Bibliography: pages 147-169. / Butyrivibrio fibrisolvens H17c is a gram-negative obligate anaerobic bacterium found in the rumen of most ruminants. The aim of this thesis was to investigate the enzymes produced by B. fibrisolvens H17c involved in the degradation of cellulose, xylan, and protein. A library of chromosomal DNA fragments from B. fibrisolvens H17c was established in the plasmid pEcoR251, an Escherichia coli positive selection vector. The library was screened for genes expressing cellulase, xylanase, and protease activity. Two genes expressing endo-β-1,4-glucanase and cellodextrinase activity were cloned in E. coli as host. The gene expressing endo-β-1,4-glucanase activity (end1) was cloned on a recombinant plasmid pES400. The end1 gene was located on a 6.8 kb DNA fragment and expressed from its own promoter in the E. coli host. It was shown that 64% of the endoglucanase activity was located in the periplasm of the E. coli host. TnphoA mutagenesis indicated the presence of a functional E. coli-like signal peptide. The nucleotide sequence of end1 was determined and the amino acid sequence (547 amino acids) deduced. The catalytic domain of End1 showed very good similarity to the catalytic domain of the Clostridium thermoceiium EGE endoglucanase. End1 also has a non-catalytic domain similar to the binding domains of the CenA and Cex cellulases from Ceilulomonas fimi The gene expressing cellodextrinase activity (ced1) was cloned on a recombinant plasmid pES500. This gene was located on a 3.55 kb fragment and was also expressed from its own promoter in the E. coli host. The Ced1 enzyme was also exported to the periplasm of the E. coli host, but did not contain a functional E. coli-like signal peptide. The nucleotide sequence was determined and the deduced amino acid sequence (547 residues) showed high similarity to the catalytic domain of the C. thermocellum EGD endoglucanase. The proteins of End1 and Ced1 showed no similarity. The End1 and Ced1 enzymes were characterized using a range of different substrates. The End1 enzyme showed optimal activity at pH 5.6 and 45°C. Optimal activity for the Ced1 enzyme was obtained at pH 6.6 and 50°C. The proteolytic activity of B. fibrisolvens H17c was characterized using gelatin-SD5-PAGE. Ten bands of protease activity with apparent molecular weights ranging between 42 000 and 101 000 were detected at different stages during the growth cycle. The effect of protease inhibitors indicated that all ten protease bands were serine proteases. Optimal activity was observed between pH 6.0 to 7.5 and at a temperature of 50°C. The proteolytic activity of B. fibrisolvens H17c varied depending on the type of carbohydrate substrate in the medium, and was positively correlated with the growth rate.

The disposal of infectious medical waste has become a major environmental concern. New disposal methods are currently being investigated; one of these is a bioremediation process which utilizes enzymes in a batch reactor to render the waste noninfectious. The success of this biological process depends on (among other things) two mechanisms: the diffusion of the disinfecting enzymes into small crevices within the waste stream and the breakdown of cellulose-derived material by cellulase enzymes. It was found that the waste stream contained a variety of small crevices which could possibly contain pathogens. Circulation in these crevices was restricted by their small openings and one must rely on diffusion of enzymes to disinfect their interiors. Numerical models for the diffusion of enzymes within one-dimensional and re-entrant crevices were developed and a method for comparing various re-entrant crevices was presented. From these models a conservative method for determining approximate disinfection times for the crevices was described. It was determined from this conservative method that most crevices within the waste will either be disinfected during the process or shortly thereafter. This biological process also utilizes cellulases to breakdown the paper within the waste stream. Small-scale simulated waste experiments were conducted with cellulases to determine the increase in maximum mixable solids concentration and the mass reduction of the waste due to cellulase activity. The addition of cellulases to the slurry more than doubled the waste concentration which could be agitated and reduced the agitator shaft power by as much as 50% when compared to the simulated waste tests with no cellulase. Significant mass reduction was also observed with the addition of cellulases to the slurries. Small-scale breakdown experiments were conducted with and without cellulases using newsprint as the substrate. These experiments were performed to determine the influence of cellulose hydrolysis by cellulases on agitator power. A simple mathematical model was developed and presented which described this phenomenon. / Master of Science

Cellulolytic responses of the Asiatic clam, Corbicula fluminea and a snail, Mudalia dilatata, to selected constituents of power plant effluents (i.e., zinc, cadmium, acidic and alkaline pH, individually and paired) were investigated in 30-day exposures. Exposures were conducted in both laboratory and field-oriented artificial streams and then validated in the river receiving system of a power plant. Cellulolytic activity was reduced by laboratory and field exposures to cadmium and zinc at all levels tested from 0.012 to 0.10 mg cadmium/L and generally at 0.025 to 1.0 mg zinc/L. Clams detected acute lethal levels of metal and used valve closure as an avoidance mechanism for 14 days. Snails, however, did not effectively avoid exposures and were more sensitive to acute stress during all exposures. These behavioral responses were corroborated by both cellulolytic activity and metal accumulation. Measurements of cellulolytic activity for both test species in laboratory exposures differed from those in field artificial streams. Reduced enzyme activity in controls by day 30 was attributed to artificially induced stress associated with the laboratory environment. This factor precluded any analysis of laboratory responses for periods of exposure longer than 20 days as well as recovery analysis. Field oriented artificial streams provided a sufficient environment to adequately assess long-term stress and recovery as measured by cellulolytic activity and metal accumulation in both clams and snails. Enzyme activity responded to metal exposure with respect to both degree and duration of exposure. Cadmium and zinc combined exposures caused significantly reduced cellulolytic activity at the same concentration as those for cadmium alone. Reduced enzyme activity caused by cadmium and zinc addition at levels that were not detectable suggested that the cellulolytic index was sensitive to sublethal stressors. This was supported by metal uptake patterns in clams and snails. Cellulolytic activity responded to zinc addition at alkaline and acidic pH in a manner that supported pH optima for cellulases and bioavailability of metals. Effects seen in macroinvertebrate assemblages (diversity, richness, and similarity) were compared with cellulolytic activity of caged Corbicula from a site specific power plant discharge. Enzyme activity inhibition was the most sensitive indicator measured. Reductions in cellulolytic activity at stations monitored for total zine content were consistent with effects seen at comparable exposures to zine in field-located artificial streams. A zine concentration of 0.05 mg/L consistently caused the first significant reductions in cellulolytic activity. This concentration is comparable to the U.S. Environmental Protection Agency's Water Quality Criteria value (0.047 mg/L zinc) for protection of aquatic life. / Ph. D.

Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.

Changes in cell wall composition and in vitro degradation of corn stover fractions (leaf, upper stem and lower stem) with advancing maturity and in response to NH; treatment were determined, and possible inhibitory mechanisms of lignin were evaluated. With advancing maturity, IVDMD decreased (P<.001), associated with decreases (P<.001) in CP and water soluble carbohydrates (WSC), and increases (P<.001) in NDF and ADF. The IVDMD of leaf was higher (P<.001) than of stems, associated with higher CP, hemicellulose:cellulose, and arabinan:xylan, and lower lignin methoxyl content. A hypothesis of formation of reactive quinone methide intermediates from lignin during rumen fermentation was tested in vitro by incubating corn stover fractions with S-containing reducing agents. Sulphur incorporation into residual fiber occurred (P<.05), indicative of nucleophilic addition to quinone methide intermediates. Degradation of NDF was highly correlated with lignin methoxyl content. The impact of lignin on cellulose degradation was studied using lignocellulosic hydrogels, in which hydroxypropylated or unmodified hardwood lignin was blended with cellulose. In vitro cellulose degradation of lignocellulose blends was higher (P<.01) than of control. Addition of lignin at incubation depressed (P<.01) cellulose degradation. Hydroxypropylation enhanced (P<.001) the increase in cellulose degradation with lignin blending, and reduced (P<.001) the inhibitory effect of lignin addition at incubation. Treatment of drought-stressed corn stover with 3% aqueous NH₃ decreased (P<.05) NDF, compared to isonitrogenous NH₃ addition and control, associated with solubilization of hemicellulose. Esterified phenolic acids were released (P<.05) by NH₃ treatment in upper stem. The IVDMD and NDF degradation increased (P <.001) after ammoniation, with higher (P<.05) values for NH₃ treatment than NH₃ added in leaf. The in vitro response to ammoniation of fractions of drought-stressed and non-drought stressed corn stover harvested in subsequent years was compared, using N-sufficient and N-limiting buffers. Response was highest (P<.001) for non-drought stressed stover fractions, and in N-limiting medium. Response appeared to be affected by high concentration of WSC in lower stalks of drought-stressed stover. / Ph. D.

Using ����C cross polarization magic angle spinning nuclear magnetic resonance techniques on 5 species of dead trees from the northwest (western hemlock, Douglas fir, Sitka spruce, lodgepole pine and ponderosa pine) I tracked the lignin and cellulose content over a 22 to 36 year period in order to determine the effects of decay fungi, if any, that is attacking certain species of tree. I had samples from the wood of the roots, the bark on the roots and, in some cases, the resin core of the roots. The Department of Forest Science at Oregon State University has studied this problem by using wet chemical analysis, and direct visual observation. Mark Harmon and Hua Chen of the Department of Forest Science believe that white rot occurred most frequently in the lodgepole pine and ponderosa pine and brown rot was more frequent in the Douglas-fir and Sitka spruce. Western hemlock seemed to have both brown and white rots active. The Douglas fir bark sample showed definite decomposition consistent with white rot during the first 10 years. The ponderosa pine sap showed decomposition consistent with white rot in the 10 to 22 year period. Sitka Spruce showed some decomposition consistent with white rot in the bark from 7 to 33 years, and the western hemlock showed some decomposition consistent with white rot in the sap in the first 10 years. The decompositions consistent with brown rot were much easier to see in this study. Virtually all the sap and bark samples showed decomposition consistent with brown rot at some point. The Douglas fir was the only species, other than lodgepole pine, not to show any decomposition consistent with brown rot in the bark of the tree, only decomposition consistent with white rot. The Douglas fir did show a decay consistent with brown rot in the sap for the first ten years. Ponderosa pine showed evidence of decay that brown rot would cause for the first 10 years in the sap and the bark. The Sitka spruce species analysis showed brown rot type decay in the bark for the first 7 years and in the sap for the entire time studied of 33 years. The lodgepole pine was the only species to not show any brown rot type decay in the sap or bark for the entire 22 year period studied. The western hemlock was distinct by not showing any definitive brown rot type decay for the first 10 years, but showed massive decay consistent with brown rot in both sap and bark during the following 26 years studied. I used an 8 Tesla magnet and the MAS frequency was at 5 kHz. The recycle time was 1.5 seconds and the contact time was 1 ms. I generally took about 10,000 acquisitions per sample, which added up to about 4 hours total acquisition time per sample. Presence of these rots shows that certain species are more susceptible than others, and also shows that local environmental conditions can contribute to rot susceptibility. / Graduation date: 2003

Lignocellulosic biomass is an abundant renewable resource targeted for biofuel production. Cellulose and hemicellulose from biomass both contain fermentable sugars and other moieties that can be converted to biofuels or other commodity chemicals. Enzymatic hydrolysis of these biopolymers is a critical step in the liberation of sugars for fermentation into desired products. In nature, anaerobic microbes produce protein nanostructures called cellulosomes that efficiently degrade cellulose substrates by combining multiple enzyme activities onto a scaffolding protein. However, current enzyme cocktails used in industry contain secretomes of aerobic microbes and are not efficient enough to be highly economical. Furthermore, most bio-processes focus on cellulose, rendering hemicellulose under-utilized. The three main objectives of this dissertation are to 1) develop multi-functional, self-assembling protein nanostructures for hemicellulose degradation using the architecture provided by cellulosomes, 2) understand the self-assembly mechanism at conditions for consolidated bioprocessing applications, and 3) compare the effectiveness of structured to non-structured hemicellulases in the hydrolysis of biomass. Xylan is a major type of hemicellulose in biomass feedstocks targeted for biofuel production. Six different xylanosomes were designed for hydrolysis of xylan within multiple biomass substrates using the cohesin-dockerin domain systems from Clostridium thermocellum, Clostridium cellulovorans, and Clostridium cellulolyticum. Each two-unit structure contained a xylanase for internal cleavage of the xylan backbone and one side-chain acting enzyme, either a ferulic acid esterase or bi-functional arabinofuranosidase/xylosidase. Expansion to three-unit xylanosomes included a family 10 or 11 xylanase, a bi-functional arabinofuranosidase/xylosidase, and bi-functional ferulic acid esterase/acetylxylan esterase. These multi-functional biocatalysts were used to degrade hemicellulose-rich wheat arabinoxylan and cellulose-containing destarched corn bran. Synergistic release of soluble sugars and ferulic acid was observed with select xylanosomes and in some cases required addition of an endoglucanase and cellobiohydrolase for enhanced hydrolysis. Furthermore, a putative ferulic acid esterase gene from the soil bacterium Cellvibrio japonicus was characterized and its role in xylan hydrolysis investigated. Information for the development of stable and functional cellulosome-like biocatalysts in metabolically-engineered microbes was collected using surface plasmon resonance. The protein-protein interaction of cohesin and dockerin domains for xylanosome self-assembly was examined at various temperatures and in the presence of ethanol to mimic different hydrolysis and fermentation processes and found to retain high affinities at the selected conditions. Moreover, the high-affinity interaction of cohesin and dockerin domains in the presence of non-specific proteins eliminated the need for protein purification for xylanosome construction. In addition to development of the first cellulosome-like biocatalysts targeted for hemicellulose degradation, this dissertation provides insight on possible improvements for the enzymatic hydrolysis of biomass, as well as the applicability of xylanosomes in consolidated bioprocessing.

Submitted by Milena Rubi (milenarubi@ufscar.br) on 2016-11-09T14:11:31Z No. of bitstreams: 1 ROSA_Wesley_2016.pdf: 8516860 bytes, checksum: ee5a7fd54adf8975660601cc883291f2 (MD5) / Approved for entry into archive by Milena Rubi (milenarubi@ufscar.br) on 2016-11-09T14:11:44Z (GMT) No. of bitstreams: 1 ROSA_Wesley_2016.pdf: 8516860 bytes, checksum: ee5a7fd54adf8975660601cc883291f2 (MD5) / Approved for entry into archive by Milena Rubi (milenarubi@ufscar.br) on 2016-11-09T14:11:50Z (GMT) No. of bitstreams: 1 ROSA_Wesley_2016.pdf: 8516860 bytes, checksum: ee5a7fd54adf8975660601cc883291f2 (MD5) / Made available in DSpace on 2016-11-09T14:11:57Z (GMT). No. of bitstreams: 1 ROSA_Wesley_2016.pdf: 8516860 bytes, checksum: ee5a7fd54adf8975660601cc883291f2 (MD5) Previous issue date: 2016-03-28 / Não recebi financiamento / The need to obtain biomaterials in order to reduce environmental impacts has been the focus of research groups in recent years, and cellulose, a dominant component at most forms of plants is a promising resource because of its abundance. In order to improve the ability processing, the chemical modification of cellulose has been widely studied. Among the most important reactions of cellulose are: etherification, esterification, acetylation and oxidation; being cellulose acetate, viscose, nitrocellulose and cellulose ethers, the main cellulose derivatives. The chemical modification with isocyanates presents some unique properties, such as absence of by-products and chemical stability of the urethane group. In this work we were synthesized gels obtained by modified cellulose acetate (CA) with a degree of substitution (DS) 2,5 by crosslinking, with 4,4' - Diphenylmethane diisocyanate (MDI) in stoichiometry of 1:1, in homogeneous by varying the humidity and the homogenization time. For characterization were used the following techniques and tests: vibrational infrared absorption spectroscopy (Fourier Transform Spectrometer - FTIR), size exclusion Chromatography (SEC), molecular absorption spectrophotometry UV-VIS, density determining of the gels by pycnometry, determination of the coefficient swelling, determination of cross-links by Flory-Rehner theory, thermogravimetry (TG) and scanning electron microscopy (SEM). Crosslink density results showed that the gel synthesized in the absence of moisture suffered greater crosslinking with an average number of repeat units between the crosslinking points of about 1000 times lower. The potential applications of these gels were tested, by using than as stationary phase in size exclusion chromatography, having been assessed its efficiency in the fractionation and separation of natural and synthetic polymers. Results showed the effectiveness of the gel as stationary phase on separation of polymers, opening up a range of opportunities, taking into consideration the simplicity of the process and lower costs attributed to it. / A necessidade de se obter biomateriais na tentativa de reduzir impactos ambientais tem sido o foco de grupos de pesquisa nos últimos anos e, a celulose, um componente dominante na maioria das formas de plantas, é um recurso promissor devido à sua abundância. A fim de melhorar a capacidade de processamento, a modificação química da celulose tem sido amplamente estudada. Dentre as reações mais importantes da celulose estão: eterificação, esterificação, acetilação e oxidação; sendo o acetato de celulose, viscose, nitrocelulose e éteres de celulose, os principais derivados da celulose. A modificação química com isocianatos apresenta algumas propriedades únicas, como ausência de produtos secundários e estabilidade química do grupo uretano. Nesse trabalho foram sintetizados géis obtidos por meio da modificação de Acetato de Celulose (AC) com grau de substituição (GS) 2,5 através da reticulação com 4,4' - Difenilmetano Diisocianato (MDI), na estequiometria 1:1, em meio homogêneo, variando a umidade e o tempo de homogenização. Para caracterização foram utilizadas as seguintes técnicas e ensaios: espectroscopia vibracional de absorção no infravermelho por Transformada de Fourier (FTIR), cromatografia de exclusão por tamanho (SEC), espectrofotometria de absorção molecular UV-VIS, determinação de densidade dos géis por picnometria, determinação do coeficiente de intumescimento, determinação de ligações cruzadas pela teoria de Flory-Rehner, termogravimetria (TG) e microscopia eletrônica de varredura (MEV). Resultados da densidade de ligações cruzadas mostraram que o gel sintetizado na ausência de umidade sofreu uma maior reticulação, com um número médio de unidades de repetição entre os pontos de reticulação cerca de 1000 vezes menor. As aplicações potenciais desses géis foram testadas como fase estacionária em cromatografia de exclusão por tamanho, tendo sido avaliada sua eficiência no fracionamento e separação de polímeros naturais e sintéticos. Resultados mostraram a eficácia do gel como fase estacionária na separação de polímeros, abrindo uma gama de oportunidades, levando-se em consideração a simplicidade do processo e os baixos custos a ele atribuídos.

CAPES / O processo kraft é o mais empregado em todo o mundo para produção de papel e celulose. Esse processo gera efluente com altas concentrações de matéria orgânica e compostos recalcitrantes, que podem causar impacto significativo no ambiente aquático. No Brasil, o tratamento mais difundido para este efluente são as Lagoas Aeradas Facultativas (LAFs). Este tipo de tratamento é eficiente na remoção da matéria orgânica biodegradável, entretanto alguns contaminantes persistem devido à sua recalcitrância. O objetivo deste trabalho foi avaliar o tratamento de efluente kraft por processo biológico na presença de Lacase comercial (NOVOZYM 51003®) e enzimas ligninolíticas produzidas em laboratório por extrato de Fungos da Podridão Branca (FPB). Foi empregado delineamento experimental com sistema aerado em batelada, para determinar os efeitos do pH, temperatura e concentração da Lacase comercial, além de testes rápidos de batelada para verificar o efeito da inoculação associada a Lacase comercial, o efeito do sistema Lacase/mediador, o efeito da Lacase comercial inativa no tratamento do efluente, entre outros. Também foi estudado o desempenho da Lacase comercial e do Extrato Enzimático de Trametes sp. (EET) em fluxo contínuo, com LAFs em escala de bancada, sob condições de temperatura ambiente, operadas por 60 dias em Carga Orgânica Volumétrica (COV) de 0,2 kg DQO m-3 d-1. Numa segunda fase do experimento com as LAFs foram realizadas variações na COV e na biodegradabilidade do efluente kraft. O experimento fatorial mostrou que o uso da Lacase comercial influencia positivamente a remoção de lignina, cor e área espectral, sendo mais eficiente em pH 4 e temperatura de 37°C e 3,9 U mL-1 de atividade de Lacase. Os testes em batelada mostraram que a inoculação não interfere na atividade enzimática e que a enzima comercial é biodegradável. O experimento com as LAFs em sistemas contínuos em COV de 0,2 kg DQO m-3 d-1, mostrou que a LAF com Lacase comercial, removeu 86 %, 52 %, 20 % e 30 % de DBO5,20, DQO, Cor e Compostos Lignínicos (CL), respectivamente. A LAF com EET removeu 90 %, 50 %, 2 % e 20 % de DBO5,20, DQO, Cor e CL, respectivamente. No entanto, os resultados anteriores não foram estatisticamente diferentes daqueles obtidos nos sistemas sem enzimas (controles). Na LAF com Lacase comercial não se verificou remoção de Compostos Fenólicos Totais (CFT), enquanto na LAF com EET houve remoção de apenas 1 %, o qual foi importante, pois nos controles e LAF com enzima comercial ocorreu geração de CFT. O aumento da COV para 0,6 ou 1,2 kg⋅DQO⋅m-3⋅d-1 não diminuiu a eficiência das LAFs, chegando a aumentar a redução dos parâmetros de DQO e CFT. Já a redução da biodegradabilidade do efluente kraft impactou mais os sistemas do que as sobrecargas de matéria orgânica biodegradável. Por conta das características do efluente e condições utilizadas, a atividade da Lacase comercial reduziu em 58 % após 24 horas de contato dentro da LAF. Já o EET na LAF, apesar de diluído, manteve a Lacase ativa e permitiu catalisar a oxidação dos CFTs no efluente kraft, possivelmente pela presença de compostos que interagem mediando estas reações. A enzima Lacase associada a LAF se manteve ativa no tempo de operação, não sofreu influência do inóculo, porém não foi capaz de melhorar o desempenho observado no sistema controle nas condições de operação, especialmente na forma pura, como Lacase comercial, já o EET se mostrou promissor pelo seu desempenho sobre os CFT. / The kraft process is the most used in the world for the production of pulp and paper. This process was done with high concentrations of organic matter and recalcitrant compounds, which can have a significant impact on the aquatic environment. In Brazil, the most widespread treatment for this effluent is as Facultative Aerated Lagoons (FALs). This type of treatment is efficient in the removal of biodegradable organic matter, however some contaminants persist because of its recalcitrance, attributing toxicity to the effluent even after treatment. The objective of this work was to evaluate the treatment of kraft effluent by biological process in the presence of commercial Laccase (NOVOZYM 51003®) and ligninolytic enzymes produced in the laboratory by extract of White Rot Fungi (WRF). An experimental study was carried out with the aerobic batch system to determine the effects of pH, temperature and commercial Laccase concentration, as well as the rapid batch tests to verify the effect of inoculation associated with commercial Laccase, the effect of Laccase/Mediator and the effect of Inactive commercial Laccase in the effluent treatment. We also study the performance of the commercial Laccase and the Enzymatic Extract of Trametes sp. (EET) in continuous flow, in lab scale FALs, under room temperature conditions, for 60 days in a constant Organic Load Rate (OLR). In a second phase of the experiment with FALs, a sudden increase in OLR was performed to verify the efficiency of shock systems. The factorial experiment showed that the use of commercial Laccase positively influences a lignin removal, color and spectral area, being more efficient at pH 4 and temperature of 37 ° C and 3.9 U mL-1 laccase. Batch tests show that an inoculation does not interfere with enzymatic activity and that a commercial enzyme is readily biodegraded within the system. The experiment with FALs at constant OLR of 0.2 kg COD m-3 d-1, showed that FAL with commercial Laccase removed 86%, 52%, 20% and 30% of BOD5,20, COD, Color and Lignin Compounds (LC), respectively with FAL with EET removed 90%, 50%, 2% and 20% of BOD5,20, COD, Color and CL, respectively. But the values of both FALs were not statistically different compared to their control. Also in the OLR of 0.2 kg COD m-3 d-1, the FAL with EET showed a 1% removal, which represents a reduction of the Total Phenolic Compound (TPC). The increase of OLR to 0.6 or 1.2 kg COD m-3d-1 does not negatively impact the efficiency of the FAL, leading to a weight increase in the removal of COD and TPC. A reduction of the BOD5,20/COD ratio caused a expressively more important shock them biodegradable organic matter overloads. Due to the effluent characteristics and conditions of use, the commercial Laccase had its activity reduced within the LAF system in 58% after 24 hours of contact. However, the FAL with EET it showed that, although diluted, the crude laccase present in the EET remained active and allowed to catalyze the removal of TPC present in the kraft pulp mill effluent, Due to the presence of other compounds Interacting with each other, mediating these reactions. This it seems expressively important, since the FAL used as control, absence of Laccase, promoted an increase of about 23% in the TPC content of the treated effluent.

CAPES / O processo kraft é o mais empregado em todo o mundo para produção de papel e celulose. Esse processo gera efluente com altas concentrações de matéria orgânica e compostos recalcitrantes, que podem causar impacto significativo no ambiente aquático. No Brasil, o tratamento mais difundido para este efluente são as Lagoas Aeradas Facultativas (LAFs). Este tipo de tratamento é eficiente na remoção da matéria orgânica biodegradável, entretanto alguns contaminantes persistem devido à sua recalcitrância. O objetivo deste trabalho foi avaliar o tratamento de efluente kraft por processo biológico na presença de Lacase comercial (NOVOZYM 51003®) e enzimas ligninolíticas produzidas em laboratório por extrato de Fungos da Podridão Branca (FPB). Foi empregado delineamento experimental com sistema aerado em batelada, para determinar os efeitos do pH, temperatura e concentração da Lacase comercial, além de testes rápidos de batelada para verificar o efeito da inoculação associada a Lacase comercial, o efeito do sistema Lacase/mediador, o efeito da Lacase comercial inativa no tratamento do efluente, entre outros. Também foi estudado o desempenho da Lacase comercial e do Extrato Enzimático de Trametes sp. (EET) em fluxo contínuo, com LAFs em escala de bancada, sob condições de temperatura ambiente, operadas por 60 dias em Carga Orgânica Volumétrica (COV) de 0,2 kg DQO m-3 d-1. Numa segunda fase do experimento com as LAFs foram realizadas variações na COV e na biodegradabilidade do efluente kraft. O experimento fatorial mostrou que o uso da Lacase comercial influencia positivamente a remoção de lignina, cor e área espectral, sendo mais eficiente em pH 4 e temperatura de 37°C e 3,9 U mL-1 de atividade de Lacase. Os testes em batelada mostraram que a inoculação não interfere na atividade enzimática e que a enzima comercial é biodegradável. O experimento com as LAFs em sistemas contínuos em COV de 0,2 kg DQO m-3 d-1, mostrou que a LAF com Lacase comercial, removeu 86 %, 52 %, 20 % e 30 % de DBO5,20, DQO, Cor e Compostos Lignínicos (CL), respectivamente. A LAF com EET removeu 90 %, 50 %, 2 % e 20 % de DBO5,20, DQO, Cor e CL, respectivamente. No entanto, os resultados anteriores não foram estatisticamente diferentes daqueles obtidos nos sistemas sem enzimas (controles). Na LAF com Lacase comercial não se verificou remoção de Compostos Fenólicos Totais (CFT), enquanto na LAF com EET houve remoção de apenas 1 %, o qual foi importante, pois nos controles e LAF com enzima comercial ocorreu geração de CFT. O aumento da COV para 0,6 ou 1,2 kg⋅DQO⋅m-3⋅d-1 não diminuiu a eficiência das LAFs, chegando a aumentar a redução dos parâmetros de DQO e CFT. Já a redução da biodegradabilidade do efluente kraft impactou mais os sistemas do que as sobrecargas de matéria orgânica biodegradável. Por conta das características do efluente e condições utilizadas, a atividade da Lacase comercial reduziu em 58 % após 24 horas de contato dentro da LAF. Já o EET na LAF, apesar de diluído, manteve a Lacase ativa e permitiu catalisar a oxidação dos CFTs no efluente kraft, possivelmente pela presença de compostos que interagem mediando estas reações. A enzima Lacase associada a LAF se manteve ativa no tempo de operação, não sofreu influência do inóculo, porém não foi capaz de melhorar o desempenho observado no sistema controle nas condições de operação, especialmente na forma pura, como Lacase comercial, já o EET se mostrou promissor pelo seu desempenho sobre os CFT. / The kraft process is the most used in the world for the production of pulp and paper. This process was done with high concentrations of organic matter and recalcitrant compounds, which can have a significant impact on the aquatic environment. In Brazil, the most widespread treatment for this effluent is as Facultative Aerated Lagoons (FALs). This type of treatment is efficient in the removal of biodegradable organic matter, however some contaminants persist because of its recalcitrance, attributing toxicity to the effluent even after treatment. The objective of this work was to evaluate the treatment of kraft effluent by biological process in the presence of commercial Laccase (NOVOZYM 51003®) and ligninolytic enzymes produced in the laboratory by extract of White Rot Fungi (WRF). An experimental study was carried out with the aerobic batch system to determine the effects of pH, temperature and commercial Laccase concentration, as well as the rapid batch tests to verify the effect of inoculation associated with commercial Laccase, the effect of Laccase/Mediator and the effect of Inactive commercial Laccase in the effluent treatment. We also study the performance of the commercial Laccase and the Enzymatic Extract of Trametes sp. (EET) in continuous flow, in lab scale FALs, under room temperature conditions, for 60 days in a constant Organic Load Rate (OLR). In a second phase of the experiment with FALs, a sudden increase in OLR was performed to verify the efficiency of shock systems. The factorial experiment showed that the use of commercial Laccase positively influences a lignin removal, color and spectral area, being more efficient at pH 4 and temperature of 37 ° C and 3.9 U mL-1 laccase. Batch tests show that an inoculation does not interfere with enzymatic activity and that a commercial enzyme is readily biodegraded within the system. The experiment with FALs at constant OLR of 0.2 kg COD m-3 d-1, showed that FAL with commercial Laccase removed 86%, 52%, 20% and 30% of BOD5,20, COD, Color and Lignin Compounds (LC), respectively with FAL with EET removed 90%, 50%, 2% and 20% of BOD5,20, COD, Color and CL, respectively. But the values of both FALs were not statistically different compared to their control. Also in the OLR of 0.2 kg COD m-3 d-1, the FAL with EET showed a 1% removal, which represents a reduction of the Total Phenolic Compound (TPC). The increase of OLR to 0.6 or 1.2 kg COD m-3d-1 does not negatively impact the efficiency of the FAL, leading to a weight increase in the removal of COD and TPC. A reduction of the BOD5,20/COD ratio caused a expressively more important shock them biodegradable organic matter overloads. Due to the effluent characteristics and conditions of use, the commercial Laccase had its activity reduced within the LAF system in 58% after 24 hours of contact. However, the FAL with EET it showed that, although diluted, the crude laccase present in the EET remained active and allowed to catalyze the removal of TPC present in the kraft pulp mill effluent, Due to the presence of other compounds Interacting with each other, mediating these reactions. This it seems expressively important, since the FAL used as control, absence of Laccase, promoted an increase of about 23% in the TPC content of the treated effluent.