Дисертації з теми "Fermentation – Productivité"

The development of insecticides resistance among many insect species and the ecological damage occasionally caused by the lack of specificity in the toxic effects of insecticides have provided the impetus to seek alternative methods of insect control. This observation led to the development of bioinsecticides based on the insecticidal action Bacillus sphaericus (Bs), Bacillus turingiensis (Bt). The discovery of biolarvicidal actions of Bacillus thuringiensis and Bacillus sphaericus opened a new perspective for insect control. In the first part of the study was initiated to determine a suitable fermentation medium formulation and optimal fermentation conditions for large scale, low cost production of Bs. Bs 2362 was tested in whey and soy flour based media. These media was reformulized form of NYSM (Nutrient Broth Yeast Extract Sporulation Medium). Soy flour based medium, SYSM, gave the promising results in terms of cell yield, sporulation frequency and toxin production. In the second part of the study, fermentation productivity anlaysis of a local isolate Bacillus thuringiensis subsp. kurstaki 81 was evaluated. In order to compare different C:N ratios (1:1, 2:1, 4:1, 8:1, 10:1 20:1 and 30:1) of YSM medium. Btk 81 were run for 72 h and cell growth, sporulation and toxin protein profile of Btk 81 were determined for each. When all the quantitative toxin data for both glucose and sucrose varying C:N ratios were compared, it was determined that the crystal protein concentrations had the highest value in sucrose based medium when C:N ratio was 10:1. Regulation by C:N ratio of crystal protein biosynthesis was investigated for improving the production of this protein by our third candidate strain Bacillus thuringiensis subsp. israelensis ONR60. The experiments were performed by using TBL medium, at three different C:N ratios, 2:1, 4:1 and 8:1 respectively. In view of the cell growth characteristics and bioassy results, TBL medium designed with 2:1 C:N ratio was chosen as the best for further steps. In addition, running time of the culture determined as 60 hours as was also determined in the previous experiment. As the last step of this study, the pre-determined optimal conditions were applied to a 30L batch type fermentor for toxin production by using Bacillus thuringiensis subsp. israelensis ONR60. Unfortunately, the toxicity was not satisfactory, being much below the level of that expected as based on the results of the laboratory scale studies.

Three Brazilian yeast strains, Saccharomyces cerevisiae 42 - F, Saccharomyces cereviaiae PLA 851 and Saccharomyces boulardli IZ 1904, all currently employed in the sugar fermentation industry, were evaluated with respect to their thermal tolerance and alcohol production kinetics. Best performance was found in Saccharomyces cerevisiae PLA 851 at temperatures up to 40 degrees (a common fermentation temperature in the Brazilian industry). This strain was further evaluated in chemostatic growth under sucrose limitation with biomass feedback on a 1 Litre scale in a specially constructed apparatus. At 30 degrees and 35 degrees under a dilution (growth) rate of 0.1 /h ethanol productivity increased by a factor of 2 with feedback and at 40 degrees by a factor of 3. The feedback factor (Beta) was 0.9. PLA 851 cells, heat - shocked at 45 degrees, resulted in a greater biomass productivity subsequently at 40 degrees coupled with a change in cell morphology. Highest ethanol productivity was found with 10% initial sucrose concentration at a dilution rate of 0.25 /h with feedback. Saccharomyces cerevisiae PLA 851 appears to be well adapted to the harsh physiological conditions in alcohol fermentations as currently practiced in Brazil.

Les experimentations ont porte sur deux filieres distinctes de traitement des eaux usees urbaines: 1) fermentation anaerobie+lagunage a microphytes+pisciculture; 2) fermentation anaerobie+lagunage a macrophytes ou sont cultivees des jacinthes d'eau+pisciculture. Le traitement primaire a ete optimise par des essais a differents temps de sejour hydraulique en reacteur anaerobie a 25c dans un reacteur de 5 m#3 equipe de support de biomasse a base de fibres synthetiques regroupees en pompons. Des abattements significatifs en dco ont ete observes. Des taux de croissance de 0,6 a 0,8 g/jour ont ete obtenus sur un elevage de tilapias dans les eaux epurees

This study aimed to evaluate the influence of concentrations of sugars in the must, on the performance of ethanol fermentation conducted in fed batch with variable flow of power to define the best concentrations of ART in the must (juice, molasses and mixed) that lead to improved efficiencies and productivity in ethanol fermentation.In the preparation of mash mix were used the following proportions (20% molasses + 80% broth, 40% molasses + 60% broth, 50% molasses + 50% broth, 60% molasses + 40% broth, 80% molasses + 20% broth ). The profile power was declining, varying the flow rate from 0.75 to 0.25 Lh-1, with time filling the fermenter 3 hours for all tests, ranging from 30 to 30 minutes to feed flow wort in fermenter 4L workload (3 liters of wine and 1 liter of inoculum), evaluating diferente concentrations of ART in three types of wine studied. We evaluated performance parameters such as fermentation and process efficiencies and productivity in ethanol. Musts were quantified in pH, sulfuric acid, Brix and ART.In the middle fermented (wine), pH, acidity, residual sugar and ethanol content and quantity of cells. The kinetic profile was defined by quantifying the concentrations of cells, substrate and ethanol (in 1 hour). The figures in this study as a starting point for industrial use are 16 to 18 Brix (ART 114.25 to 125.86 g / L), 16 to 18 ° Brix (ART 127.70 to 141.24 g / L) and around 16 ° Brix (ART 113.68 g / L to 123.30), respectively, for juice of molasses, juice and mix (juice + molasses).The fermentation efficiencies were 77.17 to 90.30% for grape juice, from 74.4 to 86.51% for wine and mixed wine from 61.84 to 84.06 for molasses. Yields were obtained from 6.85 to 8.21 g / Lh for wine broth, 5.90 to 7.77 g / Lh for wine mixed and 4.04 to 6.72 g / Lh for grape molasses. These tracks serve to subsidize recommended as a starting point, the conduct of industrial ethanol fermentation conducted in fed batch with variable flow supply, since the conditions for conducting the tests, as well as the raw materials used in the preparation of musts were similar to those used industrially.
Fundação de Amparo a Pesquisa do Estado de Alagoas
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Este estudo objetivou avaliar a influência das concentrações de açúcares nos mostos, sobre o desempenho da fermentação etanólica conduzida em batelada alimentada com vazão variável de alimentação, para a definição das melhores concentrações de ART nos mostos (de caldo, de melaço e misto) que conduzam a melhores eficiências de fermentação e produtividade em etanol. Na preparação do mosto misto foram utilizadas as seguintes proporções (20% melaço + 80% caldo, 40% melaço + 60% caldo, 50% melaço + 50% caldo, 60% melaço + 40% caldo, 80% melaço + 20% caldo). O perfil de alimentação foi decrescente, variando-se a vazão de 0,75 a 0,25 L.h-1, com tempo de enchimento do fermentador de 3 horas para todos os ensaios, variando-se de 30 em 30 minutos a vazão de alimentação de mosto, em fermentador de 4L de volume de trabalho (3 litros de mosto e 1 litro de inoculo), avaliando-se diferentes concentrações de ART nos 3 tipos de mosto estudados. Foram avaliados parâmetros de desempenho, como eficiências fermentativa e de processo e produtividade em etanol. Nos mostos foram quantificados pH, acidez sulfúrica, Brix e ART. No meio fermentado (vinho), pH, acidez, Açúcares Residuais e teor de etanol e quantidade de células. O perfil cinético foi definido, quantificando-se as concentrações de células, substrato e etanol (em intervalos de 1 hora). Os valores indicados neste estudo, como ponto de partida para utilização industrial, são Brix de 16 a 18 (ART 114,25 a 125,86 g/L), de 14 a 18 °Brix (ART de 112,90 a 141,24 g/L) e próximo de 16 °Brix (ART de 113,68 g/L a 123,30), respectivamente para mostos de melaço, caldo e misto (caldo + melaço). As eficiências de fermentação foram: 77,17 a 90,30%, para mosto de caldo, 74,4 a 86,51% para mosto misto e 61,84 a 84,06 para mosto de melaço. As produtividades obtidas foram 6,85 a 8,21g/L.h, para mosto de caldo, 5,90 a 7,77g/L.h para mosto misto e 4,04 a 6,72g/L.h para mosto d melaço. Estas faixas recomendadas servem para subsidiar, como ponto de partida, a condução da fermentação etanólica industrial conduzida em batelada alimentada com vazão variável de alimentação, visto que as condições de condução dos ensaios, assim como as matérias-primas utilizadas na preparação dos mostos, foram semelhantes às utilizadas industrialmente.

Doctor of Philosophy
Department of Grain Science and Industry
Praveen V. Vadlani
Microbial lipids derived from oleaginous yeasts are a promising alternative source of edible oils due to the following advantages: no requirement of broad lands; availability of year-round production; and no food versus fuels controversy. Oleaginous yeast has an inherent ability to accumulate lipids inside cells and their lipids are preferable as starting materials in oleo-chemical industries because of their distinct fatty acid composition. Lignocellulosic biomass is a promising substrate to supply carbon sources for oleaginous yeast to produce lipids due to the high content of polysaccharides and their abundancy. Lignocellulosic-based sugar streams, which can be generated via pretreatment and enzymatic hydrolysis, contained diverse monosaccharides and inhibitors. The major objectives of this study were: 1) to develop a novel purification method to generate clean sugar stream using sorghum stalks after acid pretreatment; 2) to optimize fermentation conditions for Trichosporon oleaginosus to achieve high yields and productivity of microbial lipids using lignocellulosic hydrolysates; 3) to investigate the potentials of sorghum stalks and switchgrass as feedstocks for microbial lipid production using oleaginous yeast strains, such as T. oleaginosus, Lipomyces starkeyi, and Cryptococcus albidus; 4) to develop an integrated process of corn bran based-microbial lipids production using T. oleaginosus; and 5) to develop bioconversion process for high yields of lipids from switchgrass using engineered Escherichia coli. In our investigation, major inhibitory compounds of lignocellulosic hydrolysates induced by pretreatment were acetic acid, formic acid, hydroxymethyl furfural (HMF) and furfural. The activated charcoal was effective in removing hydrophobic compounds from sorghum stalk hydrolysates. Resin mixtures containing cationic exchangers and anionic exchangers in 7:3 ratio at pH 2.7 completely removed HMF, acetic acid, and formic acid from sorghum stalk hydrolysates. T. oleaginosus was a robust yeast strain for lipid production. In the nitrogen-limited synthetic media, total 22 g/L of lipid titers were achieved by T. oleaginosus with a lipid content of 76% (w/w). In addition, T. oleaginosus efficiently produced microbial lipids from lignocellulosic biomass hydrolysates. The highest lipid titers of 13 g/L lipids were achieved by T. oleaginosus using sorghum stalk hydrolysates with a lipid content of 60% (w/w). L. starkeyi and C. albidus also successfully produced microbial lipids using lignocellulosic hydrolysate with a lipid content of 40% (w/w). Furthermore, corn bran was a promising feedstock for microbial lipid production. The highest sugar yields of 0.53 g/g were achieved from corn bran at the pretreatment condition of 1% acid and 5% solid loading. Microbial lipids were successfully produced from corn bran hydrolysates by T. oleaginosus with lipid yields of 216 mg/g. Engineered E. coli also effectively produced lipids using switchgrass as feedstocks. E. coli ML103 pXZ18Z produced a total of 3.3 g/L free fatty acids with a yield of 0.23 g/g. The overall yield of free fatty acids was 0.12 g/g of raw switchgrass and it was 51 % of the maximum theoretical yield. This study provided useful strategies for the development of sustainable bioconversion processes for microbial lipids from renewable biomass and demonstrated the economic viability of a lignocellulosic based-biorefinery.

L'etude cinetique et physiologique de la conversion du glycerol en dihydroxyacetone par gluconobacter oxydans montre que cette fermentation est soumise a une double inhibition. L'effet inhibiteur du substrat se manifeste par une diminution des cinetiques de croissance et de production avec l'augmentation de la teneur en glycerol, modelisee par des lois de type exponentiel. Cet effet est essentiellement lie a l'abaissement de l'activite de l'eau par les concentrations elevees en substrat. L'inhibition par la dha se manifeste tout d'abord par une diminution des cinetiques de croissance et d'oxydation du substrat (faibles concentrations) puis par un blocage du developpement cellulaire et de la synthese de dha (concentrations elevees). Ces phenomenes, modelises par des lois de levenspiel, ont ete attribues a la forte reactivite de la dha avec les enzymes cytoplasmiques et impliques dans le transport du glycerol et avec la glycerol deshydrogenase membranaire, responsable de la formation de dha. Un procede bi-phasique mono-etage, integrant ces contraintes biologiques, a alors ete mis au point, permettant d'ameliorer les performances fermentaires de cette transformation. Le metabolisme fermentaire du glycerol a ete aborde sous un angle microbiologique. Deux micro-organismes responsables de la degradation du glycerol au sein d'une flore microbienne anaerobie ont ete isoles et caracterises: clostridium butyricum cncm 1. 1211, qui transforme le glycerol en 1,3-propanediol et butyrate avec des rendements de conversion respectifs de 0,66 et 0,13; enterobacter agglomerans cncm 1. 1210, espece nouvellement decrite pour ses capacites d'assimilation du glycerol et de conversion de ce substrat en acetate et 1,3-propanediol (rendements respectifs de 0,25 et 0,7). La preponderance du role joue par le systeme accepteur final d'equivalents reducteurs sur la regulation du metabolisme fermentaire quelle que soit l'espece microbienne etudiee a egalement ete definie

碩士
臺中師範學院
環境教育研究所
90
The conventional anaerobic wastewater treatments are able to deal with various types of organic wastewater and able to recovery biogas, which is mainly methane. Based on the regulation of the Framework Convention On Climate Change, methane is the next item to be put under control due to its greenhouse effect. Hydrogen produced by anaerobic fermentation can be used as an energy source with no greenhouse effect and therefore, become a highly potential technique with a great commercial market. After considering the physiological characteristics and growth condition of anaerobic hydrogenic bacteria, this study has designed 4 typical reactors suitable for anaerobic hydrogenesis , including Sludge recycling reactor, Continuous flow stirred tank reactor (CSTR), Non-mixing conventional reactor, and Plug flow reactor. The hydrogen productivity and wastewater treatment efficiencies of these 4 reactors were compared to evaluate which is the best for anaerobic production of hydrogen. The best operation condition of each reactor was also determined. As the results shown, within the influent COD concentration being 2,000-15,000 mg/L and HRT being 6-24 hrs, the hydrogen productivity is the best with an organic loading of 60 kg-COD/m3×d. Among these 4 reactors, continuous flow stirred tank reactor is the most appropriate for anaerobic fermentation hydrogenesis with a hydrogen productivity of 150 ml/g-CODre and a hydrogen productivity of per unit reactor of 774 L-H2/m3×d（1atm,25℃）, followed by non-mixing conventional reactor with a hydrogen productivity of 129 ml/g-CODre and a hydrogen productivity of per unit reactor of 646 L-H2/m3×d（1atm,25℃）. Only at a higher organic loading, sludge recycling reactor has a higher efficiency of hydrogen production due to the higher total production with a hydrogen productivity of 74.9 ml/g-CODre（1atm,25℃） and a hydrogen productivity of per unit reactor of 1100 L-H2/m3×d（1atm,25℃）. The hydrogen productivity of each reactor was increased with the increase of organic loading and the decrease in HRT, furthermore, the effect of HRT was more significant. At an organic loading of 2 kg-COD/m3×d, all reactors have the best COD removal, ranging from 54.0 to 60.1%, due to the highly methanation at the low organic loading. At an organic loading of 60 kg-COD/m3×d, all reactors have the worst COD removal, ranging from 9.0 to 19.0%. The COD removal of each reactor was decreased with the increase in organic loading and the decrease in HRT. From the observation under the fluorescent microscope and electron microscope, it was found that yellow-orange fluorescence was emitted by a large amount of Clostridium bacteria in biomass while the anaerobic fermentation hydrogenesis was good. However, blue fluorescence, which indicated a highly methanation, was emitted while the anaerobic fermentation hydrogenesis was poor. These results may be helpful in determining the efficiency of anaerobic fermentation hydrogensis .

碩士
國立成功大學
環境工程學系
103
Selective fermentation realized by invertase-defective yeasts that convert only the reducing sugars in a mixed saccharide (e.g. sugarcane juice) into ethanol is an emerging process technology in sugarcane industry. This technology opens possibilities in stabilization and enhancement of total productivity of sugar and ethanol, as productive and stronger cultivars that have higher content of reducing sugar becomes a potential raw material in sugar mills. To trigger the system-wide innovation of this technology, the changes in stability and enhancement of productivity must be described by changes in cultivars and cropping schedules. Here, a descriptive model developed in this study highlights consequences of introduction of selective fermentation technology considering a given scenario on choice of cultivars and cropping schedules. Moreover, utilizing a prototype database, design of scenarios based on optimization techniques are demonstrated. The results from demonstrative scenario design indicate the potential advantages of selective fermentation technology in combination with a cane cultivar with high yield, high biomass and reducing sugar content on Tanegashima Island of Japan. The study also indicates the new requirement on data from sugarcane cultivation, such as a wider range of growth profiles (stalk weight, composition), growth and harvest observations of perennial ratoon and rate of physical damage by typhoon by varied rationing months. Future directions of study including directions in enhancement of the model and database are discussed.

碩士
大葉大學
生物產業科技學系
99
To avoid grain prices rising and causing inflation, production of the biomass fuel alcohol of the second era is mostly abandoned grain crops, and used the non-grain crops of abounded biomass cellulose or agriculture waste as raw materials. The saccharides that biomass fiber through preprocessing hydrolysis and producing are used by organism methods for fermentation to get alcohol, and the saccharides of the kind of raw materials obtained possess the two classes of five carbon and six carbon. In order to amply use these reducing sugars, developing co-fermentation process is an important topic. The bacteria strains that are able to ferment five carbon saccharides are quite rare in the nature world, and therefore using gene recombination to develop new bacteria strains is an important work. Besides the fermentation rate of the five carbon saccharides is quite slow, and therefore the rising of efficiency is also an important research. This research is according to the earlier established Pichia stipitis fermentation model, and analyzing the efficiency problem of two-tank continuous fermenting glucose and xylose the mixture solution to produce alcohol. The used substrate sources of two-tank continuous fermentation, in accordance with preprocessing, can be classified as 50 g/L pure glucose and the mixture solution of 8 g/L glucose/24 g/L xylose, the two sorts. Therefore, this research is designed that tank-one and tank-two are particular fed one kind of substrates, and the first tank is fed two kinds of substrates at the same time, et cetera, many kinds of operating method, to confer ethanol production rate, ethanol produced ratio, and substrate used ratio, et cetera, the variation under respective sorts of combination, and analyze the ethanol production rate. When comparing two-tank continuous fermenting and one-tank continuous fermenting, two-tank ethanol production rate is not certainly higher, but substrate used ratio is higher. For example of pure glucose fed, one-tank ethanol production rate in dilution rate 0.06 1/hr is 0.24 g/L/hr, and substrate used ratio is 0.265; and two-tank ethanol production rate, in the first and second dilution rate particularly 0.06 1/hr and 0.30 1/hr, is 0.232 g/L/hr, and substrate used ratio is 0.306. And the case of the first tank fed mixture solution substrates and the second tank fed pure glucose of two-tank continuous fermenting, mixture solution fed dilution rate 0.015 – 0.1 1/hr, and pure glucose fed dilution rate 0.050 1/hr, and dilution rate of liquid flowing from the first tank into the second tank 0.0005 1/hr, equivalent to the volume of the second tank being at least 30 times of the volume of the first tank, can get the most ideal ethanol production rate 0.267 g/L/hr, but neither ethanol produced ratio 0.323 nor substrate used ratio 0.34 are ideal. Because dilution rate is lower, ethanol production rate is lower, but ethanol produced ratio and substrate used ratio are higher. That is ethanol produced ratio and substrate used ratio needing sacrificing in order to obtain the highest ethanol production rate.

The stoichiometric relationship between carbon dioxide (CO2) generated and glucose consumed during fermentation can be utilized to predict glucose consumption as well as yeast growth by measuring the CO2 concentration. Dissolved CO2 was chosen as opposed to off-gas CO2 due to the high solubility of CO2 in the fermentation broth as well as its ability to reflect on yeast growth more accurately than off-gas CO2. Typical very-high-gravity (VHG) ethanol fermentation is plagued by incomplete glucose utilization and longer durations. Aiming to improve substrate utilization and enhance VHG fermentation performance, characteristics of dissolved CO2 concentration in fermentation broths using Saccharomyces cerevisiae were studied under batch conditions. Based on this study a novel control methodology based on dissolved CO2 was developed and its effectiveness on enhancing VHG fermentation was evaluated by measuring the fermentation duration, glucose conversion efficiency and ethanol productivity. Four different initial concentrations 150, 200.05±0.21, 250.32±0.12, and 300.24±0.28 g glucose/L were used for batch ethanol fermentation without control. Zero substrate was indicated for 150 and 200.05±0.21 g glucose/L by a characteristic abrupt drop in dissolved CO2 concentration. On the other hand sluggish fermentation and incomplete substrate utilization were witnessed for 250.32±0.12, and 300.24±0.28 g glucose/L. A material balance equation was developed to compensate for the inability of the dissolved CO2 profiles to accurately predict the different growth phases of yeast. Dissolved CO2 was controlled at three distinct levels of 500, 750 and 1000 mg/L using aeration rates of 820 and 1300 mL/min for initial concentrations of 259.72±7.96 and 303.92±10.66 g glucose/L. Enhancement of VHG fermentation was achieved in the form of complete glucose utilization and higher ethanol productivities and shorter fermentation duration in comparison to batches without control. Complete glucose utilization was facilitated under ~250 and ~300 g glucose/L in 27.02±0.91 and 36.8±3.56 h respectively. Irrespective of the control set points and aeration rates, ethanol productivities of 3.98±0.28 g/L-h and 3.44±0.32 g/L-h were obtained for 259.72±7.96 and 303.92±10.66 g glucose/L respectively. The glucose conversion efficiencies for both 259.85±9.02 and 299.36±6.66 g glucose/L when dissolved CO2 was controlled were on par with that of batches without control.

Rumen acidosis is a metabolic disorder of ruminants, characterized by a severe drop in rumen pH. This is due to an accumulation of acids, especially lactic acid in the rumen. Lactic acid is one of the strongest acids with a major effect on rumen pH. A low and / or fluctuating rumen pH can have a severe impact on the productivity and health of dairy cattle, especially during the early lactation period. Rumen acidosis can, for example, occur during the rapid change from a low concentrate to a high concentrate diet. Megasphaera elsdenii (Me) is a lactate-utilizing micro-organism that converts the lactic acid that is produced from the fermentation of starch in the rumen, to propionic acid. Based on the ability of Me to convert lactic acid to propionic acid, a study was conducted to determine the effect of dosing live sources of Me on the level of rumen acidosis, general health and productivity. Sixty high-producing multiparous dairy cows were used in a randomized complete block design experiment. Cows were blocked according to milk production during the previous lactation and, thereafter, randomly allocated, within each block, to one of the following treatments:

1. 60% concentrate TMR;
2. 60% concentrate TMR with Me dosing;
3. 70% concentrate TMR;
4. 70% concentrate TMR with Me dosing.

The experimental period was 60 days and cows were dosed on day 2, 10 and 20 post-partum. Cows were housed in a semi-intensive housing unit equipped with Calan gates for determining individual feed intake. Daily milk production and dry matter intake were measured, as well as body mass and body condition score. Milk was analyzed for fat, protein, lactose and MUN; rumen fluid for pH, volatile fatty acids and lactic acid; faecal samples for pH and starch and feed refusal samples for nutrient components. In addition the health statuses of the cows were also monitored. In general the results did not show a clear advantage of dosing Me, regardless of the level of concentrate. Dry matter intake, milk production, milk composition, feed efficiency, body mass and body condition score were not affected by treatment (P > 0.05). Contrary to expectation, treatment did not affect rumen pH, rumen lactic acid or volatile fatty acid concentrations (P > 0.05). Faecal pH, however, was higher and the starch content lower in cows dosed with Me (P < 0.05) suggesting a positive effect on rumen fermentation and more efficient total tract starch fermentation. Furthermore only two cows were culled from the dosed group, compare to eleven from the control group, suggesting a positive influence of Me on the general health of stress, early lactation cows. Further research is needed to better quantify the potential role of Me in preventing SARA.
Dissertation (MSc(Agric))--University of Pretoria, 2008.
Animal and Wildlife Sciences
unrestricted

碩士
臺中師範學院
環境教育研究所
93
Hydrogen-Productivity Comparison of connected-in-series Reactors of Anaerobic hydrogen-fermentation and Anaerobic photohydrogenesis under Various Operational Conditions Abstract The conventional anaerobic wastewater treatment can be used to treat different kinds of organic wastewater and able to recover biogas energy, which is mainly methane. Methane causes to serious green-house effect. Based on the regulation of "the Kyoto Protocol", a clean energy should be sought out in the biomass energy.Due to no-greenhouse effect ,the hydrogenic anaerobic reaction (includes: Anaerobic fermentative hydrogenesis ,anaerobic photohydrogenesis),become a highly potential marketing technique. Based on the past four-years research of our lab.,the CSTR reactor is the most suitable for anaerobic hydrogen fermentation and the plug flow reactor that is the most suitable for anaerobic photohydrogenesis. The fermentative hydrogenic bacteria came from our lab. for long-term cultivation, mainly the Clostridium.The photohydrogenic one came from the lab. of prof. Chi-Mei Lee, Depart. of Environ. Eng., National Chung–Hsing University, which was Rhodopseudomonas palustris (non-sulfur photosynthetic bacteria), coded as WP3-5 .And as the literature mentioned, it is suitable for Anaerobic photosynthetic hydrogenic bacteria to treat the effluent of Anaerobic fermentative hydrogen production process, which contain massive volatile acid. Therefore this research took the main system with the above two kinds of connected in-series reactors, the front stage used the CSTR reactor and the rear stage use the plug flow reactor separated by membrane,and conducted the comparision of hydrogenesis. The results were shown as follows: For various organic loadings and HRTs ,the CSTR of front stage had the best hydrogen-productivity in the case of HRT=2hrs,volume=1.3L. The MLSS could reach 4462mg/l, hydrogen productivity up to 465 mole/m3‧day, per gram of influent COD got up to 3.88 mmole-H2/g-CODin; The secondary highly run was that operating at HRT=4hrs, volume=2L; The third highly run was operated at HRT=6hrs, volume=3L; The fourth highly run was operated at HRT=12hrs, volume=3L.The biomass recycling system can promote MLSS concentration up to 183%,hydrogen productivity up to 233%.The 3 pHs and HRTs tests of the rear-stage plug flow reactor(pH and HRTs of 5.0 and 10hrs,6.5 and 10hrs,5.0 and 24hrs ),the results were shown the photo-hydrogenic reactor had the best hydrogen productivity in the case of pH=5.0 and HRT=10hrs.While the organic loading of 20.4kg-COD/m3‧d,the plug flow reactor had the best hydrogen productivity of 21.5mole-H2/m3‧d, per gram of influent COD got up to 2.30mmole-H2 /g-CODin.The secondary highly run was operated at pH=6.5 and HRT=10hrs and The third highly run was operated at pH=5.0 and HRT=24hrs.In the hydrogen productivity of two connected-in-series system, it had best hydrogen productivity with the front stage reactor of pH =5.25, volume=1.3L, HRT=2hrs and with the rear stage of pH =5.0, HRT=10hrs, the total hydrogen productivity up to 486.5 mole/m3‧day. The front stage hydrogen productivity of CSTR reactor could reach 465 mole/m3‧day and the rear stage of the plug flow reactor could reach 21.5 mole/m3‧day, it was the best connected-in-series system test of ten tests.So the hydrogen production rate of front stage reactor was increased with the increase in organic loading. The photohydrogenic reactor had the best hydrogen productivity in the case of pH=5.0 and HRT=10hrs and was increased with the increase in organic loading. The comparison of hydrogen productivity of connected-in-series reactors and co-cultural reactor, the hydrogen productivity of connected-in-series reactors was better than that of co-cultural reactor.The increased NH3-N concentration and the decreased Org-N concentration of the effluent of front stage reactor was increased with the increase in organic loading, the increased NH3-N and the decreased Org-N of rear stage reactor mainly was increased with the increase in organic loading of front stage reactor (regressional coefficient 0.860 and 0.962), next the influenceial factor was organic loading of next rear stage reactor and was increased with the decreased in organic loading of rear stage reactor (regressional coefficient -0.643 and -0.462). From the observation under the phrase contrast microscope and the fluorescent microscope,the bacterial consortia of the front stage reactor mainly was bacillus of the pale orange fluorescence and the was increased with the increase in organic loading.The rear stage reactor mainly was short bacillus of the faint yellow fluorescece and was increased with the increase in organic loading. From the observation under the SEM,the bacteria of the front stage reactor mainly was bacillus and was increased with the increase in organic loading. When pH=5.0 ,the bacterial consortia of rear stage reactor was more simple and more short bacillus.When pH=6.5, the bacteria was complex.

Continuous fermentations were performed in an external-recycle, biofilm reactor using glucose and CO2 as carbon substrates. The nitrogen source for the auxotrophic Actinobacillus succinogenes was a combination of yeast extract (YE) and corn steep liquor (CSL), and sometimes only YE or CSL was used. In this study, the succinic acid productivity of A. succinogenes decreased by 67% as the amount of YE in the complex nitrogen source mixture decreased from 16 g·L-1 to 0 g·L-1. Succinic acid production increased as the CSL concentration in the nitrogen source increased, and the mass ratio of succinic acid to acetic acid exceeded the theoretical maximum limit of 3,93 g·g-1 when only CSL was used as the nitrogen source. The mass ratio of formic acid to acetic acid was consistently within the theoretical yield limitations (0,77 g·g−1) and decreased as the CSL concentration in the nitrogen source increased. The highest SA concentration in this study was 22,57 g·L-1 when only YE was used as the nitrogen source in the growth medium, and the highest SA productivity obtained in this study was 1,58 g·L-1·h-1 when a combination of YE and CSL was used as a nitrogen source. The highest mass ratio of SA to AA achieved was 8,3 g·g-1 when CSL was the sole nitrogen source. The mass ratio of FA to AA was consistently less than 0,77 g·g-1, approaching 0 g·g-1, as the CSL concentration in the nitrogen source increased.
Dissertation (MSc)--University of Pretoria, 2016.
National Research Foundation (NRF)
Chemical Engineering
MSc
Unrestricted

碩士
國立臺灣大學
食品科技研究所
94
The objectives of this research were to determine the optimum fermentation condition and to enhance the 1,3-β-D-glucan production of Ganoderma lucidum in submerged culture. The immunopotentiation of the polysaccharide from G. lucidum fermentation was evaluated by the inhibitory ratio of leukemic U937 cell as an model. The basic medium consisted of 5% black soybean, 2% Huangqi and 2% glucose. Furthermore, 1% glucose was added on day 4, 6 and 8, respectively. The effect of feeding time during the 12-day cultivation in 5L fermentor was evaluated. Addition of 1% glucose on day 6 inhibited the 1,3-β-D-glucanase activity and enhanced 1.54-flod 1,3-β-D-glucan in the culture. Moreover, the addition of chitin synthase inhibitor, Polyoxin B (1.5 ppm), probably reduced the binding between the structure glucan and chitin in cell wall, and therefore enhanced 1,3-β-D-glucan content up to 1.68 fold on day 8 without affecting cell growth. The inhibitory ratio of leukemic U937 cell resulted from the G. lucidum polysaccharides was positive correlated to the percentage of 1,3-β-D-glucan, molecular weight and degree of branching. Moreover, significant increases of TNF-α, IL-1β and IFN-γ in the mononuclear condition medium treated with the polysaccharidrs of 1% glucose fed-batch on day 6 were observed.

Fatty acid components on yeast plasma membrane were critical in maintaining proper cell activity during bioethanol fermentation. The alteration of fatty acid composition on yeast plasma membrane was recognized as an adaptive response to several environmental stress including osmotic pressure, ethanol inhibition and nutrients limit. These stresses were exacerbated under very-high-gravity condition in which excessive fermentable sugar was provided in feedstock. Controlling redox potential was proved beneficial in improving yeast performance under very-high-gravity condition. Fatty acid synthesis and desaturation pathways involved dissolved oxygen as well as balance between NAD+/NADH and NADP+/NADPH which could be influenced by the regulation of redox potential in media. In this study, fatty acid composition profiles under different glucose concentrations and different redox potential control level were examined. Its connection with yeast cell growth, ethanol productivity and other metabolites’ concentrations were studied as well to reveal any causal correlation between redox potential control, membrane fatty acid composition and yeast activity. Two glucose concentrations used in this study were 200 g/L and 300 g/L which represented normal and very high gravity respectively in bioethanol fermentation. In 300 g/L fermentation, three redox conditions were adopted while two different redox conditions were used in 200 g/L fermentation. Biomass concentration, ethanol productivity and fatty acid composition were observed to be affected by both gravity and ORP control strategy. Final biomass concentrations were 4.302 g/L in 200 g/L glucose with no ORP control condition and 7.658 in 200 g/L glucose with ORP controlled at -100 mV condition. In 300 g/L glucose fermentation, final biomass concentrations were 3.400 g/L for no ORP control, 4.953 g/L for -150 mV ORP control and 5.260 for -100 mV ORP control. Ethanol productivities were 2.574 g/Lh for 200 g/L glucose without ORP control and 3.780 g/Lh for 200 g/L glucose with -100 mV ORP control. In 300 g/L glucose fermentation, ethanol productivity decreased to 1.584 g/Lh when no ORP control was imposed. ORP control at -150 mV could improve the ethanol productivity to 1.693 g/Lh while -100 mV ORP control was able to further enhance the ethanol productivity to 1.829 g/Lh. Fatty acid composition was observed to shift to more saturated components when no ORP control was applied. Such trend of saturation was increased by higher gravity condition. ORP control was shown to change this tendency to saturation and help restore fatty acid components on plasma membrane to a more balanced distribution.

碩士
國立成功大學
化學工程學系
102
Bioethanol as one of renewable energy is considered an excellent alternative clean-burning fuel to replace gasoline. Continuous bioethanol fermentation systems have offered important economic advantages in comparison with traditional systems. Fermentation rates can be significantly improved when the continuous fermentation is integrated with cell immobilization techniques to enrich the cells concentration in the fermenter. Growing cells of Zymomonas mobilis immobilized in polyvinyl alcohol (PVA) gel beads were employed in an immobilized-cell fermenter for continuous bioethanol fermentation from glucose. The glucose loading, dilution rate and cells loading were varied in order to determine the best condition employed in obtaining both high bioethanol production and low residual glucose at high dilution rates. Higher glucose fermentation rate has been considered as the most important target as it leads to higher bioethanol titer and better bioethanol productivity. To enable bioethanol fermentation at high glucose concentration with continuous-flow operations, the Z. mobilis cells were immobilized using polyvinyl alcohol (PVA) matrix modified with a certain amount of iron (III) oxide (Fe2O3) and the cell immobilization of Z. mobilis was simply performed by using the enriched cells culture media harvested at the exponential growth phase. The production of bioethanol was affected by the medium flow rates or hydraulic retention time (HRT) from 1 to 4 hour. In addition, the effects of both initial glucose loadings (100 g/L, 125 g/L and 150 g/L) and cell loadings (20% (w/v), 40% (w/v) and 50% (w/v)) were also investigated. On the other hand, the bioethanol production performance of Z. mobilis cells immobilized with cross-linked polymer of PVA with 1% (w/v), 2% (w/v) and 3% (w/v) of Fe2O3 and unmodified PVA-immobilized Z. mobilis was compared. Reusability of the immobilized Z. mobilis or stability of beads was examined and found that the immobilized cells could be utilized for more than 20 days without losing their activity. Biothanol productivity increased from 15.74 g/L/h to 31.09 g/L/h when the modified PVA-immobilized Z. mobilis with 1% (w/w) of Fe2O3 was employed and the glucose loading was 125 g/L. Moreover, the HRT of 2 hour and cell loading of 40% (w/v) were considered as the optimum conditions to obtain the best bioethanol fermentation performance. The modified PVA-immobilized Zymomonas mobilis cells were used to enhance the efficiency of bioethanol production under very high gravity (VHG) conditions. Continuous bioethanol fermentation was integrated with in-situ bioethanol removal via vacuum membrane distillation (VMD) to overcome the problems associated with product inhibition and the resulting low bioethanol productivity during bioethanol fermentation. The developed VMD-integrated VHG fermentation system can be successfully operated under a feeding glucose concentration of up to 300 g/L (or 30% (w/v)), obtaining a maximum bioethanol concentration of 127.39 g/L (or 16.14% (v/v)), an bioethanol productivity of 63.69 g/L/h, and a glucose conversion of 84.93%. In addition, the production of bioethanol by fermentation must be accompanied by some by-products, such as carbon dioxide (CO2) and organic acids. These by-products can lower the quality and usability of bioethanol as a biofuel and also increase the amount of wastes to cause environmental pollution. CO2 produced from fermentation is of high purity and is nearly a saturated gas (almost 100%). The majority of CO2 applications were dedicated to serving carbonated beverage and food processing/preservation. Beyond these traditional applications, one of the most potential ones is the production of algae-based biofuels through CO2 fixation by microalgae. The advantages of using microalgae CO2 fixation include rapid growth rate and high CO2 fixation capability when compared to conventional plants and high oil/carbohydrate production. The carbohydrate-rich microalgal biomass then can be used for bioethanol production in large scale applications. Photosynthesis is often required for CO2 fixation. The need of solar energy supply in photosynthesis reactions appears to limit the application of biological CO2 sequestration due to scale-up problems. Furthermore, many microorganisms considered for CO2 fixation have fastidious growth requirements. Succinic acid is a common natural organic acid often found in humans, animals, plants and microorganisms. As 1 mol CO2 is theoretically required for the synthesis of 1 mol succinic acid. CO2 should play an important role in succinic acid production to promote the regulation of the PEP carboxykinase pathway. Compared to carbon capture by microalgae, this developed system encourages much higher CO2 fixation rate. The highest carbon fixation rate achieved for microalgae cultivation was 2.06 g/L/d, while it was 31.92 g/L/d (15 times higher) for succinic acid production. Therefore, production of succinic acid is also a feasible way of biological CO2 removal. In the end, bioethanol has to be produced from non-food sources. Cellulosic biomass was used for bioethanol fermentation for the purpose of reducing greenhouse gas emissions and giving impacts to rural economy condition. Cellulosic resources are in general very widespread and abundant. Being abundant and outside the human food chain brings cellulosic materials such bagasse, rice straw, etc. relatively inexpensive feedstocks for bioethanol production. A 120 g/L of bagasse loading or corresponding to 62.79 g/L of glucose after hydrolysis was used to evaluate the feasibility of producing bioethanol with the modified-PVA immobilized Z. mobilis cells. The glucose conversion obtained was about 94.57% in average with a bioethanol titer and productivity of approximately 26.82 g/L and 13.41 g/L/h, respectively, during fermentation. The results demonstrate that the modified PVA-immobilized Z. mobilis cells are preferable cell immobilization system for cellulosic bioethanol production.

Tese de doutoramento em Engenharia Química e Biológica
This thesis addresses the development and optimization of a fermentation process for the production of fructooligosaccharides (FOS) by fungi. FOS are prebiotics with numerous health benefits within which the improvement of gut microbiota balance can be highlighted, playing a key role in individual health. Conventionally, FOS production is a two-stage process that requires an enzyme production/purification step in order to proceed with the chemical reaction itself. FOS production by fungi in bioreactors in a one-stage process is an emergent alternative, since the use of whole cell as the biocatalyst avoids the need for FOS-producing enzyme purification from the cell extract, thus enabling a more competitive less expensive process. Therefore, the main purpose of this thesis was to develop and optimize a one-stage fermentation process for a more competitive FOS production towards its industrial implementation. Optimization of fermentation conditions to maximize FOS production via sucrose fermentation by Aureobasidium pullulans through response surface methodology, using a compilation of mathematical and statistical techniques, was performed and a significant improvement of the total FOS production yield by using a one-stage process was obtained. Although several microorganisms have been reported to have transfructosylation activity due to fructosyltransferase (FTase) and/or fructofuranosidase (FFase) activities, the search for other fungi with higher transfructosylation activity remains a challenge. By means of a presumptive and indirect colorimetric plate assay developed for the evaluation of transfructosylation activity in fungi, several fungal strains belonging to the Aspergillus and Penicillium species were identified, showing potential as FOSproduction microorganisms. An enzymatic extraction procedure was established to determine the transfructosylation and hydrolyzing activities of the enzymes in the extracellular, intracellular and periplasmic spaces of A. pullulans. Extracellular enzymes were found to have the higher transfructosylation activity, supporting the idea of developing a one-stage FOS production process. Fed-batch sucrose fermentations by A. pullulans with two sucrose feeding profiles (constant feeding and linear feeding) were conducted in order to increase FOS production. A slight increase in FOS production yield was achieved with sucrose linear feeding profile, compared to the batch production process, however FOS productivity decreased significantly. The main advantage to operate in fed-batch mode with a linear feeding profile was shown to be the production of fructofuranosyl nystose (GF4), that can add commercial value to the FOS mixture obtained, since GF4 may exert a prebiotic effect in more distal colonic regions compared with the lower-molecular-weight FOS. The scale-up of the one-stage batch FOS production process from sucrose by A. pullulans, to a semi-pilot scale, was performed. Similar FOS production yield, productivity, composition and kinetics were obtained by a 25 times volume increase scaling up procedure, suggesting that the one-stage batch process has potential to be used at an industrial level. Moreover, based on the process design and economics of an industrial unit built to produce 10.000 tons/year of FOS using the here developed production process, it was demonstrated that lower operational costs can be achieved, when compared to the current two-stage FOS production process. An estimated investment of 20 M€ in a FOS industrial unit can achieve a NPV of 71,5 M€, with a IRR of 45,18 % and ROI of 5 years, presenting itself as a very attractive business opportunity.
Esta tese aborda o desenvolvimento e otimização de um processo de fermentação para a produção de fruto-oligossacáridos (FOS) por fungos. Os FOS são pré-bióticos com inúmeros benefícios para saúde, dos quais se pode destacar o equilíbrio da flora intestinal, desempenhando um papel fundamental na saúde do indivíduo. Convencionalmente, a produção de FOS é um processo em duas etapas, que requer uma etapa de produção/purificação enzimática para que a reação química ocorra. A produção de FOS por fungos em biorreatores, tendo por base um processo de produção por uma só etapa, é uma alternativa emergente, dado que a utilização das células inteiras como biocatalisador evita a purificação das enzimas produtoras de FOS do extrato celular, originando um processo mais competitivo e menos dispendioso. Por esse motivo, o principal objetivo desta tese foi o desenvolvimento e otimização de um processo de fermentação para produção de FOS mais competitivo, numa só etapa, com vista à sua implementação industrial. A otimização das condições de fermentação para maximização da produção de FOS por fermentação de sacarose por Aureobasidium pullulans foi efetuada usando a metodologia de otimização fatorial, com recurso a uma série de ferramentas matemáticas e estatísticas, tendo-se alcançado uma melhoria significativa do rendimento global de produção de FOS por A. pullulans com o processo de produção em uma só etapa. Vários microrganismos são reportados como tendo atividade de transfrutosilação por intermédio das enzimas frutosiltransferase (FTase) e / ou frutofuranosidase (FFase). No entanto, a procura de outros fungos com atividade de transfrutosilação mais elevada permanece um desafio. Através de um ensaio colorimétrico em placa, presuntivo e indireto, desenvolvido para a avaliação da atividade de transfrutosilação em fungos, várias estirpes de fungos dos géneros Aspergillus e Penicillium spp foram identificados como potenciais microrganismos produtores de FOS. Um procedimento de extração enzimática foi estabelecido para determinar as atividades de transfrutosilação e hidrólise das enzimas nos espaços extracelulares, intracelulares e periplasmático de A. pullulans. As enzimas extracelulares demonstraram ter a atividade de transfrutosilação mais elevada, reforçando a ideia do desenvolvimento de um processo de produção numa só etapa. Processos de fermentação semi-contínua de sacarose por A. pullulans, com dois perfis de alimentação (alimentação constante e alimentação linear) foram realizados com a finalidade de aumentar a produção de FOS. Um ligeiro aumento no rendimento de produção de FOS foi conseguido com o perfil de alimentação linear de sacarose, em comparação com o processo de produção descontínuo. No entanto, a produtividade de FOS diminuiu significativamente. A principal vantagem de operar em modo semi-contínuo com um perfil de alimentação linear é produzir frutofuranosil nistose (GF4), que pode incrementar o interesse comercial da mistura de FOS obtida, uma vez que o GF4 pode exercer um efeito pré-biótico em regiões mais distais do cólon, em comparação com os FOS de menor peso molecular. Um aumento de escala no processo de produção de FOS numa só etapa, por fermentação de sacarose por A. pullulans, a uma escala semi-piloto, foi realizado e com um aumento de escala de 25 vezes, foram obtidos semelhantes rendimentos de produção de FOS, composição, produtividade e cinéticas de fermentação, sugerindo que o processo numa só etapa tem potencial para ser usado a um nível industrial. Além disso, com base no processo de conceção e economia de uma unidade industrial para a produção de 10.000 ton/ano de FOS, tendo por base o processo de produção desenvolvido nesta tese, menores custos de operação podem ser obtidos, quando comparados com o atual processo de produção de FOS em duas etapas. Um investimento estimado de 20 M€ numa unidade industrial de FOS pode promover um VAL de 71,5 M €, com uma TIR de 45,18% e um tempo de retorno do investimento de 5 anos, apresentando-se como uma oportunidade de negócio muito atrativa.