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Orientadores: Anete Pereira de Souza, Sindélia Freitas Azzoni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T11:06:28Z (GMT). No. of bitstreams: 1 Crucello_Aline_D.pdf: 3829574 bytes, checksum: b0eb3f1e3e0030f42a0081e3bf8b5ef8 (MD5) Previous issue date: 2014 / Resumo: O Brasil é hoje referência mundial na produção de etanol produzido a partir da cana-de-açúcar, cujo consumo tem aumentado significativamente nos últimos anos. Entretanto, a produção atual de etanol a partir do suco da cana-de-açúcar é insuficiente para atender a demanda do mercado nacional e internacional. Nesse contexto, a produção de etanol celulósico (de segunda geração) emergiu como uma alternativa promissora ao bioetanol de primeira geração. O fungo filamentoso Trichoderma harzianum IOC-3844 é uma linhagem brasileira que se destaca pela alta capacidade de produção de enzimas do complexo das celulases e hemicelulases, característica de grande interesse em biocatálise para conversão de biomassa em monômeros de açúcar fermentáveis. Apesar de seu comprovado potencial, há poucos dados de literatura disponíveis a respeito de sua capacidade celulolítica. Desta forma, este projeto teve como objetivo principal contribuir para o conhecimento básico sobre regiões do genoma de T. harzianum IOC-3844 envolvidas na via de hidrólise de compostos celulósicos, através da construção de uma biblioteca genômica de BAC (bacterial artificial chromosome). A biblioteca de BACs conta com 5760 clones, com insertos de DNA de tamanho médio de 90 kb, o que dá uma cobertura de aproximadamente 12 vezes o genoma de T. harzianum. Através da seleção de clones contendo genes de interesse, foram identificadas regiões com altas concentrações de genes relacionados à hidrólise de biomassa. Além disso, a combinação de dados genômicos, obtidos através da biblioteca de BACs, juntamente com dados de transcriptoma possibilitou a identificação de novos potenciais genes regulatórios. Os resultados trazem grande contribuição para a pesquisa associada a T. harzianum e à genômica de fungos relacionada à produção de etanol de segunda geração / Abstract: Brazil is a world reference in sugarcane ethanol production, whose consumption has increased significantly in recent years. However, the current production of ethanol from sugarcane juice is insufficient to meet the demand of national and international market. In this context, the production of cellulosic ethanol (second generation) has emerged as a promising alternative to first-generation bioethanol. The filamentous fungus Trichoderma harzianum IOC-3844 is a Brazilian strain known for its high ability to produce enzymes of cellulosic and hemicellulosic complex, characteristic of great interest in biocatalysis for conversion of biomass into fermentable sugar monomers. Despite its potential, there are few published data available regarding its cellulolytic ability. Thus, this project aimed to contribute to the basic knowledge about regions of the genome of T. harzianum IOC-3844 involved in the hydrolysis of cellulosic compounds pathway, through the construction of a genomic BAC (bacterial artificial chromosome) library. The BAC library comprises 5,760 clones with an average DNA insert size of 90 kb, which represents about 12-fold coverage of the T. harzianum genome. Through the selection of clones containing genes of interest, regions containing high concentrations of genes related to biomass hydrolysis were identified. Furthermore, the combination of genomic data obtained from BAC library together with transcriptome data allowed the identification of novel potential regulatory genes. The results bring great contribution to studies related to T. harzianum and to fungal genomics regarding second generation bioethanol production / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

The waste from agricultural and food industry are accessible in large quantity anywhere in the whole world nowadays. Most of these wastes include cellulose (30 - 40 %), hemicellulose (20 - 40 %) and lignine (10 - 20 %). Therefore these waste materials have wide use as the substrates for the microbial growth and the production of the enzymes. The microorganisms are able to use organic compounds from the wastes as the source of energy for the growth and carbon for synthesis of cellular biomass [24]. Wheat and rice straw are possible to use as the substrates for cultivation of the microorganisms and following production of the enzymes. In this thesis the utilization of the wastes from food industry for the production of the enzymes by the microorganisms was studied. We observed utilization of wheat straw as source of energy for growth of tested microorganisms and investigated their ability for the production of oxidoreductase (laccase). The optimalization of growth conditions of Aureobasidium pullulans was proceeded. Further the activity of laccase was studied. Milled wheat straw was used as the substrate. The cultivation was done in the thermoregulator at the temperature of 27°C. The activity of laccase was not found in this thesis. Petri dishes were contaminated by three unknown microoganisms during optimalization of growth of Aureobasidium pullulans. One of them produced laccase in cultivation with straw.

The study of production of hydrolytic enzymes dealt with the production of cellulase and polygalacturonase by two microbial strains - Aspergillus niger and Aureobasidium pullulans. The enzymes were produced in solid-state fermentation system. The wheat straw and sugar beet pulp were used as a substrate. The substrates were moistened by water, mineral solution or by medium with glucose. The effect of mineral solution and glucose on production of these enzymes were monitored during cultivation. The highest production of polygalacturonase was achieved by Aspergillus niger during cultivation on sugar beet pulp moistened by mineral solution. The highest production of cellulase was achieved by Aspergillus niger during cultivation on wheat straw moistened by medium with glucose.

Study of the employment of waste paper to microbial production of cellulase was carried out using Aspergillus niger cultivation on carbon sources that have been waste office paper and cardboard, humidified by no – carbon medium or distiled water. Cultivation took place in the SSF way in Erlenmeyer flasks and columns. Concentration of extracellular proteins, cellulase and protease activity for selected samples were monitored. It was found that the most advantageous method of cultivation in terms of cellulase activity production is the cultivation in the column washing by no – carbon medium in three day intervals.

The theoretical part of this thesis deals with cellulolytic enzymes, their microbial producers, the possibilities of using such enzymes in the industry and how can be enzymes - not only cellulolytic - immobilized. Experimental part examines the preparations created by immobilizing various amounts of the commercially used cellulolytic complex Cellulast 1.5L onto various synthetic carriers made of polyethylene terephthalate - commercially used Sorsilen, PET carrier and glutaraldehyde-treated PET carrier. Enzyme activity of these preparations was determined by Somogyi - Nelson method by spectrophotometry. For the highest activity immobilized preparation was determined the temperature- and the pH-optimum. The difference in effects change between the free and immobilized enzyme by measuring viscosity decrease of the substrate depending on the degradation of glycosidic bonds was also studied.

This study evaluates the pre-treatment of cellulose from kenaf plant to yield sugar precursors for the production of ethanol or butanol for use as biofuel additives. In order to convert the crystalline cellulosic form to the amorphous form that can undergo enzymatic hydrolysis of the glycosidic bond to yield sugars, kenaf pulp samples were subjected to two different pre-treatment processes. In the acid pre-treatment, the pulp samples were treated with 37.5% hydrochloric acid in the presence of FeCl 3 at 50 °C or 90 °C whereas in the alkaline method, the pulp samples were treated with 25% sodium hydroxide at room temperature and with 2% or 5% sodium hydroxide at 50 °C. Microwave-assisted NaOH-treatment of the cellulose was also investigated and demonstrated to be capable of producing high glucose yield without adverse environmental impact by circumventing the use of large amounts of concentrated acids i.e., 83-85% phosphoric acid employed in most digestion processes. The treated samples were digested with the cellulase enzyme from Trichoderma reesei. The amount of glucose produced was quantified using the QuantichromTMglucose bioassay for assessing the efficiency of glucose production for each of the treatment processes. The microwave-assisted alkaline pre-treatment processes conducted at 50 °C were found to be the most effective in the conversion of the crystalline cellulose to the amorphous form based on the significantly higher yields of sugar produced by enzymatic hydrolysis compared to the untreated sample.

Cellulase-producing microorganisms are considered as a key player in various environments to degrade the plant biomass and were isolated from various environment like soils, mangroves and oceans. The Red Sea has a unique environment in terms of high seawater temperature, high salinity and low nutrients. This study aims of examining if the Red Sea is a potential resource for cellulase-producing microorganisms and cellulase genes. First, I investigated types of microbial cellulase genes in the Red Sea based on public metagenomic datasets. The analysis revealed 3,383 microbial cellulase were more abundant in shallow depth than in deep seawater, and were classified into 16 sub-GH orthologous groups. These results suggest that the Red Sea environment is potentially an excellent gene resource of microbial cellulases due to its high diversity. Next, cellulase-producing microorganisms were isolated and screened from the Red Sea. Three bacterial and one fungal strain were successfully obtained. The MLTS analysis showed that the three bacterial strains belong to Bacillus paralichiniformis. The 18S rRNA of fungal strain showed 99% similarity to Aspergillus ustus and the enzymatic assay of the four strains showed high cellulase activity. These results suggest that these four isolates secreted active cellulases. Next, I tried to identify cellulase genes actually working during their cellulolysis by conducting comparative transcriptome analysis of the candidate genes and identified cellulase genes that are highly expressed during cellulolysis. To my knowledge, it is the first attempt to find out cellulase genes functioning during their cellulolysis among distinct cellulases on genomes of microorganisms. The results showed that although all the candidate genes were upregulated in general, a limited number of cellulase genes were highly expressed, which are highly expected to have a crucial role in cellulolysis. I also identified operon structures composed of genes including cellulases. This will provide us with the information to elucidate the cellular mechanisms occurring along with the cellulolysis in bacterial strains. We can expect that the Red Sea is a potential resource for new cellulase genes applicable for the industry. These information can be significantly useful for the bio-prospecting research of microbial cellulases in the Red Sea.

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<p>  </p> <p>The potential of lignocellulose for producing fermentable sugars as feedstock to manufacture fuels, chemicals, and materials for decarbonization remains untapped due to costly logistics and conversion processes. Pelleting technology offers a solution by addressing logistical issues and impacting downstream conversion, though it comes with its own costs. An overview of recent advances in pelleting technologies and their impact on bioconversion highlights the importance of understanding variables and product attributes. The interplay between pelleting and pretreatment processes, considering various feedstocks, is crucial for future design. Practical considerations such as energy consumption, costs, and environmental impacts must not be overlooked, along with exploration of cutting-edge technologies and strategies in this field. This work further presents a comprehensive investigation into the recalcitrance of pelleted corn stover to enzymatic digestion prior to any pretreatment.</p> <p>The potential of lignocellulose for producing fermentable sugars as feedstock to manufacture fuels, chemicals, and materials for decarbonization remains untapped due to costly logistics and conversion processes. Pelleting technology offers a solution by addressing logistical issues and impacting downstream conversion, though it comes with its own costs. An overview of recent advances in pelleting technologies and their impact on bioconversion highlights the importance of understanding variables and product attributes. The interplay between pelleting and pretreatment processes, considering various feedstocks, is crucial for future design. Practical considerations such as energy consumption, costs, and environmental impacts must not be overlooked, along with exploration of cutting-edge technologies and strategies in this field. </p> <p>This dissertation further presents a comprehensive investigation into the recalcitrance of pelleted corn stover to enzymatic digestion prior to any pretreatment. One aspect focuses on the role of high moisture pelleting in enhancing the enzymatic digestibility of corn stover before pretreatment, along with the relevant substrate characteristics. The pelleting process increases the digestibility of unpretreated corn stover, resulting in a glucan conversion increase from 8.2% to 15.5% at a 5% solid loading using 1 FPU Cellic® CTec2 per gram of solids. Under the same enzymatic hydrolysis conditions, the conversion of glucan remains higher for pelleted corn stover compared to its non-pelleted counterpart, even though both samples underwent identical milling processes and passed through the same screen to minimize particle impact. Compositional analysis reveals that loose and pelleted corn stover have similar non-dissolvable compositions, albeit with differences in their extractives. Using microcrystalline cellulose (Avicel) as a substrate, the presence of corn stover extractives results in a lower sugar yield compared to using citrate buffer instead, particularly for extractives from pelleted corn stover. This indicates a more negative impact of pelleted corn stover extractives on the activity of employed enzyme, CTec2. However, pelleted corn stover still shows increased overall glucan conversion compared to loose corn stover, suggesting improved digestibility of non-dissolvable components after milling and washing. The improvement in digestibility of pelleted material can be attributed to factors such as reduced particle size, enhanced substrate accessibility, and hydrolysis of cross-linking structures induced by the pelleting process. These findings offer valuable insights for the development of processing strategies aimed at sustainable and efficient utilization of lignocellulose.</p> <p>Furthermore, this dissertation delves into the profound impact of extractives on enzymatic hydrolysis, prompting a thorough examination of the composition and characteristics of extractives derived from pelleted corn stover, as well as their effects on enzymatic conversion. In contrast to previous reports, it is discovered that water extractable materials actually enhance the enzymatic hydrolysis of extractive-free stover, while the enzyme activities diminish when using microcrystalline cellulose as a substrate. This divergent behavior of extractives is attributed to the presence of lignin, which may interact with inhibitory compounds like phenolics, thereby mitigating the detrimental effects of soluble inhibitors or insoluble lignin, or both. These findings significantly advance our fundamental understanding of the intrinsic behavior of extractives and contribute to the optimization of schemes for efficient and cost-competitive enzymatic conversion of lignocellulose. </p>