Research Journal of Biotechnology

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Research Journal of Biotechnology





Fermentative Conversion of Glycerol to Ethanol using Mixed Consortium

Vikromvarasiri Nunthaphun and Pisutpaisal Nipon

The fermentative conversion of glycerol to ethanol by a mixed consortium under various cultivation conditions in batch experiments was the main focus of the current study. Experiments were setup, using the microbial consortium obtained from anaerobic wastewater treatment facility as starting seed, with varied initial glycerol concentrations ranging from 0 -128 g L-1, types of cultural media including simple (without yeast extract and tryptone) and supplement media (with yeast extract and tryptone) and under mesophilic fermentation (30o C). Ethanol concentration of 158 mM, yield of 0.33 mol mol-1, glycerol utilization of 99.8 % was obtained at the optimal condition when the consortium was cultivated in the simple medium containing 45 g L-1 glycerol, after 3 d fermentation. Glycerol concentra­tions greater than or equal to 65 g L-1 showed apparently negative impact on the ethanol concentration, yield and the extent of glycerol utilization for both types of media. Ethanol was predominant fermentative product with minor amount of volatile fatty acids detected, when the simple medium was used. Conversely, acetic and butyric acids were the main fermentative products, when the supplement medium was used. Based on the ethanol yield and the compositions of the cultivation medium, the simple medium was suitable and advantageous for the ethanol production.

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Improvement of Plant Regeneration and Agrobacterium-mediated Genetic Transformation Efficiency in Red Clover (Trifolium pratense L.)

Khosro Mehdi Khanlou, Mansour Karimi, Asad Maroufi and Erik Van Bockstaele

Red clover (Trifolium pretense L.) is one of the important forage crops in most temperate agricultural regions. Red clover germplasm has limited genetic variation for disease resistance. Application of an efficient gene transformation protocol to introduce genes against diseases is considered the only approach for developing new resistant cultivars. In this study, in order to establish an efficient regeneration system in red clover, highly regenerative genotypes were utilized for evaluation of different media sequences for their suitability to utilize potentiality of regeneration capacity. Media sequence KBC-SPL-SPL significantly increased the efficiency of somatic embryogenesis of red clover. There was no relationship between regeneration ability and fresh callus weight. Also, no interaction was observed between genotypes and media sequences for fresh callus weight, number of embryos and number of developed shoots. To develop an efficient gene transfer system, some parameters were investigated, including evaluation of suitable concentration of selection agent, examination of the effect of pre-culturing of explants on efficiency of transformation and comparison of different Agrobacterium strains for efficiency of gene transformation. The effect of pre-culturing explants was increased slightly by the frequency of gene transferring. Highly infective Agrobacterium strain C58C1 can increase the transformation efficiency for red clover to 83%. PCR and histochemical GUS assay of transformed plants confirmed successful integration of the T-DNA into the red clover genome. Our established system will be a platform for genetic manipulation of red clover to broaden its genetic variation and for germplasm innovation.

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Antioxidant activity of Ocimum basilicum and Salvia officinalis

Yadav Sadhana Shivmurat and Mukundan Usha

In view of the free radical theory of ageing, antioxidant property is an important characteristic of herbs species. The intake of antioxidants present in food is an important health-protecting factor. In the present study, Ocimum basilicum and Salvia officinalis belonging to the family Lamiaceae were screened for their in vitro antioxidant activity. By employing DPPH (2, 2-diphenyl-1-picrylhydrazyl) and Superoxide radical scavenging assays, it was shown that the methanolic as well as aqueous extracts of leaves of both the herbs were endowed with antioxidant activity, though, as expected, their potency varied. In particular, methanolic extracts of Ocimum basilicum displayed the highest activity. The IC50 value of the extracts tested ranged from 5.38 mg/ml-7.61 mg/ml. It is generally accepted that a diet rich in plants is associated with a reduced incidence of degenerative diseases such as atherosclerosis and cancer. This study suggests that these Lamiaceae plants could be pharmaceutically exploited for their antioxidant properties.

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Optimization and Cultural Characterization of Bacillus RV.B2.90 producing Alkalophilic Thermophilic Protease

Vijayalakshmi S., Venkat Kumar S. and Thankamani V.

A newly isolated alkalophilic, thermophilic Bacillus RV.B2.90 was characterized and tested for the production of extracellular protease in 50 – 300 ml shake flask experiments. The strain was identified as Bacillus cereus by biochemical and 16S rRNA sequencing. Maximum amount of enzyme produced in basal medium at exponential phase was 1284 U/ml. The optimized medium could increase enzyme production by 30% to 4189 U/ml. Growth and enzyme production occurred in media at all initial pH ranging from 7.0 – 12.0, the maximum enzyme production occurred in the medium with initial pH 8.0. The pH increased with growth. Depending on the starting pH 7.0 – 12.0 the duration of incubation required for change of pH to 9.0-10.0 varied markedly 40 – 12 hrs. This phase coincided with maximal protease production at each pH. Production of enzyme increased significantly with temperature of incubation from 37-70˚C, with 1284 U/ml at 37˚C and 1984 U/ml at 70˚C. At 90˚C the growth and enzyme production showed a steep decline to 400 U/ml. With respect to other growth parameters, the optimum agitation speed was 50 rpm, the best carbon sources were xylose and arabinose that enhanced enzyme production to 4189 U/ml and 4016 U/ml, the best nitrogen source were casein and malt extract showing 3946 U/ml and 3182 U/ml. Inorganic nitrogen sources did not have significant effect on enzyme production. Sodium chloride at 4% (w/v) was found to be optimum for enzyme production. Among the 5 amino acids added to the medium, histidine increased enzyme production to 1853 U/ml (about 30%). Among the salts added to the medium K+, Mg2+ and Ca2+ were found to increase the enzyme production. The isolate was found to be highly alkalophilic, thermophilic, thermo stable and moderately halophilic protease producing organism.

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Screening of A. Terreus Klvb28 Mutants by Bioassay Method for Overproduction of Lovastatin (A Vital Statin in Coronary Heart Disease)

Prabhakar M. and Lingappa K.

Coronary Heart Disease (CHD) is the main clinical manifestation of atherosclerosis and is the major cause of death in modern days. One of the major risk factors for atherosclerosis and coronary heart disease is hypercholesterolemia. Lovastatin is a potent cholesterol-lowering drug. It acts by competitively inhibiting the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) which catalyses the rate-limiting step of cholesterol biosynthesis. Lovastatin is produced as a secondary metabolite by a variety of filamentous fungi including Penicillium sp., Monascus rubber and Aspergillus terreus. However these organisms suffer from low yield. Hence, in the present study the strain A. terreusKLVB28 isolated from soil was subjected to mutation for over-production of lovastatin by chemical as well as physical methods. Over all 43 mutants were obtained, amongst obtained mutants A. terreus KLVB8mu21 (obtained after EMS treatment of 5mg/ml for 5min treatment) is potent lovastatin producer when employed for bioassay determination. Confirmation of lovastatin by TLC and spectral analysis at 238 nm serves as alternative method for HPLC which is an expensive method.

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Detection of Extracellular Enzymes produced by Keratinophilic Fungi isolated from Poultry Farm Soil

Periasamy Anbu, Hilda Azariah, Kyeong Ho Min and Byung Ki Hur

A total of 25 keratinophilic fungi including five dermatophytes and closely related species and 20 other fungi were isolated in poultry farm soil from Namakkal, India. The isolated keratinophilic fungi were screened for the extracellular enzymes viz. amylase, protease, cellulase, lipase and keratinase by using plate assay. All of the fungi, with the exception of a non-sporulating fungus, were able to produce at least one of the enzymes and most of the fungi (i.e. Chrysosporium keratinophilum, Aspergillus flavus, A. niger, A. terreus, Penicillium citrinum, P. frequentans and P. purpurogenum) could produce all types of the examined enzymes. A. versicolor and A. niger synthesized the highest amounts of amylase and cellulase respectively and P. citrinum and Trichoderma viride produced the highest amount of lipase. Maximum amounts of protease and keratinase were synthesized by Scopulariopsis brevicaulis. In addition, Trichophyton mentagrophytes and A. niger were also potential producer of keratinase. Finally, these keratinophilic fungi were able to produce a wide range of industrial extracellular enzymes. Interestingly, all of the dermatophytes and closely related species produced protease and keratinase.

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Production of L-Glutaminase by Pseudomonas VJ-6

Hiremath Jyothi, Shivaveerakumar and Rathod Vandanau

Of the fifteen bacterial isolates screened for glutaminase production, four were positive for glutaminase production. Among them only one isolate Pseudomonas-VJ6 was selected based on plate assay. Glutaminase production was 51.5 U/ml by submerged fermentation. Enzyme activity of was assayed in various ranges of incubation period, temperature and pH. The enzyme activity was optimal at 72 hrs (53.3 U/ml), temperature, 30°C (56.0 U/ml) and pH of 6.0 (57.6 U/ml). Nutritional parameters, carbon and nitrogen sources also played an efficient role in enhancing the yield. Among the different carbon sources, glucose with concentration (10 g/l) showed maximum activity of 58.4 U/ml and among different nitrogen sources yeast extract (2 g/l) showed maximum activity of 58.9 U/ml. L-glutaminase was partially purified from cell free extracts of Pseudomonas-VJ6 by ammonium sulphate salt precipitation which showed activity of 56.6 U/ml at 80% precipitation and dialyzed enzyme extract having activity of 56.0 U/ml with protein content of 70 mg/ml was obtained. SDS-PAGE showed a molecular weight of 31 kDa. L-glutaminase from bacterial sources is reported to have high industrial application for their stability.

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Physicochemical and SEM Studies on Capsosiphon fulvescens powders prepared using Different Drying Methods

Son Seok Min and Lee Jun Ho

Caposiphon fulvescens is a green algae that grows in copious amounts along the southwestern coast of South Korea. A systematic study on how different drying methods namely, hot-air drying, vacuum drying and freeze drying, affect the color, browning index, degree of rehydration and water solubility is of importance for utilizing C. fulvescens as a novel ingredient in functional foods. Samples prepared by freeze drying showed a significantly higher L-value but lower a-value as compared with those prepared by hot-air drying and vacuum drying (p<0.05). The yellowness (b-value) of fresh samples was significantly lower than the other samples while that of the vacuum dried samples was significantly higher than the other samples (p<0.05). The browning index was significantly higher in samples prepared by hot-air drying than those prepared by freeze drying (p<0.05) but both were not significantly different from the vacuum dried samples (p>0.05). Freeze dried C. fulvescens powders exhibited a significantly higher degree of rehydration than the other samples (p<0.05). Water solubility was not significantly affected by the drying methods (p>0.05). Scanning electron micrographs revealed that freeze drying produced more orderly and denser samples than did vacuum and hot-air drying.

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PCR Amplification of DNA from Blood Samples in Holstein Friesian Calf and NDL cross breeds

Joshna P., Anuradha P., Jyothi K., Vinathi D. and Jyothi G.

The present study was undertaken with the primary objective, amplification of DNA isolated from blood samples of cattle breeds in Holstein Friesian calf and NDL cross breeds. Blood samples were collected in vacutainers containing EDTA as an anticoagulant from cattle breeds. Genomic DNA was extracted from blood samples using phenol: chloroform method. The quality of DNA was assessed by UV spectrophotometry. PCR was carried out in a final reaction volume of 25ul reaction mixture. Increase in the stringency during initial cycles of PCR reaction increased the sharpness and brightness of the bands. DNA samples were electrophoresed on polyacrylamide gel and observed as the DNA pattern by silver staining. Milk protein genes (β-lactoglobulin) could be useful as genetic markers for additional selection criteria in dairy cattle breeding. The polymorphism of β-lactoglobulin gene was identified by conducting PCR studies.

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Strain Improvement of Selected Strain Bacillus subtilis (MTCC No.10619) for Enhanced Production of Antimicrobial Metabolites

Radha Krishna E., Shamsher Kumar P., Sujatha P. and Veerendra Kumar B.

A total of 178 strains (bacteria, fungi, yeast, actinomycetes) were isolated from sponges, Bay of Bengal and their antagonistic activities were tested against eight pathogenic bacteria Bacillus subtilis MTCC 441, Staphylococcus aureus MTCC 96, Pseudomonas aeruginosa MTCC 424, Escherichia coli MTCC 443, Bacillus cereus MTCC 430, Proteus vulgaris MTCC 1771, Candida albigans MTCC 227 and Aspergillus niger MTCC 1344. Among these the potent bacteria Bacillus subtilis showed high antibacterial activity. The present study is focused on the improvement of Bacillus subtilis through random mutagenesis to obtain mutant having high antibacterial activity. The Bacillus subtilis was subjected for mutation study by using physical (UV radiation) and chemical (NTG) mutation methods.

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Random amplified polymorphic DNA (RAPD) molecular marker based genetic relationship study of medicinal plants collected from tribal region of Chhattisgarh, India

Tiwari K. L., Jadhav S.K. and Kumar Ashish

The objective of this study was to evaluate the genetic relationship using random amplified polymorphic DNA marker among different medicinal plants used by tribes and local healers of Chhattisgarh, India. In present study, an attempt has been made to assess the genetic diversity among 28 genotypes of medicinal plant species belonging to different family of dicot and monocot using RAPD marker. Twenty five decamer- primers generated a total of 235 RAPD fragments of which 219 (93.19%) were polymorphic. The number of amplification products produced by each primer varied from 5-13 with an average of 9.4 bands per primer. Similarity co-efficient was used to evaluate the relationship among different medicinal plant with a cluster analysis using an Unweighted Pair Group Method with Arithmetic Average (UPGMA). Similarity had estimated the range from 0.13-0.99 Jaccords coefficient among different medicinal plant. Selected 28 medicinal plants were grouped into two major clusters. Interestingly as expected different medicinal plant belonging to same family did not form well defined family wise grouping, but they formed separate group with plant belonging to different family indicating association between RAPD patterns. Present study indicated Cathranthus roseus (pink flower) and C. roseus (white flower), Euphorbia hirta and Jatropha circus, Mentha arvensis and Leucas aspera was the very closest group. Considerable level of genetic similarity was observed among different medicinal plants. The information generated from this study can be used for development of marker for particular trait in medicinal plant and also can be used to maximize selection genetically similar plants which may be very useful in field of medical biotechnology.

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Antimicrobial Activity of Some Selected Medicinal Plants on Skin Pathogens

Vitekari Hrishikesh and Pillai Meena

Eight medicinal plants Acacia concinna, Chrysopogon zizanioides, Alardostachys jatamashi, Cyperus rotundus, Phyllanthus emblica, Curcuma zedoria, Santalum album, Aloe vera were selected for antimicrobial activity against seven skin pathogens. The dry powder was extracted in methanol. The antibacterial and antifungal activity was checked in different concentrations like 10, 50,100 and 200 µg/ml. Phyllanthus emblica had shown significant zone of inhibition following Cyperus rotundus up to certain extent. All plant extracts except Acacia concinna showed inhibition against Cryptococcus neoformans. Different combinations of concentrations of Phyllanthus emblica and Cyperus rotundus were also checked for antimicrobial activity. Different concentrations of mixture of Phyllanthus emblica and Cyperus rotundus exhibited antimicrobial activity for all microorganisms.

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Trichoderma Species as Abiotic and Biotic Stress Quenchers in Plants

Bae Hanhong

Trichoderma species have recently been described as opportunistic, avirulent plant symbionts with potential to control plant diseases. Trichoderma species are soil fungi that have been studied extensively for their biological control potential in many cropping systems. Consequently, commercial products have been developed as biopesticides, biofertilizers and soil amendments. Trichoderma species use several mechanisms to prevent plant diseases including antibiosis, induced resistance niche exclusion and mycoparasitism. Antibiosis is the production of secondary metabolites or peptides with antimicrobial activity. Plants recognize Trichoderma colonization and generate an active defense reaction limiting Trichoderma spread as well as simultaneously induce resistance to plant pathogens. Trichoderma species out-compete plant pathogens for nutrients or physically exclude pathogens. Mycoparasitism is the inhibition of disease development by directly feeding on plant pathogens. Root colonization also enhances plant growth and productivity and helps plants to overcome abiotic stresses such as drought or acidic soil. These biocontrol events are consequences of Trichoderma-plant-pathogen interactions.

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