Pretratamiento de bagazo de caña de azúcar por ozonólisis para obtención de bioalcoholes: Efectos sobre la liberación de azúcares, la generación de inhibidores y las fermentaciones

En las últimas décadas, el creciente aumento de la población humana asociado al desarrollo industrial ha llevado al consumo excesivo de combustibles fósiles, que han sido los responsables del incremento en las concentraciones de gases contaminantes en la atmosfera, amenazando la calidad ambiental del planeta. Este panorama ha llevado a la búsqueda de nuevas alternativas para la sustitución de los combustibles fósiles por otras reservas energéticas ambientalmente sostenibles y provenientes de fuentes renovables. Una de las opciones más prometedoras es la producción de biocombustibles líquidos para utilización como carburantes a partir de residuos abundantes como los lignocelulósicos, ricos en azúcares. Su principal ventaja es que se trata de residuos que, en vez de ser tratados como tales, pueden ser valorizados generando productos de valor añadido, pero, además, suponen una baja huella de gases del efecto invernadero. Sin embargo, su producción es compleja, y presenta retos tecnológicos que todavía deben de ser superados para llegar a procesos que sean económicamente viables. La producción de biocombustibles lignocelulósicos, en lo que se denomina combustibles de segunda generación, puede dividirse, de manera simplificada, en tres etapas principales. La primera etapa, el pretratamiento, es la clave para la obtención de buenos rendimientos, es la responsable de la desestructuración de la biomasa y la liberación de los polímeros de azúcares de su matriz con la lignina que les protege. Una vez liberados los polímeros de azúcares, la etapa siguiente, la hidrólisis enzimática, tiene por objetivo su sacarificación y producción de azúcares más pequeños que puedan ser convertidos por microorganismos en los productos de interés. En una última etapa, los hidrolizados ricos en azúcares simples son fermentados por microorganismos especializados, convirtiéndolos en biocombustibles. En esta tesis doctoral se ha estudiado la aplicación del ozono como forma de pretratamiento para producción de biocombustibles a partir de un residuo lignocelulósico muy abundante, el bagazo de caña de azúcar. Este pretratamiento, llamado ozonólisis, es un método químico oxidativo para la remoción de la lignina por degradación y/o solubilización.

Ozonolysis: an advantageous pretreatment for lignocellulosic biomass revisited.

Ozonolysis, as a lignocellulosic biomass pretreatment, goes back to 80s; however, in the last years it is becoming widespread again owing to its efficiency and mild operation conditions. Ozone reacts preferably with lignin than carbohydrates, promoting biomass destructuration and delignification, and so the sugar release by enzymatic hydrolysis. The hydrolysate from pretreated biomass has being used as sugars source for second-generation fuels production, mainly ethanol, methane and hydrogen. Short-chain carboxylic acids are the main inhibitory compounds generated, being properly removed by water washing. The most common inhibitory compounds reported for other pretreatments, furfural and HMF (5-hydroxymethylfurfural) , are not found in ozone-pretreated hydrolysates. Composition of pretreated biomass and ozone consumption depends on several process parameters: reactor design, moisture content, particle size, pH, reaction time, ozone/air flow and ozone concentration. Additional studies are necessary to clarify process parameters effec

24th European Biomass Conference & Exhibition

As a result of the current oil crisis, there is an increasing interest in the development of alternative energy sources characterized for being renewable, economically competitive and environmentally friendly. The production of biofuels through biomass conversion from lignocellulosic materials, or more recently, from microalgae biomass, seems to be an attractive option among them. Within the biofuel production, enzymatic hydrolysis represents a key point that must be carefully considered, as the costs derived from the commercial enzymes used during the hydrolysis can compromise the economical feasibility of the process. The microalgae biomass has proved to be an effective wastewater treatment with high nutrient contents. The nutrients accumulation (N and P) in the biomass, which was produced in these processes of treatment, becomes an attractive substrate for the enzyme production. This work aims at implementing the biorefinery concept by valorising the microalgae biomass produced from agro-food industry wastewater treatment as a substrate for enzyme production and the subsequent biofuel generation through enzymatic hydrolysis. Thus, the production of cellulases and xylanases has been tested for the fungal specie Trichoderma reesei QM9414 using microalgae biomass through solid-state fermentation (SSF). The microalgae biomass was obtained from a mixture of primary wastewater and fertilizer treatment in raceway reactor and thin layer, respectively. This biomass produced was a cocktail of microalgae, principally Scenedesmus obliquus with a content of 45.03% C, 7.80% N and 1.99% P. The fungus, initially stocked at 4ºC in the private collection, was inoculated in commercial potato dextrose agar (PDA) at 28ºC during 7 days, and was suspended in water using it as pre-inoculum. Enzyme production was conducted with different substrates: microalgae, 1:1 sugarcane bagasse:microalgae and 1:1 sugarcane bagasse:wheat bran. It was worked by adding different concentrations of saline solution at 28ºC during 7 days, reducing the external nutrients supply, demonstrating the advantages of using microalgae as a substrate for enzyme production. Each 24 hours two flasks were taken and the enzymes were suspended in water at 150 rpm during 1 h and then centrifuged at 20000xg during 20 min. The supernatants were used for enzyme activity assays that were conducted according to IUPAC recommendations. The enzymes, produced during this process, were used for the enzymatic hydrolysis of microalgae biomass, and other lignocellulosic materials such as sugarcane bagasse; reporting promising results compared with the commercial enzymes

Pretratamiento de bagazo de caña de azúcar por ozonólisis para obtención de bioalcoholes: Efectos sobre la liberación de azúcares, la generación de inhibidores y las fermentaciones

En las últimas décadas, el creciente aumento de la población humana asociado al desarrollo industrial ha llevado al consumo excesivo de combustibles fósiles, que han sido los responsables del incremento en las concentraciones de gases contaminantes en la atmosfera, amenazando la calidad ambiental del planeta. Este panorama ha llevado a la búsqueda de nuevas alternativas para la sustitución de los combustibles fósiles por otras reservas energéticas ambientalmente sostenibles y provenientes de fuentes renovables. Una de las opciones más prometedoras es la producción de biocombustibles líquidos para utilización como carburantes a partir de residuos abundantes como los lignocelulósicos, ricos en azúcares. Su principal ventaja es que se trata de residuos que, en vez de ser tratados como tales, pueden ser valorizados generando productos de valor añadido, pero, además, suponen una baja huella de gases del efecto invernadero. Sin embargo, su producción es compleja, y presenta retos tecnológicos que todavía deben de ser superados para llegar a procesos que sean económicamente viables. La producción de biocombustibles lignocelulósicos, en lo que se denomina combustibles de segunda generación, puede dividirse, de manera simplificada, en tres etapas principales. La primera etapa, el pretratamiento, es la clave para la obtención de buenos rendimientos, es la responsable de la desestructuración de la biomasa y la liberación de los polímeros de azúcares de su matriz con la lignina que les protege. Una vez liberados los polímeros de azúcares, la etapa siguiente, la hidrólisis enzimática, tiene por objetivo su sacarificación y producción de azúcares más pequeños que puedan ser convertidos por microorganismos en los productos de interés. En una última etapa, los hidrolizados ricos en azúcares simples son fermentados por microorganismos especializados, convirtiéndolos en biocombustibles. En esta tesis doctoral se ha estudiado la aplicación del ozono como forma de pretratamiento para producción de biocombustibles a partir de un residuo lignocelulósico muy abundante, el bagazo de caña de azúcar. Este pretratamiento, llamado ozonólisis, es un método químico oxidativo para la remoción de la lignina por degradación y/o solubilización.

Utilização de processos oxidativos avançados baseados em ozônio como pré-tratamento para bagaço de cana-de-açúcar, e hidrólise do bagaço pré-tratado com combinado enzimático dos fungos Penicillium viridicatum RFC3 e Trichoderma reesei QM9414

Neste trabalho foi estudada a utilização do ozônio como pré-tratamento do bagaço de cana-de-açúcar para posterior hidrólise enzimática. O bagaço in natura foi caracterizado quimicamente, e após cada ensaio de pré-tratamento foram quantificados no bagaço pré-tratado: celulose, xilana, lignina ácida insolúvel (LAI), lignina ácida sóluvel (LAS) e lignina total (LT). Após os pré-tratamentos as amostras foram hidrolisadas enzimaticamente por mistura dos complexos NS50013 e NS50010 de enzimas comerciais fornecidas pela Novozymes. Foram realizados quatro ensaios de pré-tratamento, variando-se a umidade e a concentração de ozônio (gO3/h), e mantendo-se constante o fluxo 60 L/h e o tamanho de partícula em 3-5 mm. A melhor condição de ensaios encontrada foi com 40 % de umidade e 3,85 gO3/h, onde houve diminuição na quantidade de LAI de 19,54±0,03 para 6,49±0,03 %, aumento na LAS de 3,13±0,04 para 7,21±0,09 % e diminuição da LT de 22,67±0,07 para 13,70±0,12 %, comparando-se com o bagaço in natura. No mesmo ensaio houve uma conversão de hidrólise enzimática da celulose de 39,27±0,32 % no bagaço pré-tratado e 50,62±1,59 % no pré-tratado e lavado, da xilana de 55,90±0,14 % no pré-tratado e 28,14±0,75 % no pré-tratado lavado, de açúcares de 44,61±0,17 % no pré-tratado e 43,41±0,84 % no pré-tratado lavado. Houve pouco ataque do ozônio aos carboidratos em todos os ensaios, sendo o maior ataque no ensaio de melhores rendimentos supracitado, com perda de menos de 5 pontos percentuais na quantidade de celulose e pouco mais de 1 ponto percentual na quantidade de xilana. A análise de inibidores revelou baixas concentrações destes, sendo o de maior concentração o ácido acético, com 1,40040±0,07359 g·L-1 no hidrolisado. A análise das amostras pré-tratadas por micrografia eletrônica de varredura...In this work was studied the utilization of the ozone as a pretreatment of the sugarcane bagasse for enzymatic hydrolysis. The in natura bagasse was chemically characterized, and after each pretreatment test was quantified in the pretreated bagasse: cellulose, xylan, acid insoluble lignin (AIL), acid soluble lignin (ASL) and total lignin (TL). After the pretreatments the samples was enzymatic hydrolyzed by a mixture of NS50013 and NS50010 complexes, of commercial enzymes provided by Novozymes. Was realize four tests of pretreatment, changing the moisture and ozone concentration (gO3/h), fixing the flux in 60 L/h and the particle size at 3-5 mm. The best condition of test was find at 40 % of moisture and 3,85 gO3/h, where was a diminution in the AIL from 19,54±0,03 to 6,49±0,03, a increase in the ASL from 3,13±0,04 to 7,21±0,09 %, and diminution of LT from 22,67±0,07 to 13,70±0,12 %, comparing to the in natura bagasse. In the same test was a conversion of enzymatic hydrolysis of the cellulose of 39,27±0,32 % in the pretreated bagasse and 50,62±1,59 % in the pretreated and washed, of xilan 55,90±0,14 % in the pretreated bagasse and 28,14±0,75 % in the pretreated and washed, of sugar 44,61±0,17 % in the pretreated bagasse and 43,41±0,84 % in the pretreated and washed. Was a little attack to the carbohydrates in all the tests, being the major attack in the test of the best yields aforementioned, with loss of less than 5 percentage points in the cellulose and little more than 1 percentage point in the amount of xylan. The inhibitors analysis showed slow concentrations of they, being the major concentration of acetic acid, with 1,40040±0,07359 g·L-1 in the hydrolyzed. The electron micrograph analysis of the pretreated samples showed that the ozone pretreatment is able to modify microscopically the bagasse, exposing the fibers and increasing the amount and size of the poresFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Sugarcane bagasse ozonolysis pretreatment: Effect on enzymatic digestibility and inhibitory compound formation

Sugarcane bagasse was pretreated with ozone to increase lignocellulosic material digestibility. Bagasse was ozonated in a fixed bed reactor at room temperature, and the effect of the two major parameters, ozone concentration and sample moisture, was studied. Acid insoluble and total lignin decreased whereas acid soluble lignin increased in all experiments. Pretreatment barely attacked carbohydrates, with cellulose and xylan recovery rates being >92%. Ozonolysis increased fermentable carbohydrate release considerably during enzymatic hydrolysis. Glucose and xylose yields increased from 6.64% and 2.05%, for raw bagasse, to 41.79% and 52.44% under the best experimental conditions. Only xylitol, lactic, formic and acetic acid degradation compounds were found, with neither furfural nor HMF (5-hydroxymethylfurfural) being detected. Washing detoxification provided inhibitor removal percentages above 85%, increasing glucose hydrolysis, but decreasing xylose yield by xylan solubilization. SEM analysis showed structural changes after ozonization and washing. © 2013 Elsevier Ltd

Studies on the application of Myceliophthora thermophila JCP1-4 cellulases cocktail on sugarcane bagasse pretreated by different methods

The hydrolysis step for sugar production in biorefineries is crucial for the sequential processes involved and cellulases cocktails behave differently according to the pretreatment employed. In this study, the application of the cellulases cocktail produced by the fungus Myceliophthora thermophila JCP1-4 was studied on the saccharification of sugarcane bagasse pretreated by ozonolysis and thermic ferric nitrate (TFN), and the results were compared with commercial enzymes (Novozymes Celluclast 1.5L, Novozym 188). The fungal cellulases cocktail hold an activity of FPU:β-glucosidase of 1:4(U/mL); time, temperature, FPU by g of cellulose load and percentage of dry matter (DM) were studied. The analysis of central composite design of TFN pretreated showed that fungal cellulases works better in DM values of 3–3.5% (4.5% for commercial), temperatures higher than 50 °C (<45 °C for commercial) and 15FPU for both; commercial enzymes yielded 7.78 g/L of reducing sugars and the fungal enzymes 5.42 g/L. With the ozone pretreated, the fungal enzymes presented a higher thermostability with faster kinects, being able to produce 5.56 g/L of reducing sugars (60 °C, 8 h), against 5.20 g/L for commercial enzymes (50 °C, 24 h), (10FPU, 3%DM for both). The FPU derivate analysis revels better yields with 7.5FPU, and the increase of DM to 7.5% resulted 13.28 g/L of reducing sugars