Submitted by LOUISE GARBELOTTI GONÇALVES (lou_ise_gg@hotmail.com) on 2016-05-31T17:27:57Z No. of bitstreams: 1 Dissertação_GONÇALVES, Louise G._2016.pdf: 1045451 bytes, checksum: 2b2d475a38ba54ed6a247190f4fc82b0 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-05-31T17:56:58Z (GMT) No. of bitstreams: 1 goncalves_lg_me_rcla.pdf: 1045451 bytes, checksum: 2b2d475a38ba54ed6a247190f4fc82b0 (MD5) / Made available in DSpace on 2016-05-31T17:56:58Z (GMT). No. of bitstreams: 1 goncalves_lg_me_rcla.pdf: 1045451 bytes, checksum: 2b2d475a38ba54ed6a247190f4fc82b0 (MD5) Previous issue date: 2016-04-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O desenvolvimento de bioprocessos para produção de alimentos e energia, que utilizam resíduos agroindustriais como matéria-prima, é alvo de intensas pesquisas no mundo devido às limitações de novas fronteiras agrícolas e de fontes de energia renováveis. A utilização de enzimas nesses processos produtivos surge como uma forte tendência, dentre elas, as amilases. O objetivo desse estudo foi a otimização da produção de amilases empregando o fungo Rhizopus microsporus var. oligosporus, cultivado em Fermentação em Estado Sólido (FES), tendo como substrato os resíduos agroindustriais: farelo de trigo, bagaço de mandioca e bagaço de cana de açúcar. O hidrolisado obtido da hidrólise do bagaço de mandioca foi avaliado quanto à sua fermentabilidade por meio da fermentação alcoólica em batelada alimentada por Saccharomyces cerevisiae. A melhor fonte de carbono como substrato para formulação do meio de cultivo foi aquele contendo 100% de farelo de trigo. Em relação às fontes de nutrientes para produção de amilases, a maior atividade amilolítica (418 U/g) foi para a solução de nutrientes contendo (%): 1,34 de sulfato de amônio, 0,54 de monofosfato de amônio (MAP) e 1,86 de ureia. Porém, foi observado que, ao acrescentar um mínimo de 0,01 % (m/m) de extrato de levedura na solução de nutrientes, obteve-se aumento na atividade amilolítica de 418 U/g para 551 U/g. A hidrólise enzimática do bagaço de mandioca foi feita com a associação de celulases comerciais (Novozyme) (10 FPU/g no tempo zero horas) e com o extrato amilolítico (12 U/g no tempo 10 horas de reação) produzido em FES para a obtenção de um hidrolisado concentrado. Com estas condições foi possível converter 63% de bagaço em açúcares redutores com predomínio de glicose. Por meio da fermentação alcoólica em batelada alimentada por Saccharomyces cerevisiae, obteve-se eficiências de 74,6 e 76,8%, e produtividades etanólicas com 3,14 g L-1h-1 e 1,61 g L-1h-1 em 12 e 24 h, respectivamente. A produção de etanol a partir de um hidrolisado de bagaço de mandioca nas condições de hidrólise proposta foi possível, o que corresponde a uma alternativa à tecnologia da cana-de-açúcar para produção deste biocombustível. / The development of bioprocesses for food and energy production, which makes use of agricultural waste as raw material, is focus of intense research around the world due to limitations on new agricultural frontiers and on renewable energy sources. The use of enzymes in these processes emerges as a strong trend, among them, amylases. The aim of this study was the amylase production optimization employing fungus Rhizopus microsporus var. oligosporus, grown by Solid Station Fermentation (SSF), using as substrate these agricultural waste: wheat bran, cassava bagasse and sugarcane bagasse. The hydrolizate obtained from cassava bagasse hydrolysis was evaluated with regards to its fermentability through fed-batch alcoholic fermentation by Saccharomyces cerevisiae. The best carbon source as substrate for the culture medium formulation was the one containing 100% wheat bran. Regarding salts sources for amylases production, the higher amylolytic activity (418 U/g) was for the nutrient solution containing (%): ammonium sulfate 1.34; ammonium monophosphate 0.54 and urea 1.86. However, the addiction of 0.01% (w/w) yeast extract in salts solution, amylolytic activity increased from 418 U/g to 551 U/g. The enzymatic hydrolysis of cassava bagasse was performed with the combination of commercial cellulases (Novozyme) (10 FPU/g at time zero hours) and the amylolytic extract (12 U/g at time 10 hours) produced by SSF to obtain a hydrolizate concentrate. With these conditions it was possible to convert 63% of bagasse in reducing sugars predominantly glucose. Through the fed-batch alcoholic fermentation tests by Saccharomyces cerevisiae, efficiencies were determined as 74.6 and 76.8%, and the ethanolic productivities with 3.14 gL-1h-1 and 1.61 gL-1h-1, at 12 and 24 h, respectively. Ethanol production from cassava bagasse hydrolyzate in hydrolysis conditions proposal was possible, which corresponds to an alternative technology of sugarcane for production of biofuel.

This master thesis explored washing with water grades and lipase as environmentally friendly alternatives to conventional detergents containing surfactants. On hydrophilic surfaces, purified water can remove fat through roll-up mechanism, initiated by electrostatic repulsion forces. On hydrophobic surfaces, purified water alone cannot remove fat as there are no electrostatic repulsion forces. However, addition of lipase might promote degreasing through solubilization. Lipases are only activated when encountering an oil-water interface. Once activated, lipases can hydrolyze carboxylic ester bonds in fats. The aim of this project was to evaluate if addition of lipase from Rhizopus niveus (RNL) to water grades (such as ultrapure water, DIRO, and tap water) can enhance their cleaning efficiency to remove fat-based stains from hydrophilic and hydrophobic surfaces. An interesting phenomenon was observed in contact angle measurements. On hydrophilic surface, some solutions with high RNL concentration caused the oil droplet to divide into several droplets. The involved mechanisms are yet unknown. Gravimetric analysis showed a significant increase in cleaning efficiency in most samples (except tap water) after addition of RNL. Also, the effect of interfacial changes became significant. Multiple cycle washing, with repeated interfacial changes and high rate of fat removal, was more efficient than single cycle washing. In the quartz crystal microbalance with dissipation (QCM-D) measurements, RNL had a significant effect on the frequency and dissipation data. Observed changes when washing with RNL, suggest that apart from the cleaning promoted by interfacial changes, also enzymatic cleaning was occurring. Unfortunately, the calculated cleaning efficiencies reveal that addition of RNL did not increase the cleaning efficiency in this specific washing test. To obtain extensive understanding of RNL’s behavior and activity in water grades, as well as the effect of RNL on different surfaces (without or with fats involved), further experiments are necessary.

Rhizopus oryzae is a filamentous fungi which produces lactic acid and ethanol in fermentations. R. oryzae has numerous advantages for use industrial production of L-(+)-lactic acid but the yield of lactic acid produced on the basis of carbon consumed is low. Metabolic flux analysis of R. oryzae has shown that most of the pyruvate produced at the end of the glycolysis is channelled to ethanol, acetyl-CoA and oxaloacetate production. This study aimed to answer some questions addressed on the regulation of pyruvate branch point in R. oryzae and for this purpose biochemical characterisation of the enzymes acting at this branch point and cloning the genes coding for these enzymes have been done. Pyruvate decarboxylase was purified and characterised for the first time from R. oryzae. The purified enzyme has a Hill coefficient of 1.84 and the Km of the enzyme is 8.6 mM for pyruvate at pH 6.5. The enzyme is inhibited at pyruvate concentrations higher than 30 mM. The optimum pH for enzyme activity shows a broad range from 5.7 and 7.2. The monomer molecular weight was estimated as 59&plusmn / 2 kDa by SDS-PAGE analysis. Pyruvate decarboxylase (pdcA and pdcB) and lactate dehydrogenase (ldhA and ldhB) genes of R. oryzae have been cloned by PCR-cloning approach and the filamentous fungi Aspergillus niger was transformed with these genes. The A. niger transformed with either of the ldh genes of R. oryzae showed enhanced production of lactic acid compared to wild type. Citric acid production was also increased in these transformants while no gluconate production was observed Cloning of hexokinase gene from R. oryzae using degenerate primers was studied by the use of GenomeWalker kit (Clontech). The results of this study were evaluated by using some bioinformatics tools depending on the unassembled clone sequences of R. oryzae genome.

No description available.

Det slängs bröd i svenska matvarubutiker i Sverige samtidigt som läderproduktion är miljöförstörande. För att mildra miljöpåverkan av konventionell lädertillverkning och matsvinn i matvarubutiker odlades filamentös svamp på brödrester för att användas som ett miljövänligt alternativ till animaliskt läder. Detta arbete undersöker ifall en ny typ av läder-liknande material kan förbättras genom att tillsätta växt-tanniner och andra naturliga ämnen till filamentös svampbiomassa vid olika steg i tillverkningsprocessen. Den filamentösa svampen Rhizopus delemar odlades med hydrolyserade brödrester från matvarubutiker som substrat, och fyra olika växt-tanniner tillsattes odlingsmediumen (Tara, Myrobalan, Quebracho, Kastanje). Av de tannin-behandlade biomassorna uppvisade material som behandlats med tara tannin efter skörd av biomassan mest läderliknande mekaniska egenskaper (dragstyrka och förlängning) medan ren, obehandlad biomassa uppvisade högst dragstyrka av alla testade material. Additionen av tanniner ökade töjningsförmågan samtidigt som dragstyrkan minskade hos de resulterande materialen. Behandling med den naturliga tvärbindaren genipin resulterade i ökad dragstyrka och förlängning för flera av materialen. Resultat från HPLC analys av odlingsmediet med tara tannin indikerade även att Rhizopus delemar använde tanninet som substrat. / Bread is thrown away in Swedish grocery stores in Sweden at the same time as leather production is environmentally destructive. In order to mitigate the environmental impact of conventional leather making and to reduce food waste, fungal mycelium was grown on bread waste to be utilized as an eco-friendly leather substitute. This work investigates whether this new type of leather-like material can be improved by adding vegetable tannins and other natural compounds to filamentous fungal biomass at different stages of the production process. The filamentous fungus Rhizopus delemar was grown in shake flasks with hydrolyzed bread as substrate and four different plant tannins were added to the growth mediums. (Tara, Myrobalan, Quebracho, Chestnut). Tara and myrobalan were not inhibitory for growth during the shake flask cultivation, so they were further used in a scaled-up cultivation using 4 L bubble column reactors. However, myrobalan tannin was inhibitory when cultivating in 4 L bubble column reactors. Out of the tannin-treated biomasses, materials treated with tara tannin post cultivation exhibited the highest mechanical properties (tensile strength and elongation), while pure, untreated biomass exhibited the highest tensile strength of all materials tested. Overall, the addition of tannins to the biomasses increased the elongational capacities while lowering the tensile strengths. Treatment with the natural crosslinking-agent genipin resulted in increased average tensile strength and elongation at break for several of the materials tested. Furthermore, HPLC analysis of culture medium containing tara tannin indicated that Rhizopus delemar was able to metabolize the tannin and use it as a substrate.

Consumption of citrus fruits and citrus juice production creates wastes, which could be valorized by using it for cultivating fungi. Before cultivation, the medium needs to be sterilized though autoclavation. Larger volumes used when autoclaving requires longer heating cycles and therefore runs the risk of degrading the medium to a greater extent. This research examines the effects of the volume lemon waste medium used while sterilizing. The aim is to find the largest volume still providing good growth for the filamentous fungus used, Rhizopus Delemar. Lemon waste was provided by Herrljunga Musteri AB and was pre-treated at 45°C for 2h. The liquid was strained and autoclaved in different volumetric series ranging from 200 – 10 000 mL, that was then used in 200 mL shake flask cultivations.  A scale up in two 3,5 L bubble column reactors was also performed from the 10 000 mL autoclaved medium, after not observing severe impacts on growth. Testing was done by weighing biomass and HPLC analysis of sugars. The yield of the biomass in the shake flasks ranged from 0,11 – 0,14 g/g sugars and the biomass concentration ranged between 2,4 - 3,0 g/L. Overall, the volume of autoclavation seems to not too be of great concern when cultivating R. Delemar on lemon waste medium in the analyzed ranges.

No description available.