Dissertationen zum Thema „Xylitol Production“

Seventeen species of facultative anaerobic bacteria belonging to three genera (Serratia, Cellulomonas, and Corynebacterium) were screened for the production of xylitol; a sugar alcohol used as a sweetener in the pharmaceutical and food industries. A chromogenic assay of both solid and liquid cultures showed that 10 of the 17 species screened could grow on D-xylose and produce detectable quantities of xylitol during 24-96 h of fermentation. The ten bacterial species were studied for the effect of environmental factors, such as temperature, concentration of D-xylose, and aeration, on xylitol production. Under most conditions, Corynebacterium sp. NRRL B 4247 produced the highest amount of xylitol. The xylitol produced by Corynebacterium sp. NRRL B 4247 was confirmed by mass spectrometry. Corynebacterium sp. NRRL B 4247 was studied for the effect of initial D-xylose concentration, glucose, glyceraldehyde, and gluconate, aeration, and growth medium. Corynebacterium sp. NRRL B 4247 produced xylitol only in the presence of xylose, and did not produce xylitol when gluconate or glucose was the substrate. The highest yield of xylitol produced in 24 h (0.57 g/g xylose) was using an initial D-xylose concentration of 75 g/l. Under aerobic conditions the highest xylitol yield was 0.55 g/g while under anaerobic conditions the highest yield was 0.2 g/g. Glyceraldehyde in concentrations greater than 1 g/l inhibited Corynebacterium sp. B 4247 growth and xylitol production. Corynebacterium sp. NRRL B 4247 culture grown in the presence of potassium gluconate (96 g/l) for 48 h and on addition of D-xylose to the media increased accumulation to 10.1 g/l of xylitol after 150 h. Corynebacterium sp. NRRL B 4247 exhibited both NADH and NADPH-dependent xylose reductase activity in cell-free extracts. The NADPH-dependent activity was substrate dependent. The activity was 2.2-fold higher when DL-glyceraldehyde was used as substrate than with D-xylose. In cell-free extracts the difference in xylose reductase and xylitol dehydrogenase activity was highest at 24 h, whereas for cell cultures that were grown in gluconate and xylose, the difference in the reductase and dehydrogenase activities was highest at 12 h after xylose addition. The NAD+ dependent xylitol dehydrogenase activity was low compared to the cells grown without gluconate. The molecular weight of NADPH-dependent xylose reductase protein obtained by gel filtration chromatography was 58 kDa. Initial purification was performed on a DE-52 anion exchange column. Purification using Red Sepharose affinity column resulted in a 58 kDa protein on the SDS PAGE gel and was further purified on a Mono-Q column. The activity stained band on the native gel yielded 58, 49, 39 and 30 kDa bands on the denaturing gel. The peptides of the 58 kDa protein of Corynebacterium sp. B 4247 sequenced by mass spectrometry, identified with E2 and E3 (Bacillus subtilis) components of multi-enzyme system consisting of pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex and oxo-acid dehydrogenase complex. A 75% match was shown by the peptide â QMSSLVTRâ with E-value of 8e-04 to the Saccharomyces cerevisiae protein that was capable of reducing xylose to xylitol. The peptide â LLNDPQLILMEAâ had conserved match â LL + DPâ over several aldose reductases. The xylose reductase of the yeast Candida tropicalis ATCC 96745 was also purified. The molecular weight of the yeast NADPH-dependent xylose reductase was about 37 kDa on an SDS PAGE
Ph. D.

En vue de produire du xylitol a partir d'un hydrolysat hemicellulosique riche en xylose, nous avons tout d'abord caracterise et quantifie les reponses cinetiques de deux especes levuriennes candida guillermondii et candida parapsilosis, souches qui ont ete choisies pour leurs aptitudes a fermenter le xylose en xylitol. Cette etude, realisee en milieu synthetique et dans differentes conditions de culture, a abouti a la definition des conditions optimales pour la production de xylitol. Ainsi, avec candida guilliermondii, le rendement de production en xylitol atteint une valeur maximale de 0,67 g/g en presence de 300 g/l de xylose, a un ph de 6,0 et avec une vitesse de transfert d'oxygene de 2,2 mmol/l. H. Les conditions optimales de production de xylitol par candida parapsilosis (y#p#/#s = 0,75 g/g) sont obtenues lors de cultures realisees avec 100 g/l de xylose, a un ph initial de 4,75 et pour une vitesse de transfert d'oxygene de 0,4 mmol/l. H. En etudiant le comportement des deux souches dans un milieu reproduisant un hydrolysat hemicellulosique c'est-a-dire contenant principalement du glucose et du mannose, nous avons pu analyser l'influence de ces differents substrats sur la production de xylitol. Nous avons pu observer que la consommation du glucose en conditions aerobies par ces levures conduisait en fait a l'obtention d'une biomasse cellulaire nettement mieux adaptee pour produire du xylitol a partir de xylose. Nous nous sommes donc bases sur cette particularite pour ameliorer la fermentescibilite d'un hydrolysat hemicellulosique de peuplier. Outre cette approche physiologique, les reactions enzymatiques directement impliquees dans l'accumulation du xylitol ont ete etudiees. La comparaison de l'expression de la xylose reductase et de la xylitol deshydrogenase mesuree in vitro avec la production de xylitol mesuree in vivo a permis de souligner l'importance de l'equilibre de la balance d'oxydo-reduction dans une cellule

Ethanol from lignocellulosic biomass is a promising alternative to rapidly depleting oil reserves. However, natural recalcitrance of lignocelluloses to biological and chemical treatments presents major engineering challenges in designing an ethanol conversion process. Current methods for pretreatment and hydrolysis of lignocelluloses generate a mixture of pentose (C5) and hexose (C6) sugars, and several microbial growth inhibitors such as acetic acid and phenolic compounds. Hence, an efficient ethanol production process requires a fermenting microorganism not only capable of converting mixed sugars to ethanol with high yield and productivity, but also having high tolerance to inhibitors. Although recombinant bacteria and yeast strains have been developed, ethanol yield and productivity from C5 sugars in the presence of inhibitors remain low and need to be further improved for a commercial ethanol production. The overarching objective of this work is to transform Zymomonas mobilis into an efficient whole-cell biocatalyst for ethanol production from lignocelluloses. Z. mobilis, a natural ethanologen, is ideal for this application but xylose (a C5 sugar) is not its 'natural' substrate. Back in 1995, researches at National Renewable Energy Laboratory (NREL) had managed to overcome this obstacle by metabolically engineering Z. mobilis to utilize xylose. However, even after more than a decade of research, xylose fermentation by Z. mobilis is still inefficient compared to that of glucose. For example, volumetric productivity of ethanol from xylose fermentation is 3- to 4- fold lower than that from glucose fermentation. Further reduction or complete inhibition of xylose fermentation occurs under adverse conditions. Also, high concentrations of xylose do not get metabolized completely. Thus, improvement in xylose fermentation by Z. mobilis is required. In this work, xylose fermentation in a metabolically engineered Z. mobilis was markedly improved by applying the technique of adaptive mutation. The adapted strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, the strain produced a total of 9% (w/v) ethanol from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Investigation was done to identify the molecular basis for efficient biocatalysis. An altered xylitol metabolism with reduced xylitol formation, increased xylitol tolerance and higher xylose isomerase activity were found to contribute towards improvement in xylose fermentation. Lower xylitol production in adapted strain was due to a single mutation in ZMO0976 gene, which drastically lowered the reductase activity of ZMO0976 protein. ZMO0976 was characterized as a novel aldo-keto reductase capable of reducing xylose, xylulose, benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, but not glucose or fructose. It exhibited nearly 150-times higher affinity with benzaldehyde than xylose. Knockout of ZMO0976 was found to facilitate the establishment of xylose fermentation in Z. mobilis ZM4. Equipped with molecular level understanding of the biocatalytic process and insight into Z. mobilis central carbon metabolism, further genetic engineering of Z. mobilis was undertaken to improve the fermentation of sugars and lignocellulosic hydrolysates. These efforts culminated in construction of a strain capable of fermenting glucose-xylose mixture in presence of high concentration of acetic acid and another strain with a partially operational EMP pathway.

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碩士
大葉大學
食品工程研究所
87
Xylitol has multiple biological functions that render the sugar alcohol many potential applic-ations in the food industry. This research used the yeast, Candida subtropicalis C22 , isolated from sugar can bagasse to ferment xylose into xylitol. The strain produced mostly xylitol with very small amount of ethanol. Shaker flasks of working volume of 150ml were used for the study. The strain could produce 17.5% (w/v) xylitol with initial xylose concentration of 20% (w/v) within 9 days. The addition of surfactant (Triton X-100) was found to siguificantly speed up the fermentation,similar xylitol conc.(16% w/w) was achieved in 5 days. However, the yield was slightly decreased. The productivity was 0.0359g/hr/L/g dry cell. Key Words：Xylose、Xylitol、surfactant、Candida subtropicalis

碩士
國立雲林科技大學
工業化學與災害防治研究所碩士班
89
Production of xylitol by immobilized yeast cell Student：Kaun-Ben Chen Advisors：Dr. Wen-Chang Liaw Institute of Industrial Chemistry and Hazards Prevention National Yunlin University of Science & Technology ABSTRACT This study deals with production of xylitol from rice straw. Straw was first treated with sulfuric acid, the result indicates that the best condition for hydrolysis is using 2% H2SO4 and heat the straw at 126℃for 60min.Througth this treatment 13.3g of xylose can be obtained from 100g of rice straw. Xylose thus obtained is further decolorized with activated charcol and pH adjusted to remove the salts. Meanwhile, the yeast strain-Candida subtropicalis is immobilized and entrapped in the hydrophilic acrylic resin matrix using photopolymerization method. The immobilized cells are then used to ferment xylose to xylitol. As for immobilization with photo-crosslinking, the raw material used are acrylic monomer such as polyethylene glycol diacrylate ( PEG-DA) and 2-hydroxyethyl methacrylate (HEMA).To these substances were added with 1% Benzoin isopropyl ether(photo-semsitizer agent)and yeast cells. The membrarce thus formed has a thickness of 0.2mm and in the 10% culture medium it will product the highest amount of xylitol with a yield of 70%. The immobilized yeast cells are them treated batchwise for endurance. Result indicates that it is fairly stable for 1~2 months with a yield exceeds 60%.

Xylitol is a sugar alcohol that can replace sucrose, is tolerated by diabetics and has several clinical applications. Currently, xylitol is manufactured on a large scale through a chemical process, but there is a search for alternative processes that are environmentally friendly and more cost-effective. In this thesis, the biotechnological production of xylitol has been examined as an alternative to the chemical process, using the yeast Komagataella pastoris to produce xylitol from glucose/xylose mixtures. Different parameters, namely, pH, temperature, xylose concentration, the presence of arabinose as substrate and dissolved oxygen were tested. This work demonstrated that the best pH value for xylitol synthesis was 7.0 - 7.5 that resulted in 4.04 g/L of xylitol, with a volumetric productivity of 0.024 g/L.h and a specific productivity 0.35 gxylitol /gCDW. Cultivation with initial pH value 7.00 and 37 ºC, in fully aerobic conditions, and changing the pH to 6.4 at 72 h of cultivation, resulted in the highest xylitol concentration of this study: 12.00 g/L, concomitant with a volumetric productivity of 0.071 g/L.h and a specific productivity of 1.41 gxylitol/gCDW. Limiting oxygen conditions resulted in a xylitol concentration of 5.81 g/L, with a specific productivity of 0.33 gxylitol/ gCDW, and a volumetric productivity, of 0.035 g/L.h. Several concentrations of xylose in the glucose/xylose mixtures were tested. The highest xylitol production was obtained with 60 g/L of xylose. Reducing the xylose concentration resulted in lower xylitol production. Interestingly, it was observed for the first time that K. pastoris was able to synthesize arabitol using arabinose as substrate, with the arabitol concentration reaching 3.15 g/L on cultivation at 37 ºC under oxygen limitation. This feature opens up the possibility of using this process for the synthesis of both sugar alcohols, xylitol and arabitol, being apparently possible to tune the synthesis of each product by altering the cultivation conditions.

碩士
國立雲林科技大學
工業化學與災害防治研究所碩士班
89
Xylitol is a sugar alcohol which is widely distributed in nature and is also and intermediate of human or mammal metabolic function. Recently, it has drawn greet attention as it can be used as a sweetner in the diet for diabetics, besides, it can prevent dental caries. Some other advantages using xylitol include high solubility and high degree of sweetness. Previously xylitol was produced by chemical process which gave low yield and was difficult to recover it. Since 1980, production of xylitol was shifted to fermentation process since this method showed high yield, less by-product and fast production rate. This study utilizes Candida subtropicalis for xylitol production. Fermentation condition such as substrate concentration, nitrogen source and concentration, temperature, pH, cell concentration, and dissolved oxygen were studied in detail. After the best fermentation conditions were determined, fermentation was performed in a 4L fermentor to determine the optimum mixing rate, kLa, and the effect of dissolved oxygen concentration. The effect of fermentor operating conditions on xylitol yield, xylitol production rate and xylose consumption rate were also investigated. Optimum kLa value was obtained by varying aeration rate and impeller mixing rate in order to understand oxygen mass transfer rate on xylitol production. In batch fermentation conducted in a rotary shaker, production yield of 70% was achieved when temperature was controlled at 30-35℃ and pH was around 4-6. Yeast extract still is the best nitrogen source although 1% soy bean powder hydrolyzed by KOH at 121℃ can perform fairly well giving 62% yield. Initial cell concentration does not affect significantly on production yield. The best shaker speed found to be 150 rpm. As for fermentor operation is concerned, the mixing rate of 300 rpm and the aeration rate of 0.2 vvm can give the highest production rate with good xylitol yield. This culture is a microaerobic and prefers a kLa of 10-15 hr-1 for xylitol production.

碩士
大同大學
生物工程研究所
88
Xylitol (C5H12O5) is a sugar alcohol derived from the reduction of xylose (C5H10O5). There are several technologies available for xylitol production, which were solid-liquid extraction, chemical synthesis, and biotechnological methods. However, production of xylitol by microorganisms is more economic than others. Among 35 yeast strains isolated from natural sources, Yeast NO. 23, a Saccharomyces sp., was found to have the highest xylitol conversion yield, Y p/s = 0.68 (g/g). On the other hand, except CCRC 21945, the strains purchased from the Culture Collection and Research Center, with D-xylose consumption varied from 19 % to 80 %, accumulated low xylitol. The addition of methanol or ethanol substituting for glucose as energy source in the shaking culture of CCRC 21945 enhanced xylitol production. Whereas, the culture of Yeast NO. 23 was unaffected by the addition of methanol or ethanol. The optimum conditions of Yeast NO. 23 for fermentation of xylitol in stirred tank bioreactor were as follows: temperature, 30℃; stirring rate, 300 min-1; aeration rate, 0.02 vvm (KLa = 8.64 h-1); pH controlled above 5.0; the ratio of supplementary substrate, glucose : xylose = 1 : 3. Under these conditions, maximum xylitol production, xylitol conversion yield and volumetric productivity were 26.7 (g/l), 0.863 (g/g), and 0.477 (g/ liter h), respectively. Raising the concentration of total sugar in supplementary substrate was able to increase xylitol concentration in culture broth. But the more the concentration of total sugar the more fermentation time was required resulting in considerable amount of xylose remained in final culture broth. Therefore, it is necessary to develop a dual- or sequencing-bioreactor system to solve these problems.

碩士
大葉大學
環境工程學系研究所
105
Xylitol is a natural sugar alcohol, it can be used for food, medicine and health food, it is a natural sweetener. In this study, Candida guilliermondii BCRC 21559 and Candida tropicalis BCRC 21436 were used as experimental strains to optimize the production of xylitol. The optimum conditions for the production of xylitol from C. guilliermondii and C. tropicalis were studied by one factor at a time method. It was found that the optimum condition for C. guilliermondii was 100 rpm, 30 °C and pH 6, and for C. tropicalis it was 125 rpm, 40°C and pH 6. Xylitol production for C. guilliermondii was 10.18 g/L and for C. tropicalis, it was 25.03 g/L. In addition to one-factor-at-a-time method, response surface method (RSM) was also used to optimize the production of xylitol by C. tropicalis. In the 23 factorial design experiment, it was shown that the linear term of temperature has significant effect on xylitol production, Furthermore, it also indicated that the first order with interaction model (R2 was 0.997) was adequate. The optimal condition was then assessed by using a central composite design (CCD). However, it was found that the regression equation of the second-order response surface experiment can not fit the applicability of this model on the experimental data. To investigate the use of bagasse for xylitol production, 10g of bagasse powder was hydrolyzed in 100 ml, 0.25N H2SO4 at 131°C for 30min. The hydrolysate contained glucose 20 g/L and xylose 12 g/L was used for xylitol production, two sets of experiments were carried out ; In the first set, the commercial glucose 20 g/L was supplemented with xylose 12 g/L, 50 g/L, 75 g/L, and 100 g/L. respectively. In the second set, the bagasse hydrolysate (glucose 20 g/L, xylose 12 g/L) were supplemented with xylose to 12 g/L, 50 g/L, 75 g/L and 100 g/L, respectively. The medium of the two groups was cultured at pH 6, 125 rpm and 40 ℃ for 168h, the yields of xylitol were 5.7 g/L, 20 g/L, 20 g/L, 25.3 g/L and 0.8 g/L, 11.45 g/L, 13.2 g/L, 16 g/L, respectively. The results showed that the xylitol production was lower using bagasse hydrolysate than using commercial substrate even when glucose and xylose coutent were adjusted to the same. This might suggest there were ihhibitors of xylitol production present in the hydrolysate.

碩士
大同大學
生物工程研究所
89
Xylitol, a five-carbon sugar alcohol, was an anti-cariogenic substance and has the same sweetness of sucrose by weight. It could serve as a diabetic sweetener. About 70% chewing gums in Europe are xylitol-added. Fed-batch and continuous fermentation processes by Candida tropicalis CCRC 21436 and its mutant, M40, for the production of xylitol were investigated. Feeding glucose in fermentation process could enhance xylitol production. Intermittent glucose-feeding caused the decreasing in pH and ORP of the fermentation medium and after glucose in medium was used up, pH and ORP fluctuated. According to this property, pH-stat and ORP-stat glucose-feeding fermentation processes were carried out for the optimum production of xylitol from xylose. The processes of glucose-feeding fermentation using strain M40 via pH-stat at pH 5 and ORP-stat at -200 mv were compared. At hr 30 in the pH-stat fermentation at pH 5, 83 g xylitol was produced from 196 g xylose and xylitol conversion yield (xylitol produced/xylose consumed), xylose consumption, specific productivity were 74%, 57%, 0.065 g (xylitol)/g (cell dry weight)/hr, respectively. Whereas at hr 27 in ORP-stat fermentation at -200 mv, 105 g xylitol was produced from 196 g xylose and xylitol conversion yield (xylitol produced/xylose consumed), xylose consumption, specific productivity were 86%, 63%, 0.091 g (xylitol)/g (cell dry weight)/hr, respectively. The processes of ORP-stat fermentationat at ORP values of -100, -150, or -200 mv and at aeration rates of 0.2, 2, or 4 l/min were compared. The optimum production by Candida tropicalis CCRC 21436 was obtained at ORP of -150 mv and aeration rate of 0.2 l/min. At hr 28, 148 g xylitol was produced from 203 g xylose and xylitol conversion yield (xylitol produced/xylose consumed), xylose consumption, specific productivity were 75%, 98%, 0.095 g (xylitol)/g (cell dry weight)/hr, respectively. By the mutant M40, the optimum production was achieved under a condition with ORP of -150 mv and aeration rate of 2 l/min. At hr 28, 144 g xylitol was produced from 199 g xylose and xylitol conversion yield (xylitol produced/xylose consumed), xylose consumption, specific productivity were 74%, 98%, 0.105 g (xylitol)/g (cell dry weight)/hr, respectively. Both strains had the similar optimum productivities of xylitol via the ORP-stat fermentation under different conditions. In continuous process of fermentation, at a dilution rate of 0.022 hr-1, from hr 12 to hr 72, xylitol conversion yield (xylitol produced/xylose consumed), xylose consumption and xylitol productivity were in the ranges of 81-93%, 82-87% and 6.2-7.1 g/h, respectively. These results were better than those were obtained at the dilution rate of 0.044 hr-1.

Dissertação de mestrado em Biotecnologia
The transformation of the human diet in the last ten thousand years has led to an increase of the global average energy intake, translating into an upsurge of obesity and diabetes all over the world. One of the major problems with the current diet is the amount of sugars that it has. Efforts have been made to reverse the situation, such as the use of sugar substitutes in food products. These sweeteners can be artificially produced by biotechnology processes or naturally present in plants. Polyols are an example of the latter and xylitol represents a great alternative to sucrose due to having 40 % less calories than sucrose and its metabolism not inducing the release of insulin into the bloodstream. Additionally, the biotechnological production of xylitol may be accomplished with the use renewable lignocellulosic materials, such as agro-industrial residues. Their composition rich in cellulose and hemicellulose offers the possibility to utilize xylose as a precursor of xylitol. In this study, the xylitol production from hydrothermally pretreated corn cob was evaluated and investigated by simultaneous saccharification and fermentation (SSF) process using a recombinant Saccharomyces cerevisiae strain. In terms of pretreatment conditions, 25 % (w/w) of corn cob of solid loading in the autohydrolysis treatment presented the highest xylan recovery in liquid and solid phases. In this condition, the xylan was solubilized in the autohydrolysis liquor as xylose and xylooligosaccharides. Moreover, the enzymatic hydrolysis of whole slurry corn cob achieved a xylose final concentration of 53.8 g/L, showing an efficient conversion of xylooligosaccharides into xylose. SSF process was performed to evaluate and optimize several operational conditions, such as temperature, pre-saccharification, size of inoculum, enzyme loading and percentage of solids, for the production of xylitol. The results revealed that the optimal solid concentration was 6.76 % (w/w) and the enzyme loading was 24 FPU/g of substrate, with a potential production of 46.5 g/L of xylitol and 0.32 g/L/h of productivity. These optimized conditions were carried out to validate the proposed model and this assay produced 42.9 g/L of xylitol and 0.30 g/L/h of productivity, an error of 7.66 % and 6.66% of the theoretical values, which validated the model. Finally, complete detoxification of hydrolysate was carried out to improve xylitol productivity under previous optimized conditions. SSF process using detoxified autohydrolysis liquor and 22 g/L of wet cells resulted in 71.7 g/L of xylitol, 0.60 g/L/h of productivity and practically full conversion of xylose. Overall, in this study, the biotechnological conversion of xylose into xylitol was successful achieved.
A transformação da dieta humana nos últimos dez mil anos tem levado ao aumento da ingestão média global de energia, traduzindo-se num incremento da obesidade e diabetes por todo o mundo. Um dos maiores problemas da dieta atual é a quantidade de açúcares que possui. Esforços têm sido feitos para reverter a situação, como o uso de substitutos do açúcar em produtos alimentares. Estes adoçantes podem ser produzidos artificialmente por processos biotecnológicos ou naturalmente presentes nas plantas. Os polióis são um exemplo do segundo e o xilitol representa uma alternativa à sacarose devido a ter 40 % menos calorias que esta e ao seu metabolismo não induzir a libertação de insulina. Adicionalmente, a produção biotecnológica de xilitol tem sido atingida com o uso de resíduos agro-industriais, como materiais linhocelulósicos. A sua composição rica em celulose e hemicelulose oferece a possibilidade de utilizar xilose como um percursor do xilitol. Neste estudo, a produção de xilitol a partir de caroço de milho pré-tratrado hidrotermicamente foi avaliada e investigada por sacarificação e fermentação simultâneas (SSF) usando uma estirpe recombinante de Saccharomyces cerevisiae. Em termos de condições do pré-tratamento, 25 % (p/p) de caroço de milho durante a auto-hidrólise apresentou a maior recuperação de xilano nas fases líquidas e sólidas. Nesta condição o xilano foi solubilizado no licor da auto-hidrólise como xilose e xilo-oligossacáridos. Ainda, a hidrólise enzimática das duas frações atingiu uma concentração final de xilose de 53.8 g/L, mostrando uma eficiente conversão dos xilo-oligossacáridos em xilose. Um processo de SSF foi realizado para avaliar e otimizar várias condições operacionais, como a temperatura, a pré-sacarificação, a concentração do inóculo, de enzima e de sólidos, para a produção de xilitol. Os resultados revelaram que a concentração de sólido ótima foi 6.76 % (p/p) e de enzima foi 24 FPU/g de substrato, com uma produção potencial de 46.5 g/L de xilitol e 0.32 g/L/h de produtividade. Estas condições foram realizadas para validar o modelo proposto e este ensaio produziu 42.9 g/L de xilitol e 0.30 g/L/h de produtividade, um erro de 7.66 % e 6.66 % dos valores teóricos, o que valida o modelo. Finalmente, a destoxificação completa do hidrolisado foi feita para melhorar a produtividade de xilitol sob condições previamente otimizadas. Um processo de SSF usando licor destoxificado e com 22 g/L de massa fresca de células resultou em 71.7 g/L de xilitol, 0.60 g/L/h de produtividade e com praticamente conversão completa da xilose. De forma geral, neste estudo, a conversão biotecnológica de xilose em xilitol foi atingida com sucesso.

碩士
朝陽科技大學
應用化學系碩士班
95
Xylitol, a five carbon sugar alcohol, is one of valuable polyol sweeteners. It can use as a natural food sweetener, a dental caries reducer, and a sugar substitute for diabetics. In the past, xylitol was produced from xylose fermented by suitable microorganisms, but the cost of xylitol is expensive. Therefore, how to develop cheap materials to substitute xylose and maintain the reasonable yield efficiency of xylitol is a necessary research. In this study, Pichia caribbica isolated from soil and Candida guilliermondii collected from BCRC ( Bioresource collection and research center, Taiwan ) to compare the effects of environmental conditions on the productions of xylitol between the two strains and select suitable material to substitute xylose for producing xylitol. The experimental results showed the suitable conditions for P. caribbica and C. guilliermondii to ferment 15% xylose to produce xylitol were 35℃ and 30℃, 50-100 rpm and 100-150 rpm rotary speeds respectively. When the inoculation ratios of P. caribbica and C. guilliermondii suspensions to cultured medium raised from 1% ( v:v ) to 3% , the xylitol yield efficiencies could increase 0.09 g g-1 respectively. When xylose containing medium added with 10-20% ( v:w ) glucose and fermented with P. Caribbica the yield efficiency of xylitol increased 1.5 folds that of without adding glucose. Because the hydrolysate of white corn cob had the highest xylose concentration among the twelve organic materials hydrolyzed with 1% H2SO4, so it was selected for the candidate material of the research. 2.45% xylose was obtained when 50 g L-1 corn cobs was hydrolyzed with 1.5% sulfuric acid under 121℃, 1.1atm and 15 minute condition. After hydrolysis, the hydrolysate concentrated to 4 folds and passed through calcium saturated ion exchange resin to separate xylose from pigment and salt. The result showed when the flow rate of mobile phase was 3 mL min-1 , with loading 1mL each time at 45℃ operation temperature, the separation percentage of xylose could reach 63.25%. After passing through calcium saturated ion exchange the concentrated hydrolysate inoculated with P. caribbica and C. guilliermondii suspension could get 0.39 g g-1 and 0.48 g g-1 yield efficiency respectively.

Xylitol is a high value polyalcohol being used in pharmaceutical, hygiene, and food products due to its functional properties such as anticariogenic, antibacterial as well as low calorie and low glycemic properties. An alternative route for xylitol production is the biotechnological method in which microorganisms or enzymes are involved as catalysts to convert xylose into xylitol under mild conditions of pressure and temperature. This method is unlike the conventional chemical method that requires high pressure and temperature and results in low product yield. The goal of this research is to employ an integrated process using all fractions of an agro-industrial biomass (oat hull) for xylitol bioproduction, preferably in a repeated batch bioconversion process, with C. guilliermondii as the biocatalyst. Processes including hydrolysis, biomass delignification, hydrolysate detoxification using adsorption process, and finally free- and immobilized-cell bioconversions were employed in this study. The kinetics of acid-catalyzed hydrolysis of hemicellulose was investigated under mild conditions (temperature: 110ºC to 130ºC and catalyst (H2SO4) concentrations from 0.1 to 0.55 N) to determine the kinetic mechanism and generation of monosaccharides (xylose, glucose, and arabinose) as well as the microbial inhibitors consisting of acetic acid, furfural, and hydroxymethylfurfural (HMF) in the hydrolysate. A maximum recovery of 80% was attained for xylose as the main monosaccharide and the substrate for xylitol; its generation in the hydrolysate followed a single-phase 2-step kinetic mechanism similar to that of the HMF. However, a single-phase mechanism with no decomposition could describe the formation of arabinose, acetic acid, and furfural. Glucose generation followed a biphasic mechanism (fast and slow releasing) apparently with no decomposition. In the alkaline delignification of the hydrolysis byproduct (solid fraction) and the intact (crude) biomass, kinetic models based on biphasic mechanism consisting of bulk and terminal phases gave the best results and fit to the experimental data. In the bulk phase, where the temperature ranged from 30ºC to 100ºC, the reaction rate constant varied from 0.15 to 0.19 1/min for the intact biomass and from 0.25 to 0.55 1/min for the hydrolysis byproduct. According to the models, accelerated lignin removal with the increased operating temperature could be due to the shift of the process from the terminal phase to the bulk phase. The values obtained for the activation energies herein ( 33 kJ/mol) were less than the values reported in the literature for other lignocellulosic materials. The removal or reduction of the microbial inhibitors in the medium was carried out by activated carbon (adsorptive detoxification). According to the results using the Langmuir model with the activated carbon as the adsorbent, the maximum monolayer capacities of 341, 211, and 46 mg/g were obtained, respectively, for phenol, furfural, and acetic acid. Thermodynamic analyses indicated that the adsorption of the three abovementioned chemicals by the activated carbon was exothermic (enthalpy: H0), spontaneous (free energy: G0), and based on the affinity of the solute toward the adsorbent (entropy: S0). In the concentrated hydrolysate, the removal of phenols, as the main inhibitor, was very successful such that by activated carbon doses of 1.25%, 2.5%, and 5% (w/v) they could be reduced to 34%, 13%, and 3% of the initial concentration (8.7 g/l), respectively. During xylitol bioproduction process in the repeated batch mode using C. guilliermondii, variables of pH control, medium supplementation, and cell recycling proved to be more important than medium detoxification. Processes involving pH-controlled condition combined with nitrogen supplementation and a mild detoxification performed very well with consistent conversion parameters in the successive batches; values of over 0.8 g/g, 0.55 g/l/h, and 53 g/l were obtained respectively for xylitol yield, volumetric productivity, and final concentration. On the other hand, in a single-batch bioconversion, there was no need for supplementing the medium with the nitrogen source. Kinetic modeling of the process showed that substrate (xylose) as well as co-substrate (glucose) consumption, product (xylitol) formation, and cell regeneration could be predicted by a diauxic model. In the aerated free-cell and immobilized-cell systems, aeration rates of 1.25 vvm and 1.25-1.5 vvm were required for free-cell and immobilized-cell systems, respectively, to reach the maximum bioconversion performance. In the immobilized-cell system, cell support also played an important role in this biotransformation. Application of the support based on the delignified hydrolysis byproduct resulted in high and consistent bioconversion parameters in all batches comparable to the ones in the free-cell system. However, bioconversions using the lignin-rich material (hydrolysis byproduct) resulted in a lower efficiency in the first batch which could be partly improved in the second batch and almost fully increased in the third batch to nearly reach performance parameters comparable to the ones obtained in the free-cell system. Overall, the integrated process employed in this investigation helps fill in the knowledge gaps existing on the lignocellulosic biomass application for xylitol bioproduction and biorefinery industries.

碩士
朝陽科技大學
應用化學系碩士班
92
In the research, the yeast, Candida subtropicalis, isolated from bagasse was used to ferment xylose into xylitol. Then, the response surface methodology was used to optimise xylitol production by Candida subtropicalis in the flask culture. The factors selected, among twelve factors studied, for optimization were concentrations of xylose, glucose, and peptone. In addition, analyses of variance indicated that the quadratic terms of xylose, glucose, and peptone in the obtained quadratic model were significant. The analysis of variance also indicated that the only significant interaction term was xylose-peptone. The optimized composition of culture medium obtained from response surface methodology ( RSM ) for the shaker-flask experiments was: 15.30 % ( w/v ) xylose, 0.58 % ( w/v ) glucose and 0.77 % ( w/v ) peptone. Using this composition, the xylitol production was 10.55 %, which was very close to that predicted by the quadratic model ( 10.77 % ).

碩士
大葉大學
生物產業科技學系
93
Abstract Recently, it was cared by the most population that the functionality in food including the nutrient balance, the promotion in body, and so on. Xylitol, because of its taste of dew likes peppermint, low quantity of heat, the sweetening equals to the sucrose, and anti-cariogenic properties, has been appreciated gradually in the food of new generation. Otherwise, the agricultural residual production abounds with cellulose, hemicellulose, and lignin. When the hemicellulose was hydrolyzed, abundant raw materials like xylose, glucose, and few other sugars (e.g., galactose, mannose, and arabinose) was productive. Hence, the subject of several investigates on the fermentation process for productive of xylitol was discussed the liquid of the lignomicellulose hydrolyzed as the stock. Therefore, it not only reduces cost but also exploits nature resource by the sufficient disposal. In this study, in order to increase the yield and productivity of xylitol in ferment process by yeast, a two-stage fermentation that employ difference concentrations of dissolved oxygen was proposed due to the phenomenon of the diauxic growth when two kinds of main sugar (xylose and glucose) as substrate was used. Results explained that it is increased in xylitol’s yield and productivity when the two composite substrates were applied. According to the object of increasing the biomass yield at first-stage fermentation, the dissolved oxygen was 5~10% for glucose metabolism (μ = 0.356 hr-1). Moreover, at second-stage, metabolism of D-xylose into xylitol, the conditions of the fermentation were 0.25vvm and 130 rpm for the highest yield (0.649 g g-1) and productivity (0.263 g L-1 hr-1) of the xylitol. Based on above of results, an operation procedure that is series connection the fed-batch culture and batch culture was designed. Results of the experiment displayed that xylitol’s yield (0.246 g L-1 hr-1) had been increased effectively. Key world: two-stage fermentation, xylitol, dissolved oxygen, lignocellulose hydrolyzates, and diauxic growth.

Xilitol e arabitol são álcoois de açúcar naturais usados como alternativas à sucrose e adoçantes artificiais, que pertencem à lista de compostos de valor acrescentado a serem produzidos a partir de biomassa de baixo custo. As suas ótimas propriedades e benefícios para a saúde têm atraído a atenção das indústrias alimentares e farmacêuticas, mas as suas aplicações continuam limitadas pelo preço e baixa disponibilidade. O objetivo principal desta tese foi avaliar, pela primeira vez, a capacidade de levedura Komagataella pastoris DSM 70877 para produzir xilitol e arabitol utilizando resíduos lignocelulósicos como substrato, particularmente, cascas de banana, dreche, carolos de milho, bagaço e engaço de uva e serradura. Uma via biotecnológica promissora e alternativa à produção química em larga escala. A primeira parte do trabalho focou-se na obtenção de licores ricos em açúcares e fermentáveis, a partir dos diferentes resíduos lignocelulósicos, submetendo-os a uma hidrólise ácida diluída. Na dreche, engaço de uva e serradura revelou-se uma maior recuperação de açúcares, originando hidrolisados com um teor total de monossacarídeos entre 12.9 e 21.5 g/L e diferentes rácios de glucose, xilose e arabinose. Os inevitáveis subprodutos tóxicos do processo foram também detetados nos hidrolisados, nomeadamente, furfural, 5-HMF e ácido acético, e, portanto, na tentativa de os reduzir/eliminar recorreu-se a tratamentos com carvão ativado. Na segunda parte deste trabalho, os hidrolisados foram testados como substratos para o cultivo de K. pastoris e produção de álcoois de açúcar. Entre os ensaios em Erlenmeyer, o hidrolisado da dreche levou à maior produção de xilitol, 3.97 g/L, com um rendimento de xilitol/xilose de 0.47 g/g. Ensaios em bioreator também foram realizados, em batch e fed-batch, testando diferentes substratos e condições de arejamento. Os substratos selecionados foram dreche, cascas de banana, mistura de dreche (ou engaço de uva) com alimentação de serradura e ainda, uma mistura de três substratos: dreche (ou engaço de uva) e cascas de banana com alimentação de serradura. A levedura alcançou uma produção máxima de xilitol de 1.33 g/L (rendimento 0.18 gxilitol/gxilose) num ensaio em batch, com uma taxa de fluxo de ar de 0.5 L/min e usando como única fonte de carbono o hidrolisado de dreche concentrado 2 vezes e destoxificado. Apesar das baixas produções alcançadas, neste trabalho provou-se o potencial da levedura para produzir xilitol a partir de hidrolisados hemicelulósicos. Assim, este estudo terá de ser validado e aprofundado para desenvolver e otimizar o processo. Curiosamente, percebeu-se que a cultura estava a canalizar parte do carbono fornecido pelos hidrolisados hemicelulósicos para outras vias metabólicas e/ou produção de outros compostos, como detetado pela análise de HPLC. Abre-se assim a possibilidade da levedura K. pastoris ser capaz de produzir compostos de grande interesse e valor comercial.
Xylitol and arabitol are natural sugar alcohols used as alternatives to sucrose and artificial sweeteners, that belong to the list of value-added compounds to be produced from low-cost biomass. Their great properties and health benefits have attracted the attention of food and pharmaceutical industries, but their applications are still limited by cost and lack of availability. The main goal of this thesis was to evaluate, for the first time, the ability of the yeast Komagataella pastoris DSM 70877 to produce xylitol and arabitol by using lignocellulosic waste materials as feedstocks, namely, banana peels, brewers’ spent grains (BSG), corncobs, grape pomace, grape stalks and sawdust, a promising biotechnological route as an alternative to the chemical large-scale production. The first part of this work was focused on obtaining sugar-rich and fermentable liquors from different lignocellulosic waste materials, by subjecting them to dilute acid hydrolysis. The higher sugar recoveries were achieved for BSG, grape stalks and sawdust, leading to hydrolysates with total monosaccharides’ contents between 12.9 and 21.5g/L, with different ratios of glucose, xylose and arabinose. The inevitable toxic by-products generated were also detected in the hydrolysates, furfural, 5-HMF and acetic acid, and attempts were done to reduce/eliminate them by treatment with activated charcoal. In the second part of this work, the hydrolysates were tested as substrate for cultivation of K. pastoris and sugar alcohols production. Within shake flask assays, BSG hydrolysate attained the highest xylitol production of 3.97 g/L, with a xylitol/xylose yield of 0.47 g/g. Bioreactor cultivations were also performed in batch and fed-batch modes, testing different feedstocks and aeration conditions. The selected feedstocks were BSG, banana peels, a mixture of BSG (or grape stalks) with feeding of sawdust and triple mixture of BSG (or grape stalks) and banana peels with feeding of sawdust. The yeast achieved a maximum xylitol production of 1.33 g/L (yield of 0.18 gxylitol/gxylose) in a batch cultivation with an airflow rate of 0.5 L/min, using twofold concentrated and detoxified BSG hydrolysate as the sole carbon source. Despite the low productions achieved, this work proved the yeast potential to produce xylitol from hemicellulosic hydrolysates. Thus, further research is required in order to develop and optimize the process. Interestingly, it was noticed that the culture was channeling part of the carbon provided by the hemicellulosic hydrolysates into other metabolic pathways and/or different products, as detected by HPLC analysis. This opens the possibility of K. pastoris to be able to produce other compounds of great interest and commercial value.