Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2020 / Bioethanol production from lignocellulosic biomass is seen as an alternative source of energy. However, large-scale production of bioethanol from lignocellulosic biomass is still not feasible due to the high cost of cellulase and lack of cellulases with a high specific activity that can act on crystalline cellulose. The study aimed at screening for microbial cellulolytic enzymes using a proteomic approach. The objectives were to screen for microbial cellulases with a high specific activity and separate the cellulolytic enzymes using a combination of zymography and two dimensional (2-D) gel electrophoresis followed by tryptic digestion, matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) and bioinformatics analysis. Fungal and bacterial isolates were cultured in M9 minimal and Mandel media for a period of 168 hours at 60°C and 30°C with cellobiose and Avicel as carbon sources. Microbial cells were separated from the supernatants through centrifugation and the crude enzymes from the cultures were used for the determination of cellulase activity, zymography, SDS-PAGE and two-dimensional gel electrophoresis. Five isolates, with lytic action on carbon sources studied were a bacterial strain, (BARK) and fungal strains (VCFF1, VCFF14, VCFF17 and VCFF18). Peak cellulase production by the isolates was found to be 3.8U/ml, 2.09U/ml, 3.38U/ml, 3.18U/ml and 1.95U/ml, respectively. Beta-glucosidase zymography resulted in a dark brown band and clear zones against a dark background for endoglucanase. Affinity precipitation of the VCFF17 isolate’s crude enzyme resulted in seven glycoside hydrolases with a carbohydrate binding module (CBM). The presence of the CBM in the glycoside hydrolases produced by the VCFF17 confer the isolate’s potential to be used in the hydrolysis of plant biomass for bioethanol production. Two-dimensional gel protein maps resulted in the separation and quantitative expression of different proteins by the microbial isolates. MALDI-TOF analysis and database search showed that the expressed proteins in this study closely relate to different glycoside hydrolases produced by other microbial species (Hypocrea jecorina, Emericella nidulans, Trichoderma pseudokoningii and Trichoderma koningii). BARK, VCFF1, VCFF14, VCFF17 and VCFF18 showed great potential as cellulolytic enzyme producers for bioethanol production. The BARK isolate exhibited the highest beta glucosidase activity. The isolates studied may benefit the industry in reducing the costs associated with bioethanol production in consolidated bioprocessing system. / National Research Foundation (NRF)

O aproveitamento de células vivas de leveduras - Saccharomyces cerevisiae - como probiótico, e, componentes extraídos da parede celular desta levedura, como aditivos, para desejadas funções, têm sido objeto de interesse cada vez maior, pelos resultados promissores até então obtidos e, concomitantemente, pelo excedente de biomassa de leveduras gerado pela indústria de etanol e cerveja no Brasil. Por estes motivos, foram conduzidos dois experimentos, um com o objetivo de investigar a possibilidade de utilizar o milho como um veículo alternativo para a levedura S. cerevisiae - como probiótico, e outro, com o propósito de estudar a capacidade de reduzir danos promovidos pelas aflatoxinas, das leveduras vivas, das termolisadas e mananoligossacarídeos. O primeiro experimento foi montando em um arranjo fatorial 2x2x5, constituído de duas concentrações de leveduras (1 e 2%), grãos de milho com dois teores de umidade (16 e 20%) e 5 períodos de armazenamento (0, 15, 30, 90 e 110 dias), distribuído ao acaso, com 4 repetições. O outro estudo, foi um bioensaio, conduzido durante 28 dias, com ratos Wistar, em um experimento com delineamento inteiramente casualizado, com 7 tratamentos e 5 repetições. A concentração de células aplicadas nos grãos de milho não influenciou a viabilidade das leveduras durante o armazenamento. A umidade do substrato interferiu no desenvolvimento da viabilidade, e que, em substratos com maiores teores de umidade foram observadas menores médias de viabilidade durante o armazenamento. A viabilidade das células de leveduras permaneceu constante por 30 dias e aos 90 dias em armazenamento foram observadas reduções no número de células viáveis. As leveduras apresentaram viabilidade de 70% aos 110 dias em armazenamento. A inoculação de leveduras vivas em grãos de milho, visando a sua utilização como probiótico, é uma técnica viável. Os tratamentos com levedura termolisada e mananoligossacarídeos não foram capazes de reduzir os danos promovidos pelas aflatoxinas em nível de hepatócitos. As leveduras vivas foram capazes de reduzir os danos promovidos pelas aflatoxinas em nível celular. / The utilization of live cells of yeasts - Saccharomyces cerevisiae - as probiotic, and, extracted components of the cellular wall of this yeast, as addictive, to desired functions, have been object of interest, due the promising results until then obtained and, simultaneously, for the excess of biomass of yeasts generated by the etanol and beer industry in Brazil. For these reasons, two experiments were led, one with the objective of investigate the possibility to use the corn as an alternative vehicle for the yeast S. cerevisiae - as probiotic, and other, with the purpose of studying the capacity to reduce damages promoted by the aflatoxins of the live yeasts, termoliseds and mananoligossacarides. The first experiment was mounted in a factorial arrangement 2x2x5, constituted of two concentrations of yeasts (1 and 2%), corn grains with two moisture contents (16 and 20%) and 5 storage periods (0, 15, 30, 90 and 110 days), random arrangement, with 4 replications. The other study, was a bioassay, led during 28 days, with rats Wistar, in completely randomzed desing, with 7 treatments and 5 repetitions. The concentration of applied cells in the corn grains did not influence the viability of the yeasts during the storage. The moisture of the substrate interfered in the development of the viability, and that in substrate with bigger moisture contents smaller viability averages were observed during the storage. The viability of the cells of yeasts remains constant for 30 days and to the 90 days in storage reductions were observed in the number of viable cells. The yeasts presented viability from 70% to the 110 days in storage. The inoculation of live yeasts in corn grains, aiming its use as probiotic, is a viable technique. The treatments with yeast termolised and mananoligossacarídes were not capable of reducing the damages promoted by the aflatoxins in hepatocites level. The live yeasts were capable to reduce the damages caused by the aflatoxins in cellular level.

O aproveitamento de células vivas de leveduras – Saccharomyces cerevisiae – como probiótico, e, componentes extraídos da parede celular desta levedura, como aditivos, para desejadas funções, têm sido objeto de interesse cada vez maior, pelos resultados promissores até então obtidos e, concomitantemente, pelo excedente de biomassa de leveduras gerado pela indústria de etanol e cerveja no Brasil. Por estes motivos, foram conduzidos dois experimentos, um com o objetivo de investigar a possibilidade de utilizar o milho como um veículo alternativo para a levedura S. cerevisiae – como probiótico, e outro, com o propósito de estudar a capacidade de reduzir danos promovidos pelas aflatoxinas, das leveduras vivas, das termolisadas e mananoligossacarídeos. O primeiro experimento foi montando em um arranjo fatorial 2x2x5, constituído de duas concentrações de leveduras (1 e 2%), grãos de milho com dois teores de umidade (16 e 20%) e 5 períodos de armazenamento (0, 15, 30, 90 e 110 dias), distribuído ao acaso, com 4 repetições. O outro estudo, foi um bioensaio, conduzido durante 28 dias, com ratos Wistar, em um experimento com delineamento inteiramente casualizado, com 7 tratamentos e 5 repetições. A concentração de células aplicadas nos grãos de milho não influenciou a viabilidade das leveduras durante o armazenamento. A umidade do substrato interferiu no desenvolvimento da viabilidade, e que, em substratos com maiores teores de umidade foram observadas menores médias de viabilidade durante o armazenamento. A viabilidade das células de leveduras permaneceu constante por 30 dias e aos 90 dias em armazenamento foram observadas reduções no número de células viáveis. As leveduras apresentaram viabilidade de 70% aos 110 dias em armazenamento. A inoculação de leveduras vivas em grãos de milho, visando a sua utilização como probiótico, é uma técnica viável. Os tratamentos com levedura termolisada e mananoligossacarídeos não foram capazes de reduzir os danos promovidos pelas aflatoxinas em nível de hepatócitos. As leveduras vivas foram capazes de reduzir os danos promovidos pelas aflatoxinas em nível celular. / The utilization of live cells of yeasts - Saccharomyces cerevisiae - as probiotic, and, extracted components of the cellular wall of this yeast, as addictive, to desired functions, have been object of interest, due the promising results until then obtained and, simultaneously, for the excess of biomass of yeasts generated by the etanol and beer industry in Brazil. For these reasons, two experiments were led, one with the objective of investigate the possibility to use the corn as an alternative vehicle for the yeast S. cerevisiae - as probiotic, and other, with the purpose of studying the capacity to reduce damages promoted by the aflatoxins of the live yeasts, termoliseds and mananoligossacarides. The first experiment was mounted in a factorial arrangement 2x2x5, constituted of two concentrations of yeasts (1 and 2%), corn grains with two moisture contents (16 and 20%) and 5 storage periods (0, 15, 30, 90 and 110 days), random arrangement, with 4 replications. The other study, was a bioassay, led during 28 days, with rats Wistar, in completely randomzed desing, with 7 treatments and 5 repetitions. The concentration of applied cells in the corn grains did not influence the viability of the yeasts during the storage. The moisture of the substrate interfered in the development of the viability, and that in substrate with bigger moisture contents smaller viability averages were observed during the storage. The viability of the cells of yeasts remains constant for 30 days and to the 90 days in storage reductions were observed in the number of viable cells. The yeasts presented viability from 70% to the 110 days in storage. The inoculation of live yeasts in corn grains, aiming its use as probiotic, is a viable technique. The treatments with yeast termolised and mananoligossacarídes were not capable of reducing the damages promoted by the aflatoxins in hepatocites level. The live yeasts were capable to reduce the damages caused by the aflatoxins in cellular level.