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This master’s thesis discusses the useof enzymatic hydrolysis process of waste cardboard using simultaneous saccharification and fermentation (SSF) as a source of raw materials for production of liquid biofuels. This thesis is based on theses written by Ing. Brummer and Ing.Lepař.Thus, results gained in these works have been used and also further developed. The theoretical part summarizes the reasons for further development of SSF method and discusses, as well, the achievements reached in the processing of lignocellulosic waste materials by the SSF method so far.This section also discusses the general characteristics of lignocellulosic materials and also of the cellulolytic enzymes. It focusses also on individual pretreatment methods of lignocellulosic material and options of increasing the yield of the whole process. The experimental part verifies the particular results reached in previous theses and at the same time a further optimization of the method has been carried out because of the transfer of the whole process into a fermenter. Cardboard was set as the substrate for the experiments as it was evaluated by Ing. Brummer as the best one for enzymatic hydrolysis which was carried out by enzymes from Novozymes®. Parameters such as temperature, pH and kind of used buffer, the loading concentration of substrate and enzymes, were set according to the thesis of Ing. Lepař, which was aimed to their optimization. The SSF process done in fermenter of 2.0 l volume confirmed the previous results and furthermore it has been more effective through optimization of the added inoculum volume. It has been confirmed that the best substrate is cardboard finely grinded by vibrating mill. Also experiments with added nutrients had been done as an effort to increase the ethanol concentration, but these haven’t resulted insatisfying results. The maximal concentration of ethanol was 23,49 g/l, which was achieved after further optimization of various conditions. This result equals to experimental yield of 84,79 %.

Thesis (Ph. D. (Microbiology)) -- University of Limpopo, 2019 / In an effort to align the current research with the country’s biofuel strategy, the aim of the study was to utilize banana pseudostem in the production of fungal cellulolytic enzymes and bioethanol through fermentation of the banana pseudostem hydrolysate. The selection of microorganisms was based on the ability of the fungi to grow on agar containing Avicel (microcrystalline cellulose) followed by assaying for cellulases in the form of endoglucanase and total cellulase activity. Ten fungal isolates obtained from screening process showed positive endoglucanase activity on carboxymethyl cellulose – Congo Red agar plate. The six fungal isolates selected based on high cellulase activity belonged to Trichoderma and Aspergillus genera. In submerged fermentation (SmF), the maximum cellulase and endoglucanase production under optimal conditions by all fungal isolates was achieved in media with an initial of pH 6.5 at 30 °C. Under these conditions, the total cellulase activity was 9.79 filter paper units (FPU)/mL and endoglucanase activity 45.2 U/mL for Trichoderma longibrachiatum LMLUL 14-1 and total cellulase activity of 7.7 FPU/mL and endoglucanase activity of 32.7 U/mL for Trichoderma harzianum LMLUL 13-5. These cellulase activities were higher than in the crude enzymes system for all Aspergillus fumigatus. The production conditions for maximum β-glucosidase varied amongst the Aspergillus spp. For example, Aspergillus fumigatus LMLUL 13-4 had produced higher β-glucosidase activity in a medium with an initial pH of 6.5 and at an incubation temperature of 30 °C whereas A. fumigatus LMLUL 13-1 had produced higher β-glucosidase activity at an initial pH of 7.0 and at 35 °C. Solid state fermentation (SSF) to produce cellulase enzymes system was influenced by temperature, nature of the substrate (i.e. moisture, modification) and culturing technique/strategy (i.e. monoculture versus co-culture). Higher cellulase enzymes system was produced under the conditions of 30 °C, 75% moisture content of untreated (native) BPS and pH 6.5. All the fungi investigated, produced thermotolerant and acidophilic cellulase and endoglucanase, whilst β-glucosidase is both acidophilic and alkaliphilic. The cellulase enzymes complex of T. harzianum LMLBP07 13-5 is most stable, followed by A. fumigatus LMLPS 13-4 and the least stable cellulase enzymes complex was for T. longibrachiatum LMLULSA 14-1. For the pretreatment of BPS, the material was first subjected to three different pretreatment conditions; namely alkaline (3% NaOH), acid (5% H2SO4) and hot water (autoclave method) pretreatment to remove lignin and loosen the cellulose structure. After the pretreatments, alkaline method exposed more cellulose than other pretreatments methods. The alkaline pretreated BPS contained 52.3% cellulose, 10.8% hemicellulose and 8.7% lignin, which is 2.3-fold more cellulose and 0.48-fold less hemicellulose as well as 0.6-fold less lignin to the native BPS. The enzymatic saccharification of the alkaline pretreated BPS at different substrate loadings at 50 °C for 76 hours by an individual crude cellulase enzymes system from T. longibrachiatum LMLSAUL 14-1 and T. harzianum LMLBP07 13-5 cultures were used at a final concentration of 10 FPU/g. Saccharification released maximum glucose of 43.5 g/L and 20.1 g/L form alkaline pretreated BPS by crude cellulase enzymes from T. longibrachiatum LMLSAUL 14-1 and T. harzianum LMLBP07 13-5 measured at the highest solid loading. The production of bioethanol was carried out in separate hydrolysis and fermentation (SHF). Fermentation of nutrient supplemented BPS hydrolysate with an initial pH of 5.0 by S. cerevisiae UL01 occurred at 30 °C for 48 hours. The maximum ethanol concentration obtained after fermentation was 17.6 g/L corresponding to ethanol yield of 60% of the maximum theoretical yield. In conclusion, banana pseudostem is a suitable alternative substrate for the production of second-generation bioethanol. / National Research Foundation (NRF) and Vlaamse Interuniversitaire Raad (VLIR- UOS)

Os micro-organismos apresentam uma imensa diversidade genética e desempenham funções únicas e cruciais na manutenção de ecossistemas. Uma dessas funções é a produção de enzimas extracelulares, que ajudam na degradação da matéria orgânica e são cada vez mais procuradas e exploradas pela indústria. Essa propriedade aumenta a busca por enzimas que possam ser utilizadas nos diversos setores industriais com maior aproveitamento e baixo custo. A celulase pertence a essa classe de enzimas e é formada por um complexo multienzimático capaz de hidrolisar celulose por meio da quebra da ligação β,1-4. A partir dessa característica da celulase, foi realizada uma expressão heteróloga relacionada com a hidrólise da celulose em biblioteca metagenômica de solo da Caatinga. Foram realizados testes de produção enzimática por meio dos quais selecionamos os clones 283/A8 e 307/E11 como melhores produtores de endoglicanases. Com o objetivo de analisar a cinética de produção de celulases pelos clones, estes foram inoculados em diferentes fontes de celulose, pH e temperatura. A faixa ideal de pH foi 5,0 e de temperatura, 50º C, verificada para as enzimas Celulase Total, Endoglicanase e β-glicosidase. Quanto à termoestabilidade, as enzimas presentes mantiveram mais de 60% da atividade inicial após 2 horas de incubação a 50º C. O perfil de proteínas analisado por SDS-PAGE demonstrou que os clones secretam um conjunto de enzimas celulolíticas com 25 a 100 KDa, quando cultivado em farelo de trigo, e 30 a 60 KDa, quando cultivados em CMC. No ensaio de cromatografia para o clone 307/E11, foram selecionadas 5 frações que obtiveram melhores resultados na dosagem enzimática e testados frente ao pH e à temperatura. O resultado obtido foi que o complexo enzimático bruto, extraído do sobrenadante produzido pelo clone, possui melhor atividade frente ao pH e à temperatura do que as frações parcialmente purificadas. / Microorganisms are distinguished by a wide genetic diversity and they perform unique and crucial functions concerning the maintenance of ecosystems. One of those functions is the production of extracellular enzymes, which help in the degradation of organic matter and which are increasingly wanted and explored by industry. Such feature boosts the search of enzymes that can be exploited in several industrial sectors with improved full use and low cost. Cellulase belongs to such class of enzymes and it is composed of a multi-enzymatic complex which is able to hydrolyse cellulose through the breaking of the chemical bond β,1-4. Considering such attribute of the cellulase, a heterologous expression, related to cellulose hydrolysis in a metagenomic inventory of the soil of Caatinga, was realized. Tests of enzymatic production were performed, and through them, we could elect the clones 283/A8 and 307/E11 as the best endoglicanase producers. Those clones were inoculated in different cellulose sources, pH and temperature, so that we could analyse the kinetic of cellulase production between them. The ideal pH range was 5.0 and the ideal temperature range was 50º C (122º F), verified for the enzymes Total Cellulase, Endoglicanase and β-glucosidase. With regard to thermostability, the present enzymes kept more than 60% of the initial activity after 2 hours of incubation at 50º C (122º F). The proteins profile analysed with the help of SDS-PAGE proved that the clones secrete a group of cellulolytic enzymes with a weight average of 25 to 100 KDa, when cultivated in wheat bran, and of 30 to 60 KDa, when cultivated in CMC. Five fractions with the best results, regarding enzymatic dosage and tested before pH and temperature, were chosen by the chromatography research for the clone 307/E11. The achieved result proved that the raw enzymatic complex, extracted from the supernatant produced by the clone, develops a better activity before pH and temperature than the fractions partially purified.

O álcool, considerado um subproduto de grande importância da cultura de cana-deaçúcar, tem apresentado grande interesse nos últimos anos devido a questões econômicas e ambientais. A estimativa de produção para a safra 2007-2008, de acordo com a Companhia Nacional de Abastecimento (Conab), é de 251,59 milhões de toneladas de álcool. Por ser considerada uma fonte de energia alternativa (em substituição aos combustíveis fósseis) e renovável, muitos estudos estão sendo direcionados à cultura da cana-de-açúcar como, por exemplo, o aproveitamento do bagaço considerado um resíduo do setor sucroalcooleiro. O intuito deste trabalho foi o de realizar a hidrólise do bagaço de cana-de-açúcar por meio de enzimas celulolíticas comerciais visando identificar e quantificar açúcares fermentescíveis. Com essa finalidade, primeiramente, foram selecionadas as seguintes enzimas comerciais HPL1800, CL3708, P1250 e P4500; as quais apresentaram maior atividade celulolítica total em papel de filtro. Posteriormente foram testados dois pré-tratamentos do bagaço (ácido ou alcalino) e verificadas a atuação dessas enzimas em cada pré-tratamento do bagaço de cana-de-açúcar pelas metodologias do ácido dinitrosalicílico (DNS) e da cromatografia líquida (HPAEC - PAD). Os dados obtidos por ambas as metodologias foram analisados estatisticamente e concluiu-se que a ação conjunta do pré-tratamento ácido 0,5%, autoclave a 121ºC por 30 minutos e a enzima P4500 foram as melhores formas de tratamento para a obtenção de açúcares. / Ethanol is considered an important by-product from sugar cane culture. Nowadays it has been shown great importance in economics and environmental questions. The estimate ethanol production 2007-2008 is about 251.59 millions of tons, according to Companhia Nacional de Abastecimento (Conab). Alcohol is considered an alternative and renewable source of energy; this has lead several new studies on development of sugarcane culture and its derivatives, such as bagasse which is considered a residue from sugarcane industry. The aim of this research is the enzymatic hydrolysis of sugarcane bagasse for sugar production and its quantification and identification. The first step consisted at the selection of higher cellulolytic activity commercial enzymes in filter paper. The four enzymes selected were: HPL1800, CL3708, P1250 and P4500. After that, their performances were tested with two different pre-treated (acid and alkali) bagasse. The total sugars presents in the hydrolyzed were measured by dinitrosalicilic acid (DNS) and liquid chromatography (HPAEC - PAD) methodologies. The results were analyzed with statistics program. The datelines showed that joint action of 0.5% acid pre-treatment, 121ºC per 30 minutes and enzyme P4500 were the best treatment to sugars attainment.

O álcool, considerado um subproduto de grande importância da cultura de cana-deaçúcar, tem apresentado grande interesse nos últimos anos devido a questões econômicas e ambientais. A estimativa de produção para a safra 2007-2008, de acordo com a Companhia Nacional de Abastecimento (Conab), é de 251,59 milhões de toneladas de álcool. Por ser considerada uma fonte de energia alternativa (em substituição aos combustíveis fósseis) e renovável, muitos estudos estão sendo direcionados à cultura da cana-de-açúcar como, por exemplo, o aproveitamento do bagaço considerado um resíduo do setor sucroalcooleiro. O intuito deste trabalho foi o de realizar a hidrólise do bagaço de cana-de-açúcar por meio de enzimas celulolíticas comerciais visando identificar e quantificar açúcares fermentescíveis. Com essa finalidade, primeiramente, foram selecionadas as seguintes enzimas comerciais HPL1800, CL3708, P1250 e P4500; as quais apresentaram maior atividade celulolítica total em papel de filtro. Posteriormente foram testados dois pré-tratamentos do bagaço (ácido ou alcalino) e verificadas a atuação dessas enzimas em cada pré-tratamento do bagaço de cana-de-açúcar pelas metodologias do ácido dinitrosalicílico (DNS) e da cromatografia líquida (HPAEC - PAD). Os dados obtidos por ambas as metodologias foram analisados estatisticamente e concluiu-se que a ação conjunta do pré-tratamento ácido 0,5%, autoclave a 121ºC por 30 minutos e a enzima P4500 foram as melhores formas de tratamento para a obtenção de açúcares. / Ethanol is considered an important by-product from sugar cane culture. Nowadays it has been shown great importance in economics and environmental questions. The estimate ethanol production 2007-2008 is about 251.59 millions of tons, according to Companhia Nacional de Abastecimento (Conab). Alcohol is considered an alternative and renewable source of energy; this has lead several new studies on development of sugarcane culture and its derivatives, such as bagasse which is considered a residue from sugarcane industry. The aim of this research is the enzymatic hydrolysis of sugarcane bagasse for sugar production and its quantification and identification. The first step consisted at the selection of higher cellulolytic activity commercial enzymes in filter paper. The four enzymes selected were: HPL1800, CL3708, P1250 and P4500. After that, their performances were tested with two different pre-treated (acid and alkali) bagasse. The total sugars presents in the hydrolyzed were measured by dinitrosalicilic acid (DNS) and liquid chromatography (HPAEC - PAD) methodologies. The results were analyzed with statistics program. The datelines showed that joint action of 0.5% acid pre-treatment, 121ºC per 30 minutes and enzyme P4500 were the best treatment to sugars attainment.

Os micro-organismos apresentam uma imensa diversidade genética e desempenham funções únicas e cruciais na manutenção de ecossistemas. Uma dessas funções é a produção de enzimas extracelulares, que ajudam na degradação da matéria orgânica e são cada vez mais procuradas e exploradas pela indústria. Essa propriedade aumenta a busca por enzimas que possam ser utilizadas nos diversos setores industriais com maior aproveitamento e baixo custo. A celulase pertence a essa classe de enzimas e é formada por um complexo multienzimático capaz de hidrolisar celulose por meio da quebra da ligação β,1-4. A partir dessa característica da celulase, foi realizada uma expressão heteróloga relacionada com a hidrólise da celulose em biblioteca metagenômica de solo da Caatinga. Foram realizados testes de produção enzimática por meio dos quais selecionamos os clones 283/A8 e 307/E11 como melhores produtores de endoglicanases. Com o objetivo de analisar a cinética de produção de celulases pelos clones, estes foram inoculados em diferentes fontes de celulose, pH e temperatura. A faixa ideal de pH foi 5,0 e de temperatura, 50º C, verificada para as enzimas Celulase Total, Endoglicanase e β-glicosidase. Quanto à termoestabilidade, as enzimas presentes mantiveram mais de 60% da atividade inicial após 2 horas de incubação a 50º C. O perfil de proteínas analisado por SDS-PAGE demonstrou que os clones secretam um conjunto de enzimas celulolíticas com 25 a 100 KDa, quando cultivado em farelo de trigo, e 30 a 60 KDa, quando cultivados em CMC. No ensaio de cromatografia para o clone 307/E11, foram selecionadas 5 frações que obtiveram melhores resultados na dosagem enzimática e testados frente ao pH e à temperatura. O resultado obtido foi que o complexo enzimático bruto, extraído do sobrenadante produzido pelo clone, possui melhor atividade frente ao pH e à temperatura do que as frações parcialmente purificadas. / Microorganisms are distinguished by a wide genetic diversity and they perform unique and crucial functions concerning the maintenance of ecosystems. One of those functions is the production of extracellular enzymes, which help in the degradation of organic matter and which are increasingly wanted and explored by industry. Such feature boosts the search of enzymes that can be exploited in several industrial sectors with improved full use and low cost. Cellulase belongs to such class of enzymes and it is composed of a multi-enzymatic complex which is able to hydrolyse cellulose through the breaking of the chemical bond β,1-4. Considering such attribute of the cellulase, a heterologous expression, related to cellulose hydrolysis in a metagenomic inventory of the soil of Caatinga, was realized. Tests of enzymatic production were performed, and through them, we could elect the clones 283/A8 and 307/E11 as the best endoglicanase producers. Those clones were inoculated in different cellulose sources, pH and temperature, so that we could analyse the kinetic of cellulase production between them. The ideal pH range was 5.0 and the ideal temperature range was 50º C (122º F), verified for the enzymes Total Cellulase, Endoglicanase and β-glucosidase. With regard to thermostability, the present enzymes kept more than 60% of the initial activity after 2 hours of incubation at 50º C (122º F). The proteins profile analysed with the help of SDS-PAGE proved that the clones secrete a group of cellulolytic enzymes with a weight average of 25 to 100 KDa, when cultivated in wheat bran, and of 30 to 60 KDa, when cultivated in CMC. Five fractions with the best results, regarding enzymatic dosage and tested before pH and temperature, were chosen by the chromatography research for the clone 307/E11. The achieved result proved that the raw enzymatic complex, extracted from the supernatant produced by the clone, develops a better activity before pH and temperature than the fractions partially purified.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As pesquisas acerca de fontes alternativas de combustÃveis tem se concentrado principalmente na biomassa composta de celulose, hemicelulose e lignina. Os carboidratos estruturais contidos na matriz celulÃsica representam o substrato que pode ser utilizado para produÃÃo de biocombustÃveis por processos fermentativos. A opÃÃo pela hidrÃlise enzimÃtica de biomassa vegetal, utilizando celulases, pode gerar uma opÃÃo de menor impacto ambiental frente a outros processos de hidrÃlise. Estudos sobre novas fontes microbianas e, anÃlises mais acuradas das etapas que compÃem a produÃÃo de celulases sÃo essenciais como estratÃgias para diminuir os custos gerados pelo uso de celulases nos processos de obtenÃÃo de aÃÃcares fermentescÃveis. Portanto, este trabalho tem como objetivo a seleÃÃo de fungos filamentosos, produtores de enzimas do complexo celulolÃtico, assim como a investigaÃÃo dos parÃmetros que envolvem a produÃÃo de celulases atravÃs do processo de fermentaÃÃo submersa. Em uma etapa inicial foram selecionados os micro-organismos por metodologia em placas, onde se averiguou o potencial celulolÃtico atravÃs do Ãndice enzimÃtico em meio especÃfico. Foram selecionadas as linhagens que atingiram um diÃmetro de crescimento de colÃnia de 150 mm, em placas de Petri, em menor tempo de incubaÃÃo (48 horas). Da etapa de seleÃÃo inicial, foram escolhidos os micro-organismos com maior potencial enzimÃtico que foram entÃo submetidos aos testes em fermentaÃÃo submersa. O primeiro teste consistiu em selecionar os micro-organismos como melhores produtores de celulases totais (FPase) em erlenmeyer nÃo aletados contendo 100 mL de meio de cultura especÃfico. O teste subsequente analisou as trÃs melhores linhagens da etapa anterior, que foram entÃo submetidos à fermentaÃÃo submersa em frascos contendo aletas. A etapa seguinte teve o intuito de investigar a produÃÃo enzimÃtica em fermentaÃÃo com quatro tipos de aÃÃcares solÃveis: glicose, lactose, sacarose e xilose. A linhagem fÃngica selecionada nas etapas anteriores foi utilizada nos testes em biorreator, onde foram analisados trÃs diferentes estratÃgias de inoculaÃÃo. Dentre os fungos analisados na etapa inicial, destacaram-se as linhagens Fusarium sp. SAP 09, Lasiodiplodia theobromae CNPAT 040, Trichoderma sp. LCB 79, Trichoderma, sp. INPA 666, Trichoderma sp. INPA 1014 e Trichoderma sp. INPA 1218. Na etapa de seleÃÃo por fermentaÃÃo submersa em frascos nÃo aletados, a melhor atividade de FPase foi apresentada pela linhagem Trichoderma sp. INPA 666 (48,0 FPU/L) e CMCase pelo fungo Lasiodiplodia theobromae CNPAT 040 (350,0 U/L). Na comparaÃÃo com erlenmeyers aletados, houve uma maior produÃÃo tanto de FPase quanto de CMCase em frascos sem a presenÃa de aletas, apontando que as forÃas de cisalhamento aplicadas nas culturas fÃngicas possivelmente foram deletÃrias para a produÃÃo enzimÃtica. O uso de sacarose mostrou-se ser a melhor opÃÃo dentre os aÃÃcares solÃveis testados, apresentando os maiores valores de atividade de FPase (49,9 FPU/L) e CMCase (119,7 U/L). A melhor estratÃgia de inoculaÃÃo para o biorreator foi uma suspensÃo de esporos obtidos a partir de uma fermentaÃÃo semi-sÃlida de farelo de trigo, no tempo de 72 horas de fermentaÃÃo. / The research about alternative sources of fuels has been mainly focused on biomass composed of cellulose, hemicellulose and lignin. The structural carbohydrates contained in the cellulosic matrix represent the substrate that can be used for biofuel production by fermentative processes. The choice of the biomass enzymatic hydrolysis of plants using cellulases, can generate an option less environmental impact compared to other processes of hydrolysis. Studies about microbial new sources, and more accurate analysis of the steps that make up the production of cellulases are essential as a strategy to reduce the costs generated by the use of cellulases in the process of obtaining fermentable sugars. This study aims at the selection of filamentous fungi producers of cellulolytic enzymes, as well as investigating the parameters of cellulase production by submerged fermentation process. In the initial stage were selected micro-organisms by methodology in plates. Was investigated the potential cellulolytic enzyme through the index in a specific medium and were selected strains that reached a diameter of colony growth of 150 mm in Petri dishes in a shorter time of incubation (48 hours). Were selected micro-organisms with the greatest potential enzymatic tests that were then tested in submerged fermentation. The first test consisted in selecting the best microorganisms as producers of FPase in non-baffled flask containing 100 mL of culture medium specific. In the subsequent test were examined the three best strains from the previous step, which were then subjected to submerged fermentation in baffled flasks. The next step was designed to investigate the enzyme production in fermentation with four types of soluble sugars: glucose, lactose, sucrose and xylose. The fungal strain was selected in the previous steps used in the tests in a bioreactor, which analyzed three different strategies of inoculation. The strains Fusarium sp. SAP 09, Lasiodiplodia theobromae CNPAT 040, Trichoderma sp. LCB 79, Trichoderma sp. INPA 666, Trichoderma sp. INPA 1014 and Trichoderma sp. INPA 1218 were selected from the first stage. In the selection stage by submerged fermentation in non-baffled flasks, the best FPase activity was achieved by the strain Trichoderma sp. INPA 666 (48.0 FPU /L) and CMCase by the fungus Lasiodiplodia theobromae CNPAT 040 (350.0 U /L). In comparison with baffled flasks, there was a greater production both FPase and CMCase in Erlenmeyer without baffles, indicating that the shear forces applied to the fungal cultures were potentially harmful for enzyme production. The use of sucrose proved to be the best option among soluble sugars tested, with higher rates of FPase activity (49.9 FPU / L) and CMCase (119.7 U / L). The best strategy for the inoculation was a spore suspension obtained from a solid state fermentation of wheat bran, in the time of 72 hours of fermentation.

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.

In this thesis the use of waste materials for the microbial production of important metabolites is reported. The first part is focused on the use of waste paper (a lignocellulosic material) as a non-traditional source for the production of bioethanol. The second part is focused on the immobilization of cellulolytic enzymes, which are used for the hydrolysis of lignocellulosic materials. First, the waste paper (cardboard) was pre-treated using a blender and a vibratory mill. The pre-treated cardboard was used for the production of ethanol by the method of simultaneous saccharification and fermentation. This method was optimized with free cells of Saccharomyces cerevisiae. Then strains suitable for the immobilization were selected. Strains of S. cerevisiae and Pichia kudriavzevii were immobilized by encapsulation into the polyvinyl alcohol carrier and tested again for the ethanol production by simultaneous saccharification and fermentation. In the second part of the work a carrier from waste polyethylene terephthalate bottles was prepared and used for the immobilization of the cellulolytic complex. The basic characteristics were determined, such as optimal pH and optimal temperature, storage, operational and thermal stability, enzyme kinetics and the mode of action of the enzyme. Compared to two other commercial carriers this carrier showed to be suitable for the immobilization of the cellulolytic complex.