Development of a loop-mediated isothermal amplification assay targeting lmo0753 gene for detection of Listeria monocytogenes in wastewater.

Contaminated wastewater plays an important role in the transmission of Listeria monocytogenes in the environment. In this study, a loop-mediated isothermal amplification (LAMP) assay for sensitive detection of L.  monocytogenes in wastewater from treatment plants was developed, validated and compared to conventional PCR. The lmo0753 gene which codes for a Crp/Fnr family transcription factor, was targeted to design four specific primers to detect L.  monocytogenes in 60 min at 63°C in a water bath. Amplification products were visualized by agarose gel electrophoresis. The detection limit of the LAMP assay was 65 fg µl-1 of DNA and 38 CFU per ml. Conventional PCR was 10 times less sensitive than LAMP assay with primers targeting the HlyA gene. A total of 70 crude wastewater samples collected at different treatment stages (aeration tank, pre chlorination and post chlorination), were tested directly by LAMP and PCR. Samples from aeration and pre-chlorination stages tested positive with LAMP and culture method but not with conventional PCR. LAMP assay was tolerant to inhibitors present in wastewater and circumvented the need for isolation of pure DNA for detection. Both LAMP assay and culture method failed to detect L.  monocytogenes in post-chlorinated wastewater, confirming the efficiency of the treatment process in the removal of L.  monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated wastewater effluent contains Listeria monocytogenes which survives conventional wastewater treatment processes and can re-enter human food chain, thus it is imperative to detect L.  monocytogenes using a rapid and an inexpensive method. To the best of our knowledge, this is the first report of a loop-mediated isothermal amplification (LAMP) assay, targeting the lmo0753 gene for detection of L.  monocytogenes in wastewater from treatment plants. The LAMP assay detects L.  monocytogenes in 60 min at 63°C in a water bath. LAMP does not require isolation of pure genomic DNA hence it is a user friendly method for L.  monocytogenes detection.

Isolation, Identification and High-Throughput Screening of Neutral Lipid Producing Indigenous Microalgae from South African Aquatic Habitats.

Exploring indigenous microalgae capable of producing significant amounts of neutral lipids through high-throughput screening is crucial for sustainable biodiesel production. In this study, 31 indigenous microalgal strains were isolated from diverse aquatic habitats in KwaZulu-Natal, South Africa. Eight superior lipid-producing strains were selected for further analysis, based on Nile red fluorescence microscopy screening. The microalgal isolates were identified to belong to the genera Chlorella, Neochloris and Chlamydomonas via morpho-taxonomic and molecular approach by 18S rRNA gene sequencing. Chlorella vulgaris PH2 had the highest specific growth rate (µ) and lowest doubling time of 0.24 day-1 and 2.89 ± 0.05 day-1, respectively. Chlorella vulgaris T4 had the highest biomass productivity of 35.71 ± 0.03 mg L-1day-1. Chlorella vulgaris PH2 had the highest lipid content of 34.28 ± 0.47 and 38 ± 9.2% (dcw) as determined by gravimetric analysis and the sulfo-phospho-vanillin (SPV) method, respectively. Chlorella vulgaris PH2 exhibited a high content of saturated fatty acids, while Chlorella sp. T4 exhibited a high total content of saturated and monounsaturated fatty acids with a low content of polyunsaturated fatty acids. The preponderance of neutral lipids suggests that Chlorella sp. T4 is a suitable candidate for biomass feedstock for biodiesel production.

Microbial enzymatic production and applications of short-chain fructooligosaccharides and inulooligosaccharides: recent advances and current perspectives.

The industrial production of short-chain fructooligosaccharides (FOS) and inulooligosaccharides is expanding rapidly due to the pharmaceutical importance of these compounds. These compounds, concisely termed prebiotics, have biofunctional properties and hence health benefits if consumed in recommended dosages. Prebiotics can be produced enzymatically from sucrose elongation or via enzymatic hydrolysis of inulin by exoinulinases and endoinulinases acting alone or synergistically. Exoinulinases cleave the non-reducing ß-(2, 1) end of inulin-releasing fructose while endoinulinases act on the internal linkages randomly to release inulotrioses (F3), inulotetraoses (F4) and inulopentaoses (F5) as major products. Fructosyltransferases act by cleaving a sucrose molecule and then transferring the liberated fructose molecule to an acceptor molecule such as sucrose or another oligosaccharide to elongate the short-chain fructooligosaccharide. The FOS produced by the action of fructosyltransferases are 1-kestose (GF2), nystose (GF3) and fructofuranosyl nystose (GF4). The production of high yields of oligosaccharides of specific chain length from simple raw materials such as inulin and sucrose is a technical challenge. This paper critically explores recent research trends in the production and application of short-chain oligosaccharides. Inulin and enzyme sources for the production of prebiotics are discussed. The mechanism of FOS chain elongation and also the health benefits associated with prebiotics consumption are discussed in detail.

Haloalkane and haloacid dehalogenases from aerobic bacterial isolates indigenous to contaminated sites in Africa demonstrate diverse substrate specificities.

Five bacteria were isolated from contaminated sites in Nigeria and South Africa using the culture enrichment technique. They were subjected to standard cultural, biochemical and microbiological techniques and identified to be species of Bacillus, Burkholderia, Corynebacterium, Micrococcus and Pseudomonas. Axenic cultures of the bacterial isolates utilized 1,2-dichloroethane (1,2-DCE) as the sole carbon source up to a final substrate concentration of 10 mM. Their mean generation time in 1,2-DCE ranged significantly (P<0.05) from 9.77 to 15.72 h with the maximum chloride release ranging between 59% and 86%. All the bacterial isolates produced two different dehalogenases, viz. one which is heat labile and specific for halogenated alkanes with optimum activity at a pH of 7.5 and the other which is more heat stable with a higher pH optimum of 9.0 and specific for halogenated alkanoic acids. However, the two enzyme types when tested demonstrated wide substrate specificities. It is therefore adjudged that these organisms may play a vital role in the bioremediation of sites polluted with chlorinated hydrocarbons.

Aerobic dehalogenation potentials of four bacterial species isolated from soil and sewage sludge.

Four bacterial species each were isolated from soil and a sewage oxidation pond using enrichment culture technique, and the bacterial isolates were identified to belong to the two genera Bacillus and Corynebacterium. The axenic cultures of the isolates utilized monochloroacetic acid (MCA), trichloroacetic acid (TCA), CHCl3 and CCl4 for growth up to 1 g substrate l(-1) (w/v) and growths were enhanced in the mixed cultures of the isolates. The specific growth rate constants in the media ranged from 0.144 to 0.475. This is lower than the comparatively high values observed for the glucose medium, which varied significantly (P < 0.05) between 0.699 and 0.792 h(-1). Serial adaptation of the individual isolates in the organochloride media significantly (P < 0.05) affected the bacteria growth yields. The dehalogenase specific activity observed in the cell-free extracts of the mixed cultures of the isolates was significantly higher (P < 0.05) than those of their respective monocultures. Optimal pH of the dehalogenase activity was between 7.6 and 8.0, while temperature optima were between 30 degrees C and 35 degrees C.