Research Journal of Biotechnology

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Research Journal of Biotechnology





Solubilization of inorganic phosphate (Pi) and plant growth-promotion (PGP) activities by root-nodule bacteria isolated from cultivated legume, mothbean (Vigna aconitifolia L.) of the Great Indian Thar desert

Sharma S., Gaur R.K.* and Choudhary D. K.

In the present study, endophytic root-nodule bacterial strains were isolated from cultivated legume, mothbean (Vigna aconitifolia L.). Isolates VL-1, VN-1, VN-2 were found positive for IAA production wherein VN-2 showed maximum IAA production. It was observed that tricalcium phosphate served as better phosphorus source for seeds inoculated with VN-2 with an increase in shoot length (18.9%) over control. Plant growth parameters (root and shoot length) showed significant increase in response to bacterial inoculation. In unsterilized soil system, the effect of VN-2 was highest with an increase in shoot length (24.4%) and root length (20.5%), followed by VL-1 showing an increase in shoot (14.3%) and root length (13.8%). Isolates were identified by partial 16S rDNA sequence analysis. A comparison of 16S rDNA sequence with the reference strain, to which they matched, was performed. Sequencing data showed that most isolates belonged to genus, Rhizobium and Pseudomonas spp. being a dominant species. Isolates VL-2, VL-3, VS-2, and VS-3 together with VL-1 and VN-2 showed similarity with reference strain Rhizobium sp. B5. An understanding of the diversity of these endophytes that interact with plants will be worthwhile to fully achieve the biotechnological potential of efficient plant-endophyte partnerships for a range of applications.

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Production of Carboxymethyl Cellulase by Bacillus sphaericus JS1 Strain in Low Cost Agriculture Waste Medium

Singh Jagtar

Utilization of agriculture byproducts can contribute to efficient solid-waste management. Production of CMCase using different agricultural byproducts such as coconut seed meal, sesame seed meal, cotton seed meal, mustard seed meal, maize bran and wheat bran was studied with Bacillus sphaericus JS1 (MTCC Accession No 3243). It worked best at pH 7.0 in the presence of 12h seed culture of 6.0% (v/v) for the production of CMCase at 40oC. ≥85% of relative activity of enzyme was produced in the range of pH 7-9. Coconut seed meal media worked best at all experimental concentrations (142, 158, 142, 158% relative activity at 0.25, 0.50, 0.75 and 1.0% w/v respectively) for the production of enzyme. Sesame seed meal media reported marginally higher production of enzyme (110, 135, 110, 105% relative activity at 0.25, 0.50, 0.75 and 1.0% w/v respectively) as compared to control. Cotton seed meal media observed higher production of CMCase (119, 131% relative activity at 0.50 and 0.75% w/v respectively). Coconut seed meal, cotton seed meal and sesame seed meal worked better for the production of CMCase than the branded chemicals/control media. Optimum production of JS1 CMCase can be achieved by utilizing agriculture byproducts at cost effective rates at large scale.

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Induction of β-Glucuronidase Gene Fusions in Arabidopsis thaliana by Erwinia chrysanthemi

Eswara Reddy N.P.1* and Shaik Thahir Basha2

A study was focused on the interaction of Erwinia chrysanthemi with Arabidopsis thaliana C24. Erwinia chrysanthemi has shown compatible interaction and caused soft rot by complete maceration of the tissues and collapse of the infected plant part. Induction of random β-glucuronidase (GUS) gene fusions in transgenic Arabidopsis after infection with E. chrysanthemi was observed. Transgenic Arabidopsis lines At1082 and At1275 have shown increased GUS expression after infection with E. chrysanthemi. Histochemical staining results indicated localized GUS expression in the vascular bundles of the leaves present above the site of infection. DNA analysis results showed the presence of a single T- DNA copy in transgenic lines At1085 and At1275. The possibility of cloning E. chrysanthemi induced A. thaliana plant promoter sequences is discussed.

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Antifungal Bacillus subtilis AFB22 from Pomegranate with Potential to control Fruit Rot

Gajbhiye M.H.,1* Sathe S.J.,2 Marathe R.J.3 and Deshmukh R.B.4

In all, 200 antifungal Bacillus spp. (AFB) were isolated from aerial surfaces of pomegranate plant and 50 inhibited the growth of major fruit rot pathogens viz., Phomopsis varsoniana, Aspergillus flavus, A. clavatus, Beltraniella humicola, Fusarium graminearum and Rhizopus stolonifer. The AFB isolates exhibited maximum antifungal activity at pH 7 to 9 at 30oC and 40oC in malt extract broth. The most potent isolate AFB22 was identified as B. subtilis by 16S rDNA sequencing. Crude antifungal compound was recovered from isolate, after 80% saturation by ammonium sulphate, dialysis and freeze drying, was heat stable but sensitive to proteolytic enzymes and low pH. This isolate also demonstrated plant growth promoting traits like phosphate solubilization and production of indole acetic acid, siderophore and chitinase. Cells and cell free supernatant (CFS) of the AFB22 prevented rotting in pomegranates when challenged with spores of P. varsoniana. According to Kaplan-Meier procedure of survival analysis and Tarone-Ware statistics test, the incidence of rot was significantly lower (P <0.05) in pomegranates treated with cells and CFS, when compared with fruits inoculated with pathogen only. Mean survival time of pomegranates was 6.74, 6.68 and 4.33 days in treatments of cells, CFS and fungal pathogen, respectively. This study demonstrates the bio-protection of pomegranates by B. subtilis AFB22 from the fungal fruit rot pathogens, an alternative to synthetic fungicides.

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Effect of AM fungi, Azotobacter and Phosphate solubilizing bacteria in improvement of Amaranthus paniculatus L. - a leafy vegetable

Madgaonkar Shwetha C.* and Lakshman H.C.

Arbuscular mycorrhizal fungi interact with a wide range of other microorganisms in the root and in the rhizosphere. There is a contradictory statement of mycorrhizal colonization in amaranthaceae. Hence an attempt was made to explore its association with AM fungi (Glomus macrocarpum), Azotobactor and Phosphate solubilizing bacteria (PSB). Green house experiment on Amaranthus paniculatus L.was carried out with following treatments (1) control (2) Glomus macrocarpum (3) Glomus macrocarpum + Azotobacter (4) Glomus macrocarpum + PSB (Bacillus polymyxa) (5) Glomus macrocarpum + Azotobacter + PSB in which triple inoculation (Glomus macrocarpum + Azotobacter + PSB) shows higher shoot, root length and dry weight of shoot and root followed by dual and single inoculation. The experimental plant results more number in per cent of root colonization, spore number and P uptake of shoot with triple inoculation compared to control plants. The result suggests that there is a synergistic interaction with different bioinoculants and the increase of biomass with the application of these bioinoculants.

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Isolation, Characterization and Extraction of antimicrobial compound from marine actinomycete Streptomyces hygroscopicus BDUS 49

Parthasarathi S.,1* Sathya S.,2 Bupesh G.,3 Manikandan M.,4 Kim C.J.,5 Manikandan T.1 and Balakrishnan K.1

A total of eight actinomycetes colonies were isolated from near sea shore marine environment locations of Bigeum Island, South West coast of South Korea. Among them, 4 isolates were morphologically distinct on the basis of spore mass colour, reverse side colour, aerial and substrate mycelia formation and production of diffusible pigment. The majority of these isolates were assigned to the genus Streptomyces of which one isolate showing broad spectrum of antimicrobial was on the basis of their morphological, physiological and biochemical properties. Phylo genetic analysis of a 16S rRNA gene sequence showed that strain Streptomyces hygroscopicus BDUS 49 forms a distinct clade within the Streptomyces 16S rRNA gene tree and is closely related to Streptomyces hygroscpicus subsp sub species. This strain possessed a broad spectrum of antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. The UV spectra of the active compounds in ethyl acetate showed peaks between 200 to 295 nm. The bioactive region was detected on the TLC plate (Rf 0.40). The structure of the bioactive components was further determined using FTIR, MS, 13C NMR and 1H NMR. The molecular formula of the given compound was identified as 7, demethoxy rapamycin C50H75NO12+NA (MW 905.12).

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Bio-demineralization of Indian Bituminous Coal by Aspergillus niger and characterization of the products

Manoj B.

The effect of demineralization on an Indian bituminous coal has been investigated by filamentous fungus Aspergillus niger. X-ray diffraction profile reveals the presence of inorganic components in the sample. Bio-Solubilization using Aspergillus niger significantly reduced the ash content in the coal sample (10.23wt% to 5.21wt %). Leaching process removed silicate and pyrite minerals where as aluminates were decreased considerably. The carbon content showed an increase of 19.94% where as the oxygen content decreased by 52.3%. During biosolubilization the fungus produced acids like gluconic acid, oxalic acid and citric acid along with oxalates which are responsible for the demineralization in coal by the formation of mineral salts. The broad diffraction peak at 2θ~ 25.5o is due to the crystalline carbon in the sample which is mainly due to the typical (002) plane reflection of graphite.

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In vitro protocol standardization for Turmeric Multiplication in Jammu Division

Sharma Satish K.,* Sharma Mamta and Raina Vishal

A protocol for the in vitro multiplication of turmeric (Curcuma longa L.) has been developed using rhizome bud as explants. The explants were surface sterilized with a combination of sterilants (Bavistin 2% and HgCl2 0.1%). Maximum per cent of aseptic cultures were obtained when 0.1% HgCl2 was used for 3 minutes. The sterilized explants were inoculated on MS medium with or without growth regulators. The cultures kept in dark (2 weeks) responded well in comparison to those kept in light. Sprouted shoots sub cultured on medium with different concentrations and combination of growth regulators resulted in varying degrees of multiple shoots. Maximum proliferation of shoots was observed in MS medium augmented with BAP (2.5 mg L-1) and NAA (0.5 mg L-1) within 2 weeks and average number of shoots per explant was 5.6.

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Studies on Purification of Alkaline Protease from a mutant Aspergillus flavus AS2

Roja Rani M.,* and Prasad N.N.

Alkaline protease from a mutant of Aspergillus flavus AS2 was purified from the culture supernatant by employing different methods such as ammonium sulphate precipitation, DEAE cellulose chromato graphy followed by Gel filtration using Sephadex G-100. The yield of the enzyme after purification was found to be 10%. Protease was found to be homogenous when examined by SDS-PAGE and the enzyme showed that it has a molecular weight of 33 KDa . Characterization studies were carried out using the purified enzyme. optimum pH and temperature for the activity of alkaline protease was found to be 8.5 and 550C and stable in the pH range 5.0 - 12.0. The thermo stability exhibited by protease ranges from 35-600C. Among various protease inhibitors PMSF strongly inhibited the enzyme activity revealing that the enzyme in the present study is serine alkaline protease.

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Bioefficacy of leaf extracts of Biophytum sensitivum (L.) DC. against Aedes aegypti (Diptera : Culicidae)

Shivakumar M.S.,* Natarajan D. and Srinivasan R.

A novel bioactive molecule for use in vector control is the need of the hour. Plants hold the key to such molecules which provide an alternative approach for producing a larvicidal product from plant sources. In the present study the larvicidal activity of leaf extracts of B. sensitivum at different doses were analyzed on fourth instar Aedes aegypti larvae. Three different concentrations (200, 300 and 500 mg/L) of extracts were used to determine larvicidal and consequent effects on adult emergence. Larvicidal activities against A. aegypti have been determined in the different solvent leaf extracts of B. sensitivum. Results show that acetone extract had a dose-dependent effect and produced higher mortality (LC50 = 21.79 and LC99 = 139.50 µg/ml) in larvae. Pupicidal activities of acetone extract showed the highest effect (LC50 = 13.05 and LC99 = 137.75 µg/ml). Acetone extracts also interfered with the normal development of adult mosquitoes (LC50 = 9.77 and LC99 = 11.83 µg/ml). The study shows that acetone extracts of B.sensitivum is effective in controlling the Aedes aegypti larvae.

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Biosynthesis of silver nanoparticles using Pseudomonas fluorescens

Silambarasan S. and Abraham Jayanthi*

Biosynthesis of nanoparticles employing microbes has emerged as an alternative method to complex chemical synthesis. In the present work evaluation of extracellular biosynthesis of silver nanoparticles (Ag NPs) by Pseudomonas fluorescens was investigated. The silver nanoparticles were characterized by means of UV-VIS spectroscopy, Fourier Transform Infrared (FTIR) and Atomic Force Microscopy (AFM). The UV-VIS spectroscopy showed an absorption peak at 420 nm which corresponds to the plasmon resonance of silver nanoparticles. The Fourier Transform Infrared Spectroscopy confirmed the presence of protein as the stabilizing agent surrounding the silver nanoparticles. Atomic Force Microscopy showed topographical image of irregular silver nanoparticles and the size is 85.46 nm. The Silver Bio-nanoparticles were evaluated for their antimicrobial activities against Staphylococcus aureus and Escherichia coli. The highest antimicrobial activities have been recorded against Staphylococcus aureus and for Escherichia coli lesser activity was recorded.

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Cross-amplification polymorphism of rice microsatellites in Zingiberaceae

Kar Basudeba, Joshi Raj Kumar* and Nayak Sanghamitra

Crop plants of the family Zingiberaceae are cultivated in different habitat and are mainly appreciated for their use as spices and traditional medicine. However, limited characterization at DNA level and lack of availability of appropriate genetic markers is a drawback in their improvement. Cross-amplification potential of 40 BAC based rice microsatellite markers among 11 genotypes of Zingiberaceae was tested to facilitate genetic characterization and comparative mapping to assess the genetic diversity. IR64 was taken as control. Two primers failed to amplify fragments while another two primers were monomorphic. Of the 181 bands, 165 fragments were polymorphic and each primer amplified an average of 4.58 polymorphic fragments. Although the amplified fragments were large compared to the target bands amplified by rice, the efficiency of rice microsatellites in terms of amplification of larger number of fragments, better coverage of the genome and efficient fingerprint is higher and better as compared to RAPD markers. The 12 genotypes were classified into three clusters based on molecular data. Cluster I comprised only IR64, Cluster II represented all the six genotypes of Zingiber and the only one genotype of Kaempferia while cluster III represented all the four species of Curcuma genotypes. Thus the present set of rice microsatellites exhibits high degree of polymorphism in Zingiberaceae and can be used as valuable source for germplasm conservation and improvement of novel varieties.

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Bioelectricity Production using Microbial Fuel Cell

Shah Chirag K. and Yagnik B.N.*

Energy with its ever increasing demand is becoming more and more scarce resource. Microbial Fuel Cells (MFC) hold promise as an alternative not only to meet the widening gap in the energy sector but can be an effective mean for the sustainable utilization of reduced compounds in an eco-friendly way. The bacteria within the MFC oxidize complex substrates within the waste by removing electrons. The electrons generated by the bacteria growing in the oxygen-deficient anodic chamber of the MFC, are captured by the cathode exposed to the air. The potential difference generated can be optimized and developed into an independent electricity production unit. For this reason, better understanding of the electron coupling reactions and carriers within the bacteria is attaining wider attention. The MFC can be customized to suite diverse substrate range by choosing specific bacterial species having matching metabolic repertoire and accordingly chosen operational parameters of the MFC.

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