Simultaneous saccharification and fermentation of broken rice: an enzymatic extrusion liquefaction pretreatment for Chinese rice wine production

Broken rice, pretreated by enzymatic extrusion liquefaction, was used to produce Chinese rice wine by simultaneous saccharification and fermentation (SSF) process in this study. The study compared the novel process and traditional process for Chinese rice wine fermentation utilizing broken rice and head rice, respectively. With the optimum extrusion parameters (barrel temperature, 98 °C; moisture content, 42 % and amylase concentration, 1 ‰), 18 % (v/v at 20 °C) alcoholic degree, 37.66 % fermentation recovery and 93.63 % fermentation efficiency were achieved, indicating enzymatic extrusion-processed rice wine from broken rice exhibited much higher fermentation rate and efficiency than traditional-processed rice wine from head rice during SSF. The starch molecule distribution data indicated that the alcoholic degree was related to the oligosaccharides’ formation during enzymatic extrusion. Sum of amino acid (AA) in the extrusion-processed wine was 53.7 % higher than that in the traditional one. These results suggest that the enzymatic extrusion pretreatment for broken rice is a feasible and alternative process in the fermentation of Chinese rice wine.     

Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast

Simultaneous saccharification and fermentation (SSF) studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells. The ability of a thermotolerant yeast, Kluyveromyces fragilis NCIM 3358, was compared with Saccharomyces cerevisiae NRRL-Y-132. K. fragilis was found to perform better in the SSF process and result in high yields of ethanol (2.5-3.5% w/v) compared to S. cerevisiae (2.0-2.5% w/v). Increased ethanol yields were obtained when the cellulase was supplemented with beta-glucosidase. The conversions with K. fragilis were completed in a short time. The substrates were in the following order in terms of fast conversions: Solka floc > A. leptopus > sugar cane.     

Simultaneous saccharification and fermentation of straw to ethanol using the thermotolerant yeast strain Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was grown at 45°C on media containing 2, 4 and 6 % (w/v) pulverized barley straw and supplemented with 2% (v/v) cellulase. Maximum ethanol concentrations produced were 2, 3 and 3.6g/l, respectively. When the pulverized straw was replaced by NaOH pretreated straw (at 2, 4 and 6% (w/v); based on original untreated straw), ethanol concentrations increased to maxima of 3.9, 8, and 12g/l, respectively. The ethanol yields amount to 20g ethanol from 100g of straw.     

Simultaneous saccharification and fermentation of cellulose to lactic acid

Recent interest in the industrial manufacture of ethanol and other organic chemicals from biomass has led to the utilization of surplus grain and cane juice as a fermentation feedstock. Since those starting materials are also foods, they are expensive. As an alternative, cellulosic substances-the most abundant renewable resources on earth(1)-have long been considered for conversion to readily utilizable hydrolyzates.(2, 3)For the production of ethanol from cellulose, we have proposed the simultaneous saccharification and fermentation (SSF) process.(4) In SSF, enzymatic cellulose hydrolysis and glucose fermentation to ethanol by yeast proceed simultaneously within one vessel. The process advantages-reduced reactor volume and faster saccharification rates-have been confirmed by many researchers.(5-8) During SSF, the faster saccharification rates result because the glucose product is immediately removed, considerably diminishing its inhibitory effect on the cellulase system.(9)To effectively apply the SSF method to produce substances fermented from glucose, several conditions should be satisfied. One is coincident enzymatic hydrolysis and fermentation conditions, such as pH and temperature. The other is that cellulase inhibition by the final product is less than that by glucose and/or cellobiose. One of us has reported that acetic acid, citric acid, itaconic acid, alpha-ketoglutaric acid, lactic acid, and succinic acid scarcely inhibit cellulase.(10) This suggests that if the microorganisms which produce these organic acids were compatible with cellulase reaction conditions, the organic acids could be produced efficiently from cellulosic substrates by SSF.In this article, the successful application of SSF to lactic acid production from cellulose is reported. Though there have been several reports of direct cellulose conversion to organic acids by anaerobes such as Clostridium, only trace amounts of lactic acid were detected in the fermentation medium among the low-molecular-weight fatty acid components.(11-13) Lactic acid is one of the most important organic acids and has a wide range of food-related and industrial applications.     

Simultaneous saccharification and fermentation of cellulose for lactic acid production

Lactic acid production from α-cellulose by simultaneous saccharification and fermentation (SSF) was studied. The cellulose was converted in a batch SSF using cellulase enzyme Cytolase CL to produce glucose sugar andLactobacillus delbrueckii to ferment the glucose to lactic acid. The effects of temperature, pH, yeast extract loading, and lactic acid inhibition were studied to determine the optimum conditions for the batch processing. Cellulose was converted efficiently to lactic acid, and enzymatic hydrolysis was the rate controlling step in the SSF. The highest conversion rate was obtained at 46°C and pH 5.0. The observed yield of lactic acid from α-cellulose was 0.90 at 72 hours. The optimum pH of the SSF was coincident with that of enzymatic hydrolysis. The optimum temperature of the SSF was chosen as the highest temperature the microorganism could withstand. The optimum yeast extract loading was found to be 2.5 g/L. Lactic acid was observed to be inhibitory to the microorganisms’ activity.     

碱预处理玉米芯米根霉同步糖化发酵产富马酸

以碱预处理玉米芯渣为原料,采用单因素优化方法优化米根霉同步糖化发酵产富马酸。在此基础上,研究米根霉利用碱预处理玉米芯渣的同步糖化发酵,并与纯糖发酵进行对比。结果表明:在50 g/L底物、(NH4)2SO4质量浓度0.71 g/L、纤维素酶用量20 FPIU(以1 g纤维素计)、Ca CO3加入量30 g/L、接种量10%(体积分数)和装液量50 m L的条件下,米根霉同步糖化发酵过程产富马酸13.78 g/L,而纯糖发酵富马酸生成量仅6.21 g/L。     

Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation

Production of d-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueckii IFO 3202 produced 28 kgm(-3) lactic acid from 100 kgm(-3) rice bran after 36 h at 37 degrees C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kgm(-3) from 100 kgm(-3) of the bran) was 78%. The optical purity of produced d-lactic acid was 95% e.e.     

Simultaneous enzymatic wheat starch saccharification and fermentation to lactic acid by Lactococcus lactis

Simultaneous saccharification of starch from whole-wheat flour and fermentation to lactic acid (SSF) was investigated. For saccharification the commercial enzyme mixture SAN Super 240 L, having α-amylase, amyloglucosidase and protease activity, was used, and Lactococcus lactis ssp. lactis ATCC 19435 was used for the fermentation. SSF was studied at flour concentrations corresponding to starch concentrations of 90 g/l and 180 g/l and SAN Super concentrations between 3 μl/g and 8 μl/g starch. Kinetic models, developed for the saccharification and fermentation, respectively, were used for simulation and data from SSF experiments were used for model verification. The model simulated SSF when sufficient amounts of nutrients were available during fermentation. This was achieved with high wheat flour concentrations or with addition of yeast extract or amino acids. Nutrient release was dependent on the level of enzyme activity. Received: 26 January 1999 / Accepted: 20 February 1999     

Simultaneous saccharification and fermentation of acid-pretreated rapeseed meal for succinic acid production using Actinobacillus succinogenes

Rapeseed meal was evaluated for succinic acid production by simultaneous saccharification and fermentation using Actinobacillus succinogenes ATCC 55618. Diluted sulfuric acid pretreatment and subsequent hydrolysis with pectinase was used to release sugars from rapeseed meal. The effects of culture pH, pectinase loading and yeast extract concentration on succinic acid production were investigated. When simultaneous saccharification and fermentation of diluted acid pretreated rapeseed meal with a dry matter content of 12.5% (w/v) was performed at pH 6.4 and a pectinase loading of 2% (w/w, on dry matter) without supplementation of yeast extract, a succinic acid concentration of 15.5 g/L was obtained at a yield of 12.4 g/100g dry matter. Fed-batch simultaneous saccharification and fermentation was carried out with supplementation of concentrated pretreated rapeseed meal and pectinase at 18 and 28 h to yield a final dry matter content of 20.5% and pectinase loading of 2%, with the succinic acid concentration enhanced to 23.4 g/L at a yield of 11.5 g/100g dry matter and a productivity of 0.33 g/(Lh). This study suggests that rapeseed meal may be an alternative substrate for the efficient production of succinic acid by A. succinogenes without requiring nitrogen source supplementation.     

Dilute acid pretreatment, enzymatic saccharification, and fermentation of rice hulls to ethanol

Rice hulls, a complex lignocellulosic material with high lignin (15.38 +/- 0.2%) and ash (18.71 +/- 0.01%) content, contain 35.62 +/- 0.12% cellulose and 11.96 +/- 0.73% hemicellulose and has the potential to serve as a low-cost feedstock for production of ethanol. Dilute H2SO4 pretreatments at varied temperature (120-190 degrees C) and enzymatic saccharification (45 degrees C, pH 5.0) were evaluated for conversion of rice hull cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from rice hulls (15%, w/v) by dilute H2SO4 (1.0%, v/v) pretreatment and enzymatic saccharification (45 degrees C, pH 5.0, 72 h) using cellulase, beta-glucosidase, xylanase, esterase, and Tween 20 was 287 +/- 3 mg/g (60% yield based on total carbohydrate content). Under this condition, no furfural and hydroxymethyl furfural were produced. The yield of ethanol per L by the mixed sugar utilizing recombinant Escherichia colistrain FBR 5 from rice hull hydrolyzate containing 43.6 +/- 3.0 g fermentable sugars (glucose, 18.2 +/- 1.4 g; xylose, 21.4 +/- 1.1 g; arabinose, 2.4 +/- 0.3 g; galactose, 1.6 +/- 0.2 g) was 18.7 +/- 0.6 g (0.43 +/- 0.02 g/g sugars obtained; 0.13 +/- 0.01 g/g rice hulls) at pH 6.5 and 35 degrees C. Detoxification of the acid- and enzyme-treated rice hull hydrolyzate by overliming (pH 10.5, 90 degrees C, 30 min) reduced the time required for maximum ethanol production (17 +/- 0.2 g from 42.0 +/- 0.7 g sugars per L) by the E. coli strain from 64 to 39 h in the case of separate hydrolysis and fermentation and increased the maximum ethanol yield (per L) from 7.1 +/- 2.3 g in 140 h to 9.1 +/- 0.7 g in 112 h in the case of simultaneous saccharification and fermentation.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Simultaneous saccharification and L-(+)-lactic acid fermentation of protease-treated wheat bran using mixed culture of lactobacilli

Protease-treated wheat bran (20% w/v) of particle size less than 300 μm containing 65% (w/w) starch was used for the simultaneous saccharification and l-(+)-lactic acid fermentation by the mixed cultures of Lactobacillus casei and Lactobacillus delbrueckii. Maximum lactate yield after various process optimizations was 123 gl⁻¹ with a productivity of 2.3 gl⁻¹ h⁻¹ corresponding to a conversion of 0.95 g lactic acid per gram starch after 54 h at 37°C. By using protease-treated wheat bran around tenfold decrease in supplementation of the costly medium component, like yeast extract, was achieved together with a considerable increase in the production level.     

Simultaneous saccharification and ethanol fermentation of oxalic acid pretreated corncob assessed with response surface methodology

Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 2³ full factorial design with six axial points. Temperatures ranged from 132 to 180 °C, time from 10 to 90 min and oxalic acid loadings from 0.01 to 0.038 g/g solids. Separate maxima were found for enzymatic saccharification and hemicellulose fermentation, respectively, with the condition for maximum saccharification being significantly more severe. Ethanol production was affected by reaction temperature more than by oxalic acid and reaction time over the ranges examined. The effect of reaction temperature was significant at a 95% confidence level in its effect on ethanol production. Oxalic acid and reaction time were statistically significant at the 90% level. The highest ethanol concentration (20 g/l) was obtained after 48 h with an ethanol volumetric production rate of 0.42 g ethanol l⁻¹ h⁻¹. The ethanol yield after SSF with P. stipitis was significantly higher than predicted by sequential saccharification and fermentation of substrate pretreated under the same condition. This was attributed to the secretion of β-glucosidase by P. stipitis. During SSF, free extracellular β-glucosidase activity was 1.30 pNPG U/g with P. stipitis, while saccharification without the yeast was 0.66 pNPG U/g.     

Simultaneous saccharification and ethanol fermentation of sago starch using immobilized Zymomonas mobilis

Simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglucosidase (AMG) and immobilized Zymomonas mobilis ZM4 on sodium alginate was studied. The immobilized Zymomonas cells were more thermo-stable than free Zymomonas cells in this system. The optimum temperature in the SSF system was 40°C, and 0.5% (v/w) AMG concentration was adopted for the economical operation of the system. The final ethanol concentration obtained was 68.3 g/l and the ethanol yield, Y_p/s, was 0.49 g/g (96% of the theoretical yield). After 6 cycles of reuse at 40°C with 15% sago starch hydrolysate, the immobilized Z. mobilis retained about 50% of its ethanol fermenting ability.     

Simultaneous saccharification and fermentation of Kanlow switchgrass by thermotolerant Kluyveromyces marxianus IMB3: the effect of enzyme loading, temperature and higher solid loadings

Switchgrass (Panicum virgatum) was subjected to hydrothermolysis pretreatment and then used to study the effect of enzyme loading and temperature in a simultaneous saccharification and fermentation (SSF) with the thermotolerant yeast strain Kluyveromyces marxianus IMB3 at 8% solid loading. Various loadings of Accellerase 1500 between 0.1 and 1.1 mL g(-1) glucan were tested in SSF at 45 °C (activity of enzyme was 82.2 FPU mL(-1)). The optimum enzyme loading was 0.7 mL g(-1) glucan based on the six different enzyme loadings tested. SSFs were performed at 37, 41 and 45 °C with an enzyme loading of 0.7 mL g(-1) glucan. The highest ethanol concentration of 22.5 g L(-1) was obtained after 168 h with SSF at 45 °C, which was equivalent to 86% yield. Four different batch and fed-batch strategies were evaluated using a total solid loading of 12% (dry basis). About 32 g L(-1) ethanol was produced with the four strategies, which was equivalent to 82% yield.     

Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluyveromyces marxianus IMB4

A thermotolerant yeast strain named Kluyveromyces marxianus IMB4 was used in a simultaneous saccharification and fermentation (SSF) process using Kanlow switchgrass as a feedstock. Switchgrass was pretreated using hydrothermolysis at 200 degrees C for 10 min. After pretreatment, insoluble solids were separated from the liquid prehydrolyzate by filtration and washed with deionized water to remove soluble sugars and inhibitors. Insoluble solids were then hydrolyzed using a commercial cellulase preparation and the released glucose was fermented to ethanol by K. marxianus IMB4 in an SSF process. SSF temperature was 37, 41, or 45 degrees C and pH was 4.8 or 5.5. SSF was conducted for 7 days. Results were compared with a control of Saccharomyces cerevisiae D(5)A at 37 degrees C and pH 4.8. Fermentation by IMB4 at 45 and 41 degrees C ceased after 3 and 4 days, respectively, when a pH 4.8 citrate buffer was used. Fermentation continued for all 7 days using IMB4 at 37 degrees C and the control. When pH 5.5 citrate buffer was used, fermentation ceased after 96 h using IMB4 at 45 degrees C, and ethanol yield was greater than when pH 4.8 citrate buffer was used (78% theoretical). Ethanol yield using IMB4 at 45 degrees C, pH 5.5 was greater than the control after 48, 72, and 96 h (P < 0.05).     

Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a single process, is a promising strategy for effective ethanol production from lignocellulosic materials because of the resulting reduction in utilities, the substrate and other raw materials and simplification of operation. CBP requires a highly engineered microbial strain capable of hydrolyzing biomass with enzymes produced on its own and producing high-titer ethanol. Recently, heterologous production of cellulolytic enzymes has been pursued with yeast hosts, which has realized direct conversion of cellulose to ethanol. Specifically, the development of cell surface engineering, which provides a display of cellulolytic enzymes on the yeast cell surface, facilitates effective biomass hydrolysis concomitantly with ethanol production. On the other hand, the difference in optimum temperature between saccharification and fermentation is a drawback of efficient ethanol production in the simultaneous saccharification and fermentation (SSF). The application of thermotolerant yeast strains engineered to the SSF process would overcome the drawback by performing hydrolysis and fermentation at elevated temperature. In this review, we focus on the recent advances in the application of thermotolerant yeast to CBP and SSF of lignocellulosic material to ethanol. The development of thermotolerant and ethanologenic yeast strains with the ability to hydrolyze lignocellulosic materials is emphasized for high-temperature CBP.     

Biotechnological production of l(+)-lactic acid from wood hydrolyzate by batch fermentation of Enterococcus faecalis

The inhibition of lactic acid fermentation by wood hydrolyzate was decreased (approx. 20%) by adaptation of Enterococcus faecalis RKY1 to wood hydrolyzate-based medium whereby lactic acid productivity and cell growth were enhanced by 0.5 g l(-1) h(-1) and 2.1 g l(-1), respectively. When the diluted or concentrated wood hydrolyzate (equivalent to 25-100 g glucose l(-1)) was supplemented with 15 g yeast extract l(-1), 24-93 g lactic acid l(-1) was produced at a rate between 1.7 g l(-1) h(-1) and 3.2 g l(-1) h(-1).     

木薯粉同步糖化发酵(SSF)产丁二酸

【目的】通过优化产琥珀酸放线杆菌GXAS137同步糖化发酵木薯粉产丁二酸的发酵培养基,提高丁二酸产量,降低生产成本。【方法】在单因素试验的基础上,先利用Plackett-Burman试验设计筛选出影响丁二酸发酵的重要参数,再采用正交试验确定重要参数的最佳水平。【结果】价格低廉玉米浆可用作氮源,影响丁二酸产量的重要参数是木薯粉、玉米浆、碱式碳酸镁和糖化酶浓度。最佳条件为(g/L):木薯粉100,玉米浆14,糖化酶2.0 AGU/g底物,碱式碳酸镁75。优化后丁二酸产量达到69.31 g/L,丁二酸得率为90.01%,生产强度为1.44 g/(L·h)。与初始条件(52.34 g/L)相比,丁二酸浓度提高了32.42%。并利用1.3 L发酵罐对SSF与SHF两种发酵工艺进行了比较,SSF丁二酸产量(72.21 g/L)远高于SHF(56.86 g/L)。【结论】产琥珀酸放线杆菌同步糖化发酵木薯粉丁二酸产量高,生产成本低,具有较好的工业化应用前景。