Research Journal of Biotechnology

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Vol. 15(1) January 2020

Selection of stress tolerant Saccharomyces cerevisiae strains isolated from sloughing off soil for bioethanol production using Prosopis juliflora

Chandrasekhar Banoth, Srinivas Naik Kethavath, Praveen Kumar Keshav and Bhima Bhukya

Page No: 1-13

Abstract: Yeasts in industrial ethanol production require superior strains exhibiting multiple tolerance that result in economic feasibility. In the present study, various strains of Saccharomyces cerevisiae were isolated from soils of different regions of India and screened for stress tolerance and higher ethanol yield. The selected yeast strains were characterized by 18s rRNA sequencing. The HSP104 gene involved in thermotolerance was analyzed for selected isolates using polymerase chain reaction (PCR).

The sequences were BLAST searched in National Center for Biotechnology Information (NCBI) nucleotide database for sequence similarity and phylogenetic evolutionary analysis. The yeast isolates OBC9, OBC14 and OBC15 showed 100% similarity with Saccharomyces cerevisiae and OC521 with Kluveromyces marxianus. Fermentation of lignocellulosic hydrolysate of Prosopis juliflora by the selected yeast isolate OBC14 resulted in 30 g/L ethanol production with 0.46 ±0.02 g/g yield, 0.625 g/l/h productivity with 90% fermentation efficiency.

Influence of Fermented Fish waste (Gunapaselam) on nodulation (nodD) and nitrogen fixation (nifH) transcripts of Bradyrhizobium japonicum – Vigna radiata

Balraj Thendral Hepsibha and Arumugam Geetha

Page No: 14-24

Abstract:The agricultural sector was confronted with challenges to increase the food production and seek an alternative to chemical fertilizers. Fermented Fish waste – Gunapaselam (FFW-G) has agronomic values. Vigna radiata, a legume usually enters into a symbiotic relationship with Bradyrhizobium to fix atmospheric nitrogen. A study was conducted to examine the influence of FFW-G on the release of flavonoids by Vigna radiata, leghemoglobin content and the transcription profile of nodD and nifH genes. The treatments groups were: Group I – Water control; Group II – Chemical Fertilizer (25kg N2: 50kg P2O5: 25kg K2O: 20kg S/ha); Group III – Fermented Fish waste – Gunapaselam (FFW-G) (1:100 fold dilution).

Fermented Fish waste – Gunapaselam (FFW-G) has optimized the release of flavonoids by Vigna radiata root and increased the transcription of nodD gene. FFW-G treatment has increased the leghemoglobin content and also up-regulated the nifH transcript level. The results indicate that FFW-G is a potential value-added product and it could be amended in the unfertile soil as organic fertilizer to improve nitrogen-fixing ability of legumes.

Morphological and molecular diversity among Phytophthora spp. infecting arecanut

Prathibha V.H., Gangaraj K.P. and Hegde Vinayaka

Page No: 25-34

Abstract:Phytophthora causing diseases such as fruit rot, bud rot and crown rot are very serious owing to heavy economic loss. Fruit rot is a major yield limiting factor while bud rot and crown rot diseases cause death of arecanut palms. Phytophthora isolates infecting arecanut were collected from major arecanut growing areas. Diversity among the Phytophthora isolates was assessed based on morphological, pathological and molecular tools. In total, forty-one isolates of Phytophthora spp. designated as P1 to P41 were purified after isolation.

All the Phytophthora isolates exhibited considerable variation in phenotypic characters and pathogenecity. Morphological traits coupled with sequencing of ITS region of rDNA revealed association of homothallic species, Phytophthora heveae along with the dominant species, Phytophthora meadii. RAPD analysis of selected isolates of Phytophthora, clearly distinguished P. meadii and P. heveae isolates by forming two major clusters in dendrogram. However, clustering is not congruent with either geographical origin or virulence type; this indicates existence of wide variability particularly in P. meadii isolates.

Plant growth promotion and biocontrol activity of some typical harsh environment rhizo-microbes and their effects on Amaranthus cruentus plants

Mitra Debasis, Ganeshamurthy A.N., Sharma Komal, Radha T.K. and Rupa T.R.

Page No: 35-45

Abstract: Microbial synergism is an important factor in improving plant growth that promotes microbes and thus improves the development of plant. The different groups of typical harsh environmental rhizo-microbes were screened and their efficacy on plant growth promoting attributes viz. phytohormones, ammonia, HCN, siderophore, enzymes production and phosphate solubilization was evaluated. Pseudomonas sp. KB9, Streptomyces sp. A6 and Aspergillus niger FA8 showed potential for growth promoting attributes and biocontrol activity against Fusarium oxysporum, Phytophthora capsici, Colletotrichum glocoporioides and Xanthomonas axonopodis. The results of antibiotic diversity showed that the isolates were therefore very diverse and that the NT-SYS analysis showed many differences in intrinsic antibiotic resistance. For the consortium, highly compatible isolates FA8, A6 and KB9 were selected.

The results of pot experiments showed that consortium soil drenching treatments (KB9+A6+FA8) and A6 significantly enhanced A. Cruentus growth compared to control and IIHR - AMC. As a biofertilizer, the consortium and A6 isolate can therefore be used for development in horticultural crops and help in plant protection.

Phylogenetic analysis of Trichophyton mentagrophytes isolated from Tinea Patients

Sharma V., Kumawat T.K., Seth R. and Sharma A.

Page No: 46-52

Abstract:In the Asian subtropical region, the dermatophytic infections are important public health issues which are increasing day by day in an insidious manner. Especially in India, about 20-25% of the population has been affected by Tinea or other dermatophytic infections. This study was aimed to explore the molecular characterization of Trichophyton mentagrophytes isolated from clinical specimens of Tinea patients and to analyze the phylogenic relationship between isolates.

The study was extended to examine the effect of environmental factors on the growth of T. mentagrophytes. The culture isolates were identified based on macroscopic and microscopic character and pure culture was confirmed by DNA sequencing by PCR amplification using ITS1 and ITS4 primers. The fungal species were identified as T. mentagrophytes by the nBLAST using fungal DNA sequences against the data available in NCBI/GenBank. Phylogenic trees were constructed using MEGA7 software with the help of reference data retrieved from NCBI GenBank data to study the phylogenic relationship between the isolated fungi.

Assessment of Genetic diversity in Sesamum indicum by EST-SSR: problems and prospects

Kumari Rakhi and Arumugam Neelakantan

Page No: 53-61

Abstract:Aim of this study was to assess the genetic diversity in the cultivated sesame germplasm using expressed sequence tagged – simple sequence repeat (EST-SSR) markers. Of the thirty-four EST – SSRs tested nine revealed polymorphism. Four genomic – simple sequences repeat (G-SSR) tested additionally, however, showed almost cent percent polymorphism. In total there were sixty-seven EST SSR bands and thirty-seven G-SSR bands were obtained. PIC value of the polymorphic primers ranged from 0.45 to 0.85. Blast analysis of the primer sequence with the available sesame genome sequence revealed association of the chosen SSRs with gene regions encompassed with metabolic functions. The selected SSRs were found to spread over all the linkage groups excepting the linkage groups 9 and 13 of sesame available, with some of them associated with more than one linkage group. Lack of polymorphism for several of the SSRs confirmed that the genomic regions covered by them are highly conserved. It is concluded that inclusion of additional markers in the analysis may lead to a complete understanding of the genetic structure of Sesamum that would be useful in its breeding.

Purification and biochemical characterization of Trypsin-like enzyme from Antarctic Hydrobiont Adamussium Colbecki

Raksha Nataliia, Halenova Tetiana, Kravchenko Olga, Vovk Tetiana, Savchuk Olexiy and Ostapchenko Lydmila

Page No: 62-68

Abstract:A two-step high-yielding procedure was described for the purification of a trypsin-like enzyme from hydrobiont Adamussium colbecki, consisting of affinity chromatography on SBTI-sepharose 4B followed by size exclusion chromatography on Superdex 75 PG column. After the final purification step, trypsin-like enzyme was purified 7.52-fold with 50.3 % yield. The purified enzyme (24 kDa) was homogeneous as confirmed by SDS-PAGE.

The pH and temperature optimum were estimated to be 9.0 and 24°C. Trypsin-like enzyme was unstable above pH 12.0 and below pH 5.0. The enzyme was partially inhibited by PMSF, fully inhibited by SBTI and was not inhibited by EDTA, characterizing it as serine protease. The trypsin-like enzyme kinetic constant Km was lower at 8ºC compared to result at 24ºC while the catalytic efficiency kcat/Km was the same at both temperatures.

Beta carotene inhibiting HIV-1 reverse transcriptase, an in silico approach

Nag Anish and Das Reema

Page No: 69-73

Abstract: Due to the outspread of various emerging diseases, research on the discovery of new drugs is being carried out extensively. Several phytochemicals with medicinal importance are now being used for this purpose due to its effectiveness and safety in comparison to the conventional synthetic ones. Computational docking is further being used for the fast and cost-effective screening. Reverse transcriptase is a key enzyme involved in the conversion of viral RNA sequence into complementary DNA (cDNA) sequence leading to various retroviral diseases like HIV/AIDS. Patchdock docking server was used in this study to perform in silico enzyme-inhibitor binding experiment between twenty phytocompounds and HIV-1 reverse transcriptase enzyme.

Beta-carotene was found to have strongest binding potential in comparison to other phytochemicals. The results suggested that this compound can be used as therapeutics in the future as naturally occurring HIV-1 reverse transcriptase inhibitor.

Study of expression of glucose transporters and insulin like growth hormone in the testes of Non-Obese Diabetic mice

Kathirvel N., Dhana Sekaran G. and Arulvasu C.

Page No: 74-81

Abstract: Recent research on Diabetes Mellitus (DM) suggested that it affects the functions of all major vital organs which included male reproductive systems. Less secretion of insulin leads to less intake of glucose from blood. Hence the patient will suffer with hyperglycemia that will cause heavy stress to individual cells including developing germ cells. Glucose transporters and insulin like growth factors are known to play important role in hyperglycemia. Therefore, we would like to study the expression of some of the glucose transporters such as GLUT1, GLUT3 and GLUT8 as well as insulin like growth factor (IGF) in the testes tissues of Non-Obese Diabetic (NOD) mice by PCR, Western blot and Immunofluorescence analysis.

PCR analysis results showed the higher expression of glucose transporters (GLUT1, 3 and 8) in the testes of NOD mice than control mice. But the IGF expressed in the testes was in lesser amount when compared to the control. The expression pattern of these molecules was also confirmed by the Western blot analysis. Moreover, the Immunofluorescence analysis of GLUT1, 3, 8 and IGF in NOD mice testes samples revealed that glucose transporters and IGF expression were less on surface and distributed on the cytoplasm of testes tissues. When compared to control, these proteins were present in the cell surface suggesting the role of glucose transporters and IGF during germ cells development in male.

An efficient in vitro propagation protocol for Catharanthus roseus (L.)

Sharma Vandana, Kumar Ashwini, Kumar Arun and Kumar Sumit

Page No: 82-87

Abstract: Catharanthus roseus (L.) is an important medicinal plant and has several commercially valuable secondary metabolites which are used by the pharmaceutical industry for the treatment of severe diseases including cancer and Hodgkin's disease etc. Major limiting factor for its large-scale cultivation is only 30% seed germination rate and 70% success rate in stem cuttings and vegetative splits.

For its fast regeneration, an efficient and reproducible protocol has been developed through standardization of surface sterilization and shoot and root regeneration. Treatment with 0.1% HgCl2 solution was found to be best with maximum survival rate of explants. Shoot induction starts in 5 to 6 days at 3.0 mg/l BAP and it takes 11 to 12 days at 0.5 mg/l BAP concentration in both MS and Gamborg media. Higher numbers of shoots (3.8) and shoot length (4.0cm) were recorded in MS medium than Gamborg medium (0.6 shoots per explants along with 3.5cm length) at BAP (3.0 mg/l) + NAA (1.0 mg/l) concentration. Regenerated shoots were transferred for rooting on half strength MS medium containing IBA and NAA and best rooting was found at 0.5mg/l IBA. However, MS medium was found best for propagation of C. roseus.

Pigment production by an extreme halophilic archaea on Halorubrum sp. J4.2.2 from little Rann of Kutch, Gujarat, India

Pandya R.D. and Singh S.P.

Page No: 88-100

Abstract:Pigments are produced by a variety of microorganisms from various habitats. The halophilic archaea accumulate pigments under certain conditions of the salt concentrations exceeding 250g/l and characterized by dense color of the concerned habitat. The chlorophylls, scytonemin, carotenoids including α-bacterioruberin, salinixanthin and spirilloxanthin derivatives are reported in the cell pellets of the model organisms belonging to the genera Haloferax, Haloarcula and Halobacterium. In the current study, pigments were extracted from Halorubrum sp J4.2.2 (KR857507) isolated from the saline desert soil on archaeal medium, pH 8. After growth, the pigment was extracted with acetone-methanol (7:3 v/v) and identified by UV-VIS Spectroscopy and FTIR. The spectrophotometric analysis of the pigments revealed their carotenoid nature.

The phenotypic attributes of the isolate were studied and the organisms were further studied by 16S rRNA sequencing. The 16S rRNA gene sequence was submitted to Gene bank with the accession number of KR857507. The 16S rRNA gene sequence analysis confirmed the identification of the organisms as Halorubrum sp., J4.2.2. The cultural conditions with respect to the salt concentrations and carbon sources were optimized for the pigment production. The characteristics of the pigments can provide important clues for the identification of the archaea.

Combining ability and heterosis studies for some economic traits in rice (Oryza sativa L.)

Gowayed Salah M.H., Abd El-Moneim Diaa, Metwali Ehab M.R. and El-Malky Mohamed M.

Page No: 101-111

Abstract:Ten rice genotypes were crossed in a line x tester mating design using four testers and six lines to produce 24 hybrids. Analysis of variance showed highly significant differences among genotypes for all the studied traits indicating a wide range of genetic diversity, different origin of these parents and prevalence of additive variance. However, mean sum of squares due to line x tester was significant for the studied traits except plant height. Sakha106, Eg-N-9 and Sakha101 showed negative highly significant GCA effects for duration revealing that these varieties could be good combiners for developing early duration genotypes. While for plant height, Sakha101, Sakha 106, Eg-N-1, Eg-N-2 and Eg-N-9 exhibited highly significant negative GCA effects for short plant height.

The best hybrid combinations were IRBL3-CP4 x Eg-N-9, IRBL5-M x Sakha101, Sakha106 x Al-Ahsa Type 1 and Sakha106 x Al-Ahsa Type 2. The best hybrids produced from different crosses were IRBL3-CP4 x Eg-N-9, IRBL5-M x Sakha101, Sakha106 x Al-Ahsa Type 1 and Sakha106 x Al-Ahsa Type 2. These crosses could be involved in the rice breeding program. The cluster analysis classified the ten parental genotypes into four clusters. The similarity was higher between the genotypes within cluster showing same origin.

Eradication of Microbial Biofilms by Tannin Rich Crude Extracts of Indian Medicinal Plants

Shukla Varsha and Lokur Anushree

Page No: 112-117

Abstract:Ability of pathogens to form sessile adherent structures on indwelling medical devices poses a major threat because these structures called biofilms are not only impermeable to antibiotics but also can disperse to cause multiple foci and systemic manifestations. S. aureus shows high incidence of post-operative implant-based infections. Recently high incidence of nosocomial infections by A. baumannii is also reported.

In the search for new pharmacological agents that would be effective in overcoming biofilm-based infections, we explored tannin rich plants used in traditional Indian medicine for their ability to eradicate biofilms (MBEC) and also destroy liberated bacteria (BBC). All the six extracts tested showed ability to eradicate biofilms, T. chebula, P. granatum and M.indica being the most promising against both the tested organisms. Moreover, the extracts of these plants demonstrated this ability against both antibiotic sensitive as well as resistant strains of both the organisms at fairly low concentrations, as compared to antibiotics, ampicillin and chloramphenicol making them good candidates for curbing implant-based infections.

Morinda tinctoria Roxb. as potential source of biopesticides against the cotton bollworm Helicoverpa armigera

Praveena A. and Sanjayan K.P.

Page No: 118-124

Abstract:The cotton bollworm Helicoverpa armigera is a well-known pest in many agricultural cropping systems worldwide. The extensive and widespread use of synthetic insecticides against H.armigera has led to the development of resistance against almost all commercially used compounds including new insecticides. The underlying possible effects of Morinda tinctoria against H.armigera were investigated based on the biological and biochemical studies. Root-bark and fruit extracts were tested for mortality of 3rd instar larvae of H.armigera at concentrations of 1, 3 and 5%. 1% ethyl acetate fruit and root-bark extract were the most potent bringing the maximum mortality.

The maximum feeding deterrence index of 79.832% was observed in the 5% ethyl acetate extract of root-bark. A significant percentage of the pupa failed to emerge as adult and the emerged ones displayed structural abnormalities in the form of deformed wings. The adults that emerged from larvae fed with extracts produced lesser number of eggs than the control group. There was a decrease in the activity of Carboxylesterase and Acetylcholinesterase compared to the control. Protease activity was least affected by the extracts of the fruit and root bark.

Varietal identification in rice (Oryza sativa L.) hybrids and parental lines using biochemical and molecular markers

Kumar Sunil, Singh Shailaja, Misra Pragati, Shukla Pradeep Kumar, Rai Prashant Kumar and Pradhan S.K.

Page No: 125-132

Abstract:Limitations in the morphological variation among the majority of rice varieties due to narrow genetic base make it compulsory for the development of expeditious and reliable methods or varietal identification categorically for those working in seed certification and quality maintenance. Identification of felicitous rice genotypes is of prime paramountcy to ascertain quality seed required for achieving ecumenical victuals demand. Rice hybrids such as NARENDRA SANKAR DHAN 2, RAJALAXMI and AJAY their parental lines such as IR 58025A, NDR3026-3IR, CRMS32A, IR42266-29-3R, CRMS31A and IR42266-29-3R were taken for the present experiment. Albeit, unifications of different rapid chemical tests were subsidiary in identification of individual genotypes.

However, all these cultivars were identified by SDS-PAGE and hence could be utilized as a puissant implement to identify every genotype in a short period of time at primary functional level. A set of 16 SSR markers distributed across the genome was utilized for screening the rice hybrids and their parental lines. Eight primers namely RM202, RM234, RM228, RM6100, RM258, RM18, RM335 and RM334 could not differentiate among the four hybrids, however, other eight primers i.e. RM202, RM234, RM228, RM6100, RM258, RM18, RM335 and RM334 engendered paramount differences, hence, were taken for further analysis. The cluster analysis predicated on Jaccard’s kindred attribute co-efficient utilizing UPGMA grouped eight parental lines into two clusters. Within the cluster, the parental lines of hybrids shared a mundane genetic background. The eight parental lines along with their hybrids were clustered with reverence to the genotyping data utilizing the microsatellite markers. It could limpidly distinguish two hybrids NDRH2 and Rajlaxmi and their parents into two distinct clusters.

Antibacterial activity of biogenic silver nanoparticles synthesized using Phycobiliproteins of Anabaena iyengarii

Thangaraj Ramasamy, Mubarak Ali Davoodbasha and Thajuddin Nooruddin

Page No: 133-139

Abstract:The present study reported the biogenic synthesis of silver nanoparticles using paddy inhabitant cyanobacteria Anabaena iyengarii. The synthesized silver nanoparticles were characterized by UV-vis, FT-IR, SEM and XRD analysis. The UV-Vis spectrum revealed a characteristic surface plasmon resonance peak at 423 nm which corresponds to the absorption band of silver nanoparticles. FTIR spectrum and X-ray diffraction patterns confirmed the formation of silver nanoparticles. The SEM results showed that synthesized silver nanoparticles were spherical shaped with size of 54nm.

In this study, the efficiency of silver nanoparticles as an antibacterial against maximum zone of inhibition on Staphylococcus aureus followed by Pseudomonas aeroginosa and E. coli was evaluated. This study elaborated the process of biogenic silver nanoparticles formation via green chemistry approach. Antibacterial activity was also analyzed.

Downregulation of Lactate Dehydrogenase Expression in Lactobacillus Brevis ATCC367

Devi Alka, Kumar Narayan and Khanna Sunil

Page No: 140-147

Abstract:Chemical mutagenesis with ethyl methansulphate (EMS) was used to develop mutants of Lactobacillus brevis ATCC367 that have downregulated lactate dehydrogenase activity. Three mutants (M-43, M-135 and M-200) showed 1, 1.6 and 4 fold reduction in LDH activity as compared to the wild-type Lactobacillus brevis but growth rate and substrate utilization were unaltered as compared to the control.

Lactate dehydrogenase from mutant M-200 which had 4 folds decreases in enzymatic activity was purified to homogeneity with a molecular size of 35KDa, maximum activity of 0.0046 IU at pH- 7.0 and temperature 250C.

Transcriptome mining and functional annotation of Coscinium fenestratum (Gaertn.) Colebr by Next Generation Sequencing

Ashalatha and Gopinath S.M.

Page No: 148-163

Abstract:Coscinium fenestratum (Gaertn.) Colebr, a yellow vine which belongs to Menispermaceae family is rich with bioactive secondary metabolites that might signify valuable leads in the production of new pharmaceutical agents. This non-model plant is already at the verge of extinction and the little genetic information is accessible for this species, particularly concerning with the putative gene and its expression pathway in the alkaloids present. In the present study, the alkaloids present were explored by LC-MS and the major alkaloids like berberine, jatrorrhizine, palmatine, tetrahydropalmatine, tetrahydroberberine, magnoflorine, isocorydine, glaucine were reported. Next generation sequencing (NGS) was employed as a powerful tool for identifying the molecular signatures related to physiological functions of this plant. In view of exploring the candidate genes responsible for the biosynthesis of various alkaloids in C.fenestratum, the de novo transcriptome sequencing of leaf and stem of C.fenestratum was done using Illumina Hiseq 2500 platform. Around 17,342,427 raw reads from leaf and 19,277,386 raw reads from stem sample were deciphered.

These raw reads on quality filtration, assembly and clustering obtained 17,783 transcripts unique for leaf and 3,284 transcripts unique for stem and 31,731 transcripts which were conserved among the stem and leaf sample. The transcripts were then annotated with all available databases; of this, around 144 candidate genes were annotated for the enzyme related to protoberberine and aporphine biosynthesis. Total 10,949 SSRs and 3,930 transcription factors at 60% coverage and 60% identity were also annotated. Thus the information obtained from this work will provide an important information about gene expression, genomics and proteomics research in C.fenestratum.

Microbial Bioremediation of Heavy Metals: Emerging Trends and Recent Advances

Zango Usman U., Yadav Mukesh, Sharma Vandana, Sharma J.K., Panwar Sanjay, Dan Sidhartha and Sharma Anil K.

Page No: 164-178

Abstract:Significant amounts of heavy metals are being added into the soil and effluents each and every day due to enhanced industrialization and other anthropocentric activities. Many heavy metals are even non-degradable in nature and hence once released into the environment remain in circulation. Upon increased concentrations beyond the threshold limit, these heavy metals may not only result in chronic and lethal diseases in humans but also affect the animal and plant metabolism. Many of the physico-chemical methods employed as of today such as electrochemical treatment, ion exchange, precipitation and reverse osmosis for heavy metal removal from waste streams have not been proved cost effective, therefore a biological approach could prove as an alternative remediation technology for heavy metals contamination.

Microbes have evolved mechanisms such as active efflux or sequestration with proteins or insoluble compounds through which they may resist, detoxify or metabolize these heavy metals. Current review highlights the recent advancements made in understanding the heavy metals-microbes interaction and applications thereof in heavy metal remediation.