P. Khanna

Enhanced cellulase production by a mutant of Trichoderma reesei

The need for renewable energy resources has focused attention on the renewable polysaccharide cellulose, which can be enzymatically hydrolyzed to yield sugars. Trichoderma reesei QM9414, a cellulolytic microorganism, was subjected to mutagenesis using a combination of ultraviolet light and sodium nitrite (45 min, 0.5 mg ml−1). A cellulase hyperproducing mutant, whose enzyme is more resistant to catabolite repression by glucose and cellobiose, was isolated. Enhancement in cellulase yields (1.5- to 1.75-fold) and hydrolytic potential (1.2-fold) was achieved. The mild chemical mutagen used in this research has not been reported before.

Osmotolerance and hydrocarbon degradation by a genetically engineered microbial consortium

Abstract Most bacteria characterized as hydrocarbon degrading are isolated from the terrestrial environment and have very limited or no application under estuarine/hyper-saline conditions. The collective efforts of these hydrocarbon-degrading bacteria help in the preservation of the environment under man-made disasters. However, the process has a long lag phase, since the contaminated ecosystem undergoes a process of natural selection of efficient hydrocarbon degrading bacteria. This paper uses a microbial consortium designed from a library of soil isolates, composed of four members; strain Pseudomonas NCC.DSS6, P. NCC.DSS8, P. NCC.GSS3 and P. putida, which can attack various fractions of crude oil. To extend the capacity of the consortium for degradation of hydrocarbons in marine environments or estuarine conditions the key is the requirement of an osmotolerance function. Hence, in the present study, to provide osmotolerance to these soil isolates, the E.coli pro U operon was subcloned into a broad-host range vector and transferred into the members of the microbial consortium. The non-specific basal expression of the pro U operon, under the control of tac-lac promoter was demonstrated by the ability of the transformed organisms to grow under hyper-saline conditions. The degradation capacity of the engineered consortium was also studied using a designed model petroleum mixture. The study underscores the potential of the osmoregulatory function for degradation of anthropogenic molecules in marine niches.

De-emulsification of oil-in-water emulsions by Bacillus subtilis

Bacillus subtilis grown on glucose produced a de-emulsifier that broke down model oil-in-water emulsions. The deemulsification increased with contact time and the recovery of oil was 98% with 0.6 mg de-emulsifier/ml emulsion. GLC and i.r. analysis of the molecule extracted from cell-free broth demonstrated that the de-emulsifier was acetoin. It may be better to use acetoin, a metabolite of glucose metabolism, to recover oil from oily sludges instead of microbial cells, because of its stability and de-emulsification activity.

Techno-economic evaluation of soil-aquifer treatment using primary effluent at Ahmedabad, India.

A pilot study was carried out in Sabarmati River bed at Ahmedabad, India for renovation of primary treated municipal wastewater through soil aquifer treatment (SAT) system. The infrastructure for the pilot SAT system comprised of two primary settling basins, two infiltration basins and two production wells located in the centre of infiltration basins for pumping out renovated wastewater. The performance data indicated that SAT has a very good potential for removal of organic pollutants, nutrients as well as bacteria and viruses. The SAT system was found to be more efficient and economical than the conventional wastewater treatment systems and hence recommended for adoption. A salient feature of the study is the introduction of biomat concept and its contribution in the overall treatment process.

Microbial production of 2,3-butanediol from water hyacinth

Abstract A scheme for microbial production of 2,3-butanediol from water hyacinth (WH), a nuisance weed, is presented. It consists in sun-drying WH followed by crushing, alkaline treatment at high temperature, enzymatic hydrolysis, and fermentation. A caustic soda concentration of 1% and steam pressure of 1 kg/cm 2 for 1 h were optimal for delignification and yielded 35 g delignified water hyacinth (DLWH)/100 g crude water hyacinth. A slurry concentration of 5%, an enzyme concentration yielding filter-paper cellulase activity (FPCA) of 14 IU/g DLWH, a temperature of 50°C, a pH of 4·8, and an incubation period of 48 h were optimal for enzymatic hydrolysis yielding 19 g/litre reducing sugars. Prefermentation hydrolysis followed by combined hydrolysis and fermentation (PH-CHF) was most efficient and yielded 15 g 2,3-butanediol per 100 g DLWH. An HRT of 4 h corresponding to a productivity of 1·4 g/litre/h of 2,3-butanediol with anaerobic-fixed-film and upflowanaerobic-sludge-blanket (UASB) reactor systems was achieved on WH hydrolysate in a continuous mode.

Stress Survival of a Genetically Engineered Pseudomonas in Soil Slurries: Cytochrome P-450cam-Catalyzed Dehalogenation of Chlorinated Hydrocarbons

Biological treatment of hazardous chemical wastes has potential as an effective, practical, and economically viable process in above the ground treatment systems that consist of both genetically engineered microorganisms (GEMs) and bioreactors with process control instruments to create ideal conditions for biodegradation. A strain of Pseudomonas putida coexpressing cytochrome P‐450cam and luciferase (lux) that provides both the reductive detoxification potential of the hemoprotein and a mechanism for its reduction in the absence of “normal” P‐450 redox partners was evaluated for its ability to survive and remain metabolically competent under nutrient stress in soil slurry microcosms. More than 74% of the cells of engineered Pseudomonas were culturable after 7 days of multiple nutrient (C,N,P) starvation. The diagnostic luminescence and carbon monoxide‐difference spectra for the two engineered traits could be detected in a significant fraction of the surviving population. The GEM could be revived after repeated desiccation and starvation using Luria broth, benzoate, or citrate as nutrients. Soil slurries inoculated with the GEM transformed hexachloroethane (HCE) to tetrachloroethylene (tetraCE) 8–10‐fold faster than uninoculated slurries. The GEM also transformed the insecticide, γ‐HCH (γ‐3,4,5,6‐hexachlorocyclhexene), to γ‐3,4,5,6‐tetrachlorocyclohexene (γtetraCH) in soil slurries under subatmospheric conditions. These results indicate that GEMs can be constructed with broad substrate range detoxification catalysts such as cytochrome P‐450 for remediation.

A simple elution and reconcentration technique for viruses concentrated on membrane filters from drinking water samples

Abstract An optimum concentration of urea (1.5 M)-arginine phosphate (0.2:0.008 M) buffer (U-APB) has been designed in this research as an eluent at pH 9.0 for effective desorption and elution of viruses from negatively charged membrane filters. The primary eluate is further reconcentrated by the precipitation of MgHPO 4 (s) on addition of MgCl 2 . The flocs are centrifuged, the pellet is dissolved in McIlvaines buffer (pH 5) and neutralized with sodium bicarbonate (8.8%) prior to assay on cell culture. The efficacy of the method has been tested at different inoculum levels and also for different volumes of water samples seeded with viruses. U-APB gives 92–100 and 88–93% recovery for Poliovirus 1 and bacteriophage, respectively, as against a 30–40% recovery of Poliovirus 1 and bacteriophage, respectively, and a meagre 30–40% recovery of Poliovirus 1 in the organic flocculation of beef extract method presently in use. Further, the eluent (U-APB) is easy to constitute and it performs elution and reconcentration of viruses without any pH adjustment.

Concentration and detection of rotavirus in water samples using polymerase chain reaction during a gastroenteritis epidemic outbreak in Madras city

Water samples collected from different parts of North Madras city revealed the presence of rotavirus genome in 7 out of 9 water samples analysed using reverse transcription polymerase chain reaction (RT‐PCR) during gastroenteritis epidemic outbreak. Retrospective epidemiological studies brought out that the outbreak occurred due to ineffective sewerage system and surface run off contamination of water supplies during the rainy season.

Evaluation of urea-arginine phosphate buffer (U-APB) for reconcentration of viruses in field samples

Urea‐Arginine Phosphate Buffer (U‐APB), a composition identified by the authors for virus elution and reconcentration through membrane filter, is readily available and appropriate for concentration process during viral assay in drinking water samples. In the present study, contaminated lake water samples have been analysed through a method incorporating U‐APB. IR spectral analyses of precipitates indicate the presence of HPO4 band and that the adsorption of viruses during reconcentration step is attributable to formation of MgHPO4 in the reconcentration processes.

A SIMPLE AND RAPID TECHNIQUE FOR CONCENTRATION OF ENTAMOEBA HISTOLYTICA CYSTS FROM CONTAMINATED WATER

Entamoeba histolytica, the causative agent of amebiasis, has world wide distribution. A simple and rapid technique was developed for the concentration of Entamoeba histolytica cysts from contaminated water. The efficiency of the procedure is 80% and it requires five hours. The procedure was used to determine Entamoeba histolytica cysts in water samples from various sampling points on river Kanhan and the lakes in and around Nagpur City. The cyst recovery in environmental samples varied from 0.3 to 31 cysts/L. This methodology can prove useful for rapid detection of the cysts in contaminated water by microscopic or nucleic acid probe techniques.