sulfidogenic bacteria in two oil–water separation tanks in the north-eastern oil fields of India

Sulfidogenic communities in the production waters of onshore oil fields in north-eastern India were examined using a culturing approach. Production water samples were inoculated into medium selective for Sulfate reducing bacteria (SRB) and Thiosulfate Reducing Bacteria (TRB). The total number of viable sulfidogenic microorganisms in the samples obtained from the two production water tanks was approximately 10(5) MPN ml(-1) (most probable number per ml). Most of the isolates were thermo-tolerant and could be grown between 40 and 45 degrees C. Hydrogen sulfide production by TRB was significantly higher than by SRB. Based on 16S rRNA gene sequencing, the isolates were grouped in nine different phylotypes. Phylogenetic analysis indicated that most of the SRB were affiliated with the phylum Proteobacteria, encompassing Gram-negative bacteria, belonging to the genera Desulfovibrio, Desulfomicrobium, and Desulfobulbus. However, five isolates grouped with the genus Desulfotomaculum were found to be gram-positive SRB. Most of the thiosulfate reducing isolates was affiliated with the phylum Firmicutes, including Clostridium and Fusibacter and also with the phylum Proteobacteria, including the genera Enterobacter and Citrobacter. Phylotypes related to Clostridium (69%) and Desulfovibrio (53%) dominated the community in the production water samples. This study demonstrates the diversity of the TRB and SRB that play a critical role in the souring mediated corrosion of the oil-water separation tanks in the north-eastern India oil fields.

Isolation of a novel yeast strain Candida digboiensis TERI ASN6 capable of degrading petroleum hydrocarbons in acidic conditions.

A novel yeast species Candida digboiensis TERI ASN6 was isolated from soil samples contaminated with acidic oily sludge (pH 1-3) from the Digboi refinery (Northeast India). The strain TERI ASN6 could degrade 73% of the total petroleum hydrocarbons present in the medium at pH 3 in a week. This strain presents a dimorphic behaviour and showed mycelia morphology when grown under stressed conditions such as low pH and in a medium containing petroleum hydrocarbons. The C. digboiensis strain could efficiently degrade the aliphatic and aromatic fractions of the acidic oily sludge at pH 3 as confirmed by gas chromatography. During the growth of TERI ASN6 in dibenzothiophene (DBT), DBT-sulfone and biphenyl-2-ol were detected. An active cytochrome P450 system, implicated in hydrocarbon oxidation, was also detected in this yeast using degenerated primers based on its conserved regions. This yeast is a potential candidate for petroleum bioremediation treatment of hydrocarbon contaminated acidic soils. Its physiological behaviour allows the strain to work efficiently where other hydrocarbon-degrading bacteria may not survive.

Biodegradation of asphalt by Garciaella petrolearia TERIG02 for viscosity reduction of heavy oil

Petroleum hydrocarbon is an important energy resource, but it is difficult to exploit due to the presence of dominated heavy constituents such as asphaltenes. In this study, viscosity reduction of Jodhpur heavy oil (2,637 cP at 50°C) has been carried out by the biodegradation of asphalt using a bacterial strain TERIG02. TERIG02 was isolated from sea buried oil pipeline known as Mumbai Uran trunk line (MUT) located on western coast of India and identified as Garciaella petrolearia by 16S rRNA full gene sequencing. TERIG02 showed 42% viscosity reduction when asphalt along with molasses was used as a sole carbon source compared to only asphalt (37%). The viscosity reduction by asphaltene degradation has been structurally characterized by Fourier transform infrared spectroscopy (FTIR). This strain also shows an additional preference to degrade toxic asphalt and aromatics compounds first unlike the other known strains. All these characteristics makes TERIG02 a potential candidate for enhanced oil recovery and a solution to degrading toxic aromatic compounds.

Enhanced performance of sulfate reducing bacteria based biocathode using stainless steel mesh on activated carbon fabric electrode

An anoxic biocathode was developed using sulfate-reducing bacteria (SRB) consortium on activated carbon fabric (ACF) and the effect of stainless steel (SS) mesh as additional current collector was investigated. Improved performance of biocathode was observed with SS mesh leading to nearly five folds increase in power density (from 4.79 to 23.11 mW/m(2)) and threefolds increase in current density (from 75 to 250 mA/m(2)). Enhanced redox currents and lower Tafel slopes observed from cyclic voltammograms of ACF with SS mesh indicated the positive role of uniform electron collecting points. Differential pulse voltammetry technique was employed as an additional tool to assess the redox carriers involved in bioelectrochemical reactions. SRB biocathode was also tested for reduction of volatile fatty acids (VFA) present in the fermentation effluent stream and the results indicated the possibility of integration of this system with anaerobic fermentation for efficient product recovery.