With recent emphasis on development of alternatives to fossil fuels, sincere attempts are being made on finding suitable lignocellulosic feedstocks for biochemical conversion to fuels and chemicals. Sweet Sorghum is among the most widely adaptable cereal grasses, with high drought resistance, and ability to grow on low quality soils with low inputs. It is a C4 crop with high photosynthetic efficiency and biomass yield. Since sweet sorghum has many desirable traits, it has been considered as an attractive feedstock. Large scale sweet sorghum juice extraction results in excessive amounts of waste sweet sorghum bagasse (SSB), which is a promising low cost lignocellusic feed stock. The ability of two pretreatment methods namely conventional oven and microwave oven pretreatment for disrupting lignocellulosic structures of sweet sorghum bagasse with lime [Ca(OH)2] and sodium hydroxide [NaOH] was evaluated. The primary goal of this study was to determine optimal alkali pretreatment conditions to obtain higher biomass conversion (TRS yield) while achieving higher lignin reduction for biofuel production. The prime objective was achieved using central composite design (CCD) and optimization of biomass conversion and lignin removal simultaneously for each alkali separately by response surface method (RSM). Quadratic models were used to define the conditions that separately and simultaneously maximize the response variables. The SSB used in this study was composed of cellulose, hemicellulose, and lignin in the percentage of 36.9 + 1.6, 17.8 + 0.6, and 19.5 + 1.1, respectively. The optimal conditions for lime pretreatment in the conventional oven at 100 °C was 1.7 (% w/v) lime concentration (=0.0024 molL-1), 6.0% (w/v) SSB loading, 2.4 hr pretreatment time with predicted yields of 85.6% total biomass conversion and 35.5% lignin reduction. For NaOH pretreatment, 2% (w/v) alkali (=0.005 molL-1), 6.8% SSB loading and 2.3 hr duration was the optimal level with predicted biomass conversion and lignin reduction of 92.9% and 50.0%, respectively. More intensive pretreatment conditions removed higher amount of hemicelluloses and cellulose. Microwave based pretreatments were carried out in a CEM laboratory microwave oven (MARS 6-Xpress Microwave Reactions System, CEM Corporation, Matthews, NC) and with varying alkali concentration(0.3 - 3.7 % w/v) at varying temperatures (106.4 - 173.6 °C), and length of time (6.6 - 23.4 min). The NaOH pretreatment was optimized at 1.8 (% w/v) NaOH, 143 °C, 14 min time with predicted yields of 85.8% total biomass conversion and 78.7% lignin reduction. For lime pretreatment, 3.1% (w/v) lime, 138 °C and 17.5 min duration was the optimal level with predicted biomass conversion and lignin reduction of 79.9% and 61.1%, respectively. Results from this study were further supported by FTIR spectral interpretation and SEM images.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20766号 / 農博第2249号 / 新制||農||1054(附属図書館) / 学位論文||H29||N5086(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 金山 公三, 教授 矢野 浩之, 教授 吉村 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The first part of the dissertation presented a potentially novel aerobic, thermophilic, and cellulolytic bacterium identified as Brevibacillus sp. Strain JXL which was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between the effects caused by these two simple sugars. After the end of the 24 hour period, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also observed when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and optimal pH range of 6-8. These cellulases also had high thermotolerance as demonstrated by retaining more than 50% activity after 1 h at 100°C. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose. In the second part of the dissertation, the effect of Cellulose- and Organic-Solvent based Lignocellulose Fractionation (COSLIF) (Zhang, Y.-H. P.; Ding, S.-Y.; Mielenz, J. R.; Elander, R.; Laser, M.; Himmel, M.; McMillan, J. D.; Lynd, L. R. Biotechnol. Bioeng.2007, 97 (2), 214−223) pretreatment conditions on sweet sorghum bagasse (SSB) feedstock was studied using Response Surface Methodology (RSM). Batch experimental matrix was set up based on response surface method's central composite design in two factors to determine the effects of reaction time and temperature on the yield of simple sugars after a sequential pretreatment-enzyme hydrolysis process. Accordingly, changes in delignification, total reducing sugar (TRS) yield, glucan retention, digestibility and overall sugar yields resulting from various combinations of reaction times and temperatures were determined. The results suggested that both pretreatment temperature and reaction time were significant factors, although temperature was more so than reaction time. COSLIF pretreatment conditions of 50°C and 40 min were found to be the optimum pretreatment conditions for the saccharification of SSB. At the end of pretreatment and enzymatic hydrolysis, maximum values of 51.4% delignification, 85% overall glucose yield, and 44% overall xylose yield at an ACCELERASE®1500 loading of 0.25 mL/g sweet sorghum bagasse were achieved. Optimum ACCELERASE®1500 dosage of 0.1 mL/g of sweet sorghum bagasse was identified which resulted in an overall glucose yield of 82.2%±1.05. An effort has also been made to prescribe predictive models which represented the correlation between independent variables (reaction time and temperature), and dependent variables (delignification, and overall glucose yield) using RSM. The significance of the correlations and adequacy of these models were statistically tested for the selected objective functions. The outcomes suggested very competent and statistically adequate regression models which provided quantitative information both for delignification and overall glucose yield for the batch experiments studied.

Succinic acid is a compound used for manufacturing lacquers, resins, and other coating chemicals. It is also used in the food and beverage industry as a flavor additive. It is predominantly manufactured from petrochemicals, but it can also be produced more sustainably by fermentation of sugars from renewable feedstocks (biomass). Bio-based succinic acid has excellent potential for becoming a platform chemical (building block) for commodity and high-value chemicals. In this study, we focused on the production of bio-based succinic acid from the fiber of sweet sorghum (SS), which has a high fermentable sugar content and can be cultivated in a variety of climates and locations around the world. To avoid competition with food feedstocks, we targeted the non-edible ‘bagasse’, which is the fiber part after extracting the juice. Initially, we studied various conditions of pretreating SS bagasse to remove most of the non-fermentable portions and expose the cellulose fibers containing the fermentable sugars (glucose). Concentrated (83%) phosphoric acid was utilized at mild temperatures of 50-80 °C for 30-60 minutes at various bagasse loadings (10-15%) using a partial factorial experimental design. After pretreatment, the biomass was subjected to enzymatic hydrolysis with commercial cellulase enzyme (Cellic® Ctec2) to identify the pretreatment conditions that lead to the highest glucose yield that is critical for the production of succinic acid via fermentation with the bacterium Actinobacillus succinogenes. As the pretreatment temperature and duration increased, the bagasse color changed from light brown to dark brown-black, indicating decomposition, which ranged from 15% to 72%. The pretreatment results were fitted with an empirical model that identified 50 °C for 43 min at 13% solids loading as optimal pretreatment conditions that lead to the highest glucose release from sweet sorghum bagasse. Biomass pretreated at those conditions and subjected to separate enzymatic hydrolysis and fermentation with A. succinogenes yielded almost 18 g/L succinic acid, which represented 90% of the theoretical yield, a very promising performance that warranties further investigation of bio-based succinic acid production from sweet sorghum bagasse, as a more sustainable alternative to succinic acid produced from fossil sources, such as oil.

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.

The growing demand for energy in the world, the implications of climate change, the increasing damages to our environment and the diminishing fossil fuel reserves have created the appropriate conditions for renewable energy development. Biofuels such as bioethanol can be produced by breaking down the lignocellulosic structure of plant materials to release fermentable sugars. Sweet sorghum bagasse has been shown to be an important lignocellulosic crop residue and is potentially a significant feedstock for bioethanol production. The aim of this study was to investigate suitable microwave assisted pretreatment conditions of sweet sorghum bagasse for bioethanol production. A chemical pretreatment process of sweet sorghum bagasse, using different concentrations (1 to 7 wt%) of sulphuric acid (H2SO4) and calcium hydroxide (Ca (OH)2) was applied to break up the lignocellulosic matrix of sweet sorghum bagasse. The pretreated broth, which contained pentose and hexose sugars, was fermented using a combination of Zymomonas mobilis ATCC31821 and Saccharomyces cerevisiae to produce bioethanol at pH 4.8 and 32oC for 24 hours. The highest reducing sugar yield of 0.82 g/g substrate was obtained with microwave irradiation at 180 W for 20 minutes in a 5 wt% sulphuric acid solution. The highest ethanol yield obtained was 0.5 g/g from 5 wt% H2SO4 pretreated bagasse at 180 W using a 10:5% v/v of Saccharomyces cerevisiae to Zymomonas mobilis ratio, whereas for 3 wt% Ca (OH)2 microwave pretreatment, a sugar yield of 0.27 g/g substrate was obtained at 300 W for 10 minutes. Thereafter, an ethanol yield of 0.13 g/g substrate was obtained after 24 hours of fermentation when using a 10:5% v/v of Saccharomyces cerevisiae to Zymomonas mobilis ratio. The effect of microwave pretreatment on the bagasse was evaluated using Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) analysis. The reducing sugars formed were quantified using High Performance Liquid Chromatography (HPLC). The results showed that microwave pretreatment using 5 wt% H2SO4 is a very effective pretreatment that can be used to obtain sugars from sweet sorghum bagasse. The analytic results also showed physical and functional group changes after microwave pretreatment. This confirms that microwave irradiation is very effective in terms of breaking up the lignocellulose structure and improving fermentable sugar yield for fermentation. Bioethanol yields obtained from microwave pretreatment using different solvents also show that Saccharomyces cerevisiae and Zymomonas mobilis ATCC31821 is a good combination for producing ethanol from sweet sorghum bagasse. Sweet sorghum bagasse is clearly a very effective and cheap biomass that can be used to produce bioethanol, since very high yields of fermentable sugars were obtained from the feedstock. / Thesis (MSc (Engineering Sciences in Chemical Engineering))--North-West University, Potchefstroom Campus, 2013.

The growing demand for energy in the world, the implications of climate change, the increasing damages to our environment and the diminishing fossil fuel reserves have created the appropriate conditions for renewable energy development. Biofuels such as bioethanol can be produced by breaking down the lignocellulosic structure of plant materials to release fermentable sugars. Sweet sorghum bagasse has been shown to be an important lignocellulosic crop residue and is potentially a significant feedstock for bioethanol production. The aim of this study was to investigate suitable microwave assisted pretreatment conditions of sweet sorghum bagasse for bioethanol production. A chemical pretreatment process of sweet sorghum bagasse, using different concentrations (1 to 7 wt%) of sulphuric acid (H2SO4) and calcium hydroxide (Ca (OH)2) was applied to break up the lignocellulosic matrix of sweet sorghum bagasse. The pretreated broth, which contained pentose and hexose sugars, was fermented using a combination of Zymomonas mobilis ATCC31821 and Saccharomyces cerevisiae to produce bioethanol at pH 4.8 and 32oC for 24 hours. The highest reducing sugar yield of 0.82 g/g substrate was obtained with microwave irradiation at 180 W for 20 minutes in a 5 wt% sulphuric acid solution. The highest ethanol yield obtained was 0.5 g/g from 5 wt% H2SO4 pretreated bagasse at 180 W using a 10:5% v/v of Saccharomyces cerevisiae to Zymomonas mobilis ratio, whereas for 3 wt% Ca (OH)2 microwave pretreatment, a sugar yield of 0.27 g/g substrate was obtained at 300 W for 10 minutes. Thereafter, an ethanol yield of 0.13 g/g substrate was obtained after 24 hours of fermentation when using a 10:5% v/v of Saccharomyces cerevisiae to Zymomonas mobilis ratio. The effect of microwave pretreatment on the bagasse was evaluated using Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) analysis. The reducing sugars formed were quantified using High Performance Liquid Chromatography (HPLC). The results showed that microwave pretreatment using 5 wt% H2SO4 is a very effective pretreatment that can be used to obtain sugars from sweet sorghum bagasse. The analytic results also showed physical and functional group changes after microwave pretreatment. This confirms that microwave irradiation is very effective in terms of breaking up the lignocellulose structure and improving fermentable sugar yield for fermentation. Bioethanol yields obtained from microwave pretreatment using different solvents also show that Saccharomyces cerevisiae and Zymomonas mobilis ATCC31821 is a good combination for producing ethanol from sweet sorghum bagasse. Sweet sorghum bagasse is clearly a very effective and cheap biomass that can be used to produce bioethanol, since very high yields of fermentable sugars were obtained from the feedstock. / Thesis (MSc (Engineering Sciences in Chemical Engineering))--North-West University, Potchefstroom Campus, 2013.

The present study investigated the feasibility of production of sophorolipids (SLs) using yeast Candida bombicola grown on hydrolysates derived lignocellulosic feedstock either with or without supplementing oil as extra carbon source. Several researchers have reported using pure sugars and various oil sources for producing SLs which makes them expensive for scale-up and commercial production. In order to make the production process truly sustainable and renewable, we used feedstocks such as sweet sorghum bagasse, corn fiber and corn stover. Without oil supplementation, the cell densities at the end of day-8 was recorded as 9.2, 9.8 and 10.8 g/L for hydrolysate derived from sorghum bagasse, corn fiber, and corn fiber with the addition of yeast extract (YE) during fermentation, respectively. At the end of fermentation, the SL concentration was 3.6 g/L for bagasse and 1.0 g/L for corn fiber hydrolysate. Among the three major sugars utilized by C. bombicola in the bagasse cultures, glucose was consumed at a rate of 9.1 g/L-day; xylose at 1.8 g/L-day; and arabinose at 0.98 g/L-day. With the addition of soybean oil at 100 g/L, cultures with bagasse hydrolysates, corn fiber hydrolysates and standard medium had a cell content of 7.7 g/L; 7.9 g/L; and 8.9 g/L, respectively after 10 days. The yield of SLs from bagasse hydrolysate was 84.6 g/L and corn fiber hydrolysate was15.6 g/L. In the same order, the residual oil in cultures with these two hydrolysates was 52.3 g/L and 41.0 g/L. For this set of experiment; in the cultures with bagasse hydrolysate; utilization rates for glucose, xylose and arabinose was recorded as 9.5, 1.04 and 0.08 g/L-day respectively. Surprisingly, C. bombicola consumed all monomeric sugars and non-sugar compounds in the hydrolysates and cultures with bagasse hydrolysates had higher yield of SLs than those from a standard medium which contained pure glucose at the same concentration. Based on the SL concentrations and considering all sugars consumed, the yield of SLs was 0.55 g/g carbon (sugars plus oil) for cultures with bagasse hydrolysates. Further, SL production was investigated using sweet sorghum bagasse and corn stover hydrolysates derived from different pretreatment conditions. For the former and latter sugar sources, yellow grease or soybean oil was supplemented at different doses to enhance sophorolipid yield. 14-day batch fermentation on bagasse hydrolysates with 10, 40 and 60 g/L of yellow grease had cell densities of 5.7 g/L, 6.4 g/L and 7.8 g/L, respectively. The study also revealed that the yield of SLs on bagasse hydrolysate decreased from 0.67 to 0.61 and to 0.44 g/g carbon when yellow grease was dosed at 10, 40 and 60 g/L. With aforementioned increasing yellow grease concentration, the residual oil left after 14 days was recorded as 3.2 g/L, 8.5 g/L and 19.9 g/L. For similar experimental conditions, the cell densities observed for corn stover hydrolysate combined with soybean oil at 10, 20 and 40 g/L concentration were 6.1 g/L, 5.9 g/L, and 5.4 g/L respectively. Also, in the same order of oil dose supplemented, the residual oil recovered after 14-day was 8.5 g/L, 8.9 g/L, and 26.9 g/L. Corn stover hydrolysate mixed with the 10, 20 and 40 g/L soybean oil, the SL yield was 0.19, 0.11 and 0.09 g/g carbon. Overall, both hydrolysates supported cell growth and sophorolipid production. The results from this research show that hydrolysates derived from the different lignocellulosic biomass feedstocks can be utilized by C. bombicola to achieve substantial yields of SLs. Based upon the results revealed by several batch-stage experiments, it can be stated that there is great potential for scaling up and industrial scale production of these high value products in future.