Ramesh Chander Kuhad

Microbial Cellulases and Their Industrial Applications

Microbial cellulases have shown their potential application in various industries including pulp and paper, textile, laundry, biofuel production, food and feed industry, brewing, and agriculture. Due to the complexity of enzyme system and immense industrial potential, cellulases have been a potential candidate for research by both the academic and industrial research groups. Nowadays, significant attentions have been devoted to the current knowledge of cellulase production and the challenges in cellulase research especially in the direction of improving the process economics of various industries. Scientific and technological developments and the future prospects for application of cellulases in different industries are discussed in this paper.

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.

Lignocellulose biotechnology: current and future prospects.

Lignocellulose is the most abundant biomass available on Earth. It has attracted considerable attention as an alternate feed stock and energy resource because of the large quantities available and its renewable nature. The potential uses of lignocelluloses are in pulp and paper industries, production of fuel alcohol and chemicals, protein for food, and feed using biotechnological means. The current industrial activity of lignocellulosic biomass fermentation is limited mainly because of the difficulty in economic bioconversion of these materials to value-added products. Considerable improvement in many processes related to lignocellulose biotechnology appeared during the last decade. Current uses of lignocellulosic biomass, process constraints, and areas of future research are discussed here.

Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, a woody substrate, for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498

Prosopis juliflora (Mesquite) is a raw material for long-term sustainable production of cellulosics ethanol. In this study, we used acid pretreatment, delignification and enzymatic hydrolysis to evaluate the pretreatment to produce more sugar, to be fermented to ethanol. Dilute H(2)SO(4) (3.0%,v/v) treatment resulted in hydrolysis of hemicelluloses from lignocellulosic complex to pentose sugars along with other byproducts such as furfural, hydroxymethyl furfural (HMF), phenolics and acetic acid. The acid pretreated substrate was delignified to the extent of 93.2% by the combined action of sodium sulphite (5.0%,w/v) and sodium chlorite (3.0%,w/v). The remaining cellulosic residue was enzymatically hydrolyzed in 0.05 M citrate phosphate buffer (pH 5.0) using 3.0 U of filter paper cellulase (FPase) and 9.0 U of beta-glucosidase per mL of citrate phosphate buffer. The maximum enzymatic saccharification of cellulosic material (82.8%) was achieved after 28 h incubation at 50 degrees C. The fermentation of both acid and enzymatic hydrolysates, containing 18.24 g/L and 37.47 g/L sugars, with Pichia stipitis and Saccharomyces cerevisiae produced 7.13 g/L and 18.52 g/L of ethanol with corresponding yield of 0.39 g/g and 0.49 g/g, respectively.

Optimization of cellulase production by a brown rot fungus Fomitopsis sp. RCK2010 under solid state fermentation

Culture conditions for enhanced cellulase production from a newly isolated brown rot fungus, Fomitopsis sp. RCK2010 were optimized under solid state fermentation. An initial pH of 5.5 and moisture ratio of 1:3.5 (solid:liquid) were found to be optimal for maximum enzyme production. Of the different carbon sources tested wheat bran gave the maximum production of CMCase (71.526 IU/g), FPase (3.268 IU/g), and β-glucosidase (50.696 IU/g). Among the nitrogen sources, urea caused maximum production of CMCase (81.832 IU/g), where as casein and soyabean meal gave the highest FPase (4.682 IU/g) and β-glucosidase (69.083 IU/g) production, respectively. Among amino acids tested glutamic acid gave the highest production for CMCase (84.127I U/g); however 4-hydroxy-l-proline stimulated maximum FPase production (6.762 IU/g). Saccharification of pretreated rice straw and wheat straw by crude enzyme extract from Fomitopsis sp. RCK2010 resulted in release of 157.160 and 214.044 mg/g of reducing sugar, respectively.

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.

Bioethanol production from Gracilaria verrucosa, a red alga, in a biorefinery approach.

In this study, Gracilaria verrucosa, red seaweed has been used for production of agar and bioethanol. The algae harvested at various time durations resulted in extraction of ~27-33% agar. The leftover pulp was found to contain ~62-68% holocellulose, which on enzymatic hydrolysis yielded 0.87 g sugars/g cellulose. The enzymatic hydrolysate on fermentation with Saccharomyces cerevisiae produced ethanol with an ethanol yield of 0.43 g/g sugars. The mass balance evaluation of the complete process demonstrates that developing biorefinery approach for exploiting Gracilaria verrucosa, a red alga, could be commercially viable.

Bioethanol production from Lantana camara (red sage): Pretreatment, saccharification and fermentation

Lantanacamara contains 61.1% (w/w) holocellulose and can serve as a low-cost feedstock for bioethanol production. Acid hydrolysis (3.0%, v/v H(2)SO(4), 120 degrees C for 45 min) of L. camara produced 187.14 mg/g total sugars along with fermentation inhibitors such as phenolics (8.2mg/g), furfurals (5.1mg/g) and hydroxy methyl furfurals (6.7 mg/g). Sequential application of overliming (pH 10.0) and activated charcoal (1.5%, w/v) adsorption was used to remove these toxic compounds from the acid hydrolysate. The acid-pretreated biomass of L. camara was further delignified through combined pretreatment of sodium sulphite (5.0% w/v) and sodium chlorite (3.0% w/v), which resulted in about 87.2% lignin removal. The enzymatic hydrolysis of delignified cellulosic substrate showed 80.0% saccharification after 28 h incubation at 50 degrees C and pH 5.0. Fermentation of acid and enzymatic hydrolysates with Pichiastipitis and Saccharomycescerevisiae gave rise to 5.16 and 17.7 g/L of ethanol with corresponding yields of 0.32 and 0.48 g/g after 24 and 16 h, respectively.

Advances in Applied Bioremediation

Biological Remediation of Soil: An Overview of Global Market and Available Technologies.- Local Gain, Global Loss: The Environmental Cost of Action.- Bioavailability of Contaminants in Soil.- Biosurfactants in Bioremediation.- The Diversity of Soluble Di-iron Monooxygenases with Bioremediation Applications.- Bioremediation of Polluted Soil.- Soil Bioremediation Strategies Based on the Use of Fungal Enzymes.- Anaerobic Metabolism and Bioremediation of Explosives-Contaminated Soil.- Biological Remediation of Petroleum Contaminants.- Bioremediation of Benzene-contaminated Underground Aquifers.- Microbial Remediation of Metals in Soils.- Transformations of Toxic Metals and Metalloids by Pseudomonas stutzeri Strain KC and its Siderophore Pyridine-2,6-bis(thiocarboxylic acid).- Biomining Microorganisms: Molecular Aspects and Applications in Biotechnology and Bioremediation.- Advances in Phytoremediation and Rhizoremediation.- Phytoremediation for Oily Desert Soils.- Heavy Metal Phytoremediation: Microbial Indicators of Soil Health for the Assessment of Remediation Efficiency.- The Environment and the Tools in Rhizo- and Bioremediation of Contaminated Soil.- Molecular Tools for Monitoring and Validating Bioremediation.

Properties and application of a partially purified alkaline xylanase from an alkalophilic fungus Aspergillus nidulans KK-99.

An alkalophilic Aspergillus nidulans KK-99 produced an alkaline, thermostable xylanase (40 IU/ml) in a basal medium supplemented with wheat bran (2% w/v) and KNO3 (at 0.15% N) pH 10.0 and 37 degrees C. The partially purified xylanase was optimally active at pH 8.0 and 55 degrees C. The xylanase was stable in a broad pH range of 4.0-9.5 for 1 h at 55 degrees C, retaining more than 80% of its activity. The enzyme exhibited greater binding affinity for xylan from hardwood than from softwood. The xylanase activity was stimulated (+25%) by Na+ and Fe2+ and was strongly inhibited (maximum by 70%) by Tween-20, 40, 60, SDS, acetic anhydride, phenylmethane sulphonyl fluoride, Triton-X-100. The xylanase dose of 1.0 IU/g dry weight pulp gave optimum bleach boosting of Kraft pulp at pH 8.0 and temperature 55 degrees C for 3 h reaction time.