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.

Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production.

Global petroleum reserves are shrinking at a fast pace, increasing the demand for alternate fuels. Microalgae have the ability to grow rapidly, and synthesize and accumulate large amounts (approximately 20-50% of dry weight) of neutral lipid stored in cytosolic lipid bodies. A successful and economically viable algae based biofuel industry mainly depends on the selection of appropriate algal strains. The main focus of bioprospecting for microalgae is to identify unique high lipid producing microalgae from different habitats. Indigenous species of microalgae with high lipid yields are especially valuable in the biofuel industry. Isolation, purification and identification of natural microalgal assemblages using conventional techniques is generally time consuming. However, the recent use of micromanipulation as a rapid isolating tool allows for a higher screening throughput. The appropriate media and growth conditions are also important for successful microalgal proliferation. Environmental parameters recorded at the sampling site are necessary to optimize in vitro growth. Identification of species generally requires a combination of morphological and genetic characterization. The selected microalgal strains are grown in upscale systems such as raceway ponds or photobireactors for biomass and lipid production. This paper reviews the recent methodologies adopted for site selection, sampling, strain selection and identification, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail.

Controlled production of fructose by an exoinulinase from Aspergillus ficuum.

An exoinulinase has been isolated, purified and characterised from a commercially available broth of Aspergillus ficuum. The enzyme was purified 4.2-fold in a 21% yield with a specific activity of 12,300 U mg(-1)(protein) after dialysis, ammonium sulphate fractionation and Sephacryl S-200 size exclusion and ion exchange chromatography. The molecular weight of this enzyme was estimated to be 63 kDa by SDS-PAGE. It exhibited a pH and temperature optima of 5.4 and 50 degrees C respectively and under such conditions the enzyme remained stable with 96% and 63.8% residual activity after incubation for 12 h and 72 h respectively. The respective K (m) and V (max) values were 4.75 mM and 833.3 micromol min(-1) ml(-1), respectively. Response surface methodological statistical analysis was evaluated for the maximal production of fructose from the hydrolysis of pure commercial chicory inulin. Incubation of the dialyzed crude exoinulinase (100 U/ml, 48 h, 50 degrees C, 150% inulin, pH 5.0) produced the highest amount of fructose (106.4 mg/ml) under static batch conditions. The purified exoinulinase was evaluated for fructose production and the highest amount (98 mg/ml) was produced after 12 h incubation at 50 degrees C, 150% inulin pH 5.0. The use of a crude exoinulinase preparation is economically desirable and the industrial production of fructose from inulin hydrolysis is biotechnologically feasible.

Response surface methodology: Synthesis of short chain fructooligosaccharides with a fructosyltransferase from Aspergillus aculeatus.

A transferase was isolated, purified and characterised from Aspergillus aculeatus. The enzyme exhibited a pH and temperature optima of 6.0 and 60 degrees C, respectively and under such conditions remained stable with no decrease in activity after 5h. The enzyme was purified 7.1 fold with a yield of 22.3% and specific activity of 486.1Umg(-1) after dialysis, concentration with polyethyleneglycol (30%) and DEAE-Sephacel chromatography. It was monomeric with a molecular mass of 85kDa and K(m) and V(max) values of 272.3mM and 166.7micromolmin(-1)ml(-1). The influence of pH, temperature, reaction time, and enzyme and sucrose concentration on the formation of short-chain fructooligosaccharides (FOS) was examined by statistical response surface methodology (RSM). The enzyme showed both transfructosylation and hydrolytic activity with the transfructosylation ratio increasing to 88% at a sucrose concentration of 600mgml(-1). Sucrose concentration (400mgml(-1)) temperature (60 degrees C), and pH (5.6) favoured the synthesis of high levels of GF(3) and GF(4). Incubation time had a critical effect on the yield of FOS as the major products were GF(2) after 4h and GF(4) after 8h. A prolonged incubation of 16h resulted in the conversion of GF(4) into GF(2) as a result of self hydrolase activity.