>Magister Scientiae - MSc / Due to economic and environmental concerns associated with use of fossil fuels, humanity is seeking alternative fuels. Ethanol is one of the alternative fuels produced commercially. Current ethanol production technologies using first generation ethanol processes is criticised for depleting the food supply and escalating food prices. Biomass is a target feedstock for use in bioethanol production and would resolve the criticism associated with the current bioethanol industry. Bacterial strains such as Geobacillus thermoglucosidasius NCIMB 11955 can be used to produce ethanol from biomass because they assimilate hexose and pentose sugars, a property that is lacking in first generation ethanol producing microbes (Saccharomyces cerevisiae and Zymomonas mobilis) (Riyanti and Rogers, 2009). Due to the low ethanol tolerance (4 % v/v (maximum)) of G. thermoglucosidasius, use of this species for bioethanol production is not economically viable. GroES and GroEL genes are involved in stress tolerance in bacteria: activation of these genes has been observed in stress induced bacteria (Rasouly and Ron, 2009). In this study the ethanol tolerance of G. thermoglucosidasius NCIMB 11955 was characterised by culturing at 45 ºC and 55 ºC in the presence of ethanol. A greater ethanol tolerance was observed at the sub-optimal growth temperature of 45 ºC. Escherichia coli metabolic systems are well understood. Aiming to improve the ethanol tolerance G. thermoglucosidasius NCIMB 11955, the GroES and GroEL genes of the organism were cloned in an expression vector and expressed in E.coli before testing their ability to confer an increased tolerance to ethanol. Proteomic analysis of the recombinant E. coli strain showed that GroES was over-expressed while GroEL was not. After over expression of GroES, the optical density of cultures was periodically measured. Over-expression of the G. thermoglucosidasius NCIMB 11955 GroES gene improved the ethanol tolerance of E. coli Rosetta pLySs growing in 4% (v/v) ethanol.

>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.

Bioethanol, produced from organic waste as a second-generation biofuel, is an important renewable energy source. Here, recalcitrant carbohydrate sources, such as municipal and agricultural waste, and plants grown on land not suitable for food crops, are exploited. The thermophilic, Gram-positive bacterium Geobacillus thermoglucosidasius is naturally very flexible in its growth substrates and produces a variety of fermentation products, including lactate, formate, acetate and ethanol. TMO Renewables Ltd. used metabolic engineering to enhance ethanol production, creating the production strain TM242 (NCIMB 11955 ∆ldh, ∆pfl, pdhup). Ethanol yield has been increased to 82% of the theoretical maximum on glucose and up to 92% of the theoretical maximum on cellobiose. However, this strain still produces acetate, presumably derived from the overproduction of acetyl-CoA through the upregulated pdh gene encoding the pyruvate dehydrogenase complex. An alternative to the mixed fermentation pathway found in G. thermoglucosidasius is to introduce a homoethanologenic pathway. Yeast and a very limited range of mesophilic bacteria use the homoethanol fermentation pathway, which employs pyruvate decarboxylase (PDC) in conjunction with alcohol dehydrogenase (ADH), to convert pyruvate to ethanol. Despite extensive screening, no PDC has yet been identified in a thermophilic organism. Using the thermophile G. thermoglucosidasius as a host platform, we endeavoured to develop a thermophilic version of the homoethanol pathway for use in Geobacillus spp. This Thesis reports the in vitro characterization and crystal structure of one of the most thermostable bacterial PDCs from the mesophile Zymobacter palmae (ZpPDC) and describes strategies to improve expression of active PDC at high growth temperatures. This includes codon harmonization and the successful development of a PET (producer of ethanol) operon. Furthermore, ancestral sequence reconstruction was explored as an alternative engineering approach, but did not yield a PDC more thermostable than ZpPDC. In vitro ZpPDC is most active at 65°C with a denaturation temperature of 70°C, when sourced from a recombinant mesophilic host. Codon harmonization improved detectable PDC activity in G. thermoglucosidasius cultures grown up to 65°C by up to 42%. Pairing this PDC with G. thermoglucosidasius ADH6 produced a PET functional up to 65°C with ethanol yields of 87% of the theoretical maximum on glucose. This increase in yield at temperatures of up to 15°C higher than previously reported for any PDC expressed.

Due to the depletion of petroleum reserves and environmental concerns, bioethanol has been identified as an alternative fuel to petrol. Bioethanol is a fuel of bio-origin derived from renewable biomass. Starch and sugar containing materials are the primary sources of carbon for bioethanol production. Starch is firstly hydrolysed into simple sugars which are later fermented to bioethanol using Saccharomyces cerevisiae (S. cerevisiae). The fermentation of sugars to bioethanol is however limited by inhibition of S. cerevisiae by the major product of the process, bioethanol. The challenge is thus in keeping the bioethanol concentration at levels which are not harmful to the fermenting organism. Keeping bioethanol concentration low in the broth will provide a suitable environment for yeast to grow and thus increase the overall production. Currently bioethanol producers use high water dilution rates to keep the bioethanol concentrations in the broth low enough so that yeast is not harmed. This excess water has to be removed in the downstream process, which is expensive. The use of excessive amounts of water in the fermentation can be avoided by continual removal of bioethanol from the broth. During this investigation the experimental conditions for the hydrolysis process were determined. A pH of 5.5 was determined as the best pH for Termamyl SC at 95°C with a pH of 5.0 for Spirizyme Fuel at 55°C during the liquefaction and the saccharification step, respectively. During the fermentation process the influence of yeast concentration on bioethanol production was investigated by varying the yeast concentration between 2 g.L-1 and 7 g.L-1. A yeast concentration of 5 g.L-1 produced the highest bioethanol yield of 0.48 g.g-1 after 48 hours of fermentation using S. cerevisiae. Later during the investigation a coupled fermentation/pervaporation system was employed in a batch system for continual removal of bioethanol in the fermentation broth in a process called simultaneous fermentation and separation (SFS). Through the continuous removal of bioethanol from the fermentation broth, the bioethanol concentration in the broth was kept low enough so that it was not harmful to the fermenting organism but the overall fermentation yield was not improved. Pervaporation is a membrane separation process used to separate azeotropic mixtures such as bioethanol and water. It is highly efficient, cost effective and uses less energy than distillation. During the SFS process a bioethanol yield of 0.22 g.g-1 was obtained. The SFS process yield for bioethanol was low compared to 0.45 g.g-1 of the traditional batch fermentation process. The lower overall bioethanol yield obtained in the SFS process could be attributed to only the supernatant being used in the SFS process and not the entire fermentation broth as in the traditional process. The results from this study proved that the SFS process was less efficient compared to the traditional batch fermentation process with respect to the bioethanol yield, but that the fermentation could be carried out without the necessity for additional process water. / Thesis (M.Sc. Engineering Sciences (Chemical and Minerals Engineering))--North-West University, Potchefstroom Campus, 2010.

Due to the depletion of petroleum reserves and environmental concerns, bioethanol has been identified as an alternative fuel to petrol. Bioethanol is a fuel of bio-origin derived from renewable biomass. Starch and sugar containing materials are the primary sources of carbon for bioethanol production. Starch is firstly hydrolysed into simple sugars which are later fermented to bioethanol using Saccharomyces cerevisiae (S. cerevisiae). The fermentation of sugars to bioethanol is however limited by inhibition of S. cerevisiae by the major product of the process, bioethanol. The challenge is thus in keeping the bioethanol concentration at levels which are not harmful to the fermenting organism. Keeping bioethanol concentration low in the broth will provide a suitable environment for yeast to grow and thus increase the overall production. Currently bioethanol producers use high water dilution rates to keep the bioethanol concentrations in the broth low enough so that yeast is not harmed. This excess water has to be removed in the downstream process, which is expensive. The use of excessive amounts of water in the fermentation can be avoided by continual removal of bioethanol from the broth. During this investigation the experimental conditions for the hydrolysis process were determined. A pH of 5.5 was determined as the best pH for Termamyl SC at 95°C with a pH of 5.0 for Spirizyme Fuel at 55°C during the liquefaction and the saccharification step, respectively. During the fermentation process the influence of yeast concentration on bioethanol production was investigated by varying the yeast concentration between 2 g.L-1 and 7 g.L-1. A yeast concentration of 5 g.L-1 produced the highest bioethanol yield of 0.48 g.g-1 after 48 hours of fermentation using S. cerevisiae. Later during the investigation a coupled fermentation/pervaporation system was employed in a batch system for continual removal of bioethanol in the fermentation broth in a process called simultaneous fermentation and separation (SFS). Through the continuous removal of bioethanol from the fermentation broth, the bioethanol concentration in the broth was kept low enough so that it was not harmful to the fermenting organism but the overall fermentation yield was not improved. Pervaporation is a membrane separation process used to separate azeotropic mixtures such as bioethanol and water. It is highly efficient, cost effective and uses less energy than distillation. During the SFS process a bioethanol yield of 0.22 g.g-1 was obtained. The SFS process yield for bioethanol was low compared to 0.45 g.g-1 of the traditional batch fermentation process. The lower overall bioethanol yield obtained in the SFS process could be attributed to only the supernatant being used in the SFS process and not the entire fermentation broth as in the traditional process. The results from this study proved that the SFS process was less efficient compared to the traditional batch fermentation process with respect to the bioethanol yield, but that the fermentation could be carried out without the necessity for additional process water. / Thesis (M.Sc. Engineering Sciences (Chemical and Minerals Engineering))--North-West University, Potchefstroom Campus, 2010.

Orientador: Fabio Marcio Squina / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T21:44:36Z (GMT). No. of bitstreams: 1 Buchli_Fernanda_M.pdf: 4527154 bytes, checksum: 3262f15a20dc87dfa1272cb774a61703 (MD5) Previous issue date: 2014 / Resumo: A Biomassa vegetal tem sido reconhecida como uma potencial fonte de açúcares fermentescíveis para a produção de biocombustível, principalmente pelo crescente incentivo do uso de fontes renováveis de combustíveis e sustentabilidade. Atualmente, no Brasil, o etanol é quase exclusivamente produzido pela fermentação da sacarose, um açúcar que pode ser facilmente extraído da cana-de-açúcar, esse etanol produzido a partir da sacarose é chamado de etanol de primeira geração. O processo de extração dos açúcares da biomassa da cana, através da hidrólise enzimática para a produção do chamado etanol de segunda geração, ainda apresenta um baixo rendimento e elevado custo de produção. O objetivo deste trabalho foi à busca por enzimas capazes de promover uma degradação mais eficiente, contribuindo para a viabilidade da produção do bioetanol de segunda geração. Para este estudo foi utilizada uma abordagem de metagenômica e metaproteômica. A análise metagenômica baseou-se em uma amostra de solo de canavial a qual teve seu DNA extraído e sequenciado. Em paralelo utilizou-se este solo para o estabelecimento de dois consórcios microbianos utilizando o bagaço de cana-de-açúcar como única fonte de carbono, estes consórcios também foram sequenciados. As sequências foram anotadas e analisadas na plataforma MG-Rast. Para a abordagem metaproteômica foram utilizadas proteínas extraídas diretamente do solo e o secretoma de ambos os consórcios. A análise do sequenciamento revelou a predominância de bactérias, que representaram 94,86% do metagenoma de solo de canavial, sendo o filo Proteobacteria o grupo mais abundante em todos os metagenomas avaliados. Durante as análises foi possível observar mudanças populacionais entre os metagenomas, a exemplo, as classes Bacteroidia, Alphaproteobacteria e Gammaproteobacteria se mostraram mais abundantes nos metagenomas dos consórcios do que do solo. Analisando as proteínas identificadas nas análises de metaproteômica pertencentes à família das glicosil hidrolases nota-se uma predominância das hemicelulases seguida das celulases entre as enzimas mais abundantes identificadas para as três comunidades analisadas. Dentre as celulases identificadas as mais abundantes foram a GH1, GH3 e GH9, entres as hemicelulases as mais abundantes foram GH2, GH43 e GH51. As análises de metagenômica e metaproteômica sugerem que os consórcios apresentam um enriquecimento das enzimas de interesse e revelam o potencial destas comunidades para prospecção de novas enzimas envolvidas na degradação da biomassa lignocelulósica / Abstract: Plant Biomass has been recognized as a potential source of fermentable sugars for biofuel production, mainly by the growing concern with renewable fuels and sustainability. Ethanol is currently almost exclusively produced by fermentation of sucrose, a sugar that can be easily extracted from sugar cane and thus, this process is called first generation ethanol. The process of extracting the sugars from sugarcane biomass, through enzymatic hydrolysis to produce the so-called second-generation ethanol, still has a low yield and high cost process. The objective of this study was to search for enzymes capable of promoting a more efficient degradation, making possible the production of second generation bioethanol. We used the metagenomic and metaproteomic approaches. The metagenomic analysis was based on a soil sample of sugar cane which had its DNA extracted and sequenced. In parallel the soil was used to establish two microbial consortia using sugarcane bagasse as a sole carbon source, these consortia were also sequenced. The sequences were annotated and analyzed in MG-Rast platform. Proteins extracted directly from soil and the secretome of both consortia were used for metaproteomic approach. The sequencing analysis revealed the predominance of bacteria, representing 94.86 % of the soil metagenome, phylum Proteobacteria is the most abundant group in all metagenomas reviews. During the analysis it was observed population changes between the metagenomes, we notice some groups that seem to be more abundant in consortia¿s metagenomes than in soil. Between these enriched classes of microorganisms we have the Bacteroidia, Alphaproteobacteria and Gammaproteobacteria classes. Among the proteins identified in the metaproteomic 61% of the soil¿s proteins represent glycosyl hydrolases and 25% glycosyl transferases, the consortia presented a similar profile. Analyzing the enzymes belonging to the family of glycoside hydrolases we can notice a predominance of hemicellulases then cellulases among the most abundant enzymes identified for the three communities. Among the most abundant cellulases identified were the GH1, GH3 and GH9, among hemicellulases the most abundant were GH2, GH43 and GH51. The metagenomic and metaproteomic analyzes suggest that consortia have an enrichment of the enzymes of interest and reveal the potential of these communities to search for new enzymes involved in the degradation of lignocellulosic biomass / Mestrado / Bioquimica / Mestra em Biologia Funcional e Molecular

>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.

>Magister Scientiae - MSc / Biofuels are currently recognised as the most viable source of energy to replace depleting fossil fuel reserves, with bioethanol the most popular alternative alcohol fuel. Producing bioethanol from agricultural waste residues is a feasible socio-economic industrial process. Lignocellulose, from which plant material is composed, is highly recalcitrant to enzymatic degradation and therefore requires a suite of enzymes for complete hydrolysis of the biomass. Metagenomes, particularly from extreme environments, represent an unlimited resource for the discovery of novel biocatalysts for inclusion in industrial processes. Here we report on the cloning and functional characterisation of a novel thermophilic cellulase identified by the functional screening of a Malawian, hotspring sediment metagenomic library. The gene encoding the cellulase, celMHS, composed of 2,705 nucleotides and encoded a polypeptide of 905 amino acids with a predicted molecular mass of about 98 kDa. The in silico translated protein, CelMHS, contained a putative transmembrane domain, a family 4 carbohydrate binding motive (CBM 4), a truncated glycoside hydrolase family 42 (GH42) domain and a N-terminal region that does not have sequence similarity to any previously described domains. Functional characterisation of the recombinant CelMHS demonstrated that the protein displayed an optimal pH of 6.0 and temperature of 100°C. CelMHS had high specific activity toward substrates comprising of β-1,4 linked glucose subunits such as carboxymethyl cellulose, β-D-glucan from barley and lichenan, however, some activity was also observed against avicel, a crystalline cellulose substrate. HPLC analysis of the hydrolysis products produced by CelMHS indicates that this particular enzyme prefers longer chain oligosaccharides. This is, to the best of our knowledge, the first investigation describing the cloning and characterization of a carbohydrate hydrolysing enzyme comprised of the unique sequence architecture: a partial GH42 catalytic domain, a CBM 4 and a unique N-domain sequence. Key words: cellulose, cellulases, lignocellulosic biomass, bioethanol, saccharification, hydrolysis, metagenomic library, thermophilic

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第23292号 / エネ博第417号 / 新制||エネ||79(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー社会・環境科学専攻 / (主査)教授 河本 晴雄, 教授 石原 慶一, 教授 上髙原 浩 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

Non-ionic surfactants are known to enhance the biochemical conversion of lignocellulosic (LC) biomass to bioethanol. Their mechanisms of action, however, are incompletely understood. This research was conducted to elucidate the effects of the non-ionic surfactant Tween 80 on the enzymatic hydrolysis of cellulose and LC substrates. Model cellulose substrates were prepared from cellulose nanocrystals (CNCs) obtained by sulfuric acid hydrolysis of wood pulp. Two methods were developed for the removal of the sulfate groups on the CNCs, resulting from the use of sulfuric acid in their preparation. The effect of sulfate groups, which may be introduced into LC biomass during pretreatment with sulfuric acid, on the enzymatic hydrolysis of cellulose was studied with model cellulose substrates prepared from CNCs with different sulfate group densities. Adsorption of cellulases onto sulfated substrates increased with increasing sulfate group density but their rate of hydrolysis decreased. The decrease indicated an inhibitory effect of sulfate groups on the enzymatic hydrolysis of cellulose, possibly due to non-productive binding of the cellulases onto the substrates through electrostatic interactions instead of their cellulose binding domains. The effect of Tween 80 on the adsorption of cellulases onto lignin, often present as residual lignin in pretreated biomass, was studied with model lignin substrates, prepared from kraft lignin, organosolv lignin, and milled wood lignin. Cellulases appeared to adsorb onto the lignin substrates via both hydrophobic and polar interactions. Tween 80 molecules on the lignin substrates seemed to hinder cellulase adsorption via hydrophobic interactions and reduced the adsorption rate. Finally, the effects of lignin and Tween 80 on the enzymatic hydrolysis of cellulose and LC substrates were studied. Lignin hindered both the adsorption of cellulases onto the substrates and the enzymatic hydrolysis of the substrates. Tween 80 was found to form surfactant–protein complexes with cellulases in solution without compromising cellulase activity. Either substrate-adsorbed or in solution, Tween 80 had no effect on the hydrolysis of cellulose by cellulases. Substrate-adsorbed Tween 80 increased the apparent enzymatic hydrolysis rates of LC substrates but the ability of Tween 80 to increase their apparent hydrolysis rate depended strongly on their structural properties and the chemical properties of the lignin. Hence, Tween 80 may be able to mitigate the inhibitory effect of lignin on the enzymatic hydrolysis of pretreated biomass. / Ph. D.