Gainful Utilisation Of Crop Residues/Waste Materials
Conversion of cellulosic wastes in to value added products:Processes for conversion of cellulosic plant materials such as rice straw, sugarcane bagasse, groundnut shells, corncobs, etc. to fungal biomass (single cell protein with 30-40% crude protein) were developed. A process for conversion of alkali-treated rice straw to SCP, by growing Penicillium chrysogenum was scaled up to pilot plant stage. Cellulase enzymes were also produced,by utilizing different plant materials. All these processes have high potential for commercial exploitation.
Conversion of starchy wastes in to value added products:
Processes for saccharification of cassava fibrous waste (tippi) obtained after starch extraction were developed by (i) acid hydrolysis (ii) acid-enzyme hydrolysis and (iii) enzyme-enzyme hydrolysis to obtain a reducing sugar yield of 92-95% from the 50-60% of the starch present in the material.
A process for production of confectioner’s syrup from cassava starch processing fibrous waste was developed by hydrolysis with sulphuric acid.
A process for production of cell biomass of yeasts such as Candida utilis and Saccharomyces cerevisiae for use as single cell protein has been developed utilising saccharified cassava fibrous waste as a carbon and energy source.
Processes for production of alcohol, utilising the hydrolysate of the cassava fibrous waste prepared either by (i) enzyme-enzyme process (involving alpha-amylase and amyloglucosidase) or (ii) an acid-enzyme process (involving a mild acid treatment followed by hydrolysis with amyloglucosidase) were developed.
A process was developed for production of confectioner’s syrup by hydrolysis of potato biomass either by hydrochloric acid or sulphuric acid. This obviates the economic loss encountered due to the surplus availability of potatoes during glut season and also through value addition to culled variety of potatoes.These processes have already been commercialized.
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Processes For Microbial Degradation Of Organochlorine Pesticide Residues And Other Hazardous Compounds The industrial production of man-made chemicals has been on the increase during the last few decades. Most of these compounds, especially the halogenated ones, are highly recalcitrant and persist in the environment for several years, disturbing the ecological balance. The more alarming fact is that these compounds reach human body through almost every item of food that one consumes, in a concentrated form through the process of bio-magnification at different trophic levels. Efforts are on, throughout the world to find ways to eliminate/abate these pollutants from environment. Among the different methods, microbial degradation is thought to be the most effective. Studies have been done in our laboratory to develop treatment technologies for the elimination of xenobiotics and other hazardous chemicals from industrial effluents and contaminated soils, using the degradative potentials of microorganisms.
Processes for bio-mineralization of alpha-, beta-, gamma-and delta-HCH, either singly or in mixtures, by microbial consortia containing bacteria and a fungal strain. Up to 100 ppm each of alpha– and gamma-isomers can be degraded in bioreactors within 2-3 days. 25 ppm of beta– and delta-isomers can also be degraded within 4-5 days. Up to 200 ppm of technical HCH containing all the isomers can also be degraded effectively within 4-5 days. These processes will be very useful for decontamination of old stocks of HCH and also for cleanup of waste dump sites.
Processes for decontamination of DDT by bacterial degradation. Four bacterial strains capable of degrading DDT residues were isolated and acclimated. All the strains could degrade up to 25 ppm, p,p’-DDT by 72 h in liquid cultures. One of the strains, Pseudomonas aeruginosa strain DT-Ct-2 could bio-remediate up to 25 ppm of DDT in soil. These cultures also showed the ability to degrade DDE, which is a more toxic and more recalcitrant intermediary metabolite of DDT.
Processes for bio-elimination of phenolic compounds from wastewaters through microbial degradation. Five strains of Pseudomonas capable of degrading phenol were isolated and detailed studies on degradation kinetics have been carried out. Pseudomonas sp. CP4 can degrade up to 1.5 g/L of phenol by free cells and up to 4.0 g/L by agar-agar encapsulated (immobilized) whole cells. This strain and other strains, CPC-1, CoPC-4 and SoPC-5 also degraded all the three isomers of cresol and 2,3-dimethyl phenol (2,3-xylenol) and several other aromatic compounds. All these strains followed a meta-cleavage pathway.
Processes for continuous degradation of phenol in a fluidized bed bioreactor using Pseudomonas sp. CP4 immobilized by entrapment in Ca-alginate and agar gel beads (separately) have been developed. Agar-entrapped cells showed far superior efficiency in the degradation of phenol.
Another strain, P. stutzeri SPC-2 degraded phenol (up to 1.2 g/L) through an ortho-cleavage pathway, which was the first report on such a phenomenon in a pseudomonad.
A microbial consortium that can efficiently degrade the three isomers of nitro-phenol also has been developed.
A process for bioremediation of chlorobenzoate-contaminated soils through inoculation of soil with a bacterial strain Pseudomonas aeruginosa 3mT has been developed. This helps in the reversal of inhibition of seed germination in 3-CBA/4-CBA-contaminated soils. This strain can also be used for removal of 3-CBA (up to 8 g/L) and 4-CBA (up to 12 g/L) from waste- waters.
A process for bioremediation of 2,4,5–trichlorophenoxyacetic acid–contaminated soils by inoculation with Burkholderia cepacia AC1100 has been developed, which revokes the inhibitory effect of the toxic chemical on germination of crop seeds.
Production And Application Of Ribonuclease Of Aspergillus candidus M16AAprocess for reduction of nucleic acid content of yeast biomass, to be used as single cell protein, by exogenous application of a potent ribonuclease from Aspergillus candidus M16a has been developed. For the economic production of the enzyme through solid-state fermentation cultural conditions such as nutritional and physical parameters were optimized by growing the fungus in wheat bran solid medium. Methods of extraction and purification of the enzyme has also been developed. The enzyme has been analyzed for its physico-chemical characteristics and reaction kinetics and specificities.
Algal Biomass Production ─ Cost Reduction By Utilising Waste Materials The cost of production of biomass of Scenedesmus acutus and Spirulina platensis for use as SCP could be significantly reduced by substituting certain nutrient input into the medium with agricultural and domestic wastes. Urine and bone-meal reduced the inputs of nitrate, calcium and phosphate. Sheep’s blood showed good growth promotion of algal cultures. Carbon dioxide enriched air generated by cow dung composts could be used as a good carbon source.
STUDIES ON BIOPEPTIDES DERIVED FROM FOOD PROTEINS FOR ACE-INHIBITORY, ANTIOXIDANT, ANTICANCER & ANTIBACTERIAL ACTIVITIES. Functional foods and nutraceuticals for use as anti-hypertensive agents:Hypertension is a major risk factor for the development of cardiovascular diseases. Several synthetic drugs have been developed to combat this problem and most of these drugs have been shown to have drastic side effects. Some of these drugs function by inhibiting Angiotensin converting enzyme I (ACE-I), the key enzyme involved in the biochemical pathway, renin-angiotensin-aldosterone system (RAAS). Several natural materials have been successfully tried to replace these synthetic drugs, which include peptides derived from foods as well as some plant seed proteins.
It was demonstrated by in vitro tests that peptides derived from seeds of tamarind (Tamarindus indica L.), papaya (Carica papaya), watermelon (Citrullus lanatus), and long-podded cowpea (Vigna unguiculata subsp. Sesquipedalis) inhibited ACE I activity. Different fractions of digests obtained by partial hydrolysis of the proteins of these seeds by proteases such as tyrosine, pepsin, pancreatin, and papain were tested and were shown to possess varying levels of ACE I-inhibitory activity. Peptides derived from meat proteins of the three-spotted crab, Portunus sanguinolentusi> also revealed good ACE I-inhibitory activity. These peptides also exhibited antioxidant and antibacterial activities. Peptides derived from watermelon seeds also revealed strong anticancer and anti-cell migratory activities.
Further analysis of the active peptide fractions from the above sources and development of functional food formulations and nutraceutical preparations have to be carried out.
Antibacterial potentials of plant extracts particularly against multi-drug resistant pathogenic strains.
The studies included the following:
Antibacterial activity of Myristica fragransand Crysopogon zizanioides against multidrug resistant organisms.
Antimicrobial activity of Stachytarpheta jamaicensis and its synergy with chloramphenicol.
Antibacterial and synergistic effects of leaf extracts of Chromolaena odorata and Hyptis suaveolens against multi drug resistant strain Staphylococcus aureus.
Phytochemical Analysis, Antibacterial Activity and Antioxidant Potential of Aerial Parts of Cleome rutidosperma.
Evaluation of Antimicrobial Activity of Leaf and Flower Extracts of Wedelia trilobata.
Antimicrobial Activity of Chromolaena odorata, Hemigraphis colorata Leaf extracts and Punica granatum Peel Extracts against Multi-drug Resistant Staphylococcus aureus.
Antibacterial Activity of Myristica fragrans against Multidrug Resistant Organisms
Evaluation of Antibacterial Activity of Crysopogonziz anioides Leaf and Root Extracts against Multi-drug Resistant Organisms.
Others:
Besides the above areas, Dr Kunhi has worked on isolation of bacteria for production of flavour nucleotides such as 5′-IMP, 5′-XMP, 5′-GMP, etc. He also has screened hundreds of fungal strains for extra-cellular production of 5′-nucleases with a view to use them for flavour nucleotides production
For improving alcohol yield from molasses by Saccharomyces cerevisiae fermentation, the effect of clarification of molasses and the immobilization of yeast cells in Ca-alginate gels etc. have been attempted.
Production and partial characterization of keratinase enzyme produced by Pseudomonas sp. S-CSR-0004.
Elimination of inhibitory effect of phenol on germination of Cicer arietinum seeds through bioremediation by Pseudomonas aeruginosa S-CSR-0013.
Tamarind (Tamarindus indica L.) seed protein-derived peptides as Angiotensin-I-Converting Enzyme (ACE-1) Inhibitors.
MOLECULAR BIOLOGICAL AND OTHER BASIC STUDIES
Cloning and expression of argF and argG genes of Pseudomonas aeruginosa. The genes coding for two key steps in the biosynthesis of arginine were cloned in E. coli as a project work under the one year course work he did at the Institute for Biotechnological Studies, London, UK.
Cloning and expression of alpha-amylase gene. α-Amylase gene of Bacillus laterosporus was cloned in E. coli HB101 and DH5α through plasmid vectors pGEM-4 and pKT240. The gene got expressed and 90% of the gene product was secreted to the medium. This is an interesting phenomenon that 90% of the gene product is secreted by a generally non-secreting organism.
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Cloning and expression of ATP citrate lyase gene of Rhodotorula gracilis. Work on microbial fat was undertaken for (1) its commercial interest, as an alternative source of edible oil, and (2) for elucidation of basic mechanism in biological overproduction of lipids. A strain of red yeast, Rhodotorula gracilis capable of synthesizing and accumulating fat (up to 60-70% of the total biomass) was isolated in this laboratory. Efforts were made to understand the molecular genetics of fat over production by this strain. With this in view conditions were standardized for isolation of DNA, in vitro cloning of genomic fragment into a shuttle vector, YEp13, and transformation of the rec. plasmid into E. coli, Saccharomyces cerevisiae as well as into Rh. gracilis. Mutants of Rh. gracilis with impaired lipid synthesis and pigment production were also isolated for this purpose.
A rapid semi-quantitative method for estimation of fat in oleaginous microorganisms by employing fat staining dyes such as Sudan IV B or Sudan Fat Red 7B was developed Attempts were made to construct hybrids of the oleaginous strain of Rh. gracilis and fast growing S. cerevisiae or Aspergillus niger. None of the hybrids obtained was found to be promising in terms of the desired traits of fat over-production combined with fast growth and high biomass-build up. ATP: Citrate lyase is a key enzyme in the overproduction of fat in oleaginous yeasts, such as Rh. gracilis. The gene encoding this enzyme was cloned and got expressed in a non-oleaginous mutant of the same organism lacking in ATP: citrate lyase.
Development of a modified method for assay of alpha-amylase. Commonly used bacterial growth media such as Luria broth, nutrient broth, etc. interfere with the assay of dextrinizing activity of α-amylase, due to the presence of thiol groups. A modified method for assay of α-amylase enzyme based on starch-iodine colour reaction has been developed which obviates the interference of thiol groups.
