Biogas production through co-digestion of enzymatically pretreated corn bran and cow manure

Biogas production from wastes is an alternative that contributes positively to the environment and minimize the dependence on fossil energy sources. Additionally, the reuse of biomasses helps to reduce the waste production, but a pretreatment is required to use it in the anaerobic digestion. Here biogas was produced through co-digestion of enzymatically pretreated corn bran and cow manure. Firstly, it was selected the most hydrolysable waste (barley bagasse, sugar cane bagasse, elephant grass, thick orange pie, average orange pie, wheat bran, coffee grounds, orange peel, white sludge, vinasse, corn bran, soy bran, soy peel, cotton bran, cassava husk, cassava flour, banana peel, corn bran, sorghum stem, sorghum seed, total sorghum and wet distiller grain) by the crude extracts containing amylase (secreted by Aspergillus brasiliensis), xylanase (Aspergillus tamarii Kita) and cellulase (Trichoderma reesei, Novozymes®). Later on, different mixtures of these enzymes were studied using simplex-centroid designs. The most hydrolyzed waste by each enzyme individually (measured by reducing sugar using dinitrosalicylic acid, DNS) at 50°C, 120 rpm and 24 h were corn bran, banana peel and sorghum seed. Then, the simplex-centroid designs resulted in model equations and respective response surface contours. Amylase extract had a significant positive influence on corn bran hydrolysis by maximizing the reducing sugar yield when it was used individually (35g/L of reducing sugar). After it, the pretreated corn bran and a cow manure (1:2 g of volatile solids) were employed for biogas production in batch assays. It was found a biogas accumulation of 326 mL in the 12nd day of anaerobic codigestion, which were similar to the control (containing 35 g/L of glucose alone) and 53% higher than that found with corn bran without enzymatic pretreatment. In conclusion, it was observed that the crude extract optimized for amylase production affected significantly the corn bran hydrolyses and consequently the biogas production in a co-digestion with cow manure.CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico process 142139/2017-3)FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo process 2018/07522-6)FCT (Fundação para a Ciência e Tecnologia)info:eu-repo/semantics/publishedVersio

Comparison in the Trichoderma longibrachiatum xyloglucanase production using tamarind (Tamarindus indica) and jatobá (Hymenaea courbaril) seeds: factorial design and immobilization on ionic supports

in the control of the stretching and expansion of the plant cell wall. There are five types of enzymes known to be capable of cleaving the linear chain of xyloglucan, the most famous of them being the xyloglucanase (XEG). The immobilization can be used to solve problems related to stability, besides the economic benefits brought by the possibility of repeated use and recovery, decreasing the costs of production. Therefore, this study aims the optimization of the production of a xyloglucanase from Trichoderma longibrachiatum, with the aid of factorial design, using tamarind (Tamarindus indica) and jatobá (Hymenaea courbaril) seeds as carbon source; and the immobilization of the enzyme on ionic supports, such as MANAE (monoamino-N-aminoethyl), DEAE (diethylaminoethyl)-cellulose, CM (carboxymethyl)-cellulose and PEI (polyethyleneimine). High concentrations of carbon source in the culture medium, especially tamarind seeds, were the most favorable conditions for the greater activity of the xyloglucanase from T. longibrachiatum. The scaling up from Erlenmeyer flasks to the bioreactor was an essential strategy to increase the content of secreted enzyme. Regarding the biochemical characterization of the crude extract, the optimal temperature was 50-55 °C and the optimal pH 5.0. Regarding the stabilities to pH and to temperature, the enzyme was not stable for prolonged periods, which was crucial for the performing of immobilization on ionic resins (CM-cellulose, DEAE-cellulose, MANAE, and PEI), being the first time described in literature the immobilization of a xyloglucanase on these supports.We thank the Fundação de Amparo à Pesquisa do estado de São Paulo (process 2018/07522-6; 2014/50884-5), and Conselho Nacional de Dsenvolvimento Científico (process 301963/2017-7; 465319/2014-9).info:eu-repo/semantics/publishedVersio

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Educomunicação, Transformação Social e Desenvolvimento Sustentável

Esta publicação apresenta os principais trabalhos dos GTs do II Congresso Internacional de Comunicação e Educação nos temas Transformação social, com os artigos que abordam principalmente Educomunicação e/ou Mídia-Educação, no contexto de políticas de diversidade, inclusão e equidade; e, em Desenvolvimento Sustentável os artigos que abordam os avanços da relação comunicação/educação no contexto da educação ambiental e desenvolvimento sustentável

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

Production of enzymatic cocktails with potential into cellulose pulp biobleaching for paper production through lignocellulosic wastes and secondary fibres

Este trabalho teve como objetivo a prospecção e caracterização de enzimas fúngicas, que degradam a biomassa, visando à aplicação no biobranqueamento da polpa de celulose. Para isto, foram selecionados 13 fungos filamentosos da Micoteca e entre estes Aspergillus versicolor e A. brasiliensis foram aqueles que se sobressaíram quanto à produção de xilanase, amilase, CMCase, avicelase e ?-glucosidase. As melhores condições de produção enzimática corresponderam à utilização de bagaço de cevada como fonte de carbono em meio SR (Segato Rizzatti) para xilanase, amilase e ?- glucosidase e meio M5 para CMCase, avicelase e FPase, nos cultivos incubados por tempos variáveis de 72 a 168 horas. Para otimizar a concentração da fonte de carbono e a temperatura nos cultivos foi realizado um Delineamentro Composto Central Rotacional. Como resultante da otimização do bioprocesso foram obtidos os extratos brutos: (i) meio BSR, para a produção de xilanase por A. brasiliensis, com 96 horas de cultivo estático, a 30°C, com bagaço de cevada 3% em meio SR; (ii) Meio VSR para amilase e ?-glucosidase a partir do A. versicolor, com 120 horas de cultivo estático, a 35°C, com bagaço de cevada 3,41% em meio SR; (iii) Meio VM5 para CMCase, avicelase e FPase de A. versicolor com 120 horas de cultivo estático, a 30°C, com bagaço de cevada 2% em meio M5. Lacase foi produzida por Trametes versicolor (iv), cultivado por quinze dias em Fermentação Submersa estática, a 30°C, em meio contendo vinhaça e água destilada (1:5 v/v), 1% de algodão e 0,1% de peptona. Paralelamente, a produção de xilanases de A. tamarii Kita (v) com bagaço de cevada 2,9% foi otimizada em meio ADAMS por 129 horas (recebendo a denominação - meio TKADAMS). Quanto à caracterização das enzimas de A. versicolor (xilanase) e A. brasiliensis (demais enzimas) a temperatura ótima de xilanase foi 70°C; amilase 60-65°C; CMCase 65°C; avicelase 50°C; FPase e lacase 60°C e ?-glucosidase 70-75°C. Quanto à estabilidade térmica, xilanase mostrou-se com 60% de atividade relativa por até 24 horas à 40°C e 30 minutos à 50°C. A 60°C mostrou-se pouco estável. A amilase mostrou-se estável por 24 horas a 40 e 50°C, com 80% da atividade relativa. CMCase e FPase mostraram-se pouco estável nas temperaturas citadas. Avicelase apresentou ativação quando exposta ao aquecimento de 40, 50 e 60°C. ?-glucosidase foi estável a 40°C por até 24 horas, com atividade relativa de 80%; a 50°C apresentou 60% de atividade relativa por 180 minutos e a 60°C exibiu 40% por até 30 minutos. Lacase foi estável a 50°C, com um t50 de 60 minutos; a 60°C teve atividade relativa próxima de 30%, por até 240 minutos e a 70°C apresentou 40% de atividade por 30 minutos. Quanto ao pH, xilanase apresentou uma faixa ótima de 4,0-5,0 e também entre 7,0-8,0. Amilase, CMCase, avicelase, FPase, ?-glucosidase e lacase apresentaram atividades mais expressivas na faixa de pH 4,0-5,5. Com relação à estabilidade ao pH em 24 horas, xilanase foi mais estável nos pH 5,5-7,0, amilase nos pH 5,0-6,5, CMCase, FPase e lacase em pH 4,5, avicelase em pH 3,0 e ?-glucosidase em pH 5,0-5,5. Quanto ao efeito de íons, CMCase e ?-glucosidase foram ativadas por K+, Zn+ e Ba2+; CMCase, ?-glucosidase e lacase foram ativadas por NH4+ e Ca2+; amilase, CMCase, ?-glucosidase e lacase foram ativadas por Co2+; amilase, CMCase e ?- glucosidase foram ativadas por Al3+ e Fe2+; xilanase, avicelase, CMCase e ?-glucosidase foram ativadas por Mn+; avicelase e CMCase foram ativadas por Ag+; lacase por EDTA; ?-glucosidase e lacase por Mg2+, CMCase por Hg2+ e xilanase, ?-glucosidase e lacase por Cu2+. Os extratos foram utilizados na formulação de coquetéis enzimáticos para biobranqueamento da polpa de celulose. A aplicação dos extratos BSR, VSR, VM5 sobre a polpa marrom não resultou em uma redução significativa do número Kappa quando comparados ao controle, uma vez que todos os extratos apresentavam uma coloração muito escura, a qual fora originada por componentes e pigmentos provenientes dos meios de cultivo dos micro-organismos com bagaço de cevada e que, consequentemente, interferiram na determinação do número Kappa. Desta forma, o coquetel otimizado teve como formulação: 20,3 mL do meio TKADAMS e 10 mL do extrato do meio produtor de lacase para cada 4 gramas de polpa tratada, pH 5,5; 35,9ºC, 48 horas. Este tratamento resultou na redução de 1,83 pontos no número Kappa da polpa marrom, representando uma eficiência de 20,3%, e aumento de 4,65 na alvura, em relação ao controle. A aplicação do coquetel nos resíduos lignocelulósicos ocasionou a formação máxima de 85 mg/mL de açúcares redutores, em 24 horas no tratamento do bagaço de cevada, e 25 mg/mL de açúcares redutores, em 3 horas no tratamento do bagaço de cana. A aplicação do coquetel nas polpas de papel reciclado ocasionou um maior destintamento. A aplicação do coquetel desenvolvido na polpa de celulose, nos resíduos lignocelulósicos e fibras secundárias mostrou-se promissora para biobranqueamento, biodegradação e destintamento destes, respectivamente. A aplicação de enzimas no processo de biobranqueamento da polpa de celulose é uma alternativa viável e que auxilia a redução de custos, água, energia e colabora com o meio ambiente. A lacase foi importante no biobranqueamento da polpa de celulose sendo que o aumento da escala de produção do T. versicolor para biorreator levou a uma produção 6,25 vezes maior comparada aquela em Erlenmeyer, provavelmente devido a aeração constanteThis work aimed to prospect and characterize fungal enzymes, which break biomass, looking for the cellulose pulp biobleaching application. For this, 13 filamentous fungi of the Fungi Library were selected and among them Aspergillus versicolor and A. brasiliensis were those that stood out as inducers of xylanase, amylase, CMCase, avicelase and ?-glucosidase production. The use of barley bagasse as carbon source was the best condition for xylanase, amylase and ?-glucosidase production in SR medium (Segato Rizzatti) and M5 medium was the best for CMCase, avicelase and FPase production, in cultures incubated for 72-168 h. In order to optimize the concentration of the carbon source and the temperature of the cultures, a Central Composite Rotational Design was elaborated. (i) BSR extract, in SR medium for the xylanase production by A. brasiliensis, 96 hours, in static culture, at 30°C with barley bagasse 3%; (ii) VSR extract, in SR medium, for the amylase and ?-glucosidase from A. versicolor, 120 hours in static culture, at 35°C, with barley bagasse 3.41%. (iii) VM5 extract, in M5 medium for CMCase, avicelase and FPase from A. versicolor, 120 hours of static culture, at 30°C with barley bagasse 2%. Lacase was produced by Trametes versicolor (iv), in a medium containing vinasse and distilled water (1:5 v/v), cotton 1% and peptone 0.1%, for 15 days at 30ºC. In parallel, the production of a xylanase from A. tamarii Kita (v) with barley bagasse 2.9% was optimized in ADAMS medium for 129 hours (designated - TKADAMS medium). The thermal stability study showed that xylanase was stable with 60% of relative activity at 40°C for up to 24 hours and for 30 minutes at 50°C. At 60°C the enzyme was poorly stable. Amylase was stable for 24 hours at 40 and 50°C, with 80% of relative activity. CMCase and FPase was poorly stable at all temperatures tested. Avicelase was activated by the exposure at 40, 50 and 60°C. ?-glucosidase was stable at 40°C for up to 24 hours, with 80% of relative activity; at 50°C it showed a relative activity of 60% for 180 minutes and at 60° it showed 40% of relative activity for 30 minutes. Laccase was stable at 50°C with t50 for 60 minutes. At 60°C it showed an activity of 30% for up to 240 minutes and at 70°C it showed 40% of activity for 30 minutes. As for pH, xylanase showed the best activity in a range of pH 4.0-5.0 and 7.0-8.0. Amylase, CMCase, avicelase, FPase, ?-glucosidase and laccase showed expressive activities in a range of pH 4.0-5.5. The tests of pH stability in 24 hours showed that xylanase was stable at pH 5.5-7.0, amylase at pH 5.0-6.5, CMCase, FPase and laccase at pH 4.5, avicelase at pH 3.0 and ?-glucosidase at pH 5.0-5.5. The effect of ions showed that CMCase and ?-glucosidase were activated by K+, Zn+ and Ba2+; CMCase, ?-glucosidase and lacase were activated by NH4+ and Ca2+; amylase, CMCase, ?-glucosidase and lacase were activated by Co2+; amylase, CMCase and ?-glucosidase by Al3+ and Fe2+; xylanase, avicelase, CMCase and ?-glucosidase by Mn+; avicelase and CMCase by Ag+; lacase by EDTA; ?-glucosidase e lacase by Mg2+, CMCase by Hg2+ and xylanase, ?-glucosidase and lacase by Cu2+. The extracts were used in the formulation of enzymatic cocktails for cellulose pulp biobleaching. The application of BSR, VSR and VM5 extracts on the brown pulp did not result on a significant reduction of the Kappa number when compared to the control, on account of the dark coloration of these extracts caused by the components and pigments from the cultivation with the microorganisms and barley bagasse, which as a consequence, interfered in the determination of the Kappa number. Thus, an optimized cocktail was formulated: for each 4 grams of treated pulp 20.3 mL of TKADAMS extract and 10 mL of laccase extract, pH 5.5; 35.9°C, 48 hours. This treatment resulted in the reduction of 1.83 points in the Kappa number of the brown pulp, representing an efficiency of 20.3%, and a brightness increase of 4.65 when compared to the control. The application of the cocktail in the lignocellulosic residues resulted in the formation of 85 mg/mL of reducing sugars in barley bagasse treatment for 24 hours and, 25 mg/ml of reducing sugars in sugarcane bagasse treatment for 3 hours. The application of the cocktail in the recycled paper pulps caused a greater deinking. The application of the formulated cocktail in the cellulose pulp, lignocellulosic residues and secondary fibres was promising for biobleaching, biodegradation and deinking, respectively. The enzyme application in cellulose biobleaching is a viable alternative, which helps reducing costs, water, energy and collaborates with the environment. Laccase was important in the cellulose biobleaching and the increase of its production by T. versicolor through bioreactor led to a production 6.25 times higher than that in Erlenmeyer, probably due to the constant aeratio

Potencial biotecnológico da biomassa influência do pré-tratamento enzimático para a digestão anaeróbia e fermentação alcoólica

O consumo de energia aumentou à medida que a população mundial cresceu e os países se industrializaram. A energia é vital para praticamente todas as atividades humanas, por exemplo, iluminação, produção alimentícia, aquecimento, cozimento de alimentos, produção industrial e transporte. Atualmente, o bem-estar mundial e a competitividade industrial dependem da sustentabilidade e acessibilidade a energia. Os biocombustíveis são alternativas ecológicas de energia renovável que minimizam a dependência de fontes de energia fóssil. A biomassa pode ser utilizada como substrato para esses combustíveis renováveis, porém o pré-tratamento do substrato é uma etapa útil neste processo, pois facilita e acelera a hidrólise de materiais compostos. O objetivo deste trabalho foi (1) triar, selecionar, estudar e otimizar a formulação de coquetéis enzimáticos para a hidrólise de biomassa, onde as enzimas fornecidas foram tanto de origem comercial (Trichoderma reesei, Novozymes®) quanto de cultivo de micro-organismos (Aspergillus brasiliensis e Aspergillus tamarii Kita); (2) estudar a digestão anaeróbia da biomassa selecionada (i) quando hidrolisada ou não e (ii) em co-digestão com dejeto bovino; e (3) comparar a fermentação alcoólica da biomassa selecionada quando a hidrólise enzimática e fermentação são simultâneas ou separadas. Planejamentos experimentais de mistura foram realizados para otimizar a conversão de substratos enzimáticos em açúcares simples. O extrato bruto rico em amilase (AMI) teve uma influência positiva significativa na hidrólise do farelo de milho (Zea mays), maximizando a liberação de Açúcar Redutor (AR) (173 µmol/mL). CelluclastTM, rico em celulase, teve um efeito significativo na hidrólise da casca de banana (Musa ssp.), maximizando a produção de AR (175 µmol/mL). Estudos posteriores mostraram que o pré-tratamento enzimático de farelo de milho por AMI, contendo principalmente enzimas degradantes de amido, quando numa carga enzimática de 1:10 m/v (g de massa seca do substrato/ mL de extrato enzimático adicionado) a 45 °C por 48 h, resultou na hidrólise de 79 ± 5% dos carboidratos do substrato. Além disso, os testes de produção de metano mostraram que houve um aumento significativo na produção específica desse gás durante os ensaios em batelada quando as enzimas foram adicionadas diretamente ao digestor ou quando o hidrolisado de farelo de milho foi digerido. Além disso, foram encontrados efeitos sinérgicos ao co-digerir farelo de milho e dejeto bovino, levando a uma maior produção de metano (280 NmL / g SV) do que (200 NmL / g SV) a calculada com base no potencial de CH4 dos substratos individuais. Com relação aos efeitos a longo prazo, os experimentos semi-contínuos em escala laboratorial também demonstraram que a co-digestão de farelo de milho e dejeto bovino (1:1 na base de SV) levou a um processo estável ao longo do período de tempo estudado de 140 dias, atingindo uma taxa de carga orgânica de 3 g SV/L/dia e atingindo uma produção diária de metano de 1280,12 ± 99,4 NmL CH4/dia. No entanto, quando o farelo de milho foi investigado em mono-digestão, ou quando AMI foi adicionado diretamente durante a digestão semi-contínua do farelo, o acúmulo de ácidos graxos voláteis foi observado, o que levou a uma redução no pH. Por fim, a fermentação alcoólica a partir do farelo de milho pré-sacarificado com extrato enzimático bruto produzido por fungo filamentoso selvagem apresentou 100% de potencial etanólico com relação ao potencial teórico, o que mostra a promissora aplicação das enzimas brutas em processos industriais bioenergéticos.Energy consumption has enhanced as the world population has grown and countries became industrialized. Energy provides vital power for practically all human activities, e.g., lighting, food production, heating, cooking, industrial production, and transportation. Currently, the overall world well-being and industrial competitiveness are dependent on the sustainability and affordability of energy. Biofuels are environmental-friendly renewable energy alternatives which minimize the dependence on fossil energy sources. Biomass can be used as substrates for renewable fuels, nevertheless the substrate pretreatment is a step useful to this process, since it facilitates and accelerates the hydrolysis of compounded materials. The aim of this work was (1) to screen, select, study and optimize a cocktail formulation for biomass hydrolysis, where the enzymes were provided from both commercial source (Trichoderma reesei, Novozymes®) and microorganism cultivation (Aspergillus brasiliensis and Aspergillus tamarii Kita); (2) to study the anaerobic digestion of the selected biomass (i) when hydrolyzed or not and (ii) in co-digestion with cow manure; and (3) to compare the alcoholic fermentation of the selected biomass when enzymatic hydrolysis and fermentation are simultaneous or separated. Mixture experimental designs were performed to optimize the enzymatic substrates conversion into simple sugars. Crude extract rich in amylase (AMI) had a significant positive influence on cornmeal (Zea mays) hydrolysis by maximizing the Reducing Sugar (RS) yield (173 µmol/mL) and CelluclastTM, rich in cellulase had a significant effect on banana (Musa spp) peel hydrolysis by maximizing the RS yield (175 µmol/mL). Further studies, showed that the enzymatic pretreatment of cornmeal by AMI, containing mainly starch-degrading enzymes, with an enzyme load of 1:10 w/v (g of dry weight of substrate/ mL of enzyme extract added) at 45 °C for 48 h resulted in the hydrolysis of 79 ± 5% of substrate carbohydrates. Furthermore, the methane production tests showed that there was a significant enhancement in the specific gas production observed during the batch assays, both when enzymes were directly added to the digester or when cornmeal hydrolysate was digested. Additionally, synergetic effects were found when co-digesting cornmeal and cow manure, leading to higher methane yield (280 NmL/g VS) than that (200 NmL/g VS) calculated on the basis of CH4 potential of the individual substrates. Concerning long term effects, the laboratory-scale semi-continuous experiments similarly demonstrated that the co-digestion of cornmeal and cow manure (1:1 volatile solid (VS) basis) led to a stable process over the studied time period of 140 days reaching an organic loading rate of 3 g VS/L/day and reaching a daily methane production of 1280.12 ± 99.4 NmL CH4/day. Nevertheless, when cornmeal was investigated in mono-digestion, along with when the enzyme extract was directly added during semi-continuous digestion of cornmeal, volatile fatty acids accumulation was observed leading to a reduction in pH. Finally, the alcoholic fermentation from pre-saccharified cornmeal with crude enzymatic extract produced by wild filamentous fungus showed a 100% ethanolic potential in relation to the theoretical potential, which shows the promising application of crude enzymes in bioenergetic industrial processes

Production of enzymatic cocktails with potential into cellulose pulp biobleaching for paper production through lignocellulosic wastes and secondary fibres

Este trabalho teve como objetivo a prospecção e caracterização de enzimas fúngicas, que degradam a biomassa, visando à aplicação no biobranqueamento da polpa de celulose. Para isto, foram selecionados 13 fungos filamentosos da Micoteca e entre estes Aspergillus versicolor e A. brasiliensis foram aqueles que se sobressaíram quanto à produção de xilanase, amilase, CMCase, avicelase e ?-glucosidase. As melhores condições de produção enzimática corresponderam à utilização de bagaço de cevada como fonte de carbono em meio SR (Segato Rizzatti) para xilanase, amilase e ?- glucosidase e meio M5 para CMCase, avicelase e FPase, nos cultivos incubados por tempos variáveis de 72 a 168 horas. Para otimizar a concentração da fonte de carbono e a temperatura nos cultivos foi realizado um Delineamentro Composto Central Rotacional. Como resultante da otimização do bioprocesso foram obtidos os extratos brutos: (i) meio BSR, para a produção de xilanase por A. brasiliensis, com 96 horas de cultivo estático, a 30°C, com bagaço de cevada 3% em meio SR; (ii) Meio VSR para amilase e ?-glucosidase a partir do A. versicolor, com 120 horas de cultivo estático, a 35°C, com bagaço de cevada 3,41% em meio SR; (iii) Meio VM5 para CMCase, avicelase e FPase de A. versicolor com 120 horas de cultivo estático, a 30°C, com bagaço de cevada 2% em meio M5. Lacase foi produzida por Trametes versicolor (iv), cultivado por quinze dias em Fermentação Submersa estática, a 30°C, em meio contendo vinhaça e água destilada (1:5 v/v), 1% de algodão e 0,1% de peptona. Paralelamente, a produção de xilanases de A. tamarii Kita (v) com bagaço de cevada 2,9% foi otimizada em meio ADAMS por 129 horas (recebendo a denominação - meio TKADAMS). Quanto à caracterização das enzimas de A. versicolor (xilanase) e A. brasiliensis (demais enzimas) a temperatura ótima de xilanase foi 70°C; amilase 60-65°C; CMCase 65°C; avicelase 50°C; FPase e lacase 60°C e ?-glucosidase 70-75°C. Quanto à estabilidade térmica, xilanase mostrou-se com 60% de atividade relativa por até 24 horas à 40°C e 30 minutos à 50°C. A 60°C mostrou-se pouco estável. A amilase mostrou-se estável por 24 horas a 40 e 50°C, com 80% da atividade relativa. CMCase e FPase mostraram-se pouco estável nas temperaturas citadas. Avicelase apresentou ativação quando exposta ao aquecimento de 40, 50 e 60°C. ?-glucosidase foi estável a 40°C por até 24 horas, com atividade relativa de 80%; a 50°C apresentou 60% de atividade relativa por 180 minutos e a 60°C exibiu 40% por até 30 minutos. Lacase foi estável a 50°C, com um t50 de 60 minutos; a 60°C teve atividade relativa próxima de 30%, por até 240 minutos e a 70°C apresentou 40% de atividade por 30 minutos. Quanto ao pH, xilanase apresentou uma faixa ótima de 4,0-5,0 e também entre 7,0-8,0. Amilase, CMCase, avicelase, FPase, ?-glucosidase e lacase apresentaram atividades mais expressivas na faixa de pH 4,0-5,5. Com relação à estabilidade ao pH em 24 horas, xilanase foi mais estável nos pH 5,5-7,0, amilase nos pH 5,0-6,5, CMCase, FPase e lacase em pH 4,5, avicelase em pH 3,0 e ?-glucosidase em pH 5,0-5,5. Quanto ao efeito de íons, CMCase e ?-glucosidase foram ativadas por K+, Zn+ e Ba2+; CMCase, ?-glucosidase e lacase foram ativadas por NH4+ e Ca2+; amilase, CMCase, ?-glucosidase e lacase foram ativadas por Co2+; amilase, CMCase e ?- glucosidase foram ativadas por Al3+ e Fe2+; xilanase, avicelase, CMCase e ?-glucosidase foram ativadas por Mn+; avicelase e CMCase foram ativadas por Ag+; lacase por EDTA; ?-glucosidase e lacase por Mg2+, CMCase por Hg2+ e xilanase, ?-glucosidase e lacase por Cu2+. Os extratos foram utilizados na formulação de coquetéis enzimáticos para biobranqueamento da polpa de celulose. A aplicação dos extratos BSR, VSR, VM5 sobre a polpa marrom não resultou em uma redução significativa do número Kappa quando comparados ao controle, uma vez que todos os extratos apresentavam uma coloração muito escura, a qual fora originada por componentes e pigmentos provenientes dos meios de cultivo dos micro-organismos com bagaço de cevada e que, consequentemente, interferiram na determinação do número Kappa. Desta forma, o coquetel otimizado teve como formulação: 20,3 mL do meio TKADAMS e 10 mL do extrato do meio produtor de lacase para cada 4 gramas de polpa tratada, pH 5,5; 35,9ºC, 48 horas. Este tratamento resultou na redução de 1,83 pontos no número Kappa da polpa marrom, representando uma eficiência de 20,3%, e aumento de 4,65 na alvura, em relação ao controle. A aplicação do coquetel nos resíduos lignocelulósicos ocasionou a formação máxima de 85 mg/mL de açúcares redutores, em 24 horas no tratamento do bagaço de cevada, e 25 mg/mL de açúcares redutores, em 3 horas no tratamento do bagaço de cana. A aplicação do coquetel nas polpas de papel reciclado ocasionou um maior destintamento. A aplicação do coquetel desenvolvido na polpa de celulose, nos resíduos lignocelulósicos e fibras secundárias mostrou-se promissora para biobranqueamento, biodegradação e destintamento destes, respectivamente. A aplicação de enzimas no processo de biobranqueamento da polpa de celulose é uma alternativa viável e que auxilia a redução de custos, água, energia e colabora com o meio ambiente. A lacase foi importante no biobranqueamento da polpa de celulose sendo que o aumento da escala de produção do T. versicolor para biorreator levou a uma produção 6,25 vezes maior comparada aquela em Erlenmeyer, provavelmente devido a aeração constanteThis work aimed to prospect and characterize fungal enzymes, which break biomass, looking for the cellulose pulp biobleaching application. For this, 13 filamentous fungi of the Fungi Library were selected and among them Aspergillus versicolor and A. brasiliensis were those that stood out as inducers of xylanase, amylase, CMCase, avicelase and ?-glucosidase production. The use of barley bagasse as carbon source was the best condition for xylanase, amylase and ?-glucosidase production in SR medium (Segato Rizzatti) and M5 medium was the best for CMCase, avicelase and FPase production, in cultures incubated for 72-168 h. In order to optimize the concentration of the carbon source and the temperature of the cultures, a Central Composite Rotational Design was elaborated. (i) BSR extract, in SR medium for the xylanase production by A. brasiliensis, 96 hours, in static culture, at 30°C with barley bagasse 3%; (ii) VSR extract, in SR medium, for the amylase and ?-glucosidase from A. versicolor, 120 hours in static culture, at 35°C, with barley bagasse 3.41%. (iii) VM5 extract, in M5 medium for CMCase, avicelase and FPase from A. versicolor, 120 hours of static culture, at 30°C with barley bagasse 2%. Lacase was produced by Trametes versicolor (iv), in a medium containing vinasse and distilled water (1:5 v/v), cotton 1% and peptone 0.1%, for 15 days at 30ºC. In parallel, the production of a xylanase from A. tamarii Kita (v) with barley bagasse 2.9% was optimized in ADAMS medium for 129 hours (designated - TKADAMS medium). The thermal stability study showed that xylanase was stable with 60% of relative activity at 40°C for up to 24 hours and for 30 minutes at 50°C. At 60°C the enzyme was poorly stable. Amylase was stable for 24 hours at 40 and 50°C, with 80% of relative activity. CMCase and FPase was poorly stable at all temperatures tested. Avicelase was activated by the exposure at 40, 50 and 60°C. ?-glucosidase was stable at 40°C for up to 24 hours, with 80% of relative activity; at 50°C it showed a relative activity of 60% for 180 minutes and at 60° it showed 40% of relative activity for 30 minutes. Laccase was stable at 50°C with t50 for 60 minutes. At 60°C it showed an activity of 30% for up to 240 minutes and at 70°C it showed 40% of activity for 30 minutes. As for pH, xylanase showed the best activity in a range of pH 4.0-5.0 and 7.0-8.0. Amylase, CMCase, avicelase, FPase, ?-glucosidase and laccase showed expressive activities in a range of pH 4.0-5.5. The tests of pH stability in 24 hours showed that xylanase was stable at pH 5.5-7.0, amylase at pH 5.0-6.5, CMCase, FPase and laccase at pH 4.5, avicelase at pH 3.0 and ?-glucosidase at pH 5.0-5.5. The effect of ions showed that CMCase and ?-glucosidase were activated by K+, Zn+ and Ba2+; CMCase, ?-glucosidase and lacase were activated by NH4+ and Ca2+; amylase, CMCase, ?-glucosidase and lacase were activated by Co2+; amylase, CMCase and ?-glucosidase by Al3+ and Fe2+; xylanase, avicelase, CMCase and ?-glucosidase by Mn+; avicelase and CMCase by Ag+; lacase by EDTA; ?-glucosidase e lacase by Mg2+, CMCase by Hg2+ and xylanase, ?-glucosidase and lacase by Cu2+. The extracts were used in the formulation of enzymatic cocktails for cellulose pulp biobleaching. The application of BSR, VSR and VM5 extracts on the brown pulp did not result on a significant reduction of the Kappa number when compared to the control, on account of the dark coloration of these extracts caused by the components and pigments from the cultivation with the microorganisms and barley bagasse, which as a consequence, interfered in the determination of the Kappa number. Thus, an optimized cocktail was formulated: for each 4 grams of treated pulp 20.3 mL of TKADAMS extract and 10 mL of laccase extract, pH 5.5; 35.9°C, 48 hours. This treatment resulted in the reduction of 1.83 points in the Kappa number of the brown pulp, representing an efficiency of 20.3%, and a brightness increase of 4.65 when compared to the control. The application of the cocktail in the lignocellulosic residues resulted in the formation of 85 mg/mL of reducing sugars in barley bagasse treatment for 24 hours and, 25 mg/ml of reducing sugars in sugarcane bagasse treatment for 3 hours. The application of the cocktail in the recycled paper pulps caused a greater deinking. The application of the formulated cocktail in the cellulose pulp, lignocellulosic residues and secondary fibres was promising for biobleaching, biodegradation and deinking, respectively. The enzyme application in cellulose biobleaching is a viable alternative, which helps reducing costs, water, energy and collaborates with the environment. Laccase was important in the cellulose biobleaching and the increase of its production by T. versicolor through bioreactor led to a production 6.25 times higher than that in Erlenmeyer, probably due to the constant aeratio

Trametes versicolor laccase production using agricultural wastes: a comparative study in Erlenmeyer flasks, bioreactor and tray

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00449-019-02245-z) contains supplementary material, which is available to authorized users.Laccases are very interesting biocatalysts of recognized importance for several industrial applications. Its production by Trametes versicolor, a white-rot fungus, was induced by a combination of cotton gin wastes (1\%), a lignocellulosic waste, and vinasse (15\%), an industrial by-product from sugarcane industry. The use of these agro-industrial wastes are interesting, since it helps in reducing the enzyme production costs, due to their low cost and wide availability, as well as the environmental contamination issues, due to their improper disposal. Thus, laccase production was studied in submerged fermentation of T. versicolor using these agro-industrial wastes (cotton gin waste and vinasse) as carbon source and an additional nitrogen source (0.1\\% peptone). Three different bioreactors were evaluated for laccase production, such as BioFlo 310 bioreactor, aluminium tray and Erlenmeyer flasks to achieve high levels of laccase production. The highest specific production of laccase was found in BioFlo 310 bioreactor with 12 days of fermentation (55.24 U/mg prot.), which has been shown to be closely related to the oxygen supply to the microorganism through aeration of the fermentation medium. This study brings new insights into green biotechnology regarding vinasse utilization, which is frequently discharged in soils, rivers, and lakes causing adverse effects on agricultural soils and biota, as well as the cotton gin waste recovery.The authors are grateful to Mariana Cereia and Maurício de Oliveira for their technical assistance. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, processes 2010/52322-3; 2014/50884-5; 2018/07522-6), and V. E. P. was the recipient of a FAPESP fellowship (Process 2015/23200-0).info:eu-repo/semantics/publishedVersio