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.

The growth of Rhodotorula glutinis in sweet sorghum juice in three levels of three factors of temperature, carbon to nitrogen ratio and pH was evaluated. Accompanying of this growth was the generation of lipids converted to fatty acid methyl ester (FAMEs) and carotenoids. The optimized condition for maximum biomass and carotenoid accumulation was determined to be at 25C, pH of 5.5 and carbon to nitrogen ratio of 10. This condition yielded 22.7 g/L biomass with specific growth rate of 0.213 hr-1. At this condition the carotenoids generation was also maximum with 2.6 mg/gram biomass, comprising of torularhodin, beta-carotene and torulene. The accumulation of lipids following generation of biodiesel was highest at same temperature and pH but carbon to nitrogen ratio of 70, generating 96.3 mg of FAMEs/gram of biomass containing methyl ester of oleic acid, linoleic acid, palmitic acid, stearic acid and linolenic acid.

Sweet sorghum has potential as an energy crop in the Southwest since, compared to corn, it requires less fertilizer and water, is cheaper to grow, and requires less energy to process into ethanol. The purpose of this study is to determine the feasibility of obtaining two crops of sweet sorghum from a single seeding. Two cultivars of sweet sorghum were seeded at early and late dates at the Maricopa Agricultural Center in 2006. Two crops of sweet sorghum were obtained in our study with a short season cultivar Bundle King, but not with the longer season cultivar MMR 327/36. The ethanol yield of Bundle King of 213 gal/acre from two crops planted on April 7 was not significantly greater statistically than the ethanol yield of 162 gal/acre from a single crop planted on June 1. Bundle King is an inherently low yielding variety, as are most short season sweet sorghum cultivars that may be used for double cropping. Thus, the problem with double cropping is identifying a suitable cultivar along with increased harvest costs, despite the advantage of providing a more even supply of feedstock to an ethanol plant.

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%.

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 achieved 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%.

Doctor of Philosophy / Department of Agronomy / P. V. Vara Prasad / Sweet sorghum (Sorghum bicolor L. Moench) is an important bioenergy crop. There is a wide array of genetic diversity in sweet sorghum germplasm collections. However, information on traits associated with sugar yield, optimum harvesting time for maximum sugar yield, effects of abiotic stresses on sugar yield is scarce. The objectives of the present study were: to identify traits that are associated with sugar yield, to determine the optimum harvesting time for maximum sugar yield and to understand the physiological responses of different sweet sorghum genotypes to drought and high temperature. In order to meet these objectives, five independent field and greenhouse studies were conducted. Field experiments were conducted using 280 sweet sorghum germplasm and were evaluated for 2 years. From this study, 30 genotypes representing high and low sugar yielders were selected for the subsequent experiment. We observed a significant variation in physiological, morphological and sugar yield traits associated with biofuel production. In the selection experiment, investigations on the morphological, physiological attributes helped to identify those characters which influence or limit sugar yield in the sweet sorghum. Another field study was conducted to optimize the harvesting time for obtaining highest sugar and juice yields in sweet sorghum. Sweet sorghum variety M81E was harvested at ten growth stages. Our results suggest that the optimum time for harvesting of sweet sorghum cultivar M81E is between milk and hard dough stages when highest sugar yield was observed. Studies on different levels of water stress were studied under greenhouse conditions. Four sweet sorghum genotypes (Awanlek, Smith, Tracy and Wray) were subjected to three water stress treatments (100% pot capacity (PC); 70% PC and 30% PC) for 20 days at early seed filling (Milk) stage. The results showed that genotypes differed significantly for all growth and yield, biochemical and physiological traits. Severe water stress significantly decreased juice and sugar yields by decreasing net photosynthetic rate, transpiration rate, stomatal conductance and sucrose content in the stem juice. Genotypes Tracy and Wray produced significantly highest brix, stem fresh weight, juice and sugar yield under both irrigated and water stress conditions. In another greenhouse study, we quantified the effects of drought, high temperature, and their combinations on growth, physiology and yield of sweet sorghum genotypes. The same four genotypes above were subjected to four treatments, T1 - control, T2 - drought stress, T3 - high temperature stress and T4 - combination of drought and high temperature for 16 days after anthesis. The result showed that significant difference was observed for growth and yield traits, physiological traits and non-reducing and total sugar content in juice for genotypes and treatments. Among the genotypes Tracy recorded higher juice and sugar yield. Among the various treatments, combination of drought and high temperature was found to be more deleterious in reducing most of the biofuel traits followed by drought and high temperature stress. The above studies gave significant findings with regards to the identification of superior sweet sorghum germplasm, their tolerance capacity to different abiotic stresses, which allows better selection for the use of bioenergy production.

O etanol é utilizado como combustível desde o início do século XX, porém foi a partir da década de 1970 que sua utilização em larga escala foi concretizada pela primeira vez no mundo. Através do PROÁLCOOL foi estabelecido bases para sua produção, distribuição e comercialização. A cultura da cana-de-açúcar prevaleceu em relação às demais como mandioca e babaçu. Análises do potencial do sorgo sacarino também foram realizadas, mas devido ao desconhecimento desta cultura no Brasil não houve muito progresso na sua utilização. No início do século XXI, motivado por razões ambientais e estratégicas, surge o etanol brasileiro como exemplo de utilização de combustíveis alternativos aos derivados do petróleo. A aceitação do veículo com gerenciamento eletrônico para alimentação de combustíveis diferenciados, popularmente denominados de Flex, impulsionou o uso de etanol em território nacional chamando a atenção do mundo. A constatação de mudanças climáticas mundiais despertou a consciência do uso de hidrocarbonetos e suas conseqüências. A expectativa de um mercado mundial de etanol leva a procura de novas fontes de matérias-primas, uma vez que não se pode e não se deve plantar cana-de-açúcar em todos os lugares ou regiões do planeta. Surge o sorgo sacarino como uma das alternativas. Cultura milenar em vários paises demonstra grande potencial para produção de etanol, seguindo os mesmos procedimentos utilizados para cana, porém com menor ciclo de cultivo e menor necessidade hídrica e tolerância ao tipo de terra a ser cultivado. Seu aproveitamento é apoiado pela FAO em diversos paises, entre eles a China. A adoção por parte dos Estados Unidos do etanol em substituição ao metanol e as metas estabelecidas para a adição à gasolina nos próximos anos, provocou um acelerado aumento na produção de etanol, sendo esta baseada em milho. Diversos paises já se espelham nas experiências brasileiras para obtenção de maior independência energética. As necessidades para o abastecimento, dos mercados interno e externo, refletem as iniciativas de investimentos em novos projetos de novas usinas produtoras de etanol. Paises como China, Suécia, Japão já demonstraram amplo interesse na adoção do etanol como aditivo junto à gasolina. As pesquisas em novos sistemas de produção de etanol motivam instituições e empresas a uma busca acelerada para obtenção de processos mais rentáveis e economicamente viáveis. / Ethanol is being used as combustible since the beginning of century XX. However, since the decade of seventy it has been used in large scale in the world. The PROÁLCOOL program established bases for its production, distribution and commercialization. The culture of the sugar cane prevailed in relation to cassava and babaçu. Analyses of the potential of sweet sorghum had been also carried through, but due to the unfamiliarity of this culture in Brazil it did not have much progress in its use. At the beginning of century XXI, motivated for environmental and strategy reasons, Brazilian ethanol appears in the world scenario as an example of use of alternative fuels as substitutes for oil derivatives. The acceptance of vehicles with electronic management for differentiated fuel feeding, known as Flex cars, stimulated the use of ethanol in Brazil calling the attention the world. The knowledge of the worldwide climate changes brought the conscience of the use of hydrocarbons and its consequences. The expectation of a worldwide market of ethanol leads to the search for new sources of fuels. Since sugar cane cannot be planted all over the world due to climate differences, sweet sorghum appears as a promising alternative. Millenarian culture in several countries, it demonstrates a great production potential for the production of ethanol. The same procedures employed for sugar cane can be used. However, the sorghum crops require a lesser cycle of culture and minor water needs and tolerance when compared against sugar cane. Its exploitation is supported by FAO in several countries, being China among them. The adoption of ethanol the United States in substitution to methanol and the goals established for the addition to the gasoline in the next years, has been leading to the increase in the production of ethanol, manufactured from maize. Several countries already have been following the Brazilian path for the attainment of bigger energy independence. The necessities for the supplying of the domestic and external markets reflect the initiatives of investments in new projects of new producing plants of ethanol. Countries such as China, Sweden and Japan already had demonstrated a great interest in the adoption of ethanol as a gasoline additive. The research for new systems of production of ethanol motivates institutions and companies to search for the attainment of more income-producing and economically viable processes.

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.

Στην παρούσα εργασία μελετήθηκαν οι μεταβολές του ολικού φωσφόρου σε φυτά γλυκού σόργου (Sorghum bicolor (L) Moench) ποικιλίας Keller και του διαθέσιμου φωσφόρου στο έδαφος. Ακολουθήθηκαν δύο χειρισμοί διαχείρισης, ο βιολογικός και ο συμβατικός. Σκοπός της εργασίας ήταν να διαπιστωθεί εάν οι πρακτικές βιολογικής διαχείρισης της γονιμότητας του εδάφους που εφαρμόστηκαν ήταν επαρκείς για την κάλυψη των αναγκών του γλυκού σόργου σε φώσφορο, προκειμένου να επιτευχθούν αποδόσεις που να προσεγγίζουν τις αποδόσεις του συμβατικού τρόπου καλλιέργειας. Τα πειράματα πεδίου πραγματοποιήθηκαν σε αγρόκτημα στο χώρο του Πανεπιστημίου Πατρών (θέση: ΓΠ: 38° 25΄ Β, ΓΜ: 21° 8΄ Α) την περίοδο 2003-2004. Η συσσώρευση φωσφόρου από τα φυτά του γλυκού σόργου ακολούθησε σιγμοειδή μεταβολή με το χρόνο και στους δυο χειρισμούς όπως αναφέρεται στη βιβλιογραφία. Με τα φυτά του συμβατικού να προσλαμβάνουν λίγο περισσότερο φώσφορο. Η αύξηση της ξηρής βιομάζας ήταν επίσης σιγμοειδής μεταβολή με το χρόνο και στους δύο χειρισμούς, σύμφωνα με τη βιβλιογραφία. Οι τελικές αποδόσεις ήταν της τάξεως των 24,47 t/ha για το βιολογικό χειρισμό και 23,42 t/ha για το συμβατικό. Τιμές που βρίσκονται στα επίπεδα που αναφέρονται στη βιβλιογραφία για τη συμβατική καλλιέργεια του γλυκού σόργου στη νότια Ευρώπη. Επομένως ο τρόπος διαχείρισης του εδαφικού φωσφόρου δεν επηρέασε τις τελικές αποδόσεις σε ξηρή υπέργεια βιομάζα στην καλλιέργεια του γλυκού σόργου. Η βιολογική διαχείριση του φωσφόρου στην καλλιέργεια του γλυκού σόργου, σε έδαφος με επάρκεια φωσφόρου έδωσε ικανοποιητικές τελικές αποδόσεις βιομάζας. Ενώ οι επιπλέον προσθήκες φωσφορικών λιπασμάτων στη συμβατική διαχείριση πιθανόν να ήταν πλεονασματικές και να επιβάρυναν το περιβάλλον. / In the present work was studied the total phosphorus fluctuation of sweet sorghum plants (Sorghum bicolor (L) Moench, variety Keller) and the available phosphorus in the soil. Two treatment practices were followed, the biological and the conventional one. Aim of the study was to ascertain whether the applied soil fertility practices of the biological management were sufficient in terms of the crop’s phosphorus nutrition, in order to achieve yields approaching those of the conventional one. Field experiments were conducted at the Patras’ University farm (lat. 38o 25׳ B, long. 21o 8׳A) during the period 2003-2004. The phosphorus uptake of sweet sorghum plants followed a sigmoid variation with time at both treatments as mentioned in literature with plants under conventional treatment taking up slightly higher amounts of phosphorus. Dry matter growth followed the same sigmoid variation in time at both treatments, according to literature. The total yields reached 24.47 t/ha for the biological treatment and 23.42 t/ha for the conventional one. These values were within the levels reported in literature for conventional treatment of sweet sorghum in southern Europe. Thus, soil phosphorus management practices had no effect on total above the ground dry matter yields of sweet sorghum. Biological management of phosphorus in the cultivation of sweet sorghum in a soil sufficient in phosphorus gave satisfactory total dry matter yields. Further addition of phosphates during conventional treatment was possibly excessive and may have harmed the environment.