Ethanol Production from Oil Palm Trunk: A Combined Strategy Using an Effective Pretreatment and Simultaneous Saccharification and Cofermentation

Background. Oil palm trunk (OPT) with highly cellulose content is a valuable bioresource for bioethanol production. To produce ethanol from biomass, pretreatment is an essential step in the conversion of lignocellulosic biomass to fermentable sugars such as glucose and xylose. Several pretreatment methods have been developed to overcome biomass recalcitrance. In this study, the effects of different pretreatment methods such as alkali pretreatment, microwave-alkali, and alkaline peroxide combined with autoclave on the lignocellulosic biomass structure were investigated. Moreover, ethanol production from the treated biomass was performed by simultaneous saccharification and cofermentation (SSCF) under different temperatures, fermentation times, and cell ratios of Saccharomyces cerevisiae NCYC 479 and pentose-utilizing yeast, Pichia stipitis NCYC 1541. Results. Pretreatment resulted in a significant lignin removal up to 83.26% and cellulose released up to 80.74% in treated OPT by alkaline peroxide combined with autoclave method. Enzymatic hydrolysis of treated OPT resulted in an increase in fermentable sugar up to 93.22%. Optimization of SSCF by response surface method showed that the coculture could work together to produce maximum ethanol (1.89%) and fermentation efficiency (66.14%) under the optimized condition. Conclusion. Pretreatment by alkaline peroxide combined with autoclave method and SSCF process could be expected as a promising system for ethanol production from oil palm trunk and various lignocellulosic biomass.

Conversion of corn fiber into fuel ethanol

Corn fiber due to its chemical composition (up to 20% starch, 50 - 60% non-starch polysaccharides) and availability has potential to serve as a substrate for manufacture of various products, including fuel ethanol. This paper deals with assessment of fiber-to-ethanol conversion. The water/dry fiber ratio in suspensions was 10/1. Enzyme liquefaction and saccharification of residual starch in corn fiber was carried out in two steps with thermostable α-amylase (20 min, 120°C) and mixture of pullulanase and glucomalyse (24 hours, 60°C). Procedures resulted in release of 57.7±1.6 mg of glucose per gram of dry fiber basis. It responds to the dextrose equivalent expression to 96.7±2.2%. By fermentation of the starch hydrolysates by yeasts Saccharomyces cerevisiae CCY-11-3 (5% v/v inoculum, 28°C, 72 hours) 0.48 g of ethanol per gram of glucose in hydrolysates was obtained. The solids after starch hydrolysis were separated by filtration and processed by acid pretreatment (0.1 g of conc. HCl/g of biomass/5 ml of water, 120°C, 20 min) with subsequent enzyme hydrolysis (24 hours, 60°C) by the multienzyme preparations containing cellulases and hemicellulases. Overall yield of reducing sugars after these two steps was 740.7±3.9 mg/gram of dry corn fiber basis. Fermentation of lignocellulosic hydrolysates by yeasts Pichia stipitis CCY-39-50-1 and Candida shehatea CCY-29-68-4 (in both cases 5% v/v inoculum, 28°C, 72 hours) resulted in 0.38 and 0.12 g of ethanol per gram of reducing sugars. The results indicate that applied pretreatment methods and used microorganisms are able to produce ethanol from corn fiber.

Studies on Bioethanol Production with Thermo Tolerant Yeast Isolates and their Co-Cultures using African Wild Cocoyam as Feedstock

In this work different ways of optimally producing bioethanol at various pH with thermotolerant yeasts and their cocultures using a non-human edible starchy food as feedstock was examined. African wild cocoyam, Xanthosoma roseum, sourced from abandoned farmlands in Obukpa, Nsukka, Nigeria was used as the substrate, while strains of Kluyveromyces marxianus and Pichia stipitis were used to ferment them. First the tubers were gelatinized by boiling under pressure above 100oC before hydrolysis with concentrated H2SO4. The hydrolysates were then fermented at 35oC with the thermotolerant yeasts for five days at different pH. Results obtained showed that gelatinized sample of the substrate gave optimum glucose yield when hydrolysed with 1M H2SO4 for 60 minutes. Kluyveromyces marxianus produced more ethanol than Pichia stipitis at all the four fermentation pH values tested. However, optimum ethanol production was obtained when the two yeast strains were used as coculture at pH 4.5. The peak time for ethanol production was 96 hours for the individual yeast cultures while that of their coculture was 72 hours. The results of the study indicated that wild cocoyam is an excellent feedstock for bioethanol production with many advantages including being non-edible, thereby eliminating concerns for food security, and containing high amount of carbohydrate. The study also revealed that fermenting sugar hydrolysates with a coculture of microorganisms during bioethanol production is a more efficient process than using individual cultures.

Bioethanol Production by Pichia stipitis Immobilized on Water Hyacinth and Thin-Shell Silk Cocoon

Cell immobilization technique was applied in this study in order to examine effect of immobilized Pichia stipitis TISTR5806 on bioethanol production. Water hyacinth (WH) and thin-shell silk cocoon (CC) were used as cell carriers. Characteristics of the cell carriers were examined to explain the mechanism of bioethanol production. Carrier sizes and weights were optimized to improve bioethanol production. Moreover, stabilities of immobilized cells and carriers were evaluated. Because of high porosity, high surface area and good swelling ability of WH, cell immobilized on 1 g WH with 1 cm length produced the highest ethanol concentration at 13.3 g/L. Five cycles of a repeated batch of immobilized cell (IC) system on WH showed stable performance in ethanol production (8.2–10.4 g/L) with large numbers of the immobilized cells. The interaction between the immobilized cells and the WH surface were discovered.

Strain improvement of native Saccharomyces cerevisiae LN ITCC 8246 strain through protoplast fusion to enhance its xylose uptake

Co-utilization of xylose and glucose and subsequent fermentation using Saccharomyces cerevisiae could enhance ethanol productivity. Directed engineering approaches have met with limited success due to interconnectivity of xylose metabolism with other intrinsic, hidden pathways. Therefore, random approaches like protoplast fusion were used to reprogram unidentified mechanisms. Saccharomyces cerevisiae LN, the best hexose fermenter, was fused with xylose fermenting Pichia stipitis NCIM 3498. Protoplasts prepared using glucanex were fused under electric impulse and fusants were selected using 10% ethanol and cycloheximide (50 ppm) markers. Two fusants, 1a.23 and 1a.30 showing fast growth on xylose and tolerance to 10% ethanol, were selected. Higher extracellular protein expression observed in fusants as compared to parents was corroborated by higher number of bands resolved by twodimensional analysis. Overexpression of XYL1, XYL2, XKS and XUT4 in fusants as compared to S. cerevisiae LN as observed by RT-PCR analysis was substantiated by higher specific activities of XR, XDH and XKS enzymes in fusants. During lignocellulosic hydrolysate fermentation, fusants could utilize glucose faster than the parent P. stipitis NCIM 3498 and xylose consumption in fusants was higher than S. cerevisiae LN.