Philosophiae Doctor - PhD (Biotechnology) / The major aim of this study is to determine and compare the distribution of bacteria and actinobacteria in Marion Island terrestrial habitats. / South Africa

>Magister Scientiae - MSc / Heterologous metagenomic screening strategies have relied largely on the construction of DNA libraries and screening in Escherichia coli to access novel enzymes. There is an increased demand for the identification of novel lignocellulose degrading enzymes with enhanced biochemical properties which are suitable for applications in industrial processes; biofuels being one of them. The use of heterologous gene expression in function based metagenomic studies has resulted in the discovery of enormous novel bioactive compounds. However, there are limitations associated with using E. coli as a heterologous host which does not allow transcription and translation of all genes in the metagenome. E. coli can only express 40% of the environmental DNA because of promoter recognition, codon usage, and host toxicity of gene products. Therefore alternative strategies for expressing or producing novel enzymes are needed, which can also be employed in metagenomic gene discovery. In vitro protein synthesis is an important tool in molecular biology and used to obtain proteins from genes for functional and expression studies. These systems may hold the key to unlock more of the potential in metagenomic DNA. The broader aim of the study is to develop non- E. coli based cell-free protein synthesis systems to further the metagenomics screening. In this study, Rhodococcus erythropolis H8 was evaluated for its suitability in cell-free expression. Crude extracts containing the macromolecular components (70S or 80S ribosomes, tRNAs, initiation, elongation and termination factors) fromR. erythropolis were prepared using existing crude extract based cell-free protein synthesis (CFPS) protocols. Three genes were selected and used as templates for synthesis: cell11, xp12 and acetyl xylan esterase (axe10), all previously isolated from metagenomic libraries screened inE. coli. As judged by zymograms and enzyme assays, all enzymes were successfully expressedfrom their native promoters and in recombinants clones using the PtipA promoter, and wereactive. Furthermore, the amounts of XP12 protein produced using pFos-XP_12 was 1.2mg/mlfrom E. coli and 1.67mg/ml from R. erythropolis CFPS, showing that the R. erythropolismachinery was more efficient in the expression of XP12 than the E. coli machinery. To the best of our knowledge this is the first demonstration of a cell-free expression using an actinomycete.

Bioflocculants produced by two identified bacteria: Actinobacterium sp. Mayor and Methylobacterium sp. Obi were investigated with regard to their physicochemical and flocculating characteristics. The two strains were later combined to form a consortium for further studies. The optimum culture conditions for the bioflocculant production were similar for all strains except in the case of Actinobacterium sp. Mayor and the consortium, where glucose was replaced by sodium carbonate as a carbon source. Multi-nitrogen source was the best nitrogen source compare to individual sources for both strains. The divalent cation, Ca2+ proved to be a better flocculating activity stimulus for all produced bioflocculants in this study. The optimum flocculating activities obtained for both individual strains and the consortium were all at alkaline pH. The yield of purified bioflocculant produced by the consortium was 8.203 g/l, while 4.190 g/l and 4.610 g/l were recovered for single strains of Actinobacterium sp. Mayor and Methylobacterium sp. Obi respectively. Further characterization of pure bioflocculants revealed that a bioflocculant dosage of 0.3 mg/ml resulted in the highest flocculating activity for both individual strains while 1.0 mg/ml of the bioflocculant produced by the consortium was required to enhance maximum flocculating efficiency. These bioflocculants proved to be all thermo stable at a temperature range of 20 to 900°C with a heating rate of 10oC/min under a constant flow of nitrogen gas. The presence of functional groups normally required for bioflocculation such as hydroxyl, carboxyl and amino was also detected. The findings of this study suggest that the producedbioflocculants can be utilized as excellent substitutes for harmful synthetic flocculants in both water and wastewater treatments as well as in other industrial applications.

It is the purpose of this investigation to improve upon techniques to enhance the taste of municipal water and attempt to devise one that could desorb the compounds from carbon in an unaltered state. It was decided that the odor character would be the criterion used to decide if the eluted compounds were the same as those in the raw water.

Infectious disease has always been and will continue to be a heavy burden on human society worldwide. Terrestrial actinobacteria, notable as a source of antibiotics, have been well investigated in the past. In constrast, marine actinobacteria, especially sponge-associated species, have received much less attention and isolates are sparse. With the aim of studying and discovering novel marine actinobacteria, 11 different species of sponges were collected from the Central Red Sea in Saudi Arabia and cultured with three different types of media. 16S rRNA gene-sequencing revealed that among all 75 isolated bacterial strains 13 belonged to the order actinomycetales. These 13 actinomycetes fall into four different families and can be assigned to six different genera. Antibiotic activity tests using disc diffusion assay were performed against Gram-positive bacteria (Bacillus sp.), Gram-negative bacteria (Escherichia coli), fungi (Fusarium sp.) and West Nile virus NS3 protease. Nine strains presented different level of bioactivity against these pathogens. These findings provide evidence that actinomycetes are presented in marine sponges and that they have the potential to be good candidates in the search for new effective antibiotic, antifungal, and antiviral compounds.

No description available.

No description available.

The purpose then of this paper has been to expand our knowledge of these aquatic forms by investigating the cultural, physiological, morphological, and chemical characteristics of an actinomycete isolated from the water supply, Lake Waco, of Waco, Texas.

Several bacteria were isolated from the bottom sediments of Tyume River and investigated for bioflocculant production potentials. Kaolin clay suspension (4 g/l) was used to measure the flocculating activity and three of the positive isolates were identified by 16S rRNA gene nucleotide sequence analyses and the sequences deposited in GenBank as Streptomyces sp Gansen (accession number HQ537129), Brachybacterium sp UFH (accession number HQ537131.), and Cellulomonas sp Bola (accession number HQ537132). Streptomyces sp Gansen exhibited its maximum flocculating activity using lactose (85% activity), peptone (76.3% activity), Ca2+ as sole sources of carbon, nitrogen and cations respectively, and at a neutral pH of 7.0, while, the bioflocculant produced by Brachybacterium sp UFH with glucose, urea and Ca2+ as carbon, nitrogen and cations sources yielded 82% and 97% flocculation activity respectively at a neutral pH. Also, glucose (73.2% activity), ammonium chloride (78.2% activity) and Ca2+ resulted in optimal production of bioflocculant by Cellulomonas sp Bola, also at a neutral pH. Chemical analysis confirmed that bioflocculant produced by Streptomyces Gansen is a polysaccharide while Brachybacterium sp UFH and Cellulomonas sp Bola produces a glycoprotein compound. This freshwater actinomycetes appears to have a tremendous potential as sou rces of new bioflocculants.

As actinobactérias endofíticas estão presentes nos tecidos das plantas e, por meio da produção de metabólitos ativos, às protegem e auxiliam em condições de estresse. Esses microrganismos tem sido amplamente estudados no controle de doenças fitopatogênicas, como a mancha marrom causada por Bipolaris sorokiniana. Este fungo é o agente causal da podridão comum da raiz, manchas foliares, morte de plântulas e ponto preto das sementes de trigo e cevada, causando redução significativa na produtividade. Neste contexto, os objetivos do presente estudo foram avaliar a virulência de isolados de B. sorokiniana e a atividade antifúngica de actinobactérias contra este fitopatógeno. Os antagonistas com elevada atividade contra o fitopatógeno foram caracterizados quanto à produção enzimática, fisiologia, condições de crescimento e produção de metabólitos, bem como sequenciamento do 16S rRNA para identificação dos antagonistas. A caracterização parcial dos metabólitos foi realizada por meio de sistemas de Cromatografia de Camada delgada (CCD) contendo diferentes solventes. Os resultados mostraram que os isolados de B. sorokiniana apresentaram elevada virulência às plântulas e sementes de trigo, sendo que a maior agressividade foi relatada à semente. Por outro lado, 69,6% das actinobactérias apresentaram elevada atividade antifúngica contra isolados de B. sorokiniana em meio sólido, e 17% a mantiveram em cultura submersa. A maior produção ocorreu a 30°C após 72h de incubação, para a maioria dos isolados. A detecção da produção de catalase, amilase, pectinase, lipase e esterase foi observada para a maioria das actinobactérias (100, 95,6, 91,30, 95,6, 100%, respectivamente). Enquanto que a degradação de caseína, carboximetilcelulose e gelatina foi realizada por 60,8, 34,78 e 47,82% dos isolados, respectivamente. Os isolados 6(2), 6(4), 16(3) e R18(6), selecionados devido à elevada atividade antifúngica e enzimática, apresentaram reação positiva para produção de compostos voláteis, quitinase e glucanase, sideróforos, fixação de nitrogêno, AIA e colonização de raizes. Somente isolado R18(6) não apresentou capacidade de solubilizar fosfatos. A caracterização molecular determinou que estes isolados pertencem ao gênero Streptomyces. Os metabólitos produzidos pelo isolado R18(6) foram mais estáveis a mudanças de temperatura e pH, bem como para a ação das proteases e EDTA, quando comparado aos demais. Os solventes acetato de etila e hexano foram mais eficientes na extração de metabólitos do extrato bruto, porém a melhor separação de metabólitos em CCD foi obtida com misturas de solventes. / Endophitic actinobacterias are present in plant tissues and by means of active metabolites they protect and help them in stress conditions. These microorganisms have been widely used in the control of phytopathogenic diseases, such as the spot blotch caused by Bipolaris sorokiniana. This fungus is a causal agent of common root rot, leaf spots, death of seedlings and black point of the seeds of wheat and barley, causing significant reduction in productivity. In this context, the aim of this study was to evaluate the virulence of the B. sorokiniana isolates and the antifungal activity of actinobacterias against this phytopathogen. The antagonists with the higher activity against the phytopatogen were characterize taking in consideration their physiology, enzyme production, growth conditions and metabolites production and 16S rRNA sequencing for identification of the antagonists. Partial characterization (nós não purificamos) of the metabolites was performed using thin layer chromatography (TLC) systems containing different solvents. The results showed that the isolates of B. sorokiniana have a high virulence on wheat seed and seedlings, however the greater aggressiveness was observed to seed. On the other hand, 69.6% of actinomycetes showed high antifungal activity against of B. sorokiniana isolates on solid medium, and 17% maintained this behavior in submerged culture. The highest yield happened, for most isolates, when grown at 30°C with agitation after 72h of incubation. The detection of catalase, starch, pectin, lipase and esterase production was observed for most of the actinomycetes (100, 95.6, 91.30, 95.6, 100%, respectively). While the hydrolysis of casein, carboxymethylcellulase and gelatin was performed by 60.8, 34.78 and 47.82% of the isolates, respectively. Isolates 6(2), 6(4), 16(3) e R18(6), selected due to the high antifungal and enzyme activity, showed a positive reaction for the production of volatile compounds, chitinase and glucanase, siderophores, nitrogen fixation, AIA and colonization of the roots. Only the isolated R18(6) showed no ability to solubilize phosphates. Molecular characterization of the isolates determined that they belong to the genus Streptomyces. The metabolites produced by isolate R18 (6) were more stable to temperature and pH changes, as well to the action of proteases and EDTA, when compared to the others. The solvents ethyl acetate and hexane were more efficient for the extraction of the metabolites from the crude extract, however a better separation of the metabolites in the TLC was obtained with mixture of solvents.