Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2020 / In nature, saccharification is done by a variety of microorganisms, secreting a variety of cellulase in addition to other proteins. Co-culturing enables the production of more efficient enzyme preparations that would mimic the natural decomposition of lignocelluloses. During the decay of banana (Musa paradisiaca) pseudostem, a potential feedstock for second-generation biofuels, there may be a number of microorganisms producing cellulolytic enzymes, and other factors, which in combination might decompose the lignocelluloses more efficiently. The aim of the study was to establish a microbial co-culture for the production of highly active cellulase preparations. Banana pseudostems (BPS) and microbial samples from decaying banana pseudostems were collected in the Mopani District Allesbeste Nursery, Limpopo Province, South Africa. Fungi and bacteria were isolated using CMC agar plates. The best cellulase producing fungi and bacteria were tested for cellulase activity in monocultures and in various combinations (fungi-fungi, fungi-bacteria, bacteria-bacteria, fungi-live bacterial cells and fungi-dead bacterial cells) in submerged fermentation, using Avicel™ as a carbon source. Solid-state fermentation was also performed using banana pseudostem as a carbon source. Zymography was done in studying the variety of cellulase in the secretions from co-cultures/ mixed cultures. Identification of the bacterial and fungal isolates from decomposing banana pseudostems was also done using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) or DNA sequencing. A mixed culture of fungi in combination with dead bacterial cells was the best combination to produce higher levels of endoglucosidase and β-glucosidase activities in both submerged fermentation and solid-state fermentation. During SmF, endoglucosidase was (0.229 after 144 h) and β-glucosidase (4.519 after 96 h) activities and SSF, endoglucosidase (12.793 after 48 h) and β-glucosidase (37.45 after 144 h). Endoglucosidase zymography showed that monocultures and co-cultures produced four active bands for endoglucanase, except for the monoculture Trichoderma longibrachiatum 1B that produced a faint or unclear band. The current study demonstrated that three fungal strains namely, T longibrachiatum 1B, Aspergillus fumigatus 5A, and Aspergillus flavus 2A and one bacterial strain Enterobacter asburiae 1 are capable of producing a variety of endoglucanases. It seems that a combination of fungi with dead cells could significantly improve endoglucosidase and v β-glucosidase activities. The use of A. fumigatus in mixed cultures is highly recommended in order to produce high levels of β-glucosidases, no matter the combination used. / Foodbev Seta

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)

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / A utilizaÃÃo de materiais polimÃricos oriundos do petrÃleo na fabricaÃÃo de produtos de difÃcil decomposiÃÃo leva a estudos e desenvolvimento de materiais parcialmente ou completamente biodegradÃveis e de fontes renovÃveis. A celulose à um grande alvo destas pesquisas, nÃo somente por suas diversas fontes, mas tambÃm pela vasta aplicabilidade, principalmente em nanocompÃsitos. O Brasil, um paÃs do agronegÃcio, possui uma grande fonte de biomassa proveniente dos resÃduos do setor agroindustrial. Uma dessas fontes à a fibra do pseudocaule da bananeira, que à utilizada como adubo e cobertura de solo no prÃprio bananeiral. Tendo a grande geraÃÃo de resÃduos no bananeiral como oportunidade, o desenvolvimento de novas alternativas de aproveitamento amplia as opÃÃes de agregaÃÃo de valor e contribui para reduzir os seus impactos negativos. No presente trabalho, fibras do pseudocaule da bananeira (PCB), variedade Pacovan, foram avaliadas como possÃvel fonte para obtenÃÃo de nanocelulose para elaboraÃÃo de nanocompÃsitos em matriz de poli(Ãlcool vinÃlico) (PVOH), um polÃmero hidrofÃlico e biodegradÃvel. Inicialmente, o PCB foi dividido em quatro fraÃÃes: fraÃÃo externa, central, interna e nÃcleo para posterior caracterizaÃÃo quÃmica, tÃrmica e morfolÃgica. Em razÃo do maior conteÃdo de celulose e cristalinidade, utilizaram-se as fraÃÃes externas como matÃria-prima para a obtenÃÃo de nanocelulose. ApÃs branqueamento em meio alcalino, a fibra foi submetida à hidrÃlise Ãcida (H2SO4 62% m/m, 70 min, 45 ÂC) para obtenÃÃo dos nanocristais de celulose. A nanocelulose obtida do PCB apresentou-se como uma suspensÃo estÃvel de coloraÃÃo marrom. Tipicamente, os nanocristais apresentaram comprimentos (L) de 135,0 nm e diÃmetros (d) situados em torno de 7,2 nm; o que reproduziu razÃes de aspecto (L/d) de 21,2. A nanocelulose foi aplicada em uma matriz polimÃrica biodegradÃvel e solÃvel em Ãgua, o poli(Ãlcool vinÃlico), para obtenÃÃo de filmes nanocompÃsitos de diferentes concentraÃÃes (0, 1, 3 e 5% em base seca de matriz). A adiÃÃo de nanocelulose melhorou as propriedades mecÃnicas dos filmes atà a concentraÃÃo de 3%, diminuiu as propriedades tÃrmicas em todas as concentraÃÃes, melhorou a propriedade de barreira ao vapor de Ãgua gradualmente, com pequenas mudanÃas nas propriedades Ãpticas evidenciando uma oportunidade de aplicaÃÃo desse filme nanocompÃsito para embalagem. AlÃm disso, representando uma alternativa de agregaÃÃo de valor a um relevante resÃduo do agronegÃcio. / The use of polymeric materials from petroleum in the manufacture of difficult decomposition products leads to studies and development of partially or completely biodegradable materials from renewable sources. Cellulose is a major target of this research, not only for its various sources, but also by the wide applicability, especially in nanocomposites. Brazil, a country of agribusiness, has a large source of waste biomass from the agribusiness sector. One such source is the fiber from the banana pseudostem , which is used as fertilizer and soil cover in bananeiral own. Having a big residue generation in banana crop as an opportunity, the development of new alternative utilization expands the options of adding value and helps to reduce the negative impacts. In the present work, fibers from the banana pseudostem (PCB), Pacovan variety, were evaluated as a possible source for obtaining nanocelulose for preparation of nanocomposites in matrix of polyvinyl alcohol (PVOH), a hydrophilic and biodegradable polymer. Initially, the PCB was divided into four fractions: external fraction, central fraction, inner fraction and core for subsequent chemical, thermal and morphological characterization. Because of the higher cellulose content and crystallinity, we used the external fractions to obtain nanocelulose. After bleaching in alkaline environment, the pulp was subjected to acid hydrolysis (H2SO4 62% m / m, 70 min, 45  C) and reduced to cellulose nanocrystals. The nanocelulose extracted from the PCB appeared as a stable brown suspension . Typically, the crystals exhibited lengths (L) of 135.0 nm, and diameters (d) situated around 7.2 nm, which reproduces aspect ratios (L/d) 21.2. The nanocellulose was applied to a biodegradable polymeric matrix and water-soluble polyvinyl alcohol, to obtain nanocomposite films of different concentrations (0, 1, 3 and 5% on dry basis matrix). The addition of nanocellulose improved the mechanical properties of the films to 3% concentration, diminished the thermal properties at all concentrations, improved barrier property to water vapor gradually with small changes in the optical properties evidencing an opportunity to apply this nanocomposite film for packaging. Moreover, representing an alternative of adding value to a relevant agribusiness residue.

Made available in DSpace on 2016-08-19T12:56:38Z (GMT). No. of bitstreams: 1 dissertacao Nubia.pdf: 1577562 bytes, checksum: f392d5054fc1b5a19f04963529814a0c (MD5) Previous issue date: 2011-12-06 / FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO / In this work was investigated the potential of banana tree pseudostem, an agricultural waste used in natura (BIN) and treated with methanol (BTM), as adsorbents in removal of textile dyes Violet Brilliant Remazol and Turquoise Remazol from aqueous solution. The adsorbents were characterized by infrared spectroscopy, 13C nuclear magnetic resonance, elemental analysis, thermogravimetry and X-ray diffraction. The zero point of charge (pHzpc) the materials were 5.1 (BIN) and 4.3 (BTM). The pH study showed that the maximum amount adsorbed occurred in pH's 1.0 and 2.0. The kinetics was performed at pH 2.0, 25°C and was evaluated at concentrations of 250 and 1000 mg.L-1. The contact times required for both adsorbents reach equilibrium in the two concentrations studied were 120 and 300 minutes for the dyes Violet and Turquoise, respectively. The kinetics sorption data were fitted to pseudo-first order, second order, and intraparticle diffusion models and the equilibrium data were fitted to the Langmuir and the Freundlich isotherm models. Taking into account correlation coefficients, the data were best fitted to the second order kinetic model (R2> 0.999). The intraparticle diffusion model is also involved in the mechanism of adsorption, which showed that the adsorption takes place in three steps. With the exception of Turquoise Remazol by BIN, all other systems were best fitted to Freundlich isotherms. The adsorption isotherms were evaluated at four different temperatures (10, 25, 40 and 55 °C) by varying the concentration of the dye from 100 to 1000 mg.L-1 in the best conditions of pH and equilibrium time. The thermodynamic parameters indicated that the process is endothermic (Turquoise) and exothermic (Violet). The spontaneity of the sorption processes for all dyes was also confirmed by the favorable negative values of Gibbs free energy and by positive entropic data. Desorption of dyes was carried out in alkaline (pH 8.0), being recovered 44% (BIN) and 38% (BTM) for the Violet Brilliant Remazol and 34% (BIN) and 21% (BTM) for the dye Turquoise Remazol. This value suggests the application of this biopolymer as a favorable agent for textile dyes removal from a given system. / Neste trabalho foi investigado o potencial do pseudocaule de bananeira, um resíduo agrícola utilizado in natura (BIN) e tratado com metanol (BTM), como adsorventes na remoção dos corantes têxteis Violeta brilhante remazol e Turquesa remazol de soluções aquosa. Os adsorventes foram caracterizados por espectroscopia no infravermelho, ressonância magnética nuclear de 13C, análise elementar, termogravimetria e difratometria de raios X. O ponto de carga zero (pHzpc) dos materiais foram 5,1 (BIN ) e 4,3 (BTM). O estudo do pH mostrou que a quantidade máxima adsorvida ocorreu nos pH s 1,0 e 2,0. As cinéticas foram realizadas em pH 2,0, temperatura de 25°C e foram avaliados nas concentrações de 250 e 1000 mg.L-1. Os tempos de contato necessários para ambos os adsorventes atingirem o equilíbrio, nas duas concentrações estudadas foram 120 e 300 minutos para os corantes Violeta e Turquesa, respectivamente. Os dados cinético de adsorção foram ajustados aos modelos de pseudo-primeira ordem, segunda ordem e difusão intrapartícula e os dados de equilíbrio foram ajustados aos modelos de isoterma de Langmuir e Freundlich. Levando em consideração os coeficientes de correlação, os dados cinéticos foram melhor ajustados ao modelo de segunda ordem (R2 > 0,999). O modelo de difusão intrapartícula também está envolvido no mecanismo de adsorção, o qual mostrou que a adsorção acontece em três etapas. Com exceção do Turquesa remazol pelo BIN, todos os outros sistemas foram melhor representados pela isoterma de Freundlich. As isotermas de adsorção foram avaliadas em quatro temperaturas diferentes (10, 25, 40 e 55 ºC) variando-se a concentração do corante de 100 a 1000 mg.L-1 nas melhores condições de pH e tempo de equilíbrio. Os parâmetros termodinâmicos indicaram que o processo é endotérmico (Turquesa), exotérmico (Violeta). A espontaneidade dos processos de sorção de todos os corantes também foi confirmada pelos valores negativos da energia livre de Gibbs e pelos dados positivos de entropia. A dessorção dos corantes foi realizada em meio alcalino (pH 8,0), sendo recuperado 44% (BIN) e 38% (BTM) para Violeta brilhante remazol e 34% (BIN) e 21% (BTM) pra o corante Turquesa remazol. Este valor sugere a aplicação deste biopolímero como agente favorável para a remoção de corantes de efluentes têxteis a partir de um determinado sistema.