Rapid depletion of fossil fuel stock with a simultaneous rise in greenhouse gas emissions has led to an increase in the need for alternative energy. Cellulose based biofuels, especially bioethanol is a form of alternative energy that has the potential to replace petrol. The first step in cellulosic bioethanol production is the release of fermentable sugars via pre-treatment. Conventionally, physico-chemical and biological pre-treatment methods are energy intensive, environmentally unfavourable and expensive. This study, however reports on the use of a less energy consuming, cheap and environmental friendly alternative; photocatalysis, to produce fermentable sugars from cellulose. To achieve this, a range of photocatalysts were first screened based on their OH radical production rates using coumarin as a probe. TiO2 P25 was the photocatalyst that was found to have the highest OH radical production rate of 35.6 μM/hr, followed by Pt-C3N4 (0.88 μM/hr) and WO3 (0.28 μM/hr). LaCr-SrTiO3, Cr-SrTiO3 and yellow TiO2 did not produce any OH radicals due to their unsuitable electronic structure. P25 was further used for photocatalytic fermentable sugar production from cellulose. Photocatalytic cellulose I breakdown produced 0.04 % fermentable sugars whereas, with cellulose II feedstock the yield increased to 0.2 %. To further improve the yield, membrane bags were deployed which improved the sugar yields to 0.43 % and 0.71 % respectively from cellulose and cellulose II feedstocks. Photonic efficiencies followed the same trends as the sugar yields. Engineering design was further opted to enhance the sugar yields and hence a stacked frame photocatalytic reactor (SFPR) was designed. Various mixer configurations were designed and their mixing regime was determined using COMSOL Multiphysics 5.1 simulations. Amongst the mixers simulated, an 8-blade Rushton impeller was found to be the best configuration due its superior radial mixing profile and higher fluid velocity. The SFPR was then fabricated and operated with the impeller or a plus shaped magnetic bar as the mixer and the sugar yields were determined. Highest sugar yield and photonic efficiency was obtained from the cellulose II-impeller setup and was calculated to be 2.61 % and 9.45 % respectively. Respective lowest yields were obtained with cellulose I-stirrer bar setup and calculated to be 1.71 % and 5.64 %. Furthermore, the effect of H2O2 on fermentable sugar production was also tested. The cellulose II-stirrer bar configuration yielded 3.15 % fermentable sugars with the addition of 0.01 wt% H2O2 to the reaction mixture. The yield improved significantly to 14.1 % when the concentration of H2O2 was increased to 0.1 wt%.

Doctor of Philosophy / Department of Biological & Agricultural Engineering / Donghai Wang / Sweet sorghum accumulates high concentrations of fermentable sugars in the stem, produces significant amount of starch in the grain (panicle) and has shown to be a promising energy feedstock. Sweet sorghum has a short growing season so adding it to the sugar cane system would be good. The overall goal of this dissertation is to enhance the attractiveness of biofuel production from sweet sorghum to fully utilize fermentable sugars in the juice, starch in the panicle and structural carbohydrates in the stalk for high efficiency and low-cost ethanol production. Sweet sorghum juice was incorporated into the dry-grind process which increased ethanol yield by 28% increase of ethanol yield compared to the conventional ethanol method and decreased enzymatic hydrolysis time by 30 minutes. A very high gravity fermentation technique was applied using sweet sorghum juice and sorghum grain yielded 20.25% (v/v) of ethanol and 96% fermentation efficiency. Response surface methodology was applied in order to optimize diffusion conditions and to explore effects of diffusion time, diffusion temperature, and ratio of sweet sorghum biomass to grain on starch-to-sugar efficiency and total sugar recovery from sweet sorghum. Starch hydrolysis efficiency and sugar recovery efficiency of 96 and 98.5% were achieved, respectively, at an optimized diffusion condition of 115 minutes, 95 °C, and 22% grain loading. Extraction kinetics based on the optimized diffusion parameters were developed to describe the mass transfer of sugars in sweet sorghum biomass during the diffusion process. Ethanol obtained from fermented extracted sugars treated with granular starch hydrolyzing enzyme and those with traditional enzymes were comparable (14.5 – 14.6% v/v). Ethanol efficiencies also ranged from 88.92 –92.02%.

The utilization of lignocellulosic residues to produce renewable energy is an interesting alternative to meet the increasing demand of fuels while at the same time reducing greenhouse gas emissions and climate change. Sweet sorghum bagasse is a lignocellulosic residue composed mainly of cellulose, hemicellulose, and lignin; and it is a promising substrate for ethanol production because its complex carbohydrates may be hydrolyzed and converted into simple sugars, and then fermented into ethanol. However, the utilization of lignocellulosic residues is difficult and inefficient. Lignocellulose is a very stable and compact complex structure, which is linked by β-1,4 and β-1,3 glycosidic bonds. Furthermore, the crystalline and amorphous features of cellulose fibers and the presence of hemicellulose and lignin make the conversion of lignocellulose into fermentable sugars currently impractical at commercial scale. The bioconversion of lignocellulose in nature is performed by microorganisms such as fungi and bacteria, which produce enzymes that are able to degrade lignocellulose. The present study evaluated the bioconversion of lignocellulosic residues of sweet sorghum into simple sugars using filamentous fungi directly in the hydrolysis of the substrate, without prior isolation of the enzymes. The fungus Neurospora crassa and some wild fungi (that grew naturally on sweet sorghum bagasse) were used in this investigation. The effect of the fungi on substrate degradation and the sugars released after hydrolysis were evaluated, and then compared with standard hydrolysis performed by commercial enzymes (isolated cellulases). In addition, different combinations of fungi and enzymes were used to determine the best approach. The main goal was to verify if the fungi were able to attack and break down the lignocellulose structure directly and at a reasonable rate, rather than by the current method utilizing isolated enzymes. The main finding of this study was that the fungi (N. crassa and wild fungi) were able to degrade sweet sorghum bagasse directly; however, in all of the cases, the hydrolysis process was not efficient because the hydrolysis rate was much lower than the enzymatic hydrolysis rate. Hydrolysis using a combination of fungus and commercial enzymes was a good approach, but still not efficient enough for practical use. The best results of combined hydrolysis were obtained when the substrate was under the fungus attack for three days and then, commercial enzymes with low enzymatic activity (7 FPU/g and 25 CBU/g) were added to the solution. These enzymes represent 10% of the current enzymatic activity recommended per gram of substrate. This process reached reasonable levels of sugars (close to 85% of sugars yield obtained by enzymatic hydrolysis); however, the conversion rate was still slower, making the process impractical and more expensive since it took twice the amount of time as commercial enzymes. Furthermore, the wild fungi able to degrade cellulose were isolated, screened, and identified. Two of them belong to the genus Aspergillus, one to the genus Acremonium, and one to the genus Rhizopus. Small concentration of spores-0.5mL- (see Table 4, CHAPTER III- for specific number of spores per mL) did not show any sugar released during hydrolysis of sweet sorghum bagasse. However, when the concentration of spores was increased (to 5mL and 10mL of solution), citric acid production was detected. This finding indicates that those wild fungi were able to degrade lignocellulose, even though no simple sugars were measured, citric acid production is an indicator of fungi growing and utilization of lignocellulose as nutrient. It is assumed that the fungi consume the sugars at the same time they are released, thus they are not detected. The maximum concentration of citric acid (~14.50 mg/mL) was achieved between days 8-11 of hydrolysis. On the other hand, before using lignocellulose, the substrate needed to be pretreated in order to facilitate its decomposition and subsequent hydrolysis. Sweet sorghum bagasse was washed three times to remove any soluble sugars remaining after the juice was extracted from the stalks. Then, another finding of this study was that the first wash solution could be used for ethanol production since the amount of sugars present in it was close to 13°Brix. The ethanol yield after 48 hours of fermentation was in average 6.82mg/mL, which is close to the theoretical ethanol yield. The other two washes were too dilute for commercial ethanol production. In terms of pretreatments, the best one to break down sweet sorghum bagasse was 2% (w/v) NaOH. This pretreatment shows the highest amounts of glucose and xylose released after hydrolysis. Unwashed and untreated bagasse (raw bagasse) did not show any sugar released. In terms of ethanol, 74.50% of the theoretical yield was reached by enzymatic hydrolysis, while 1.10% was reached by hydrolysis using the fungus N. crassa. Finally, it is important to remark that further investigation is needed to improve the direct conversion of lignocellulose into fermentable sugars by fungal enzymes. This approach is a promising technology that needs to be developed and improved to make it efficient and feasible at commercial scale.

In this paper, replacing complete or partly of growth mediums with secondarily wastewater was studied. Lipid content of Neochloris oleoabundans grown in a 0.3 X SE medium and autoclaved secondarily treated wastewater mixture was 22.27 % (w/w). The maximum biomass concentration of N. oleoabundans grown in wastewater with no additional nutrients was 0.636 g/L with 33% (w/w) glucose. Two culture lines, MA, and NA were isolated within our laboratory and could grow in secondarily treated wastewater with no additional nutrients. The maximum biomass concentration of MA in batch culture was 0.860 g/L and the sum of glucose and xylose was 40% (w/w). The maximum biomass concentration of NA was 1.562 g/l and the sum of glucose and xylose was 33.8% (w/w). The maximum specific growth rates of NA and MA were determined to be 0.0566 and 0.0337 per hour.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work aimed to study the enzymatic hydrolysis of malt bagasse, using mechanical agitation, ultrasonic probe and supercritical CO2. Enzymatic hydrolysis was performed on granular starch. For this, was used a commercial amylolytic complex suitable for this type of hydrolysis, STARGENTM 002. For each technology were studied different variables. The first planning was carried out for the hydrolysis assisted by mechanical agitation and the variables studied were temperature (°C), enzyme concentration (%, m/m) and substrate concentration in the medium (m/m). TRS concentrations were found up to 75.5 g per kg of substrate and all variables had a significant effect on the response. This concentration of TRS was defined as mass yield of the process and this yield was corroborated by a kinetic, developed under the same conditions, with slight increase in temperature (70°C). From this initial planning, the temperature variables (70°C), enzyme concentration (8.2%) and substrate concentration (170 g.L-1) were fixed to carry out the hydrolysis assisted by direct and indirect ultrasound. The variables were investigated in the second planning were amplitude (%) and pulse factor (-) for 2 hours of reaction. With the application of direct sonication, it was possible to achieve 100% efficiency in the starch conversion process. The TRS for the best essay (5) was 370.86 g.kg-1. For indirect sonication TRS concentration at the best condition (run 6) was 162.96 g / kg of substrate. A kinetic assay for the best condition under direct sonication was carried out for 3 hours, confirming that the ultrasound increases the reaction rate resulting in better yields in less time compared to the other techniques. For reactions with supercritical CO2 was studied the influence of the moisture content, temperature and pressure, where the best result among all the reactions was using at pressure 175 bar, 40 °C for temperature and moisture content of 80%, resulting in 104.28 g of TRS per kg of dry pulp. Ultrasound showed better results than other technologies investigated in this study. / Este trabalho teve como objetivo estudar a hidrólise enzimática do bagaço de malte, utilizando agitação mecânica, sonda de ultrassom e CO2 supercrítico. A hidrólise enzimática foi realizada sobre o amido granular. Para tanto, foi utilizado um complexo amilolítico comercial próprio para este tipo de hidrólise, STARGENTM 002. Para cada tecnologia foram estudadas diferentes variáveis. O primeiro planejamento foi realizado para a hidrólise assistida por agitação mecânica e as variáveis estudadas foram temperatura (ºC), concentração de enzima (%, m/m) e concentração de substrato no meio (m/m). Foram encontradas concentrações de ART de até 75,5 g por kg de substrato e todas as variáveis apresentaram efeito significativo sobre a resposta. Essa concentração de ART, foi definida como rendimento mássico do processo e este valor foi corroborado com uma cinética, desenvolvida nas mesmas condições do melhor ensaio, com leve aumento apenas na temperatura (70ºC), e indicou valor concernente ao anterior além de provar que o tempo de 4 horas de hidrólise foi suficiente. A partir deste primeiro planejamento, as variáveis temperatura (70°C), concentração de enzima (8,2%) e concentração de substrato (170 g.L-1) foram fixadas para a realização da hidrólise assistida por ultrassom, de forma direta e indireta. As variáveis investigadas neste segundo planejamento foram amplitude (%) e fator de pulso (-) durante 2 horas de reação. Com aplicação de sonicação direta, foi possível alcançar 100% de eficiência no processo de conversão do amido. A resposta encontrada para o melhor ensaio (5) foi de 370,86 g.kg-1. Já com aplicação de sonicação indireta, a eficiência do processo caiu pela metade e o melhor resultado foi para o ensaio de número 6, com concentração de 162,96 g ART/ kg de substrato. Uma cinética para o melhor ensaio de sonicação direta foi desenvolvida durante 3 horas, atestando que o ultrassom aumenta a velocidade da reação resultando no melhor rendimento em menor tempo comparado às outras técnicas. Para as reações com CO2 supercrítico, foi estudada a influência da umidade, da temperatura e pressão, onde o melhor resultado obtido, entre todas as reações, foi com a utilização de pressão de 175 bar, temperatura de 40ºC e 80% de água adicionada ao bagaço, resultando em 104,28 g de ART por kg de bagaço seco. Esse valor corresponde a 11,53% de eficiência da reação de hidrólise do amido em açúcares redutores totais. De um modo geral, os processos obtiveram um bom desempenho na obtenção de açúcares fermentescíveis, destacando-se o ultrassom em relação as demais tecnologias testadas.