Studies on the microbial degradation of complex substrates

Microorganisms, mainly bacteria and fungi, are key agents involved in the breakdown and decomposition of plant and animal polymers in ecosystems. The aim of this research project was to study the mechanisms of degradation of four complex substrates; keratin, pectin, alginate and chitin. In this study, keratinophylic fungi were isolated from agricultural soil via a hair-baiting technique (HBT) using wool and hair as baits; additionally, keratinophylic species were isolated from contaminated feathers. The isolates from hair, wool and feathers were grown on solid media supported by keratin azure as a source of carbon and nitrogen. Keratinolytic activities were observed by the formation of a clearing zone in the medium. A study of keratinolytic assay in shaking culture was made by measuring the activity of keratinase (release keratin azure). In addition, scanning electron microscopy (SEM) studies were included in this study. Qualitative assays of pectin degradation, using apple pectin as a carbon source are reported. Pectin degradation in plates was detected using a solution of iodine-potassium iodide. Pectinase activity was determined in the supernatants by release of reducing sugars (galacturonic acid) using dinitrosalicylate reagent (DNS). Antimicrobial activities of pectin esterified potassium salt against some pathogens partically the bacteria which cause infection in wounds was determined by measuring inhibition zones around the wells. Alginolytic microorganisms were isolated from two fresh seaweeds, namely Fucus and Laminaria. The enzymatic activities were quantified by the formation of new unsaturated non-reducing ends and as reducing sugar (RS). The amount of reducing sugar formed was determined using 3, 5-dinitrosalicylic acid (DNS) methods. Crab shell chitin was hydrolysed by acid to produce colloidal chitin. Fungal and bacterial isolates were tested to determine chitinolytic properties in plates by measuring purple zones against yellow background. The supernatants derived from selected isolates were then used to determine chitinase activity by measuring reducing sugars (RS). RS calculated as glucose using Nelson and DNS methods. The fertilizer-potential of the substrates was determined by measuring nitrification and the oxidation of sulphur in soil amendment with the individual complex substrates. A variety of bacteria and fungal isolates were identified using molecular identification techniques. Finally, four enzymes were isolated and partially purified using ammonium sulphate in order to determine their molecular weight using SDS polyacrylamide gel electrophoresis (SDS-PAGE). In addition, liquid chromatography mass spectrometry (LC-MS/MS) has been used to identify three enzymes namely; keratinase, pectinases and chitinases.