Research Journal of Biotechnology

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Vol. 9(1) January 2014

Study on a malate-producing Rhizopus delemar strain utilizing xylose

Wu Yue, Li Xingjiang*, Li Shuo and Pan Lijun

Malic acid, as one of the most important organic acids in food and chemical field, was paid great attention to its microbial fermentation. The high-production of malic acid was expected to be obtained by an isolated strain with xylose as carbon source in this paper. Phylogenetic tree of ITS region analysis was carried out for identification and then the single factor experiments and response surface methodology were employed to optimize the fermentation process parameters. Identification showed that the strain HF-119 for malic acid production belonged to the Rhizopus delemar species. The optimal medium composition for malic acid production were as follows: xylose, yeast extract powder, (NH4)2SO4, MnSO4.H2O, FeSO4.7H2O, MgSO4, KH2PO4 and CaCO3 were 80.0g/L, 2.97g/L, 2.0g/L, 0.21 g/L, 0.07g/L, 0.27g/L, 0.4g/L and 70g/L, respectively; the malic acid yield was 57.42g/L under this condition. The isolated strain Rhizopus delemar HF-119 can be used for malic acid production. The yield of malic acid was enhanced effectively through the fermentation process optimization.

An Efficient In Vitro Regeneration System in Lentil (Lens culinaris) using Cotyledons with Half Embryonic Axes

Sharma Richa, Acharjee Sumita and Sarmah Bidyut Kumar*

Lentil (Lens culinaris Medik) crops are affected by a number of stresses such as drought, diseases (Fusarium wilt, Ascochyta blight) and pests (Etiella zinckenella and aphids). Lack of resistant sources within lentil gene pool prevents development of improved lentil varieties to resistant to these stresses. Gene technology could be an alternative approach for the development of elite genotypes resistant to the above stresses. However, one of the pre-requisites of genetic transformation methods is availability of an efficient and reliable in vitro regeneration and transformation protocol. We optimized an in vitro regeneration system in lentil using cotyledons with half embryonic axes as explants. For in vitro shoot regeneration, SIM medium (SIM6) supplemented with 1mg/L of 6-Benzyl amino purine (BAP) and kinetin (1mg/l) was found suitable. While shoot elongation was achieved on medium containing 0.5mg/L BAP (SEM3), the cotyledonary explants produced highest (8.3) number of multiple shoots per explant on SIM6 followed by SEM3 medium. The healthy shoots were rooted on MS basal medium after dipping the base of shoots in 1 mg/l IBA and rooted plantlets were established in soil. The protocol showed in vitro regeneration efficiency of 90.6%. Thus we developed an efficient in vitro regeneration protocol in lentil.

Genetic Transformation and Plant Regeneration in Cotton (Gossypium hirsutum L.)

Ezhilarasi T.* and Nallathambi G.

An efficient protocol has been developed for regeneration of complete plants through somatic embryogenesis in cotton. For the development of highly reproducible regeneration system through somatic embryogenesis seven days old hypocotyl of Coker 310 and Coker 312 were used. Pale yellow colored calli were obtained on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l 2, 4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l Kinetin (KT) after 30 days. Pale yellow coloured calli turned grayish white in the callus maturation medium containing MS medium without plant growth harmones for the next 30 days. 60 days old calli were infected with the Agrobacterium strain 611 harbouring plasmid pBI 121 for 30 minutes followed by 48 h length of co-cultivation. After co-cultivation, the calli were transferred to the somatic embryo induction medium MS containing 3.8 g/l Potassium Nitrate (KNO3), 50 mg/l kanamycin. The bipolar torpedo stage and cotyledon stage embryos were transferred to the regeneration medium MS supplemented with 0.1 mg/l Gibberellic acid (GA3) and 1.0 mg/l 3-Indoleacetic acid (IAA) for shoot elongation and root development. The regenerated plantlets produced 3-5 leaves and sufficient root system within 8 weeks period.

Evaluation and Selection of native fungal isolates for cellulase enzyme production

Ganesan Sasikala and Nellaiappan Olaganathan Gopal

Cellulases are complex hydrolytic enzymes capable of hydrolyzing cellulose into glucose units. They have immense potential in many industries and also in saccharification of the lignocellulosic substrates. This study was aimed to screen the cellulolytic fungi from native environmental sources. About 21 fungal cultures were isolated and screened for cellulolytic activity using plate screening technique and cellulase assays. Preliminary screening was done based on the clear zone formation. The clear zone ratio ranged from 1.246 to 0.138 after 5 days after incubation (DAI). The isolates FD3, FP1 and FP2 showed higher zone ratio of 1.246, 1.193 and 0.950 respectively. Out of 21 isolates, only 18 exhibited cellulolytic activity. The screened isolates were also subjected to enzyme assays like cellulase, exoglucanase, endoglucanase and cellobiase after 3DAI, 6DAI and 9DAI. Higher cellulase, exoglucanase and endoglucanase activity was found from the mean results of isolate FD3 which exhibited 5.659, 5.120, 5.887 units per ml after 9DAI followed by isolates FP1 and FP2. But the cellobiase activity was found higher in FP1 isolate (5.102 units per ml after 9DAI). Based on microscopic characters, these particular efficient isolates FD3 and FP1 were identified as Trichoderma and Aspergillus. This can be chosen for the cellulase production to saccharify the cellulosic substrates having higher value in ethanol production.

Purification and composition analysis of polysaccharide from edible seaweed Enteromorpha prolifera and polysaccharides depolymerized enzymes from microorganisms

Chao Zhao

Enteromorpha prolifera, an important member of marine green algae, contains many bioactive compounds and polysaccharide is the major component. A water-soluble polysaccharide was extracted from E. prolifera and isolated by ion-exchange chromatography and gel-filtration chromatography. Its primary monosaccharide compositions were determined by gas chromatography. The data obtained indicated that the polysaccharide was mainly composed of rhamnose, galactose and xylose in a molar ratio of 43:46:10. Treatment with the polysaccharolytic enzymes to produce oligosaccharides can improve the value-added utilization remarkably. A new method was reported herein for the detection of extracellular E. prolifera polysaccharase production by way of plate assay. It was based on the formation of haloes on the petri dish containing the Trypan blue dye labeled E. prolifera polysaccharide as substrate. The plate assay could provide an easy and successful way for screening large numbers of microbial cultures exhibiting polysaccharide- degrading activities.

Photocatalyst-Incorporated Nonthermal Plasma Sterilization System for the Treatment of Biologically Contaminated Water

Lee S. B.*, Jang D. I. and Mok Y. S.

Sterilization of Escherichia coli (E. coli) in synthetically prepared wastewater was investigated with a dielectric barrier discharge (DBD) plasma reactor. To take advantage of the ultraviolet (UV) light emitted by the plasma for the sterilization, tinanium dioxide (TiO2) and graphene oxide (GO) were used as photocatalysts. The DBD reactor emitted at various wavelengths in the range of 316-380.5 nm and the experimental results indicated that the combination of the DBD plasma with the photocatalysts resulted in substantial improvements in the sterilization of E. coli. At a voltage of 21.5 kV, about 3 log reduction in the E. coli population density (water volume: 1,000 mL; initial E. coli concentration: 107 colony forming units (CFU) mL-1) was achieved in 6 min with the incorporation of the photocatalysts (content: 0.1 g L-1) whereas in the DBD-alone case it decreased by 102. Compared to the TiO2, the GO exhibited better sterilization performance. The present DBD plasma was found to be more energy-efficient than other sterilization methods such as pulsed electrical discharge, UV irradiation and photocatalysis. On the basis of an initial E. coli concentration of 107 CFU mL-1, the energy requirement for 4 log reduction was about 2.2 J per mL of water volume.

Bioinformatics Analysis of Metallothionein Gene from Agaricus bisporus, Garnoderma lucidum, Taiwanofungus camphoratus and Paxillus involutus

Qiang Cheng-kui, Zhou Bao-ya, Yang Xiao-zheng, Wei Feng, Lu Zheng-li, Wang Song-song3 and Qin Yue-hua*

Based on the published sequences of MT genes from A. bisporus, G. lucidum, T. camphoratus and P. involutus in NCBI database, their molecular structures, physiological functions and evolutionary relationship were analyzed using the bioinformatics methods. Results showed that AbMT, GlMT, TcMT and PiMT genes contained complete ORF and their theoretical points of the last three encoding proteins were higher than 7.0. AbMT, GlMT, TcMT and PiMT were relatively rich in random coil and extended strand but transmembrane helices and signal peptides were not found. 4 MTs were mainly localized in cell nucleus (over 60%) and their cellular functions might have some relation to central intermediary metabolism. Multiple sequence alignment indicated relatively high identity (more than 52%) and short genetic distances (lower than 0.900) among 4 MT nucleotide sequences. Abundant genetic diversity and strong codon bias were found based on the halotype diversity, average number of nucleotide differences, nucleotide diversity, effective number of codons, codon bias index and scaled chi-square. Simultaneously, we deduced that 4 MT genes during molecular evolution were under positive selection. The present study might provide basis for further investigation of MTs molecular mechanisms and genetic laws.

Bioleaching enhancement of copper mine tailings by oxalic acid

Li Dou and Li Dongwei*

Copper mine tailings are harmful to environment because of generating acid mine drainage (AMD). Bioleaching is not only the cause of AMD but also can be regarded as an appropriate disposal of tailings if the toxic elements were oxidized by bacteria and then dissolved in the treatment facilities before discharging into the tailings ponds. The residual floatation agents, such as oxalic acid, are ubiquitous in copper mine tailings and the information of their effects on bioleaching is few. Acidithiobacillus ferrooxidans was used in bioleaching tests with oxalic acid at 200 mg/L and 500 mg/L respectively. The results in determination of Cu, Mn, As, Fe, pH, Eh and cell numbers in leachate during the 30 days indicated that oxalic acid enhanced the metals leaching effects. The morphology of surface on copper mine tailings was observed by scanning electron microscope after the bioleaching. The results show the aggregates and tracks of acid leaching could prove the evidence of bioleaching enhancement.

Effect of Cyclophosphamide on the immune status of chickens vaccinated with Newcastle disease vaccine

Varalakshmi S.* and Kirubaharan J. John

The effect of Cyclophosphamide (CY) on the immune status of birds was studied by administering the chickens with live Newcastle disease vaccine. Normal and immunosuppressed chickens were vaccinated with the Thermostable live Newcastle disease vaccine by ocular-nasal route at 10th day of age. The immune suppressed chickens had significantly low serum antibody titers and histopathological studies showed that there is depletion of bursal tissue in the CY treated chickens compared to CY untreated chickens. There is no significant difference in the cell mediated immunity between ablated and non ablated group. This study concludes that there is suppression of only humoral immunity by administering the cyclophosphamide and there is no effect on cell mediated immunity. CY will be used to study the role of cell mediated immunity and mucosal immunity in the absence of humoral immunity.

The development of strain dependent mechanical anisotropy in chicken skin

Cilingir Ahmet C.*, Yilmazcoban I. Kutay, Nursel Kiratli-Yilmazcoban and Durmaz Ufuk

Mechanical anisotropy is critical to the function of many load-bearing soft tissues such as skin. Biaxial tensile experiments were performed to investigate the tensile stress-strain response of chicken skin in both longitudinal and transverse directions. This testing system was also used to study the development of mechanical anisotropy in the tissue by applying uniaxial remodelling load in transverse direction. The skin samples were found mechanically anisotropic with longitudinal axis stiffer than the transverse axis. When the sample loaded uniaxially in transverse direction, the mechanical anisotropy also changed the direction by incubation time and the speed of remodelling was increased by increasing the load. Since the skin samples were kept in saline solution during the remodelling period, collagen fibrils were not able to synthesize, therefore, the skin was broken down after a period of incubation according to remodelling load. Not only the initial anisotropic behaviour but also the change of the direction of the mechanical anisotropy of the skin could be easily studied by using this simple biaxial tensile testing system.

High radical scavenging activity of camphor rich Artemisia nilagirica essential oil growing in Eastern plain areas of India

Sreeram Sandip, Parida Reena, Sahoo Santilata, Nayak Sanghamitra and Mohanty Sujata*

Essential oil extracted from leaves of Artemisia nilagirica growing on eastern plain area was analyzed by extensive GC–FID and GC–MS. Twenty two constituents amounting to 98.9% of the total essential oil contents were identified. The major constituents of the oil were camphor (32.56%), borneol (12.59%), caryophyllene (9.6%) beta-pinene (9.4%) β- transocimene (6.14%) and germacrene-D (5.34%) respectively. The other characteristic compounds observed in the plants were eucalyptol, caryophyllene oxide, humulene, bornyl ester, terpeneol, T-Muurolol and δ-Cadinene etc. The essential oil screened for possible antioxidant activity using DPPH (2,2-diphenylpicrylhydrazyl) showed high free radical scavenging activity (6.72 μg/ ml) while the IC50 value of standard antioxidant ascorbic acid was 6.58μg/ml. This study indicated that A. nilagirica essential oil having high antioxidant activity bears immense potential for future potential uses.

Dynamic Degradation of p-Nitrophenol using Immobilized Cells of Pseudomonas aeruginosa HS-D38 in Beads of Acticarbon-Ca-alginate Gel

Zheng Yongliang

p-Nitrophenol (PNP) is distributed as herbicide and hydrolytic product of some organophosphorus insecticides or industrial waste in various environments. In this study the beads of calcium alginate with acticarbon (BCAAs) were employed as a gel matrix to immobilize Pseudomonas aeruginosa HS-D38 cells for PNP degradation. Dynamic analysis by UV-Vis spectrophotometer showed that free cells displayed better PNP-removing efficiencies than that by cells immobilized in beads of calcium alginate (BCAs) at lower initial PNP concentration of less than 100 mg/L whereas the cells immobilized in BCAAs showed an efficient PNP removing performance compared to free cells. When the initial PNP concentration exceeded 200 mg/L, the BCAAs-immobilized cells possessed higher PNP degrading activity and stronger tolerance against the inhibitory effect of PNP even at a high concentration of 600 mg/L. HPLC analysis indicated that PNP was completely degraded by BCAAs-immobilized cells of HS-D38. A wider range of temperature (250C-450C) and pH conditions (pH 5.0-10.0) were observed in the immobilized cells system. Storage stability and reusability experiments revealed that the PNP degradation functions of BCAAs-immobilized cells were stable after reused for 30 times or storing at 40C for 5 weeks suggesting that immobilized cells of HS-D38 in BCAAs have a potential application prospect in bioremediation of p-nitrophenol-contaminated water.

Molecular Characterization, Sequence Analysis and Tissue Expression of a Porcine Gene-MOSPD1

Xie Xuru and Liu Yonggang*

The full-length cDNA sequences of one porcine gene, MOSPD1, was amplified using the rapid amplification of cDNA ends (RACE) method based on one pig EST sequence which was highly homologous to the coding sequence of human MOSPD1 gene. Sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 213 amino acids that has high homology with the motile sperm domain containing 1 (MOSPD1) of eight species—rhesus monkey (98%), human (98%), sumatran orangutan (98%), horse (98%), cattle (97%), mouse (96%), rat (96%) and dog (96%)—so that it can be defined as porcine MOSPD1 gene. This novel porcine gene was assigned to GeneID: 100359355. This gene is structured in five exons and four introns as revealed by computer-assisted analysis. The phylogenetic analysis revealed that the porcine MOSPD1 gene has a closer genetic relationship with the MOSPD1 gene of horse. Tissue expression analysis indicated that the porcine MOSPD1 gene is generally and differentially expressed in detected tissues including spleen, muscle, skin, kidney, lung, liver, fat and heart.

In vitro multiple shoot induction and plantlet formation from cotyledon and hypocotyl explants in tomato

Kurra Archana, Pamulaparthi Archana, Marka Rajinikanth and Nanna Rama Swamy*

An efficient, reproducible protocol for multiple shoot induction and plantlet formation from cotyledon and hypocotyl explants of Solanum lycopersicum cv Arka Vikas is reported. The cotyledon and hypocotyl explants were cultured on MS medium supplemented with 0.5-4.0 mg/L 6-Benzlaminopurine (BAP) / Kinetin (KIN) individually. Maximum number of multiple shoots formation was observed at 2.0 mg/L BAP in cotyledon and 1.5 mg/L KIN in hypocotyl explants. The newly formed micro-shoots were multiplied further on MS medium supplemented with 2.0 mg/L BAP for cotyledon and 1.5 mg/L KIN for hypocotyl explants. For elongation, micro-shoots were cultured on MS medium containing 0.5 mg/L GA3. The elongated shoots were transferred on to MS medium supplemented with varying concentrations of IAA/NAA (0.5 to 2.0 mg/L) for in vitro rooting. Profuse rhizogenesis was observed at 1.0 mg/L IAA. The in vitro regenerated plants were acclimatized in walk-in-chamber at 250±10C and maintained in the green house. Later these were shifted to field conditions and the survival percentage was found to be 80%. These regenerated plants were found to be similar to that of donor plants in all aspects. This reproducible regeneration protocol can be used for genetic transformation studies in tomato cv Arka Vikas.

Aspergillus sydowi Metabolites Efficacies against the Mosquito Larval (Culexpipiens and Aedescaspius) Population and Cytotoxicity after Purification with Column Chromatography

Abutaha Nael, Al-mekhlafi Fahd A., Semlali Abdelhabib, Baabbad Almohannad and Wadaan Mohammed A.

The present study was carried out to evaluate the larvicidal activity and cytotoxicity of Aspergillus sydowi extract. The LC50 calculated of methanol crude extract and fraction 6 was 250.86±4.15 and 56.61±0.89 µg/ml for Aedes caspius and 271.02±7.01 and 103.74±0.99 for Culex pipina respectively after 24 hours of exposure. Larvae of Ae. caspius treated with fraction 6 of A. sydowi methanol extract underwent a lethal disruption of the peritrophic membrane and an extensive cellular microvillar disruption. Expression of Bcl-2 representative of antiapoptotic protein was greatly reduced in control when compared to treated group conversely; Bax the pro-apoptotic protein was higher in treated group. The cytotoxicity of fraction 6 and 7 showed 80.25% and 86.3 % survival at the highest concentration (37µg/ml) tested against Hepg2. On the other hand, Jurkat cells lines showed higher sensitivity to the fraction tested. Fraction 6 showed 45.17% survival where as fraction 7 showed 86.34 % survival at the highest concentration tested. The HPLC chromatogram revealed that fraction 6 obtained is not pure and requires further purification and characterization to examine larvicidal and cytotoxic effect.

Review Paper:

Two-photon fluorescence microscopy-a prospective tool in the research of Traditional Medicine

Wang Changming, Yu Guang, Jian Tunyu, Yang Niuniu and Tang Zongxiang*

Two-photon fluorescence microscopy is a new technique combined with the laser scanning confocal microscopy and two photon excitation techniques. It is increasingly being applied to the research of modern medicine because it has the advantages of deep tissue imaging and less tissue injuries. The technology can help researchers to achieve the imaging of the living animals without organ harvesting and sectioning. This kind of body imaging technology with no damage to the organization is very suitable for study of the characteristics of traditional medicine (TM). Here we reviewed the characteristics, the advantages and the applications of two-photon fluorescence microscopy in several living tissues and discussed the possibility in the TM fields.