This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.

Thesis (Ph. D. (Microbiology)) -- University of Limpopo, 2020 / The use of lignocellulosic biomass for bioethanol production is largely dependent on cost effective production of cellulase enzymes and most importantly, the availability of cellulases with sufficient β-glucosidase activity for complete hydrolysis of cellulose to glucose. Commercial cellulase preparations are often inefficient in the complete hydrolysis of cellulose to glucose. The addition of β-glucosidases to commercial cellulase preparations may enhance cellulolytic activity in the saccharification of cellulose to fermentable sugars. A β-glucosidase producing thermophilic bacterium, Anoxybacillus sp. KTC2 was isolated from a hot geyser in the Zambezi Valley, Zimbabwe. The bacterium identified through biochemical tests and 16S rDNA sequencing, had an optimal growth temperature and pH of 60˚C and pH 8, respectively. The β-glucosidase enzyme had an optimal temperature of 60˚C and a broad pH range for activity, between 4.5 and 7.5 with an optimum at pH 7. The β-glucosidase enzyme retained almost 100% activity after 24 hours’ incubation at 50˚C. The Anoxybacillus sp. KTC2 β-glucosidase was partially purified and a partial amino acid sequence obtained through MALDI-TOF analysis. The whole genome of Anoxybacillus sp KTC2 β-glucosidase was sequenced and a β-glucosidase gene identified. The deduced amino acid sequence corresponded to the peptide sequences obtained through MALDI-TOF, confirming the presence of the a β glucosidase on the genome of Anoxybacillus sp KTC2. Analysis of the deduced amino acid sequence revealed that the β-glucosidase enzyme belongs to the GH family 1. The β-glucosidase gene was isolated by PCR and successfully cloned into an E. coli expression system. The saccharification efficiency of the β-glucosidase enzyme was evaluated through the creation of enzyme cocktails with the commercial cellulase preparation, CelluclastTM. CelluclastTM with the Anoxybacillus sp KTC2 β-glucosidase were used to hydrolyse pure Avicel cellulose, at 50˚C over a 96 hour reaction time. The Anoxybacillus sp KTC2 β-glucosidase enabled a 25% decrease in the total cellulose loading without a decrease in the amount of glucose released. / University of Limpopo staff development programme and VLIR