Simultaneous saccharification and fermentation of pretreated sugar cane leaves to ethanol

The simultaneous saccharification and fermentation (SSF) of pretreated sugar cane leaves to produce ethanol using a cellulolytic enzyme complex from Trichoderma reesei QM 9414 and Saccharomyces cerevisiae NRRL-Y-132 was optimized. Enzymic saccharification parameters were evaluated prior to SSF studies. A 92% conversion of 2·5% substrate (alkaline hydrogen peroxide pretreated) to sugars was achieved at 50°C and pH 4·5, using T. reesei cellulase (40 FPU/g substrate), in 48 h. The pretreated substrate was then subjected to an SSF process using the cellulase complex and S. cerevisiae cells. Optimization of the SSF system is described.     

Ethanol production from lignocellulosic biomass of energy cane

Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum based liquid fuels. The release of many new sugarcane varieties by the United States Department of Agriculture to be used as energy crops is a promising feedstock alternative. Energy cane produces large amounts of biomass that can be easily transported, and production does not compete with food supply and prices because energy cane can be grown on marginal land instead of land for food crops. The purpose of this study was to evaluate energy cane for lignocellulosic ethanol production. Energy cane variety L 79-1002 was pretreated with weak sulfuric acid to remove lignin. In this study, 1.4 M sulfuric acid pretreated type II energy cane had a higher ethanol yield after fermentation by Klebsiella oxytoca without enzymatic saccharification than 0.8 M and 1.6 M sulfuric acid pretreated type II energy cane. Pretreated biomass was inoculated with K. oxytoca for cellulose fermentation and Pichia stipitis for hemicellulose fermentation under simultaneous saccahrification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) conditions. For enzymatic saccharification of cellulose, the cellulase and ??-glucanase cocktail significantly increased ethanol production compared to the ethanol production of fermented acid pretreated energy cane without enzymatic saccharification. The results revealed that energy cane variety L 79-1002 produced maximum cellulosic ethanol under SHF (6995 mg/L) and produced 3624 mg/L ethanol from fermentation of hemicellulosic sugars.     

Evaluation of pretreatment methods for lignocellulosic ethanol production from energy cane variety L 79-1002

Approximately half of the 80 billion tons of crop produced annually around the world remains as residue that could serve as a renewable resource to produce valuable products such as ethanol and butanol. Ethanol produced from lignocellulosic biomass is a promising renewable alternative to diminishing oil and gas liquid fuels. Sugarcane is an important industry in Louisiana. The recently released variety of “energy cane” has great potential to sustain a competitive sugarcane industry. It has been demonstrated that fuel-grade ethanol can be produced from post harvest sugarcane residue in the past, but optimized ethanol production was not achieved. Optimization of the fermentation process requires efficient pretreatment to release cellulose and hemicellulose from lignocellulosic complex of plant fiber. Determining optimal pretreatment techniques for fermentation is essential for the success of lignocellulosic ethanol production process. The purpose of this study was to evaluate three pretreatment methods for the energy cane variety L 79-1002 for maximum lignocellulosic ethanol production. The pretreatments include alkaline pretreatment, dilute acid hydrolysis, and solid-state fungal pretreatment process using brown rot and white rot fungi. Pretreated biomass was enzymatically saccharified and subjected to fermentation using a recombinant Escherichia coli FBR5. The results revealed that all pretreatment processes produced ethanol. However, the best result was observed in dilute acid hydrolysis followed by alkaline pretreatment and solid-state fungal pretreatment.     

Enhanced poly(L-malic acid) production from pretreated cane molasses by Aureobasidium pullulans in fed-batch fermentation

Poly(L-malic acid) (PMA) is a natural polyester with many attractive properties for biomedical application. However, the cost of PMA production is high when glucose is used as a carbon source. To solve this problem, cane molasses as a low-cost feedstock was applied for the production of PMA. Six pretreatment methods were applied to cane molasses before fermentation. Pretreatment with combined tricalcium phosphate, potassium ferrocyanide, and sulfuric acid (TPFSA) removed significant amounts of metal ions from cane molasses. The PMA concentration increased from 5.4?g/L (untreated molasses) to 36.9?g/L (TPFSA-pretreated molasses) after fermentation in shake flasks. A fed-batch fermentation strategy was then developed. In this method, TPFSA-pretreated cane molasses solution was continuously fed into the fermentor to maintain the total sugar concentration at 20?g/L. This technique generated approximately 95.4?g/L PMA with a productivity of 0.57?g/L/hr. The present study indicated that fed-batch fermentation using pretreated cane molasses is a feasible technique for producing high amounts of PMA.     

Enzyme hydrolysis and ethanol fermentation of liquid hot water and AFEX pretreated distillers' grains at high-solids loadings

The dry milling ethanol industry produces distiller's grains as major co-products, which are composed of unhydrolyzed and unfermented polymeric sugars. Utilization of the distiller's grains as an additional source of fermentable sugars has the potential to increase overall ethanol yields in current dry grind processes. In this study, controlled pH liquid hot water pretreatment (LHW) and ammonia fiber expansion (AFEX) treatment have been applied to enhance enzymatic digestibility of the distiller's grains. Both pretreatment methods significantly increased the hydrolysis rate of distiller's dried grains with solubles (DDGS) over unpretreated material, resulting in 90% cellulose conversion to glucose within 24h of hydrolysis at an enzyme loading of 15FPU cellulase and 40 IU beta-glucosidase per gram of glucan and a solids loading of 5% DDGS. Hydrolysis of the pretreated wet distiller's grains at 13-15% (wt of dry distiller's grains per wt of total mixture) solids loading at the same enzyme reduced cellulose conversion to 70% and increased conversion time to 72h for both LHW and AFEX pretreatments. However, when the cellulase was supplemented with xylanase and feruloyl esterase, the pretreated wet distiller's grains at 15% or 20% solids (w/w) gave 80% glucose and 50% xylose yields. The rationale for supplementation of cellulases with non-cellulolytic enzymes is given by Dien et al., later in this journal volume. Fermentation of the hydrolyzed wet distiller's grains by glucose fermenting Saccharomyces cerevisiae ATCC 4124 strain resulted in 100% theoretical ethanol yields for both LHW and AFEX pretreated wet distiller's grains. The solids remaining after fermentation had significantly higher protein content and are representative of a protein-enhanced wet DG that would result in enhanced DDGS. Enhanced DDGS refers to the solid product of a modified dry grind process in which the distiller's grains are recycled and processed further to extract the unutilized polymeric sugars. Compositional changes of the laboratory generated enhanced DDGS are also presented and discussed.     

Ethanol production from oil palm trunks treated with aqueous ammonia and cellulase

Oil palm trunks are a possible lignocellulosic source for ethanol production. Low enzymatic digestibility of this type of material (11.9% of the theoretical glucose yield) makes pretreatment necessary. An enzymatic digestibility of 95.4% with insoluble solids recovery of 49.8% was achieved after soaking shredded oil palm trunks in ammonia under optimum conditions (80 °C, 1:12 solid-to-liquid ratio, 8 h and 7% (w/w) ammonia solution). Treatment with 60 FPU of commercial cellulase (Accellerase 1000) per gram of glucan and fermentation with Saccharomyces cerevisiae D₅A resulted in an ethanol concentration of 13.3 g/L and an ethanol yield of 78.3% (based on the theoretical maximum) after 96 h. These results indicate that oil palm trunks are a biomass feedstock that can be used for bioethanol production.     

The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol

Autohydrolysis and ethanol-alkali pulping were used as pretreatment methods of wheat straw for its subsequent saccharification by Trichoderma reesei cellulase. The basic hydrolysis parameters, i.e., reaction time, pH, temperature, and enzyme and substrate concentration, were optimized to maximize sugar yields from ethanol-alkali modified straw. Thus, a 93% conversion of 2.5% straw material to sugar syrup containing 73% glucose was reached in 48 h using 40 filter paper units/g hydrolyzed substrate. The pretreated wheat straw was then fermented to ethanol at 43 degrees C in the simultaneous saccharification and fermentation (SSF) process using T. reesei cellulase and Kluyveromyces fragilis cells. From 10% (w/v) of chemically treated straw (dry matter), 2.4% (w/v) ethanol was obtained after 48 h. When the T. reesei cellulase system was supplemented with beta-glucosidase from Aspergillus niger, the ethanol yield in the SSF process increased to 3% (w/v) and the reaction time was shortened to 24 h.     

Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation

AIM: To evaluate the solid-state fermentation (SSF) production of cellulase and hemicellulases (xylanases), by Penicillium echinulatum 9A02S1, in experiments carried out with different concentrations of the pretreated sugar cane bagasse (PSCB) and wheat bran (WB). METHODS AND RESULTS: This study reports the production of xylanolytic and cellulolytic enzymes by P. echinulatum 9A02S1 using a cheap medium containing PSCB and WB under SSF. The highest amounts of filter paper activity (FPA) could be measured on mixtures of PSCB and WB (32.89 +/- 1.90 U gdm(-1)). The highest beta-glucosidase activity was 58.95 +/- 2.58 U gdm(-1) on the fourth day. The highest activity for endoglucanases was 282.36 +/- 1.23 U gdm(-1) on the fourth day, and for xylanases the activity was around 10 U gdm(-1) from the second to the fourth day. CONCLUSIONS: The present work has established the potential of P. echinulatum for FPA, endoglucanase, beta-glucosidase and xylanase productions in SSF, indicating that WB may be partially substituted by PSCB. SIGNIFICANCE AND IMPACT OF THE STUDY: The incorporation of cheap sources, such as sugar cane bagasse, into media for the production of lignocellulose enzymes should help decrease the production costs of enzymatic complexes that can hydrolyse lignocellulose residues for the formation of fermented syrups, thus contributing to the economic production of bioethanol.     

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.     

Evaluation of pelleting as a pre-processing step for effective biomass deconstruction and fermentation

Densification of bulky forages by pelleting reduces their transportation, handling, and storage costs. Because of high shearing force and frictional heating during the pelleting process, it is hypothesized that pelleting of lignocellulosic biomass could also partially deconstruct its complex structure and facilitate bioethanol production. In this study, pelleted wheat straw, corn stover, big bluestem, and sorghum stalk were evaluated for sugars and ethanol production, and compared with those of unpelleted biomasses. Mass recovery after alkali pretreatment increased by 14%, 11%, 2%, and 5%, respectively, in unpelleted biomasses. Lignin content reduced significantly more in pelleted samples for all types of biomass, except sorghum stalk. Volumetric productivity of enzymatic hydrolysis was 23%, 21%, 20% and 12% higher, respectively, in pelleted samples; ethanol yield on the basis of released sugars did not differ significantly between pelleted and unpelleted samples. These results indicate that the pelleting process led to better enzymatic hydrolysis of pretreated biomasses without affecting the quality of sugars for fermentation. However, overall yield of ethanol from the raw biomass was not significantly higher in pelleted biomasses because of higher mass loss during pretreatment process. In our study, we propose a schematic for complete utilization of various byproducts for enhanced economic viability.     

Enzymatic digestion of alkaline‐sulfite pretreated sugar cane bagasse and its correlation with the chemical and structural changes occurring during the pretreatment step

Sugar cane bagasse is recalcitrant to enzymatic digestion, which hinders the efficient conversion of its polysaccharides into fermentable sugars. Alkaline‐sulfite pretreatment was used to overcome the sugar cane bagasse recalcitrance. Chemical and structural changes that occurred during the pretreatment were correlated with the efficiency of the enzymatic digestion of the polysaccharides. The first 30 min of pretreatment, which removed approximately half of the initial lignin and 30% of hemicellulose seemed responsible for a significant enhancement of the cellulose conversion level, which reached 64%. After the first 30 min of pretreatment, delignification increased slightly, and hemicellulose removal was not enhanced; however, acid groups continued to be introduced into the residual lignin. Water retention values were 145% to the untreated bagasse and 210% to the bagasse pretreated for 120 min and fiber widths increased from 10.4 to 30 μm, respectively. These changes were responsible for an additional increase in the efficiency of enzymatic hydrolysis of the cellulose, which reached 92% with the 120 min pretreated sample. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:890–895, 2013     

沙柳原料高浓度底物酶解发酵乙醇工艺

为了提高沙柳生物转化过程的经济可行性,考察了沙柳原料经过蒸爆、超微粉碎+稀酸、超微粉碎+稀碱预处理后高浓度底物补料酶解的效果,并对其高浓度水解糖液进行了乙醇发酵。结果表明:蒸爆处理法水解效果最好,通过补料酶解,底物质量分数可以达到30%,酶解液中总糖质量浓度达到132 g/L,葡萄糖质量浓度105 g/L;超微粉碎+稀酸预处理原料底物质量分数可以达到22%,酶解液中总糖质量浓度达到123 g/L,葡萄糖质量浓度73 g/L;超微粉碎+稀碱预处理原料底物质量分数可以达到22%,酶解液中总糖质量浓度133 g/L,葡萄糖质量浓度77 g/L。3种预处理使沙柳原料的酶解糖液都可以较好地被酿酒酵母利用发酵产乙醇,蒸爆处理原料的酶解糖液乙醇发酵效果最好,乙醇质量浓度达到47 g/L。     

Two-stage pretreatment of rice straw using aqueous ammonia and dilute acid

Liberation of fermentable sugars from recalcitrant lignocellulosic biomass is one of the key challenges in production of cellulosic ethanol. Here we developed a two-stage pretreatment process using aqueous ammonia and dilute sulfuric acid in a percolation mode to improve production of fermentable sugars from rice straw. Aqueous NH? was used in the first stage which removed lignin selectively but left most of cellulose (97%) and hemicellulose (77%). Dilute acid was applied in the second stage which removed most of hemicellulose, partially disrupted the crystalline structure of cellulose, and thus enhanced enzymatic digestibility of cellulose in the solids remaining. Under the optimal pretreatment conditions, the enzymatic hydrolysis yields of the two-stage treated samples were 96.9% and 90.8% with enzyme loadings of 60 and 15FPU/g of glucan, respectively. The overall sugar conversions of cellulose and hemicellulose into glucose and xylose by enzymatic and acid hydrolysis reached 89.0% and 71.7%, respectively.     

Rheological characterization of dilute acid pretreated softwood

Large-scale bioethanol production from lignocellulosic biomass will require high solids loading in the enzymatic hydrolysis step. However, slurries of pretreated lignocelluloses are complex fluids due to the fibrous nature, especially at high concentrations of water insoluble solids (WIS). A prerequisite for dealing with transport issues and for developing efficient full-scale processes is a fundamental understanding of the flow properties of pretreated lignocellulose. A comprehensive rheological characterization of dilute acid pretreated spruce has been carried out in this study, accounting for the effects of WIS concentration, particle size distribution (PSD), and the degree of enzymatic hydrolysis. The rheology of pretreated spruce slurries was found to be strongly dependent on the WIS concentration. The storage modulus (G'(LVR)) and yield stress showed typical power-law dependencies on volume fraction and WIS content. Milling of the pretreated material resulted in significantly higher yield stress and viscosity, likely due to narrower PSD, which suggests that the strength of the network of the coarsest fibers determines the rheology of these materials to a large extent. During enzymatic hydrolysis, yield stress and viscosity decreased dramatically, partly due to decreasing WIS content, but possibly also due to changes in fiber properties such as the chemical composition.     

Aqueous ammonia pretreatment of oil palm empty fruit bunches for ethanol production

Oil palm empty fruit bunches (EFB) were pretreated by aqueous ammonia soaking for ethanol production. Pretreated EFB, which were pretreated at the optimal conditions of 60 °C, 12 h, and 21% (w/w) aqueous ammonia, showed 19.5% and 41.4% glucose yields during an enzymatic digestibility test for 96 h when using 15 and 60 FPU of cellulase, respectively. Using the pretreated EFB, simultaneous saccharification and fermentation for 168 h with 5% (w/v) glucan loading and 60 FPU of cellulase and 30 CBU of β-glucosidase per gram glucan resulted in ethanol production of 18.6 g/L titer, 65.6% of theoretical maximum yield, and 0.11 g/L/h of productivity.     

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.     

Enzyme production by the mixed fungal culture with nano‐shear pretreated biomass and lignocellulose hydrolysis

Cellulase, xylanase, and β‐glucosidase production was studied on novel nano‐shear pretreated corn stover by the mixed fungi culture. The high shear force from a modified Tayor‐Couette nano‐shear mixing reactor efficiently disintegrated corn stover, resulting in a homogeneous watery mash with particles in much reduced size. Scanning electron microscope study showed visible mini‐pores on the fiber cell wall surface, which could improve the accessibility of the pretreated corn stover to microorganisms. Mixed fungal culture of Trichoderma reesei RUT‐C30 and Aspergillus niger produced enzymes with higher cellulolytic and xylanolytic activities on corn stover pretreated with nano‐shear mixing reactor, in comparison with other pretreatment methods, including acid and ammonia fiber explosion (AFEX) pretreatment. The hydrolytic potential of the whole fermentation broth from the mixed fungi was studied, and the possibility of applying the whole cell saccharification concept was also investigated to further reduce the cost of lignocellulose hydrolysis. Biotechnol. Bioeng. 2013; 110: 2123–2130. © 2013 Wiley Periodicals, Inc.     

Enzyme characterization for hydrolysis of AFEX and liquid hot-water pretreated distillers' grains and their conversion to ethanol

Dried distillers' grains with solubles (DDGS), a co-product of corn ethanol production, was investigated as a feedstock for additional ethanol production. DDGS was pretreated with liquid hot-water (LHW) and ammonia fiber explosion (AFEX) processes. Cellulose was readily converted to glucose from both LHW and AFEX treated DDGS using a mixture of commercial cellulase and beta-glucosidase; however, these enzymes were ineffective at saccharifying the xylan present in the pretreated DDGS. Several commercial enzyme preparations were evaluated in combination with cellulase to saccharify pretreated DDGS xylan and it was found that adding commercial grade (e.g. impure) pectinase and feruloyl esterase (FAE) preparations were effective at releasing arabinose and xylose. The response of sugar yields for pretreated AFEX and LHW DDGS (6wt%/solids) were determined for different enzyme loadings of FAE and pectinase and modeled as a response surfaces. Arabinose and xylose yields rose with increasing FAE and pectinase enzyme dosages for both pretreated materials. When hydrolyzed at 20wt%/solids with the same blend of commercial enzymes, the yields were 278 and 261g sugars (i.e. total of arabinose, xylose, and glucose) per kg of DDGS (dry basis, db) for AFEX and LHW pretreated DDGS, respectively. The pretreated DDGS's were also evaluated for fermentation using Saccharomyces cerevisiae at 15wt%/solids. Pretreated DDGS were readily fermented and were converted to ethanol at 89-90% efficiency based upon total glucans; S. cerevisiae does not ferment arabinose or xylose.     

The use of tamarind waste to improve ethanol production from cane molasses

Tamarind wastes such as tamarind husk, pulp, seeds, fruit and the effluent generated during tartaric acid extraction were used as supplements to evaluate their effects on alcohol production from cane molasses using yeast cultures. Small amounts of these additives enhanced the rate of ethanol production in batch fermentations. Tamarind fruit increased ethanol production (9.7%, w/v) from 22.5% reducing sugars of molasses as compared to 6.5% (w/v) in control experiments lacking supplements after 72 h of fermentation. In general, the addition of tamarind supplements to the fermentation medium showed more than 40% improvement in ethanol production using higher cane molasses sugar concentrations. The direct fermentation of aqueous tamarind effluent also yielded 3.25% (w/v) ethanol, suggesting its possible use as a diluent in molasses fermentations. This is the first report, to our knowledge, in which tamarind-based waste products were used in ethanol production. Received 2 April 1998/ Accepted in revised form 13 November 1998     

The enzymatic hydrolysis and fermentation of pretreated wood substrates

Aspenwood chips were pretreated by steam explosion. The various wood fractions obtained were assayed for their ability to act as substrates for growth and cellulase production of different Trichoderma and Clostridium thermocellum species. Steam exploded aspenwood was as efficiently utilized as solka floc and correspondingly high cellulase activities were detected in the various culture filtrates. When T. harzianum E58 was grown on increasing concentrations of solka floc, highest cellulase and xylanase activities were detected at 1% substrate concentrations while high substrate concentrations (10-20%) inhibited growth and enzyme production. When the cellulosic substrates were supplemented with increasing amounts of glucose, cellulase and xylanase production were inhibited when the glucose concentration exceeded 0.1%. Highest xylanase activities were detected after growth of T. reesei C30 and T. harianum E58 on xylan and solka floc respectively. All of the steam exploded fractions were at least partially hydrolyzed by the T. harzianum E58 cellulase system. The extent of the pretreatment also influenced the ability of Zymomonas mobilis and Saccharomyces cerevisiae to ferment the liberated sugars to ethanol. About 85% of the theoretical yield of ethanol from cellulose could be obtained from the combined hydrolysis and fermentation of pretreated aspenwood.