>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.

Given the increasing demand for energy, climate change, and environmental concern of fossil fuels, it is becoming increasingly significant to find alternative renewable energy sources. Bioethanol as one sort of cellulosic biofuel produced from lignocellulosic biomass feedstocks has shown great potential as a renewable resource. Delivering a competitive, sustainable biofuel product requires comprehensive supply chain planning and design. Developing economically and environmentally optimal supply chain models is necessary in this context. Also, designing biomass bioethanol supply chain (BBSC) models addressing social issues requires using second-generation biomass which is not a source of food for humans. Currently, corn as a first-generation feedstock is the primary source of bioethanol in the United States which has given growth to new social issues such as the food versus fuel debate. Considering incentives for first-generation bioethanol producers to switch to second-generation biomass and associated production technologies will help to address such social issues. The scope of this study focuses on analyzing economic and environmental market incentives for second-generation bioethanol producers while considering different carbon policies as penalties and restrictions for emissions coming from BBSC activities. First, we develop an integrated life cycle emission and energy optimization model for analyzing an entire second-generation bioethanol supply chain using switchgrass as the source of biomass while finding the most appropriate potential locations for building new cellulosic biorefineries in North Dakota. Second, we propose a supply chain model by comparing a first-generation (corn) and a second-generation (corn stover) bioethanol supply chain to analyze how policymakers can incentivize first-generation bioethanol producers to switch their technology and biomass supply from first-generation to second-generation biomass. Third, we develop the model further by investigating the impact of four different carbon policies including the carbon tax, carbon cap, carbon cap-and-trade, and carbon offset on the supply chain strategic and operational decisions. This research will help to design robust BBSCs focused on sustainability in order to optimally utilize second-generation biomass resources in the future. The findings can be utilized by renewable energy policy decision makers, bioethanol producers, and investors to operate in a competitive market while protecting the environment.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第20479号 / エネ博第348号 / 新制||エネ||69(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 坂 志朗, 教授 梅澤 俊明, 准教授 河本 晴雄 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

L'objectif global de cette étude était de produire du bioéthanol à partir de biomasse lignocellulosique en utilisant des enzymes libres ou immobilisées de type xylanase, cellulase et β-1,3-glucanase. L'isolement de la souche AUKAR04 de Trichoderma citrinoviride a permis de produire par fermentation solide ces trois enzymes à un taux de 55 000, 385 et 695 UI / gd, respectivement. L’activité biochimique des enzymes libres a été caractérisée en faisant varier différents paramètres : pH, température et concentration en cations métalliques, et les paramètres cinétiques correspondants ont été identifiés. Par la suite, les enzymes ont été immobilisées en phase solide, soit sous forme d’agrégats sans support de type (combi-CLEA), soit par association avec des nanoparticules magnétiques bifonctionnalisées (ISN-CLEA). Ces dernières ont fourni de meilleures performances en termes de stabilité thermique, d’activité et d’aptitude à réutilisation après un temps de conservation prolongé. Le substrat végétal utilisé (SCB : bagasse de canne à sucre) a été prétraité chimiquement par cuisson à l'ammoniac, permettant d’éliminer 40% de la lignine initiale tout en préservant 95% de glucane, 65% de xylane et 41% d'arabinane. L’hydrolyse enzymatique du substrat prétraité a permis une conversion de la cellulose en 87% de glucose, et une conversion des hémicelluloses (arabinoxylanes) en 74% de xylose et 64% d'arabinose, chiffres notoirement supérieurs à l'activité des enzymes libres. L'analyse chimique et structurale du substrat a été faite par spectrométrie ATR-FTIR et DRX, et par analyse TGA. L’étude FTIR a prouvé l’efficacité du traitement enzymatique en montrant que les hémicelluloses et la cellulose subissent une dépolymérisation partielle par l’action simultanée des trois enzymes immobilisées dans les ISN-CLEA. L’étude TGA a montré que la stabilité thermique des échantillons prétraités à l'ammoniac puis traités par des enzymes est notoirement améliorée. L’analyse DRX a montré que l'indice de cristallinité du substrat prétraité à l’ammoniac puis traité par l'ISN-CLEA a augmenté de 61,3 ± 1%, par rapport au substrat avant traitement enzymatique. La fermentation par la levure Saccharomyces cerevisiae LGP2Y1 utilisée en monoculture, à partir d’un hydrolysat enzymatique contenant 103,8 g / L de glucose, a produit 42 g / L d'éthanol en 36 h de fermentation. Le rendement métabolique global atteint ainsi environ 79% du rendement théorique. La fermentation en co-culture avec Saccharomyces cerevisiae LGP2Y1 et Candida utilis ATCC 22023 d’un hydrolysat à 107,6 g / L de glucose et 41,5 g / L de xylose a produit 65 g / L d'éthanol en 42 h de fermentation. Ainsi, en co-culture fermentaire, le rendement métabolique global atteint environ 88 % du rendement théorique. / The overall objective of the study was to produce bioethanol from lignocellulosic biomass by using free and immobilized xylanase, cellulase and β-1, 3-glucanase. Specifically, this study was focused on the isolation of Trichoderma citrinoviride strain AUKAR04 and it produces xylanase (55,000 IU/gds), Cellulase (385 IU/gds) and β-1, 3-glucanase (695 IU/gds) in solid state fermentation. Then the free enzymes were biochemically characterized such as effect of pH, temperature and metal ion concentration and kinetics parameters. Then the enzymes were subjected to two types of immobilization using carrier-free co-immobilization (combi-CLEAs) method and immobilized on bifunctionalized magnetic nanoparticles (ISN-CLEAs) with higher thermal stability, extended reusability and good storage stability. Liquid ammonia pretreatment removed 40% lignin from the biomass and retained 95% of glucan, 65% of xylan and 41% of arabinan in sugarcane bagasse (SCB). SCB was enzymatically hydrolyzed and converted to 87% glucose from cellulose and 74% of xylose, 64% of arabinose from the hemicelluloses which is remarkably higher than the activity of the free enzymes. Chemical and structural analysis of SCB was done by ATR-FTIR, TGA and XRD. FTIR result showed a successful pretreatment of the SCB raw material. It showed that hemicelluloses and cellulose are partially depolymerized by the action of xylanase, cellulase and β-1,3-glucanase in ISN-CLEAs. TGA studies showed that the thermal stability of the ammonia pretreated and enzymatically treated samples have improved remarkably. XRD results showed that the crystallinity index of the ISN-CLEAs treated SCB increased to 61.3±1% when compared to the ammonia-treated SCB. Mono-culture fermentation using Saccharomyces cerevisiae LGP2Y1 utilized SCB hydrolysate containing 103.8 g/L of glucose and produced 42 g/L ethanol in 36 h of fermentation. The overall metabolic yield achieved was about 79% of theoretical yield. Co-culture fermentation using Saccharomyces cerevisiae LGP2Y1 and Candida utilis ATCC 22023 utilized SCB hydrolysate containing 107.6 g/L of glucose and 41.5 g/L xylose and produced 65 g/L ethanol in 42 h of fermentation. The overall metabolic yield in co-culture fermentation achieved was about 88 % of the theoretical yield.

Thesis (Ph. D. (Microbiology)) -- University of Limpopo, 2020 / The use of lignocellulosic biomass for bioethanol production is largely dependent on cost effective production of cellulase enzymes and most importantly, the availability of cellulases with sufficient β-glucosidase activity for complete hydrolysis of cellulose to glucose. Commercial cellulase preparations are often inefficient in the complete hydrolysis of cellulose to glucose. The addition of β-glucosidases to commercial cellulase preparations may enhance cellulolytic activity in the saccharification of cellulose to fermentable sugars. A β-glucosidase producing thermophilic bacterium, Anoxybacillus sp. KTC2 was isolated from a hot geyser in the Zambezi Valley, Zimbabwe. The bacterium identified through biochemical tests and 16S rDNA sequencing, had an optimal growth temperature and pH of 60˚C and pH 8, respectively. The β-glucosidase enzyme had an optimal temperature of 60˚C and a broad pH range for activity, between 4.5 and 7.5 with an optimum at pH 7. The β-glucosidase enzyme retained almost 100% activity after 24 hours’ incubation at 50˚C. The Anoxybacillus sp. KTC2 β-glucosidase was partially purified and a partial amino acid sequence obtained through MALDI-TOF analysis. The whole genome of Anoxybacillus sp KTC2 β-glucosidase was sequenced and a β-glucosidase gene identified. The deduced amino acid sequence corresponded to the peptide sequences obtained through MALDI-TOF, confirming the presence of the a β glucosidase on the genome of Anoxybacillus sp KTC2. Analysis of the deduced amino acid sequence revealed that the β-glucosidase enzyme belongs to the GH family 1. The β-glucosidase gene was isolated by PCR and successfully cloned into an E. coli expression system. The saccharification efficiency of the β-glucosidase enzyme was evaluated through the creation of enzyme cocktails with the commercial cellulase preparation, CelluclastTM. CelluclastTM with the Anoxybacillus sp KTC2 β-glucosidase were used to hydrolyse pure Avicel cellulose, at 50˚C over a 96 hour reaction time. The Anoxybacillus sp KTC2 β-glucosidase enabled a 25% decrease in the total cellulose loading without a decrease in the amount of glucose released. / University of Limpopo staff development programme and VLIR

This thesis is concerned with combustion of liquid biofuels, and possibility of using liquid biofuels for lower heat output power units. Overview of basic usable liquid biofuels in Czech Republic is executed in the beginning of the thesis. This part is focused especially on production method and energy effectivity of rape-oil methyl ester (RME) and bioethanol production. Overview of basic atomization method of liquid fuels is executed in next chapters. The focus is stressed on pneumatic atomization, especially effervescent atomization method, which was used in practical experiment. Practical part contains fossil fuel and selected biofuel (RME) combustion test executed on burner testing device. Basic combustion properties was found and test plan was made before executing the test. Various operating conditions are compared in terms of atomization quality, combustion quality and geometrical characteristics of flame. Usability of tested liquid biofuels for lower heat output power units is evaluated in the conclusion.

The search for other sources of energy has encouraged the development of research and innovation in the production of biofuels, such as the second generation ethanol. Biomass from agricultural residues has advantages such as reuse, solves the disposal problem and also offers a low cost of production. In this context, this paper evaluates the best pretreatment (acid, alkaline and hydrothermal) waste from processing fruits (orange, passion fruit and soursop) to obtain bioethanol. The waste collected, stored at -20°C were thawed at room temperature, sanitized in 100 ppm sodium hypochlorite for 15 min and dried in an oven with air circulation at 55 ± 5°C until constant weight, ground and subsequently on a knife mill type Willye 30 mesh and packed in airtight plastic bottles at room temperature. Determinations of lipid, protein, moisture, ash, fiber, pectin and carbohydrate were performed. Pretreatments were designed according to experimental design where, for the acid, time 15 to 120 min, Cacid from 1 to 5% and Cbiomass 1 to 9%. For the alkali, it was used the same conditions as the acid, changing only the Cbasis, from 0.5 to 2.5%. For the hydrothermal only Cbiomass and time were evaluated. As answers, the mass yield, the amount of total reducing sugars (TRS) and total soluble solids in the liquid fraction. For the enzymatic hydrolysis it was employed cellulase Sigma-Aldrich in a 2:1 by enzyme mL:g pretreated biomass in 60 mL of 50 mM citrate buffer at 50°C and 100 rpm, evaluating the concentration of total reducing sugars. In ethanol fermentation, was used the hydrolyzate complemented with mineral solution and the yeast Saccharomyces cerevisiae, the main responses analyzed were ethanol concentration and yield of fermentation. The waste orange, passion fruit and soursop higher content of sugar in the liquor pretreatment occurred in the acid using low biomass concentration and longer pretreatment (65%), whereas at higher acid concentrations was sugars decreased, probably because undergo degradation. In alkaline pretreatment, was lower than the saccharification acid pretreatment (35%) and lower yields mass, indicating that some component of the lignocellulosic matrix was solubilized lignin probably characteristic of alkaline treatments. In hydrothermal there was the lowest saccharification liquor from both the pretreatment and in the enzymatic hydrolysis, possibly because the time and temperature used were not effective in destroying the lignocellulosic matrix. In enzymatic hydrolysis, alkali was more efficient than the acid, achieving, in the best conditions, around 35% of ART, except for the residue of passion fruit (<10% hydrolysis for the three pre-treatments), suggesting negative relationship between the amount of pectin and action of cellulases. The yield of fermentation behaved differently among trials for obtaining pre-treatment acid with shorter (15 minutes) the highest rates, while for these alkaline and hydrothermal stood in a longer time (120 min) pretreatment. These observations suggest that in the case of residual soursop optimizations must be carried out using lower heating times and higher biomass concentrations. However, for the orange peel and passion fruit residue, the intermediate condition seems to be more appropriate, and more efficient enzyme complexes and the presence of enzymes that break down pectin. / A busca por outras fontes de energia tem incentivado o desenvolvimento de pesquisas e a inovação na produção de biocombustíveis, a exemplo do etanol de segunda geração. A biomassa proveniente de resíduos agroindustriais apresenta como vantagens seu reaproveitamento, resolve o problema de descarte e, também, oferece um baixo custo de produção. Neste contexto, o presente trabalho avalia o melhor pré-tratamento (ácido, alcalino e hidrotérmico) de resíduos do processamento de frutas (laranja, maracujá e graviola) para a obtenção de bioetanol. Os resíduos coletados, armazenados em freezer a -20°C, foram descongelados à temperatura ambiente, sanitizados em hipoclorito de sódio 100 ppm por 15 min e secos em estufa de recirculação de ar a 55±5°C até peso constante, sendo posteriormente triturados em um moinho de facas do tipo Willye a 30 mesh e acondicionados em frascos plásticos herméticos à temperatura ambiente. Foram realizadas determinações de lipídios, proteínas, umidade, cinzas, fibra, pectina e carboidratos totais. Os pré-tratamentos foram idealizados de acordo com delineamentos experimentais, sendo para o ácido, tempo de 15 a 120 min, Cácido de 1 a 5% e Cbiomassa de 1 a 9%. Para o alcalino, utilizaram-se as mesmas condições do ácido, mudando apenas a Cbase, de 0,5 a 2,5%. Para o hidrotérmico, somente os tempos e Cbiomassa foram avaliados. Como respostas, o rendimento mássico, a quantidade de açúcares redutores totais (ART) e sólidos solúveis totais na fração líquida. Para a hidrólise enzimática, empregou-se celulase Sigma-Aldrich® na proporção 2:1, em mL enzima:g biomassa pré-tratada em 60 mL de tampão citrato 50 mM a 50°C e 100 rpm, avaliando-se a concentração de açúcares redutores totais. Na fermentação etanólica, empregou-se o hidrolisado, solução mineral e a levedura Saccharomyces cerevisiae, tendo como principais respostas a concentração de etanol e o rendimento da fermentação. Nos resíduos de laranja, graviola e maracujá as maiores sacarificações no licor do pré-tratamento ocorreram no ácido, utilizando-se menor concentração de biomassa e maior tempo de pré-tratamento (65%), ao passo que nas concentrações mais elevadas de ácido houve uma diminuição de açúcares, provavelmente porque sofreram degradação. No pré-tratamento alcalino, houve menor sacarificação que o ácido (35%) e menor rendimento mássico, indicando que algum componente da matriz lignocelulósica foi solubilizado, provavelmente a lignina, característica de tratamentos alcalinos. No hidrotérmico, houve a menor sacarificação tanto no licor do pré-tratamento quanto na hidrólise enzimática, possivelmente porque o tempo e temperatura usados não foram eficientes na destruição da matriz lignocelulósica. Na hidrólise enzimática, o alcalino foi mais eficiente que o ácido, conseguindo-se, nas melhores condições, em torno de 35% de ART, à exceção do resíduo do maracujá (< 10% de hidrólise para os três pré-tratamentos), sugerindo relação negativa entre quantidade de pectina e ação das celulases. O rendimento de fermentação se comportou de modo diverso entre os ensaios, obtendo-se para o pré-tratamento ácido com menor tempo (15 min) as maiores taxas, enquanto que para o alcalino e o hidrotérmico estas se situaram em um maior tempo (120 min) de pré-tratamento. Essas observações sugerem que, no caso do resíduo de graviola, as otimizações devem ser realizadas empregando menores tempos de aquecimento e maiores concentrações de biomassa. Entretanto, para o bagaço de laranja e resíduo de maracujá, a condição intermediária parece ser mais adequada, além de complexos enzimáticos mais eficientes e com a presença de enzimas que quebrem a pectina.