Browsing by Author "Ladeira, S. A."
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- Characterization of thermophile xylanase produced by anoxybacillus sp. strain 3M in submerged fermentation using brewers’ spent grainPublication . Alves, Luís; Ladeira, S. A.; Paixão, Susana M.The main goal of this study was the characterization of the extremophile xylanases produced by by Anoxybacillus sp. strain 3M, a thermophilic bacterium isolated from terrestrial hot springs (temperature of 90°C) samples collected on S. Miguel, Azores, Portugal. In this context, several batch fermentations using different agro-industrial wastes as inducer substrates (BSG – Brewers’ spent grain, wheat straw, sugarcane bagasse, and corn cobs) were performed towards an optimal enzyme production. The results for xylanase production showed that the higher enzymatic levels were obtained in the growth medium containing 1% (w/v) BSG (1.34 U mL-1), indicating that the BSG was the best inducer substrate, but the xylanase activity was also observed when wheat straw (1.33 U mL-1), sugarcane bagasse (0.80 U mL-1), corn cobs (0.30 U mL-1) and commercial xylan (0.21 U mL-1) were used as inducers. The extracellular crude enzymatic extract produced by Anoxybacillus sp. 3M in optimized submerged fermentation with BSG, attaining a maximal xylanase activity of 1.41 U mL-1 (i.e. 5-fold higher than with xylan), was further characterized for its optimal temperature and pH and stability. The highest enzyme activity was observed at a temperature of 60ºC and pH 5.3, but the enzyme retained 100% of its original activity after 96 h at 60°C and pH 7.0 and it still maintained 46% activity after 28 days at the same conditions. At 60ºC, the enzyme also retained its activity for pH ranging from 7.0 to 10.5. In addition, a zymogram of native gel analysis of the different culture supernatants revealed the presence of an enzymatic complex with an apparent molecular weight ranging from 400-500 kDa. Further application of strain 3M enzymatic extract to commercial oat spelts xylan revealed the presence of xylose and xylooligosaccharides (XOS), mainly X2 and X3, in the hydrolyzates produced. So, the thermostable and alkalistable extracellular xylanases from Anoxybacillus sp. 3M are biocatalysts with high potential for the development of processes where lignocellulosics are converted to precursors for several biotechnological applications.
- KOH for enhanced sugarcane bagasse delignification and further production of sugar-rich hydrolyzates by enzymes applicationPublication . Paixão, Susana M.; Ladeira, S. A.; Arez, B. F.; Martins, M. L. L.; Roseiro, J. Carlos; Alves, LuísLignocellulosic biomass is envisaged as an important raw material for bioethanol production due to its low cost and high availability. Sugarcane bagasse (SCB), a fibrous residue of cane stalks left over after crushing and extraction of the juice from sugarcane; it is one of the largest cellulosic agro-industrial by-products. Tons of SCB are produced in Brazil as a waste of sugar and ethanol industries. This lignocellulosic by-product is a potential renewable source for 2G-bioethanol production. Usually, SCB is pretreated using alkaline and/or acid treatments viewing higher ethanol yields. The main goal of this study was to optimize the delignification of SCB towards the higher availability of glucans and xylans for further enzymatic hydrolysis to obtain sugar-rich syrups that will be more readily fermented to bioethanol. The delignification was carried out by autoclaving the biomass with KOH and the influence of KOH concentration (1-10%) and the autoclave time (10-60 min) were evaluated through a statistical design. Experimental distribution for two factors according to the Doehlert uniform design was used to produce response surfaces. The responses studied in this design were the percentage of hemicellulose, lignin and total polysaccharides. The results showed that from the two factors evaluated, the KOH concentration was the one that most influenced the response observed and that the treatments of SCB with KOH 5-10% for 35 minutes of autoclave at 121ºC and 1 atm led to the highest rates of lignin extraction. Using KOH treatment, a significant reduction of lignin content in SCB was observed, namely from 19% to 5%. Scanning electron micrographs of SCB pre-treated with 10% KOH for 35 minutes demonstrated a change in the structure of the material, with the appearance of broken structures, which can be attributed to the alkaline treatment. To validate the experiments, the SCB pretreated in the optimal conditions (95% of total polysaccharides) was further hydrolyzed with commercial enzymes and the enzymatic hydrolysis performance was evaluated.
- Properties of Anoxybacillus sp. 3M Xylanases and further application towards sugar rich hydrolysatesPublication . Alves, Luís; Ladeira, S. A.; Arez, B. F.; Paixão, Susana M.This study aimed to optimize the production of xylanases by Anoxybacillus sp. strain 3M, a thermophilic bacterium isolated from terrestrial hot springs (temperature of 90°C) samples collected on S. Miguel, Azores, Portugal, in batch fermentation testing several agroindustrial byproducts as inducer substrates (BSG - Brewer's spent grain, wheat straw, sugarcane bagasse, and corn cobs). In addition, the xylanases produced by this bacterium with the best inducer substrate were characterized for their optimal pH, temperature and stability. The results for xylanase production showed that the higher levels of xylanases were obtained in growth medium containing 1% (w / v) BSG (1.35 U/mL), but the xylanolytic activity was also observed when wheat straw (1.32 U/mL), sugarcane bagasse (0.80 U/mL), corn cobs (0.30 U/mL) and commercial xylan (0.21 U/mL) were used as substrates. The extracellular crude enzymatic extract from Anoxybacillus sp. 3M was then characterized for its optimal temperature and pH and stability. The best enzyme activity was observed at a temperature of 60 ºC and pH 5.3, and the enzyme retained 100% of its original activity after 96 h at 60 °C and pH 7.0. Zymogram of native gel analysis of the different culture supernatants revealed the presence of an enzyme complex with a molecular weight of 420 kDa. This xylanase may be considered as a biocatalyst thermotolerant and it is interesting for biotechnological applications. Further application of Anoxybacillus 3M crude enzymatic extract to BSG and commercial xylan revealed the presence of xylose and xylooligosaccharides, mainly X2 and X3, in the hydrolizates produced.
- Sugarcane bagasse delignification with potassium hydroxide for enhanced enzymatic hydrolysisPublication . Paixão, Susana M.; Ladeira, S. A.; Silva, Tiago; Arez, B. F.; Roseiro, J. Carlos; Martins, M. L. L.; Alves, LuísThe optimization of an alkaline pretreatment process for the delignification of sugarcane bagasse (SCB) to enhance the subsequent enzymatic hydrolysis was performed according to the Doehlert uniform shell design. In this experimental design, the effect of two factors—potassium hydroxide (KOH) concentration and autoclaving time at 121 C (1 atm)—on cellulose, hemicellulose, or the total polysaccharide and lignin content in SCB was evaluated. This response surface methodology revealed that KOH concentration is the factor that most influences the chemical characteristics of treated SCB (SCBt), with optimal conditions for the highest delignification being KOH in the range 5–10% (w/v) and an autoclaving time of 35 min, which provides an average of 97% total polysaccharides without inhibitor accumulation (furfural, 5-hydroxymethyl furfural) and #5% lignin. SCBt samples from two pretreatment conditions (KOH 3.25% – 13 min; KOH 10% – 35 min) were selected, based on the greatest delignification (70–74%) and polysaccharide availability (95–97%) after pretreatment, and further hydrolysed for fermentable sugar production. High sugar yields were obtained from both the pretreated samples (866 to 880 mg sugar per g biomass, respectively) in contrast with the 129 mg sugar per g raw biomass obtained from untreated SCB. These results demonstrate the effectiveness of KOH alkali pretreatments, which improves the overall digestibility of raw SCB polysaccharides from about 18% up to 91%. However, harsh alkali treatment (KOH 10%) is the most effective if the highest glucose/xylose ratio in the final sugar-rich hydrolysate is the aim. Hence, the use of sugar-rich hydrolysates obtained from SCBt as the carbon source for industrial purposes may provide a sustainable and economic solution for the production of bio-based added-value products, such as second generation (2G) bioethanol.