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Ph. D.

Solar radiation influences virtually all biological process on earth. Yeasts, the microbial driver of ethanol fermentation, evolved on the surface of vegetation and had to adapt to survive photonic assault. Past research has demonstrated that white light affects yeast metabolism along with the ability to entrain circadian rhythms, although no known genetic mechanism accounts for this. High intensity narrow wavelength light-emitting diodes were employed to illuminate synthetic cultures under fermentation. Multiple colors along the visible spectrum were used, corresponding to the peak absorbance wavelengths of Saccharomyces sp. yeast. Impacts in primary metabolite evolution were found, dependent on wavelength. Longer wavelengths produced higher amounts of acetic acid and glycerol; shorter wavelengths produced more ethanol. Because past research showed light timing had pronounced effects, illumination schemes on the scale of milliseconds to hours were tested for ethanol production. Light schemes on the scale of enzymatic reactions, yeast generation times, and circadian rhythms produced the most ethanol. Discrete blocks and duration of illumination were used to elucidate where light had the most influence over yeast metabolism and fermentation. Late lag phase and mid log phase illumination impacted ethanol fermentation more than any other period of time. Light effects were tested on apple juice to see if they extended from synthetic media to natural products. Significant impacts on ethanol production were discovered and flavor/aroma impacts were noted. Light, color, intensity, and timing have all been shown to control and affect fermentation with both positive and negative effects established. / Doctor of Philosophy / Sun light influences virtually all biological process on earth. Yeasts, the microbial drivers of ethanol fermentation, evolved on the surface of vegetation and had to adapt to survive destructive effects of the sun. Past research has demonstrated that white light affects yeast metabolism along with the ability to develop growth cycles similar to day / night patterns, although it is currently not believed this possible due to the biology of yeast. High intensity single color light-emitting diodes were employed to illuminate laboratory formulated cultures under fermentation. Multiple colors along the visible spectrum were used, corresponding to the peak absorbance wavelengths of Saccharomyces sp. yeast. Green/yellow/red wavelengths produced higher amounts of acetic acid (vinegar) and glycerol; blue and ultraviolet wavelengths produced more ethanol. Because past research showed light timing could change how yeast grow and consumed carbohydrates, light timing on the scale of milliseconds to hours were tested for ethanol production. Light timing on the scale of milliseconds, hours, and daylight cycles produces the most ethanol. Discrete blocks and duration of illumination were used to find where during fermentation light had the most impact. It was found that from immediately after the beginning of fermentation to the middle of fermentation is where yeast responded the most strongly. Light effects were tested on apple juice to see if they extended laboratory cultures to natural products. Significant changes in the amount of ethanol produced were discovered and changes in the taste and smell of fermented apple juice were noted. Light, color, intensity, and timing have all been shown to control and affect fermentation with both positive and negative effects established.

The ability of a self-contained denitrification system, using fermentation products from waste fish solids, to maintain reliable performance was studied. Denitrification performance was described kinetically and stoichiometrically under different initial nitrate-nitrogen and soluble organic carbon to nitrate-nitrogen ratios. Characterization of soluble organic carbon (measured as soluble chemical oxygen demand, sCOD) indicated that volatile fatty acids (VFA) were generated during the fermentation of the waste fish solids. The results from batch experiments showed that over the range of initial nitrate concentrations studied, complete denitrification was achieved within 6 hrs. sCOD, nitrite, and nitrate profiles across several batch experiments showed that transient nitrite accumulations occurred, but the maximum measured concentrations never completely inhibited nitrate removal. The results suggested that the rate of denitrification was influenced by the initial sCOD to nitrate-nitrogen ratio when transient nitrite concentrations remained below 20 mg/L. However, when nitrite-nitrogen exceeded 25 mg/L, the rate of denitrification was negatively correlated with the maximum measured nitrite-nitrogen concentration. The stoichiometric carbon requirement was not correlated to any parameters believed to influence carbon consumption. After complete denitrification was achieved residual sCOD was still measured, which could not be identified as VFAs. Batch aerobic treatment of denitrified effluent resulted in a 60 to 70 % removal of the residual sCOD when allowed to react for 8 days. It was further determined that the residual sCOD exerted an oxygen of 5.81 on g COD/g C. Additional studies were conducted to maximize sCOD production during fermentation. Increasing the fermentation temperature from 28 oC to 40 oC facilitated a 36 % increase in the specific sCOD production rate (g sCOD/ g fish solids applied). In addition to sCOD production, ammonia production increased 20 % when the fermentation was conducted at the elevated temperature. An analysis comparing the cost of methanol addition to support denitrification to the cost associated with fermenting waste fish solids indicated that supplementing fermentation products with methanol resulted in the least costly strategy for promoting denitrification of an aquaculture waste stream. / Master of Science

Omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA, C20:5, n-3) and docosahexaenoic acid (DHA, C22:6, n-3), have many medically established benefits against cardiovascular diseases, cancers, schizophrenia, and Alzheimer's. Currently, fish oil is the main source of omega-3 fatty acids, but there are many problems associated with it such as undesirable taste and odor, and heavy metal contamination. As a result, it is necessary to seek alternative production sources based on various microorganisms. In this thesis we have developed a novel microfungal culture process to produce EPA from the crude glycerol byproduct generated in biodiesel industry. This process provides both an alternative source of omega-3 fatty acids and a benefit to the biodiesel industry. Indeed, as oil prices reach historical highs, biodiesel has attracted increasing interest throughout the United States. The disposal of the crude glycerol byproduct has been a challenge faced by the biodiesel producers. Crude glycerol presents a cheap carbon source for growth of many microorganisms. In this thesis, we tested the feasibility of using crude glycerol for producing eicosapentaenoic acid (EPA, 20:5, n-3) by one algal species, Phaeodactylum tricornutum and two fungal species, Mortierella alpina and Pythium irregulare. We observed that the algal growth is inhibited in the crude glycerol while the fungi can grow very well in crude glycerol-containing medium. The fungus M. alpina produced significant amount of ARA but negligible amount of EPA. P. irregulare produced significant amount of biomass as well as a relatively high level of EPA. The maximum dry biomass for the P. irregulare culture was 2.9 g/L with an EPA productivity of 7.99 mg/L-day. Based on these results, we concluded that P. irregulare was a promising candidate for EPA production from biodiesel derived crude glycerol. Further optimization work showed that P. irregulare grown 30 g/L crude glycerol and 10g/L yeast extract results in the highest level of EPA production. A temperature of 20o C is optimal for high fungal biomass and EPA levels. Addition of vegetable oil (at 1%) enhanced the EPA production and almost doubled the amount of biomass reached. Soap inhibits growth as well as EPA production severely even in small amounts. Methanol completely inhibits growth. The final optimized growth conditions for the fungus P.irregulare were a medium with 30g/L of crude glycerol, 10 g/L of yeast extract at a pH of 6 with 1% supplementation of oil, at a temperature of 20o C for a period of 7 days.Thus we have established that the fungus P.irregulare can be used successfully to produce high mounts of EPA from crude glycerol. / Master of Science

Xylitol can be produced from xylose or hemicellulose hydrolysates by either chemical reduction or microbial fermentation. Current technology for commercial production is based on chemical reduction of xylose or hemicellulose, and xylitol is separated and purified by chromatographic methods. The resultant product is very expensive because of the extensive purification procedures. Microbial production of xylitol is being researched as an alternative method for xylitol production. Apart from the chromatographic separation method and activated carbon treatment, no other separation method has been proposed for the separation of xylitol from the fermentation broth. Membrane separation was proposed as an alternative method for the recovery of xylitol from the fermentation broth because it has the potential for energy savings and higher purity. A membrane separation unit was designed, constructed, tested, and successfully used to separate xylitol from the fermentation broth. Eleven membranes were investigated for xylitol separation from the fermentation broth. A 10,000 nominal molecular weight cutoff (MWCO) polysulfone membrane was found to be the most effective for the separation and recovery of xylitol. The membrane allowed 82.2 to 90.3% of xylitol in the fermentation broth to pass through while retaining 49.2 to 53.6% of the Lowry's method positive material (such as oligopeptides and peptides). Permeate from the 10,000 MWCO membrane was collected and crystallized. Crystals were analyzed by HPLC for xylitol and impurities and determined to have purity up to 90.3%. / Master of Science