Tesis sobre el tema "Fungal bioreactors"

Wastewater, recycled irrigation water, and agricultural runoff can contain high levels of pathogenic bacteria, which pose a threat to human and ecosystem health. The use of a bioreactor containing mycelial mats of filamentous fungi is a novel treatment technology that incorporates physical, biological, and biochemical processes to remove bacterial pathogens from influent water. Although a relatively new concept, fungal bioreactors have demonstrated the ability to dramatically reduce fecal coliform bacteria in water, but no studies have attempted to explicitly identify the bacterial pathogen removal mechanisms exhibited by the fungi. This study evaluated several different species of fungi for use in fungal bioreactor systems and aimed to identify the modes of action responsible for the removal of bacterial pathogens. The species evaluated were Daedaleopsis confragosa, Pleurotus eryngii, and Piptoporus betulinus. Experimental results showed that all species of fungi assessed were capable of removing E. coli in a synthetic water solution. Significant concentrations of hydrogen peroxide, an antiseptic, were produced by all species of fungi evaluated. The fungal bioreactors containing P. eryngii produced the highest concentrations of hydrogen peroxide, generating a maximum concentration of 30.5 mg/l or 0.896 mM. This maximum value exceeds reported minimum concentrations required to demonstrate bacteriostatic and bactericidal effects when continually applied, providing evidence that a major bacterial removal mode of action is the production of antimicrobial compounds. In addition to its promising application to improve water quality, fungal bioreactors are a low cost and passive treatment technology. The development a hyper-functional system could be a have a substantial impact on the use of recycled irrigation water and on the water/wastewater treatment industry, for both municipal and agricultural wastewater.
Master of Science

Thesis (MTech (Chemical Engineering))--Peninsula Technikon, 2001
Certain fungi have been shown to excrete extracellular enzymes, including peroxidases, laccases, etc. These enzymes are useful for bioremediation of aromatic pollutants present in industrial effluents (Leukes, 1999; Navotny et aI, 1999). Leukes (1999) made recent significant development in the form of a capillary membrane gradostat (fungal) bioreactor that offers optimal conditions for the production of these enzymes in high concentrations. This system also offers the possibility for the polluted effluent to be treated directly in the bioreactor. Some operating problems relating to continuous production of the enzymes and scale-up of the capillary modules, were, however, indentified. In an attempt to solve the above-mentioned identified problems the research group at Peninsula Technikon considered a number of alternative bioreactor configurations. A pulsed packed bed bioreactor concept suggested by Moreira et at. (1997) was selected for further study. Their reactor used polyurethane pellets as the support medium for the fungal biofilm and relied upon pulsing of the oxygen supply and recycle of nutrient solution in order to control biomass accumulation. These authors reported accumulation due to the recycle of proteases that were believed to destroy the desired ligninases. We experimented with a similar concept without recycle to avoid backrnixing and thereby overcome protease accumulation. In our work, a maximum enzyme productivity of 456 Units.L1day·1 was attained. Since this was significantly greater than the maximum reported by Moreira et aI, 1997 (202 Units.L-1day-I) it appeared that the elimination of recycle had significant benefits. In addition to eliminating recycle we also used a length / diameter (L / D) ratio of 14: 1 (compared with 2.5: 1 used by Moreira et aI, 1997) in order to further reduce backrnixing. Residence time distributions were investigated to gain insight into mechanisms of dispersion in the reactor. It was found that the pulsed packed bed concept presented problems with regard to blockage by excess biomass. This led us to consider the advantages of a fluidized bed using resin beads. Accordingly, growth of fungi on resin beads in shake flasks was investigated with favorable results. An experimental program is proposed to further investigate the fluidized bed concept with a view to extending the operation time of the bioreactor. From our literature survey to date, packed bed fungal bioreactors are still the best reactor configuration for continuous production ofligninolytic enzymes. An interesting study of the application of laccases to the degradation of naphthalene and MTBE is described in an addendum to this thesis.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xviii, 246 p.; also includes graphics (some col.) Includes bibliographical references (p. 207-222).

A significant by-product of bioethanol plants is whole stillage, commonly used to produce animal feed due to its nutritious value, has a potential to be used to produce various value-added products while eliminating a costly process step is an alternative approach. In this study, production and separation of additional ethanol, fungal biomass and enzyme were successfully achieved with the cultivation in membrane bioreactors in batch process condition. Process optimization studies regarding fermentation and filtration conditions were carried out. Up to 10.4 g/l ethanol per litre of used whole stillage can be produced in simultaneous saccharification and fermentation (SSF) condition without any pH adjustment and additional pretreatment step. Also, 50% diluted whole stillage provided 87% higher ethanol production comparing to non-diluted medium. Moreover, 71 % higher biomass production was obtained with the filtrate of 50% diluted whole stillage comparing to 25% diluted one. Considering the achieved results, a two-stage cultivation using SHF (Separate Hydrolysis and Fermentation) strategy in membrane bioreactors for separation of ethanol, lignin-rich stream, protein-rich fungal biomass and enzymes was proposed. The present thesis showed that the integration of filamentous fungi with membrane bioreactors can increase the range of products that can be produced from whole stillage.

Penicillium ochro-chloron (ATCC strain # 36741), a filamentous fungus with the capability for removing copper ions from aqueous solutions, was studied as a possible biological trap (biotrap) for remediation of heavy metal contaminants in industrial wastewaters. This research demonstrated that in shake flasks the fungus removed copper from surrogate wastewater with 100mg/L copper contamination by as much as 99%. These results did not translate to the bioreactor configuration of a packed bed column, as channeling occurred through the bed, shown by conductivity tracer studies. A fluidized bed configuration was studied and resulted in copper removal of 97%, with a capacity of 149 mg[Cu]/g dry weight biomass, under the conditions of 50% dissolved oxygen. For dissolved oxygen concentrations below the critical oxygen concentration for the fungus (20% saturation) there was minimal copper removal. Mixing studies in the fluidized bed reactor showed that the system was diffusion limited. Mathematical modeling using first order kinetics associated with diffusion limited reactions resulted in rate constants for Cu 2+ uptake of approximately 0.031 h -1 , which were dependent on the dissolved oxygen concentration. Modeling of the reaction with a second order kinetic equation showed that there are possibly factors regulating copper uptake besides oxygen. Electron microscopy showed that in some instances the copper removed was retained as large porous spherical extracellular precipitates. Energy Dispersive X-ray (EDX) analysis has shown similar complexes to be copper phosphate precipitates (Crusberg, 1994). Removal of heavy metal contaminants from wastewater discharge is a necessity for many industries, due to environmental concerns and federal regulations. The use of a biological system for the removal and recycling of heavy metals could prove more economical than currently used physio-chemical processes.

Stale bread contributes to the biggest volume of food waste in Sweden. Current method on recovering bread waste is by producing biogas or bioethanol. Despite advantages in the energy sector, the bread which still has relatively high quality could be recovered into new products with higher value, such as food for human consumption. Development of a product, termed ‘fungal cake’ by solid state fermentation on bread waste using Neurospora intermedia in small scale petri dishes have previously been successfully conducted. This study aims to scale up the production of fungal cake into bench scale production. Two systems using different bioreactors were used in this study. The first system operated in batch mode using a tray bioreactor, in which the effect of particle size, mixing, and bread loading were evaluated. The fermentation was conducted during 5 days. Bread crumb with a larger particle size of 2 mm resulted in similar outcomes as bread crumb with a smaller particle size of 0.5 mm in terms of CO2 evolution rate, cumulative CO2 production, starch, and protein content of the final product. However, larger particle size resulted in a more homogeneous growth of the fungus throughout the product, which is preferred. The presence of daily mixing had no significant effect compared to static condition for all measured variables. Thus, mixing could be introduced to promote product homogeneity. Likewise, bread loading had no significant effect on the measured variables, which implies that a higher productivity can be achieved using a higher bread loading. The second system operated in continuous mode using a newly developed continuous tubular bioreactor with product recycle. Two experiments, in which the residence time (48h and 24h) and recycle ratio (10/65 and 20/55) were conducted. Both experiments yielded product with stable starch and protein content, indicated by a stable CO2 evolution rate over time. The performance using continuous tubular bioreactor was compared to batch fermentation in tray bioreactor using the same ratio of inoculum and both system yielded product with the same starch and protein content. Successful operation in continuous bioreactor certainly improved the productivity of fungal cake production.

Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Proteases are essential constituents of all living beings, since they are involved in essential biological processes such as blood clotting, cell death, tissue differentiation, protein transport across the membrane etc. They also have important biotechnological applicability, because they can be used in food processing, manufacture of detergents, leather processing, meat softening, drug formulation, in the textile industry etc. These enzymes represents about 60% of the global market for industrial enzymes; so, they are considered an important group of enzymes. This work was divided into two stages and aimed to isolate filamentous fungi collected from coconut trees and soil from a coconut located in Varzeas de Sousa, Paraiba, Brazil and do a screening for the production of proteases and to evaluate, in a bioreactor, the production of proteases by the yeast Rhodotorula mucilaginosa L07. In all, 32 fungi were isolated in Paraiba semiarid. They were grown in rotary shaker and sent to the analysis of proteolytic activity. The specie, originally called Fung1, showed better results in the qualitative stage and was taken to the molecular identification and selected for production in rotary shaker (30°C / 200rpm / 240h). R. mucilaginosa L07, originally from Antarctica, was cultivated in a bioreactor (25°C / 72h), varying agitation and aeration. The maximum enzyme activity by the Fung1, identified as Aspergillus tubingensis, was 29 U.mL^-1, after 144h cultivation. This fungus is not a fumonisin B2 and ochratoxin A producer. The greatest value of proteolytic activity of R. mucilaginosa L07 was 124.88 U.mL^-1 with agitation of 500rpm and aeration 1,0vvm. The results indicated that A. tubingensis produces proteases, but other studies are needed to optimize production and classify proteases. The supply of oxygen to R. mucilaginosa L07 were positive for proteolytic activity, because it increased from 33.36 to 124.88 U.mL^-1 in rotary shaker and bioreactor, respectively.
Proteases são constituintes essenciais em todos os seres vivos, pois estão envolvidas em processos biológicos essenciais como coagulação sanguínea, morte celular, diferenciação de tecidos, transporte de proteínas através da membrana etc. Também possuem importante aplicabilidade biotecnológica, pois podem ser usadas no processamento de alimentos, formulação de detergentes, processamento de couro, amaciamento de carnes, formulação de medicamentos, na indústria têxtil etc. Por representarem aproximadamente 60% do mercado mundial de enzimas industriais, são consideradas um importante grupo de enzimas. Este trabalho foi dividido em duas etapas e objetivou isolar fungos filamentosos coletados em coqueiros e solo de um coqueiral localizado nas Várzeas de Sousa, Paraíba, Brasil e fazer um screening quanto à produção de proteases, além de avaliar, em biorreator, a produção de proteases pela levedura Rhodotorula mucilaginosa L07. Ao todo, 32 fungos foram isolados no semiárido paraibano, cultivados em agitador rotatório e encaminhados à análise da atividade proteolítica. A espécie inicialmente denominada Fung1 apresentou melhor resultado na etapa qualitativa, foi encaminhada à identificação molecular e selecionada para a produção em agitador rotatório (30°C/ 200rpm/ 240h). A R. mucilaginosa L07, coletadada na Antártida, foi cultivada em biorreator (25°C/ 72h), variando agitação e aeração. A atividade enzimática máxima do Fung1, identificado como Aspergillus tubingensis, foi 29 U.mL , após 144h de cultivo. Este fungo não é produtor de fumonisina B2 e ocratoxina A. O maior valor de atividade proteolítica da R. mucilaginosa L07 foi de 124,88 U.mL^-1 , com agitação de 500rpm e aeração de 1,0vvm. Os resultados indicaram que A. tubingensis produz proteases, porém outros estudos são necessários para otimizar a produção e classificar as proteases. O fornecimento de oxigênio em cultivos da R. mucilaginosa L07 foi positivo para a atividade proteolítica, pois a mesma aumentou de 33,36 para 124,88 U.mL^-1, em agitador rotatório e biorreator, respectivamente.

Orientador: João Cláudio Thoméo
Coorientador: Fernanda Perpétua Casciatori
Banca: Vanildo Luiz Del Bianchi
Banca: Fabio Bentes Freire
Resumo: O presente trabalho teve como objetivo principal o desenvolvimento de um biorreator de bandeja capaz de produzir esporos do fungo entomopatogênico Metarhizium anisopliae. Para esse fim, foi utilizado um biorreator de bandeja para a produção de esporos, que por sua vez foi avaliada em função da carga de substrato empregada. As variações de temperatura no biorreator foram simuladas e observadas experimentalmente. Para o desenvolvimento do biorreator, inicialmente foram realizados ensaios em embalagens plásticas contendo 10g e 500g de substrato, de modo a avaliar como o aumento da carga de meio fermentativo poderia influenciar na produção de esporos. Nestes ensaios, foram medidas as temperaturas no centro geométrico das embalagens e a elevação de temperatura foi relacionada à produção de esporos. Realizou - se uma análise de área superficial do arroz tipo 1 e da quirera de arroz para avaliar a interferência desta propriedade na produção de esporos. A partir dos resultados em embalagens plásticas foram realizados experimentos em um biorreator de 40 cm de comprimento, 29 cm de largura por 12 cm de altura, escoando ar sobre as partículas no sentido da maior dimensão. Foram empregadas cargas de 1, 2 e 3kg de arroz tipo 1, correspondente à altura de leito de 2, 4 e 6 cm, respectivamente, e 1 kg de quirera, correspondendo a 2 cm de altura. A temperatura nas posições de entrada e saída de ar e no meio geométrico do meio de cultivo foram medidas ao longo dos ensaios. A fermentação nas embalagens plásticas e no biorreator foram realizadas a 28oC. Para estimar a geração de calor metabólico do meio de cultivo, foram coletados dados de consumo de O2 e liberação de CO2 durante o período de incubação. Para tal, foi realizada a fermentação em um biorreator de leito empacotado cilíndrico, sendo os gases provenientes do leito e conduzidos a um analisador de...
Abstract: This study aimed to develop a bioreactor tray capable of producing spores of the fungus Metarhizium anisopliae entomopathogenic. To this end, we used a tray bioreactor for the production of spores, which in turn was evaluated according to the employed substrate load. Temperature variations in the bioreactor were simulated and observed experimentally. For the development of bioreactor assays were carried out in plastic bags containing substrate 10g and 500g in order to evaluate how increasing fermentative medium loading could influence the production of spores. In these tests we measured the temperature at the geometric center of the packages and the temperature rise was related to spore production performed -. A surface area analysis of rice Type 1 and broken rice to evaluate the interference at this property in the production of spores. From the results in plastic containers experiments were performed in a bioreactor of 40 cm long, 29 cm wide by 12 cm, air flowing onto the particles in the direction of the largest dimension. Loads were applied to 1, 2 and 3 kg of rice type 1, corresponding to the bed height of 2, 4 and 6 cm, respectively, and 1 kg of grits, corresponding to 2 cm. The temperature at the inlet position and the air outlet and the geometric mean of the culture medium were measured throughout the tests. Fermentation in plastic packaging and the bioreactor were carried out at 28 C. To estimate the generation of metabolic heat of culture medium were collected consumption data The O2 and CO2 release during the incubation period. To this end, the fermentation was performed in a bioreactor cylindrical packed bed, and the gases from the bed and driven to a gas analyzer. A onedimensional mathematical model capable of predicting the temperature profile and fungal growth during the process at any position of the bioreactor is proposed. The experimental results in plastic ...
Mestre

Orientador: João Cláudio Thoméo
Resumo: Este trabalho tem como objetivo produzir esporos do fungo Metarhizium anisopliae IBCB 425 em biorreator de leito empacotado visando o aumento da escala, propondo ainda alternativas para a extração dos esporos do meio de cultivo. Também se propõe compreender a preferência do fungo pelo arroz como substrato, procurando-se alternativas para redução dos custos de produção relativos a este substrato. Para isso, realizou-se o cultivo e o reuso do substrato em embalagens plásticas, onde se observou que o cultivo em arroz tipo 1 ainda é o melhor substrato para o fungo, quando comparado com os utilizados neste trabalho sendo, quirera de arroz e farelo de arroz, porém para o aumento de escala e reuso do substrato, a melhor alternativa é a mistura de arroz com bagaço de cana-de-açúcar na proporção de 9:1. Também se realizou ensaios em biorreatores de leito empacotado de 7.62 cm e 20 cm de diâmetro, utilizando a mistura de arroz e bagaço como substrato, e observou-se que, para ambas as configurações, o bagaço evitou a compactação do leito, não se observando temperaturas elevadas que interferissem no desenvolvimento do microrganismo. Com as análises de degradação do amido e da atividade das enzimas amilase total, alfa – amilase e protease, foi possível concluir que o fungo se desenvolve de maneira distinta a cada uso dos grãos e que, o maior consumo de amido ocorre no primeiro cultivo (R1), reduzindo-o em cerca de 30%. Análises de microscopia óptica foram realizadas nos grãos cultivados, ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: This work aimed to produce spores of the entomopathogenic fungus Metarhizium anisopliae IBCB 425 in packed-bed bioreactors targeting the scale-up, and also to propose alternatives to the extraction of the spores from the cultivation medium. The understanding of the preference of the fungus for the long rice as substrate was also verified in order to reduce the production costs associated to the rice. Therefore, the rice was cultivated in plastic packages and it was noticed that rice is still the best substrate for the fungal growth, even though for the scale-up and reutilization of the substrate it was necessary to mix it with sugarcane bagasse at a proportion 9:1 (rice:bagasse). Experiments in packed-beds of 7.62 and 20 cm internal diameter have been carried out using the mixture rice:bagasse and for both sizes no compaction was noted and no excessive temperature took place. From starch analysis and from the activities of total amylase, alpha-amylase and protease it was noticed that the microbe develops distinctly in each consecutive cultivation of rice, and that after the first cultivation (R1), the starch content was reduced by 30%. Optical microscopy of the cultivated grains revealed that during R1 the fungal hyphaes penetrated the grains, but during the second (r2) and third (R3) cultivations the fungal growth is restricted to the grain surface. It was observed a substrate mass reduction in the packed-bed experiments of 24%, 20% and 17% after R1, R2 and R3, respectively.... (Complete abstract click electronic access below)
Doutor

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O presente trabalho teve como objetivo principal o desenvolvimento de um biorreator de bandeja capaz de produzir esporos do fungo entomopatogênico Metarhizium anisopliae. Para esse fim, foi utilizado um biorreator de bandeja para a produção de esporos, que por sua vez foi avaliada em função da carga de substrato empregada. As variações de temperatura no biorreator foram simuladas e observadas experimentalmente. Para o desenvolvimento do biorreator, inicialmente foram realizados ensaios em embalagens plásticas contendo 10g e 500g de substrato, de modo a avaliar como o aumento da carga de meio fermentativo poderia influenciar na produção de esporos. Nestes ensaios, foram medidas as temperaturas no centro geométrico das embalagens e a elevação de temperatura foi relacionada à produção de esporos. Realizou – se uma análise de área superficial do arroz tipo 1 e da quirera de arroz para avaliar a interferência desta propriedade na produção de esporos. A partir dos resultados em embalagens plásticas foram realizados experimentos em um biorreator de 40 cm de comprimento, 29 cm de largura por 12 cm de altura, escoando ar sobre as partículas no sentido da maior dimensão. Foram empregadas cargas de 1, 2 e 3kg de arroz tipo 1, correspondente à altura de leito de 2, 4 e 6 cm, respectivamente, e 1 kg de quirera, correspondendo a 2 cm de altura. A temperatura nas posições de entrada e saída de ar e no meio geométrico do meio de cultivo foram medidas ao longo dos ensaios. A fermentação nas embalagens plásticas e no biorreator foram realizadas a 28oC. Para estimar a geração de calor metabólico do meio de cultivo, foram coletados dados de consumo de O2 e liberação de CO2 durante o período de incubação. Para tal, foi realizada a fermentação em um biorreator de leito empacotado cilíndrico, sendo os gases provenientes do leito e conduzidos a um analisador de gases. Foi proposto um modelo matemático unidimensional, capaz de prever os perfis de temperatura e o crescimento fúngico ao longo do processo em qualquer posição do biorreator. Os resultados experimentais em embalagens plásticas e no biorreator mostraram que menores quantidades de substrato são favoráveis à maior estabilidade da temperatura do meio de cultivo, resultando em maiores quantidades de esporos. No teste de área superficial, foi possível observar um aumento na área da quirera de arroz e consequentemente aumento da quantidade de esporos. Nos testes de cinética de crescimento, pode se observar que o consumo de O2 é baixo comparado a outros fungos. Os dados de simulação não demonstraram sobreaquecimento do meio de cultivo no biorreator com o aumento de carga, o que não afetaria a produção de esporos. Também pode se observar com os dados de simulação que a temperatura da superfície do meio de cultivo não sofreu sobreaquecimento. Porém, com os dados experimentais, foi possível observar um sobreaquecimento no meio de cultivo, em virtude da geração de calor metabólico. Os dados experimentais demonstraram que os melhores resultados obtidos no biorreator, para produção de esporos do fungo Metarhizium anisopliae, foram com cargas de 1 Kg, correspondente a 2 cm de altura de substrato e que com cargas maiores os perfis de temperatura apresentaram elevado aquecimento no meio de cultivo.
This study aimed to develop a bioreactor tray capable of producing spores of the fungus Metarhizium anisopliae entomopathogenic. To this end, we used a tray bioreactor for the production of spores, which in turn was evaluated according to the employed substrate load. Temperature variations in the bioreactor were simulated and observed experimentally. For the development of bioreactor assays were carried out in plastic bags containing substrate 10g and 500g in order to evaluate how increasing fermentative medium loading could influence the production of spores. In these tests we measured the temperature at the geometric center of the packages and the temperature rise was related to spore production performed -. A surface area analysis of rice Type 1 and broken rice to evaluate the interference at this property in the production of spores. From the results in plastic containers experiments were performed in a bioreactor of 40 cm long, 29 cm wide by 12 cm, air flowing onto the particles in the direction of the largest dimension. Loads were applied to 1, 2 and 3 kg of rice type 1, corresponding to the bed height of 2, 4 and 6 cm, respectively, and 1 kg of grits, corresponding to 2 cm. The temperature at the inlet position and the air outlet and the geometric mean of the culture medium were measured throughout the tests. Fermentation in plastic packaging and the bioreactor were carried out at 28 C. To estimate the generation of metabolic heat of culture medium were collected consumption data The O2 and CO2 release during the incubation period. To this end, the fermentation was performed in a bioreactor cylindrical packed bed, and the gases from the bed and driven to a gas analyzer. A one-dimensional mathematical model capable of predicting the temperature profile and fungal growth during the process at any position of the bioreactor is proposed. The experimental results in plastic bottles and bioreactor showed that smaller amounts of substrate are conducive to increased stability of the temperature of the medium, resulting in larger amounts of spores. In the test surface area, it was possible to observe an increase in the area of rice grits and therefore increasing the amount of spores. In the growth kinetics tests, it can be seen that the O 2 consumption is low compared to other fungi. Simulation data showed no overheating of the culture medium in the bioreactor with the increased load, which would not affect the production of spores. It can also be observed with the simulation data as the temperature of the surface of the medium has not suffered overheating. However, with the experimental data, it was possible to observe overheating in the culture medium by virtue of the generation of metabolic heat. The experimental data show that the best results obtained in the bioreactor for the production of Metarhizium anisopliae spores of the fungus were to loads of 1 kg, corresponding to 2 cm substrate height and with larger loads temperature profiles showed high heat in the middle cultivation.

Le sélénium et le tellurium partagent des propriétés chimiques communes et appartiennent à la colonne des éléments chalcogènes de la classification périodique des éléments. Ces métalloïdes ont des propriétés physico-chimiques remarquables et ils ont été utilisés dans un grand nombre d'applications dans le domaine des hautes technologies (électronique, semi-conducteurs, alliages). Ces éléments, qui se retrouvent généralement sous formes d'oxyanions, sont extrêmement solubles dans l'eau et présentent une forte toxicité. Leur libération dans l'environnement est donc d'un enjeu capital. Différentes méthodes physico-chimiques ont été développées pour la récupération de ces metalloïdes, en particulier pour le sélénium. Néanmoins, ces méthodes requièrent un équipement lourd et couteux et ne sont pas très recommandables sur le plan écologique. Le traitement biologique est donc une bonne alternative pour la récupération de Se et de Te provenant des effluents pollués. Cette approche réside dans la bioréduction des différents oxyanions sous formes métalliques. Ceux-ci sont moins toxiques et d'intérêts commerciales notables surtout lorsqu'ils se présentent sous forme nanométrique. L'utilisation de micro-champignons comme microorganismes catalyseur de la réduction de Se et de Te a été démontrée dans cette étude. La réactivité du champignon responsable de la pourriture blanche, Phanerochaete chrysosporium en présence de sélénite et de tellurite a été évaluée, ainsi que son application potentielle pour le traitement des eaux contaminées et la production de nanoparticules. La présence de Se et de Te a une influence importante sur la croissance et la morphologie du champignon. Il s'avère que P. chrysosporium est très sensible à la présence de sélénites. La synthèse de Se° et de Te° sous forme de nanoparticules piégées dans la biomasse fongique a été observée, ainsi que la formation de nano-composites Se-Te lorsque le champignon était cultivé simultanément en présence des deux métalloïdes. L'usage potentiel de biofilm fongiques pour le traitement des effluents semi-acides (pH 4.5) contenant du Se et du Te a été suggéré. De plus, le traitement en mode continu de sélénite dans un réacteur à biofilm fongique granulaire a été évalué. Le réacteur a montré un rendement d'élimination du sélénium en régime permanent de 70% pour differentes conditions opératoires. Celui-ci s'est montré efficace pendant une période supérieure à 35 jours. La bonne sédimentation du biofilm granulaire facilite la séparation du sélénium de l'effluent traité. L'utilisation du biofilm granulaire contenant du sélénium élémentaire comme bio-sorbant a également été étudiée. Cet adsorbant hybride s'est montré prometteur pour l'immobilisation du zinc présent dans les effluents semi-acides. La plupart des recherches effectuées se sont focalisées sur l'utilisation des biofilms granulaires. Toutefois, la croissance du champignon suite à l'exposition à des concentrations différentes de sélénites a également été étudiée. Des micro-électrodes à oxygène et un microscope confocal à balayage laser ont été utilisées pour évaluer l'effet du sélénium sur la structure des biofilms fongiques. Quel que soit le mode de croissance de P. chrysosporium, le mécanisme de réduction du sélénite semble être toujours le même tout en menant à la formation de sélénium élémentaire. Cependant, l'architecture des biofilms et l'activité en oxygène sont influencées par la présence de sélénium
Selenium (Se) and tellurium (Te) are particular elements, they are part of the chalcogens (VI-A group of the periodic table) and share common properties. These metalloids are of commercial interest due to their physicochemical properties, and they have been used in a broad range of applications in advanced technologies. The water soluble oxyanions of these elements (i.e., selenite, selenate, tellurite and tellurate) exhibit high toxicities, thus their release in the environment is of great concern. Different physicochemical methods have been developed for the removal of these metalloids, mainly for selenium. However, these methods require specialized equipment, high costs and they are not ecofriendly. The biological treatment is a green alternative to remove Se and Te from polluted effluents. This remediation technology consists on the microbial reduction of Se and Te oxyanions in wastewater to their elemental forms (Se0 and Te0), which are less toxic, and when synthesized in the nano-size range, they can be of commercial value due to their enhanced properties. The use of fungi as potential Se- and Te-reducing organisms was demonstrated in this study. Response of the model white-rot fungus, Phanerochaete chrysosporium, to the presence of selenite and tellurite was evaluated, as well as their potential application in wastewater treatment and production of nanoparticles. The presence of Se and Te had a clear influence on the growth and morphology of the fungus. P. chrysosporium was found to be more sensitive to selenite. Synthesis of Se0 and Te0 nanoparticles entrapped in the fungal biomass was observed, as well as the formation of unique Se-Te nanocomposites when the fungus was cultivated concurrently in the presence of Se and Te. Potential use of fungal pellets for the removal of Se and Te from semi-acidic effluents (pH 4.5) was suggested. Moreover, the continuous removal of selenite in a fungal pelleted reactor was evaluated. The reactor showed to efficiently remove selenium at steady-state conditions (~70%), and it demonstrated to be flexible and adaptable to different operational conditions. The reactor operated efficiently over a period of 35 days. Good settleability of the fungal pellets facilitated the separation of the selenium from the treated effluent. The use of elemental selenium immobilized fungal pellets as novel biosorbent material was also explored. This hybrid sorbent was promising for the removal of zinc from semi-acidic effluents. The presence of selenium in the fungal biomass enhanced the sorption efficiency of zinc, compared to Se-free fungal pellets. Most of the research conducted in this study was focused on the use of fungal pellets. However, the response of the fungus to selenite in a different kind of growth was also evaluated. Microsensors and confocal imaging were used to evaluate the effects of selenium on fungal biofilms. Regardless of the kind of fungal growth, P. chrysosporium seems to follow a similar selenite reduction mechanism, leading to the formation of Se0. Architecture of the biofilm and oxygen activity were influenced by the presence of selenium

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The main challenge on the conversion of lignocellulosic biomass into liquid fuels is the economic viability of this process. Thus, the commercialization of lignocellulosic ethanol is hindered mainly by the high costs of the enzyme preparations currently available cellulases - enzymes used in the saccharification step. Some strategies that can be adopted to reduce the enzymes costs include selecting microorganisms, use of cheaper raw materials and more efficient fermentation strategies such as the solid state fermentation (SSF) and efficient techniques for saccharification and fermentation. The aim this work was evaluate the use of the whole fermentation medium containing lignocellulosic biomass, fungal mycelium and enzymes in the hydrolysis of sugarcane bagasse pretreated by steam explosion for cellulosic ethanol production. In this context, a selection of filamentous fungi highly producing cellulases and hemicellulases, optimization operating conditions, such as humidity and temperature, were carried out for in house enzyme production using an instrumented bioreactor. Then, the efficiency of the whole fermentation medium and enzyme extract in enzymatic hydrolysis of lignocellulosic biomass for cellulosic ethanol production was evaluated. Among the 40 fungal strains evaluated, two strains of A. oryzae (P6B2 and P27C3A) stood out. In addition, one strain of A. niger 3T5B8 and another of T. reesei RUT C30 were also evaluated in this study. The influence of the substrate initial moisture content and temperature on efficiency of cellulase and xylanase production by strains of A. oryzae, A. niger and T. reesei grown in SSF under conditions of forced aeration and static were evaluated. The initial moisture content of the substrate did not affect the production of cellulases and xylanases by strain of A. oryzae P27C3A, however higher moisture was better for enzyme production by strains of A. oryzae P6B2 and A. niger and lower moisture were better for the production of cellulases and xylanases by T. reesei in both cultive systems. Temperature 28°C was best for xylanase production by all the fungal strains, while higher temperatures was better cellulases production in both culture systems. The use of whole fermented medium of A. niger or T. reesei obtained in the bioreactor were better in the hydrolysis sugarcane bagasse pretreated by steam explosion (BPSE) than the enzymatic extract with a final conversion of 41.3 and 24.9% of theoretical, respectively. The combination of whole fermentation medium of strains of A. oryzae (P6B2 or P27C3A) obtained in flasks and ½ commercial enzyme hydrolysis also were efficient on BPSE hydrolysis (26.1 and 42.4% of theoretical, respectively). Nevertheless, the combination of whole fermented medium of A. oryzae P6B2 and enzymatic extract of A. niger obtained in flasks promoted a conversion of 65% and an ethanol yield of 84% of the theoretical value. As overall conclusion it was found that the use of whole fermented medium produced by fungi cultivated under solid state fermentation (SSF) in the BPSE hydrolysis resulted in similar or higher yields compared to the hydrolysis using the enzyme extract, giving clear indication that the extraction/filtration step of the enzyme can be eliminated. The use of the enzyme complex of A. oryzae P6B2 in combination with the enzymes of A. niger resulted in a BPSE hydrolysis more efficient when compared with other combinations, showing the importance of selecting microorganisms for high enzymes production. Moreover, the use of a single reactor system for performing enzyme production steps by SSF, saccharification and alcoholic fermentation may be performed, avoiding the need for steps separation.
A discussão dominante sobre a transformação da biomassa lignocelulósica a combustível líquido é a sua viabilidade econômica. Assim, a comercialização do etanol a partir de biomassa lignocelulósica é dificultada principalmente pelos custos proibitivos das preparações de celulases enzimas usadas na sacarificação. Algumas estratégias que podem ser adotadas para a redução do custo das enzimas utilizadas na degradação da biomassa incluem a seleção de micro-organismos altamente produtores de celulases e hemicelulases, utilização de matéria-prima mais barata e estratégias de fermentação a um custo efetivo - como a fermentação em estado sólido (FES) e técnicas mais eficientes de sacarificação e fermentação alcoólica. O objetivo deste trabalho foi avaliar a utilização do meio fermentado integral (MFI), contendo biomassa lignocelulósica, micélio fúngico e enzimas na hidrólise do bagaço de cana pré-tratado por explosão a vapor para produção de etanol celulósico. Neste contexto, realizou-se a seleção de fungos filamentosos isolados do solo de madeira em decomposição da Região Amazônica produtores de celulases e hemicelulases, otimizou-se as condições operacionais, como umidade e temperatura para a produção de enzimas in house utilizando biorreator de coluna instrumentado e por fim, avaliou-se a eficiência do MFI e (EE) na hidrólise enzimática da biomassa lignocelulósica para produção de etanol celulósico. Entre os 40 fungos caracterizados quanto à produção de enzimas envolvidas na degradação da lignocelulose, duas linhagens de A. oryzae (P6B2 e P27C3A) se destacaram em relação às demais. Além das linhagens de A. oryzae outras duas linhagens de fungos, uma de A. niger 3T5B8 e outra de T. reesei RUT C30 foram avaliadas neste trabalho, a fim de verificar a eficiência das linhagens isoladas do solo da Floresta Amazônica. A umidade inicial do substrato não influenciou na produção de celulases e xilanases pela linhagem de A. oryzae P27C3A, no entanto umidades elevadas foram melhores para a produção de enzimas pelas linhagens de A. oryzae P6B2 e A. niger e umidades baixas foram melhores para a produção de celulases e xilanases por T. reesei em ambos os sistemas de cultivo, forçado e estático. Com relação à temperatura de fermentação, 28ºC foi melhor para a produção de xilanases por todas as linhagens fúngicas e temperaturas mais elevadas favoreceram a produção de celulases pelos fungos. A utilização do MFI de A. niger ou T. reesei obtido em biorreator de coluna instrumentado foram melhores na hidrólise do bagaço de cana pré-tratado por explosão a vapor (BEX) do que o EE, com uma conversão final de 41,3 e 24,9% do valor teórico, respectivamente. A combinação de MFI das linhagens de A. oryzae (P6B2 ou P27C3A) obtida em Erlenmeyer e ½ de enzima comercial também favoreceram a hidrólise do BEX (26,1 e 42,4% do valor teórico, respectivamente). No entanto, a combinação de MFI de A. oryzae P6B2 e EE de A. niger obtido em Erlenmeyer promoveram uma conversão final de 65% e um rendimento de etanol de 84% do valor teórico. Vale salientar que foi utilizado na fermentação alcoólica o meio hidrolisado na íntegra, contendo açúcares, enzimas, biomassa lignocelulósica e micélio fúngico. Como conclusões gerais, constatou-se que a utilização de MFI produzido pelos fungos por FES na hidrólise do BEX resultou em rendimentos semelhantes ou mais elevados quando comparado com a hidrólise do BEX utilizando EE, dando a clara indicação de que o passo de extração/filtração das enzimas pode ser eliminado; a utilização do complexo enzimático de A. oryzae P6B2 em combinação com o complexo enzimático de A. niger resultou em uma hidrólise mais eficiente do BEX quando comparado com outras combinações, mostrando a importância da seleção de micro-organismos produtores de enzimas envolvidas na degradação da lignocelulose, para que a produção de etanol celulósico possa se tornar economicamente viável; e por fim, a utilização de um único sistema de reator para a realização das etapas de produção de enzimas por FES, sacarificação e fermentação alcoólica pode ser realizada, evitando-se a necessidade de etapas de filtração.

En el darrer segle, la contaminació de l'aigua s'ha convertit en un problema important que afecta a una gran part de la població i al medi ambient. La contaminació es deu principalment a les descàrregues d'aigües residuals no tractats o tractats inadequadament. Les plantes de tractament d'aigües residuals convencionals solen eliminar els compostos orgànics, però no estan dissenyades per eliminar altres contaminants com els microcontaminants, de manera que aquests es podem descarregar directament al medi ambient juntament amb el efluents. Entre els possibles tractaments, els fongs de podridura blanca s'han convertit en una tecnologia prometedora per al tractament d'aigües residuals, ja que poden eliminar una àmplia gamma de contaminants complexos a causa del seu sistema enzimàtic ligninolític inespecífic. S'han estudiat els fongs de podridura blanca per a l'eliminació de diferents microcontaminants en aigües residuals, però el creixement de microorganismes natius de les aigües residuals ha fet que l'operació en continu en reactor duri poc temps degut a la disminució en les eficiències d'eliminació. Com alternativa a aquest problema, la present tesi proposa l'aplicació d'un bioreactor utilitzant el fong de podridura blanca T. versicolor immobilitzat sobre un suport lignocel·lulòsic per resoldre aquest problema i permetre així el tractament en continu de les aigües residuals durant llargs períodes de temps. En primer lloc, es va realitzar una selecció del substrat amb l'objectiu de seleccionar el millor material lignocel·lulòsic per al creixement fúngic i la fusta de palets es va seleccionar per als següents experiments. Es van realitzar estudis d'immobilització en un bioreactor de llit fluïditzat. Es van obtenir bons resultats amb pellets formats amb un cor de fusta, però el procés resultant no es escalable, per això es van proposar nous sistemes. Es va desenvolupar i operar un bioreactor de filtre percolador i un bioreactor de llit fix. Ambdós van operar en continu durant llargs períodes de temps amb T. versicolor immobilitzats sobre fusta de pallets i van ser emprats per al tractament d'aigües residuals procedents de diferents fonts: aigües residuals hospitalàries amb compostos farmacèutics actius, aigües residuals industrials de processament d'aliments amb àcids húmics i aigües residuals de zones rurals amb plaguicides. L'optimització de les condicions operatives és un tema clau per millorar el rendiment del reactor. Al bioreactor de filtre percolador, es van optimitzar la relació de recirculació i el volum total. D'altra banda, en el bioreactor del llit fix es van realitzar estudis preliminars de pH, biomassa fúngica, sorció sobre la fusta i aeració.
En el último siglo, la contaminación del agua se ha convertido en un problema importante que afecta a una gran parte de la población y al medio ambiente. La contaminación se debe principalmente a las descargas de aguas residuales no tratadas o tratadas inadecuadamente en cuerpos de agua. Las plantas convencionales de tratamiento de aguas residuales generalmente eliminan los compuestos orgánicos, pero no están diseñadas para la eliminación de otros contaminantes como los microcontaminantes, por lo que estos pueden ser descargados junto con los efluentes directamente al medio ambiente. Entre las posibles tecnologías para el tratamiento de aguas residuales, los hongos de podredumbre blanca se han convertido en una alternativa prometedora porque pueden eliminar una amplia variedad de microcontaminantes debido a que presentan un sistema enzimático ligninolítico inespecífico. Los hongos de podredumbre blanca se han estudiado para la eliminación de una amplia gama de microcontaminantes en aguas residuales, pero el crecimiento excesivo de bacterias nativas del agua residual por lo general produce una disminución en las eficiencias de eliminación acortando la operación en continuo de los biorreactores. Como alternativa a este problema, la presente tesis propone la aplicación de un biorreactor utilizando el hongo de podredumbre blanca T. versicolor inmovilizado sobre un soporte lignocelulósico. Esta estrategia permitiría el tratamiento en continuo de aguas residuales durante largos periodos de operación. En primer lugar, se realizó un estudio con el objetivo de seleccionar el material lignocelulósico óptimo para el crecimiento de T. versicolor eligiéndose la madera de palé para los siguientes experimentos. Los posteriores estudios de inmovilización se realizaron en un biorreactor de lecho fluidizado. Se obtuvieron buenos resultados con el hongo auto-immobilizado sobre madera formado un pellet, pero el proceso no resultó escalable por lo cual se propusieron nuevos sistemas alternativos. Se desarrolló y operó un biorreactor de filtro percolador y un biorreactor de lecho fijo utilizando T. versicolor inmovilizado sobre madera de palé para el tratamiento en continuo de aguas residuales durante largos periodos de operación. Ambos reactores se emplearon para el tratamiento de aguas residuales de diferentes orígenes: aguas residuales hospitalarias con compuestos farmacéuticos activos, aguas residuales industriales de procesadoras de alimentos con ácidos húmicos y aguas residuales de áreas rurales con pesticidas. La optimización de las condiciones operacionales resulta una cuestión clave para mejorar el rendimiento de los reactores. Por un lado, en el biorreactor de filtro percolador, se optimizaron la relación de recirculación y el volumen total de trabajo. Por otro lado, en el biorreactor de lecho fijo se realizaron estudios preliminares de pH, cantidad de biomasa, sorción en la madera y aireación. En conclusión, ambos sistemas con T. versicolor inmovilizados sobre madera de palé resultaron ser una buena alternativa para el tratamiento en continuo de diferentes aguas residuales durante largos periodos de tiempo. El biorreactor de filtro percolador logró eliminar el 61% de los compuestos activos farmacéuticos presentes en aguas residuales hospitalarias durante 85 días; el 50% de eliminación de ácido húmico presentes en aguas residuales industriales durante 26 días; y la eliminación del 84% de diuron durante 18 días utilizando agua sintética. En el biorreactor de lecho fijo se obtuvo más del 90% de eliminación de diuron durante 50 días operando con agua real de origen rural.
Over the last century, the water pollution has become a major problem which affects a large part of population and the environment. It is originated principally due to the discharges of untreated or inadequately treated wastewater in water bodies. Conventional wastewater treatment plants (WTTPs) typically remove organic compounds, but they are not designed to remove other pollutants such as micropollutants, so they can be discharged into the environment together with the effluents from the WTTPs. Among the possible treatments, white-rot fungi (WRF) have become a promising alternative for the wastewater treatment because it can remove a wide range of micropollutants due to their nonspecific ligninolytic enzymatic system. WRF have been studied for the removal of a wide range of micropollutants in real wastewater, but the bacteria overgrowth usually produced a decline in removal efficiencies and consequently short-term operations are obtained. The present thesis proposes the application of a bioreactor system using the WRF Tramentes versicolor immobilized on a lignocellulosic support, to solve this problem and to allow the continuous long-term wastewater treatment. First of all, a substrate screening was performed in order to select the best lignocellulosic material for fungal growth. The pallet wood was selected for the following experiments. Immobilization studies were performed in a fluidized bed bioreactor. Good results were obtained with complex wood pellets, but the process results not scalable, hence new systems were proposed. A trickle-bed bioreactor and a packed-bed bioreactor were developed and operated in a continuous long-term treatment with T. versicolor immobilized on pallet wood. Both reactors were employed for the treatment of wastewater from different sources: hospital wastewater with pharmaceutical active compounds, food-processing industrial wastewater with humic acids and rural area wastewater with pesticides. The optimization of operational conditions is a key issue to improve the reactor performance. In the trickle-bed bioreactor, the recycling ration and the total volume were optimized. Meanwhile, in the packed-bed bioreactor preliminary studies of pH, fungal biomass, wood sorption and aeration were carried out. In conclusion, both bioreactors systems with T. versicolor immobilized on pallet wood are a good alternative for the continuous long-term treatment of different wastewaters. The trickle-bed bioreactor achieved 61% of PhACs removal from hospital wastewater during 85 days; 50% humic acid removal from industrial wastewater for 26 days; and 84% diuron removal from synthetic tap water during 18 days. In the packed-bed bioreactor treating real wastewater, more than 90% removal of diuron was obtained during 50 days

La biotechnologie exploite depuis de très nombreuses années une importante collection de micro-organismes pour fabriquer des produits dans des secteurs tels que l’alimentation et la santé. Elle s’inscrit aujourd’hui parfaitement dans la transition écologique du secteur de la chimie et de l’environnement, en concevant des produits performants pour substituer les produits d’origine pétrolière. Les champignons filamenteux représentent d’importants organismes en biotechnologies industrielles, largement utilisés pour la production de métabolites d’intérêts commerciaux tels que les antibiotiques, les enzymes ou encore les acides organiques. Leurs mises en œuvre en milieu liquide attirent continuellement de nouvelles recherches afin de surmonter les problèmes associés à leur comportement. En effet ce mode de culture engendre différentes morphologies fongiques, dépendantes principalement de leur propriétés biologiques intrinsèques mais aussi des conditions physico-chimiques au sein de leur environnement. Cette caractéristique conditionne bien souvent leur mise en œuvre et leur productivité.Cette thèse propose une étude originale dont l’objectif est d’identifier et de contrôler les phénomènes sous-jacents qui influent sur la morphogénèse des champignons. Pour cela nous proposons des recherches couplant des études fondamentales et appliquées sur plusieurs souches de fort intérêt industriel. Nous avons investigué, mis au point et développé différentes techniques pionnières dans la maitrise de la morphologie fongique. La mise en œuvre de microparticules et le développement d’une technique d’encapsulation par microfluidique ont permis de confirmer et d’approfondir les mécanismes relatifs à l’agrégation des conidies de champignon. D’autre part, l’ajout de sels minéraux dans le milieu de culture a montré l’affranchissement de l’agrégation des hyphes de champignons. Pour concrétiser une extrapolation des résultats vers un procédé industriel, ces outils ont également été implémentés au cours des premières étapes de changement d’échelle. Ainsi ces travaux apportent des résultats et des perspectives concrètes dans la maîtrise des bioprocédés impliquant les champignons filamenteux
Biotechnology has for many years operated a large collection of microorganisms in food and health sectors. Today, this field fits perfectly into the ecological transition of chemical and environmental sectors, by designing high-performance processes to replace petroleum products. Filamentous fungi represent important organisms in industrial biotechnologies, widely used for the production of metabolites with strong commercial interests such as antibiotics, enzymes or organic acids. Their implementation in submerged environment is continually attracting new research to overcome issues associated with their behavior. Indeed, this mode of cultivation generates different fungal morphologies, mainly dependent on their intrinsic biological properties but also on physico-chemical conditions within their environment. This characteristic of filamentous fungi very often influences their implementation and their productivity.This thesis proposes an original study to identify and control the underlying phenomena that influence fungal morphogenesis. To this end, we propose research combining fundamental and applied studies on several strains of strong industrial interest. We have investigated and developed various pioneering techniques for the control of fungal morphology. Microparticles implementation and development of a microfluidic encapsulation technique allowed us to confirm and deepen the aggregation mechanisms of fungal conidia. On the other hand, the addition of mineral salts in the culture medium prevented hyphae aggregation. To validate an extrapolation of these results towards an industrial process, these tools were also implemented during the first steps of scale-up. These works bring results and perspectives in the control of bioprocesses involving filamentous fungi

Tannins are polyphenolic compounds produced by plants that are used in the vegetable tanning of leather at an industrial scale. Tannins differ from most other natural phenols since they precipitate proteins and are used in the tanning process to bind to the collagen proteins of the animal skin to make leather more durable and not putrescible. Tannins represent one of the low-biodegradability substances in tannery wastewaters with a highly recalcitrant soluble chemical oxygen demand; moreover, at high concentrations they can inhibit biological treatment. This soluble recalcitrant fraction of tannery effluents is usually removed by means of chemical processes. Therefore, a biological treatment that could remove effectively this fraction would have both environmental and economic advantages. Despite the antimicrobial properties of tannins, there are organisms that are able to grow on them; in fact their biodegradation in the environment is mainly associated with fungi rather than bacteria. Fungi could thus be exploited for the bioremediation of wastewater streams of the tanning industry. However, in environmental biotechnology applications, fungi tend to be outcompeted by bacteria. The application of a fungal-based bioreactor, that has a similar performance under sterile and non-sterile conditions in long-term operations, is still a challenging task. The present thesis was aimed at developing and testing technologies to remove tannins with an engineered ecosystem based on fungi and bacteria for application in typical tannery wastewater treatment trains (non-sterile conditions). Aspergillus tubingensis MUT 990 was selected as a suitable fungal strain. Literature research and previous experiences were used to design and build lab-scale (4 litres volume) and pilot-scale (1.5 m3 volume) reactors. The selected fungal strain was immobilised in polyurethane foam cubes carriers and inoculated in a novel rotating, submerged, packed bed reactor. The first test in bioreactors was carried out to evaluate the effect of rotation and the co-substrate addition on tannin removal. The second test in bioreactors was carried out in two submerged packed bed reactors run in parallel, the first one was fed with a condensed tannin (Quebracho tannin), and the second with hydrolysable tannin (Tara tannin), both were inoculated with Aspergillus tubingensis in attached form. The hydraulic retention time and the concentration of tannins in the medium were varied to maximise the removal capacity and to analyse the substrate inhibition. A stable biofilm was maintained in the first reactor during the 180 days of operation. On the other hand, in the second reactor there was a biomass detachment during the start-up and then grown as a suspended culture throughout the operational period (226 days). Soluble chemical oxygen demand removal up to 53% and 90% were achieved in the first and second bioreactor, respectively, without the addition of co-substrates. The microbial communities of the reactors, made up of fungi and bacteria, were characterised with a metagenomic analysis. In addition, an innovative technique, the heterogeneous respirometry, was applied to assess the biological activity of immobilised cells (biofilm). The data obtained were used to develop a mathematical model and to perform a kinetic and stoichiometric characterisation of the biomass. Since fungal biomass is poorly characterised with modelling and respirometry, dedicated experiments were set-up along with a respirometric procedure. The lab-scale results were used to design a pilot-scale reactor. Finally, after a start-up phase the pilot-scale reactor was fed with real tannin-rich wastewater collected from tanneries. At the end of the experimentation the biofilm was stable and an encouraging performance had been achieved. We believe the results obtained represent the first step for a future real-scale application to reach an efficient biological removal of tannins from tannery wastewater.

Recent evaluations have demonstrated the ability of the bacteria Rhodococcus rhodochrous DAP 96253 to inhibit the growth of molds associated with plant and animal diseases as well as post-harvest loss of fruits, vegetables and grains. Pre-pilot-scale fermentations (20-30L) of Rhodococcus rhodochrous DAP 96253 were employed as a research tool with the goal of producing a practical biological agent for field-scale application for the management of white-nose syndrome (WNS) in bats and post-harvest fungal losses in several fruit varieties. Several key parameters within the bioreactor were evaluated for the potential to increase production efficiency as well as activity of the biocatalyst. These parameters included elapsed fermentation time, dissolved Oxygen, and carbohydrate concentration of which increased carbohydrate concentration at the time of harvest was shown to have a negative impact on the catalyst activity. In addition, process improvements including utilization of a liquid inoculum, an autoinduction feed strategy, and increased glucose concentration in the feed medium increased fermentation yields to 100-150g/L, while the biocatalyst efficiency was increased from previous work. To increase production efficiency, a multi-bioreactor scheme was developed that used a seed bioreactor and subsequent production tank, which doubled run yields per production cycle. Amidase, cyanidase, urease, and alkene-monoxygenase activity were monitored throughout the study as potential indicators for the multi-faceted mechanism of fungal antagonism. Of these amidase, cyanidase, and urease were demonstrated to be more elevated in cells that showed antifungal activity than those that did not. This study represents the first example of a reproducible pre-pilot plant-scale biomanufacturing process for a contact-independent biological control agent for established and emerging fungal pathogens of plants and animals, and facilitates large-scale production for broad application.

The effect of fungal pre-treatment using Trametes pubescens on the anaerobic digestion ultrafiltration treatment of wine distillery wastewater (WDW) was studied. The downstream biological treatment system, consisting of four individual reactors, was operated for 30 days. pH buffering was achieved by mixing the pre-treated system feed with CaCO3 and K2HPO4; this proved significant for optimum performance of the system in removal of soluble chemical oxygen demand (CODS). The experimental system was shown to eliminate an average of 86 (± 4) % of CODS present in the pre-treated WDW. Treatment in a submerged membrane bioreactor (SMBR) and subsequent secondary digester, together with pH buffering using CaCO3 and K2HPO4, led to the stabilisation of CODS removal. The residual CODS levels in the final effluent were approximately 400 mg/l, significantly lower than the concentrations observed when treating raw WDW, indicating that fungal pre-treatment might have provided additional nutrients for removal of recalcitrant components of the wastewater. The resulting effluent of the system is rich in nitrates and phosphates. Together with the residual organic content it might be used as a fertiliser. Alternatively, if water management of the wine distillery is an issue, a membrane process, such as reverse osmosis or nanofiltration could be applied to bring the parameters of the water to meet the technological needs.