Research Journal of Biotechnology

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Morphological phenotypes of an Aspergillus niger strain with high citric acid production capacity

Xie Hui, Wang Fengqing and Wei Dongzhi

The study investigated morphological phenotypes of an industrial Aspergillus niger YX-1217 which is regarded as a pre-requisite to ensure high and stable citric acid productivities and to understand the complicated relationship between morphology and productivity. Multiple buds were formed on the surface of spores of A. niger YX 1217 which resulted in tightly wrapped pellets in submerged cultivation. Morphology structure analysis of A. niger YX-1217 and YX-1217G – a degenerated strain by scanning and transmission electron microscopies revealed that the surface melanin granules of the spores of the two strains have obvious differences. The melanin layer of the former was very integrated and compact, but that of the latter was partly missing and became thinner and relatively loose. Under the optimal condition, growth of A. niger YX-1217 led to a mean pellet diameter of about 140.0 µm and a yield of citric acid of 179.6 g L-1. The increased citric acid productivity by A. niger YX-1217 was related to unique budding formation of spores, integrated and compact of the spore melanin layer and the reduced mycelial pellet size and increased pellet number. It shed light on a kind of important mechanism of high yield of citric acid in A. niger and provided a novel view to understand and develop strains with high yield citric acid or other organic acids.

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Histopathological studies with Newcastle disease virus strain R2B having an altered fusion protein cleavage site amino acid motif

- Yadav Kalpana, Pathak C. Dinesh, Debnath Ashis, Kumar Asok M., Dey Sohini and Chellappa Madhan Mohan

NDV strains vary greatly in virulence. NDV tissue tropism and virulence have been attributed to the cleavage site of the fusion protein. Avirulent strains have dibasic amino acid residues whereas virulent strains have multi-basic residues at cleavage site leading to systemic infection. The effect of avirulent cleavage site motif within a mesogenic viral backbone (rNDV-R2B) was studied experimentally in SPF chickens. It was found that the birds infected with a mesogenic virus with an altered cleavage site (rNDV-R2B-FPCS) had no apparent clinical signs and the histology of the organs studied was normal as compared to birds infected with rNDV-R2B which had clinical signs with mild histopathological changes in various organs. Thus, our findings suggest that presence of lentogenic FPCS within a mesogenic backbone reduces the severity of clinicopathologic disease in terms of minimal histopathological alterations and mounts a protective immune response when challenged with a virulent virus.

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High genetic diversity and population structure of lacquer cultivars (Toxicodendron vernicifluum) in Shaanxi province, China revealed by SSR markers

Thi Tuyet-Xuan Bui, Dinh-Duy Vu, Quang-Hung Dang, Yi Zhang and Xiao-Hua Huang

Lacquer tree or varnish tree Toxicodendron vernicifluum (Stokes) F. A. Barkley (formerly Rhus verniciflua Stokes, Anacardiaceae), a member of the Anacardiaceae family is a flowering plant species found in Asia, especially in China and Japan. In order to develop conservation strategies for this species, we investigated the level of genetic variability within and among lacquer cultivars to provide guidelines for the conservation, management and restoration of this species. Twenty-five SSR markers were used to analyze 109 individuals from five lacquer cultivars in China. Results indicated that Gaobazhi cultivar had greater genetic diversity (Ne = 1.899, I = 0.638, Ho = 0.612 and He = 0.410) compared to Huangmao (Ne = 1.878, I = 0.643, Ho = 0.568 and He = 0.418), Huangrongguizhou (Ne = 1.710, I = 0.538, Ho = 0.555 and He = 0.356), Dahongpao (Ne = 1.659, I = 0.466, Ho = 0.573 and He = 0.322) and Damu (Ne = 1.536, I = 0.468, Ho = 0.315 and He = 0.297), with an average of 1.736, 0.55, 0.525 and 0.361 respectively. In all the five cultivars, Fis value had negative values (Fis < 0, P < 0.05) and suggested a remarkable excess in heterozygosity genotypes within these cultivars. Genetic differentiation among cultivars was high (Fst = 0.427 to 0.526), indicating limited gene flow (Nm = 0.278). Analysis of molecular variance (AMOVA) showed low genetic variation within cultivars (46%) compared to among cultivars. The unweighted pair group method average (UPGMA) detected the optimal five clear clusters. Gaobazhi cultivar is closer to the group containing the Dahongpao and Huangrongguizhou cultivars than Damu and Huangmao cultivars.

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Comparative Evaluation of Immobilization of Lipase from Bacillus subtilis AKL13 on Celite-545 and Chicken Egg Shell Powder and its Catalytic Property in Ethyl Ferulate Synthesis

Karthikumar Sankar and Anant Achary

In the present study, lipase from Bacillus subtilis AKL13 (BsL) was produced and immobilized on Celite-545 (CE) and eggshell powder (ESP) at various immobilization process conditions. The optimum condition for the immobilization process was found to be of initial enzyme load, pH 7.5 and 50mM of phosphate buffer. The maximum enzyme load (qmax) and specific activity were found to be 41.8±0.7 mg.g-1 of support and 425±19 U/mg of protein respectively for celite 545 where as in eggshell powder, it was 45.4±0.6 mg.g-1 and 353±32 respectively. The adsorption isotherm curve for both the matrix followed Freundlich isotherm with R2 0.993. The high intra particle diffusion kinetic constant (Kid 5.9) was determined in ESP whereas CE showed 3.5. The thermal stability and organic solvent stability of the free enzyme increased upon immobilization. The lipase immobilized on eggshell powder showed enhanced catalytic activity in the esterification of ferulic acid and ethanol towards the ethyl ferulate synthesis. The GC-MS and RP-HPLC chromatogram confirmed the formation of ethyl ferulate. Hence it was concluded that the low-cost bio-ceramic, chicken eggshell powder could be an ideal solid support than the commercial Celite 545 for lipase immobilization.

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Phylogenetic diversity of drought tolerant Bacillus spp. and their growth stimulation of Zea mays L. under different water regimes

Sajid Moon, Raheem Asif and Ali Basharat

The objective of this study was to evaluate the potential of drought tolerant rhizobacteria to enhance the growth of Zea mays L. under different moisture levels. Drought tolerant bacterial strains that included Bacillus subtilis AB-21, B. subtilis AB-61, B. aryabhatti AB-51, B. toyonesis AM-25, B. licheniformis AM-21 and B. pumilus AB-33 were isolated from a semi-arid region of Pothohar located in North-Eastern Pakistan. Taxonomic status of Bacillus strains was confirmed by 16S rRNA gene sequencing. Strains were screened for in vitro plant growth promoting traits such as auxin synthesis, phosphate solubilization and ammonia production. Colorimetric analysis for in vitro auxin production recorded 142 and 135 µg ml-1 respectively for B. aryabhatti AB-51 and B. pumilus AB-33. After screening, strains were used to mitigate the water stress of Z. mays under axenic conditions. Pot trials were conducted at 21% and 14% field capacity (FC) of the loamy soil under single and mixed culture inoculations. Loamy soil naturally has FC around 28% that was used as control. At highest water stress (14% FC), significant response for fresh (25%) and dry weight (45%) was recorded with B. subtilis AB-21 over respective control. For shoot length, mixed culture combination C-3 (B. licheniformis AM-21 B. pumilus AB-33 and B. subtilis AB-61) recorded 46% increase, over control at 14% FC. On the other hand, B. subtilis AB-21 and B. aryabhatti AB-51 recorded 15% and 50% increases respectively for root length and number of roots at 14% and 21% FC. This study showed that auxin production by different Bacillus strains is associated to enhance the vegetative growth parameters of maize under water stress conditions.

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Determination of Aflatoxin M1 Levels in Some Cheese Types and Retail Yoghurt Samples

Serap Kılıç Altun, Füsun Karaçal Temamoğulları, Mustafa Atasever and Mehmet Demirci

In this study, we investigated the Aflatoxin M1 levels in samples of 130 cheese varieties and 50 yogurt varieties sold in markets and bazaars of Diyarbakır, Erzurum, Mardin and Şanlıurfa provinces by using the ELISA method. Aflatoxin M1 was identified in all of the 130 cheese samples (100%; mean: 260.26 ± 259.46 ng/kg; range: 10.00-800.00 ng/kg) and 50 yogurt samples (100%; mean: 67.1200 ± 135.23 ng/kg; range: 11.60-685.80 ng/kg). It was determined that 16.92% (n=22) of the 130 cheese samples and 2% (n=1) of the yogurt samples contained AFM1 levels above the legal limits defined in the Turkish Food Codex (>500 ng/kg for cheese and yogurt). No significant difference was identified between the different provinces with respect to the AFM1 levels in cheese and yogurt samples. Although products without packaging contained higher level of AFM1 than product with packaging, the difference was not statistically significant. In conclusion, there is a need for increased measures and legal obligations for reducing the levels of aflatoxin in cheese and yogurt.

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Induced resistance in tomato plants to Helicoverpa armigera by mixed formulation of Bacillus subtilis and Beauveria bassiana

Prabhukarthikeyan S.R., Keerthana U., Archana S. and Raguchander T.

In the present study, efforts were made to understand the resistant mechanism mediated by biocontrol agents in tomato plants against Helicoverpa armigera. Defense enzymes viz. peroxidase, phenylalanine ammonia-lyase, polyphenol oxidase, catalase, super oxide dismutase were analysed by Polyacrylamide Gel Electrophoresis (PAGE). SDS-PAGE was used for studying the protein profile as well as Western Blotting for chitinase. Application of a mixture of B. subtilis and B. bassiana significantly enhanced the defense-related enzymes. The results showed higher induction of PO and PPO in plants treated with combination of B2 + EPC8 than the plants treated with individual strains, chemical and untreated controls. In addition, B2 + EPC8 combination showed a greater accumulation of CAT and SOD which plays a vital role in induced resistance. Western blot analysis of chitinase revealed the induction chitinase isoform (29 KDa) in tomato plants treated with a mixture of B2 + EPC8 strains against fruit borer pest. The combined application of bioformulation also increased the accumulation of phenol as well as the defence related proteins in tomato plants. Moreover, the outcome of the present study has a potential to be used for sustainable and eco-friendly tomato production.

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Purification and antimicrobial activity of Cobalt Nano particles from Neurospora crassa

Naveena Lavanya Latha Jeevigunta and Venkata Ramana Navva

The potential impact of cobalt nanoparticles on the living beings is very high as cobalt is known in involved in the vitamin B12 synthesis. The present study is aimed to investigate the role of Co NPs purified from Neurospora crassa. The MTT and anti-microbial assays revealed that Co NPs exhibited a significant role in causing a cytotoxic effect by 24 h. Further, the MMT-060562 cells exposed to Co NPs exhibited significant decline in cobalt-containing enzymes prolidase, nitrile hydratase, glucose isomerase etc. Co NPs induced cytotoxicity and oxidative stress which serve as baseline studies for future studies.

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Screening of laccase producing fungi from mound-building termite in Phatthalung province, Southern of Thailand

Pimprapa Chaijak, Monthon Lertworapreecha and Chontisa Sukkasem

Laccase are well-known industrial enzyme that play role in lignin-degradation. They are wildly distributed in various microbe, especially fungi. Termite associated fungi are the highly potential decomposer of plant biomass degradation in the tropical rain forest because they can digest the complex structure of lignocellulosic substrate by their enzyme system. In this work, the fungal strains were isolated from the nest of mound-building termites. All fungal strains were screened for the extracellular laccase secretion by ABTS assay. The laccase producing strains were identified using 18S rRNA gene. Laccase production was obtained by solid state fermentation of agricultural waste. For biotechnological application, the palm oil mill effluent was used for screening the potential of wastewater treatment. Only fungal strain CHAIJAK-MBT01 expressed the laccase activity. We found the strain CHAIJAK-MBT01 was 99% matched with yeast Galactomyces geotrichum. For laccase production, rice bran provided the maximum yield with 130.38 U/mL. The result of wastewater treatment process suggested that CHAIJAK-MBT01 has a potential to apply for decolorizing the palm oil mill effluent.

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A Molecular Dissection of Non-Host Resistance in Plants

Roy Chowdhury Moumita, Bahadur Ranjit Prasad and Basak Jolly

Non-host resistance (NHR) in plants has gained global attention as a vital regulator of plant disease resistance during the last decade. Plant NHR is defined as the resistance developed in a plant species against all the isolates of a specific pathogen. The strength and durability of NHR in plants arise from its multi-layered defense system that includes a vast amount of gene networks and their products mediated signaling pathways. However, a comprehensive molecular mechanism behind NHR is still elusive. In this review, we have dissected molecular mechanisms underlying the NHR in plants. We have discussed about various genes and proteins involved in different signaling and metabolic pathways that evoke NHR in plants. We have also integrated the role of ROS, PAMP, bacterial effectors, secondary metabolites, enzymes and ECM in order to account for the specificity of NHR. Present state of the art also lacks the investigation on the role microRNAs involved in NHR. Further identification and characterization of genes, proteins and their interactomes will provide significant insights into the genetic and structural basis of NHR in near future. This might have significant agricultural utility in the rationale design of crops expressing long-lasting disease resistance.

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