Enzymatic hydrolysis and simultaneous saccharification and fermentation of alkali/peracetic acid-pretreated sugarcane bagasse for ethanol and 2,3-butanediol production

The enzymatic digestibility of alkali/peracetic acid (PAA)-pretreated bagasse was systematically investigated. The effects of initial solid consistency, cellulase loading and addition of supplemental β-glucosidase on the enzymatic conversion of glycan were studied. It was found the alkali-PAA pulp showed excellent enzymatic digestibility. The enzymatic glycan conversion could reach about 80% after 24 h incubation when enzyme loading was 10 FPU/g solid. Simultaneous saccharification and fermentation (SSF) results indicated that the pulp could be well converted to ethanol. Compared with dilute acid pretreated bagasse (DAPB), alkali-PAA pulp could obtain much higher ethanol and xylose concentrations. The fermentation broth still showed some cellulase activity so that the fed pulp could be further converted to sugars and ethanol. After the second batch SSF, the fermentation broth of alkali-PAA pulp still kept about 50% of initial cellulase activity. However, only 21% of initial cellulase activity was kept in the fermentation broth of DAPB. The xylose syrup obtained in SSF of alkali-PAA pulp could be well converted to 2,3-butanediol by Klebsiella pneumoniae CGMCC 1.9131.     

Alkali‐based AFEX pretreatment for the conversion of sugarcane bagasse and cane leaf residues to ethanol

Sugarcane is one of the major agricultural crops cultivated in tropical climate regions of the world. Each tonne of raw cane production is associated with the generation of 130 kg dry weight of bagasse after juice extraction and 250 kg dry weight of cane leaf residue postharvest. The annual world production of sugarcane is ～1.6 billion tones, generating 279 MMT tones of biomass residues (bagasse and cane leaf matter) that would be available for cellulosic ethanol production. Here, we investigated the production of cellulosic ethanol from sugar cane bagasse and sugar cane leaf residue using an alkaline pretreatment: ammonia fiber expansion (AFEX). The AFEX pretreatment improved the accessibility of cellulose and hemicelluloses to enzymes during hydrolysis by breaking down the ester linkages and other lignin carbohydrate complex (LCC) bonds and the sugar produced by this process is found to be highly fermentable. The maximum glucan conversion of AFEX pretreated bagasse and cane leaf residue by cellulases was ～85%. Supplementation with hemicellulases during enzymatic hydrolysis improved the xylan conversion up to 95–98%. Xylanase supplementation also contributed to a marginal improvement in the glucan conversion. AFEX‐treated cane leaf residue was found to have a greater enzymatic digestibility compared to AFEX‐treated bagasse. Co‐fermentation of glucose and xylose, produced from high solid loading (6% glucan) hydrolysis of AFEX‐treated bagasse and cane leaf residue, using the recombinant Saccharomyces cerevisiae (424A LNH‐ST) produced 34–36 g/L of ethanol with 92% theoretical yield. These results demonstrate that AFEX pretreatment is a viable process for conversion of bagasse and cane leaf residue into cellulosic ethanol. Biotechnol. Bioeng. 2010;107: 441–450. © 2010 Wiley Periodicals, Inc.     

Low temperature alkali pretreatment for improving enzymatic digestibility of sweet sorghum bagasse for ethanol production

A low temperature alkali pretreatment method was proposed for improving the enzymatic hydrolysis efficiency of lignocellulosic biomass for ethanol production. The effects of the pretreatment on the composition, structure and enzymatic digestibility of sweet sorghum bagasse were investigated. The mechanisms involved in the digestibility improvement were discussed with regard to the major factors contributing to the biomass recalcitrance. The pretreatment caused slight glucan loss but significantly reduced the lignin and xylan contents of the bagasse. Changes in cellulose crystal structure occurred under certain treatment conditions. The pretreated bagasse exhibited greatly improved enzymatic digestibility, with 24-h glucan saccharification yield reaching as high as 98% using commercially available cellulase and β-glucosidase. The digestibility improvement was largely attributed to the disruption of the lignin-carbohydrate matrix. The bagasse from a brown midrib (BMR) mutant was more susceptible to the pretreatment than a non-BMR variety tested, and consequently gave higher efficiency of enzymatic hydrolysis.     

Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses

Due to the environmental concerns and the increasing price of oil, bioethanol was already produced in large amount in Brazil and China from sugarcane juice and molasses. In order to make this process competitive, we have investigated the suitability of immobilized Saccharomyces cerevisiae strain AS2.1190 on sugarcane pieces for production of ethanol. Electron microscopy clearly showed that cell immobilization resulted in firm adsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure, and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supported-biocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sugarcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and fewer yeast cells leaked into fermentation broth. Ethanol concentrations (about 89.73–77.13 g/l in average value), and ethanol productivities (about 59.53–62.79 g/l d in average value) were high and stable, and residual sugar concentrations were low in all fermentations (0.34–3.60 g/l) with conversions ranging from 97.67–99.80%, showing efficiency (90.11–94.28%) and operational stability of the biocatalyst for ethanol fermentation. The results of this study concerning the use of sugarcane as yeast supports could be promising for industrial fermentations. L. Liang and Y. Zhang have contributed equally to this work.     

Major improvement in the rate and yield of enzymatic saccharification of sugarcane bagasse via pretreatment with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim] [Ac])

In this study, sugarcane bagasse was pretreated by six ionic liquids (ILs) using a bagasse/IL ratio of 1:20 (wt%). The solubilization of bagasse in the ILs was followed by water precipitation. On using 1-ethyl-3-methylimidazolium acetate [Emim] [Ac] at 120 °C for 120 min, 20.7% of the bagasse components remained dissolved and enzymatic saccharification experiments resulted on 80% glucose yield within 6h, which evolved to over 90% within 24 h. Moreover, FE-SEM analysis of the precipitated material indicated a drastic lignin extraction and the exposure of nanoscopic cellulose microfibrils with widths of less than 100 nm. The specific surface area (SSA) of the pretreated bagasse (131.84 m2/g) was found to be 100 times that of untreated bagasse. The ability of [Emim] [Ac] to simultaneously increase the SSA and to decrease the biomass crystallinity is responsible for the improved bagasse enzymatic saccharification rates and yields obtained in this work.     

Optimization of sugarcane bagasse conversion by hydrothermal treatment for the recovery of xylose

This work aims at the valorization of sugarcane bagasse by extracting xylose which is destined to the production of xylitol after purification and hydrogenation. Our approach consists in applying the principle of biorefinery to sugarcane bagasse because of its hemicellulose composition (particularly rich in xylan: (92%)). Optimizing of the thermal treatment was investigated. A treatment at 170 °C for 2 h was found optimal, with higher solubilzation of hemicellulose than that at 150 °C and lower degradation of sugar monomers than 190 °C. Recovery of xylose was high and the purity of xylose solution (78%) allows expecting an easy purification and separation of xylose before hydrogenation. Analysis of thermal hydrolyzates shows the presence of xylan oligomers and polymers with large distribution of DPs. This fraction should be submitted to enzymatic treatment to recover more xylose monomer.     

Fermentation of pretreated sugarcane bagasse hemicellulose hydrolysate to ethanol by <Emphasis Type="Italic">Pachysolen tannophilus</Emphasis>

Sugarcane bagasse hemicellulose hydrolysates, pretreated by either over-liming or electrodialysis and, supplemented with nutrient materials, were fermented to ethanol using Pachysolen tannophilus DW06. Compared with detoxification by over-liming, detoxification by electrodialysis decreased the loss of sugar and increased the acetic acid removal, leading to better fermentability. A batch culture with electrodialytically pretreated hydrolysate as substrate was developed giving 21 g ethanol l⁻¹ with a yield of 0.35 g g⁻¹ sugar and productivity of 0.59 g l⁻¹ h⁻¹.     

Biological pretreatment of sugarcane trash for its conversion to fermentable sugars

Pretreatment of lignocellulosic biomasses, the first step in their conversion to utilizable molecules requires very high energy (steam and electricity), corrosion resistant high-pressure reactors and high temperatures. These severe conditions not only add to the cost component of the entire process but also lead to the loss of sugars to the side reactions. Microbial pretreatments have been reported to be associated with reducing the cost factors as well as the severities of the reactions. Eight bioagents, including fungi and bacteria, were screened for their pretreatment effects on sugarcane trash. They narrowed down the C:N ratio of trash from 108:1 to a varying range of approximately 42:1 to 60:1.The maximum drop in C:N ratio of 61% was observed using Aspergillus terreus followed by Cellulomonas uda (52%) and Trichoderma reesei and Zymomonas mobilis (49%). The bioagents helped in degradation of sugarcane trash by production of cellulases, the maximum being produced by A. terreus, (12 fold) followed by C. uda (10 fold), Cellulomonas cartae (9 fold) and Bacillus macerans (8 fold). The microbial pretreatment of trash rendered the easy accessibility of sugars for enzymatic hydrolysis, which can be directed for production of alcohol.     

Enhanced saccharification kinetics of sugarcane bagasse pretreated in 1-butyl-3-methylimidazolium chloride at high temperature and without complete dissolution

Dissolution of bagasse with 1-butyl-3-methylimidazolium chloride at high temperatures (110-160 °C) is investigated as a pretreatment process for saccharification and fermentation based biofuel production. Material balances are reported and used along with enzymatic saccharification data to identify optimum pretreatment conditions (150 °C for 90 min). At all pretreatment temperatures, dissolved and reprecipitated material is enriched in cellulose, has a low crystallinity and the cellulose component is easily and quantitatively hydrolysed (100%, 3h, 15 FPU). At pretreatment temperatures ≤ 150 °C, the undissolved material has only slightly lower crystallinity than the starting. At pretreatment temperatures ≥ 150 °C, the undissolved material has low crystallinity and when combined with the dissolved material has a saccharification rate and extent similar to completely dissolved material. Complete dissolution is not necessary to maximise saccharification efficiency at temperatures ≥ 150 °C.     

Valorization of cashew apple bagasse using acetic acid pretreatment: Production of cellulosic ethanol and lignin for their use as sunscreen ingredients

The present study focuses on the fractionation of cashew apple bagasse via a pretreatment using acetic acid as a delignifying agent and sulfuric acid as an external catalyst. As expected, the concentrations of both acids and the incubation time dramatically affected delignification and hemicellulose solubilization. Under the optimal pretreatment conditions, recycling of the spent liquor had no apparent impact on the chemical composition of the pretreated material, yield of sugar produced via enzymatic hydrolysis (∼37 g/L reducing sugars at 7.5% (w/v) solid loading), or yield of ethanol obtained via fermentation with Saccharomyces cerevisiae (∼16 g/L at 10% (w/v) solid loading). The lignin recovered from the spent liquor showed a good ultraviolet protective effect; the addition of 5% (w/w) of the biopolymer increased the sun protection factor of a commercial sunscreen lotion from 21.62 to 40.71. The combined use of hydrogen peroxide and ultraviolet radiation reduced the organosolv lignin color (absorbance at 450 nm was four times lower) owing to aromatic ring cleavage, but cosmetics containing whitened organosolv lignin had low sun protection factor values. In summary, the results obtained in this study demonstrate the utility of organic acid pretreatment in the valorization of lignocellulosic materials.     

Direct ethanol fermentation from lignocellulosic biomass by Antarctic basidiomycetous yeast Mrakia blollopis under a low temperature condition

Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10 °C. Maximally, 48.2 g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2 g/l, 12.5 g/l and 7.2 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1–1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in cold environments.     

Exploiting the potential of plant growth promoting bacteria in decolorization of dye Disperse Red 73 adsorbed on milled sugarcane bagasse under solid state fermentation

Bioremediation of textile dyes adsorbed on agricultural solid wastes under solid state fermentation (SSF) using rhizospheric plant growth promoting microorganisms pose an ecofriendly, economically feasible and promising treatment approach. The purpose of this study was to adsorb azo dye Disperse Red 73 (DR73) on sugarcane bagasse (SCB) and its further bioremediation using consortium-RARB under SSF. The particle size of SCB 0.002 mm showed maximum adsorption (65%) for DR73. Kinetics of adsorption of DR73 on milled SCB follows pseudo-second order kinetics. The individual cultures of Rhodobacter erythropholis MTCC 4688, Azotobacter vinelandii MTCC 1241, Rhizobium meliloti NCIM 2757 and Bacillus megaterium NCIM 2054 showed 44, 28, 50 and 61% decolorization of DR73 in 48 h respectively; while the consortium-RARB showed complete decolorization in 48 h. Optimum moisture content, temperature and pH for decolorization of DR73 was found to be 90%, 30 °C and 6 respectively. DR73 biodegradation analysis was carried out using HPTLC, FTIR and HPLC. Phytotoxicity and genotoxicity studies revealed detoxification of DR73. Tray bioreactor study for decolorization of adsorbed DR73 on SCB suggests its implementations at large scale. Use of plant growth promoting bacteria's consortium under SSF for bioremediation of adsorbed dyes gives a novel ecologically sustainable approach.     

Potential of agroindustrial waste from olive oil industry for fuel ethanol production

Olive pulp (OP) is a highly polluting semi-solid residue generated from the two-stage extraction processing of olives and is a major environmental issue in Southern Europe, where 80% of the world olive oil is produced. At present, OP is either discarded to the environment or combusted with low calorific value. In this work, utilization of OP as a potential substrate for production of bioethanol was studied. Enzymatic hydrolysis and subsequent glucose fermentation by baker's yeast were evaluated for OP from 10% to 30% dry matter (i.e., undiluted). Enzymatic hydrolysis resulted in an increase in glucose concentration by 75%, giving final glucose yields near 70%. Fermentation of undiluted OP hydrolysate (OPH) resulted in the maximum ethanol produced (11.2 g/L) with productivity of 2.1 g/L/h. Ethanol yields were similar for all tested OPH concentrations and were in the range of 0.49-0.51 g/g. Results showed that yeast could effectively ferment OPH even without nutrient addition, revealing the tolerance of yeast to OP toxicity. Because of low xylan (12.4%) and glucan (16%) content in OP, this specific type of OP is not a suitable material for producing only ethanol and thus, bioethanol production should be integrated with production of other value-added products.     

The influence of pH and dilution rate on continuous production of xylitol from sugarcane bagasse hemicellulosic hydrolysate by C. guilliermondii

Continuous fermentation of sugarcane bagasse hemicellulosic hydrolysate by the yeast Candida guilliermondii FTI 20037 was used for xylitol production from xylose. Experiments were carried out in a reactor with 1.25 l of treated hydrolysate, at 30 °C and 300 rpm. A 2² full-factorial central composite design was employed for experimental study and analysis of the results. A statistical analysis of the results showed that the effects of the pH and dilution rate (D), the interactions between these variables and the second-order effect of D on the xylitol volumetric productivity (Q_p) were significant at a 95% confidence level. The second-order effect of pH was also significant at a 90% confidence level. The k_La effect on the Q_p was not significant. A volumetric productivity of 0.68 g/l h, representing 95.8% of the predicted value (0.72 g/l h), was obtained.     

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.     

Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol

Wheat straw consists of 48.57 ± 0.30% cellulose and 27.70 ± 0.12% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for production of ethanol. Dilute acid pretreatment at varied temperature and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from wheat straw (7.83%, w/v, DS) by dilute H₂SO₄ (0.75%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase and esterase was 565 ± 10 mg/g. Under this condition, no measurable quantities of furfural and hydroxymethyl furfural were produced. The yield of ethanol (per litre) from acid pretreated enzyme saccharified wheat straw (78.3 g) hydrolyzate by recombinant Escherichia coli strain FBR5 was 19 ± 1 g with a yield of 0.24 g/g DS. Detoxification of the acid and enzyme treated wheat straw hydrolyzate by overliming reduced the fermentation time from 118 to 39 h in the case of separate hydrolysis and fermentation (35 °C, pH 6.5), and increased the ethanol yield from 13 ± 2 to 17 ± 0 g/l and decreased the fermentation time from 136 to 112 h in the case of simultaneous saccharification and fermentation (35 °C, pH 6.0).     

液泡蛋白酶B对酿酒酵母高温乙醇发酵效率的影响

【目的】提高酿酒酵母的高耐温性,从而提高菌株在高温下的乙醇发酵性能。【方法】利用染色体整合过表达酿酒酵母液泡蛋白酶B编码基因PRB1。【结果】在41 °C高温条件下进行乙醇发酵,过表达PRB1基因的重组酿酒酵母菌株可在31 h内消耗全部的葡萄糖,而对照菌株在相同时间内仅消耗不到一半的葡萄糖。【结论】利用蛋白酶B基因过表达可构建耐高温酿酒酵母菌株,提高在高温条件下乙醇的发酵效率。     

Efficient pretreatment of lignocellulose in ionic liquids/co-solvent for enzymatic hydrolysis enhancement into fermentable sugars

Ionic liquids (ILs) have been widely used as alternative solvents for biomass pretreatment, however, efficient methods that enable economically use of ILs at large scale have not been established. In this study, a new method in which ILs and polar organic solvents (ILs/co-solvent systems) was proposed for efficient pretreatment of lignocellulosic materials. The combination use of appropriate ILs and organic co-solvents can significantly enhance the solubility of lignocellulose due to the lower viscosity of ILs/co-solvent mixture as compared to those of pure ILs while the hydrogen bond basicity was maintained. In addition, the solubility of lignocellulosic materials in ILs/co-solvent system was found to be correlated with the Kamlet-Taft solvent parameters. Moreover, the use of microwave heating also enhances the efficiency of lignocellulose pretreatment. For example, the microwave-assisted [Emim][OAc]-DMSO (1:1 volume ratio) treated-rice straw could be hydrolyzed at least 22 times faster than that of untreated-rice straw by cellulase from Trichoderma reesei. This enhancement was attributed by several factors including more efficient lignin extraction, less crystalline cellulose and lower residual ILs in treated-rice straw. The produced sugars can be effectively fermented by Pichia stipitis for ethanol production. Moreover, [Emim][OAc]-DMSO mixture could be reused at least 5 times without significantly decrease in effectiveness demonstrated that the use of ILs/co-solvent was potential alternative method for large-scale biomass pretreatment.     

Characterization of the steam-exploded spent Shiitake mushroom medium and its efficient conversion to ethanol

Spent Shiitake mushroom medium was subjected to steam explosion followed by simultaneous saccharification and fermentation (SSF) using Meicelase and Saccahromyces cerevisiae AM12. Water extraction of the medium exposed to steam at 20 atm for 5 min enhanced the saccharification rate by about 20% compared to steam-exploded medium before water extraction and resulted in the production of 23.8 g/l ethanol from a substrate concentration of 100 g/l. This corresponded to 87.6% of the theoretical ethanol yield, i.e., 15.9 g ethanol was obtained from 100 g of spent Shiitake mushroom medium. Spent Shiitake mushroom medium subjected to steam explosion and then water extraction appears to be a candidate for efficient bioconversion to ethanol.     

Conversion of dextrinized cassava starch into ethanol using cultures of Aspergillus awamori and Saccharomyces cerevisiae

Aspergillus awamori and Saccharomyces cerevisiae have been used to convert dextrinized cassava root flour into ethanol. A batch culture of the combined microorganisms produced 4.3% alcohol by weight from 15% cassava flour slurry in 39 h. Two-stage continuous fermentation was done using A. awamori in an airlift fermenter and yeast in a tower fermenter. A residence time of 12.5 h for the first stage resulted in 12.5% sugar concentration and a saccharification efficiency of 88%. A residence time of 5.6 h for the second stage gave an alcohol concentration of 5.3% alcohol and a starch-into-ethanol conversion efficiency of 72.5%.