Satyendra Kumar Garg

Chromate tolerant bacteria isolated from tannery effluent

The occurrence of metal tolerant and antibiotic resistant organisms was investigated in tannery effluent. Seventy-seven isolates comprising heterotrophs (41) and coliforms (36) which were tolerant to chromate level of > 50 microg/ml were selected for detailed study. The majority of the coliforms were resistant to higher levels of chromate (200 microg/ml) whereas around 3% of the heterotrophs were resistant to Cr6+ at a level of > 150 microg/ml. All chromate tolerant heterotrophs were also tolerant to Cu2+ (100%) whereas only 58.53% coliforms were tolerant to Cu2+. Except in the case of Cd2+ a higher number of heterotrophs were found tolerant to other heavy metals tested. Both groups of isolates were found sensitive to mercury. Resistance to cephaloridine was more abundant (P < 0.001) in coliforms as compared to heterotrophs. On the other hand a significantly higher number (P < 0.01) of heterotrophs showed resistance to streptomycin and carbencillin. All coliforms were sensitive to chloramphenicol. Around 80%) and 31.70% of coliforms and heterotrophs exhibited a relationship to the combination of metals and antibiotics. Both heterotrophs and coliforms tolerant to Hg2+ were also resistant to polymixin-B.

Decolorization of Pulp-Paper Mill Effluents by White-Rot Fungi

ABSTRACT: Phenolic effluents are waste products of pulp and paper, coal conversion, dying, textile, and olive oil industries. Such effluents impose coloration and toxicity problems in the receiving waters, causing serious environmental hazards. The pulp and paper mill effluent is highly colored, imparting black/brown color to the water body. The color is mainly due to lignin and its derivatives released during various stages in the paper-making process. The complex nature of such lignin compounds and their phenolic nature make them extremely resistant to microbial degradation. Conventional treatment methods such as aerated lagoons and activated sludge process are ineffective in removing color. However, physical and chemical treatment methods, including ultrafiltration, ion-exchange, and lime precipitation, are expensive and less efficient. Therefore, alternate low-cost biotreatment processes are now being considered, most of which are based on lignin-degrading fungi. Depending on the treatment process, th...

An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease.

BackgroundMany workers have reported halotolerant bacteria from saline conditions capable of protease production. However, antibiotic resistance and heavy metal tolerance pattern of such organisms is not documented very well. Similarly, only a few researchers have reported the pattern of pH change of fermentation medium during the course of protease production. In this study, we have isolated a halotolerant Bacillus cereus SIU1 strain from a non-saline environment and studied its antibiotic and heavy metal resistance pattern. The isolate produces a thermoalkaline protease and changes the medium pH during the course of fermentation. Thermostability of protease was also studied for 30 min.ResultsSeventy bacterial strains isolated from the soils of Eastern Uttar Pradesh, India were screened for protease production. All of them exhibited protease activity. However, 40% bacterial isolates were found good protease producers as observed by caseinolytic zones on milk agar plates. Among them, culture S-4 was adjudged as the best protease producer, and was identified as Bacillus cereus by morphological, biochemical and 16 S rDNA sequence analyses. The isolate was resistant to heavy metals (As2+, Pb2+, Cs1+) and antibiotics (penicillin, lincomycin, cloxacillin, pefloxacin). Its growth behavior and protease production was studied at 45°C and pH 9.0. The protease units of 88 ml-1 were noted in unoptimized modified glucose yeast extract (GYE) medium during early stationary phase at 20 h incubation period. The enzyme was stable in the temperature range of 35°-55°C.ConclusionsAn antibiotic and heavy metal resistant, halotolerant Bacillus cereus isolate is capable of producing thermoalkaline protease, which is active and stable at pH 9.0 and 35°-55°C. This isolate may be useful in several industrial applications owing to its halotolerance and antibiotic and heavy metal resistance characteristics.

Comparative one-factor-at-a-time, response surface (statistical) and bench-scale bioreactor level optimization of thermoalkaline protease production from a psychrotrophic Pseudomonas putida SKG-1 isolate

BackgroundProduction of alkaline protease from various bacterial strains using statistical methods is customary now-a-days. The present work is first attempt for the production optimization of a solvent stable thermoalkaline protease by a psychrotrophic Pseudomonas putida isolate using conventional, response surface methods, and fermentor level optimization.ResultsThe pre-screening medium amended with optimized (w/v) 1.0% glucose, 2.0% gelatin and 0.5% yeast extract, produced 278 U protease ml-1 at 72 h incubation. Enzyme production increased to 431 Uml-1 when Mg2+ (0.01%, w/v) was supplemented. Optimization of physical factors further enhanced protease to 514 Uml-1 at pH 9.0, 25°C and 200 rpm within 60 h. The combined effect of conventionally optimized variables (glucose, yeast extract, MgSO4 and pH), thereafter predicted by response surface methodology yielded 617 U protease ml-1 at glucose 1.25% (w/v), yeast extract 0.5% (w/v), MgSO4 0.01% (w/v) and pH 8.8. Bench-scale bioreactor level optimization resulted in enhanced production of 882 U protease ml-1 at 0.8 vvm aeration and 150 rpm agitation during only 48 h incubation.ConclusionsThe optimization of fermentation variables using conventional, statistical approaches and aeration/agitation at fermentor level resulted in ~13.5 folds increase (882 Uml-1) in protease production compared to un-optimized conditions (65 Uml-1). This is the highest level of thermoalkaline protease reported so far by any psychrotrophic bacterium.

Strategies for Chromium Bioremediation of Tannery Effluent

Bioremediation offers the possibility of using living organisms (bacteria, fungi, algae,or plants), but primarily microorganisms, to degrade or remove environmental contaminants, and transform them into nontoxic or less-toxic forms. The major advantages of bioremediation over conventional physicochemical and biological treatment methods include low cost, good efficiency, minimization of chemicals, reduced quantity of secondary sludge, regeneration of cell biomass, and the possibility of recover-ing pollutant metals. Leather industries, which extensively employ chromium compounds in the tanning process, discharge spent-chromium-laden effluent into nearby water bodies. Worldwide, chromium is known to be one of the most common inorganic contaminants of groundwater at pollutant hazardous sites. Hexavalent chromium poses a health risk to all forms of life. Bioremediation of chromium extant in tannery waste involves different strategies that include biosorption, bioaccumulation,bioreduction, and immobilization of biomaterial(s). Biosorption is a nondirected physiochemical interaction that occurs between metal species and the cellular components of biological species. It is metabolism-dependent when living biomass is employed, and metabolism-independent in dead cell biomass. Dead cell biomass is much more effective than living cell biomass at biosorping heavy metals, including chromium. Bioaccumulation is a metabolically active process in living organisms that works through adsorption, intracellular accumulation, and bioprecipitation mechanisms. In bioreduction processes, microorganisms alter the oxidation/reduction state of toxic metals through direct or indirect biological and chemical process(es).Bioreduction of Cr6+ to Cr3+ not only decreases the chromium toxicity to living organisms, but also helps precipitate chromium at a neutral pH for further physical removal,thus offering promise as a bioremediation strategy. However, biosorption, bioaccumulation, and bioreduction methods that rely on free cells for bioremediation suffer from Cr6 toxicity, and cell damage. Therefore, immobilization of microbial cell biomass enhances bioremediation and renders industrial bioremediation processes more economically viable from reduced free-cells toxicity, easier separation of biosorbents from the tannery effluent, ability to achieve multiple biosorption cycles, and desorption (elution) of metal(s) from matrices for reuse. Thus, microbial bioremediation can be a cost competitive strategy and beneficial bioresource for removing many hazardous contaminants from tannery and other industrial wastes.

Strategies for Decolorization and Detoxification of Pulp and Paper Mill Effluent

The potential hazards associated with industrial effluents, coupled with increasing awareness of environment problems, have prompted many countries to limit the indiscriminate discharge of untreated wastewaters. The pulp and paper industry has been among the most significant of industrial polluters of the waterways, and therefore has been one of the industries of concern. The pulp and paper industry produces large quantities of brown/black effluent that primarily result from pulping, bleaching, and paper-making production stages. The dark color and toxicity of pulp-paper mill effluent comes primarily from lignin and its chlorinated derivatives (e.g., lignosulphonic acid, resins, phenols, and hydrocarbons) that are released during various processing steps of lignocellulosic materials. The color originates from pulping and pulp bleaching stages, while adsorbable organic halides (AOX) originates exclusively from chlorine bleaching. Discharge of untreated effluent results in increased BOD/COD, slime growth, thermal problems, scum formation, discoloration, loss of aesthetic quality and toxicity to the aquatic life, in the receiving waterbodies. The dark brow color of pulp-paper effluent is not only responsible for aesthetic unacceptability, but also prevents the passage of sunlight through colored waterbodies. This reduces the photosynthetic activity of aquatic flora, ultimately causing depletion of dissolved oxygen. The pulp-paper organic waste, coupled with the presence of chlorine, results in the generation of highly chlorinated organic compounds. These toxic constituents of wastewater pose a human health risk through long term exposure. via drinking water and\or through consumption of fish that can bioaccumulate certain pollutants from the food chain. Therefore, considerable attention has been focused by many countries on decolorization of paper mill effluents , along with reduction in the contaminants that pose human health or other environmental hazards. Various physicochemical remediation treatments in the pulp-paper industry are now used, or have been suggested, but often are not implemented, because of the high cost involved. More recently, the paper and pulp industry has been investigating the use of biological remediation steps to replace or augment current treatment strategies. Certain biological treatments offer opportunities to reduce cost (both capital and operating), reduce energy consumption, and minimize environmental impact. Two primary approaches may be effective to curtail release of toxic effluents: first, development of pulping and bleaching processes that emphasize improved oxygen delignification or biopulping, plus partial or complete replacement of chlorine treatment with hydrogen peroxide or with biobleaching; second, implementation of biological processing that involves sequential two-step anaerobic-aerobic or three-step aerobic-anaerobic treatment technologies at end of pipe. The selection of the specific process will depend upon the type of pollutants/toxicants/mutagens present in the effluent. The use of environmental-friendly technologies in the pulp and paper industry is becoming more popular, partly because of increasing regulation, and partly because of the availability of new techniques that can be used to economically deal with pollutants in the effluents. Moreover, biotechnology research methods are offering promise for even greater improvements in the future. The obvious ultimate goal of the industry and the regulators should be zero emission through recycling of industrial wastewater, or discharge of the bare minimum amount of toxicants or color.

Effect of ecological factors on conjugal transfer of chromium-resistant plasmid in Escherichia coli isolated from tannery effluent

The influence of total organic carbon (TOC), pH, and mating temperature on transfer of chromium-resistant plasmid between Escherichia coli strains in terms of variation in the number of transconjugants formed and variation in transfer frequency was investigated. In vitro transfer was studied in five chromate-tolerant E. coli strains isolated from tannery effluent using E. coli K12 J62 (Nalr Lac−) as a recipient. Conjugal transfer of different selection markers was observed in three strains. The study was carried out in sterile wastewater. A gradual decrease was observed both in the number of transconjugants and in transfer frequencies as the concentration of TOC in the mating medium descended from 10,095 to 1.2 mg of C/L, obtaining the maximum values with a TOC concentration of 10,095 mg of C/L. The number of transconjugants and the transfer frequency were maximum at 30°C. However, neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. The strains were also found resistant to different heavy metals and antibiotics. Curing of these strains resulted in loss of one or more resistance markers indicating the plasmid-borne resistance. It is inferred that plasmid transfer by conjugation occurs in wastewater bodies within a wide range of conditions.

Evaluation of Simple Microbiol Tests for Detection of Fecal Coliforms Directly at 44.5 °C

Simple microbial test comprising H2S paper strip test,presence-absence (PA) test, and fluorogenic brila broth (BB)test performed directly at 44.5 °C were evaluated andcompared with the standard most probable number (MPN) method fordetection of fecal coliforms in 173 drinking water sources. BBand PA test were comparable with standard MPN method, whereas,poor compliance was noted for H2S test. PA test whencompared with standard MPN test only 15%; disagreement wasdetected, whereas, highest disagreement of 40%; was observed incase of H2S test. BB test was found to be highly sensitiveas only 7.8% disagreement with that of standard MPN test wasfound. Three hundred cultures obtained from positive tests wereidentified in order to evaluate the specificities of test usedin detection of fecal indicator Escherichia coli. BB testwas also found highly specific in detection of indicatororganism as compared to PA and H2S test. Among theorganisms isolated from BB test 84.4%; of them were identifiedas E. coli as compared to 43.4 and 33.3 in PA and H2Stest, respectively. The low incidence of recovery of E.coli (18.1%) for the standard MPN method places doubt on thevalidity of its application in tropical areas. The result ofthis investigation suggest that BB performed directly at 44.5 °C could be suitable cost effective test to assess themicrobiological quality of drinking water in India and other tropical countries.

Response surface methodology for optimization of process variable for reactive orange 4 dye discoloration by Pseudomonas putida SKG-1 strain and bioreactor trial for its possible use in large-scale bioremediation

AbstractIn this study, Pseudomonas putida SKG-1 isolate employed earlier for pulp-/paper-mill effluent discoloration, was used for bioremediation of reactive orange 4 azo dye under varied cultural and nutritional conditions. The optimization through one-factor-at-a-time approach revealed maximum growth (A620 1.31) and dye discoloration (95.2%) at optimum temperature 35°C, pH 8.0, inoculum dose 5.0%, sucrose 0.7%, peptone 0.25%, and 50 mg reactive orange 4 dye L−1 within 72 h of incubation. Under response surface methodology (RSM; using Box–Behnken design) approach, the dye discoloration enhanced to 97.8% at reactive orange 4 concentration of 50 mg L−1, sucrose 0.7%, and peptone 0.28% during 72 h of incubation. In bioreactor trial, the maximum dye discoloration (98% within 60 h) was achieved in 12 h advance compared to RSMs trial.

Shake-flask and bench-scale stirred tank bioreactor production optimization of a thermoalkaline protease from Bacillus cereus SIU1 using one-factor-at-a-time and response surface (statistical) methodologies

Abstract We report the optimization of production of a halotolerant, thermoalkaline protease by Bacillus cereus SIU1, at shake-flask and bench-scale bioreactor level, using conventional and response surface methods. The basal medium supplemented with optimized (w/v) 0.8% glucose, 1.5% peptone, and 0.4% yeast extract produced 224 Uml− 1 alkaline protease after 20 h incubation. Enzyme yield was further increased to 491 Uml− 1 when the fermentation broth was supplemented with 0.02% (w/v) Ca2+. Optimization of physical factors resulted in still higher protease level of 651 Uml− 1 within 18 h fermentation at initial pH 9.0, 50°C, and 150 rpm agitation. Statistically designed experiments revealed significant effects of peptone and CaCl2 on protease production. A maximum of 749 protease Uml− 1 was produced at optimum factor levels (w/v) of peptone 1.75%, yeast extract 0.4%, CaCl2 0.025%, and pH 9.0 after 18 h incubation. Optimization of agitation and aeration rates in bench-scale bioreactors further enhanced the enzyme yield to 941 protease Uml− 1 at 125 rpm and 2.0 vvm aeration. Optimization of protease production by conventional and statistical approaches resulted in a ∼10.7-fold increase (941 Uml− 1) compared to un-optimized conditions (88 Uml− 1).