Tesi sul tema "White-rot fungi"

New Zealand has a large number (approx. 8000) of sites contaminated by persistent chemicals, of which approximately 10% arecontaminated with pentachlorophenol (PCP) aa a legacy of former timber treatment sites. The fungicide PCP was used extensively by the forestry industry from the late 1940s to prevent sapstaining of wood. New Zealand was a heavy user of industrial grade PCP because of the predominance of radiata pine (Pinus radiata) which is a soft timber and more susceptible than most tree species to sapstain fungi. International research has shown that soils contaminated by such xenobiotics may be ameliorated using white-rot fungi. To avoid the uncertainties associated with the release of foreign organisms into the New Zealand environment, as legislated by the Hazard Substances and New Organisms Act (HSNO) and governed by the Environmental Risk Management Authority (ERMA), a national research initiative was undertaken in 1996 to study the potential of New Zealand native white-rot fungi for bioremediation. Native white-rot isolates were (1) collected (bioprospecting), (2) selected for their ability to degrade xenobiotics - in the initial phase using PCP as the model compound and (3) studied for their mechanisms and pathways of degradation. Organic waste materials were also evaluated for their suitability to serve as a carrier for fungal augmentation to polluted soil. This PhD study formed part of this larger national research programme, with very close interaction between the different researchers and research activities. The aim of this thesis was to optimise white-rot bioremediation of New Zealand isolates. The work described here was led by me, and the principal results are mine. Selected organisms were evaluated for PCP loss and breakdown in soil. Soil limiting factors (such as soil type, moisture, temperature, pollutant concentration) affecting colonisation of augmented isolates were identified. These laboratory results then were transferred into the field and PCP degradation studied using proto-type biopiles.

The focus of this research was to examine the potential for using white rot fungi to degrade pentachlorophenol (PCP) in water. Experiments were designed to determine the optimum growth conditions for 4 species of fungi, quantify toxicity of PCP to 18 species, and examine PCP degradation by both extracellular enzymes and whole cultures of 4 species. Optimum growth temperatures ranged from 25°C for G. oregonense to 40°C from P. chrysosporium with I. dryophilus and T. versicolor at approximately 30°C. Optimum growth pH were 4.5 for Phanerochaete chrysosporium and 6.0 for the other 3 species. Eighteen species tested for PCP sensitivity were inhibited by 10 mg-PCP/L when grown on agar plates. Within 2 weeks, 17 of the 18 species grew in the inhibition zones. In liquid phase toxicity experiments, all 18 species were killed by 5 mg-PCP /L. Further liquid testing showed that P. chrysosporium and G. oregonense were among the most sensitive species while I. dryophilus and T. versicolor were more tolerant species, having lethal dosages of 17-34, 25-50, >41, and >85 μg-PCP/mg-biomass, respectively. Extracellular enzymes produced in shallow batch cultures by P. chrysosporium and T. versicolor, degraded up to 50% and 75% of the PCP, respectively, when 40 mg-PCP/L was added to mycelia free culture broth. The pattern of chloride ion release resulting from dehalogenation of PCP was bimodal for both species. PCP was degraded by 10 species when PCP was added to whole cultures. Further testing with 4 species showed P. chrysosporium and T. versicolor were the more efficient at reducing aqueous organic chlorine concentrations. Trametes versicolor consistently dehalogenated the most PCP with over 60% of the chlorine being released as chloride ion in 8 days. Comparisons of PCP degradation between species growing as fixed films in rotating tube reactors (RTRs) verified this observation. Degradation in RTRs was superior to degradation in shallow batch reactors on the basis of PCP removal, organic chlorine reductions, and dehalogenation.

Proefschrift Universiteit van Amsterdam.
Research carried out as part of the Innovation oriented research programme on catalysis (IOP Katalyse, no. IKA 94046) Met lit. opg. - Met samenvatting in het Nederlands.

The objective of this study was to examine the potential for degradation of mixtures of pesticides (chlorpyrifos, linuron, metribuzin) by a range of bacteria and fungi and to relate this capability to enzyme production and quantify the rates of degradation of the components of the mixture of xenobiotic compounds. Overall, although bacteria (19 Bacillus and 4 Pseudomonas species) exhibited tolerance to the individual and micture of pesticides actual degradation was not evident. Five species of white rot fungi were grown on minimal salts agar plates amended with 0, 10 and 30 mg L-1 of chlorpyrifos, linuron and metribuzin, individually and as a mixture with a total concentration 15 and 30 mg L-1. Four of these, T. versicolor, P. gigatea, P.coccineus and P.ostreatus, exhibited very good tolerance to the pesticides. They were also grown on a nutritionally poor soil extract agar amended with a mixture of the pesticides at different concentrations (0-70 mg L-1). Subsequently, the ability of T. versicolor, P. gigatea, P. coccineus to degrade lignin and production of laccase in the presence of mixture of the pesticides was examined as well as their capacity to degrade the pesticide mixture at different concentrations (0-50 mg L-1) in soil extract broth was quantified using HPLC. This showed that only T.versicolor had the ability to degrade linuron, after three weeks incubation although all tested species produced laccase. Subsequently, the temporal degradation rates of T.versicolor was examined in relation to temporal degradation of a mixture of the pesticides chlorpyrifos, linuron and metribuzin with total concentrations 0-50 mg L-1 and the temporal laccase production was quantified over a six week period in relation to ionic and non-ionic water potential stress (-2.8 MPa). These studies showed that the test isolate had the ability to produce very high levels of laccase at -2.8 MPa water potential adjusted non-ionically by using glycerol and quite lower levels in soil extract broth without stress while T.versicolor did not produce laccase at -2.8 MPa when the medium was modified ionically. Finally, T.versicolor was able to degrade the pesticide linuron in all tested water regimes, after five weeks incubation, regardless of the concentration of the mixture. In contrast, about 50% of the metribuzin was degraded, only at at -2.8 MPa water potential adjusted non-ionically with glycerol. Chlorpyrifos and its main metabolite TCP were not detected, possibly, due to a combination of hydrolysis, photolysis and volatilization degradation. The capacity of T.versicolor to degrade linuron in mixtures of pesticides and the production of high levels of laccase, in a nutritionally poor soil extract broth, even under water stress suggests potential application of this fungus in bioremediation.

Three species of white-rot fungi, Phlebia radiata, Phanerochaete chrysosporium, and Trametes versicolor, were monitored for laccase and lignin peroxidase production in a defined medium. P. radiata was selected for fed-batch reactor experiments due to its early laccase production, which was determined to be growth-associated. A second peak in laccase activity was observed after several days of nutrient deprivation and was attributed to autolysis of the culture. The effect of protease activity on the accumulation of extracellular laccase activity differed during primary and secondary metabolism, as observed under various conditions of nitrogen and glucose availability. A mixed culture of P. radiata and P. chrysosporium was grown on ammonium lignosulfonate, a by-product of the pulp and paper industry, as sole source of carbon and nitrogen. Under these conditions, laccase production appeared to exceed laccase production by P. radiata in defined culture medium.

Natural organic matter (NOM), a complex mixture of organic compounds, influences drinking water quality and water treatment processes. The presence of NOM is unaesthetic in terms of colour, taste and odour, and may lead to the production of potentially carcinogenic disinfection by-products (DBPs), as well as biofilm formation in drinking water distribution systems. Some NOM removal processes such as coagulation, magnetic ion exchange resin (MIEXTM) and membrane filtration produce sludge and residuals. These concentrated NOM-containing sludges from alum precipitation, membrane treatment plants and MIEX regeneration must therefore be treated prior to disposal. The white-rot fungi possess a non-specific extracellular oxidative enzyme system composed of lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and laccase (Lac) that allows these organisms to mineralise lignin and a broad range of intractable aromatic xenobiotics. Rojek (2003) has shown the capabi lity of Phanerochaete chrysosporium ATCC 34541 to remove 40-50% NOM from solution, however, this was found to be mainly due to adsorption and to be a partially metabolically linked activity. Consequently, the bioremediation of NOM wastes by selected white-rot fungi was further investigated in the present study. The P. chrysosporium seemed to preferentially remove the very hydrophobic acid (VHA) fraction, and so was most effective for a NOM preparation with a high proportion of hydrophobic content (and so high in colour and specific UV absorbance (SUVA)). The extent of NOM decolourisation by P. chrysosporium in three growth media with different C:N ratios followed the trends: Waksman (C:N = 6) > Fahy (C:N = 76) > Fujita medium (C:N = 114), such that the lower the C:N ratio, the greater NOM removal. This was consistent with the findings of Rojek (2003), who used a different NOM preparation and demonstrated that the removal of NOM increased with decreased C:N ratio (1.58-15.81). As removals of NOM with P. c hrysosporium ATCC 34541 were low, and little biodegradation occurred, this organism was compared with P. chrysosporium strain ATCC 24725, Trametes versicolor ATCC 7731, and three strains of yeast (Saccharomyces species arbitrarily denoted 1, 2 and 3). T. versicolor gave the greatest removal (59%) which was attributed largely to degradation, whereas the NOM removal by the two strains of P. chrysosporium (37%) and the yeast was predominantly due to adsorption as indicated by the deep brown colouration of the biomass. Saccharomyces sp. 1, 2 and 3 removed 12%, 61% and 23% of the colour, respectively. Although Saccharomyces sp. 2 had similar high colour reduction to T. versicolor, the specific removal values differed markedly: 0.055 compared to 0.089 mg NOM/mg biomass, respectively. The low level of the ligninolytic enzymes secreted by both strains of P. chrysosporium corresponded with the low degree of NOM removal by biodegradation as shown by high performance size exclusion chromatography (HPSEC). The high NOM removal attained by T. versicolor was attributed to the activities of the ligninolytic enzymes, especially laccase. The NOM removal was attributed to the breakdown of the high molecular weight compounds to form a pool of low molecular weight materials, which were then most likely utilised by the T. versicolor. Growth of T. versicolor cultures at 36oC caused inhibition or denaturation of the activity of the phenoloxidase enzymes compared to those grown at 30oC. The low activity of LiP in both cultures suggested that this enzyme may not play much of a role in NOM removal. The higher levels of MnP and Lac activities at 30oC were responsible for the greater NOM removal (73% vs. 59%) and thus the cleavage of aromatic rings, conjugated and C-Cβ αbonds in phenolic moieties, as well as catalysing alkyl-aryl cleavage in the NOM structures. T. versicolor cultured in Waksman medium with higher initial glucose (5 g/L cf. 2 g/L) led to lower ligninolytic enzyme activities and a lower degree of NOM removal (25% less colour reduction), probably due to preferential use of glucose over NOM as carbon source. NOM removal (mg removed) increased linearly with NOM concentration up to 600 mg C/L (62 mg (A446); 31 mg (A254)), above which removal decreased markedly. This trend coincided with increasing total ligninolytic enzyme activity, where the level of Lac increased up to 600 mg C/L NOM although MnP decreased gradually across the range while LiP was only detected for 100 and 300 mg C/L NOM. Hence, the removal of NOM from solution by T. versicolor was associated with high oxidative enzyme activity, particularly of laccase. Laccase was the major extracellular enzyme secreted by T. versicolor and by deduction, played a major role in NOM removal. The optimum temperature for Lac activity secreted by T. versicolor cultured in Waksman medium supplemented with 4.5 g/L wheat bran plus 0.5% Tween 80 was determined to be 50oC. The optimum pH for the Lac activity for guaiacol and NOM was identified as pH 4.0-4.5. Although the optimum enzyme activity occurred at 50oC, 30oC was recommended for enzymatic removal of NOM as the phenoloxidase enzyme activity may be denatured if the NOM removal process were considered to run for long period at high temperature. Although agitation led to apparent enzyme denaturation, fermentations with continuous agitation promoted enzyme activity faster than those with occasional agitation (agitated every 6 hours for 30 minutes at 130 rpm and 30oC) as it provides better mass transfer. However, it seemed that continuous agitation had an adverse effect on the fungal growth and enzyme production over extended fermentation periods. Addition of 4.5 g/L wheat bran to modified Waksman medium in the absence of NOM led to high production of Lac activity compared with LiP and MnP activities, showing its great potential as a laccase inducer. Addition of Tween 80 alone to the cultures led to a small improvement in Lac activity; however, with the presence of wheat bran it caused marked increases in LiP, MnP and Lac activit ies. When NOM was added to cultures of T. versicolor with the two supplements, it led to markedly reduced Lac activity, but increased LiP and MnP activities, and no improvement in NOM removal compared with the cultures in the absence of supplements (12 mg (or 61%) cf. 15 mg (or 73%) for 100 mg C/L after corrected for colour from and adsorption by wheat bran).

The natural breakdown of three pesticides on the UK Red List (dieldrin, simazine and trifluralin) in water and soil varied with environmental conditions. In both sterile and unsterile water, trifluralin was degraded to some extent at 20 and 30°C. In contrast, dieldrin and simazine were stable over the 42 days incubation period. A gradient HPLC method was developed for the simultaneous quantification of the three pesticides in soil. In field capacity soil mixtures of the three pesticides (5 and 10 ppm) showed a similar stability with limited degradation at 20°C but increased rates of degradation at 30°C. At the higher concentration the pesticides naturally degraded at a slower rate. Simazine and trifluralin degradation was significantly enhanced with increasing temperature from 20 to 30°C. Water potential (field capacity~ -0.065 MPa~ and - 0.28 MPa) had little effect on the natural breakdown rate of dieldrin. Simazine showed a greater breakdown in the mid-wetness soil~ while trifluralin was degraded rapidly in the field capacity soil, but not at all in the driest treatment over the 70 day experimental period. In vitro studies on solid agar media overlayed with cellophane showed that of four fungi examined~ Trametes cingulata, Trametes socotrana (tropical species) and Phanerochaete chrysosporium and Polystictus versicolor (temperate species) all except P.chrysosporium were able to grow in the presence of 5 ppm of any of the three pesticides at 20 and 30°C, with the latter only growing at 30°C. At 10 ppm concentration P. chrysosporium did not grow, regardless of temperature or time of incubation (up to 56 days). HPLC was used to quantify the temporal rates of degradation in the solid agar media and this showed that P. versicolor and T. socotrana were very effective at breaking down the three pesticides, at 20 and 30°C. The chosen fungi were grown on chopped straw as a carrier and incorporated into soil microcosms in the ratio of 1:10 containing mixtures of the three pesticides (5, 10 ppm) at 20 and 30°C, and subsequently under different water potential regimes at 20°C only, over periods of 70 days. P. versicolor alone significantly increased breakdown of 5 ppm dieldrin by 26% over untreated controls, while simazine breakdown was increased by 16%. However, for simazine at 30°C there was no difference between temporal rates of natural breakdown and those containing fungal inocula, regardless of concentration. With 5 ppm trifluralin, a maximum breakdown in untreated soil was 67% after 70 days. By contras~ this pesticide was undetectable after 28 days in the presence of the inoculant P . versicolor. This increased to 42 days where a mixture of the two fungi were used. Generally the mixture of fungi used in this study were not as effective in bioremediation of these pesticides as a single species. Field capacity soil appeared to be the best condition for P. versicolor to degrade dieldrin and trifluralin added at 10 ppm. However, for simazine this occurred in the driest water potential (-0.28 MPa) used.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第11635号
農博第1491号
新制||農||908(附属図書館)
学位論文||H17||N4028(農学部図書室)
UT51-2005-D384
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 渡邊 隆司, 教授 島田 幹夫, 教授 東 順一
学位規則第4条第1項該当

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.

Approximately 10,000 different dyes and pigments are produced annually worldwide and used extensively in the dye and printing industries. This has resulted in the generation of large volumes of highly polluted wastewater. Apart from the aesthetic deterioration of the natural water bodies, dyes also cause harm to the flora and fauna in the natural environment. Therefore, wastewater containing dyes must be treated prior to their discharge into the environment.

Different methods can be applied for the treatment of synthetic dyes from aqueous solutions, such as ozonation, coagulation, flocculation, reverse osmosis and adsorption. However, biological treatments are promising alternatives with different approaches going from the complete immobilization of microorganisms to the pure enzyme utilization. Among all enzymes, laccases are an interesting alternative for the dye degradation due to their low affinity and wide specificity for the substrates. Laccases are multicopper oxidases found in higher plant and microorganisms, like white-rot-fungi; and carry out one-electron oxidation of phenolic and related compounds, and reduce O2 to water.

Thus, this work proposes different strategies based on the use of laccases for the discoloration of synthetic dyes from aqueous solutions. These strategies include studies in different fields to promote eco-friendly solutions for different assets of the whole process. These studies include: the selection of substrates for the production of laccase by the white-rot fungus Trametes pubescens, the possible reutilization of these substrates in the discoloration process, the optimization of the laccase production per culture, the scale up of the laccase production, the use of free and immobilized laccase in the discoloration of dyes and the use of different immobilization techniques to increase the reutilization of the immobilized laccase for the treatment of synthetic dyes.
Aproximadamente 10,000 tintes y pigmentos diferentes son producidos anualmente y tienen un uso extendido en las industrias de teñido e impresión. Esto ha generado grandes cantidades de aguas residuales altamente contaminadas. A parte del deterioro estético que sufren los cuerpos de agua, los tintes también causan daño a la flora y fauna presentes en el medio ambiente. Por ello, las aguas residuales que contienen tintes deben ser tratadas antes de su descarga al ambiente.
Distintos métodos pueden ser empleados para el tratamiento de tintes sintéticos en soluciones acuosas, tales como la ozonización, coagulación, floculación, osmosis inversa y la adsorción. Sin embargo, los tratamientos biológicos resultan una alternativa prometedora con distintas aproximaciones que van desde la inmovilización de microorganismos hasta el uso de las enzimas puras. Entre todas estas enzimas, las lacasas resultan ser muy interesantes para la degradación de tintes debido a su baja afinidad y amplia especifidad por los substratos. Las lacasas son oxidasas de multicobre que se encuentran en plantas, microorganismos, como los hongos de putrefacción-blanca, y llevan a cabo la oxidación del fenol y compuestos relacionados, y reducen el O2 a agua.
Debido a esto, en este trabajo se proponen distintas estrategias basadas en la utilización de las lacasas para la decoloración de tintes sintéticos en medios acuosos. Estas estrategias incluyen estudios en distintas áreas para promover soluciones amigables con el medio ambiente en las distintas etapas del proceso. Estos estudios incluyen: la selección de substratos para la producción de lacasa con el hongo de putrefacción-blanca Trametes pubescens, la posible reutilización de estos substratos en los procesos de decoloración, la optimización de la producción de la lacasa por cultivo, el escalado de la producción de la lacasa, el uso de lacasa libre e inmovilizada en la decoloración de tintes y el uso de distintas técnicas de inmovilización para incrementar la reutilización de la lacasa inmovilizada en el tratamiento de tintes sintéticos.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第22854号
農博第2437号
新制||農||1082(附属図書館)
学位論文||R2||N5314(農学部図書室)
京都大学大学院農学研究科地域環境科学専攻
(主査)教授 本田 与一, 教授 田中 千尋, 准教授 坂本 正弘
学位規則第4条第1項該当

Le pré-traitement biologique représente une option peu couteuse et éco-compatible bien qu’elle nécessite un temps plus long en comparaison avec des méthodes chimiques. Les champignons de la pourriture blanche producteurs de laccases (phénoloxydases) comme Pycnoporus sanguineus sont de candidats pour de tels traitements biologiques. Les objectifs de ce travail sont de définir i) les conditions de cultures (sources azotée, inducteurs aromatiques des laccases, inoculum et le temps d’incubation) et ii) la préparation du substrat (pasteurisation) qui favorisent les activités laccases et la déphénolisation. De plus, les communautés microbiennes thermotolérantes peuvent agir comme antagonistes de P. sanguineus et induire laccases. Des plans d’expérience ont été utilisés pour tester quelles que soient les concentrations pour obtenir des conditions de culture appropriées. L’urée comme source d'azote, a un effet négatif sur les activités laccases. Le meilleur taux de déphénolisation (47,2%) associé aux plus fortes activités laccases et à une réduction de la cristallinité de la cellulose (suivi par RMN du solide du 13C) ont été obtenus après 40 jours en utilisant 9 % d’extrait de levure, 10% de pulpe de café et 5 % d’inoculum. Des températures entre 70 et 75 °C et des temps de 5 à 10h de pasteurisation ont favorisé les laccases. De plus les profils cataboliques des communautés microbiennes du substrat pour ces conditions de pasteurisation ont montré leur potentiel de dégradation de polysaccharides. Ceci démontre la capacité de ces microorganismes à coloniser le substrat, pour limiter les contaminations et à favoriser les étapes ultérieures du procédé
Biological pre-treatment represents a cheap, mild and eco-friendly option, though it requires longer residence time -compared to physicochemical pretreatments-. White-rot fungi producing laccases (phenoloxidases) such as Pycnoporus sanguineus are good candidates for such biological treatment. The objectives of this work were to define i) culture conditions (nitrogen sources, aromatic laccase inducers, inoculum and incubation time) and ii) substrate preparation (pasteurisation) to favour laccase activities and substrate dephenolisation. Moreover, thermotolerant microorganisms may act as antagonists of P. sanguineus and enhanced laccase. Experimental designs were used to obtain suitable culture conditions. Urea as nitrogen source led to a decrease in laccaseswhatever the concentrations used. The best dephenolisation yield (47.2%) associated with higher laccase activities and the major reduction of cellulose crystallinity according to solid-state NMR-13C, were found after 40 days of SSF using 9% yeast extract, 10% coffee pulp and 5% inoculum. Temperature from 70 to 75 °C and pasteurisation time for 5 to 10 h, favoured laccase activities. Moreover, for these pasteurisation conditions, catabolic profiles of substrate microbial communities showed the ability to degrade polysaccharides. This indicates that these microorganisms can actively colonise the substrate to limit contamination and also probably enhanced further steps of the process

Els contaminants emergents són un ampli grup de compostos orgànics detectats en diversos compartiments ambientals. Tot i que la seva concentració normalment està compresa entre els ng/L fins a pocs µg/L (força inferior que els contaminants orgànics convencionals), poden representar una amenaça per a la salut humana i el medi ambient. D’entre tots els contaminants emergents, els principis actius dels fàrmacs (PhACs) i els compostos disruptors endocrins (EDCs) generen una especial preocupació. Per altra banda, està àmpliament acceptat que la seva principal font d’entrada al medi ambient són els efluents de les plantes depuradores, on els tractaments convencionals de llots actius no són capaços de degradar-ne la majoria. Per tant, s’han de buscar tractaments alternatius. Una d’aquestes alternatives podria ser l’ús de fongs ligninolítics, aprofitant el seu sistema enzimàtic que els hi confereix l’habilitat de degradar un rang molt ampli de contaminants. Aquesta tesi avalua diferents aspectes relacionats amb la degradació de contaminants emergents per part de fongs. El fong de podridura blanca Trametes versicolor, àmpliament estudiat, és el que s’ha triat per a dur a terme els experiments d’aquesta tesi. Primer de tot s’ha estudiat la degradació individual de determinats contaminants. Tenint en compte que la degradació dels EDCs ha estat menys estudiada que la dels PhACs, es van seleccionar sis EDCs pertanyents als grups dels filtres UV (benzofenona-3 (BP3), benzofenona-1 (BP1) i 3-(4-metilbenzilidè) càmfor (4-MBC)) i dels benzotriazols (1H-benzotriazol (BTZ) i toliltriazol, una mescla de 4-metilbenzotriazol (4-MBTZ) i 5-metilbenzotriazol (5-MBTZ)). S’ha fet, doncs, un seguiment de la seva degradació per part de T. versicolor, la toxicitat aguda i les activitats estrogènica i de tipus dioxina, s’han identificat els metabòlits generats pel fong i s’han suggerit els primers passos de la via de degradació. A més a més, el destí de determinats contaminants (la BP3 i l’analgèsic i antiinflamatori diclofenac) durant la seva degradació per part del fong ha estat avaluada a través de compostos marcats amb l’isòtop estable 13C. La combinació d’anàlisis de la composició isotòpica del C del CO2 i de la biomassa total i el sondeig d’isòtops estables en aminoàcids (aa-SIP) han permès la discriminació entre simple transformació, mineralització oxidativa o incorporació del carboni a la biomassa. Pel que fa als dos compostos estudiats, els dos s’han mineralitzat però s’ha vist que només la BP3 s’utilitza com a font de carboni i és incorporada a la biomassa del fong. Per una altra banda, es van tractar dos efluents reals (l’aigua residual d’un hospital veterinari i el concentrat d’osmosi inversa d’una planta pilot de tractament d’aigües residuals urbanes) en un bioreactor de fongs fluïditzat per polsos d’aire i operat sota diferents condicions operacionals (estèril/no estèril i discontinu/continu) en vistes a una possible implementació futura. Amb aquesta intenció, aquesta tesi apunta a la importància de l’addició externa de nutrients i al control de l’aeració, els quals haurien de ser optimitzats per a obtenir una eliminació eficient de contaminants per part del fong inoculat. En aquesta tesi també es remarca la importància dels processos de conjugació i desconjugació. Per una banda, són una una restricció en l’avaluació de la degradació en efluents reals a causa de la seva absència en els mètodes analítics i, per l’altra, els conjugats representen uns metabòlits intermedis importants durant la degradació per part del fong dels contaminants seleccionats . També es van realitzar anàlisis de biologia molecular (anàlisi dels àcids grassos dels fosfolípids (PLFA), PCR quantitativa (qPCR) i gel d’electroforesis en gradient desnaturalitzant (DGGE)) amb l’objectiu de trobar alguna correlació entre l’operació dels bioreactors i el comportament del fong inoculat i els altres microorganismes que es desenvolupen en els bioreactors no estèrils. Els resultats suggereixen que els paràmetres de seguiment clàssics (com poden ser l’activitat lacasa) podrien no ser uns bons indicadors de la supervivència i predominança del fong inoculat.
Emerging contaminants are a wide group of organic compounds detected in many environmental compartments. Even though their environmental concentration is usually in the range of ng L-1 to low µg L-1 (much lower than conventional organic pollutants), they still represent a threat to human health and the environment. Among emerging contaminants, pharmaceutically active compounds (PhACs) and endocrine disrupting compounds (EDCs) are of special concern. It is widely accepted that the main source to the environment are the effluents of wastewater treatment plants (WWTPs), where conventional activated sludge treatments are not able to degrade most of them. Therefore, alternative treatments should be found. One of those alternatives might be the use of ligninolytic fungi by taking advantage of their enzymatic system, that conferes them the ability to degrade a broad range of contaminants. The present thesis assesses different factors related to the fungal degradation of emerging contaminants. The widely studied white-rot fungus Trametes versicolor has been chosen to carry out all the experiments of this thesis. First of all, individual degradation of selected contaminants was studied. Taking into account that EDCs degradation has been less studied than PhACs, six EDCs belonging to the groups of UV filters (benzophenone-3 (BP3), benzophenone-1 (BP1) and 3-(4-methylbenzylidene) camphor (4-MBC)) and benzotriazoles (1H-benzotriazole (BTZ) and tolyltriazole, a mixture of 4-methylbenzotriazole (4-MBTZ) and 5-methylbenzotriazole (5-MBTZ)) were selected. Their degradation by T. versicolor, acute toxicity, estrogenic and dioxin-like activities were monitored, the fungal metabolites were identified and the first steps of the degradation pathway were suggested. Moreover, the fate during fungal degradation of certain contaminants (BP3 and the analgesic and anti-inflammatory diclofenac) was assessed by means of compounds labelled with the stable isotope 13C. Combination of analyses of carbon isotopic composition of CO2, bulk biomass and amino acids-stable isotope probing (aa-SIP) allowed the distinction between simple transformation, oxidative mineralization or carbon incorporation into the biomass. Regarding the two studied compounds, both of them were mineralized, but only BP3 was found to be used as carbon source and incorporated in the fungal biomass. On the other hand, two real effluents (veterinary hospital wastewater and a reverse osmosis concentrate from a pilot plant treating urban wastewater) were treated in fungal air-pulsed fluidized bioreactors under different operational conditions (sterile/non-sterile and batch/continuous) in view of a possible future implementation. With respect to that, the present thesis points out the importance of an external addition of nutrients and the control of aeration, which should be further optimized for an efficient removal of contaminants by the inoculated fungus. The importance of conjugation and deconjugation processes is also highlighted in this thesis. They are a restriction in the assessment of emerging contaminants degradation in real effluents due to the absence of conjugates in the analytical methods and, at the same time, conjugates are important intermediate metabolites in the fungal degradation of the selected contaminants. Molecular biology analyses (phospholipid fatty acids analysis (PLFA), real-time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE)) were performed as well with the aim of finding some correlation between the operation of the bioreactors and the performance of the inoculated fungus and the other microorganisms that could develop in the non-sterile bioreactors. Results suggest that the classical parameters monitored (i.e. laccase activity) might not be good indicators of inoculated fungus survival and predominance.

Els microcontaminants són un ampli grup de compostos orgànics que s'han detectat a la majoria de compartiments del medi ambient. La seva concentració ambiental està compresa entre pocs ng·L-1 fins a μg·L-1, però es mantenen biològicament actius fins i tot a concentracions tan baixes, poden ser acumulats a través de la cadena tròfica i suposen una amenaça per al medi ambient, la fauna i la salut humana. D'entre els microcontaminants, els fàrmacs (PhACs) generen una especial preocupació. És acceptat que la principal font d'entrada de fàrmacs al medi ambient és via efluents de les estacions depuradores d'aigües residuals (WWTP), on els mecanismes convencionals de llots activats no són suficients per degradar-ne la majoria. En resposta a aquestes preocupacions, la comunitat científica ha destinat molta recerca a mètodes per a la degradació, transformació i/o eliminació de microcontaminants d'aigües residuals d'hospital, on els fàrmacs estan presents a major concentració. D'entre els tractaments possibles, els fongs de podridura blanca (WRF) es presenten com una possibilitat atractiva gràcies al seu baix cost en comparació amb tractaments físics i químics i la seva capacitat de transformar la majoria de compostos gràcies a la seva versàtil maquinària enzimàtica. Els WRF han estat estudiats per a la degradació de fàrmacs en aigües residuals, però la contaminació per microorganismes presents a l'aigua residual ha produït que les operacions en reactor fossin molt curtes. Aquesta tesi aborda aquest problema i proposa diverses estratègies per allargar el tractament. També serveix com a prova que una operació prolongada amb WRF tractant aigua residual d'hospital no estèril és possible. Primer de tot, diversos reactors de llit fluïditzat per polsos d'aire es van operar per estudiar l'efecte que tenien l'addició d'un pre-tractament de coagulació-floculació, l'addició d'un pre-tractament amb llum UV i l'efecte de l'operació com a batch seqüencial (SBR) i en continu en la llargada del tractament. La millor alternativa va ser un reactor en continu amb un pre-tractament de coagulació-floculació. Aquest tren de tractament va ser evaluat en un tractament d'aigua residual d'hospital no dopada amb renovació parcial de la biomassa, i va permetre una operació de dos mesos de durada. A més, diverses variables de procés, a saber, mida del pèl·let, aeració i la ràtio carboni-nitrogen es van estudiar i els valors que van suposar una operació més llarga van ser seleccionats. Aquests estudis previs van permetre per primera vegada una operació prolongada d'un reactor fúngic de llit fluïditzat tractant aigua residual d'hospital no estèril. Aquesta tesi també remarca la importància dels processos de conjugació i desconjugació. Les tècniques analítiques actuals no solen detectar els microcontaminants conjugats, i això impedeix una precisa mesura de la concentració del contaminant estudiat. Per tant, s'haurien de destinar esforços a l'anàlisi de les formes conjugades de compostos. Si s'aconsegueix, podria significar un gran avenç que faciliti l'estudi de l'eliminació de microcontaminants en aigües reals. Les anàlisis de biologia molecular com gel d'electroforesi en gradient desnaturalitzant (DGGE), seqüenciació d'ADN i PCR quantitativa (qPCR) es van aplicar els experiments no dopats per donar informació sobre les comunitats microbiològiques formades durant els tractaments en reactor i per confirmar la presència de T. versicolor durant l'operació. Els resultats suggereixen que el fong es mantenia actiu fins i tot quan l'activitat de l'enzim lacasa no es detectava.
Micropollutants are a wide group of organic compounds that are detected at most compartments of the environment. Their environmental concentration is usually in the range of few ng·L-1 to μg·L-1, but remain biologically active even at such low concentrations, may be accumulated through the food chain and pose a threat to the environment, fauna and human health. Among micropollutants, pharmaceutical active compounds (PhACs) are of special concern. It is accepted that the main sources of PhACs to the environment are effluents from wastewater treatment plants (WWTPs), where conventional activated sludge processes are not able to degrade most of them. Answering to these concerns, the scientific community has devoted extensive research into mechanisms to degrade, transform and /or remove micropollutants from hospital wastewater, where PhACs are present at higher concentrations. Among the possible treatments, white-rot fungi (WRF) are regarded as a cost-effective possibility due to their relatively low cost in comparison with physical and chemical treatments and their capacity to transform most of the compounds thanks to their versatile enzymatic machinery. WRF have been studied for the removal of pharmaceuticals in wastewater, but contamination by wastewater-native microorganisms has produced very short-term reactor operations. The present thesis tackles this problem and proposes several strategies to lengthen the fungal treatment. It also serves as proof of concept of a long-term white-rot fungal operation treating non-sterile real hospital wastewater. First of all, several air-pulsed fluidized bed bioreactors were set up in order to study the effect of the addition of a coagulation-flocculation pretreatment, of the addition of a UV pretreatment and the effect of operating the reactors as a sequencing batch reactor (SBR) or in a continuous fashion on the length of operation. The chosen alternative was a continuous reactor with a coagulation-floculation pretreatment. This treatment train was then evaluated in a non-spiked hospital wastewater treatment with partial biomass restoration, leading to a two-month operation. Additionally, several process variables, namely, pellet size, aeration and carbon-to-nitrogen ratio were studied and the values that led to a longer operation were selected. Those previous studies enabled for the first time a long-term operation of a fungal fluidized bed bioreactor treating real non-sterile wastewater. The importance of conjugation and deconjugation processes is highlighted in this thesis. Conjugated microcontaminants are not usually detected by the current analytical techniques, thus undervaluing the concentration of the pollutant studied. Therefore, an effort should be made to analyze conjugated forms of compounds. If successful, it could be a breakthrough that greatly facilitates the study of removal of micropollutants in real wastewater. Molecular biology analyses such as denaturing gradient gel electrophoresis (DGGE), DNA sequencing and real-time PCR (qPCR) were performed in the non-spiked experiments to give insight on the microbiological communities arisen during the reactor treatment and to confirm the presence of T. versicolor throughout the operation. Results suggested that the fungus was active even when no laccase activity was detected.

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Tyrosinase (E.C.1.14.18.1) is an enzyme of industrial interest that catalyses the ohydroxylation of monophenols (monophenolase activity) and the oxidation of o-diphenols to reactive o-quinones (diphenolase activity), both reactions using molecular oxygen. Pycnoporus sanguineus (L. ex Fries) Murril, is a white rot fungi capable of producing tyrosinase and widely distributed in nature. It is found in regions of mild climate and in tropical forest. The production and characterization of tyrosinase from P. sanguineus were investigated. The selection of inductors, determination of the luminosity influence, biomass and culture media in the production of tyrosinase and the effect of inhibitors on enzyme activity were determined. The fungus produced intracellular tyrosinase and the higher activity was observed using 0.15% L-tyrosine as inducer, in the presence of light, with inoculum of 10 mycelium discs, medium malt extract broth 2%, incubation at 30°C, and constant agitation of 150 rpm, during 2 days. 6 mmol.L-1 salicylhydroxamic acid (SHAM) and 6 mmol.L-1 phenylthiourea (PTU) inhibited 100% of the tyrosinase activity. 0.1 mmol.L-1 sodium azide inhibited 4.15% of tyrosinase activity, while no inhibition was observed after addition of 0.1 mmol.L-1 of phenylmethanesulfonyl fluoride (PMSF). Using L-dopa as substrate, the intracellular crude extract presented optimum pH of 6,6, optimum temperature of 45°C, low stability at 50°C, maintaining about 50% of the activity after 15 min of incubation. The tyrosinase production was confirmed by non-denaturing polyacrylamide gel electrophoresis, using commercial fungal tyrosinase as positive control
A tirosinase (E.C.1.14.18.1), também conhecida como polifenoloxidase ou catecolase, é uma enzima de interesse industrial que catalisa a o-hidroxilação de monofenóis (atividade monofenolase) e a subseqüente oxidação do o-difenol resultante em o-quinonas reativas (atividade difenolase), usando oxigênio molecular. O Pycnoporus sanguineus (L. ex Fries) Murril, um fungo de decomposição branca (white rot) capaz de produzir a tirosinase, é amplamente distribuído na natureza, sendo encontrado em regiões de clima mais ameno e em florestas tropicais. A produção e caracterização da tirosinase produzida por P. sanguineus foi investigada. Foi realizada seleção de indutores, determinação da influência da luminosidade, biomassa e meios de cultura na produção de tirosinase, bem como o efeito de inibidores sobre a atividade enzimática. O extrato bruto enzimático foi caracterizado quanto ao pH ótimo, temperatura ótima e termoestabilidade. Os resultados obtidos demonstraram que o fungo produziu tirosinase intracelular e a mais alta atividade ocorreu com a utilização de 0,15% de L-tirosina como indutor, na presença de luz, com inóculo de 10 discos de micélio fúngico, meio caldo extrato de malte 2%, incubação à 30°C e agitação de 150 rpm, durante 2 dias. Ácido salicilhidroxâmico (SHAM) 6 mmol.L-1 e feniltiouréia (PTU) 6 mmol.L-1 inibiram 100% da atividade de tirosinase. Azida sódica 0,1 mmol.L-1 inibiu 4,15% da atividade de tirosinase, enquanto nenhuma inibição foi observada após adição de fluoreto de fenilmetanosulfonil (PMSF) 0,1 mmol.L-1. Utilizando a L-dopa como substrato, o extrato bruto intracelular apresentou pH ótimo de 6,6, temperatura ótima de 45°C, baixa estabilidade à temperatura de 50°C, mantendo apenas cerca de 50% de atividade após 15 minutos de incubação. A produção da tirosinase foi comprovada através de eletroforese em gel de poliacrilamida não desnaturante, tendo como controle positivo tirosinase fúngica comercial

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The increase in world population demands ever more food and consumer goods, with a consequent increase in the production of agroindustrial residues natives from logging, alcohol fuel plants and industries for beneficiary eligibility of agricultural products. Several studies have demonstrated the potential of white rot fungi to decompose of lignocellulosic substrates, but the use of such residues in ruminant diets has not been properly examined. The objective of this study was to determine the chemical composition and in vitro digestibility of agroindustrial residues (eucalyptus bark, sawdust, sugarcane bagasse, corn kernels, coffee bark, coconut fiber and lump of cotton) inoculated with white rot fungi (Pleurotus ostreatus and Lentinula edodes), and determine the activity of cellulase, xylanase and laccase in ruminal fluid incubated in vitro with corn kernels or sugarcane bagasse ground at different particle sizes. When the residues were treated with L. edodes the content of CP in eucalyptus bark increased 91 % and 78 % in the treatments added with rice bran or urea, respectively. The content of ADL decreased 70% in fructified sawdust added with rice bran compared to the controls. The NDF of sugarcane bagasse decreased 5 % when added with rice bran and 21 % when added with urea. When the residues were treated with P. ostreatus the levels of CEL and ADL of eucalyptus bark decreased 22 % and 137 %, respectively. With sawdust, the concentrations of NDF and ADF decreased 19 % and 27 %, respectively, after fruit body formation. The EE increased 402 % in sugarcane bagasse treated with P. ostreatus. With corn kernels, the content of ash increased 130 % compared to controls in the treatment showing fruit body formation. There was a reduction of 60 % in the LDA content of coffee barks after fructification. Fungus fructification in the coconut fiber reduced the levels of LDA and CEL in 25 % and 20 %, respectively. There were no significant differences between treatments for CP, ADF, CEL, HEM and ADL contents in the lump of cotton. However, there was significant difference in IVDMD between the enriched fructified substrates treated with L. edodes. An average increase 111 % and 98 % was observed for the fructified fungus added with rice bran or urea, respectively, compared to controls. The biggest increase in IVDMD after fructification in residues treated with P. ostreatus was obtained for the eucalyptus bark (200 %), followed by corn kernels (67 %) and sugar cane bagasse (13 %). The highest cellulase activity in corn kernels residues was obtained when using particles with a diameter of 0.6 mm. The xylanase activity was higher than the activity of cellulase for all treatments and sizes of particles tested. The particle size of 0.6 mm in the inoculated treatment yielded maximum xylanase activity after 48 hours of incubation (118.17 U/mL). The activity of cellulase in sugarcane bagasse was superior for all treatments and particle sizes when compared to corn kernels residues. The highest activity of xylanase (78.89 U/mL) in the fructified sugarcane bagasse was obtained after 48 hours of incubation and particles size of 0.6 mm. The changes in chemical composition and IVDMD showed that L. edodes (UFV 73) and P. ostreatus (PLO 06) could improve the nutritional quality of ruminant rations by increasing the content of CP and IVDMD and reducing the levels of NDF, ADF and ADL of agroindustrial residues, allowing its use in ruminant rations.
O aumento da população mundial demanda cada vez mais alimentos e bens de consumo, com conseqüente aumento na produção de resíduos agroindustriais oriundos de madeireiras, usinas de álcool combustível e indústrias de beneficiamento de produtos agrícolas. Diversos estudos têm demonstrado o potencial de fungos da podridão branca em decompor substratos ligninocelulósicos, mas a utilização desses resíduos na alimentação de ruminantes ainda tem sido pouco explorada. O objetivo deste trabalho foi determinar a composição bromatológica e a digestibilidade in vitro de resíduos agroindustriais (casca de eucalipto, serragem de eucalipto, bagaço de cana-de-açúcar, sabugo de milho, casca de café, fibra de coco e casca de caroço de algodão desengordurado) inoculados com fungos causadores da podridão branca (Pleurotus ostreatus e Lentinula edodes), assim como determinar a atividade enzimática de celulase, xilanase e lacase em três diferentes tamanhos de partículas dos resíduos sabugo de milho e bagaço de cana-de-açúcar incubados com líquido ruminal in vitro, visando avaliar o potencial hidrolítico das comunidades microbianas do rúmen. Quando os resíduos foram tratados com L. edodes o teor de PB do resíduo casca de eucalipto aumentou 91 % e 78 % no tratamento enriquecido com farelo de arroz e uréia, respectivamente. O teor de LDA diminuiu 70 % na serragem de eucalipto frutificada e enriquecida com farelo de arroz comparada ao controle. A FDN do resíduo bagaço de cana diminuiu 5 % quando enriquecido com farelo e 21 % quando enriquecido com uréia. Quando os resíduos foram tratados com P. ostreatus os teores de CEL e LDA da casca de eucalipto diminuíram 22 % e 137 %, respectivamente, após o tratamento. Na serragem de eucalipto as concentrações de FDN e FDA diminuíram 19 % e 27 % no tratamento frutificado, respectivamente. O EE aumentou 402 % no tratamento frutificado no bagaço. No sabugo o conteúdo de cinzas aumentou 130 % no tratamento frutificado em relação ao controle. Houve redução de 60 % no teor de LDA da casca de café após a frutificação. A frutificação fúngica reduziu os teores de LDA e CEL em 25 % e 20 %, respectivamente na fibra de coco. Não houve diferenças significativas entre os tratamentos para os teores de PB, FDA, CEL, HEM e LDA no resíduo casca de caroço de algodão desengordurado. Houve diferença significativa na DIVMS entre os enriquecimentos dos resíduos tratados com L. edodes, tendo sido observado aumento médio de 111 % e 98 % quando frutificado e adicionado de farelo de arroz e uréia, respectivamente, em relação ao controle O maior incremento de DIVMS no tratamento frutificado nos resíduos tratados com P. ostreatus foi obtido para a casca de eucalipto (200 %), sabugo de milho (67 %) e bagaço de cana-de-açúcar (13 %). As maiores atividades de celulase no resíduo sabugo de milho foram obtidas quando se utilizou partículas com 0,6 mm de diâmetro. A atividade de xilanase foi maior quando comparada à atividade de celulase em todos os tratamentos e tamanhos de partículas testados. O tamanho de partícula 0,6 mm no tratamento inoculado foi o que apresentou atividade máxima de xilanase após 48 horas de incubação (118,17 U/mL). A atividade de celulase no resíduo bagaço de cana-de-açúcar foi superior em todos os tratamentos e tamanhos de partículas quando comparado ao resíduo sabugo de milho. O tamanho de partícula 0,6 mm apresentou as maiores concentrações de xilanase no bagaço de cana-de-açúcar, tendo sido observado valor máximo após 48 horas de incubação de 78,89 U/mL no tratamento frutificado. As mudanças na composição química e na DIVMS indicam que L. edodes (UFV 73) e P. ostreatus (PLO 06) melhoram o valor nutricional dos resíduos tratados, aumentando o teor de PB e DIVMS e reduzindo os teores de FDN, FDA e LDA dos resíduos agroindustriais, com possibilidade de utilização na alimentação de ruminantes.

Les actuals activitats humanes han comportat la contaminació del medi ambient, la qual cosa ha esdevingut una amenaça global. En concret, l’ineficient eliminació de productes d’ús diari a les plantes de tractament d’aigües residuals (EDARs) es una font de contaminació en si mateixa; especialment quan els llots no tractats i l’aigua resultant són valoritzats en activitats agrícoles. En el present treball s’han desenvolupat dues tècniques de bioremediació amb Trametes versicolor per tal de tractar diferents llots. A més a més, s’ha proposat i estudiat un post-tractament físic per tal d’incrementar la qualitat de l’efluent final d’una EDAR. En tots dos casos, l’eliminació de fàrmacs en cada corrent ha estat avaluada. En la primera secció, es va estudiar l’ús de Trametes versicolor en biopiles a escala de laboratori per tractar llots d’EDAR secs; tot avaluant la seva capacitat per eliminar fàrmacs i l’evolució de les comunitats microbianes. • Es va escollir un substrat lignocel·lulosic per les biopiles inoculades amb el fong en base a la revisió de dades experimentals publicades i no publicades. • Es va avaluar l’eliminació total de fàrmacs a concentració real en llots de depuradora tractats en biopiles inoculades i no inoculades amb el fong, provant si la re-inoculació de les biopiles milloraria l’eliminació final. • L’estudi de les comunitats bacterianes i fúngiques de les biopiles inoculades i no inoculades amb el fong va mostrar que totes dues comunitats van acabar evolucionant cap a poblacions similars. En la segona secció, es va avaluar el creixement i la capacitat per eliminar fàrmacs de Trametes versicolor en llots provinents d’un reactor biològic de membrana (MBR) en sistemes de bioslurry. • Es va avaluar la capacitat del fong per eliminar compostos en medis líquids dopats, provant diferents medis de cultiu i condicions. • Es va avaluar l’eliminació d’un gran ventall de fàrmacs per part del fong en bioslurries no estèrils i no dopats. Les anàlisis microbiològics van demostrar un increment de la diversitat microbiana després de 15 dies de tractament. • Es va incrementar l’escala dels bioslurries de Erlenmeyer a reactor de 5L i el seu corrent de sortida es va digerir anaeròbicament, per tal de comprovar si el tractament amb el fong podia ser re-valoritzat. • Els resultats finals mostren que Trametes versicolor pot eliminar PPCPs en bioslurry en condicions no esterils. En la tercera secció, es va avaluar l’eliminació de fàrmacs en aigua sintètica mitjançant l’esmena de sòls amb adsorbents de baix cost, per tal de determinar si era posible millorar la qualitat de l’efluent d’una EDAR. • Es va determinar la capacitat d’adsorció del sòl sense esmenar amb experiments en discontinu de 24h per tal de determinar els coeficients de distribució dels fàrmacs seleccionats. • Per tal de determinar quina proporció d’esmena seria la mes adequada, es va estudiar l’adsorció en 24h de 3 compostos amb un coeficient octanol/aigua similar en experiments en discontinu. • Es va mesurar el percentatge d’eliminació de cadascun dels fàrmacs provats per cada esmena de baix cost (biochar i NUA) utilitzada. Els resultats van mostrar alts percentatges d’eliminació per tots els fàrmacs, demostrant l’eficàcia dels adsorbents de baix cost com a esmenes de sòl. Els experiments de les dues primeres seccions es va realitzar al grup d’investigació BioremUAB (Grup de Biodegradació de Contaminants Industrials i Valorització de Residus) del departament d’Enginyeria Química, Biològica i Ambiental de la Universitat Autònoma de Barcelona (UAB). L’objectiu d’aquest grup es el desenvolupament de nous processos biològics pel tractament de contaminants emergents en diferents matrius. Els experiments de l’última secció es van desenvolupar al centre Land & Water del CSIRO. L’objectiu d’aquest centre és el de proporcionar solucions innovadores als complexos desafiaments que sorgeixen de les activitats humanes sobre el medi ambient.
Las actuales actividades humanas han conllevado a la contaminación del medio ambiente, lo que se ha convertido en una amenaza global. En concreto, la ineficiente eliminación de productos de uso diario en las plantas de tratamiento de aguas residuales (EDARs) es una fuente de contaminación en sí misma; especialmente cuando los lodos no tratados y el agua resultante son valorizados en actividades agrícolas. En el presente trabajo se han desarrollado dos técnicas de biorremediación con Trametes versicolor para tratar diferentes lodos. Además, se ha propuesto y estudiado un post-tratamiento físico para incrementar la calidad del efluente final de una EDAR. En ambos casos, se ha evaluado la eliminación de fármacos en cada corriente. En la primera sección, se estudió el uso de Trametes versicolor en biopilas a escala de laboratorio para tratar lodos de una EDAR secos; evaluando su capacidad para eliminar fármacos y la evolución de las comunidades microbianas. • Se escogió un sustrato lignocelulósico para las biopilas inoculadas con el hongo en base a datos experimentales publicados y no publicados. • Se evaluó la eliminación total de fármacos a concentración real en lodos de depuradora tratados en biopilas inoculadas y no inoculadas con el hongo, probando si la re-inoculación de las biopilas mejoraría la eliminación final. • El estudio de las comunidades bacterianas y fúngicas de las biopilas inoculadas y no inoculadas con el hongo mostraron que ambas comunidades acabaron evolucionando hacia poblaciones similares. En la segunda sección, se evaluó el crecimiento y la capacidad para eliminar fármacos por parte de Trametes versicolor en lodos provenientes de un reactor biologico de membrana (MBR) en sistemas de bioslurry. • Se evaluó la capacidad del hongo para eliminar compuestos en medios líquidos dopados, probando diferentes medios de cultivo y condiciones. • Se evaluó la eliminación de un amplio número de fármacos por parte del hongo en bioslurries no estériles y no dopados. Los análisis microbiológicos demostraron un incremento de la diversidad microbiana después de 15 días. • Se incrementó la escala de los bioslurries: de Erlenmeyer a reactor de 5L; y su corriente de salida se digerió anaeróbicamente, a fin de comprobar si el tratamiento con el hongo podía ser re-valorizado. • Los resultados finales muestran que Trametes versicolor puede eliminar fármacos en bioslurry en condiciones no estériles. En la tercera sección, se evaluó la eliminación de fármacos en agua sintética mediante la enmienda de suelos con adsorbentes de bajo coste, con el objetivo de determinar si era posible mejorar la calidad de un efluente de EDAR. • Se determinó la capacidad de adsorción del suelo sin enmendar con experimentos en discontinuo de 24h para determinar los coeficientes de distribución de los fármacos seleccionados. • Para determinar qué proporción de enmienda sería la más adecuada, se estudió la adsorción en 24h en experimentos en discontinuo de 3 compuestos con un coeficiente octanol / agua similar. • Se midió el porcentaje de eliminación de cada uno de los fármacos probados por cada enmienda de bajo coste (biochar y NUA) utilizada. Los resultados indicaron altos porcentajes de eliminación para todos los fármacos, demostrando la eficacia de los adsorbentes de bajo coste como enmiendas. Los experimentos de las dos primeras secciones se realizó en el grupo de investigación BioremUAB (Grupo de Biodegradación de Contaminantes Industriales y Valorización de Residuos) del departamento de Ingeniería Química, Biológica y Ambiental de la Universitat Autònoma de Barcelona (UAB). El objetivo de este grupo es el desarrollo de nuevos procesos biológicos para el tratamiento de contaminantes emergentes en diferentes matrices. Los experimentos de la última sección se desarrollaron en el centro Land & Water del CSIRO. El objetivo de este centro es el de proporcionar soluciones innovadoras a los complejos desafíos que surgen de las actividades humanas sobre el medio ambiente.
Current human activities have led to the pollution of the environment, which has aroused as a global threat. In particular, the inefficient treatment of every-day products in wastewater treatment plants (WWTPs) is an important source of contaminants; especially when untreated sewage sludge and reclaimed water are valorised for agricultural porpoises. The present work describes the development of two bioremediation processes mediated with Trametes versicolor in order to treat different types of sludge. Additionally, one physical post-treatment has been proposed and studied so as to improve the final quality of a WWTP effluent. In both cases, the removal of pharmaceuticals (PhACs) in each stream has been assessed. In the first section, the use of Trametes versicolor in biopiles at lab scale with dried sewage sludge from the WWTP of El Prat de Llobregat was studied, evaluating its capacity to remove Pharmaceutical and Personal Care Products (PPCPs) and assessing the evolution of the biopiles microbial communities. • Appropriate lignocellulosic substrate for fungal biopiles was selected according to the review of published and unpublished experimental data. • Total removal of drugs at real concentrations from sewage sludge was assessed for non-inoculated and fungal inoculated biopiles, testing if the re-inoculation of the biopiles would improve the removal yields. • The study of the bacterial and fungal communities revealed that fungal inoculated and non-inoculated biopiles evolved to similar communities. In the second section, the growth of Trametes versicolor on Membrane Biological Reactor (MBR) sludge in bioslurry systems was assessed, and its capacity to remove PPCPs was evaluated. • The ability of the fungus to remove a spiked compound in liquid medium cultures was assessed, testing different media and conditions for the bioslurry. • In non-spiked Erlenmeyer fungal bioslurries under non-sterile conditions, the removal of a wide set of PhACs was assessed and microbial analysis were performed, showing that microbial diversity increased after 15 days of treatment. • The fungal bioslurry treatment scale was improved to reactor scale and coupled to an anaerobic digestion process. • Results showed that Trametes versicolor can remove PPCPs in bioslurry systems under non-sterile conditions in matrices as complex as an MBR sludge. In the third section, the efficiency removal of PhACs in synthetic water was assessed in soil amended with low-cost sorbents in order to determine if it was possible to improve the final quality of a WWTP effluent. • The capacity of bare soil to adsorb the PhACs was assessed in 24h batch experiments in order to quantify the distribution coefficient of the drugs. • The adsorption of three drugs with similar octanol/water coefficient at different soil:amendment ratios was assessed and carried out in 24h batch experiments, in order to determine which ratio would be the most suitable. • The removal percentage of each pharmaceutical compound was measured in soil amended with two low-cost sorbents (biochar and NUA) during 21 days. The results showed high removal percentages for all the tested PhACs, proving the efficiency of sorbents to remove emerging pollutants from water by amending the soil. The experiments of the two first sections were performed in the research group BioremUAB (Grup de Biodegradació de Contaminants Industrials i Valorització de Residus) from the Department of Chemical, Biological and Environmental Engineering in Universitat Autònoma de Barcelona (UAB). The aim of this group is the development of novel biological techniques to degrade emerging pollutants in different matrices. The experiments of the last section of this thesis were carried out in the centre Land & Water from the CSIRO. The scope of this centre is to deliver innovative solutions to the complex challenges that arise from the demands and impacts of human activities.

La méthanisation de la biomasse lignocellulosique est un des moyens les plus efficients pour la production d’énergie renouvelable. Cependant, la lignine présente dans cette biomasse est difficile à hydrolyser. Cette limite peut être surmontée grâce aux prétraitements. Parmi eux, les prétraitements peu couteux par pourritures blanches sont attrayants mais ils ont été peu appliqués pour la digestion anaérobie. La présente étude explore les prétraitements par pourritures blanches de la paille de blé afin d’en améliorer sa méthanisation. Tout d’abord, une étape de sélection a révélé l’efficacité de la souche Polyporus brumalis BRFM 985 puisque 43% de méthane supplémentaire ont été obtenus par gramme de matières volatiles par comparaison avec la paille témoin. En prenant en compte les pertes de matières occasionnées par le prétraitement, cela correspondait à 21 % d’amélioration par gramme de matière sèche initiale. De plus, il a été montré que l’addition de glucose durant le prétraitement limitait la délignification et donc la production de méthane du substrat. Puis, des échantillons prétraités furent obtenus lors d’un plan d’expérience visant à optimiser le prétraitement par P. brumalis BRFM 985 ; les paramètres du prétraitement testés étaient : la durée et la température de culture, l’humidité initiale du substrat et l’addition de métaux. Les surfaces de réponse de la production de méthane à partir de ces échantillons furent construites. La production optimale de méthane ne fut pas atteinte dans le domaine expérimental testé mais l’impact positif de l’addition de métaux fut démontré, ainsi que l’importance de choisir une durée de culture adaptée. Ensuite, l’usage de la technique de la pyrolyse-GC-MS pour évaluer l’efficacité du prétraitement fut étudié. Une estimation de la quantité de biomasse fongique avec cette méthode apparaît possible. Le ratio polysaccharides/lignine déterminé par py-GC-MS a permis de classer des échantillons prétraités selon leur biodégradabilité anaérobie. La digestion anaérobie en voie sèche (DAVS) de paille de blé prétraitée en réacteur pilote fut menée en batch avec recirculation des lixiviats. Durant le démarrage de la DAVS, un trop fort S/I mène à une accumulation d’acides gras volatils (AGV) et parfois à la défaillance de la DAVS. Néanmoins, de forts S/I permettent de traiter plus de substrat et augmentent la production de méthane par volume de réacteur. Avec la paille de blé, des S/I entre 2 et 3 (en matières volatiles) permettent un bon démarrage de la DAVS. Alors qu’un ratio AGV totaux/alcalinité inférieur à 0,6 correspond à des réacteurs stables en digestion anaérobie voie liquide ; cette limite semble mal adaptée à la DAVS. Il fut observé que la DAVS pouvait récupérer d’une phase d’acidification tant que le ratio AGV totaux/alcalinité était inférieur à 2 et que la concentration en AGV était inférieure à 10 g/L dans les lixiviats. Malgré une amélioration de la biodégradabilité et une phase de démarrage facilitée, le prétraitement fongique non optimisé ne permit pas d’améliorer la production de méthane après prise en compte des pertes de matière occasionnées par le prétraitement
Anaerobic digestion of lignocellulosic biomass is one of the most efficient ways to produce renewable energy. However, lignin contained in this biomass is difficult to hydrolyze. This limitation can be overcome by pretreatments. Among them, low-cost white-rot fungi pretreatments seem attractive but were scarcely applied for anaerobic digestion. The current study investigates white-rot fungi pretreatments of wheat straw to improve its methane production. Firstly, a selection step has revealed the efficiency of Polyporus brumalis BRFM 985 since 43% more methane per gram of pretreated volatile solids were obtained compared to the control straw. Taking into account the dry weight loss occurring during the pretreatment, it still corresponded to 21% more methane per gram of initial total solids. Moreover, glucose addition during the pretreatment was shown to limit delignification and thus methane production from the substrate. Secondly, pretreated samples were obtained in an experiment device aiming to optimize the pretreatment with P. brumalis BRFM 985; tested pretreatments parameters were: culture duration, temperature, initial substrate moisture content and metals addition. Response surfaces of methane production from those samples were built. Optimum methane production was not reached in the experimental domain but the positive impact of metals addition was demonstrated, so as the importance to choose adequate culture duration. Then, the use of pyrolysis-GC-MS technic to access pretreatment efficiency was studied. Estimation of fungal biomass amount on wheat straw with this method appeared possible. Polysaccharides/lignin ratio determined with py-GC-MS allowed to classify some pretreated samples according to their anaerobic degradability. Solid State Anaerobic Digestion (SSAD) of wheat straw pretreated in pilot-reactor was carried out in batch with leachate recycle. During SSAD start-up phase, too high Substrate/Inoculum (S/I) ratio leads to Volatile Fatty Acid (VFA) accumulation and sometimes to reactor failure but with high S/I more substrate can be treated and methane production per reactor volume increases. With wheat straw, S/I between 2 and 3 (Volatile Solid basis) allow a successful start-up in SSAD. Whereas Total VFA/alkalinity ratio under 0.6 corresponds to stable wet anaerobic digestion; this limit seems not well adapted to SSAD. It was observed that SSAD reactors were able to recover from acidification phase when Total VFA/alkalinity was lower than 2 and with VFA concentrations inferior to 10 g/L in leachate. Despite the improvement of biodegradability and the facilitation of start-up phase, non-optimized fungal pretreatment did not improve methane production after taking into account mass losses occurring during the pretreatment

This diploma thesis focuses on the determination of sulfonamide antibiotics especially the possibility of elimination of these substances from the aquatic ecosystem. Nowadays, environmental contamination of the pharmaceuticals and their residues is a serious concern. Main sources of this contamination are wastewater treatment plants (WWTPs), where these compounds are not effectively removed by contemporary conventional technology. For this reason, new methods are being developed and tested that could eliminate the number of contaminants entering the environment in this way. There is a possibility to use the potential of the enzymatic system of wood-decay fungi, especially white rot fungi. Six representatives of sulfonamide antibiotics were selected and isolated from the aquatic matrix via solid phase extraction. The final identification and quantification method was high performance liquid chromatography with mass spectrometric detection. Monitoring of the concentration level of selected sulfonamide antibiotics at the inflow and effluent at the Brno-Modřice WWTP was carried out weekly. Moreover, the effectiveness of elimination of selected antibiotics from the aquatic ecosystem by the use of Trametes versicolor wood-decay fungi cultured on a suitable carrier was verified.

La bioconversion en méthane de biomasses lignocellulosiques est l’une des alternatives les plus prometteuses pour la production de méthane issu de la digestion anaérobie. Toutefois, les biomasses lignocellulosiques présentent des caractéristiques bio-physico-chimiques très variables en raison leur composition biochimique et de l’organisation structurale très diverses. Par ailleurs, leur faible biodégradabilité en conditions anaérobie nécessite de les prétraiter avant méthanisation pour optimiser la production de méthane. Ce travail vise à évaluer l’influence des caractéristiques d’une large gamme de substrats lignocellulosiques sur leur biodégradabilité anaérobie et les corrélations entre leurs caractéristiques bio-physico-chimiques et le potentiel biométhanogène, et d’étudier les effets du prétraitement fongique en présence de Ceriporiopsis subvermispora sur le potentiel biométhanogène de biomasses lignocellulosiques sélectionnées dans la présente étude et de caractériser les changements de leurs caractéristiques après le prétraitement fongique. La caractérisation de 36 biomasses lignocellulosiques représentatives d’une large gamme de gisements potentiellement mobilisables a permis de mettre en évidence les corrélations linéaires entre le potentiel biométhanogène des biomasses et certaines de leur caractéristiques bio-physico-chimiques, dont la teneur en lignine et la demande biochimique en oxygène. Les biomasses sylvicoles et agricoles ont montré des caractéristiques distinctes de la biodégradabilité aérobie et anaérobie. Les résultats de prétraitement fongique sur les 5 biomasses ont permis de mettre en évidence que le champignon de pourriture blanche Ceriporiopsis subvermispora réagit distinctement selon la biomasse prétraitée. Pour certaines biomasses, le prétraitement fongique conduit à augmenter significativement la production de méthane et la vitesse de bioconversion en méthane. Cette espèce présente la capacité de dégrader sélectivement la lignine sur certaines biomasses et, sur d’autres, celle de dégrader de manière non-sélective des polysaccharides et des lignines. De plus, pour les deux souches de Ceriporiopsis subvermispora testées, des métabolismes différents ont été mis en évidence sur une même biomasse. Les résultats de compositions et ceux de l’analyse structurale des biomasses (initiales, autoclavées, contrôles, et prétraitées par Ceriporiopsis subvermispora) ont montré que leur structure peut être modifiée sans toutefois observer une transformation significative de leur composition biochimique
Bioconversion to methane lignocellulosic biomass is one of the most promising alternatives for the production of methane from anaerobic digestion. However, lignocellulosic biomass has various bio-physicochemical characteristics due to their biochemical composition and diverse structural organization. Moreover, their low biodegradability in anaerobic condition requires pretreatment before methanation to optimize methane production. This work aims to evaluate the influence of the characteristics of a wide range of lignocellulosic substrates on their anaerobic biodegradability and correlations between their bio-physical-chemical characteristics and biomethane potential, and study the effects of fungal pretreatment in the presence of Ceriporiopsis subvermispora on the biogas potential of lignocellulosic biomass selected in this study and characterize their changes of their characteristics before and after the fungal pretreatment. The characterization of 36 representative lignocellulosic biomass of a wide range of potentially mobilized deposits allowed to highlight the linear correlations between biomethane potential of biomass and some of their bio-physical-chemical characteristics, of which the lignin content and biochemical oxygen demand. The forest and agricultural biomass exhibited distinct characteristics of the aerobic and anaerobic biodegradability. The results of fungal pretreatment of the 5 biomass indicated that the white rot fungus Ceriporiopsis subvermispora reacts distinctly depending on the pretreated biomass. For some biomass, fungal pretreatment leads to significant increase of methane production and the bioconversion rate of methane. This species presents the ability to selectively degrade lignin on some biomasses, in others, the ability to non-selectively degrade polysaccharides and lignins. In addition, for both strains of Ceriporiopsis subvermispora tested, different metabolisms were highlighted on the same biomass. The results of compositions and those of the structural analysis of biomass (initials, autoclaved, controls, and pretreated with Ceriporiopsis subvermispora) showed that their structure can be modified without observing a significant transformation of their biochemical composition

Wastewater from the textile industry is difficult to treat effectively due to the prevalent use and wide variety of synthetic dyes that are resistant to conventional treatment methods. White-rot fungi, such as Trametes versicolor, have been found to be effective in decolorizing many of these synthetic dyes and current research is focusing on their application to wastewater treatment. Although numerous studies have been conducted on the ability of both living and nonliving Trametes versicolor to separately decolorize textile dyes, no studies were found to have investigated the use of a mixture of live and dead fungus for decolorization. This study explored potential interactions between live and dead, autoclaved Trametes versicolor biomass in a mixed system by utilizing a series of batch tests with two structurally different synthetic textile dyes. Samples were analyzed by spectrophotometer and compared with controls to determine the effect of any interactions on decolorization. The results of this study indicate that an interaction between living and nonliving biomass occurred that affected the specific dye removal for both Reactive Blue 19, an anthraquinone textile dye, and Reactive Orange 16, an azo textile dye. This interaction was seen to improve the specific dye removal during the first 10-46 hours of experimentation but then diminish the specific dye removal after this period. This effect could be due to hydrophobins, which are surface-active proteins excreted by live fungi that may alter hydrophobicity. Additionally, the presence of adsorptive dead biomass could affect dye contact with degrading enzymes released from the live fungus. By expanding current knowledge of the interactions that take place in a fungal bioreactor and their effect on textile dye decolorization, this research aims to inspire more effective and less costly bioreactor designs for the treatment of textile wastewater.
B.S.Env.E.
Bachelors
Engineering and Computer Science
Environmental Engineering

In Waldökosystemen ist Totholz von zentraler Bedeutung, indem es zahlreichen Organismen einen Lebensraum bietet oder als Substrat dient, Bestandteil des Kohlenstoff- und Nährstoffkreislaufs ist sowie als ein wichtiges strukturelles Element fungiert. Für seine Zersetzung ist die Überwindung der Ligninbarriere von besonderer Bedeutung. Dazu sind lediglich saprobionte Pilze aus den Phyla der Basidiomycota und Ascomycota in der Lage, die verschiedene Strategien – die Fäuletypen – entwickelt haben, um Lignin abzubauen oder zu modifizieren und somit Zugang zu den vom Lignin inkrustierten Polysachariden (Zellulose und Hemizellulosen) zu erhalten. Eine besondere Rolle spielen dabei Weißfäulepilze, die mit ihren extrazellulären oxidativen Enzymen, wie Laccasen und verschiedenen Peroxidasen, Lignin komplett bis zum Kohlendioxid (CO2) mineralisieren. Trotz der Bedeutung des Ligninabbaus für die Totholzzersetzung sind extrazelluläre oxidative Enzyme im natürlichen Totholz kaum erforscht. Ziel dieser Arbeit war es, die Rolle der oxidativen Enzyme für die Totholzzersetzung unter Realbedingungen zu verifizieren, ihre räumlichen und zeitlichen Muster zu beschreiben und ihre Abhängigkeiten von verschiedenen Totholzvariablen sowie der pilzlichen Artengemeinschaft in und auf Totholz zu ermitteln. Weiter wurde die Veränderung der Totholzvariablen über den Zersetzungsprozess für unterschiedliche Baumarten vergleichend beschrieben und der Einfluss der Waldbewirtschaftung auf den Prozess untersucht. Dazu wurden 197 natürliche Totholzstämme (coarse woody debris, CWD) von Fagus sylvatica (Rotbuche), Picea abies (Gemeine Fichte) und Pinus sylvestris (Gemeine Kiefer) in unterschiedlich stark bewirtschafteten Wäldern in Deutschland untersucht. Insgesamt wurden 735 Proben genommen und darin die Aktivität von Laccase (Lacc), Genereller Peroxidase (GenP) und Mangan-Peroxidase (MnP) gemessen. Weiterhin wurden Variablen wie Dichte, Wassergehalt, pH-Wert, wasserlösliche Ligninfragmente, die Gehalte an Lignin und Extraktiven sowie an Nährstoffen und Metallen (N, Al, Ca, Cu, K, Mg, Mn und Zn) ermittelt. Die pilzliche Artengemeinschaft wurde anhand genetischer Fingerprints (F-ARISA) und mittels Fruchtkörperkartierung erfasst. In 79 % der untersuchten Totholzproben wurden oxidative Enzymaktivitäten festgestellt. Sie waren hoch variabel über den Zersetzungsverlauf sowie in Bezug auf die Probenahmepositionen innerhalb der einzelnen Stämme. Generell waren die Aktivitäten im F.-sylvatica-Totholz höher als im Koniferentotholz. Lineare und logistische Modelle zeigten, dass die pilzliche Artengemeinschaft, gefollgt von den wasserlöslichen Ligninfragmenten, die wichtigste Einflussgröße hinsichtlich der oxidativen Enzyme war. Ein saurer pH-Wert unterstützte die Funktion von Lacc und MnP; Mangan, Eisen und Kupfer waren in ausreichenden Konzentrationen vorhanden, um die Funktion und Bildung der Enzyme zu gewährleisten. Die holzabbauenden Pilze erwiesen sich als optimal an das niedrige Stickstoffangebot im Totholz angepasst, sodass ein erhöhter Stickstoffeintrag über zwei Jahre die oxidativen Enzymaktivitäten nicht weiter beeinflusste. Der pH-Wert sowie die Gehalte an Lignin, Extraktiven und Nährstoffen waren im Vergleich der drei Baumarten signifikant verschieden, obwohl die zeitlichen Veränderungen der Variablen über den Zersetzungsprozess vergleichbar waren. Die Anzahl operativer taxonomischer Einheiten (OTUs ~ molekulare Artenzahl) nahm im Verlauf der Holzzersetzung zu, während die Zahl fruktifizierender Arten für mittlere Zersetzungsgrade am höchsten war. Beide Artenzahlen nahmen zusammen mit dem Stammvolumen zu. Die Weißfäulepilze dominierten über den gesamten Zersetzungsprozess die fruchtkörperbasierte Artenzahl aller drei Baumarten, was mit dem Vorhandensein oxidativer Enzymaktivitäten einhergeht. Generell nahmen der massebezogene Gehalt des Lignins, der Extraktive und der Nährstoffe über die Zersetzung zu, während der volumenbezogene Gehalt abnahm. Der pH-Wert im Holz aller drei Baumarten sank kontinuierlich im Verlauf der Zersetzung. Eine Erhöhung der Waldbewirtschaftungsintensität hatte einen negativen Effekt auf das Stammvolumen und darüber vermittelt auf die Zahl fruktifizierender Pilzarten, jedoch kaum auf andere untersuchte Totholzvariablen. Aufgrund des häufigen Vorkommens von Weißfäulepilzen, der gleichzeitigen Präsenz oxidativer Enzymaktivitäten und des substanziellen Ligninabbaus kann auf eine fundamentale Bedeutung von Laccasen und Peroxidasen für die Zersetzung des Totholzes geschlossen werden. Nicht zuletzt die charakteristische Molekularmassenverteilung der wasserlöslichen Ligninfragmente deutete darauf hin, dass die Mn-oxidierenden Peroxidasen (MnPs) die dominierenden oxidativen Enzyme des Ligninabbaus sind. Das hoch variable Muster der oxidativen Enzymaktivitäten ist jedoch das Resultat eines komplexen Zusammenspiels der Holzeigenschaften und der pilzlichen Artengemeinschaft. Die dabei bestehenden funktionellen Abhängigkeiten müssen weiter im Detail in zukünftigen Studien analysiert und aufgeklärt werden
In forest ecosystems, deadwood is an important component that provides habitat and substrate for numerous organisms, contributes to the carbon and nutrient cycle as well as serves as a structural element. Overcoming the lignin barrier is a key process in deadwood degradation. Only specialized saprotrophic fungi of the phyla Basidiomycota and Ascomycota developed different strategies – the rot types – to degrade lignin or to modify it in way, which allows them to get access to the polysaccharides (cellulose and hemicelluloses) that are incrusted within the lignocellulosic complex. In this context, basidiomycetous white rot fungi secreting oxidative enzymes (especially laccases and peroxidases) are of particular importance, since they are the only organisms that are able to substantially mineralize lignin to carbon dioxide (CO2). Although lignin degradation is such an important process for deadwood degradation, oxidative enzyme activities have been only poorly studied under natural conditions in deadwood. The aim of this work was to verify the importance of oxidative enzymes for deadwood degradation in the field, to describe their temporal and spatial patterns of occurrence and to identify dependencies from deadwood variables as well as from the fungal community within and on deadwood. Furthermore, the changes of different deadwood variables were studied over the whole period of degradation and compared among three tree species. Last but not least, the influence of forest management intensity on the process of deadwood degradation was evaluated. Therefor, 197 logs of naturally occurring deadwood (coarse woody debris, CWD) of Fagus sylvatica (European beech), Picea abies (Norway spruce) and Pinus sylvestris (Scots pine) were monitored and sampled in forests with different management regimes across three regions in Germany. A total of 735 samples were taken from the logs and analyzed regarding activities of laccase (Lacc), general peroxidase (GenP) and manganese peroxidase (MnP). Wood density, water content, content of lignin and extractives as well as of nutrients and metals (N, Al, Ca, Cu, K, Mg, Mn und Zn) were determined in the samples, too. The fungal community was assessed based on sporocarps (fruiting bodies) and molecular fingerprints (F-ARISA). Oxidative enzyme activities were present in 79 % of all samples. The activities were found to be highly variable both regarding the time course of degradation and their distribution within the logs. Activities were generally higher in wood samples of F. sylvatica than in samples of conifers. Linear and logistic models revealed that the fungal community structure was the most important determinant for oxidative enzyme activities in the samples, followed by the amount of water-soluble lignin fragments. Moreover, the prevalent acidic pH determined in deadwood was suitable to facilitate the function of laccase and peroxidases. Concentrations of metals (manganese, copper, iron) were sufficient to ensure synthesis and functioning of the enzymes. Deadwood-dwelling fungi turned out to be well adapted to low nitrogen concentrations and thus, an elevated nitrogen deposition over a period of two years did not affect the oxidative enzyme activities. The pH as well as the content of lignin, extractives and nutrients significantly differed among the tree species; however, their trend over the course of degradation was rather similar. Molecular species richness (determined by F-ARISA as OTUs) increased over the whole course of degradation, while the number of fruiting species was highest in the intermediate stage of degradation. Both types of species richness increased with increasing volume of the CWD logs. Over the entire degradation period, white rot fungi – based on the identification of sporocarps – were the most abundant group of wood rot fungi in and on all three tree species. This corresponds well with the overall presence of oxidative enzyme activities. During degradation, the mass-related content of lignin, extractives and nutrients frequently increased, although the volume-related content decreased. The pH of all three tree species decreased in deadwood over the whole period of degradation. Higher forest management intensity had a negative effect on the log volume of deadwood and in consequence on fungal species richness (fruiting bodies), but hardly to other analyzed variables. Based on the widespread occurrence of white rot fungi, the concomitant presence of oxidative enzyme activities as well as the substantial loss of lignin, it can be concluded that laccases and peroxidases are highly relevant for deadwood decomposition. Not least, the detected characteristic molecular size distribution of water-soluble lignin fragments points to a key role of Mn oxidizing peroxidases (MnPs) in enzymatic lignin degradation. The variable patterns of oxidative enzymes observed in wood samples is therefore the result of a complex array of wood variables and the fungal community structure, which will have to be resolved in more detail in future studies

博士
國立臺灣大學
森林學系
81
The major results of the studies are described as follow: (1) Traditional methods of growth rate, weight loss evaluation, Klason lignin analysis and on - media chemical discolored re- action had their shortages in screening suitable fungi for bio- bleaching. Combined spot test method and colorimetry method of- fered good and fast screening for the purpose of bio-bleaching. (2) The flow chart of the reagents and reactions in spot test method forbio-bleaching fungi screening with slices of fungal cultured wood-MEA were as below: a.TMPD 0∼5 min Light pinky purple → purple or blue b.Syringaldazine 20∼30 min Light yellowish green → pink c.Guaiacol 20 min Colorless → reddish brown Pyrogallol 10 min Slight yellow → orange brown d.GA 1 hr Light tea brown → deep brown Guaic 5 min Colorless → guaicum blue Phenol (30 min)∼1 h Colorless → Brown or purple e.Guaic + MEA 4 days Yellowish brown→deep blue→decolored Fugus for biobleaching (3) The obove screened fungi gave higher whiteness in colorimetry method. Different woody materials with different specific fungi while Coriolus versicolor (L.:Fr.) Quel. gave the highest white- ness in all three woody species owing to its highly degraded ab- ility on most components of wood. With suitable culture condit- ions and controlled period, this fungus had potential for apply- ing to non-species specific bio- pulping in subtropical Taiwan. (4) A reverse study in this research by spot testing on commerc- ial enzymes which related to lignin modification or degradation gave the results that traditional used Bavendam method was not suitable for screening and the fungal enzyme system in woody ma- terial was multiple - function.

碩士
東吳大學
微生物學系
83
The capacities of various white rot fungi to decolorize serveral azo dyes, mainly Orange G, were studied in the pre- sent study. Among 15 different isolates, isolate 7 showed the best decolorization ability, which could remove 93% of the added dye in 2 hours. When isolate 7 was precultured in media buffered differently(i.e., potassium phosphate、sodium acetate and DMS buffered medium), the highest dry weight was obtained in potassium phosphate buffered medium. But, we failed to de- tect any significant of its decolorization activity. On the other hand, different medium compositions do show the effect on the decolorization ability of various white rot fungi. But we did not observe positive correlation between decolorization ability and the amount of fungi biomass been produce. When studying the effect of pH, the best decolorization activity was observed between 5 and 6. The effect of medium composition on the production of Laccase, Lignin per-oxidsae, Manganese dependent peroxidase, and Glyoxal oxidase was also studied. Regardless, whether it is in the filtrate or on the hyphae,the highest enzymatic activities were always detected when 1/2 PDB was used as the cultural medium. Unfortunatelly, I was unable to detect a correlation between the decolorization ability of isolate 7 and the production of the above enzymes.

碩士
中華科技大學
健康科技研究所在職專班
106
Dyes released by the textile industries pose threat to environment. In this study, the white-rot fungus has been evaluated for the removal of textile dyes containing methyl orange and methyl red. A full factorial central composite design was employed for experimental design and optimization of results. The effect of concentrations of azo dye, potato dextrose broth, temperature and pH on dye removal was evaluated. Response surface methodology (RSM) was applied to optimize the decolorization of the methyl orange and methyl red dyes. The removal of methyl orange and methyl red dyes increased with the temperature and pH. Results show that the optimum temperature and pH for decolorization process were found to be 30℃ and pH 7.0, respectively.

碩士
國立臺灣大學
森林學研究所
91
Wastewater treatment by immobilization of microorganisms received more attention than suspended system. Among variety of immobilization techniques, entrapment shows great promise for its simplicity and stability. Due to simpler immobilization procedure, high solubility, excellent diffusivity, good growth and good long-term biodegradability, natural polymers, such as alginate and carrageenan, are superior to synthetic polymers for microbial entrapment bedding purpose. Capability to degrade recalcitrant lignin-related pollutant by white rot fungi is well studied, however, inferior mycelial growth pattern under stirred suspended system significantly reduce their applicability for industrial scale wastewater treatment. Hence, calcium alginate and ployureathane foam were used as bedding materials to entrap two white rot fungus: Phanerochaete chrysosporium ATCC 36319 and Trametes versicolor ATCC 37501, and bleaching effluent, from mixture of chlorination and alkali extraction, was treated in this study. For treating effluents, optimal conditions using Phanerochaete chrysosporium were pH 4.0-5.5, 37℃, glucose or starch, 5g/L carbon concentration, with 0.1g/L nitrogen. Optimal conditions using Trametes versicolor were pH 4.5-6.5, 30℃, glucose or starch, 10 g/L carbon concentration, where nitrogen content was unrelated. Immobilized system were able to decolourize for longer period, although they exhibit lower optimal rate than suspended ones. In conclusion, the feasibility to treat bleaching effluent by white rot fungus entrapment is demonstrated.

碩士
中華科技大學
健康科技研究所
104
The purpose of this experiment is how to take advantage of Phanerochaete chrysosporium BCRC 36201 white rot fungi to deal with an aqueous solution of heavy metal, because Taiwan area is small, so little arable land can be used, if the land was subjected to heavy metal pollution and water irrigation, has accumulated in the soil, the residues of heavy metals in the soil, resulting in reduced the area of cultivated land; therefore this experiment, the main is to assess whether P. chrysosporium can handle heavy metals, so the experiment began use this strain to treatment solution containing heavy metals, the results are bad, only in containing copper ion and lead ion solution has high survival rate, therefore, for subsequent experiments the improved growth medium, and ultimately to SDB as basic medium, carbon source respectively sawdust and starch, and adding calcium sulfate 0.5 g and calcium carbon 1 g in the medium was the best, tolerance test of white rot fungi. The reason is that white rot fungus inoculation on heavy metal in liquid culture, growth rate of all bad; if in the general culture medium cultured for 5-7 days later, then add heavy metal solution, not only can improve the survival rate of white rot fungi directly, but also increase the white rot fungi on heavy metal liquid metabolism rate. The experiment of adding calcium in the test for heavy metal metabolism influence, calcium ion in the liquid culture that can promote the degradation of white rot fungus of heavy metals, especially for copper ion and lead ion, copper ion concentration in 10 ppm degradation to 1 ppm and 20 ppm concentration in the lead ion degradation to 1.5 ppm, respectively. Discussion on degradation ability of zinc, brass. Mercury and lead, all have good results, only the zinc ion exception, but in the zinc and copper ions in the mixed solution, zinc ions can be degradation by white rot fungi; also it is worth exploring the value of DNS, in the white rot fungus liquid mixed into the liquid metal, DNS the value will be greatly decreased, presumably because of white rot fungi in order to resist the foreign body, and reducing sugar bacteria in liquid consumed, produce enzymes required to resist adversity. Experiment that starch medium than in sawdust medium, can provide more nutrients, prompting the white rot fungi to produce catalase, may be because in adversity, the white rot fungi can secrete more peroxide dismutase to help themselves to resist adversity and Sawdust Medium although superoxide enzyme activity was lower, but the degradation rate better and more shock in acid pH value, and a stable and continuous supply of nutrients.

碩士
東吳大學
微生物學系
93
White rot fungi produce three main extracellular enzymes involved in ligninolysis, including laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP). These enzymes are capable of initiating the oxidation of lignin by a free-radical mechanism which also led to the degradation of a wide variety of normally very recalcitrant environmental pollutants. Laccase is a glycosylated polyphenol oxidase which contains four copper ions per molecule and has shown good potential to be used in bioremediation, biopulping, biobleaching, medicine, and textile industry. Forty-five isolates of white rot fungi were screened for their ability to degrade congo red. Based on the decoloration ability, twelve isolates were selected for further study. They were grown in PDB and phosphate buffer culture medium (PBCM) for laccase activity assay. Five isolates were chosen for their high level of laccase production (＞10000 U/L). In order to find out the best nitrogen source for laccase production, we have tested 7 different nitrogen sources in PBCM. With yeast extract and NH4Cl as nitrogen source, isolate TFRI 707 and A 8 have shown the highest laccase production, respectively. When study the effect of temperature on their growth, the data indicated that TFRI 707 and A 8 had higher growth rats on 40 ℃ and 30 ℃respectively. Various factors, including pH (3.5-5.5), metal ions (Mg, Mn, Ca, Cu, Fe, Zn) and inducer (ethanol, veratryl alcohol, ferulic acid, 2,5-xylidine) on laccase production were also studied. The optimal pH for laccase production was 5.5 for both isolates. When Cu supplement was deleted from the medium, decreases in laccase production were observed for both of them. As for the inducers, only veratryl alcohol had a substantial effect on laccase production. With ABTS as the substrate, laccase has an optimum activity at pH 3, and an optimum temperature at 30 ℃. In liquid and semi-solid medium, isolate A8 produce 3 and 4 isoenzymes of laccase, respectively, while 4 and 3 isoenzymes were detected for isolate TFRI 707. Molecular weight was determined using Native PAGE, and laccases secreted by isolate A 8 were between 50-90 kDa, and those excreted by TFRI 707 were between 40-50 kDa. When assaying with SDS-PAGD, the molecular weight of laccases secreted by A 8 were between 50-60 kDa and by TFRI 707 were between 30-40 kDa.

博士
國立臺灣大學
森林學研究所
91
Kraft pulps prepared from softwood Pinus taiwanensis and hardwood Eucalyptus grandis, milled wood lignin and lignin-carbohydrate complex were prepared according to Björkman 1956 Standard Method in laboratory. The basic physical and chemical properties of these materials were analyzed. Nine common white-rot fungi in Taiwan were selected through screening of biobleaching activity. After examining of physiological properties and enzyme activity such as lignin peroxidase in vitro, Phanerochaete chrysosporium, Trametes versicolor and Lenzites betulina showed high selection index. It showed that there three white-rot fungi were capable to degradate lignin. Lenzites betulina could grow between pH 3 to 10 with optimum value between pH 8 and 10. Lenzites betulina had fast growing rate, it could grow between 15 to 45℃, the optimum temperatures ranged from 30 to 45℃, it was suitable for biobleaching study. Unbleached kraft pulp of softwood and hardwood directly treated with white-rot fungi in a static state. Comparing the differences from chemical and physical properties of kraft pulps before and after biobleaching, the kappa number of kraft pulp decreased, the brightness increased and the viscosity decreased slightly. Pulps after fungi pretreatment was extracted with 0.5N NaOH, comparing with the controls, pulps with different alkalinity, the soluble lignin of alkali extraction increased. i.e. The bleachability of Pulps after white-rot fungi pretreatment increased significantly. Pulps were treated with mannase （2 IU/g）or/and xylanase （2-4 IU/g）. After a single hemicellulase sequence, the kappa number of pulps decreased slightly, the brightness increased and the viscosity could be improved. That was because of the mannase or/and xylanase could remove the residual hemicellulose on the pulps and preserve abundant high molecular weight cellulose fragment. After the alkali extraction of kraft pulps pretreated with xylanase,there were no absorbance spectra in the visible range 400-600nm. Comparing with the controls, xylanase—released materials absorbed strongly at 237nm and 280nm. This indicated that xylanase improved the removal of residual lignin and release of chromophores.The UV/VIS spectra of released materials were different between xylanase treatment and alkali-extraction. Alkali- extraction released materials absorbed at 205nm more strongly than at 237nm.Xylanase could enhance the removal of residual lignin after alikali extraction and release of chromophores. Milled wood lignin and lignin-carbohydrate complex were isolated to (Netural) C-I-M, (Lignin-rich) C-I-R and (Acidic) C-I-A fragments by DEAE-Sephadex A-50 at room temperature. Each fragment was treated with white-rot fungi. The molecular weight of isolated lignin shifted significantly by Gel Permeation Chromatography during the process of biobleaching. Lignin-carbohydrate complex of Eucalyptus grandis showed polymerization phenomenon as shown by GPC spectrum after P. chrysosporium treatment. Fungal immobilization of white-rot fungus Trametes versicolor grew on polyurethane foam after 4 days under 25℃ and 150 rpm condition. Kraft pulp ( 4 g o.d., consistency 2.0 %w/v) contained 30 immobilized polyurethane foam cubes could increase some brightness and reduce kappa number after 14 days. After cell immobilization by calcium alginate-entrapment , hyperplasia was observed on the surface and inside of polyurethane foam and calcium alginate as shown by SEM. Comparing immobilization with biobleaching, the immobilization of white-rot fungus would get mycelium free pulp. The yield of pulp was high by cell immobilization by calcium alginate-entrapment. Characteristics of immobilized pulp are similar to those biobleached ones. Immobilization of fungus on polyurethane foam allows the repeated use after storage at 4℃ for 7 days in a growth chamber.

碩士
明道大學
光電暨能源工程學系碩士班
101
The purpose of this study was to investigate the nutrient value of rice straw solid-state fermented by white rot fungi, and the feasibility of solid-state fermentation of rice straw (SFRS) in laying hen diet. Firstly, the solid-state fermentation condition was established by collecting rice straw samples from SFRS at 0, 1, 3, 5, 7, 9 day during the fermentation period to analyze the content of functioning components, In Vitro cellular immunity and DPPH scavenging capacity. The results showed that the regarding enzyme activity, rice straw 7-day fermented with white-rot fungi produce multiple hydrolases. α-Amylase activity, casein activity, and Cellulase activity at 1.45 U/ ml, 11.92 U/ ml and 110.39 U/ ml, respectively. The antioxidative activity aspect, flavonoids (1.92 μg/g), phenolic compousterol (3.45 μg/g), adenosine (18.79 μg/g), ergosterol (8.19 μg/g), polysaccharide (0.54 mg/g), triterpenoids (0.85 mg/g), the release of NO (4.39 μ mol/ ml) and MTT cell proliferation (175%) content was higher in 7-day. Therefore, the SFRS fermented by whit rot fungi for 7-day could be used as dietary supplementation. Sixty 22-week old Hendrix hens were used and randomly allocated into five groups: control (2% corn grits), negative control (2% rice straw powder), and three experimental groups (0.5%, 1.0%, and 2.0% SFRS) for 16-week feeding experiment. Egg production was recorded daily and eggs were collected to measure egg quality during the experimental period. Blood sample was taken for serum antioxidant activity measurement at every 4-week interval. The results showed that the dietary supplementation of SFRS not significantly affected in yolk weight, egg production, haugh unit, strength of eggshell and yolk index. However, feed conversion ratio in different Concentration SFRS was significantly higher than control; SFRS supplementation (0.5%, 1.0%, and 2.0% SFRS) significantly decreased egg yolk cholesterol content compared with control; superoxide dismutase in 2.0% SFRS was significantly higher than control. In conclusion, the rice straw inoculated with white rot fungi could be used as feed additive in laying hen diet, and it might has potentials in improving egg quality and decreased the egg cholesterol of laying hens.

碩士
東吳大學
微生物學系
103
Emerging contaminants are either new or unrecognized chemical that pose risks on human health and ecological environment. It can be divided into four groups: "Pharmaceuticals and personal care products, PPCPs", "Endocrine disrupting chemicals, EDCs" "industrial chemicals "and “Biosynthesis chemicals". Tetracycline is one of the emerging contaminants and belongs to PPCPs. Tetracycline is widely used in the treatment of humans and animals. General sewage system cannot effectively remove tetracycline; also, tetracycline can be detected in most surface water. Traditionally, bacteria are used to break down pollutants, but there is a risk that it might create super bacterial resist with antibiotics. White rot fungi can break down most of the lignin and polycyclic compounds by lignin-degrading enzyme. Lignin-degrading enzyme has been studied for many years. It is widely used in the degradation of compounds which are difficult to degrade. In this study, we used Perenniporia tephropora TFRI707 and laboratory isolates Cerrena unicolor A8 to produce laccase and manganese peroxidase degrade tetracycline. Isolate A8 and TFRI707 could produce highest concentration of enzyme in the 1mM Mn2+ GPCSL medium. Isolate A8 and TFRI707 could degrade 100ppm tetracycline in the GPCSL and GPL, and their degradation rates in these two different mediums were the same. After high-temperature sterilization, Isolate A8 and TFRI707 could not degrade tetracycline in the GPL. This result confirmed that their mycelium do not adsorb tetracycline. After washing mycelium of Isolate A8 and TFRI707 by sterile water, their mycelium was put into tetracycline and executed degradation. The results showed that the degradation rates of both mycelium were slower without medium. Enzymes extracted from the culture medium of GPL and GPCSL had poor degradation ability and tetracycline still could inhibit growth of E.coli 11848. Using manganese peroxidase standard (SIGMA) and laccase standard (SIGMA) to degrade tetracycline, we found that manganese peroxidase could degrade tetracycline in sodium tartrate buffer, and laccase could degrade in 1-hydroxybenzotriazole (HBT) buffer in 1 hour. According to the results above, C. unicolor A8 and P. tephropora TFRI707 could degrade tetracycline, and their mycelium do not absorb tetracycline. Enzymes from mediums, laccase and manganese peroxidase also could degrade tetracycline when appropriate medium and correct mediator is involved. That is, it could be more effective to degrade tetracycline.

碩士
國立中興大學
動物科學系所
103
White rot fungi is a group of filamentous fungi able to degrade lignocellulosic biomass. Besides the secretion of cellulose enzyme groups, it contains rich phenolic compounds and other secondary metabolites. These ingredients can protect cells from the damage of free radicals, and improve antioxidant effects. The purpose of this study was to investigate the effects of solid fermented wheat bran by white rot fungi on growth performance, antioxidant ability and the gene expression in broilers. The white rot fungi was used in this study is Pleurotus eryngii. First, the wheat bran was used as a substrate and inoculated with Pleurotus eryngii particles at a ratio of 9:1 in order to conduct solid-state fermentation for 12 day, with 50% moisture at 30℃. Fermented wheat bran contained 4.68 mg gallic acid equivalent/g dry weight (DW), 4.95 mg quercetin equivalent/g DW of total phenolics, 73.4 μg Glucose/g DW of polysaccharide, and the fermented wheat bran was twice higher than the unfermented bran (P<0.001). In addition, the scavenging action of fermented wheat bran was 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical increased significantly by about 1.5 times (P<0.05). In vitro, and under the stimulus of fermented wheat bran, the antioxidant gene expression (HO-1, GST) of chicken peripheral blood mononuclear cells was significantly higher than PBS, ascorbic acid and unfermented wheat bran, by more than 2 times (P<0.05). For genes of NOX1 and ROMO1, the expression of fermented wheat bran was the lowest. The chicken PBMC have better cell alive rate on the MTT lymphocyte proliferation assay. In animal trial, 300 day-old broilers were allocated into three treatments including the basal diet, 10% of wheat bran replaced with corn (WB group), and 10% of fermented wheat bran replaced with corn (FWB group). The results showed that all groups had no significant effect on the growth performance of broilers. The malondialdehyde (MDA) production of the serum in the FWB group was significantly decreased than that of the control group at the age of 21 d and 35 d (P<0.05). The antioxidant gene expression of chicken peripheral blood mononuclear cells was significantly higher than control group. The NO production in FWB was higher than other treatments (P<0.05). In conclusion, fermented wheat bran by white rot fungi could regulate the expression of antioxidant molecular targets, in addition improve their immune function.

O desenvolvimento deste trabalho teve como objectivo a aumentar o conhecimento do potencial lenhinocelulolítico de extractos enzimáticos obtidos através de fermentação sólida de quatro fungos da podridão branca (Trametes versicolor, Bjerkandera adusta, Ganoderma applanatum e Phlebia rufa) utilizando a palha de trigo como substrato durante 28 dias. As actividades enzimáticas da manganês-peroxidase (MnP), lenhina peroxidase (LiP), lacase, carboximetilcelulase (CMCase), avicelase, xilanase e feruloíl esterase foram analisadas com intervalos de 7 dias. A composição química da parede celular da palha de trigo, em cada um dos intervalos de tempo previamente mencionados, foi analisada medindo a concentração de lenhina e de ácidos hidroxicinâmicos esterificados. Os resultados obtidos mostraram que a actividade da MnP era predominante, relativamente às restantes actividades lenhinocelulolíticas Do mesmo modo, a actividade enzimática da xilanase era superior à das outras hidrolases. Todos os fungos produziram a enzima feruloíl esterase, enquanto que a LiP só foi detectada no extracto que continha B. adusta. Como não foram observadas diferenças significativas (P> 0.05) relativamente ao teor de lenhina da palha de trigo após a fermentação, a actividade da LiP não deverá ser considerada como um factor limitante do processo enzimático envolvido na ruptura das unidades constituintes da lenhina. Relativamente aos ácidos hidroxicinâmicos esterificados, especificamente na palha de trigo fermentada com os fungos T. versicolor e P. rufa, o ácido ferúlico e o ácido p-cumárico apresentaram valores de degradação mais elevados especialmente durante os primeiros sete dias de incubação. Foi possível identificar um mecanismo sinergético de degradação dos diferentes constituintes da parede celular em que a hidrólise dos ácidos hidroxicinâmicos e de parte das hemiceluloses poderá ser determinante para a degradação posterior da molécula de lenhina. Os níveis de degradação dos ácidos hidroxicinâmicos esterificados foram mais elevados quando comparados com outros estudos com material lenhinocelulósico. Os dados indicam que o fungo P. rufa, poderá revelar-se bastante promissor, visto que apresentou valores elevados na degradação do ácido ferúlico e p-cumárico nos tempos iniciais da incubação.
This work looks for a better understanding of the lignocellulolitic potential of crude enzyme extracts obtained from solid-state cultivation of four white-rot fungi (Trametes versicolor, Bjerkandera adusta, Ganoderma applanatum and Phlebia rufa) using the lignocellulosic substrate wheat straw. At different fermentation times, enzyme activities such as manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), laccase, carboxymethylcellulase (CMCase), avicelase, xylanase and feruloyl esterase were screened and the content of lignin as well as hydroxycinnamic acids in fermented straw were determined. In general, our results showed that MnP predominate among ligninolytic activities and xylanase among polysaccharide hydrolase activities. All fungi secreted feruloyl esterase while LiP was only detected in crude extracts from B. adusta. Since no significant differences (P>0.05) were observed in remaining lignin content of fermented straw, LiP activity was not a limiting factor of enzymatic lignin removal process. Among hydroxycinnamic acids, ferulic acid content of T. versicolor and P. rufa fermented straw exhibited significant lower titers. The levels of esterified hydroxycinnamic acids degradation were considerably higher than previous reports with lignocellulosic biomass. The data show that Phlebia rufa, may be considered for more specific studies as higher ferulic and p-coumaric acids degradation was observed for earlier incubation times.

碩士
東吳大學
微生物學系
94
White-rot fungi is the most common degrader in lignin degradation. They produce three major extra cellular lignolytic enzymes-laccase, lignin peroxidase and manganese peroxidase. Manganese peroxidase (MnP) is a heme containing glycoprotein which has a high redox potential, could be applied in oxidizing and degrading many recalcitrant pollutants such as PAHs (polycyclic aromatic hydrocarbons), azo dyes. There are eight isolates were screened for the production of MnP. TFRI 20, TFRI 554 and TFRU 691 cultivated in low nitrogen phosphate (1.2 mM) buffer medium has the highest MnP activities (13631 U/L, 21931 U/L, 37619 U/L, respectively). In the later experiment different nitrogen sources were applied in cultivation with TFRI 554 and TFRI 691. The MnP activity of TFRI 554 was suppressed by ammonium chloride and potassium nitrate; TFRI 691 under ammonium tartrate and potassium nitrate exhibited high amount of MnP activity (around 6,0000 U/L), but the activity was also reduced slightly while cultivating under ammonium chloride. In the analysis of MnP activities, different pH of analysis buffer were applied, the MnP of TFRI 554 and TFRI 691 both exhibited highest activity under pH 6, but the MnP activities decreased as the pH of analysis buffer decreased. When study the effect of temperature on MnP stability, MnP activity of TFRI 691 raised slightly after one hour treatment under 40℃ even 45℃. MnP of both TFRI 554 and TFRI 691 did not exhibit the stability to high temperature. Whether the addition of milled rice straw (0.5% W/V) or veratryl alcohol (1, 2 mM), these inducers could be suitable for inducing higher MnP production of TFRI 554 and TFRI 691, but an excess addition of veratryl alcohol (4 mM) could conversely inhibit the MnP production. In the semi-solid state study, both TFRI 554 and TFRI 691 produce high amounts of MnP (74700 U/L; 45553 U/L, respectively), even higher than which in liquid cultivation. MnP activity of TFRI 554 maintained stable amount in air pumping experiment until the eighteenth day of cultivation, much longer than that of non-air pumping cultivation. According the SDS-PAGE analysis, the MnP zymogram of TFRI 554 and TFRI 691 are different. There were three bands of MnP found in TFRI 554 (65 kDa, 40 kDa, 35 kDa respectively); and there were three bands found in TFRI 691 (40 kDa, 38 kDa, 35 kDa respectively).

碩士
國立雲林科技大學
環境與安全衛生工程系
104
The objective of this research is to develop an air biocathode microbial fuel cell (AB-MFC), with cultures of laccase-producing white rot fungi-Ganoderma lucidum (BCRC 36123) planted at the cathode. The developed AB-MFC was then applied to investigate the enhancement of the decomposition of azo dyes in wastewater as well as its power generation. The motivation of this study was that the azo dye acid orange 7 (AO7) in the wastewater contained within the anode chamber of the AB-MFC could diffuse to the cathode through a polyvinyl alcohol hydrogel (PVA-H), serving as a source of carbon for the Ganoderma lucidum and sustain its growth. The Ganoderma lucidum would continuously generate laccase which would serve as a catalyst for the oxygen reaction at the cathode, further improving voltage output of the MFC system. PVA-H also provided the additional functions of water retention and moisture absorption, helping to moisturize the cathode and facilitate growth of the Ganoderma lucidum as well as to receive protons generated through biodegradation of AO7 at the anode. Results of the study showed: (1) Ganoderma lucidum could utilize AO7 as a source of carbon to release laccase. AO7 concentration being 50 mg/L could be decolorized by 77% in 19 days, and the laccase activity reached a maximum of 20.3 ± 0.2 U/L. (2) The system of planting Ganoderma lucidum at the cathode to release active laccase by repeatedly adding 180 mg/L AO7 as the sole source of carbon was capable of achieving a maximum open-circuit voltage of 821 mV, maximum closed-circuit voltage of 394 mV (external resistance of 1000 Ω), maximum power density of 13.38 mW/m2, maximum current density of 33 mA/m2, and highest decolorization of 82%, all of which were superior to the air cathode system or the white-rot fungi system with inactive laccase. (3) Changes to voltage potential at the cathode of the AB-MFC system monitored using cyclic voltammetry scanning by a potentiostat revealed that planting laccase-releasing Ganoderma lucidum at the cathode allowed the anode to receive electrons generated through microbial decomposition. These electrons could then be transmitted to the cathode through external circuitry. At the cathode, these electrons could either use AO7 or guaiacol as the mediator and be delivered by the anode to the cathode to the active site of the laccase and finally reacted with oxygen. Therefore, laccase could serve as a catalyst for oxygen and accelerate the oxygen transport from atmosphere to cathode. Overall research outcomes of this AB-MFC showed that growing Ganoderma lucidum on the air cathode surface could generate laccase that catalyzes the cathode oxygen reaction of the MFC. Ganoderma lucidum could also utilize AO7 brought from anode as a carbon source for hyphal growth. The growing hyphae would generate more laccase, helping to further improve the overall voltage output and contaminant removal capabilities of the entire MFC system.