Biotechnology Journal International

Aims: Through the National Botanical Research Institute's phosphate growth medium (NBRIP) and 16S rDNA sequence analysis were used to isolate and identify the bacterial groups that actively solubilized phosphates in vitro from rhizosphere soil for three cultvited leguminous in agricultural soils from Meknes region. 

Study Design: Rhizosphere soil samples for three cultivated legumes in different sites from Meknes region were collected for the study.

Place and Duration of Study: Department of biology (Soil & Environment Microbiology Unit) Faculty of Sciences, Moulay Ismail University, Meknes, Morocco; between January and July 2014.

Methodology: Out of several hundred colonies that grew on NBRIP medium eight best isolates were selected based on the solubilization of insoluble phosphates in solid medium with solubilizing index (SI) and Phosphate concentration solubilized in liquid medium; The bacterial isolates were identified based on their phenotypic and 16S rDNA genes sequencing.

Results: P solubilization index of these isolates ranged from 2.51 to 6. Drop in pH of the medium ranged from 6.8 to 3.2 with the continuous growth of these isolates for seven days. P-solubilized ranged from 50.95 to 113.11 mg L^-1. They were clustered under the genera Enterobacter, Pantoea, Rhizobium, Klebsiella, Rahnella, Bacillus and Burkholderia.

Conclusion: This research extends the knowledge on Phosphate solubilizing bacteria in the rhizosphere of some cultivated legumes from Meknes region and development of environmentally friendly bio-Phosphate fertilizers.

Aims: Study was carried out to investigate the effect of imidacloprid on biochemical parameters and growth of soil isolate. The imidacloprid degradation by the soil isolate was also studied.

Study Design:  The soil isolate was identified and used for toxicity testing. The isolate of Bacillus weihenstephanensis was further tested for its ability to degrade imidacloprid in minimal salt medium (MSM) and tryptic soya medium (TSB). The role of plasmid in imidacloprid degradation was established by curing experiments.

Place and Duration of Study: Department of Biotechnology and Microbiology, Karnatak University, Dharwad, India between June 2011 and December 2012.

Methodology: The soil isolate was identified by morphological, biochemical characters and 16s rDNA identification. Effect of imidacloprid on DNA, RNA, protein, glucose and growth in soil isolate was studied with 10^-3 to 10^-7 molar imidacloprid for 96 h. Imidacloprid degradation was determined in MSM and TSB for 28 days with samples taken on 7, 14, 21 and 28^th day. The insecticide concentration was tested by HPLC. Plasmid curing was performed.  

Results: The soil isolate was identified as Bacillus weihenstephanensis. The study involving soil isolate Bacillus weihenstephanensis with 10^-3 to 10^-7 molar imidacloprid showed significant (P<0.05)  decrease in content of DNA, RNA, protein, glucose and growth. Bacillus weihenstephanensis in MSM and TSB showed 46 and 78 % imidacloprid degradation in four weeks. The plasmid of Bacillus weihenstephanensis was cured in fourth generation. 18.80% and 75% degradation observed in cured and non cure cells of Bacillus weihenstephanensis in TSB.

Conclusion: Study showed that imidacloprid affects the biochemical contents and intern growth of soil isolate Bacillus weihenstephanensis. Study also revealed that Bacillus weihenstephanensis was able to degrade imidacloprid in MSM and TSB. Further plasmid curing revealed that the genes for imidacloprid degradation are located both in plasmid and chromosome.

Aims: To investigate cell growth profile of Aspergillus niger in a batch submerged culture medium and then evaluation of cell kinetic behavior using some different non-structured kinetic models.

Methodology: Experiments of cell growth and substrate utilization were conducted in batch submerged cultures with identified medium composition. Fitness assessment of experimental data on the cell growth and glucose consumption by models was performed using the curve-fitting tool in Mat Lab software. This report is the first in the kinetic investigation of Aspergillus niger PTCC 5010 with the studied models. This work was performed in Department of Chemical Engineering, Islamic Azad University, Qaemshahr Branch between April 2013 to September 2013.

Results: Based on the obtained results; Moser kinetic model with R2 equal to 0.913 and Gompertz kinetic model with R2 equal to 0.949 were the best fitted models to describe the growth behavior of Aspergillus niger PTCC 5010 in the applied culture condition. Maximum specific cell growth rate with Moser and Gompertz kinetic models were 0.024 and 0.003h-1, respectively. Other kinetic constants for all studied models were also determined at the applied culture conditions. The consistency of the experimental data with Monod, Verhulst and Contois kinetic models wasn't in an acceptable range.

Conclusion: In scale up biochemical projects by Aspergillus niger PTCC 5010 for some industrial products, Moser and Gompertz kinetic models are able to demonstrate cell growth behavior and its substrate uptake profile. In continuous processes, dilution rate could be determined based on obtained maximum specific cell growth rate equal to 0.024 h-1.  

Establishment of Efficient System for Isolation and Manipulation of Single Protoplasts Containing Aposporous Embryo Sac Initial Cell in Guinea Grass (Panicum maximum)

Aims: In order to do the molecular analysis of mechanism of aposporous embryo sac initial cell (AIC) appearance, as the first step, we attempted to establish the system of isolating and manipulating single cells containing AIC using different methods in guinea grass (Panicum maximum).

Study Design: At first, single protoplasts were isolated from the ovaries staged in different developmental stages using different pre-treatments and different concentrations of enzyme solutions on ovaries; Based on it, the single protoplasts containing AIC were manipulated with ultra particle electronic syringe picopipet (UPESP) machine set onto microscope.

Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2012 and December 2013.

Methodology: The ovary of facultatively apomictic guinea grass (Panicum maximum) was used as materials in this study for single protoplast isolation. The ovaries were classified and collected from young buds and flowers staged in different developmental stages, according to the colors of stigma and the ovary length. And then, the ovaries were pre-treated with needle into different shapes, and treated with different kinds of enzyme solution with different concentrations of mannitol to increase efficiency of protoplast isolation.  In final, the single protoplasts containing AIC isolated from different stages of ovaries were manipulated by handle control.

Results: 1) The ovaries in different stages of before AIC appearance, AIC appearance, and AIC-derived embryo sac formation were collected successfully and respectively, indicating that the stigma colors and length of ovaries are proportionate to stages of ovary mature. And single protoplasts containing AIC were isolated from the ovaries staged in white to yellow color. 2) The ovaries be flooded in enzyme solution, were pre-treated with needle in 4 types, that is, (1) Cut in micropylar end; (2) Cut in chalazal end; (3) Cut in middle part; (4) after 1hr of (3) treatment, cut in micropylar end. As a result, the efficiency of protoplast isolation of (3) and (4) was 1-2 hrs shorter than that of (1) and (2). 12%, 11%, 10% and 9% were proper enzyme concentrations for obtaining perfect shingle protoplasts from the ovaries with white, yellow, peach and purple colors, respectively. 3) The single protoplasts containing AIC were collected and manipulated with UPESP in the performance of controlled aspiration and spit.   

Conclusion: In this study, in order to do molecular analysis of the mechanism of AIC appearance, we focus on that, the key points were to isolate AIC single protoplasts from apomictic guinea grass using different methods, and then to establish the method of controlling a single protoplast using UPESP machine. These results obtained in this study will be a useful tool for molecular analysis of AIC, and provide important information for clarification of apomixis reproductive mode.

Characterization of Trypsin-Like Serine Protease from Lethocerus indicus Salivary Venom and its Cytotoxic Effect against Human Epidermoid Carcinoma Cell, A431

Objective: Lethocerus indicus salivary venom characterization and evaluation of extracellular degradation activity and cytotoxic effect against native human collagen type 1 and epidermoid carcinoma cell, A431.

Method: Salivary venom extract was collected from adult insects by injecting 2% pilocarpine of 50 µml. Enzyme presence was detected by the apiZYM assay. The proteolytic activity was tested by the photometric and zymogram methods using specific fluorescent substrates and inhibitors. The cytotoxic activity was determined by the MTT assay and Trypan blue exclusion method. Apoptosis induction was observed using AO/EB staining solution. Digestion of extracellular matrix protein was detected against native human type I collagen.

Result: L. indicus salivary venom presents amylases, proteases, carbohydrases, phosphatases and lipases. Among them, protease enzyme showed highest composition. The highest rate of proteolytic activity observed at pH 8 in 35ºC (100 %). Serine proteases present predominantly in salivary venom. Cysteine and metalloproteases are also detected. The activation energy of salivary venom is 49.86 kJ. Use of serine inhibitor, PMSF inhibited 92.77% which indicated that the maximum activity was due to serine protease. Detection of trypsin-like protease was confirmed by using PMSF and TLCK with specific substrate, BApNA. It shows significant inhibitions, 82% and 78% respectively suggesting maximum influence in salivary venom. Degradation of the fibrillar native state collagen Type I into 8 smaller peptide bands showed it importance in medical application. IC50 concentration of venom that induces cytotoxicity in epidermoid carcinoma cells, A431was 2.3 µg/ml only. It gives prominent apoptotic features such as cytoplasmic membrane blebbing, nuclear contraction, nuclear fragmentation and contact inhibition.

Conclusion: We suggest that further investigation of the venom will lead to identification of active compound in L. indicus salivary venom for its potential use in therapeutic application.

Optimization of Chromatographic Criterion to Recover Indian White Shrimp (Fenneropenaeus indicus) Alkaline Phosphatase from Hepatopancreas

Commercially important alkaline phosphatase can be recovered from the hepatopancreatic tissue of Indian white shrimp with optimum yield, activity, and purity by carefully designing the criterions of ion exchange chromatographic purification. The tissues were homogenized, clarified, and concentrated. Concentrated homogenate was purified with optimum yield, activity and purity by optimising criterions of chromatography such as pH and ionic strength of the binding buffer, and ionic gradient and the flow rate of the elution buffer in DEAE-cellulose column. The enzyme was optimally bound to the column at pH 8.4 and ionic strength 0.1 M NaCl at flow rate of 1mL/min, and eluted with high resolution at ionic gradient of 0.10-0.35 M NaCl in 25 min at a flow rate of 1.5mL/min, and hence is the optimum criterions of chromatographic purification.

AmpC-BETA Lactamases among

Enterobacteriaceae Isolated at a Tertiary Hospital, South Western Uganda

Aim: To characterize AmpC-beta lactamases among Enterobacteriaceae isolates from clinical samples at Mbarara Regional Referral Hospital.

Study Design:  Laboratory-based descriptive cross-sectional study

Place and Duration of Study: Microbiology Department, Mbarara Regional Referral Hospital and MBN clinical Laboratories, between May to September 2013.

Methodology: This study included 293 Enterobacteriaceae isolates recovered from clinical specimens that included blood, urine, stool and aspirates. AmpC Beta lactamase production was determined  using disc placement method for cefoxitin at a break point of <18mm. Common AmpC plasmid mediated genes were   EBC, ACC, FOX, DHA, CIT and MOX were; was determined by Multiplex PCR as described by Hanson and Perez-Perez.

Results: Plasmid mediated AmpC phenotype was confirmed in 107 of the 293 (36.5%) cefoxitin resistant isolates with 30 isolates having more than one gene coding for resistance. The commonest source that harbored AmpC beta lactamases was urine and E. coli was the most common AmpC producer (59.5%).   The genotypes detected in this study, included EBC (n=36), FOX (n=18), ACC (n=11), CIT (n=10), DHA (n=07) and MOX (n=1).

Conclusion: Our findings showed that prevalence of AmpC beta- lactamase at MRRH  was high (39.6), with EBC as the commonest genotype among Enterobacteriaceae Urine and E. coli were the commonest source and organism respectively that harbored AmpC beta-lactamases. There‘s rational antimicrobial therapy and antibiotic susceptibility tests should be requested by health workers especially patients presenting with urinary tract infections and bacteraemias.

Aims: To study the diversity of traditional rice genotypes in Sri Lanka using cluster analysis and principle component analysis.

Study Design:  The experiment was carried out using one hundred rice genotypes with six modern rice cultivars and ninety four traditional rice cultivars. Rice genotypes were planted according to a randomized complete block design with four replications and 20 plants per plot with 15 cm X 20 cm spacing.

Place and Duration of Study: A field experiment was carried out during 2011/2012 Maha season and 2012 Yala season at Faculty of Agriculture, University of Ruhuna, Sri Lanka.

Methodology: Plant height (cm), number of tillers/plant and number of productive tillers/plant were measured before harvesting. Panicle length (cm), panicle weight (g), number of spikelets/panicle, number of fertile spikelets/panicle, 100 grain weight (g) and yield/plant (g) were measured after harvesting and drying of grains for 14% moisture content. Principal component analysis (PCA) and cluster analysis were performed using SPSS statistical software.

Results: Among nine studied variables three principal components exhibited more than one Eigen value and showed about 89.6 % variability. The principal components (PC) 1, 2 and 3 had 51.07%, 22.08% and 16.46% variability among the genotypes for the evaluated traits respectively. The first PC was more related to panicle weight, number of spikelets/panicle, number of fertile spikelets/panicle, spikelet fertility percentage and yield (g/plant). Number of tillers/plant, number of productive tillers/plant and yield (g/plant) were more related traits in the second principal component. The highest contribution in third principal component was from the panicle weight, 100 grain weight and yield (g/plant). Based on the nine yield and yield attributing characters, the genotypes were grouped in to seven clusters in cluster analysis. The genotypes under cluster V recorded the highest divergence among them as it exhibited the highest intra-cluster distance. The lowest intra-cluster distance was recorded in the cluster VI. The modern rice cultivar BG 379/2 was fallen in to the cluster VI with 3 traditional rice cultivars namely Karayal I, Bathkiri el and Hondarawala.

Conclusion: One hundred rice genotypes were grouped in to divergent groups by principle component analysis and cluster analysis. This clustering pattern can be used for the selection of parental materials with diverse characters.