Development of the bioprocess of ethanol production from starch residues using amylolytic enzymes produced by Rhizopus oligosporus CCT 3762
Carregando...
Data
2019-12-16
Autores
Escaramboni, Bruna [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Esta tese propõe algumas rotas biotecnológicas considerando os desafios envolvidos na produção e aplicação de enzimas fúngicas e glicose, bem como novas perspectivas para a produção de etanol utilizando matérias-primas alternativas. Inicialmente, a análise dos dados por mínimos quadrados parciais (PLS) demonstrou a correlação positiva entre a produção de amilase por Rhizopus oligosporus e a suplementação do meio de fermentação em estado sólido (FES) com nitrogênio orgânico. O extrato amilolítico apresentou desempenho competitivo e maior produtividade na hidrólise do amido do que uma glucoamilase comercial. A amilase produzida foi aplicada (15 U/g) no bagaço de mandioca (BM) para obtenção do hidrolisado de bagaço de mandioca (HBM) resultado de 42% de conversão do BM em açúcares redutores com uma hidrólise em etapa única de 10 h à 55°C. O HBM concentrado foi utilizado para fermentação alcoólica em batelada alimentada, produzindo 89,2% de rendimento. Além disso, comparando-se apenas o custo das matérias-primas, o HBM demonstrou ser mais econômico para a fermentação alcoólica do que a cana-de-açúcar. Em sequência, foi aplicado um planejamento fatorial em três níveis para otimizar a proporção enzimática de 0-30 U/g na hidrólise. Na condição otimizada 61,67% de BM foi convertido em glicose. Uma mistura sinérgica adequada reduziu 2 vezes a proporção de amilases (15 U/g) e 3 vezes para celulases (5 FPU/g), convertendo 51,79% do BM. Modificações na superfície do BM causadas pela ação enzimática foram evidenciadas por microscopia eletrônica de varredura. A hidrólise de 15% BM em biorreator produziu elevado teor de glicose (90,87 g/L; YP/S = 0,50 g/g). Além disso, o HBM foi aplicado para produzir 35,25 g/L de etanol (eficiência de 78,3%). Com isso, o bioprocesso proposto produziria 254,1 L de etanol por tonelada de BM seco em 48 h, incluindo hidrólise e fermentação alcoólica. Finalmente, um tratamento biológico de resíduos alimentares via FES foi proposto para a produção de amilases e proteases por R. oligosporus. A maior produção ocorreu com: 50% de resíduo alimentar (RA), 10% de bagaço de cana (BC) e 40% de farelo de trigo (FT), suplementado com solução salina, para amilases, e acrescido de 20% (v/m) de água de maceração de milho, para produção de proteases. A suplementação com água de maceração de milho foi adequada como alternativa à solução de sais e o reciclo de biomassa e fibras não hidrolisadas foi capaz de aumentar o rendimento de amilase e protease. O extrato enzimático foi efetivo para hidrolisar o RA, alcançando 36,44% de conversão e um hidrolisado com 47,10 g/L de açúcares redutores.
This thesis proposes some biotechnological routes considering the challenges involved in the production and application of fungal enzymes and glucose, as well as new perspectives for the production of ethanol using alternative raw materials. Initially, partial least square (PLS) data analysis demonstrated the positive correlation between the amylase production by Rhizopus oligosporus and the organic nitrogen supplementation of the solid state fermentation (SSF) medium. Crude amylase extract had competitive performance features giving higher productivities in starch hydrolysis than a commercial glucoamylase. The amylase produced was applied (15 U/g) in the cassava bagasse (CB) to obtain cassava bagasse hydrolysate (CBH). More than 42% conversion in reducing sugars was achieved with an efficient 10 h single-step hydrolysis at 55°C. The concentrated CBH was subsequently used in fed-batch process producing 89.2% ethanol yield. Furthermore, comparing just the cost of the raw materials sugarcane and CHB, the latter demonstrated to be a lower-cost feedstock for ethanol fermentation. In sequence, a three-level factorial design was applied to optimize enzymatic proportion from 0-30 U/g in hydrolysis. Optimized enzyme mixture efficiently converted 61.67% CB into glucose. An appropriated synergistic mixture was responsible for decreasing the proportion of amylases 2 times (15 U/g) and cellulases 3 times (5 FPU/g), converting 51.79% of the CB. Scanning electron microscopy image evidenced modifications on CB surface caused by the enzymatic action. Hydrolysis of 15% CB in bioreactor released great level of glucose content (90.87 g/L; YP/S = 0.50 g/g). In addition, CBH was applied to produce 35.25 g/L ethanol (78.3% efficiency). Therefore, proposed bioprocess would produce 254.1 L of ethanol per ton of dry CB in 48 h, including the hydrolysis and alcoholic fermentation period. Finally, a biological treatment of food waste by SSF culture was proposed for the production of amylases and proteases by R. oligosporus. The highest enzymatic production using food waste as the main substrate was using the blend: 50% food waste, 10% sugarcane bagasse and 40% wheat bran supplemented with salt solution for amylases, and added by corn steep liquor 20% (v/w) for proteases production. The SSF supplementation with corn steep liquor was suitable as an alternative to replace the salts solution and the recycle of the biomass and non-hydrolyzed fibers of the wastes was able to increase amylase and protease yields. The enzymatic extract produced was able to hydrolyze food waste achieving 36.44% conversion yield and a hydrolysate with 47.10 g/L reducing sugars.
This thesis proposes some biotechnological routes considering the challenges involved in the production and application of fungal enzymes and glucose, as well as new perspectives for the production of ethanol using alternative raw materials. Initially, partial least square (PLS) data analysis demonstrated the positive correlation between the amylase production by Rhizopus oligosporus and the organic nitrogen supplementation of the solid state fermentation (SSF) medium. Crude amylase extract had competitive performance features giving higher productivities in starch hydrolysis than a commercial glucoamylase. The amylase produced was applied (15 U/g) in the cassava bagasse (CB) to obtain cassava bagasse hydrolysate (CBH). More than 42% conversion in reducing sugars was achieved with an efficient 10 h single-step hydrolysis at 55°C. The concentrated CBH was subsequently used in fed-batch process producing 89.2% ethanol yield. Furthermore, comparing just the cost of the raw materials sugarcane and CHB, the latter demonstrated to be a lower-cost feedstock for ethanol fermentation. In sequence, a three-level factorial design was applied to optimize enzymatic proportion from 0-30 U/g in hydrolysis. Optimized enzyme mixture efficiently converted 61.67% CB into glucose. An appropriated synergistic mixture was responsible for decreasing the proportion of amylases 2 times (15 U/g) and cellulases 3 times (5 FPU/g), converting 51.79% of the CB. Scanning electron microscopy image evidenced modifications on CB surface caused by the enzymatic action. Hydrolysis of 15% CB in bioreactor released great level of glucose content (90.87 g/L; YP/S = 0.50 g/g). In addition, CBH was applied to produce 35.25 g/L ethanol (78.3% efficiency). Therefore, proposed bioprocess would produce 254.1 L of ethanol per ton of dry CB in 48 h, including the hydrolysis and alcoholic fermentation period. Finally, a biological treatment of food waste by SSF culture was proposed for the production of amylases and proteases by R. oligosporus. The highest enzymatic production using food waste as the main substrate was using the blend: 50% food waste, 10% sugarcane bagasse and 40% wheat bran supplemented with salt solution for amylases, and added by corn steep liquor 20% (v/w) for proteases production. The SSF supplementation with corn steep liquor was suitable as an alternative to replace the salts solution and the recycle of the biomass and non-hydrolyzed fibers of the wastes was able to increase amylase and protease yields. The enzymatic extract produced was able to hydrolyze food waste achieving 36.44% conversion yield and a hydrolysate with 47.10 g/L reducing sugars.
Descrição
Palavras-chave
Etanol, Amilase, Glicose, Fermentação em estado sólido, Rhizopus oligosporus, Ethanol, Glucose, Amylase, Solid state fermentation, Bioenergia, Bioenergy