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Dependence on foreign oil remains a serious issue for the U.S. economy. Additionally, automobile emissions related to petroleum-based, fossil fuel has been cited as one source of environmental problems, such as global warming and reduced air quality. Using agricultural and forest biomass as a source for the biofuel ethanol industry, provides a partial solution by displacing some fossil fuels. However, the use of high cost enzymes as an input is a significant limitation for ethanol production.Economic analyses of cellulase enzyme production costs using solid state cultivation (SSC) are performed and compared to the traditional submerged fermentation (SmF) method. Results from this study indicate that the unit costs for the cellulase enzyme production are $15.67 per kilogram ($/kg) and $40.36/kg, for the SSC and SmF methods, respectively, while the market price for the cellulase enzyme is $36.00/kg. Profitability analysis and sensitivity analysis also provide positive results.Since these results indicate that the SSC method is economical, ethanol production costs may be reduced, with the potential to make ethanol a viable supplemental fuel source in light of current political, economic and environmental issues.

ABSTRACT PRODUCTION OF TANNASE BY ASPERGILLUS NIGER &Ouml / zt&uuml / rk, Alev Deniz M.S., Department of Chemical Engineering Supervisor: Prof. Dr. Ufuk Bakir Co-Supervisor: Prof. Dr. B. Z&uuml / mr&uuml / t &Ouml / gel August 2006, 90 pages In this study, a filamentous fungus, Aspergillus niger was evaluated in terms of extracellular tannase production. The effect of tannic acid, glucose and nitrogen sources on tannase and biomass productions was investigated and their concentrations were optimized. The highest enzyme activity was recorded as 316 U/ml in the optimized medium containing 8% Tannic acid, 1% Glucose, 0.4% (NH4)2HPO4, 0.1% K2HPO4, 0.1% MgSO4.7H2O, 0.01% ZnSO4.7H2O, 0.0005% NaCl in a shake-flask bioreactor at 35oC and 175 rpm. The bioreaction profile including tannic acid, gallic acid, pyrogallol, glucose concentrations, pH, biomass and extracellular tannase production were determined under the optimized conditions. The maximum extracellular tannase activity (316 U/ml) was observed on the 4th day of cultivation. However, biomass continued to increase up to the 9th day of fermentation. Increase in biomass concentration during the first two days and after the 7th day was high. The microorganism used tannic acid and glucose during the first two days by considering the sharp decrease in tannic acid and glucose concentrations. The increase in biomass concentration after the 7th day was directly proportional to the decrease in pyrogallol concentration in this period of time. The pH of the cultivation medium decreased from 5.5 to 2.3 owing to the assimilation of glucose and the production of gallic acid. Keywords: Tannase, Aspergillus niger, Enzyme production, Cultivation profile, Tannic acid.

The aim of this study was to analyze the effect of hydrogen peroxide and selected phenolic compounds on growth and catalase production of Aspergillus fumigatus. As a result of growing A. fumigatus at different temperatures it was observed that, growth and catalase production of this species were highest at 37 &deg / C. Catalase production was highest in the presence of 1 mM H2O2, yielding a significant 3 fold increase with respect to the control. Biomass was also increased by 1,44 fold with respect to the control sample. H2O2 increased catalase production possibly by inducing oxidative stress as biomass production significantly increased after the depletion of H2O2. Both gallic acid and trans-resveratrol significantly enhanced biomass generation of A. fumigatus (1,17 fold increase at 10 mM gallic acid and 1,45 fold increase at 3 mM resveratrol with respect to controls) and decreased extracellular catalase production (4,33 fold at 25 mM gallic acid and 16,7 fold decrease at 3 mM resveratrol with respect to controls) especially in the first 5 or 6 days of the cultivation where the anti-oxidant activity of the compounds were possibly at their maximum. A sudden and significant rise was observed in extracellular catalase activity between 5th and 7th days of the cultivation in phenolic compound applied samples, possibly owing to the depletion of the antioxidant activity of gallic acid and resveratrol followed by fungal cells&rsquo / response to a sudden increase of oxidative stress by boosting catalase production.

Polyphenol oxidases are enzymes that catalyze the oxidation of certain phenolic substrates to quinones in the presence of molecular oxygen. Polyphenol oxidases are widely used in several applications. In food industry, they are used for enhancement of flavor in coffee, tea and cocoa production, and determination of food quality. In medicine, they have several uses in treatments of Parkinson&rsquo / s disease, phenlyketonurea and leukemia. In wastewater treatment, they are used for the removal of phenolic pollutants from wastewaters. In pharmaceutical industry, differentiation of morphine from codeine is possible by means of polyphenol oxidase immobilized electrodes. In this study, a thermophilic fungus, Thermomyces lanuginosus was evaluated in terms of poyphenol oxidase production. The effect of different nutrient sources, inducers and fermentation parameters on enzyme production were investigated and maximum PPO activity of 97 U/ml was observed in bioreactor experiments at 50&deg / C, 400 rpm and pH 8.0 in a fermentation medium containing 1.4% yeast extract, 0.3% MgSO4, 1% KH2PO4, 0.003% CuSO4, 0.032% gallic acid. Type of polyphenol oxidase produced by Thermomyces lanuginosus was determined as laccase. For biochemical characterization studies, the enzyme was enriched by electrophoresis. Temperature and pH optima for the enzyme were determined as 60&deg / C and 8.0, respectively. Enzyme retained 67% activity after 1 h incubation at 80&deg / C and retained 87% of its activity after 1 hour of incubation at pH 9.0 at room temperature. The enzyme obeys Michealis-Menten kinetics with Km and Vmax values being 5 mg /ml catechol and 38 U/ml, respectively. Molecular weight of the enzyme was determined as 29 kDa and isoelectric point of enzyme was found to be approximately 6.0.

Lignocellulose, mostly from agricultural and forestry resources, is a potential renewable material for sustainable development of biorefineries. From previous studies, reducing sugar production through biological pretreatment involves two steps: solid-state fermentation (SSF) for delignification, followed by enzymatic hydrolysis by adding celluloytic enzymes (cellulase and xylanase etc.). In the process described in this thesis, the necessary enzymes are produced in-situ and the hydrolysis proceeds directly after the solid-state fermentation. Enzyme hydrolysis releases free amino nitrogen (FAN), reducing sugar and many other potential nutrients from the fermented materials. This method additionally avoids the need for removal of inhibitors compared with conventional chemical pretreatment processes. A range of solid-state fermentations were carried out to investigate the effect of washing procedure, particle size and nitrogen supplement on Trichoderma longibrachiatum growth. From these preliminary studies it was concluded that nitrogen supplementation is a crucial factor to improve significantly the fungi growth and production of feedstock using sugarcane bagasse as raw material. In order to evaluate the influence of environmental humidity on petri dish experiments, moist environments were investigated, with over 75% relative humidity to limit water evaporation from solid-state fermentation. The results showed that moist environments gave approximately 1.85 times the reducing sugar yield than dry environments. The process of simultaneous enzymatic hydrolysis of substrates and fungal autolysis were also studied. The degree of hydrolysis was affected by initial fermented solid to liquid ratio, temperature and pH range. The optimal conditions for subsequent hydrolysis of fermented solids were determined. The optimal solid to liquid ratio, 4% (w/w), temperature 50°C and pH 7 were established. The highest final reducing sugar, 8.9 g/L and FAN, 560 mg/L, were measured after 48 h. The fungal autolysis was identified by image analysis as well as by the consumption of nutrient and the release of free amino nitrogen and phosphorous. Solid state fermentation in a multi-layer tray bioreactor and a packed-bed bioreactor were also developed, with moist air supply for oxygen provision and heat removal. Fermented solids in the multi-layer bioreactor led to the highest subsequent hydrolysis yield on reducing sugar, FAN and Inorganic Phosphorous (IP), 222.85 mg/g, 11.56 mg/g and 19.9 mg/g, respectively. These series of fermentation experiments illustrate the feasibility for the application of consolidated bioprocessing, through simultaneous pretreatment and enzyme production as a more economic and environment-friendly process compared with those reported for chemical pretreatment followed by commercial enzyme process. A growth kinetic model regarding both growth and respiration is also proposed. Ethanol production was studied using the generic feedstock produced from sugarcane bagasse and soybean hulls. Total ethanol yield reached 0.31 mg g-1 (61.4% of theoretical yield) after 30 h of submerged fermentation. The result of subsequent fermentation has already shown the potential of the generic microbial feedstock to be used to produce varied products depending on the microorganism utilised.

Several yeast species had previously been isolated from water systems in the North West Province, South Africa. Some of the identified species had, in other studies, been associated with superficial mucosal infections to life threatening diseases. Antifungal drugs are used to treat such yeast infections. However, due to prophylactic usage and continuous exposure some yeast species have developed resistance to some antifungal agents. The aim of this study was to determine the diversity and antifungal susceptibility of yeasts in selected rivers, Mooi River and Harts River in the North West Province, South Africa. Waters samples were collected from the rivers in summer and winter seasons. Physico-chemical parameters such as pH, temperature, total dissolved solids, chemical oxygen demand, nitrates and phosphates were measured to determine the water quality. Yeast colonies were enumerated at room temperature and 37°C using yeast-malt-extract agar (containing 100 ppm chloramphenicol). Pure isolates from 37°C were identified by biochemical tests and 26S rRNA gene sequencing. Yeast sequences of isolated yeasts were sent to Genbank. Phylogenetic tree was conducted to determine phylogenetic relationship between the yeast isolates. Disk diffusion antifungal susceptibility tests were conducted on the yeast species. Physico-chemical parameters of the water were within target water quality range for livestock farming but in most sampling sites out of range for irrigation use. pH, Nitrates, phosphates and chemical oxygen demand levels ranged from 7.40 to 8.64, 0 to 5.4 mg/L, 0 to 7.14 mg/L and 31 to 43 mg/L, respectively. Elevated levels of total dissolved solids were measured in all the sampling sites. Total yeast counts ranged between 320-4200 cfu/L and 27-2573 cfu/L for room temperature and 37˚C. All the yeast colonies isolated were non-pigmented. Diazonium Blue B tests determined the yeasts isolates as ascomycetes. Haemolysin and extracellular enzyme production tests were negative on all the isolates. Yeasts isolates were identified and belonged to the genera Arxiozyma, Candida, Clavispora, Cyberlindnera, Lecythophora, Pichia, Saccharomyces, and Wickerhamomyces. Saccharomyces cerevisiae and Candida glabrata were mostly isolated species. Furthermore, the results indicated that levels of yeast could be correlated to physico-chemical quality of water. A large number of isolates were resistant to azoles, especially fluconazole as well as other antifungal classes. Most of the Candida species were resistant to almost all the antifungals. Several of the isolated yeast species are opportunistic pathogens. They could cause infections in sensitive individuals during occasional direct contact especially immune compromised people. Resistance of these yeast species to antifungal agents is a major health concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Several yeast species had previously been isolated from water systems in the North West Province, South Africa. Some of the identified species had, in other studies, been associated with superficial mucosal infections to life threatening diseases. Antifungal drugs are used to treat such yeast infections. However, due to prophylactic usage and continuous exposure some yeast species have developed resistance to some antifungal agents. The aim of this study was to determine the diversity and antifungal susceptibility of yeasts in selected rivers, Mooi River and Harts River in the North West Province, South Africa. Waters samples were collected from the rivers in summer and winter seasons. Physico-chemical parameters such as pH, temperature, total dissolved solids, chemical oxygen demand, nitrates and phosphates were measured to determine the water quality. Yeast colonies were enumerated at room temperature and 37°C using yeast-malt-extract agar (containing 100 ppm chloramphenicol). Pure isolates from 37°C were identified by biochemical tests and 26S rRNA gene sequencing. Yeast sequences of isolated yeasts were sent to Genbank. Phylogenetic tree was conducted to determine phylogenetic relationship between the yeast isolates. Disk diffusion antifungal susceptibility tests were conducted on the yeast species. Physico-chemical parameters of the water were within target water quality range for livestock farming but in most sampling sites out of range for irrigation use. pH, Nitrates, phosphates and chemical oxygen demand levels ranged from 7.40 to 8.64, 0 to 5.4 mg/L, 0 to 7.14 mg/L and 31 to 43 mg/L, respectively. Elevated levels of total dissolved solids were measured in all the sampling sites. Total yeast counts ranged between 320-4200 cfu/L and 27-2573 cfu/L for room temperature and 37˚C. All the yeast colonies isolated were non-pigmented. Diazonium Blue B tests determined the yeasts isolates as ascomycetes. Haemolysin and extracellular enzyme production tests were negative on all the isolates. Yeasts isolates were identified and belonged to the genera Arxiozyma, Candida, Clavispora, Cyberlindnera, Lecythophora, Pichia, Saccharomyces, and Wickerhamomyces. Saccharomyces cerevisiae and Candida glabrata were mostly isolated species. Furthermore, the results indicated that levels of yeast could be correlated to physico-chemical quality of water. A large number of isolates were resistant to azoles, especially fluconazole as well as other antifungal classes. Most of the Candida species were resistant to almost all the antifungals. Several of the isolated yeast species are opportunistic pathogens. They could cause infections in sensitive individuals during occasional direct contact especially immune compromised people. Resistance of these yeast species to antifungal agents is a major health concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

In this study, two symbiotic fungi of Southern Pine Beetle (SPB), Entomocorticium peryii and Entomocorticium sp.A were evaluated in terms of polyphenol oxidase (PPO) production. The effect of different inhibitors, inducers and assay parameters such as temperature and pH on enzyme activity were investigated and maximum PPO activity was observed at 30&deg / C, pH 8.0 and when tannic acid was used as an inducer. Copper-chelator salicyl hydroxamic acid (SHAM) and pcoumaric acid, both indicated as inhibitors of tyrosinase and catechol oxidase significantly reduced the activity. For biochemical characterization studies, the enzyme was concentrated by ultrafiltration. To determine type of the enzyme, activity staining after Native-PAGE was carried out. Type of polyphenol oxidase produced by E. peryii and E. sp.A was determined as catechol oxidase by activity staining. However higher activity was observed on hydroquinone (p-diphenol) rather than catechol (o-diphenol). The enzyme obeys Michealis-Menten kinetics with Km and Vmaxvalues being 10.72 mM hydroquinone and 59.44 U/ml for E. peryii and 8.55 mM hydroquinone and 73.72 U/ml for E. sp.A respectively..