Open Access Original Research Article

Exploring Agricultural Potentiality of Serratia entomophila AB2: Dual Property of Biopesticide and Biofertilizer

Pritam Chattopadhyay, Sandipan Chatterjee, Shrikanth Gorthi, Sukanta K. Sen

Biotechnology Journal International, Page 1-12
DOI: 10.9734/BBJ/2012/778

Aims: In an attempt to explore novel agents for application in agriculture, the strain Serratia entomophila AB2 was investigated.
Study design: Pesticidal, Fertilizing and Toxicological effects.
Place and Duration of Study: Department of Botany (Microbiology Unit), Visva-Bharati, Santiniketan, West Bengal, India and M/S Ajay Biotech (India) Ltd., Pune, India, between December 2006 to August 2007.
Methodology: Pesticidal activity of the isolate Serratia entomophila strain AB2 was tested against 5 day old larvae of Heliothis armigera, Spodoptera litura, and Plutella xylostella through feeding assay. Fertilizing capacity of the strain was determined by inorganic phosphate and zinc solubilizing experiments. Conventional T-tests were performed to check effect against certainr rhizospheric organisms (Azotobactor chroococcum NCIM 2452, Rhizobium japonicum NCIM 2746, Azospirillum brasiliensis NCIM 5135, Erwinia amylovora MDVB). In animal toxicity experiments, adult male Swiss albino mice (body wt ≈ 25 g) were used to find LD50 within the experimental doses and male Sprague Dawley strain of Swiss albino rat (body wt ≈ 110 g) were used to find out the effect of feed inoculation treatment in liver and blood as general target of intoxication through standard thiobarbituric acid reactive substance (TBARS) and serum glutamic pyruvic transaminase (SGPT) assay.
Results: The mortality rate of, lepidopteron larvae tested was determined between 89.5 to 94.3% while LC50 value estimated between 0.44x105 to 1.44x105 CFU (colony forming unit) mL-1 through probit analysis. As a part of fertilizing activity, S. entomophila AB2 was found to solubilize phosphorus and zinc in in-vitro condition. In cross reactivity study with other rhizospheric bacteria, the isolate proved as non reactive. No mortality was recorded with Swiss albino mice and other toxicological data resulted from the experiment with Sprague Dawley strain of Swiss albino rat were found insignificant.
Conclusion: The isolate S. entomophila AB2 showed its potential in in vitro conditions for both pesticidal and fertilizing activity. The data generated in this study show much promise of S. entomophila AB2 for its field application in integrated crop management (ICM). These predictors, however, need further work for field validation.

Open Access Original Research Article

Isolation and Identification of Methicillin-Resistant Staphylococcus aureus from Keys of College Students Using Different Detection Methods

Razieh Amini, A.S. Abdulamir, Beh Poay Ling, Fatemeh Jahanshiri, Ali Hematian, Mohsen Zargar, Zamberi Sekawi, Farid Azizi Jalilian

Biotechnology Journal International, Page 13-25
DOI: 10.9734/BBJ/2012/756

Aims: In this study, the methicillin-resistant Staphylococcus aureus (MRSA) were isolated and identified by using biochemical tests, antibiogram and polymerase chain reaction (PCR) to explore the circulation of MRSA among college students.
Study design: Cross-sectional study.
Place and Duration of Study: Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia between June 2010 and December 2010.
Methodology: A total of 100 samples were collected from keys of college students. There were 39 isolates (39 %) Gram-positive cocci and Catalase positive. 29 (74.36%) were glucose oxidation and fermentation positive. From the 39 isolates, 16 (43.24%) were shown Mannitol Salt Agar (MSA) tests positive. The deoxyribonuclease (DNase) tests and tube coagulase tests with human and rabbit plasma were carried out to improve the efficiency of the MSA test.
Results: 7 (43.75%) DNase positive and 2 (12.5%) tube coagulase positive. Both human and rabbit plasma showed similar sensitivity for the tube coagulase tests in this study. However, both isolates with tube coagulase positive were confirmed as S. aureus but not resistant to oxacillin, methicillin, erythromycin and cefoxitin. 2 (66.67%) of 3 (18.75%) isolates which is tube coagulase negative were resistant to erythromycin and 1 (33.33%) of them was resistant to methicillin. Rare strains of S. aureus can be coagulase negative. PCR assay was used. 1 (33.33%) of the coagulase negative isolate resistant to erythromycin was found to have nuc gene, mecA gene, ermC gene, msrA gene, linA gene, and femA gene. The isolate was confirmed as MRSA.
Conclusion: In conclusion, PCR technique is more sensitive and reliable than tube coagulase test or antibiogram for the detection of MRSA. And keys were shown to be an important source of MRSA and other bacteria circulation in the community.

Open Access Original Research Article

Batch Equilibrium and Kinetic Studies of Simultaneous Adsorption and Biodegradation of Phenol by Pineapple Peels Immobilized Pseudomonas aeruginosa NCIB 950

S. E. Agarry, M. O. Aremu

Biotechnology Journal International, Page 26-48
DOI: 10.9734/BBJ/2012/902

This study was aimed to investigate the use of pineapple as a cheap, eco-friendly adsorbent and support matrix for the immobilization of microbial cell and for subsequent removal of phenol from waste water. The effects of initial phenol concentration, pH and adsorbent particle size on the simultaneous adsorption-biodegradation (SAB) of phenol were studied. The batch simultaneous adsorption and biodegradation (SAB) of phenol in simulated phenol waste water by pineapple peels immobilized Pseudomonas aeruginosa NCIB 950 has been studied with the use of glass bottles as bioreactors placed in a rotary mechanical shaker for 72 h. The results of the batch equilibrium adsorption-biodegradation studies showed that adsorption-biodegradation capacity decreased with increase in particle size. The equilibrium adsorption-biodegradation data were analyzed by the Langmuir, Freundlich and Redlich-Peterson models of adsorption. The results showed that the equilibrium data for phenol degradation sorbent systems were well fitted to the three adsorption models with Langmuir and Redlich-Peterson adsorption isotherms having the best fit. The adsorption-biodegradation kinetic data obtained at different initial phenol concentrations and pH showed that the adsorption-biodegradation capacity of the pineapple peels immobilized P. aeruginosa generally increased with increase in initial phenol concentration and pH. The kinetic data were analyzed using Lagergren pseudo-first order, pseudo second-order, Elovich and intraparticle diffusion rate equations. The rate equations fitting showed that the adsorption-biodegradation kinetic data generally fitted the four rate equations tested from which the rate constants and diffusion rate constants were estimated. However, the Lagergren pseudo first-order rate equation gave the best fit and, thus the process followed first-order rate kinetics. Therefore, pineapple peels being an agricultural waste product have the potential to be used as low-cost adsorbent and support matrix for microbial culture immobilization for the removal of organic pollutant from waste water.

Open Access Original Research Article

Bacterial Succession Studies during Fermentation of African Locust Bean (Parkia biglobosa) to Iru Using Molecular Methods

Aminat. O. Adelekan, Esiobu Nwadiuto

Biotechnology Journal International, Page 49-59
DOI: 10.9734/BBJ/2012/586

Aims: The identity, diversity and dynamics of the bacterial community involved in the fermentation of African Locust Bean (Parkia biglobosa) to “Iru”, a protein-rich condiment in Western Nigeria; was studied using the 16S rRNA gene sequence analysis.
Study Design: 16S rRNA gene was used to study bacterial succession and diversity in this solid-state fermentation with a view to develop a framework for improving quality control of this important and nutritious solid-state fermentation product and possibly develop starter cultures for commercializing the product.
Place and Duration of Study: Biotechnology Centre of University of Agriculture, Abeokuta, Ogun State, Nigeria and Biological Sciences Department, Florida Atlantic University, Davie Campus, Florida U.S.A., between July 2008 and October 2009.
Methodology: Raw seeds were prepared in the traditional African way by boiling them for 6hr to soften the seed coat; and for another 1hr to soften the cotyledon. The boiled seeds were immediately transferred into a jute-bag and wrapped tightly to prevent heat loss. They were left at ambient temperature to ferment for 72hr. Total Bacterial Community of the seed was obtained by vigorously rinsing seeds in phosphate buffered saline, before boiling and immediately after boiling (0hr), and subsequently at intervals of 24hrs for three days. To compare cultivable phyllotypes and possible non-cultivable bacteria, subsamples of the extracted bacteria were cultured on Tryptic Soy Agar. Total community small subunit (SSU) rRNA was amplified from extracted genomic DNA by Plate wash polymerase chain reaction (for cultured bacteria) and classic PCR directly from seed-buffer extract (uncultured bacteria). Genomic DNA was extracted employing a modified protocol of the freeze-thaw and Qiagen DNA extraction methods. Extracted genomic DNA was run on 1% agarose gel to rule out shearing before PCR amplification of the 16S rRNA gene with the 27F and 1492R primer pair. The amplified samples were cloned using TOPO cloning vector and transformed samples were sequenced. Identity of samples were done by aligning samples in Ribosomal database Project and close relatives was identified.
Results: The process was found to be a classic alkaline fermentation (pH 6 – 8.39). Cultivable bacterial populations changed from 120CFU/g at start of fermentation to 1630000000 CFU/g on day 3. The most abundant organism present in the raw African Locust Beans isolates (Clone 1A) had 97% match to Acinetobacter sp. Cooked Locust beans isolates (Clone 2A) shared 100% identity to Bacillus subtilis. Organisms present at 0 hr, 24 hr and 48 hr of fermentation (Clones 3A, 4A and 5A) proved to have 100% match to Bacillus anthracis relatives; Bacillus cereus; and Bacillus sp. respectively. Enterobacter sp. (99% similarity to Clone 6A) was only detected after 72 hrs; amidst the bacilli. Even less abundant clones were identified as various Bacillus phyllotypes. Cultured and non-cultured bacterial phyllotypes in this system clustered similarly and appear to be the same; confirming that Bacillus species were primarily responsible for the fermentation products of iru. While the bacterial identity and low diversity index reported here, is not surprising given the resilience of bacterial endospores to boiling; it provides convincing evidence to explore the use of endospores from these cultivable non-pathogenic bacillus strains as starter cultures for the solid state fermentation.
Conclusion: It is concluded from this study that the use of molecular method in the identification of organisms present during African locust beans fermentation to Iru, revealed varieties of organisms that had not been previously reported by culture- dependent methods. The application of these techniques in the field of microbiology allows a better understanding of the ecology of food fermentation.