Rozprawy doktorskie na temat „Fodder and bioethanol production”

Seaweed biomass has been identified as a potential fermentation substrate for third generation biofuel processes due to its high carbohydrate content and its potential for mass cultivation without competing for agricultural land, fresh water and fertilisers. This thesis aimed to develop and advance existing processes to convert brown seaweeds into bioethanol. The main kelp species chosen as biomass candidates were Laminaria digitata, Laminaria hyperborea, Saccharina latissima and Alaria esculenta due to their abundance in Scottish waters and their identified potential for mariculturing. These kelp species were chemically characterised to identify seasonal variations, to recommend suitable seaweed candidates for bioethanol production and predict best harvest times. This has only been demonstrated before on one species - L. digitata. The chemical composition analyses were carried out over a 14 months sampling period, which focused on the storage carbohydrates laminarin and mannitol and the structural carbohydrates alginate, cellulose, fucoidan and xylose. In addition to carbohydrates the protein, nitrogen, carbon, polyphenol, ash and metal content was also profiled. Chemical profiling identified all four kelps as potential fermentation candidates, where maximum carbohydrate contents coincided with lowest ash and polyphenol content, usually seen in autumn. Biomass pre-treatment and saccharification are up-stream processes aimed at enhancing extraction of carbohydrates and converting those into fermentable substrates. Conversion of seaweed biomass into fermentation substrate evaluated acids and enzymes for seaweed pre-treatment and saccharification. Methodologies focused on optimising saccharification yields were developed to identify process critical parameters and develop methods for routine analysis of seaweed biomass. Results demonstrated that dilute acid hydrolysis was were less effective in releasing fermentable sugars, and also resulted in higher salinities compared to enzymatic hydrolysis using hemicellulosic and cellulosic enzymes, which were the preferred method of saccharification. All seaweeds in this thesis were assessed as fermentation substrates using the yeasts S. cerevisiae and P. angophorae, that principally ferment glucose or mannitol, respectively. Small-scale fermentation assays were developed for both yeasts to maximise ethanol yields and achieve process robustness. Both yeasts achieved a maximum ethanol yield of 0.17 g g-1 using Laminaria spp. On the basis of results, S. cerevisiae is recommended as the most useful yeast at this present point for ethanol fermentation from seaweed hydrolysates because of its tolerance to high salinity and ethanol concentrations. As salinity can negatively affect non-halotolerant enzymes, isolation of marine microorganisms was therefore carried out with the aim to highlight their enzymatic potential in seaweed saccharification. This was achieved through the isolation of two members of the genus Pseudoalteromonas, where saccharification yields using crude intracellular enzyme preparations exceeded those of dilute acids. In addition, the fermentative potential of microbial isolates as future ethanologenic strains was also evaluated. Understanding of the metabolic pathways is needed to fully assess the potential of those strains for genetic alteration. In conclusion, this thesis has demonstrated that up to ca. 20 g l-1 of ethanol can be produced from kelp species that grow on the west coast of Scotland. The procedure developed and used to produce ethanol requires further development, specifically the need for ethanol-fermenting microorganisms that can utilize mannitol and alginate; use of marine-adapted enzymes for saccharifiction; and the development of processes to achieve substrate concentration with reduced salinities. Comparison of theoretical ethanol yields from seaweed biomass with ethanol yields from terrestrial crops showed that the complete utilisation of all three major seaweed carbohydrates (laminarin, mannitol and alginate) from kelp species is needed for the process to be able to compete with 1st generation biofuel processes.

Hydrogen constitutes a promising alternative to manage our energy supply more efficiently. Hydrogen can be stored and used in fuel cells to produce electricity, where it combines with the oxygen present in the air and generates solely water as by-product. Of the different methods available to produce hydrogen, the catalytic reaction of ethanol and water (reforming) is one of the most advantageous alternatives, since ethanol can be produced easily from biomass (bioethanol), is liquid and simple to manipulate. This doctoral thesis studies the behavior of a family of cobalt catalysts to produce hydrogen from ethanol and water; to be more precise, catalysts based on cobalt hydrotalcites. The same process could be triggered by other types of catalyst, but many of them are far more expensive due to the noble metals they contain, and others - those based on nickel and cobalt - desactivate after a short amount of time because their surface accumulate carbon. This thesis demonstrates that with the help of a precise method of preparation, one can create inexpensive catalysts from cobalt hydrotalcites, which remain quite stable under realistic operating conditions. Chapter 1 introduces the reader to the key aspects of this doctoral thesis. It explains the objectives pursued and gives an overview of the state of art and the groundwork on which the experimental work is based. Besides explaining the general characteristics of the catalysts and the reactions that will be studied, chapter 1 also informs about cordierite monoliths: what exactly are they and why are they used in this work to physically stabilize the catalysts and catalytic membrane reactors. In this way, the aim of this doctoral thesis is to acquire new scientific knowledge on the one hand and on the other, to apply this knowledge in the development of devices that can be applied in practice. The four chapters following thereafter form a compound of papers that have been published in notable international journals (three of them) and one article in process of revision. Chapter 2 describes the preparation of a family of cobalt hydrotalcites with different ratios of cobalt, magnesia and aluminum, and how these cobalt hydrotalcites behave in the ethanol steam reforming reaction to produce hydrogen. Starting from a detailed characterization using different techniques like TEM, XRD, IR, TGA, In situ XPS, magnetism, etc., the different chemical elements present are identified, and their structure in the catalysts before, during, and after reaction is analyzed. It becomes evident that the best formula (with the greatest yield of hydrogen and the least amount of coke residual) is a hydrotalcite with a relation of Co:Mg:Al=1:2:1. It is concluded that during the reaction, the hydrotalcite-based catalyst transforms itself to a mix of cobalt spinel, strongly interacting with MgO on a nanometric scale. Nevertheless, if the reaction is repeated using only cobalt spinel (synthesized specifically for this purpose), the outcome is a smaller amount of hydrogen. This shows that cobalt hydrotalcite used as a catalyst precursor plays a crucial part in the final structure of the catalyst. Hydrotalcite Co:Mg:Al=1:2:1 doped with Pt and Rh is studied in chapter 3. For this, two families of catalysts with different ratios of Pt and Rh were prepared. They were analyzed under the same conditions as explained in chapter 2 and were tested in the reaction. The objective of doping the cobalt hydrotalcite with noble metals was to facilitate the reaction of cobalt, given the fact that metallic cobalt is the active element in ethanol steam reforming. Besides this key function of metallic cobalt, chapter 2 also reveals, however, that metallic cobalt speeds up the catalyst deactivation by causing severe coke accumulation. Hydrotalcite Co:Mg:Al=1:2:1 doped with Pt and Rh is studied in chapter 3. For this, two families of catalysts with different ratios of Pt and Rh were prepared.

Kitchen waste, which is collected in large amounts from cafeterias, restaurants, dining halls, food processing plants, and household kitchens, have become a valuable material for bioprocess engineering. Due to the high carbohydrate fraction, kitchen waste has great potential to be used as a potential substrate for ethanol production. Utilization of it as a raw material in ethanol fermentation would also contribute to reduction of costs. In the first part of this study, the effect of pretreatment method and enzymatic hydrolysis on glucose production was evaluated. Dry baker&rsquo
s yeast, Saccharomyces cerevisiae, was used in fermentation experiments conducted with and without fermentation medium at pH 4.5 and 30oC for 48 hours. Close values of glucose concentration were obtained from no pretreated and hot water treated samples. The fermentation results indicated that ethanol can be produced at similar concentrations in bioreactors with and without fermentation medium addition (p >
0.05). Thus, it is concluded that use of kitchen wastes as is disposed and without fermentation medium in ethanol fermentation could lower the cost to a large extent. In the second part of this study, the effects of solid load, which is proportional to the glucose concentration (10% to 20% (w/w)), inoculum level of Saccharomyces cerevisiae (5% to 15% (v/v)), and fermentation time (48 to 96 h) on production of bioethanol from kitchen waste were studied using Response Surface Methodology (RSM). A three-factor Box Behnken design was used. Ethanol concentration was used as a response in the resulting experimental design. High Pressure Liquid Chromatography (HPLC) method was used to determine ethanol and glucose concentrations. The statistical analysis of the constructed model developed by RSM suggested that linear effects of solid load, inoculum level, and fermentation time and quadratic effects of inoculum level and fermentation time were all significant (p <
0.05) on bioethanol production. The model was verified by additional runs, which were not present in the design matrix. It was found that the constructed model could be used to determine successfully the bioethanol concentration with >
90% precision. An optimum ethanol concentration of 32.16 g/L was suggested by the model with 20% (w/w) solid load, 8.85% (v/v) inoculum level and 58.8 hours of fermentation. Further study is needed to evaluate the optimal fermentation conditions in a large scale fermentation

In the future the demand for ethanol is expected to increase greatly due to the rising energy requirements in the world. Lignocellulosic materials are a suitable and potentially cheap feedstock for sustainable production of fuel ethanol, since vast quantities of agricultural and forest residues are available in many countries. However, there are several problems involved in the utilization of lignocellulosic raw materials as sugar source. The most common way of releasing the simple sugars in the material is by dilute acid hydrolysis. This procedure is relatively simple and cheap, but in addition to the sugars it creates inhibitory compounds. These inhibitors make it very hard for the yeast to ferment the hydrolyzate and detoxification is often necessary. One way to overcome this problem is to encapsulate the yeast. Encapsulation is an attractive method since it improves the cells stability and inhibitor tolerance, increases the biomass amount inside the reactor, and decreases the cost of cell recovery, recycling and downstream processing. However, the method does not yet permit long-term cultivation since the capsules used so far are not robust enough. Therefore more studies have to be conducted in order to find methods which produce mechanically robust capsules. The main goal of this paper is to find a suitable method to produce robust capsules using different concentration of the chemicals at different pH and also implementing some modifications such as addition of cross-linkers in preparation procedure. In this paper comparison of three different encapsulation techniques were studied based on the mechanical robustness of the capsules. The three different techniques were calcium mineralized alginate-chitosan capsules, alginate capsules coated with 2% chitosan (2% AC) and genipin crosslinked alginate-chitosan (GCAC) capsules. The results indicate that GCAC capsules are most robust and were good enough for prolonged use since most of the capsules were not deformed in mechanical strength test. There were slight differences in the diameter and membrane thickness before and after swelling. No negative influence was observed on the yeast growth when applying the cross-linker. The results of this study will hopefully add valuable information and helps in further studies using other cross-linkers to prepare robust capsules.
Program: Industrial Biotechnology

Ce travail concerne l'étude de la production d'hydrogène par vaporeformage catalytique du bioéthanol brut pour mettre en évidence l’effet des impuretés de l’alcool brut sur les performances catalytiques. Les performances du catalyseur Rh/MgAl2O4/Al2O3 en vaporeformage de l'éthanol en présence ou non de différentes impuretés ont été évaluées. La nature de l’impureté joue un rôle promoteur ou entraîne la chute de l’activité catalytique. L’effet promoteur peut être expliqué par un blocage des sites responsables de la formation de C2H4 tandis que la désactivation semble au dépôt de coke. Par la suite les travaux ont porté sur l’amélioration de la formulation, et un catalyseur (RhNi/Y-Al) actif, sélectif et stable pour la production d’hydrogène à partir du bioéthanol brut a été mis au point. L’incorporation d’un oxyde de terre rare à l’alumine et l’ajout d’une deuxième phase métallique a permis d’améliorer les propriétés acido-basiques du support, permettant de limiter la production de coke lors du vaporeformage du bioéthanol brut
This work is devoted to the study of raw bioethanol catalytic steam reforming reaction to evidence the effect of impurities of raw alcohol on the catalyst performances. The Rh/MgAl2O4/Al2O3 catalyst uses evaluated in the ethanol steam reforming reaction, with or without impurities. The nature of the impurity plays a promoting effect or results in the decrease of the catalytic activity. This promoting effect can be explained by a blocking of active sites for C2H4 formation while the deactivation seems to be linked to coke deposition. Further, the study focused on the improvement of the catalyst formulation and an active, selective and stable catalyst (RhNi/Y-Al) for the hydrogen production from raw bioethanol was developped. Integration of rare earth oxide to alumina and addition of a second metal has improved the acid-base properties of the support, allowing the limitation of the coke production during raw bioethanol steam reforming

Access to modern energy services derived from renewable sources is a prerequisite, not only for economic growth, rural development and sustainable development, but also for energy security and climate change mitigation. The least developed countries (LDCs) primarily use traditional biomass and have little access to commercial energy sources. They are more vulnerable to problems relating to energy security, air pollution, and the need for hard-cash currency to import fossil fuels. This thesis evaluates sugarcane-molasses bioethanol, a renewable energy source with the potential to be used as a transport fuel in Nepal. Sustainability aspects of molasses-based ethanol have been analyzed. Two important indicators for sustainability, viz. net energy and greenhouse gas (GHG) balances have been used to assess the appropriateness of bioethanol in the life cycle assessment (LCA) framework. This thesis has found that the production of bioethanol is energy-efficient in terms of the fossil fuel inputs required to produce it. Life cycle greenhouse gas (GHG) emissions from production and combustion are also lower than those of gasoline. The impacts of important physical and market parameters, such as sugar cane productivity, the use of fertilizers, energy consumption in different processes, and price have been observed in evaluating the sustainability aspects of bioethanol production. The production potential of bioethanol has been assessed. Concerns relating to the fuel vs. food debate, energy security, and air pollution have also been discussed. The thesis concludes that the major sustainability indicators for molasses ethanol in Nepal are in line with the goals of sustainable development. Thus, Nepal could be a good example for other LDCs when favorable governmental policy, institutional set-ups, and developmental cooperation from donor partners are in place to strengthen the development of renewable energy technologies.
QC 20101029

Master of Science
Department of Grain Science and Industry
Praveen V. Vadlani
In our study, five different bioenergy crops: wheat straw (Triticum aestivum), forage sorghum stover (sorghum bicolor), switchgrass (Panicum virgatum), miscanthus (Miscanthus giganteus) and sweet sorghum baggase (Sorghum bicolor) were evaluated for bio-ethanol production at 20% (w/v) initial substrate concentration under separate hydrolysis and fermentation (SHF) process. The substrates were ground to pass through 600µm mesh size and treated with 2% (w/v) NaOH at 121oC for 30 minutes. The washed and neutralized pretreated residues were subjected to saccharification using cellulase and β-glucosidase enzymes (ratio 1:1.25) at concentrations of 25 filter paper unit (fpu)/g and 31.25fpu/g, respectively, in pH 5.0 citrate buffer in an orbital incubator shaker at 150 rpm for 72 h. The hydrolysate obtained was centrifuged and supernatant was collected for fermentation. Fermentation was performed in shake flasks using Saccharomyces cerevisiae at 10% (w/v) inoculum concentration at 100 rpm for 24 h. Alkali treatment was effective in delignification of all the biomass feedstocks. The highest percent removal on raw biomass basis was attained for sorghum stover BMR-DP (81.3%, w/w) followed by miscanthus (79.9%, w/w), sorghum stover BMR-RL (69.2 %, w/w), wheat straw (68.0 %, w/w), switchgrass (66.0%, w/w), and sorghum baggase (65.4%, w/w). Glucan saccharification varied from 56.4-72.6 % (w/w) corresponding to a glucose levels of 0.45-0.34 g/g of dry substrate. Highest saccharification was observed for wheat straw while lowest was observed for miscanthus after 48 hours of hydrolysis. A maximum final ethanol concentration of 4.3% (w/v) was observed for wheat straw followed by sorghum baggase (4.2%), sorghum RL-BMR (3.6%), miscanthus (3.4%), sorghum DP-BMR (3.4%), and switchgrass (3.2%). From our studies, it is evident that high substrate concentration used for enzymatic hydrolysis was able to provide high final ethanol concentration. The lignin content and its arrangement in different biomass feedstocks may have affected saccharification and subsequent ethanol production. Bulk density and flowability are the two major key parameters that should be addressed to reduce processing cost of biomass for bioethanol production. Pelleting of biomass can increase the bulk density, thereby reducing the handling and transportation costs. In addition to above study, I analyzed the changes in chemical composition due to pelletization and pretreatment, and its effect on ethanol production by comparing unpelleted and pelleted biomass ethanol production efficiency. Wheat straw and big bluestem pelleted and unpelleted biomass were compared for their ethanol production efficiency. Pelleted and unpelleted wheat straw (Triticum aestivum) and bigblue stem (Andropogon gerardii Vitman) at a substrate concentration of 10% (w/v) were subjected to 2% NaOH treatment at 1210C for 30 min and the resulting residues were analyzed for changes in chemical composition. Saccharification of residue was done at substrate concentration of 12% (w/v) for 48 h. The sugars obtained were fermented to ethanol using Saccharomyces cerevisiae. Pelletization did not significantly affect the chemical composition of biomass in terms of glucan, xylan and lignin content. Delignification of pelleted biomass was greater than unpelleted biomass. Pelletization did not influence final ethanol production for both substrates.

Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.

The increase of petroleum cost as well as global warming and climate change result in investigation to discover new renewable energy resources. Bioenergy is one of the most important sources that is concerning the scientists and industrial sector. Although bioethanol had to be known as one of the most important renewable energy sources in order to reduce greenhouse gases and global warming, there is a limited number of publications reporting on them. In this review, a brief overview is offered about bioethanol production from algae. It can be given a deeper insight in dificulties and promising potential of bioethanol from algae
Sự gia tăng giá nhiên liệu hóa thạch cùng với cảnh báo toàn cầu về biến đổi khí hậu hướng đến việc nghiên cứu tìm ra những nguồn năng lượng có thể tái tạo. Năng lượng sinh học là một trong những nguồn quan trọng được các nhà khoa học và doanh nghiệp quan tâm. Mặc dù ethanol sinh học đã được biết đến như là một trong những dạng năng lượng tái tạo quan trọng nhất để giảm thiểu các khí nhà kính và cảnh báo toàn cầu, nhưng chỉ có một số ít bài báo về nó. Trong bài tổng quan này, chúng tôi giới thiệu vắn tắt việc sản xuất ethanol sinh học từ tảo. Nó đưa ra cái nhìn sâu hơn về những khó khăn và tiềm năng hứa hẹn của sản xuất ethanol sinh học từ tảo

Environmental and economic issues have drawn the world’s attention to produce and utilize energy from renewable sources for sustainable development. One of the attempts includes the production of ethanol from various substrates. Many researchers have focused on utilizing lignocelluloses biomass as substrate for the production of ethanol, which mainly contains cellulose and is a cheap and abundantly available material in the world. One of the major problems faced by researchers during production of ethanol from the lignocellulosic biomass is the stress tolerance of yeast cells, due to the nature of the hydrolysed substrate (lignocellulosic material treated with Nitro methyl cellulose (NMC)). One of the solutions for this problem is to encapsulate the yeast cells. Encapsulation is an attractive method, which can enhance the stress tolerance of the yeast cells in the reactor, and also aid in maintaining a high yeast concentration inside the bioreactor and thereby increase the volumetric productivity of ethanol. This report includes a major study on the sodium chloride and ethanol stress tolerance of alginate chitosan alginate (ACA), alginate chitosan (AC) and APTES treated ACA encapsulated yeast biomass in medium containing different concentrations of glucose under anaerobic conditions. AC capsules shows significant results towards osmotic stress and ethanol stress compared with that of freely suspended cells in stress conditions.AC capsule encapsulated yeast tolerated osmotic stress better than ACA capsules in 2M of NaCl where as freely suspended yeast cells unable to tolerate 2M of NaCl . At 100th hour in AC capsules glucose consumption was 12 g/l where as in ACA capsules glucose consumption at same 100th hour was 2 g/l. At 10% ethanol concentration yeast inside ACA capsules showed 5 g/l of glucose consumption but in freely suspended yeast cells there is no glucose consumption as they cannot tolerate higher stress levels.

Mestrado em Biotecnologia - Biotecnologia Industrial e Ambiental
O objectivo desta dissertação foi a comparação da produção de bioetanol de 2ª geração por Scheffersomyces stipitis (em suspensão e imobilizada) e por duas estirpes industriais de Saccharomyces cerevisiae. Os substratos utilizados para a realização das fermentações foram os Licores de Cozimento ao Sulfito Ácido (SSLs) provenientes de madeira de resinosas (HSSL) e de madeiras de folhosas (SSSL – Domsjö hydrolysate). O HSSL e o SSSL são sub-produtos da indústria de pastas de papel, resultando do processo de cozimento ao sulfito com magnésio e sódio, respetivamente. Além de lenhosulfonatos, estes SSLs contêm monossacarídeos, destacando-se a glucose, xilose e manose. O ácido acético, composto inibidor da fermentação alcoólica por leveduras, também está presente em concentrações relativamente elevadas (≥ 5.0 g . L-1). O HSSL foi pré-tratado físico-quimicamente e submetido a uma remoção biológica de inibidores com P. variotii, enquanto o Domsjö hydrolysate foi utilizado sem qualquer tratamento. S. stipitis e S. cerevisiae são leveduras extensamente estudadas devido à sua capacidade de fermentação de pentoses e hexoses, respetivamente. Num estágio prévio às fermentações dos SSLs, as leveduras foram pré-adaptadas em 60% HSSL ou 40% SSSL. Os máximos de rendimento (0.440 g etanol . g açúcares-1) e produtividade em etanol (0.885 g etanol . L-1 . h-1) foram obtidos nas fermentações com S. cerevisiae TMB 3500 em SSSL, sendo estas as variáveis com maior potencial para aplicação industrial. Embora a cultura suspensa de S. stipitis tenha resultado numa menor produtividade (0.010 g etanol . L-1 . h-1), a optimização do fornecimento de oxigénio ao biorreator deverá conduzir ao aumento da produtividade volumétrica em etanol. A imobilização celular e o controlo do pH em 5.5 nas fermentações com S. stipitis melhoraram a eficiência fermentativa ao aumentarem a produção de etanol em 1.3 e 1.6 vezes, respetivamente. Quando aplicadas simultaneamente, estas duas condições aumentaram o rendimento em etanol 2.2 vezes, sugerindo que (i) a imobilização numa matriz de alginato de cálcio protegeu a levedura dos inibidores químicos e que (ii) o controlo de pH em 5.5 foi determinante para a produção de etanol a partir de HSSL biologicamente pré-tratado. Os resultados comprovam que ambos os SSLs são potenciais substratos para a produção de bioetanol de 2ª geração usando S. stipitis ou S. cerevisiae, sob o conceito de biorefinaria.
The aim of this work was the comparison of second-generation bioethanol production by Scheffersomyces stipitis (free-culture and immobilized) and two Saccharomyces cerevisiae industrial strains, using Hardwood Spent Sulphite Liquor (HSSL) or Domsjö hydrolysate (SSSL) as substrate. HSSL and SSSL are side products of pulp and paper industry, from magnesium and sodium-based acidic sulphite pulping, respectively. Besides sulphonated lignin, SSLs contain fermentable sugars, mainly glucose, xylose and mannose. Acetic acid, a known inhibitor of ethanol fermentation by yeasts, is also present in a relatively high content (≥ 5.0 g . L-1). HSSL was previously physico-chemically pretreated and bio-detoxified with P. variotii, whereas SSSL was used without any treatment. S. stipitis and S. cerevisiae are the most widely studied pentose and hexose-fermenting yeasts, respectively. Before fermentations in SSLs, all yeast strains were pre-adapted by growing them in 60% HSSL or 40% SSSL. The highest maximum ethanol yield (0.440 g ethanol . g sugars-1) and productivity (0.885 g ethanol . L-1 . h-1) were obtained using S. cerevisiae TMB 3500 and SSSL. Suspended S. stipitis achieved a lower ethanol productivity (0.010 g ethanol . L-1 . h-1) but further optimization on the supplied oxygen in the fermentor might lead to higher ethanol productivity. Immobilization and pH control at 5.5 on S. stipitis fermentations improved the fermentation efficiency, increasing up the ethanol production by 1.3-fold and 1.6-fold, respectively. When applied simultaneously, these two conditions increased the ethanol yield 2.2-fold, suggesting that (i) immobilization with a calcium-alginate matrix protected the yeast from the inhibitory compounds and (ii) the controlled pH at 5.5 was essential for ethanol production from bio-detoxified HSSL. Results showed that both SSLs are potential substrates for the production of 2nd generation bioethanol by S. stipitis or S. cerevisiae, under the biorefinery concept.

Doutoramento em Engenharia Química
A vida da sociedade atual é dependente dos recursos fósseis, tanto a nível de energia como de materiais. No entanto, tem-se verificado uma redução das reservas destes recursos, ao mesmo tempo que as necessidades da sociedade continuam a aumentar, tornando cada vez mais necessárias, a produção de biocombustíveis e produtos químicos. Atualmente o etanol é produzido industrialmente a partir da cana-de-açúcar e milho, matérias-primas usadas na alimentação humana e animal. Este fato desencadeou o aumento de preços dos alimentos em todo o mundo e, como consequência, provocou uma série de distúrbios sociais. Os subprodutos industriais, recursos independentes das cadeias alimentares, têm-se posicionado como fonte de matérias-primas potenciais para bioprocessamento. Neste sentido, surgem os subprodutos gerados em grande quantidade pela indústria papeleira. Os licores de cozimento da madeira ao sulfito ácido (SSLs) são uma matériaprima promissora, uma vez que durante este processo os polissacarídeos da madeira são hidrolisados originando açúcares fermentáveis. A composição dos SSLs varia consoante o tipo de madeira usada no processo de cozimento (de árvores resinosas, folhosas ou a mistura de ambas). O bioprocessamento do SSL proveniente de folhosas (HSSL) é uma metodologia ainda pouco explorada. O HSSL contém elevadas concentrações de açúcares (35-45 g.L-1), na sua maioria pentoses. A fermentação destes açúcares a bioetanol é ainda um desafio, uma vez que nem todos os microrganismos são capazes de fermentar as pentoses a etanol. De entre as leveduras capazes de fermentar naturalmente as pentoses, destaca-se a Scheffersomyces stipitis, que apresenta uma elevada eficiência de fermentação. No entanto, o HSSL contém também compostos conhecidos por inibirem o crescimento de microrganismos, dificultando assim o seu bioprocessamento. Neste sentido, o principal objetivo deste trabalho foi a produção de bioetanol pela levedura S. stipitis a partir de HSSL, resultante do cozimento ao sulfito ácido da madeira de Eucalyptus globulus. Para alcançar este objetivo, estudaram-se duas estratégias de operação diferentes. Em primeiro lugar estudou-se a bio-desintoxicação do HSSL com o fungo filamentoso Paecilomyces variotii, conhecido por crescer em resíduos industriais. Estudaram-se duas tecnologias fermentativas diferentes para a biodesintoxicação do HSSL: um reator descontínuo e um reator descontínuo sequencial (SBR). A remoção biológica de inibidores do HSSL foi mais eficaz quando se usou o SBR. P. variotii assimilou alguns inibidores microbianos como o ácido acético, o ácido gálico e o pirogalol, entre outros. Após esta desintoxicação, o HSSL foi submetido à fermentação com S. stipitis, na qual foi atingida a concentração máxima de etanol de 2.36 g.L-1 com um rendimento de 0.17 g.g-1. P. variotti, além de desintoxicar o HSSL, também é útil na produção de proteína microbiana (SCP) para a alimentação animal pois, a sua biomassa é rica em proteína. O estudo da produção de SCP por P. variotii foi efetuado num SBR com HSSL sem suplementos e suplementado com sais. A melhor produção de biomassa foi obtida no HSSL sem adição de sais, tendo-se obtido um teor de proteína elevado (82,8%), com uma baixa concentração de DNA (1,1%). A proteína continha 6 aminoácidos essenciais, mostrando potencial para o uso desta SCP na alimentação animal e, eventualmente, em nutrição humana. Assim, a indústria papeleira poderá integrar a produção de bioetanol após a produção SCP e melhorar a sustentabilidade da indústria de pastas. A segunda estratégia consistiu em adaptar a levedura S. stipitis ao HSSL de modo a que esta levedura conseguisse crescer e fermentar o HSSL sem remoção de inibidores. Operou-se um reator contínuo (CSTR) com concentrações crescentes de HSSL, entre 20 % e 60 % (v/v) durante 382 gerações em HSSL, com uma taxa de diluição de 0.20 h-1. A população adaptada, recolhida no final do CSTR (POP), apresentou uma melhoria na fermentação do HSSL (60 %), quando comparada com a estirpe original (PAR). Após esta adaptação, a concentração máxima de etanol obtida foi de 6.93 g.L-1, com um rendimento de 0.26 g.g-1. POP possuía também a capacidade de metabolizar, possivelmente por ativação de vias oxidativas, compostos derivados da lenhina e taninos dissolvidos no HSSL, conhecidos inibidores microbianos. Por fim, verificou-se também que a pré-cultura da levedura em 60 % de HSSL fez com que a estirpe PAR melhorasse o processo fermentativo em HSSL, em comparação com o ensaio sem pré-cultura em HSSL. No entanto, no caso da estirpe POP, o seu metabolismo foi redirecionado para a metabolização dos inibidores sendo que a produção de etanol decresceu.
The fossil resources are declining while the requirements of modern lifestyle for energy and materials are growing. Hence, the search for sustainable alternatives to produce fuels and chemicals from non-fossil feedstocks is increasing. Among all biofuels, ethanol is currently being industrially produced from sugar-containing biomass such as sugarcane and corn. The use of these raw-materials, belonging to human and animal feeding, resulted in the rise of prices of food all over the world and, consequently, in social disturbance. The use of industrial by-products, raw-materials outside the food chain, with polysaccharides hydrolysed to fermentable sugars, is an attractive prospect for future biotechnologies. In this context, spent sulphite liquors (SSLs), by-products from the pulp and paper industry, are promising feedstocks for bioprocessing. The composition of SSLs depends on the type of wood used by the pulp and paper industry (softwoods, hardwoods or mixture of both). Hardwood spent sulphite liquor (HSSL) is a by-product from the pulp and paper industry, rich in pentoses, which is not fully exploited for bioprocessing. The sustainable fermentation of pentoses into bioethanol is a challenge to overcome since not all the microorganisms are able to use these sugars. Scheffersomyces stipitis is one of the most efficient yeast to naturally ferment pentoses to ethanol. However, besides sugars (35-45 g.L-1), HSSL contains microbial inhibitors that limit the possibility of its bioprocessing. Therefore, the main purpose of this work was the production of bioethanol by S. stipitis from HSSL of Eucalypt globulus. To accomplish this objective two different strategies were studied. The first one was the bio-detoxification of HSSL with the filamentous fungus Paecilomyces variotii, known for growing in polluted residues. Two fermentative approaches were compared, a single batch and a sequential batch reactor (SBR). Biological treatment of HSSL to remove microbial inhibitors was more efficient in the SBR. P. variotti was able to assimilate acetic acid as well as low molecular weight phenolics such as, gallic acid and pyrogallol, recognized yeast inhibitors. This bio-detoxified HSSL was subjected to a successful fermentation by S. stipitis, attaining a maximum ethanol concentration of 2.36 g.L−1 with a yield of 0.17 g.g−1. Moreover, the biomass produced by P. variotii is a potential source of protein and other nutrients for animal feeding. Hence, SCP production by P. variotii from HSSL was studied using a SBR with and without mixed salts supplementation. The best approach for SCP production was the SBR without salts addition. The biomass produced presented 82.8 % of protein with 6 essential amino acids and 1.1 % of DNA. Therefore the produced SCP could be considered a good candidate for animal feeding and, eventually, human nutrition. This is a major advantage for a biorefinary approach, since this bio-detoxification process and the SCP production can be integrated with bioethanol production by S. stipitis. The second strategy to produce bioethanol was to improve the tolerance of S. stipitis in order to utilize the xylose present in HSSL without the removal of inhibitory compounds. A continuous reactor with increasing HSSL concentrations, between 20 % and 60 % (v/v) was operated during 382 generations of HSSL, at a dilution rate of 0.20 h-1. The resulting adapted population (POP) showed improved fermentation behaviour in 60 % HSSL when compared with the parental strain (PAR). POP achieved a maximum ethanol concentration of 6.93 g.L-1, with a maximum ethanol yield of 0.26 g.g-1. It was also showed that POP could assimilate dissolved lignin oligomers and tannins probably through activating oxidative pathways. Moreover, preculturing PAR in HSSL improved its tolerance towards the HSSL inhibitors and also the yeast fermentation ability. Nevertheless, preculturing POP in HSSL, redirected its metabolism to the assimilation of inhibitors, reducing the ethanol production.

Philosophiae Doctor - PhD (Biotechnology)
Parageobacillus thermoglucosidans is a promising “platform” organism to use in the production ofa range of useful metabolites with demonstrated ability to produce ethanol, isobutanol and polylactic acid for bio-degradable plastics. Extensive work has been done in engineering the organism for enhanced ethanol production. However, an often used and highly effective alternative pathway (pyruvate decarboxylase mediated) for ethanol production has not yet been demonstrated in P. thermoglucosidans. We first characterize two novel bacterial pyruvate decarboxylase enzymes (PDC’s) then attempt to express the more thermostable of these enzymes from Gluconobacter oxydans in P. thermoglucosidans to improve ethanol yields. Initial expression was unsuccessful. Analysis of the codon usage pattern for the gene revealed that the codon usage was suboptimal in the heterologous host P. thermoglucosidans. After codon harmonization, we could demonstrate successful expression of the enzyme at 45°C, however not at the bacterium’s optimum growth temperature of 60°C. This was concomitant with enhanced ethanol production close to the theoretical yield possible (0.5g/l).

Lignocellulosic materials are promising alternative feedstocks for bioethanol production. This dissertation focuses on 2nd generation bioethanol production from lignocellulosic biomass. The first part of this thesis examined the enzymatic production of fennentable sugars from a range of lignocellulosic biomass and agricultural residues e.g. miscanthus, switchgrass, reedcanary grass, wheat straw, cassava peel and millet straw. The 2n part analysed supply of feedstocks in Nigeria with respect to bioethanol yield and models the potential supply in specific geographical locations with a view to establishing commercial cellulosic ethanol facilities. The overall goal of the dissertation is to evaluate cellulosic feedstock availability in Nigeria and their potential for bioethanol production. In this study the Van Soest detergent fibre analysis method was used to determine the chemical composition of a range of feedstocks. Barley and wheat straw showed the highest cellulose content while switchgrass had the highest hemicellulose. The theoretical ethanol yield based on cellulose content for the herbaceous energy crops and agricultural residue showed that barley straw had the highest potential yield of 145.35 IIton and cassava peels had the lowest yield of 94.28 IItone because starch was not taken into account. The study also analysed varietal variation in chemical composition of wheat straw from a HGCA Recommended list Trial. Cordiale had the highest cellulose content. Cordiale and Alchemy had significantly higher ethanol yield than all the other varieties studied. Oakley, Deben and Consort had significantly lower ethanol yield than the other varieties. Using wheat straw as a model feedstock, biomass pre-treatment conditions eg acid vs alkali concentration, solid loading, temperature and residence time were optimized. The optimization showed that reducing sugar yield from NaOH pre-treated wheat straw was 2x higher than that from acid pre-treated wheat straw. The optimal pre-treatment conditions for wheat straw were 3% NaOH and132°C for 40 minutes A range of agricultural residues were examined regional wise based on the six zones in Nigeria for their potential for lignocellulosic ethanol production while forestry and grassland resources were evaluated on national basis. Agricultural residues were split into two including field residues and processing residues. In evaluating field residue, provisions were made to account for current uses in terms of soil cover and animal feed, which are the two existing uses for field residues. From the findings, maize stalk and cassava peel are two of the major processing residues available for use. Maize stalk when used as a single feedstock can only power 18 commercial bioethanol processing facilities in the North and only 7 facilities in the South. Cassava peel as a single feedstock, can power 13 bioethanol facility in the North where 9 of them would be based in the North central but power 20 facilities in the South, where they are evenly spread across the 3 Southern zones. When the total agricultural residues produced in each zone are put together in the scenario where the bioethanol processing facilities are based on multi-feedstocks, 44 - 54 ethanol processing facilities could be sited in each northern zone and only 16 - 19 facilities southern. Based on 2008 petrol consumption of 9.5 billion litres in Nigeria, a 10% blend would require 950 million litres of bioethanol per annum. Using the entire field and processing residues examined in this study produces 6.7 billion litres of bioethanol per annum which exceeds the 10% mandate. Therefore Nigeria has enough agricultural residues to exceed its bioethanol blending requirement but the big issue is collection and financial return to the fanner. Fuel wood and sawdust were evaluated at national level for lack of regional data. Potentially, fuelwood can power 153 ethanol processing facilities, sawdust can power 7 and the grassland resource can power 542 bioethanol processing facilities. There is considerable potential to cultivate purposely grown energy crops eg switchgrass and miscanthus for lignocellulosic ethanol production exists. This is dependent on identifying species suited to the climate, geographical zones and potential for genetic improvement in these species and potential for improving production via management practices.

2\(^n\)\(^d\)-generation bioethanol can be produced from cellulose fraction present in lignocellulosic biomass and has become a significant research focus due to its potential for replacing fossil fuels and decreasing greenhouse gases emissions. In order to produce 2\(^n\)\(^d\)-generation bioethanol, biomass processing is required in order to access cellulose within lignocellulose and convert it into glucose. However, an efficient cost-effective and environmental-friendly process has not been achieved. The aim of this work was to produce glucose from purified cellulose from \(Miscanthus\) \(x\) \(giganteus\), an energy crop. The lignocellulosic biomass was selectively fractionated into its main components, hemicellulose, lignin and cellulose, after extractions using ‘green’ processes in a biorefinery approach. Hydrolysis of the cellulose-enriched fibres into glucose was evaluated using subcritical water (SBW) in a batch reactor at temperatures from 190-320oC, residence times from 0-54min and biomass loading from 0.5-6.4% (w/v). The process used for cellulose purification had significant effect on glucose production by SBW, and higher glucose yields were achieved at higher temperatures and shorter residence times. Glucose was used for bioethanol production. Although the formation of inhibitors during SBW hydrolysis could not be prevented, fermentation could be performed in the presence of these compounds at some conditions, with high ethanol yields.

This project has investigated the production of bioethanol from duckweed (Lemna minor) biomass. The project includes four main sections: firstly, analysis of the chemical characteristics of duckweed, particularly the polysaccharides of the cell wall; secondly, exploration of suitable commercial enzymes for degrading duckweed biomass to fermentable sugars; thirdly, optimisation of pretreatments and enzymatic saccharification; finally, fermentation and optimisation of the ethanol yield. Pond-grown L. minor contained 51.2 % carbohydrate (w/w dry matter) of which 77 % (including glucose, galactose and xylose) is fermentable. A series of enzymatic hydrolyses was used to evaluate the commercial enzymes and optimise conditions for their use in the saccharification of duckweed biomass. Celluclast 1.5L (CE) and Novozyme 188 (BG) were identified as suitable for hydrolysing duckweed cell walls (prepared as alcohol insoluble residues). The additional use of thermophysical pretreatment (steam explosion) results in a dramatic decrease in the amount of enzyme required for quantitative saccharification. A more advanced commercial cellulase cocktail (Cellic® CTec 2; CTec 2) is likely to further reduce the enzyme cost. Methods for the simultaneous saccharification, using CTec 2 and BG, and fermentation of steam exploded duckweed were developed. These resulted in an 80 % ethanol yield at a diluted substrate concentration (1 % w/v). However the ethanol yield decreased dramatically at higher substrate concentrations (to 18 % at 20 % w/v substrate concentration, which is a highly viscous suspension). Further studies involved the development of approaches to address this: (i) increasing the yeast titre in the inoculum or (ii) growing the inoculum on steam-exploded duckweed. These approaches facilitated an ethanol yield of up to 70 % (w/w) at a substrate concentration of 20 % (w/v). Maximising the final ethanol yield is of great importance in reducing the costs of production. The optimized ethanol production process indicates the technical potential for industrial ethanol production from duckweed. Operating costs have also been estimated and are discussed in relation to the potential exploitation of protein as a co-product.

The aim of this project provides the use of a lignocellulosic biomass microbubble reactor in order to analyse the pre-treatment of cellulose and maize for, respectively, Bioethanol and Biogas production. Because of lignin recalcitrance, dielectric barrier discharge plasma is used to improve the solubility and accessibility of these feedstock. The novel reactor combined with this plasma source generates highly oxidative species (O3, H2O2, and OH radicals) close to the gas-liquid interface. The cellulose has been treated under different conditions such as treatment time (30 min, 60 min, 90 min and 120 min) and pH buffer solution (pH 3, pH 7 and pH 9). The maize biomass has been reacted under 5 different conditions (Control sludge, Untreated raw maize, Plasma treated washed maize, Plasma treated unwashed maize, Bubble treated unwashed maize). The optimal operating condition, for cellulose biomass, that produced the highest glucose concentration results with pH 3 and with 30 min treatments. On the other hand, maize samples treated with plasma, both washed and unwashed, generate more biogas than bubble treatment, control sludge or untreated raw maize.

Unlike first generation biofuels, those produced from ligno-cellulosic waste material (second generation) have the potential to offer sustainable fuel production without competition for food products, whilst making significant savings in terms of greenhouse gas emissions. Second generation bioethanol has the potential to offer a stop-gap between current vehicle fuelling technologies and future solutions such as biohydrogen. TMO Renewables Ltd, a leading developer of the second-generation conversion of biomass to biofuel, has engineered the organism Geobacillus thermoglucosidasius to optimise its production of ethanol. This thermophilic bacterium grows optimally at 60-65°C, on a wide range of different substrates including both C5 and C6 sugars. The enzyme responsible for ethanol production has been shown to be a highly-expressed bifunctional enzyme (ADHE) that possesses both an acetylating aldehyde dehydrogenase (aldDH) and an alcohol dehydrogenase (ADH) activity. This enzyme is responsible for catalysing the reduction of acetyl-CoA to ethanol via an acetaldehyde intermediate: acetyl-CoA + NADH + H+ → acetaldehyde + CoA-SH + NAD+ acetaldehyde + NADH + H+ → ethanol + NAD+ Here we report the characterisation of the bifunctional ADHE in terms of catalytic activity, substrate promiscuity and multimeric assembly. The properties of this enzyme in relation to competing pathways in fermentative metabolism, including its expression pattern during fermentation, have also been determined. Investigations included the sub-cloning and separate recombinant expression of the aldDH and ADH halves of the protein, followed by the determination of a high-resolution crystal structure of the active ADH domain. The structure of the aldDH domain has been modelled, including its interaction with the ADH component of ADHE. An additional aldDH gene was identified in Geobacillus thermoglucosidasius; this has also been cloned and expressed, and the recombinant enzyme characterised and its high-resolution crystal structure determined. Through fusion of ADH and aldDH genes, a series of novel ADHE enzymes have been generated and their effect on ethanol production within the engineered G. thermoglucosidasius strain evaluated.

Thesis (MScEng)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Whereas fuel used for transport and electricity production are mainly fossil–derived, there has recently been an increased focus on bio-fuels due to the impact of fossil derived fuel on the environment as well as the increased energy demand worldwide, concomitant with the depletion of fossil fuel reserves. Paper sludge produced by paper mills are high in lignocellulose and represents a largely untapped feedstock for bio-energy production. The aim of this study was to determine the composition, fermentability and optimum paper sludge loading and enzyme dosage for producing ethanol from paper sludge. This information was used to develop a model of the process in Aspen Plus®. The mass and energy balances obtained from the Aspen Plus® model were used to develop equipment specifications which were used to source equipment cost data. A techno-economic model was developed from the equipment cost data to assess the economic viability of the simultaneous saccharification and fermentation (SSF) process utilising paper sludge as feedstock. Nine paper sludge samples obtained from Nampak Tissue (Pty) Ltd. were evaluated in terms of ethanol production and those samples yielding the highest and lowest ethanol titres were selected for optimisation. This allowed for the determination of a range of ethanol concentrations and yields, expressed as percentage of the theoretical maximum, which could be expected on an industrial scale. Response surface methodology was used to obtain quadratic mathematical models to determine the effects of solid loading and cellulase dosage on ethanol production and ethanol yield from paper sludge during anoxic fed-batch fermentations using Saccharomyces cerevisiae strain MH1000. This approach was augmented with a multi response optimisation approach incorporating a desirability function to determine the optimal solid loading and cellulase dosage in fed-batch SSF cultures. The multi response optimisation revealed that an optimum paper sludge loading of 21% (w/w) and a cellulase loading of 14.5 FPU g-1 be used regardless of the paper sludge sample. The fact that one optimal enzyme dosage and paper sludge loading is possible, regardless the paper sludge feed stock, is attractive since the SSF process can be controlled efficiently, while not requiring process alterations to optimize ethanol production when different batches of paper sludge are processed. At the optimum paper sludge loading and cellulase dosage a minimum ethanol concentration of 47.36 g l-1 (84.69% of theoretical maximum) can be expected regardless of the paper sludge used. An economic assessment was conducted to ascertain whether ethanol production from paper sludge using SSF is economically viable. Three scenarios were investigated. In the first scenario revenue was calculated from the ethanol sales linked to the basic fuel price, whereas in the second and third scenarios liquefied petroleum gas (LPG) consumption at the paper mill was replaced with anhydrous and 95% ethanol respectively. In all the cases, paper sludge feed rates of 15, 30 and 50 t d-1 were used. The production of ethanol from paper sludge for ethanol sales (scenario 1) resulted in higher IRR and NPV values, as well as shorter payback periods, compared to replacement of LPG at the paper mill (scenarios 2 and 3). At an assumed enzyme cost of $ 0.90 gal-1 (R 2.01 litre-1), IRR values of 11%, 22% and 30% were obtained at paper sludge feed rates of 15, 30 and 50 t d-1. A sensitivity analysis performed on the total capital investment and enzyme cost revealed that the SSF process is only economically viable at a paper sludge feed rate of 50 t d-1 irrespective of the variation in capital investment. For the SSF process to be economically viable the enzyme costs must be lower than $ 0.70 gal-1 (R 1.56 litre-1) and $ 1.20 gal-1 (R 2.68 litre-1) for paper sludge feed rates of 30 and 50 t d-1 respectively. The SSF process at a paper sludge feed rate of 15 t d-1 was not economically viable even assuming a zero enzyme cost. A Monte Carlo simulation revealed that the SSF process is economically viable at a paper sludge feed rate of 50 t d-1 as a mean IRR value of 32% were obtained with a probability of 26% to attain an IRR value lower than 25%. The SSF process at lower paper sludge loadings is not economically viable as probabilities of 70% and 95% were obtained to attain IRR values lower than 25% at paper sludge feed rates of 30 and 15 t d-1 respectively. From this study it can be concluded that paper sludge is an excellent feedstock for ethanol production for the sales of ethanol at a paper sludge feed rate in excess of 50 t d-1 with the added environmental benefit of reducing GHG emissions by 42.5%.
AFRIKAANSE OPSOMMING: Aangesien dat brandstof vir vervoer en energie meestal vanaf fossiel afgeleide bronne kom, is daar onlangs ʼn groter fokus op bio-brandstowwe as gevolg van die impak van fossiel afgeleide brandstowwe op die omgewing en 'n verhoogde aanvraag na energie wêreldwyd, gepaardgaande met die uitputting van fossielbrandstof-reserwes. Papier slyk geproduseer deur papier meule is hoog in lignosellulose en verteenwoordig 'n grootliks onontginde grondstof vir etanol produksie. Die doel van die studie was om vas te stel wat die samestelling, fermenteerbaarheid, optimale papier slyk en ensiem ladings is vir die vervaardiging van etanol uit papier slyk. Die inligting was gebruik om 'n model van die proses in Aspen Plus® te ontwikkel. Die massa-en energiebalanse wat verkry is van die Aspen Plus® model was gebruik om toerusting spesifikasies te ontwikkel wat gebruik was om toerusting kostes te bereken. ‘n Tegno-ekonomiese model is ontwikkel om die ekonomiese lewensvatbaarheid van die gelyktydige versuikering en fermentasie proses “SSF” wat gebruik maak van papier slyk as grondstof te assesseer. Nege papier slyk monsters verkry vanaf Nampak Tissue (Pty) Ltd. is geëvalueer in terme van etanol produksie. Die monsters wat die hoogste en laagste etanol konsentrasies opgelewer het, is geselekteer vir optimalisering omdat dit toegelaat het vir die vasstelling van etanol konsentrasies en opbrengste, uitgedruk as persentasie van die teoretiese maksimum, wat verwag kan word in industrie. Reaksie oppervlak metodologie “RSM” is gebruik om wiskundige modelle te ontwikkel om die impak van papier slyk lading en sellulase dosis op etanol produksie en etanol opbrengs te assesseer. Die RSM is aangevul met 'n multi effek optimiserings benadering wat 'n wenslikheid funksie inkorporeer om die optimale papier slyk lading en sellulase dosis in gevoerde-enkellading SSF kulture te bepaal. Die multi effek optimalisering het getoon dat 'n optimale papier slyk lading van 21% (w/w) en 'n sellulase dosis van 14.5 FPU g-1 gebruik moet word, ongeag van die papier slyk monster. Die feit dat die optimale ensiem dosis en papier slyk lading dieselfde is ongeag die papier slyk monster, is aantreklik aangesien die SSF proses meer doeltreffend beheer kan word omdat proses veranderinge nie nodig is om die proses te optimaliseer nie. By die optimale papier slyk lading en sellulase dosis kan 'n minimum etanol konsentrasie van 47.36 g l-1 (84,69% van die teoretiese maksimum) verwag word ongeag van die papier slyk wat gebruik word. 'n Ekonomiese evaluasie is gedoen om vas te stel of etanol produksie vanaf papier slyk met behulp van SSF ekonomies lewensvatbaar is. Drie moontlikhede is ondersoek. In die eerste moontlikheid is die inkomste bereken vanaf etanol verkope gekoppel aan die basiese brandstofprys, terwyl in die tweede en derde moontlikhede, LPG by die papier meul vervang is met anhidriese en 95% etanol onderskeidelik. In al die gevalle was daar gebruik gemaak van papier slyk voer tempo’s van 15, 30 en 50 t d-1. Die produksie van etanol uit papier slyk vir verkope (moontlikheid 1) het gelei tot hoër IRR en die NPV waardes, sowel as korter terugverdien tydperke, in vergelyking met die vervanging van LPG by die papier meul (moontlikhede 2 en 3). Met ʼn ensiem koste van $ 0.90 gal-1 (R 2.01 litre-1) is IRR-waardes van 11%, 22% en 30% verkry teen papier slyk voer tempo’s van 15, 30 en 50 t d-1 onderskeidelik. 'n Sensitiwiteitsanalise uitgevoer op die totale kapitale belegging en ensiem koste het aan die lig gebring dat 'n SSF proses slegs ekonomies lewensvatbaar is op 'n papier slyk voer tempo van 50 t d-1 ongeag van die variasie in die kapitale belegging. Vir die SSF proses om ekonomies lewensvatbaar te wees, moet die ensiem kostes laer wees as $ 0.70 gal-1 (R 1.56 liter-1) en $ 1.20 gal-1 (R 2.68 liter-1) vir papier slyk voer tempo’s van onderskeidelik 30 en 50 t d-1. Die SSF proses was op 'n papier slyk voer tempo van 15 t d-1 nie ekonomies lewensvatbaar nie, selfs teen 'n ensiem koste van nul. 'n Monte Carlo-simulasie het getoon dat die SSF proses ekonomies lewensvatbaar is met 'n papier slyk voer tempo van 50 t d-1 omdat 'n gemiddelde IRR-waarde van 32% verkry is met 'n waarskynlikheid van 26% om 'n IRR-waarde laer as 25% te verkry. Die SSF proses teen papier slyk voer tempo’s van 30 en 15 t d-1 is nie ekonomies lewensvatbaar nie omdat waarskynlikhede van 70% en 95% onderskeidelik verkry is om IRR-waardes laer as 25% te kry. Daar kan van die studie afgelei word dat papier slyk 'n uitstekende grondstof is vir die produksie van etanol mits 'n papier slyk voer tempo van meer as 50 t d-1 bereik kan word. Die produksie van etanol vanaf papier slyk het die bykomende voordeel dat kweekhuis gasse (GHG) met 42.5% verminder word.

The exploitation of lignocellulosic materials with the aim of producing high value-added products will potentially counteract concerns related to the depletion of fossil resources or exponential population growth. Bioethanol produced from lignocellulosic agriculture residue exhibits promising alternative to the petroleum-based fossil fuel which reduces net emission of greenhouse gases (GHG). But, due to certain technological barriers, the large scale production of lignocellulosic bioethanol has not been successfully commercialized. In this thesis, membrane filtration as an energy efficient separation process with low environmental impact was chosen with a possibility of improvement. Interconnected multi-staged microfiltration submerged membrane bioreactors (MBRs) set-up has been applied in order to separate suspended solids, obtain high concentration of yeast inside the bioreactor, and recover particle-free ethanol stream in a continuous high productivity process. The MBRs were effectively optimized comparing to different constant permeate fluxes of 21.9 LMH, 36.4 LMH, and 51 LMH. Moreover, membrane bioreactor performed effectively at low flux 21.9 LMH up to 262 h comparing to other applied fluxes. During continuous hydrolysis, membrane showed the capability of lignin recovery nearly 70% of medium SS content in all applied flux. Although the conversion rate of total sugars by concentrated cells were similar, yeast cells proved the capability of inhibitor tolerance, and to co-utilize 100% of glucose and up to 89% of xylose, resulted in bioethanol volumetric productivity of 0.78 g ethanol/l per hour 1.3 g ethanol/l per hour and 1.8 g ethanol/l per hour for 21.9 LMH, 36.4 LMH, and 51 LMH respectively. Moreover, the effect of different factors such as filtration flux, medium quality and backwashing on fouling and cake-layer formation in submerged MBRs during continuous filtration was thoroughly studied.

The use of natural sources in economic activities can aid in the resource saving and recycling and reuse of wastes, contributing for a more sustainable world by providing clean technologies in the industrial and agricultural sector in both developed and developing countries. In general, increased and improved global strategies for energy safety, security and mitigation of CO2 emissions from energy production processes are required, especially those aimed at maximizing the energy efficiency by expanding the use of clean energy. This means using fuels that are able to implement the carbon cycle without changing the atmospheric balance (renewable fuels), by developing energy resources in CO2 reduced/neutral systems (Brennan and Owende, 2011; Moraes et al., 2017). The expansion of biofuels production and use is an important issue since it plays primary role in reducing global the climate change. But, in order to insert a new source/technology in the market, several factors are involved such as industrial aspects and economic feasibility, legal restrictions and incentives, international trade, land use, raw material availability and management techniques. At present, ethanol is the main biofuel produced worldwide. Between 2007 and 2015 bioethanol throughput practically doubled, reaching 25 billion gallons per year, even though after 2010 the production was stagnant (AFDC, 2016). This is the result of a number of reasons, to cite: - high dependency on the first-generation crops which need a lot of arable land and compete directly with food/feed production; - need for a complete validation of the lignocellulosic ethanol industry due to unsuitability of the large-scale process because of corrosion problems (mainly in the pretreatment), cost of enzymes, difficult/inhibition of the fermentation step; - difficulty to utilize all lignocellulosic fractions, according to a biorefinery approach, because each biomass has its biological complexity and the related lignocellulosic content/arrangement/recalcitrance changes significantly; - Lacking of investments/incentives (mainly, governmental) after the decrease of petroleum prices occurred at the end of 2014. In fact, based on the type of biomass, bioethanol production is classified as first (raw material saccharine or starch-based – sugarcane and corn); second (lignocellulosic materials); third (microalgal/macroalgal biomass) and fourth (genetically modified cyanobacteria) generation. Sugar cane ensures the lowest bioethanol production costs. In spite of its significant advantage, it is not a viable option for all the regions of the planet owing to climatic and soil limitations (Belincanta et al., 2016). Consequently, countries of the northern hemisphere have been incessantly looking for new technological routes that permit the efficient production of biofuels while respecting environmental and economic sustainability issues, and ‘new’ generations of biomass-to-ethanol processes are proposed. In addition, countries as Brazil have their sugarcane cultivation saturated, i.e., there is no new extensions of arable land to expand significantly the Brazilian ethanol industry. Low production costs are the advantage of first generation bioethanol, with the exception of corn-based one, which has a well-established and economically sustainable technology, while second generation still requires more investigations to become economically competitive, with pretreatment and hydrolysis processes needing to be more effective and largely scalable (Gupta and Verna, 2015). On the other hand, micro and macroalgae have not reached a maturity for designing and operating industrial scale plants yet. Therefore, in the case of third and fourth generation bioethanol, further studies are required to develop a competitive and consolidated technology, taking into account also issues other than technological ones. In third generation bioethanol, microalgae and/or macroalgae biomass are used, which do not have lignin in their cellular structure, and are cultivated with higher growth rates when compared to higher plants. As for this biomass, a suitable process is not available yet, and the related costs cannot be properly estimated. Researchers are currently trying for microalgae: to optimize microalgal productivity and cultivation conditions, as this represents the highest production costs, considering that hydrolysis and fermentation are instead easier compared with lignocellulosics and macroalgae (Jonh et al., 2011; Wei et al., 2013; Hong et al., 2014). Thanks to their high growth rate, and relatively simple biochemical composition (partitioned among carbohydrates, lipids and proteins), microalgae are acknowledged as very promising feedstock for bioethanol production (Chen et al., 2013). Main aspects needing to be developed in this respect are: carbohydrate cultivation (productivity), hydrolysis and ethanolic fermentation and nutrient recycling/recovery from residual medium/biomass. With regard to the open issues recalled above, the aim of this research project has been to address and study how to improve the knowledge and discuss the real potentiality of microalgal biomass as a feedstock for an effective bioethanol production, from a perspective of biomass/carbohydrate productivity (microalgal cultivation) and bioconversion process (hydrolysis and fermentation) in a context of a biorefinery concept. In fact, experimental values about fermentation applications from microalgae are not expanded yet in literature. The topics addressed by this thesis are organized and subdivided in twelve chapters as follows. In Chapter 1, a literature survey to collect and discuss the available information about bioethanol from photosynthetic microorganisms, and to delimit the main lacks to be developed, is done. Chapter 2 shows a basic analysis of an ethanol biorefinery scheme aimed to include microalgal biomass, discussing the main bottlenecks and the processes which must be developed to adequately evaluate the potentiality of this type of biomass for industrial fermentation proposes. Chapter 3 treats specifically of the carbohydrate-rich biomass cultivation from microalgae utilizing nutritional and environmental techniques. Operation mode of microalgae cultivation is discussed as well, and the importance to consider semi-continuous and continuous processes is shown, because batch mode is extensively used but less efficient. Chapter 4 develops a design procedure of a two-unit system composed by a reactor and settler, discussing the influence of operating variables and their limiting values. Specifically, recycle ratio and purge flow rate concepts and effects are extensively studied. In Chapter 5, the carbohydrate cultivation with Synechococcus PCC 7002 is optimized with respect to the carbon source and pH, because a stable pH (greatly influenced by the carbon source) is necessary for this strain and organic buffers exhibit toxicity. An inorganic buffer study (CO2-bicarbonate) is developed and detailed. Chapter 6 shows S. PCC 7002 treating urban wastewater to remove chemical oxygen demand, nitrogen and phosphorous content, thus ensuring a double gain: environmental enhancement and valorization of cyanobacterial biomass. In Chapter 7, continuous cultivation of Chlorella vulgaris in flat-plate photobioreactors to improve carbohydrate productivity is assessed and evaluated using nitrogen limitation as a combination between nitrogen concentration inlet, light intensity and residence time under constant light intensity. Chapter 8 demonstrates that a similar approach used for the continuous cultivation of C. vulgaris is applicable also to Scenedesmus obliquus. Additionally, it is proved that under outdoor conditions (seasonal regime of illumination – summer and winter), a high carbohydrate content can be produced as well. In Chapter 9, the kinetics regarding acidic hydrolysis to biomass solubilization and sugars depolymerization is studied with Chlorella vulgaris biomass. An n order kinetics for biomass solubilization and m order for acid concentration is applied for biomass solubilization, providing values of reaction order and activation energy for microalgae. In addition, a saccharification model based on the Michaelis-Menten model is proposed and validated. Chapter 10 demonstrates how the kinetics considerations determined in the previous chapter can be efficiently applied with the concept of severity factor – CSF (combination between time, temperature and acid concentration). A literature discussion about some assumptions so far considered and the importance to know the biomass nature to determine a coherent range of CSF is provided. Chapter 11 reports ultrasonication as an effective pretreatment method to improve enzyme accessibility and promote a high rate of hydrolysis from Scenedesmus obliquus biomass. Pretreatment time, ultrasonication intensity and biomass concentration are specifically studied in order to minimize the energy consumption since the bottleneck of the pretreatment method is a high energy dissipation. In Chapter 12, ethanolic fermentation is addressed with acidic and enzymatic hydrolysates. A systematic optimization of inoculum concentration and consortium between Saccharomyces cerevisiae and Pichia stipitis is determined. Then, the influence of salinity/matrix characteristics was evaluated to understand possible interferences during fermentation process and exhibited lower biochemical yields than the control conditions. Thus, further fermentations experiments are necessary.
L’obiettivo generale di questo progetto di ricerca è stato di verificare la potenzialità delle microalghe come fonte alternativa di biomassa per la produzione di etanolo. In particolare, sono state discusse teoricamente, sperimentalmente e tramite simulazione di processo la coltivazione, l’idrolisi e la fermentazione della biomassa microalgale. Inizialmente, grazie ad un’ampia ricerca bibliografica ed a prove preliminari effettuate nel Laboratorio Microalghe del Dipartimento di Ingegneria Industriale della Università di Padova si è dimostrato che le specie più promettenti da studiare erano Synechococcus PCC 7002, Chlorella vulgaris e Scenedesmus obliquus, grazie alle loro elevate velocità di crescita e capacità di accumulo di carboidrati, che costituiscono le materia-prima per la produzione di etanolo (fino al 50-60% del peso secco). In particolare, l’attenta analisi della letteratura riguardo a queste specie ha consentito di verificare che: - per la produzione di carboidrati è preferibile sviluppare un processo continuo, perché richiede un solo step, mentre il processo batch ne richiede due, e perciò consente di ottenere produttività significativamente inferiori; - sono disponibili pochi lavori sulla possibilità di usare le microalghe in un processo continuo di questo tipo, mentre sono parecchi i riferimenti al processo batch; - mancano informazioni sulla capacità di produrre carboidrati da parte di S. PCC 7002. In una prima parte del lavoro sono stati quindi pianificati e condotti esperimenti in modalità batch con S. PCC 7002, per studiare come mantenere la stabilità e vitalità della coltura durante tutto il periodo di coltivazione. Si sono rilevati problemi con il controllo del pH, ed é stato approfondito l’uso di bicarbonato come fonte di carbonio assieme ad un tampone inorganico, dimostrando in un primo lavoro che il suo impiego è efficiente per la produzione di biomassa ma insufficiente per accumulare un alto contenuto di carboidrati, a causa di una significativa inibizione osmotica causata dall’alta concentrazione di sodio in soluzione. D’altro canto, l’applicazione di un tampone con sostanze organiche, generalmente usato nella coltivazione di microalghe e cianobatteri, ha evidenziato notevoli fenomeni di tossicità per questa specie. Al contrario, il tampone inorganico CO2-bicarbonato messo a punto successivamente è stato capace di garantire la stabilità del pH durante 12 giorni di coltivazione, ed ha consentito di ottenere 6 g L-1 di biomassa (peso secco) con circa il 60% di contenuto di carboidrati. La coltivazione in continuo di C. vulgaris in un fotobioreattore piatto e sottile è stata studiata per verificare la produzione di carboidrati secondo questa modalità operativa. Il lavoro ha evidenziato l’importanza della riduzione della concentrazione di azoto in entrata al reattore, che va rapportata ai valori di intensità di luce e tempo di residenza per massimizzare la produzione di carboidrati. Si sono misurati valori massimi per la produttività di biomassa e di carboidrati pari a 0.7 e 0.37 g L-1 giorno-1. La stessa procedura é stata usata nello studio del comportamento di S. obliquus, per vedere se l’approccio era valido anche durante la coltivazione all’aperto, simulando la fornitura della luce in modo stagionale. S. obliquus ha mostrato una produttività quasi tre volte maggiore che Chlorella, raggiungendo valori di 0.8 g L-1 giorno-1 (con luce costante) e di 0.71 g L-1 giorno-1 (nell’estate). Questa produttività di carboidrati, se estrapolata a dimensioni industriali, consentirebbe di ottenere tra 45–100 tonbiomass ha-1 anno-1, ben di più di quanto prodotto con le fonti tradizionali di carboidrati. Un sistema reattore-sedimentatore con riciclo parziale di biomassa è generalmente usato a livello industriale in processi di coltivazione e/o fermentazione. Questo sistema fornisce semplicità e diversi vantaggi per la produzione su larga scala. É stato quindi messo a punto un modello per la simulazione di tale processo, nel caso specifico delle microalghe, per verificare l’influenza dei gradi di libertà (tempo di residenza, rapporto di riciclo della biomassa, età della biomassa e sua velocità di sedimentazione) sulle prestazioni. I principali risultati sono: - la definizione di un rapporto di riciclo minimo Rmin, di un intervallo operativo per la stessa variabile, e di un valore massimo per la portata di spurgo di biomassa Fwmax; - la dimostrazione che la perdita di biomassa dalle sommità del sedimentatore abbassa significativamente le prestazioni del sistema; - la costruzione di grafici adimensionali che legano R a θc/θ e FI/FW (età della biomassa/tempo di residenza, e rapporto tra le portate di ingresso e di spurgo); - il confronto fra il modello rigoroso messo a punto ed il modello semplificato generalmente considerato in letteratura. Synechococcus è stata coltivata in acque reflue urbane (sintetiche e reali, con valori di COD pari a 340.0 ± 14.1 mg L-1, di azoto totale pari a 31.0 ± 1.4 mg L-1, e di fosforo totale a 8.20 ± 0.99 mg L-1), con l’obbiettivo di ottenere la depurazione da questi inquinanti. Questa specie è stata molto efficiente nella rimozione di COD, azoto e fosforo totale, raggiungendo valori sotto i limiti di legge in due giorni di coltivazione. L’acqua reflua sintetica ha evidenziato una limitazione dei micronutrienti quando la concentrazione di COD era elevata, differentemente dell’acqua reflua reale, in cui Synechococcus è cresciuta più velocemente. Successivamente, l’idrolisi e la fermentazione di biomassa microalgale sono state studiate con riferimento ai processi di saccarificazione acida ed enzimatica, e con riferimento ai microorganismi Saccharomyces cerevisiae e Pichia stipitis, rispettivamente. L’idrolisi acida, con acido solforico 0-5% v/v, è stata condotta a diverse temperature (110-130 °C) e tempi di reazione (0-60 min) partendo da 100 g L-1 di concentrazione di biomassa (Chlorella vulgaris). Gli zuccheri idrolizzati sono stati recuperati con un valore massimo pari al 92%, ottenuto con il 3% di acido e 20 min di reazione a 120 °C. La solubilizzazione di biomassa ha esibito un ordine di reazione n = 3.63 ± 0.18 ed un’energia di attivazione pari a 41.19 ± 0.18 kJ/mol. Questi valori sono significativamente diversi di quelli trovati per l’idrolisi di matrici lignocellulosiche, generalmente considerata di primo ordine con Ea = 100-200 kJ/mol, e dimostrano che la biomassa microalgale è più suscettibile al trattamento termico catalizzato all’acido in confronto ai lignocellulosici. Un’equazione basata sulla cinetica di Michaelis-Menten modificata per tenere conto della concentrazione di acido è riuscita a modellare tutti i risultati sperimentali, con un valore della costante di semi-saturazione per la biomassa PolKM pari al 42% della concentrazione iniziale, e con una resa di fermentazione di circa il 60%. Prima di realizzare l’idrolisi enzimatica, si é reso necessario procedere ad un’ottimizzazione del pretrattamento della biomassa. È stata studiata l’ultrasonicazione applicando un piano statistico di sperimentazione su tre livelli con 3 esperimenti centrali (in tutto si sono condotte 11 prove). Le variabili ottimizzate sono state l’intensità, il tempo di pretrattamento e la concentrazione di biomassa. I risultati hanno dimostrato che l’intensità e il tempo di trattamento sono più importanti e consentono di ottenere un recupero degli zuccheri superiore al 90%, in 4-8 ore. Si é visto che l’energia spesa nel processo di ultrasonicazione non è direttamente collegata con l’efficienza dell’idrolisi, per cui questa può essere condotta efficientemente anche riducendo il consumo di energia nel pretrattamento. Infine, si sono eseguiti esperimenti di fermentazione dell’idrolizzato ad etanolo con le due specie menzionate (S. cerevisiae e P. stipitis). Si sono ottimizzati la concentrazione di inoculo (7.5 g L-1) ed il consorzio (25% Pichia + 75% Saccharomyces) per avere una produttività tra 5 e 10 g L-1 ora-1 (prossimo al valore industriale). Si è però visto che le velocità di fermentazione sono però più basse a causa di una inibizione dovuta alla accresciuta salinità dell’idrolizzato, un fattore. Per questo motivo, la parte di fermentazione necessita di essere più approfondita al fine di validare l’impiego di questo tipo di biomassa a livello industriale.

This study reveals the development of new 3-D support for ethanol production by Saccharomyces cerevisiae W303. The production of bioethanol by immobilising this organism had been demonstrated to have greater advantages over a suspended culture of free cells of S.cerevisiae W303. The production of ethanol is proportional to the growth of yeast, so preparing suitable supports with unlimited spaces that permit the cell proliferation are crucial for a long term continuous operation. The 3-D scaffold prepared from high internal phase emulsion (HIPE) offers good mechanical strength, and has a high surface area for allowing monolayer cell proliferation which was necessary in order to avoid additional stresses for the nutrient and oxygen transfer in the micro-environment. The enhanced porosity of this 3-D scaffold that was characterised by highly interconnected pores, not only promoted the dynamic condition in the monolith, but also facilitated easy flushing of dead cells and metabolic product. This study reveals that sulphonated polyHIPEs, a highly hydrophilic polymer which had pore and interconnect sizes of 45 μm and 16 μm respectively, had shown good bio-compatibility with the model organism, subsequently allowing its growth and glucose conversion. The ethanol productivity in the microreactor was greatly enhanced to 4.72 gL-1h-1, being over 12 times higher than that observed in the suspended shake flask culture (0.41 gL-1h-1) by free cell S.cerevisiae W303, despite only 60.1% of glucose being consumed. Since the remaining sugar must be kept low, the glucose utilization was further enhanced by introducing the two-stage reactor in series. The consumption of glucose was enhanced by 20.1% (compared to single stage reactor), where nearly 72.2 % of the supplied glucose was converted per pass during the pseudo steady state condition. This, on the other hand, increased the ethanol productivity to 5.84 gL-1h-1, which was 14 fold higher than the productivity obtained in the shake flask culture. The increment might be associated with the altered metabolical functions in the immobilised cells. This alteration is attributed to the reduction of the diffusion path of the growth nutrient (e.g: carbon, nitrogen and oxygen) that enhanced the availability and promoted the growth of yeast in the microreactor, thus enhancing the catalytic conversion of glucose to ethanol.

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

Social, economic and political pressures have driven the development of renewable alternatives to fossil fuels. Biofuels, such as bioethanol, have proved to be successful alternatives. Mature technologies are crop-based, but this has brought criticism due to the conflicting use of land for fuel versus food production. Therefore, bioethanol production technologies have shifted to utilising the sugars that derive from the degradation of lignocellulosic biomass. The thermophilic, Gram-positive bacterium, Geobacillus thermoglucosidasius, can naturally utilise a large fraction of these sugars, and metabolic engineering has been used to create a strain that produces ethanol as the major product of fermentation. This strain, G. thermoglucosidasius TM242 (Δldh, Δpfl, pdhup), does however, produce small but significant quantities of acetate, an undesirable by-product of fermentation. Therefore, acetate metabolism in the G. thermoglucosidasius TM242 strain was the focus of this study. During fermentation, ethanol is generated from the central metabolite acetyl-CoA through the activities of a bifunctional enzyme: aldehyde dehydrogenase/alcohol dehydrogenase (ADHE). On the other hand, acetate is generated from acetyl-CoA through catalysis by phosphotransacetylase (PTA) and acetate kinase (AK). Acetate metabolism in G. thermoglucosidasius TM242 was studied in this project by investigating the enzyme activities governing flux from acetyl-CoA, and the feasibility of reduced acetate production was investigated by a pta-deletion strategy. This thesis reports the characterisation of PTA and AK, by studying activities from both native cell lysates and recombinantly expressed proteins. The results indicate that the activities of PTA and AK are greater than those of ADHE, suggesting that the potential metabolic flux is greater towards acetate production than to ethanol. However, the ethanol yield from G. thermoglucosidasius TM242 fermentations is greater than that of acetate, suggesting the existence of a regulatory mechanism controlling acetyl-CoA flux. Several possible regulatory mechanisms were studied in this project and are reported here. The viability of creating a strain that reduces acetate accumulation, and potentially increases ethanol yields, was investigated and reported in this thesis. The gene encoding PTA was deleted from G. thermoglucosidasius TM242, and the resulting strain was characterised. The Δpta strain had approximately 5% of the PTA activity measured in TM242, but acetate was still generated from pentose and hexose fermentations. Additional phosphotransacylase (PTAC) enzymes were discovered in G. thermoglucosidasius TM242 that could catalyse the conversion of acetyl-CoA and orthophosphate to acetyl-phosphate and CoA. A series of PTAC null strains were created and analysed, the results of which indicated that phosphotransbutyrylase (PTB) could be involved in acetate production in vivo. It was discovered that the cell lysates of G. thermoglucosidasius strains carrying deletions to both pta and ptb could no longer catalyse the conversion of acetyl-CoA and orthophosphate to acetyl-phosphate and CoA. However, these strains still accumulated acetate, suggesting the presence of alternative acetate-producing pathways in this organism. In addition, G. thermoglucosidasius strains carrying deletions to both pta and ptb could ferment glucose but not xylose, suggesting that the production of ATP by the PTA-AK pathway is crucial for micro-aerobic growth on pentose sugars.

The second-generation bioethanol production in which lignocellulosic material is used as feedstock faces some difficulties. Lignocellulosic materials have to be pretreated prior to fermentation. In the pretreatment stages several inhibitory compounds, which can negatively affect the metabolic and physiologic activity of the microorganism used, Saccharomyces cerevisiae, are released. Moreover, wild strains of Saccharomyces cerevisiae cannot co-utilize the hexose and pentose saccharides present in the lignocellulosic substrate. In this study, reverse membrane bioreactor (rMBR) was applied to address the difficulties faced in the secondgeneration ethanol production. Semi-synthetic medium and pretreated wheat straw slurry containing different level of glucose, xylose and inhibitor concentrations were fermented in rMBR using genetically-modified xylose-consuming S. cerevisiae. The diffusion rate of different substrates and metabolites during fermentation were measured and analyzed. The results showed that the application of rMBR facilitated simultaneous utilization of hexose and pentose sugars and enhanced the cell tolerance of the inhibitor present in the medium.

Le remplacement des solvants organiques classiques par une nouvelle génération de solvants moins toxiques et moins polluants est un défi majeur pour l'industrie chimique. Les liquides ioniques (LIs) ont été largement identifiés comme substituts intéressants aux solvants traditionnels. Le but de ce travail est d'étudier la solubilité des sucres ou des constituants issus de la biomasse dans les liquides ioniques afin de pallier au manque de données expérimentales sur les équilibres de phases de systèmes {sucres + LIs} ou {biomasse + LIs}. Les données de solubilité ont été corrélées avec succès en utilisant les modèles thermodynamiques NRTL et UNIQUAC. Cette étude démontre que la méthode de l'antisolvant est une bonne technique pour l'extraction des sucres des LIs. Par conséquent, les liquides ioniques peuvent être facilement recyclés pour être réutilisés. Les natures fondamentales des interactions entre les sucres et les liquides ioniques ont été définies en utilisant le calcul ab initio. Les résultats obtenus par simulation sont en accord avec les données expérimentales et indiquent que les liquides ioniques interagissent avec les sucres par liaisons hydrogène. La seconde partie de ce travail met en évidence que le prétraitement du miscanthus avec les liquides ioniques permet d'obtenir une bonne production d'éthanol (jusqu'à 150 g d'éthanol par kg de miscanthus). Les résultats montrent que les liquides ioniques sont des solvants performants dans le domaine de la conversion des matières premières issues de la biomasse en biocarburant. Ainsi, l'application à l'échelle industrielle de ces procédés d'extraction de la cellulose pourrait être d'un grand intérêt
The replacement of conventional organic solvents by a new generation of solvents less toxic, less flammable and less polluting is a major challenge for the chemical industry. Ionic liquids have been widely promoted as interesting substitutes for traditional solvents. The purpose of this work is to study the solubility of carbohydrates or biomass based materials in ionic liquids in order to overcome the lack of experimental data on phase equilibria of {biomass or carbohydrate-ILs} mixtures. Solubility data were successfully correlated using NRTL and UNIQUAC thermodynamic models. It was found that the antisolvent method is a good technique for the extraction of carbohydrates from ILs. Ionic liquids could be then recycled successfully for reuse. The fundamental natures of the interaction between carbohydrates and ionic liquids were investigated using ab initio calculations. The theoretical results are in good agreement with experimental data. It was concluded that ionic liquids mainly interact with carbohydrates via hydrogen bonding formation. This confirms that the process of dissolution and regeneration of cellulose in ionic liquids is accompanied only with a physical change. The preatreatment of miscanthus with ionic liquids resulted in the regeneration of amorphous, porous cellulose almost free of lignin, which is suitable for enzymatic hydrolysis and fermentation processes. A successful ethanol production was obtained with an overall ethanol yield reached up to 150 g ethanol kg-1 miscanthus. This indicates the high performance of ionic liquids in converting biomass feedstocks into biofuel. Indeed, applying the cellulose extraction processes on the industrial scale could be of great interest

This thesis investigated the potential of sorghum as a feedstock source for bioethanol production in Nigeria. Sorghum is a cereal with high tolerance for varied environmental and climatic stresses. It can produce starch-rich grains, sweet stalk juice and high lignocellulosic biomass, depending on the crop variety and cultivation location. Nigeria is the third largest sorghum producer worldwide, but less than 10% of sorghum produced has commercial applications. For example, the grains represent a staple food source or can be utilised as a brewing adjunct. The stalk juices are used in syrup production while the green field residues (bagasse) are partly used in forage production and fencing but mostly left in field for burning. This thesis has shown that sorghum crops have alternative uses in liquid biofuel production. In this study, SSV2, KSV8 and KSV3 sorghum cultivars were cultivated under rain fed conditions without chemical fertilizers in Kano and Kaduna, Nigeria. The climate in Kano is relatively warmer and drier than Kaduna, with Kano favouring higher biomass yields and Kaduna favouring higher sugary stalk juice yield. Total dry bagasse yields in Kano were 29 t/ha, 33 t/ha and 37 t/ha for SSV2, KSV8 and KSV3 crops, respectively. For crops harvested in Kaduna, the yields were 24 t/ha and 31 t/ha for SSV2 and KSV8, respectively. Furthermore, raw stalk juice yields of 25000 L/ha, 23300 L/ha and 22600 L/ha were obtained for SSV2, KSV8 and KSV3 in Kano and 25500 L/ha and 24500 L/ha for SSV2 and KSV8 in Kaduna. Total fermentable sugar (analysed by HPLC) in Kano-grown SSV2, KSV8 and KSV3 sorghum juices were 144 g/L, 66 g/L and 104 g/L, respectively, compared with 162 g/L and 88 g/L for SSV2 and KSV8 juices from Kaduna-grown sorghum. Fermentations of different sorghum juices were performed with Saccharomyces cerevisiae (without exogenous nutrient supplementation) and produced ethanol yields (measured by GC-MS) of 65 g/L, 36 g/L and 62 g/L for SSV2, KSV8 and KSV3 juices in Kano while Kaduna juice fermentations produced 81 g/L and 52 g/L ethanol for SSV2 and KSV8, respectively. Supplementation of sorghum juices with additional nutrients improved fermentation performance. Floured husked grains from different sorghum cultivars were separately mashed with a combination of various enzyme cocktails, followed by fermentations of the mashes with S. cerevisiae. Ethanol yields of 355 L/t, 421 L/t and 379 L/t were obtained for SSV2, KSV8 and KSV3, respectively, and this fermentation performance was also verified by CO2 gas evolution as observed by the ANKOMRF gas monitoring system. Another yeast, Pichia stipitis showed lower corresponding ethanol yields when fermenting sorghum grain mashes. Experiments were also conducted to convert sorghum lignocellulose residues (bagasse) to ethanol. Pre-treatment of the bagasse fractions followed by detoxification of the enzymatic hydrolysates with calcium hydroxide over-liming and charcoal filtration showed ethanol yields of 23 g/L and 20 g/L for SSV2 and KSV3 (Kano) on fermentation with Pachysolen tannophilus (without nutrient supplementation) while S.cerevisiae yielded corresponding ethanol of 21 g/L and 19 g/L respectively Results from this research have shown that whilst sorghum cultivar SSV2 is a very favourable feedstock for bioconversion to ethanol from juice in Kaduna and bagasse in Kano, the KSV8 cultivar is better suited when exploiting husked grain starch as source for bioethanol production in Nigeria.

The continuous depletion of fossil fuel reserves, the global agenda on climate change and threats to energy security have led to increased global interest in the exploration, production and utilisation of bioenergy and biofuels. Access to modern bioenergy carriers derived from the efficient conversion of locally available biomass resources is indispensable for economic growth, rural development and sustainable development in developing countries. Deployment of bioenergy/biofuels technologies has significantly varied across the globe. The least developed countries (LDCs) and developing countries are still highly dependent on traditional biomass technologies with low conversion efficiency, which are typically associated with significant environmental and health impacts. Meanwhile, emerging economies and developed countries are progressively promoting biofuel industries and international trade. They are also engaged in making biofuels a sustainable proposition by developing sustainability criteria. The goal of this thesis is to address the sustainability of bioethanol production derived from one of the key feedstocks/energy crops: sugarcane. This will be done by analysing different development contexts and environmental constraints in terms of geopolitical situation, economic development and state-of-the-art technologies in agro-industrial development. Life cycle assessment (LCA), system studies, and techno-economic optimisation are the main methodological approaches applied in the thesis. The thesis primarily addresses three key questions for analysing the sustainability of bioethanol production. The first research question investigates the key parameters affecting the sustainability of bioethanol production and use in a low-income country using the case of Nepal. The net energy and greenhouse gas (GHG) balances are identified to be the main sustainability criteria of the sugarcane-molasses bioethanol (Paper I and II). Results of the lifecycle studies show that the production of bioethanol is energy-efficient in terms of the fossil fuel inputs required to produce the renewable fuel. Greenhouse gas (GHG) emissions from the production and combustion of ethanol are also lower than those from gasoline. The study also evaluates the socio-economic and environmental benefits of ethanol production and use in Nepal, concluding that the major sustainability indicators are in line with the goals of sustainable development (Paper III). Assessment of the biofuel (molasses-bioethanol) sustainability in Nepal is the first of its kind in low-income countries, and serves also the purpose of motivating the assessment of ethanol production potential in other LDCs, particularly in sub-Saharan Africa. The second question critically evaluates methodologies for accounting the lifecycle GHG emissions of Brazilian sugarcane ethanol in European and American regulations, depicting commonalities and differences among them (Paper IV). GHG emissions are becoming increasingly important as part of sustainability criteria in the context of the expansion of biofuel production and international trade. However, different methodologies still lead to quite different results and interpretation. To make this an operational criterion for international comparisons, it is necessary to establish unified methodological procedures for accounting GHG emissions. The thesis identifies the major issues as  N2O emissions from agricultural practices, bioelectricity credits in fuel production, and modelling approaches in estimating emissions related to direct and indirect land use change (LUC & iLUC), that need to be addressed for establishing methodological coherences. The third research question investigates how the sugarcane bioethanol industry can be developed in terms of energy security and the diversification of energy sources. The case of complementarity between bioelectricity and hydropower is evaluated in the cases of Nepal and Brazil and presented in Paper V. Bioelectricity could offer a significant share of electricity supply in both countries provided that favourable political and institutional conditions are applied. Finally, in order to find the choice of technological options for the production of second generation (2G) bioethanol and/or of bioelectricity, a techno-economic optimisation study on the bulk of sugarcane bio-refineries in Brazil is carried out in Paper VI, taking into account the entire lifecycle costs, emissions, and international trade. The study shows that it is worthwhile to upgrade sugarcane bio-refineries. Energy prices, type of power generation systems, biofuel support and carbon tax, and conversion efficiencies are the major factors influencing the technological choice and potential bioethanol trade. In short, this dissertation provides insights on the sustainability of the bioethanol production/industry and its potential role in the mitigation of climate change, improved energy security and sustainable development in different country contexts, as well as methodological contributions for assessing the sustainability of biofuels production in connection with energy and climate policies.
Intresset för ökad exploatering, produktion och användning av bioenergi och biobränslen har föranletts av den kontinuerliga utmattningen av fossila bränslen, den globala agendan för att motverka klimatförändringar samt hoten mot energisäkerheten. Tillgången till moderna bioenergibärare, effektivt framställda från lokal råvara, är grundläggande för ekonomisk tillväxt, landsbygdsutveckling samt för hållbar utveckling i utvecklingsländer. Användandet av bioenergi- och biobränsleteknologi har varierat markant världen över. De minst utvecklade länderna (LDCs) samt övriga utvecklingsländer är fortfarande beroende av traditionella biomassabaserade tekniker till stor utsträckning. Dessa tekniker har låg effektivitet och är ofta sammankopplade med stora miljö- och hälsoskador. Samtidigt främjar tillväxtekonomier och utvecklingsländer biobränsleindustrin och internationell handel progressivt. Länderna arbetar även för att biobränslen ska bli ett hållbart alternativ genom att utveckla hållbarhetskriterier. Den här avhandlingens mål är att adressera hållbarheten hos bioetanolproduktion från sockerrör, en av bioetanolens nyckelråvaror. Målet kommer att nås genom analyser av industrins nationella utvecklingsmiljö samt miljö- och klimatmässiga begränsningar som härstammar från den geopolitiska situationen och den ekonomiska tillväxten i landet, samt analyser av teknologier i den agro-industriella utvecklingen. De huvudsakliga metoder som använts är livscykelanalys (LCA), systemstudier och tekno-ekonomisk optimering. Avhandlingen adresserar primärt tre nyckelfrågor för att analysera hållbarheten hos bioetanolproduktion. Den första forskningsfrågan belyser hur nyckelparametrar påverkar hållbarheten hos produktion och användning av bioetanol i låginkomstländer, med fallstudien Nepal som utgångspunkt. Nettoenergi- och växthusgasbalanser identifieras som de huvudsakliga hållbarhetskriterierna för sockerrör-melass-baserad bioetanol (Artikel I och II). Livscykelstudiernas resultat visar att produktionen av bioetanol är energieffektiv sett från den mängd fossila bränslen som produktionen av förnybart bränsle krävt. Växthusgasutsläppen från produktion och förbränning av etanol är dessutom lägre än utsläppen från bensin. Studien utvärderar de socio-ekonomiska och miljö- och klimatmässiga fördelarna med produktion och användning av etanol i Nepal. Slutsatsen är att indikatorerna för hållbarhet ligger i linje med målen för hållbar utveckling (Artikel III). Bedömningen av biobränslens (melass-baserad etanol) hållbarhet i Nepal är den första studien i sitt slag för låginkomstländer. Studien motiverar dessutom en bedömning av potentialen för etanolproduktion i andra LDCs, speciellt i de afrikanska länderna söder om Sahara. Den andra forskningsfrågan kräver en kritisk utvärdering av metoderna för hur livscykelutsläpp från brasiliansk sockerrörsetanol redovisas i europeiska och amerikanska regleringar (Artikel IV). Artikeln, som påvisar likheter och skillnader mellan regionerna, visar att växthusgasutsläpp blir en mer och mer viktig del i hur hållbarhetskriterier definieras när expansionen av biobränsleproduktion och internationell handel diskuteras. Olika metoder för redovisningen av växthusgasutsläpp leder dock till mycket olika resultat och tolkningar. Det är nödvändigt att etablera en enhetlig metod för redovisning av växthusgasutsläpp för att skapa ett kriterium som möjliggör internationella jämförelser. Avhandlingen identifierar de mest beaktansvärda problemen för att etablera en enhetlig metod: N2O-utsläpp från jordbruksprocesser, tillgodoräknande av bioelektricitet inom bränsleproduktion, samt modelleringsmetoder för att uppskatta utsläpp relaterade till direkt och indirekt landanvändning (LUC och iLUC). Den tredje forskningsfrågan utreder hur industrin för sockerrörsbioetanol kan utvecklas från ett energisäkerhetsperspektiv, med speciell hänsyn till diversifieringen av energikällor. I Artikel V presenteras hur bioelektricitetsproduktion och vattenkraft kan komplettera varandra i fallen Nepal och Brasilien. Bioelektricitet skulle kunna bidra markant till tillförseln av elektricitet i båda länderna under förutsättning att de politiska och institutionella förutsättningarna är fördelaktiga. Slutligen utförs en tekno-ekonomisk studie för att identifiera den optimala teknologin för produktion av andra generationens (2G) bioetanol och/eller bioelektricitet. Studien görs för merparten av sockerrörsbioraffinaderierna i Brasilien och utgör Artikel VI. Studien tar fullskaliga livscykelkostnader i beaktande samt utsläpp och internationell handel. Studien visar att det är värt mödan att uppgradera befintliga sockerrörsbioraffinaderier. De dominerande påverkansfaktorerna för valet av teknologi och potentialen för bioetanolhandel är energipriser, typ av kraftproduktionssystem, biobränslestöd och koldioxidskatt, samt processernas effektivitet. Kortfattat behandlar den här avhandlingen bioetanolproduktionens och bioetanolindustrins hållbarhet. Avhandlingen ger insikt i dess potentiella roll för att motverka klimatförändringar, förbättra energisäkerhet samt främja hållbar utveckling i olika nationella sammanhang. Avhandlingen bidrar dessutom med metodutveckling i hur hållbarheten av biobränsleproduktion bedöms inom ramen för energi- och klimatpolicy.

QC 20130813

>Magister Scientiae - MSc
The depletion of oil reserves and the constant discharge of greenhouse gasses (GHG) that are associated with global warming have forced both political and scientific sectors to pursue alternative, renewable and sustainable fuels that will be blended with petrol and ultimately replace it as the fuel of choice. Bioethanol is a form of fuel that is obtained from natural materials such as biomass. Starch and sugar containing materials are the primary carbon sources for bioethanol production and a range of feedstocks are currently being exploited for this purpose worldwide.This study was aimed at measuring, comparing and analyzing fermentable sugars liberated by sorghum and three other grain crops (maize, barley and wheat) that are grown in South Africa and subsequently analyze ethanol yield after fermentation. Starch was extracted from sorghum, maize, barley and wheat via hot water treatment and hydrolyzed by use of !-amylase, gluco-amylase and a cocktail of both enzymes under various conditions to determine optimum hydrolysis conditions. The resultant liberated soluble sugars were measured with a pocket refractometer and High Performance Liquid Chromatography (HPLC) respectively. Hydrolysates obtained under optimum conditions were fermented with various ethanol producing microbial strains and a high-performing strain was selected. The selected high-performing strain (Saccharomyces cerevisiae NT 53) was used to ferment different grain hydrolysates (sorghum, maize, barley and wheat).The working volumes of the solutions were increased ten-fold (small-scale) and experiments were performed using sorghum grains as substrates and alcohol content was measured with an Alcolyzer Wine M instrument. The optimum hydrolysis conditions for the grain crops were determined and it was found that the enzymes performed well at 70°C and starch was hydrolyzed within the first hour.Sixty grams per litre (60 g/L) of grain solution produced a maximum of 50.8 g/L of glucose when treated with the cocktail treatment. However gluco-amylase facilitated a similar production, at 47.8 g/L glucose. Sorghum and maize produced high glucose amounts and subsequent ethanol amounts, and maximum fermentation efficiencies of 87 % and 98 % respectively when fermented with the high performing NT 53 strain. The NT 53 strain was compared with commercial baker’s yeast and they yielded similar ethanol amounts across the grain types. Under small-scale conditions, sorghum retained the consistency of yielding similar glucose amounts compared to laboratory-scale (50ml) conditions and when analyzed with the Alcolyzer, sorghum yielded a maximum alcohol content of approximately 2 % v/v. This study also showed that gluco-amylase alone was sufficient for starch hydrolysis and sorghum a more favourable and less expensive crop for ethanol production in South Africa.

Waste paper and cardboard is a potential resource for producing bioethanol. In this work, bioethanol production processes from various waste papers (newspaper, office paper, cardboard and magazine) using two enzyme alternatives (Celluclast 1.5L supplemented with Novozyme 188 and Cellic Ctec 1) were evaluated from technological, economic and environmental standpoints. Laboratory experiments were conducted to analyse the composition of the waste papers and to assess the carbohydrate yields from enzymatic saccharifications at relatively high-solids loading (15% w/w). Kinetic models were adapted for the glucan and xylan hydrolyses in this study and were validated by experimental data. The kinetic models provide further insights on enzymatic hydrolysis at high-solids loadings and predictions of the enzymatic digestibilities of the four types of paper. The experimental data, together with published data, were applied in process design models using the simulation software ASPEN PLUSTM to explore the techno-economic aspects of potential conversions of waste papers to bioethanol. The mass and energy flows and the pollutant emissions estimated from the process simulations were used in life cycle environmental analyses (LCAs) to provide insight on the potential environmental pros and cons of converting waste papers to bioethanol. This research has demonstrated that several pathways to bioethanol production from waste paper are economically and environmentally competitive with conventional petrol as a transport fuel. They can also offer environmentally favorable or neutral profiles when compared with the alternative waste paper management options of recycling or incineration with energy recovery. Simplistic, general assumptions that converting waste paper to bioethanol is either ‘good’ or ‘bad’ are not supported by this research. Instead, this study has demonstrated that detailed techno-economic and environmental profiling work, such as that carried out here, is essential to identifying the most beneficial approaches to developing valuable and environmentally favorable production of bioethanol from waste papers.

>Magister Scientiae - MSc
Lignocellulosic biomass, a waste component of the agricultural industry, is a promising source for use in bioethanol production. Due to a complex structure, the synergistic action of lignocellulosic enzymes is required to achieve complete digestion to fermentable sugars. This study aimed to isolate, identify and characterise novel lignocellulase producing bacteria from thermophilic straw-based compost (71°C). Colonies with different morphological characteristics were isolated and screened for lignocellulosic activity. A facultative aerobic isolate RZ1 showed xylanase, cellulase and lipase/esterase activity. In addition to these activities, it was also able to produce proteases, catalases, amylases and gelatinases. RZ1 cells were motile, rod-shaped, Gram positive and endospore forming. The growth temperature of isolate RZ1 ranged from 25-55°C with optimal growth at 37°C. The 16S rRNA gene sequence was 99% identical to that of Bacillus subtilis strain MSB10. Based on the biochemical and physiological characteristics and 16S rRNA gene sequence, isolate RZ1 is considered a member of the species B. subtilis. A small insert genomic library with an average insert size of 5 kb was constructed and screened for lignocellulosic activity. An E.coli plasmid clone harbouring a 4.9 kb gDNA fragment tested positive for xylanase activity. The xyl R gene was identified with the aid of transposon mutagenesis and the deduced amino acid sequence showed 99% similarity to an endo-1-4-β-xylanase from B. pumilus. High levels of xylanases were produced when isolate RZ1 was cultured (37°C) with beechwood xylan as a carbon source. On the other hand, the production of xylanases was inhibited in the presence of xylose. Marked xylanase activity was measured in the presence of sugarcane bagasse, a natural lignocellulosic substrate. While active at 50°C, higher xylanase activity was detected at 37°C. Isolate RZ1 also produced accessory enzymes such as β-xylosidases and α-L-arabinofuranosidases, able to hydrolyse hemicellulose.

This study investigated the potential benefit of introducing Moringa oleifera (MO) and Leucaena leucocephala (LL) leaves as supplementary feed resource for indigenous goats feeding systems in southern Mozambique. The study started with a description of smallholder goat production systems in three resource-poor districts of Mozambique and subsequently investigated the variation and seasonal fluctuations of natural fodder quality in the Changalane district throughout a year period. Thereafter, the effect of tree forage supplementation on growth and reproductive performance of Landim goats were evaluated by simulating a typical feeding system used in the study area. In study one, a survey was conducted in three villages to collect data on indigenous goats and in smallholder husbandry practices in terms of feeding, health and reproduction management. Information from 45-smallholder goat keepers were recorded using a survey, which was complemented by interviews. Results showed that goats were raised under extensive systems, under free grazing. Tethering was a common management practice, with limited supplementation during the dry season. In general, during the dry season the natural pasture were scarcity and poor in quality and consequently does not sustained the energy and protein requirements of ruminants for maintenance and other functions. In study two, the eight key species that were consumed by the goats (namely Sclerocarya birrea, Spirostachys africana, Dichrostachys cinerea, Flueggea virosa, Acacia nigrescens, Acacia nilotica, Panicum maximum and Morus alba) were collected and analysed. Daily energy intake (4.27 ± 0.17 MJ/kg DM vs 3.71 ± 0.41 MJ/kg DM) and crude protein (CP) intake (92.83 ± 16.05 g DM/head/day vs. 59.38 ± 13.12 g DM/head/day) were higher in the rainy season than in the dry season. Daily intake of calcium and phosphorus did not show significant seasonal variations and were below the requirements levels for maintenance of a 20 kg bodyweight goat during the dry season and for the pregnant goat during both seasons. These results showed a need to supplement goats with energy, protein and phosphorus for maintenance, growth and reproduction during the dry season. In study three, the impact of supplementation with LL and MO on the growth and reproduction performance of indigenous goats were evaluated. Fifty-six goats were randomly divided into seven groups, with four castrated males and four females in each group. One group was used as the control group (animals grazing on natural veld without any supplementation), while first three groups were fed with LL and the other three groups with MO tree leaves, respectively. Compared to the control group, both treatments had a significant effect, irrespective of the level of supplementation in terms of overall body weight gain and the final body weight of the bucks. All female reproduction parameters measured for the supplemented groups were superior when compared to the control group. Findings of this study suggest the benefit of using LL and MO tree leaves as supplement for Mozambican goats to overcome the adverse effects of seasonal fluctuations in feed quality on their growth and reproductive performance.
Thesis (PhD)--University of Pretoria, 2019.
Animal and Wildlife Sciences
PhD
Unrestricted

碩士
臺灣大學
生物產業機電工程學研究所
95
Bioethanol is a kind of clean and renewable energy which can be used directly or mixed with gasoline as fuel on vehicles. In this study, sugarcane bagasse which contained 33.34% cellulose, 22.11% semicellulose, and 6.49% lignin was pretreated by 0.25 M sulfuric acid under 95℃ and 1 atm pressure for 60 mins. After pretreatment, dried solid material was hydrolyzed by mixing enzymes of cellulase from Trichoderma reesei C2730 (Celluclast 1.5L) and cellobiase from Aspergillus niger (Novozyme 188) under conditions of pH 4.6, 50℃ in 80 rpm shaking water bath for 24 hours. Different enzyme loadings and substrate ratios were tested to find out the optimum parameters. Hydrolysate was then fermented with Saccharomyces cerevisiae BCRC 21685 under conditions of pH 4.6, 30℃ for 24-48 hours. The effect of additional glucose, sterilization, and detoxification were investigated in this step. As result, 0.52 mg/mL of glucose and 4.29 mg/mL of xylose concentrations were observed in liquid fraction and the content of solid material showed that 91.85% semicellulose and 1.46% cellulose was removed in pretreatment. In hydrolysis step, the enzyme loading of 5 mL Celluclast 1.5L plus 1 mL Novozyme 188 represented the best balance between economy and efficiency. 339.21 mg/mL of yield and 49.25% of conversion ratio were obtained under this enzyme loading with 1% substrate ratio and rising the substrate ratio did not help improving both of them. In fermentation step, without sterilization and detoxification, 26.7 g/L of glucose remains after 48 hours fermentation and ethanol yield was 0.367 g ethanol / g glucose, corresponding to 72% of theoretical ethanol yield. With sterilization and detoxification, glucose was fermented within 24 hours. The ethanol yield was 0.43 g ethanol/g glucose, , corresponding to 84% of theoretical ethanol yield. With evaporation to enhance the glucose concentration, the glucose concentration did not decrease to zero until after 30h. The ethanol concentration was 40.7 g/L, corresponding to 79% of theoretical ethanol yield.

碩士
國立中興大學
食品暨應用生物科技學系所
100
Pineapple is the one of important Taiwan economic fruits which can plant in all seasons. Pulp can be used to eat and process. In our research ,pineapple cake factory can produce 48,000 kilograms pineapple peel in each day. It was wasted that peel used to regard as feed and fertilizer. Our research is anticipated to use liquid squeezed from pineapple cake category peel which were wasted to ferment bioethanol. We expect to get high bioethanol conversion rate in short time and low cost by discussing the factor which can affect fermenting efficiency. The results show that it can squeeze 800 mL liquid from each kilograms pineapple peel in experiment step. And it can also show that adding medium included 10% (v/v) Saccharomyces cerevisiae BCRC 22460 ferment 24 hour can get about 4.5~ 5% (v/v) ideal ethanol concentration in condition without controlling pH value, adding nitrogen source and maintaining room temperature. The pH value have not significantly changed in period of the experiment. Then we use small stirring fermenter to imitate the condition of producing considerable bioethanol; it show that the formula made from ethanol fermenting experiment can be suitable in stirring fermentor. And finally, due to reducing the cost to incubating new medium and consuming time, we use fermentation broth originated from previous fermented experiment. Finally results show that the method is feasible. If we use our methods, factory can produce about 700,000 liters bioethanol each day. According to all experiments, we can explain that the liquid squeezed form pineapple peel can ferment right away. Besides it have potential to develop, it can be executed to produce ethanol by larger fermentor. We expect that it can evacuate by comparing with higher efficiency continuous fermenter in future and finally it can establish low cost, higher efficiency objective model to achieve both of environmental protection and solving energy problem.

碩士
國立臺灣科技大學
化學工程系
100
Bioethanol has become an important alternative energy source. Bioethanol production from the yeast Yarrowia lipolytica biomass was studied. The effects of biomass to acid solution ratio (1:8 – 1:15) temperature (90 - 150oC) and H2SO4 concentration (2 - 15% w/w) on the saccharification of biomass at a hydrolysis time of 1 h were investigated. A maximum glucose concentration of 35.89 g/L can be produced from defatted biomass, biomass to acid solution ratio equal to 1:10 and using 6% H2SO4 at 120oC. Subcritical water (SCW) pretreatment has negligible effect on maximum glucose concentration achievable. Only 14.53 g/L glucose can be produced using 6% H2SO4 at 120oC if un-defatted biomass was used. The highest ethanol concentration achieved was 13.39 g/L with a corresponding ethanol yield of 0.084 g/g dry biomass (0.38 g ethanol/g glucose).

碩士
國立臺灣科技大學
化學工程系
101
Bioethanol is the most widely used biofuel for transportation. It's an approach to reduce consumption of crude oil and environmental pollution. Lignocellulosic biomass is the most promising feedstock considering its great availability and low cost. In the process of fermentation, we use the yeast Kluyveromyces marxianus UFV-3 to produce bioethanol and the fermentation of glucose and xylose simultaneously. We establish a streamlined mathematical model to simulate bioethanol production and consider the effect of the different medium status. Parameters of model were obtained by using Genetic Algorithms. The optimization of the procedure is working in the flow reactor system and using Genetic Algorithms to find the highest productivity and the amount of bioethanol.

Tese de Doutoramento em Engenharia Química e Biológica.
The production of bioethanol operating at high substrate loading improves the overall process productivity and reduces initial capital investment and water consumption comparing to processing at normal gravity. However, there are some inhibition issues that become more severe under these operation conditions. This thesis focused on the development of efficient 1st and 2nd generation bioethanol production processes running at high substrate concentration. Driven by the promising results obtained in 1st generation Very High Gravity (VHG) processes, an approach to the 2nd generation processes was then implemented aiming a better understanding of the physiological responses of yeast under stressful conditions. Aiming the fulfilment of Saccharomyces cereviae nutritional requirements for maximal ethanol production, a factorial design approach was successfully employed to optimize a high-level glucose medium (330 g/L) based in Corn step liquor (CSL) and other low-cost nutrients. Using the optimized medium (g/L: CSL 44.3, urea 2.3, MgSO4·7H2O 3.8 and CuSO4·5H2O 0.03), PE-2 and CA1185 isolates exhibited the best overall fermentation performance, among the eleven laboratory and industrial background strains tested. PE-2 and CA1185 isolates produced high ethanol titres (up to 19 %, v/v) with high ethanol batch productivity (> 2.3 g/Lh). These outstanding ethanol titres obtained by industrial strains were accompanied by an increased content of sterols (2 to 5- fold), glycogen (2 to 4-fold) and trehalose (1.1-fold), relatively to CEN.PK 113-7D laboratory strain, which demonstrate their robustness to cope with VHG stresses. Driven by the detailed physiological information of these industrial isolates, a VHG repeated-batch fermentation system, using the PE-2 strain, was successfully operated during fifteen consecutive cycles, attaining an average ethanol titre of 17.1% (v/v) and batch productivity of 3.51 g/Lh. To further understand how the inhibitory conditions influence the physiology and metabolism of the producing cells at the genetic level, an approach for identifying key genes common to relevant stresses in bioethanol fermentations and validating the identified genes under industrial relevant fermentation conditions, was conducted. Primarily, the intersection of chemogenomic data previous obtained in single stress phenotypic analysis allowed the identification of eight genes simultaneously involved in yeast tolerance to VHG-related stresses. Comparative VHG fermentation tests, showed that five of them are required for maximal fermentation performance: genes BUD31 and HPR1 were found to lead to the increase of both ethanol yield and fermentation rate, while PHO85, VRP1 and YGL024w genes were required for maximal ethanol production. Aiming a complementary approach to identify key genes and confirm their role in inhibitor tolerance, a genome-wide survey of S. cerevisiae genes implicated in resistance to an industrial Wheat Straw Hydrolysate (WSH) was conducted. The results highlight the genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics among the newly found determinants of WSH resistance. Moreover, comparing the results of WSH fermentations, with the genes identified in WSH genome-wide survey, PRS3, VMA8, ERG2, RAV1 and RPB4 were highlighted as key genes on yeast tolerance and fermentation of industrial WSH. Robust industrial isolates were further evaluated in fermentation of Eucalyptus globulus wood hydrolysate (114 g/L glucose). PE-2 isolate was able to resourcefully degrade furfural and HMF inhibitors attaining a remarkable final ethanol titre of 6.9% (v/v) and productivity of 0.8 g/L.
A produção de bioetanol através da operação a alta concentração de substrato aumenta a produtividade global do processo e reduz o capital de investimento inicial e consumo de água comparando com o processamento a concentração normal. No entanto, existem alguns problemas de inibição que se tornam mais graves nestas condições de operação. Esta tese focou-se no desenvolvimento de processos eficientes de produção de bioetanol de primeira e segunda geração recorrendo a elevada concentração de substrato inicial. Devido aos promissores resultados obtidos nos processos “Very High Gravity, (VHG)” de primeira geração, uma abordagem aos processos de segunda geração foi então implementada visando um melhor entendimento das respostas fisiológicas da levedura em condições de stress. Com o objectivo de preencher os requisitos nutricionais da levedura Saccharomyces cerevisiae para uma máxima produção de etanol, uma metodologia de desenho factorial foi aplicada com sucesso para otimizar um meio de cultura com elevados níveis de glucose (330 g/L) baseado em “Corn steep liquor, (CSL)” e outros nutrientes de baixo custo. Usando o meio de cultura otimizado (g/L: CSL 44.3, ureia 2.3, MgSO4·7H2O 3.8 e CuSO4·5H2O 0.03), os isolados de levedura PE-2 e CA1185 apresentaram a melhor performance global de fermentação, entre as onze estirpes laboratoriais e industriais testadas. Os isolados PE-2 e CA1185 produziram elevados teores de etanol (mais de 19% v/v) com elevada produtividade (>2.3 g/Lh). Estes notáveis teores de etanol obtidos pelas estirpes industriais foram acompanhados por um aumento no teor de esteróis (2 a 5 vezes), glicogénio (2 a 4 vezes) e trealose (1.1 vezes), relativamente à estirpe laboratorial CEN.PK 113-7D, o que demonstra a sua robustez para superar o stress em condições “VHG”. Motivado pela detalhada informação fisiológica obtida destes isolados industriais, um sistema de fermentação com reciclagem de levedura em condições “VHG”, usando a estirpe PE-2, foi operado com sucesso durante quinze ciclos consecutivos, obtendo-se um teor de etanol médio de 17.1% (v/v) e produtividade de 3.51g/Lh. Para melhor compreender a forma como as condições inibitórias influenciam a fisiologia e metabolismo das células produtoras a nível genético, foi realizada uma abordagem para identificar genes chave comuns aos diferentes stresses em fermentações de bioetanol e validar os genes identificados em condições de fermentação relevantes a nível industrial. Primeiramente, o cruzamento de dados de análise quimiogenómica, previamente obtidos em análises de fenótipo a um único stress, permitiram a identificação de oito genes simultaneamente envolvidos na tolerância da levedura aos stresses relacionados com as condições “VHG”. Testes comparativos de fermentação em condições “VHG”, mostraram que cinco destes genes eram necessários para um desempenho fermentativo máximo: a presença dos genes BUD31 e HPR1 levaram ao aumento dos rendimentos em etanol e taxas de fermentação, enquanto que os genes PHO85, VRP1 e YGL024w mostraram ser necessários para uma máxima produção de etanol. Visando uma abordagem complementar para identificar genes chave e confirmar o seu papel na tolerância aos inibidores, foi realizada uma pesquisa baseada numa análise à escala do genoma de genes S. cerevisiae envolvidos na resistência a um hidrolisado de palha de trigo. Os resultados destacaram os genes associados ao metabolismo das vitaminas, funções da mitocôndria e peroxissomas, biogénese dos ribossomas e biogénese dos microtúbulos, entre os novos determinantes na resistência aos hidrolisados de palha de trigo. Além disso, comparando os resultados das fermentações em hidrolisado de palha de trigo, com os genes identificados na análise à escala do genoma, distinguiram-se os genes PRS3, VMA8, ERG2, RAV1 e RPB4 como genes chave na tolerância da levedura e fermentação de hidrolisados de palha de trigo industriais. Isolados de leveduras industriais foram avaliados na fermentação de um hidrolisado de “Eucalyptus globulus” (114 g/L). O isolado PE-2 foi capaz de degradar eficientemente os inibitórios furfural e HMF obtendo-se um notável teor de etanol final de 6.9% (v/v) e produtividade de 0.8 g/Lh.
Fundação para a Ciência e a Tecnologia PhD grant SFRH/BD/64776/2009
PROBIOETANOL PTDC/BIO/66151/2006
GlycoCBMs PTDC/AGR-FOR/3090/2012 - FCOMP-01-0124-FEDER-027948

碩士
國立臺南大學
材料科學系碩士班
97
Fermentation sugar concentrations and ethanol yield related to two kinds of energy plants, traditional agriculture wastes (bagasse and corn leaves) and local candidate plants (Pennisetum purpureum、Leersia hexandra and Phragmites communis), were investigated for their use in ethanol production. In this study, the fermentation sugar highest concentrations 9.23mg/ml from corn leaves. In addition to ethanol concentrations in Pennisetum purpureum highest then others plants because it had slightly higher ethanol yield. Traditional yeast produced 0.044g ethanol/ g raw material , corresponding to 14.19% of theoretical ethanol yield. SEM results indicated that autoclave reactor with dilute acid pretreatment increased surface area and pore size. It is postulated that these physical changes enhance fermentation sugar concentrations in the dilute acid treated raw materials.

The primary objective of this work is to identify the optimal bioethanol production plant capacity and configuration based on currently available technology for all the processing sections involved. To effect this study, a systematic method is utilized which involves the development of a superstructure for the overall technology selection, process simulation and model regression of each processing step as well as equipment costing and overall economic evaluation. The developed optimization model is also designed to incorporate various biomass feedstocks as well as realistic maximum equipment sizing thereby ensuring pragmatism of the work. For this study, the criterion for optimization is minimum ethanol price. The secondary and more interesting aim of this work was to develop a systematic method for evaluating the economics of biomass storage due to seasonal availabilities. In essence, a mathematical model was developed to link seasonal availabilities with plant capacity with subsequent integration into the original model developed. Similarly, the criterion for optimization is minimum ethanol price. The results of this work reveal that the optimal bioethanol production plant capacity is ~2800 MT biomass/day utilizing Ammonia Fiber Explosion pretreatment technology and corn stover as the preferred biomass feedstock. This configuration provides a minimum ethanol price of $1.96/gal. Results also show that this optimal pretreatment choice has a relatively high sensitivity to chemical cost thereby increasing the risk of implementation. Secondary to this optimal selection was lime pretreatment using switchgrass which showed a fairly stable sensitivity to market chemical cost. For the storage economics evaluation, results indicated that biomass storage is not economical beyond a plant capacity of ~98 MMgal/yr with an average biomass shortage period of 3 months. The study also showed that for storage to be economical at all plant capacities, the storage scheme employed should be general open air land use with a corresponding biomass loss rate as defined in the study of 0.5 percent per month.