Experimentation on artificial inoculation studies for persistence of shiga-like toxin-producing Escherichia coli (E. coli O157) in agricultural soils and vegetables using real-time PCR.

Diarrheagenic Escherichia coli O157 is an important reason for largest food borne inflectional outbreaks. E. coli O157 invades into the food chain through contaminated irrigation water and soil causing infectious diseases to humans. In our previous study, we have evaluated the persistence of E. coli O157 through plate count methods. However, conventional cultural procedures are less sensitive to discriminate the pathogenic strain and are time consuming. Therefore, in the present study we have enumerated the persistence of E. coli O157 in soil and vegetables using specific shiga toxin genes (stx1, stx2) through quantitative PCR. Initially, we have standardized a simple Sephadex-based DNA extraction protocol that could detect 2-3 cells/25g of vegetables. Further, quantitative PCR analysis showed a 103 fold difference in the enumeration of persistence as compared to simple plating techniques. Thus, qPCR-based persistence study can be used for rapid and accurate detection techniques for analyzing E. coli O157 contamination. PRACTICAL APPLICATIONS: Our experiment on E. coli O157 expression could be used as a scale for further studies on E. coli O157 pollution in the cropped soils, additionally the DNA extraction protocol experimented by us could be used in all sensitive quantitative assays, as it could detect the expression in lowest cell loads. However, our methodology is a more reliable and sensitive assay compared to normal cultural methods. Our experiment provides a strong evidence of persistence of E. coli O157 prevailing up to half or full cropping season.

Occurrence of diversified N-acyl homoserine lactone mediated biofilm-forming bacteria in rice rhizoplane.

Quorum sensing (QS)-mediated biofilm-forming rhizobacteria are indispensable due to their competitiveness in the crop rhizosphere. In the present work, we have reported on the occurrence of diversified bacterial species capable of producing N-acyl homoserine lactone (AHL) as the QS signal in the roots of a rice plant grown under field conditions. The AHL-producing bacteria were directly isolated from the rice root by the biosensor reporter (Chromobacterium violaceum CV026) overlay method and characterized for biofilm production by the microtiter plate method. A total of 48 QS-positive bacterial isolates were purified from different aged (7, 20, 24, 26, and 36 days) rice seedlings. The in vitro biofilm production and genetic diversity as revealed by BOX-PCR fingerprinting showed high variability among the isolates. Most of the best biofilm-forming isolates produced a N-butyryl dl-homoserine lactone (a C4-AHL type) signal in the medium. The 16S ribosomal RNA (rRNA) gene sequence of these putative elite isolates identified that they were close to Aeromonas hydrophila (QS7-4; QS36-2), A. enteropelongenes (QS20-8), A. veronii (QS36-3), Enterobacter sp. (QS20-11), Klebsiella pneumoniae (QS24-6), Kosakonia cowanii (QS24-21), Providentia rettigeri (QS24-2), Sphingomonas aquatilis (QS24-17), and Pseudomonas sihuiensis (QS24-20). These strains profusely colonized the rice root upon inoculation and formed biofilms on the surface of the root under gnotobiotic conditions. Developing inoculants from these strains would ensure competitive colonization on the rhizoplane of the crop through their biofilm and thereby improve plant growth and health.

Growth and metabolic characteristics of oleaginous microalgal isolates from Nilgiri biosphere Reserve of India.

BACKGROUND: Renewable energy for sustainable development is a subject of a worldwide debate since continuous utilization of non-renewable energy sources has a drastic impact on the environment and economy; a search for alternative energy resources is indispensable. Microalgae are promising and potential alternate energy resources for biodiesel production. Thus, our efforts were focused on surveying the natural diversity of microalgae for the production of biodiesel. The present study aimed at identification, isolation, and characterization of oleaginous microalgae from shola forests of Nilgiri Biosphere Reserve (NBR), the biodiversity hot spot of India, where the microalgal diversity has not yet been systematically investigated. RESULTS: Overall the higher biomass yield, higher lipid accumulation and thermotolerance observed in the isolated microalgal strains have been found to be the desirable traits for the efficient biodiesel production. Species composition and diversity analysis yielded ten potential microalgal isolates belonging to Chlorophyceae and Cyanophyceae classes. The chlorophytes exhibited higher growth rate, maximum biomass yield, and higher lipid accumulation than Cyanophyceae. Among the chlorophytes, the best performing strains were identified and represented by Acutodesmus dissociatus (TGA1), Chlorella sp. (TGA2), Chlamydomonadales sp. (TGA3) and Hindakia tetrachotoma (PGA1). The Chlamydomonadales sp. recorded with the highest growth rate, lipid accumulation and biomass yield of 0.28 ± 0.03 day-1 (µexp), 29.7 ± 0.69% and 134.17 ± 16.87 mg L-1 day-1, respectively. It was also found to grow well at various temperatures, viz., 25 °C, 35 °C, and 45 °C, indicating its suitability for open pond cultivation. The fatty acid methyl ester (FAME) analysis of stationary phase cultures of selected four algal strains by tandem mass spectrograph showed C16:0, C18:1 and C18:3 as dominant fatty acids suitable for biodiesel production. All the three strains except for Hindakia tetrachotoma (PGA1) recorded higher carbohydrate content and were considered as potential feed stocks for biodiesel production through hydrothermal liquefaction technology (HTL). CONCLUSIONS: In conclusion, the present investigation is a first systematic study on the microalgal diversity of soil and water samples from selected sites of NBR. The study resulted in isolation and characterization of ten potent oleaginous microalgae and found four cultures as promising feed stocks for biodiesel production. Of the four microalgae, Chlamydomonadales sp. (TGA3) was found to be significantly thermo-tolerant and can be considered as promising feedstock for biodiesel production.

Aerobic cyanide degradation by bacterial isolates from cassava factory wastewater.

Ten bacterial strains that utilize cyanide (CN) as a nitrogen source were isolated from cassava factory wastewater after enrichment in a liquid media containing sodium cyanide (1 mM) and glucose (0.2% w/v). The strains could tolerate and grow in cyanide concentrations of up to 5 mM. Increased cyanide levels in the media caused an extension of lag phase in the bacterial growth indicating that they need some period of acclimatisation. The rate of cyanide removal by the strains depends on the initial cyanide and glucose concentrations. When initial cyanide and glucose concentrations were increased up to 5 mM, cyanide removal rate increased up to 63 and 61 per cent by Bacillus pumilus and Pseudomonas putida. Metabolic products such as ammonia and formate were detected in culture supernatants, suggesting a direct hydrolytic pathway without an intermediate formamide. The study clearly demonstrates the potential of aerobic treatment with cyanide degrading bacteria for cyanide removal in cassava factory wastewaters.

Aerobic cyanide degradation by bacterial isolates from cassava factory wastewater

Ten bacterial strains that utilize cyanide (CN) as a nitrogen source were isolated from cassava factory wastewater after enrichment in a liquid media containing sodium cyanide (1 mM) and glucose (0.2% w/v). The strains could tolerate and grow in cyanide concentrations of up to 5 mM. Increased cyanide levels in the media caused an extension of lag phase in the bacterial growth indicating that they need some period of acclimatisation. The rate of cyanide removal by the strains depends on the initial cyanide and glucose concentrations. When initial cyanide and glucose concentrations were increased up to 5 mM, cyanide removal rate increased up to 63 and 61 per cent by Bacillus pumilus and Pseudomonas putida. Metabolic products such as ammonia and formate were detected in culture supernatants, suggesting a direct hydrolytic pathway without an intermediate formamide. The study clearly demonstrates the potential of aerobic treatment with cyanide degrading bacteria for cyanide removal in cassava factory wastewaters.

Microbial community diversity of organically rich cassava sago factory waste waters and their ability to use nitrate and N2O added as external N-sources for enhancing biomethanation and the purification efficiency.

Water shortage necessitated South Indian sago factory owners, extracting starch out of cassava tubers, to install biogas plants where a starch utilizing microbial community multiplies and reduces the biological oxygen demand (BOD) of the waste waters by presently about 30%. The purification efficiency of sago factory waste waters, rich in solid particles and having wide C/N ratios, around 250, through unstirred biogas plants needs to be improved. Our approach was to apply instead of animal slurry nitrate (NO3(-)) and nitrous oxide (N2O) as external N-sources anticipating a better N-distribution in the unstirred biogas plants. Estimated cell numbers, bacterial community changes, on the basis of 16S rRNA gene clone libraries and changing CO2-, CH4-, N2O releases due to the presence of nitrate or N2O suggest that acid tolerant Lactobacillus spp. dominate the biogas plant inflows (pH 3.5). They were very less or not found in the outflows (pH 7.3). Assumingly, the phyla Bacteroidetes (Prevotella spp.), Proteobacteria (Rhizobium spp., Defluvibacter sp.), Firmicutes (Megasphaera spp., Dialister spp., Clostridium spp.) and Synergistetes (Thermanaerovibrio spp.), not-detectable in the biogas plant inflows, replaced them. Anaerobes, about 400cellsml(-1) in the inflows, increased to about 10(6)cellsml(-1) in the outflows. The methane formation, as confirmed by the incubation experiments, suggests that methanogens must have been present among the anaerobes. In the biogas plant in- and outflows also about 300cellsml(-1) denitrifying bacteria and up to 10(4)cfu fungi were found. Despite the low number of denitrifying bacteria nitrate added to the biogas plant in- and outflows was widely consumed and added N2O decreased considerably. Thus, wide C/N ratios substrates like sago factory waste waters keep the N2O emissions low by using N2O either as electron acceptor or by incorporating it into the growing biomass what needs to be confirmed. The biogas plant inflow samples have emitted tentatively more CO2 and the outflow samples released more CH4.