Modelling ethanol production from cellulose: separate hydrolysis and fermentation versus simultaneous saccharification and fermentation

In ethanol production from cellulose, enzymatic hydrolysis, and fermentative conversion may be performed sequentially (separate hydrolysis and fermentation, SHF) or in a single reaction vessel (simultaneous saccharification and fermentation, SSF). Opting for either is essentially a trade-off between optimal temperatures and inhibitory glucose concentrations on the one hand (SHF) vs. sub-optimal temperatures and ethanol-inhibited cellulolysis on the other (SSF). Although the impact of ethanol on cellobiose hydrolysis was found to be negligible, formation of glucose and cellobiose from cellulose were found to be significantly inhibited by ethanol. A previous model for the kinetics of enzymatic cellulose hydrolysis was, therefore, extended with enzyme inhibition by ethanol, thus allowing a rational evaluation of SSF and SHF. The model predicted SSF processing to be superior. The superiority of SSF over SHF (separate hydrolysis and fermentation) was confirmed experimentally, both with respect to ethanol yield on glucose (0.41 g g^?1 for SSF vs. 0.35 g g^?1 for SHF) and ethanol production rate, being 30% higher for an SSF type process. High conversion rates were found to be difficult to achieve since at a conversion rate of 52% in a SSF process the reaction rate dropped to 5% of its initial value. The model, extended with the impact of ethanol on the cellulase complex proved to predict reaction progress accurately.     

Ethanol production from sunflower meal biomass by simultaneous saccharification and fermentation (SSF) with Kluyveromyces marxianus ATCC 36907

The lignocellulosic materials are considered promising renewable resources for ethanol production, but improvements in the processes should be studied to reduce operating costs. Thus, the appropriate enzyme loading for cellulose saccharification is critical for process economics. This study aimed at evaluating the concentration of cellulase and β-glucosidase in the production of bioethanol by simultaneous saccharification and fermentation (SSF) of sunflower meal biomass. The sunflower biomass was pretreated with 6 % H₂SO₄ (w/v), at 121 °C, for 20 min, for hemicellulose removal and delignificated with 1 % NaOH. SSF was performed with Kluyveromyces marxianus ATCC 36907, at 38 °C, 150 rpm, for 72 h, with different enzyme concentrations (Cellulase Complex NS22086-10, 15 and 20 FPU/g_substrate and β-Glucosidase NS22118, with a cellulase to β-glucosidase ratio of 1.5:1; 2:1 and 3:1). The best condition for ethanol production was cellulase 20 FPU/g_substrate and β-glucosidase 13.3 CBU/g_substrate, resulting in 27.88 g/L ethanol, yield of 0.47 g/g and productivity of 0.38 g/L h. Under this condition the highest enzymatic conversion of cellulose to glucose was attained (87.06 %).     

Study of the enzymatic hydrolysis of cellulose for production of fuel ethanol by the simultaneous saccharification and fermentation process

The biochemical conversion of cellulosic biomass to ethanol, a promising alternative fuel, can be carried out efficiently and economically using the simultaneous saccharification and fermentation (SSF) process. The SSF integrates the enzymatic hydrolysis of cellulose to glucose, catalyzed by the synergistic action of cellulase and beta-glucosidase, with the fermentative synthesis of ethanol. Because the enzymatic step determines the ethanol. Because the enzymatic step determines the availability of glucose to the ethanologenic fermentation, the kinetic of cellulose hydrolysis by cellulase and beta-glucosidase and the susceptibility of the two enzymes to inhibition by hydrolysis and fermentation products are of significant importance to the SSF performance and were investigated under realistic SSF conditions. A previously developed SSF mathematical model was used to conceptualize the depolymerization of cellulose. The model was regressed to the collected data to determine the values of the enzyme parameters and was found to satisfactorily predict the kinetics of cellulose hydrolysis. Cellobiose and glucose were identified as the strongest inhibitors of cellulase and beta-glucosidase, respectively. Experimental and modeling results are presented in light of the impact of enzymatic hydrolysis on fuel ethanol production. (c) 1993 Wiley & Sons, Inc.     

Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF,recycling of hydrolytic enzymes and yeast,and recombinant cellulase-producing Aspergillus niger

Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.     

Evaluation of nanoparticle-immobilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions

Ethanol yields were 2.1 (P = 0.06) to 2.3 (P = 0.01) times higher in simultaneous saccharification and fermentation (SSF) reactions of microcrystalline cellulose when cellulase was physisorbed on silica nanoparticles compared to enzyme in solution. In SSF reactions, cellulose is hydrolyzed to glucose by cellulase while yeast simultaneously ferments glucose to ethanol. The 35°C temperature and the presence of ethanol in SSF reactions are not optimal conditions for cellulase. Immobilization onto solid supports can stabilize the enzyme and promote activity at non-optimum reaction conditions. Mock SSF reactions that did not contain yeast were used to measure saccharification products and identify the mechanism for the improved ethanol yield using immobilized cellulase. Cellulase adsorbed to 40 nm silica nanoparticles produced 1.6 times (P = 0.01) more glucose than cellulase in solution in 96 h at pH 4.8 and 35°C. There was no significant accumulation (<250 μg) of soluble cellooligomers in either the solution or immobilized enzyme reactions. This suggests that the mechanism for the immobilized enzyme's improved glucose yield compared to solution enzyme is the increased conversion of insoluble cellulose hydrolysis products to soluble cellooligomers at 35°C and in the presence of ethanol. The results show that silica-immobilized cellulase can be used to produce increased ethanol yields in the conversion of lignocellulosic materials by SSF.     

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.     

Effect of substrate and cellulase concentration on simultaneous saccharification and fermentation of steam-pretreated softwood for ethanol production

Economic optimization of the production of ethanol by simultaneous saccharification and fermentation (SSF) requires knowledge about the influence of substrate and enzyme concentration on yield and productivity. Although SSF has been investigated extensively, the optimal conditions for SSF of softwoods have yet not been determined. In this study, SO2-impregnated and steam-pretreated spruce was used as substrate for the production of ethanol by SSF. Commercial enzymes were used in combination with the yeast Saccharomyces cerevisiae. The effects of the concentration of substrate (2% to 10% w/w) and of cellulases (5 to 32 FPU/g cellulose) were investigated. SSF was found to be sensitive to contamination because lactic acid was produced. The ethanol yield increased with increasing cellulase loading. The highest ethanol yield, 68% of the theoretical based on the glucose and mannose present in the original wood, was obtained at 5% substrate concentration. This yield corresponds to 82% of the theoretical based on the cellulose and soluble glucose and mannose present at the start of SSF. A higher substrate concentration caused inefficient fermentation, whereas a lower substrate concentration, 2%, resulted in increased formation of lactic acid, which lowered the yield. Compared with separate hydrolysis and fermentation, SSF gave a higher yield and doubled the productivity.     

Bioethanol Production from Horticultural Waste Using Crude Fungal Enzyme Mixtures Produced by Solid State Fermentation

It was desired to study efficient and simplified methods to convert organosolv-pretreated horticultural waste (HW) to ethanol fuel using cellulase produced under solid-state fermentation (SSF). The unprocessed cellulase crude (72.2 %) showed better reducing sugar yield using filter paper than the commercial enzyme blend (68.7 %). Enzymatic hydrolysis of organosolv-pretreated HW using the crude cellulase with 20 % solid content, enzyme loading of 15 FPU/g HW at 50 °C, and pH 5.5 resulted in a HW hydrolysate containing 25.06 g/L glucose after 72 h. Fermentation of the hydrolysate medium produced 12.39 g/L ethanol with 0.49 g/g yield from glucose and 0.062 g/g yield from HW at 8 h using Saccharomyces cerevisiae. This study proved that crude cellulase complex produced under SSF and organosolv pretreatment can efficiently convert woody biomass to ethanol without any commercial cellulase usage.     

Ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (SFS) with Kluyveromyces marxianus CECT 10875

Simultaneous saccharification and fermentation (SSF) process for ethanol production from various lignocellulosic woody (poplar and eucalyptus) and herbaceous (Sorghum sp. bagasse, wheat straw and Brassica carinata residue) materials has been assayed using the thermotolerant yeast strain Kluyveromyces marxianus CECT 10875. Biomass samples were previously treated in a steam explosion pilot plant to provide pretreated biomass with increased cellulose content relative to untreated materials and to enhance cellulase accessibility. SSF experiments were performed in laboratory conditions at 42 °C, 10% (w/v) substrate concentration and 15 FPU/g substrate of commercial cellulase. The results indicate that it is possible to reach SSF yields in the range of 50–72% of the maximum theoretical SSF yield, based on the glucose available in pretreated materials, in 72–82 h. Maximum ethanol contents from 16 to 19 g/l were obtained in fermentation media, depending on the material tested.     

Effects of intermittent addition of cellulase for production of L-lactic acid from wastewater sludge by simultaneous saccharification and fermentation

An attempt was made to create L-lactic acid, a precursor of poly-lactic acid, which is a biodegradable plastic, from wastewater sludge from the paper-manufacturing industry. The sludge contained a high percentage of cellulose and needed to be hydrolyzed to glucose by the action of the cellulase before being treating with lactic acid bacteria. Therefore, a method involving simultaneous saccharification and fermentation (SSF) was carried out. The optimum pH of the SSF for production of the lactic acid by the newly isolated lactic acid bacterium with a high selectively of L-lactic acid was found out to be around pH = 5.0, and the optimum temperature to be approximately 40 degrees C. On the basis of the measurement of the cell density changes in the lactic acid bacteria, it was ascertained that the bacterial activity could continue at a high level for a relatively long period of time, and that the L-lactic acid productivity was diminished by the rapid deactivation of the cellulase. With the intermittent addition of cellulase once daily for the sake of compensating for the cellulase deactivation, the L-lactic acid attained a maximum concentration of 16.9 g/L, i.e., a 72.2% yield based on the potential glucose contained in the sludge under optimum pH and temperature conditions.     

湿氧化爆破稻秆的同步糖化发酵条件研究

探讨了木质纤维素经过湿氧化爆破后在同步糖化发酵过程中酵母产乙醇的基本规律.采用单因素方法对湿氧化爆破条件、酶系组成和添加量以及预酶解时间和温度进行了优化.不同湿氧化爆破预处理条件下的稻秆对同步糖化发酵工艺的影响较大,在预处理温度160 ℃,进氧压力为4×105 Pa,碱用量为6%(w/w),反应时间为20 min的条件...     

A comparison of liquid hot water and steam pretreatments of sugar cane bagasse for bioconversion to ethanol.

Sugar cane bagasse was pretreated with either liquid hot water (LHW) or steam using the same 25 l reactor. Solids concentration ranged from 1% to 8% for LHW pretreatment and was > or = 50% for steam pretreatment. Reaction temperature and time ranged from 170 to 230 degrees C and 1 to 46 min, respectively. Key performance metrics included fiber reactivity, xylan recovery, and the extent to which pretreatment hydrolyzate inhibited glucose fermentation. In four cases, LHW pretreatment achieved > or = 80% conversion by simultaneous saccharification and fermentation (SSF). > or = 80% xylan recovery, and no hydrolyzate inhibition of glucose fermentation yield. Combined effectiveness was not as good for steam pretreatment due to low xylan recovery. SSF conversion increased and xylan recovery decreased as xylan dissolution increased for both modes. SSF conversion, xylan dissolution. hydrolyzate furfural concentration, and hydrolyzate inhibition increased, while xylan recovery and hydrolyzate pH decreased, as a function of increasing LHW pretreatment solids concentration (1-8%). These results are consistent with the notion that autohydrolysis plays an important. if not exclusive, role in batch hydrothermal pretreatment. Achieving concurrently high (greater than 90%) SSF conversion and xylan recovery will likely require a modified reactor configuration (e.g. continuous percolation or base addition) that better preserves dissolved xylan.     

Characteristics of cellulase preparations affecting the simultaneous saccharification and fermentation of cellulose to ethanol

The cellulase, Spezyme CP from Genencor, widely used for the simultaneous saccharification and fermentation (SSF) of cellulose to ethanol, contained substances inhibitory to the growth of Klebsiella oxytoca P2, emphasising the need to check for inhibition effects in SSF experimentation. Also, the preparation contained enough -glucosidase activity to prevent cellobiose accumulation in SSF with a conventional non-cellobiose fermenting yeast: this finding is relevant to attempts to evaluate novel recombinant cellobiose-fermenting microbial strains.     

Cellulose ethanol production from waste newsprint by simultaneous saccharification and fermentation using Saccharomyces cerevisiae KNU5377

A thermotolerant ethanol-fermenting yeast, Saccharomyces cerevisiae KNU5377, isolated from a sludge of a local industrial complex stream in Korea, was evaluated for its capability for lignocellulosic ethanol production from waste newsprint in high temperature. In this fermentation, most of dry-defibrated waste newspaper was first saccharified at 50 °C for 108 h using a commercial cellulase and, then with the last addition of dry-defibrated newsprints to the pre-saccharified broth, simultaneous saccharification and fermentation (SSF) of 1.0 L of reaction mixture was carried out at 40 °C, slowly being dropped from 50 °C, for further 72 h in a 5 L fermentor by inoculating the overnight culture of KNU5377. The maximum production of 8.4% (v/v) ethanol was obtained when 250 g (w/v)/L of dry-defibrated waste newspaper was used for ethanol production by SSF. These results suggest that S. cerevisiae KNU5377 is very useful for cellulose ethanol production by the SSF system.     

One-pot bioethanol production from cellulose by co-culture of Acremonium cellulolyticus and Saccharomyces cerevisiae

ABSTRACT: BACKGROUND: While the ethanol production from biomass by consolidated bioprocess (CBP) is considered to be the most ideal process, simultaneous saccharification and fermentation (SSF) is the most appropriate strategy in practice. In this study, one-pot bioethanol production, including cellulase production, saccharification of cellulose, and ethanol production, was investigated for the conversion of biomass to biofuel by co-culture of two different microorganisms such as a hyper cellulase producer, Acremonium cellulolyticus C-1 and an ethanol producer Saccharomyces cerevisiae. Furthermore, the operational conditions of the one-pot process were evaluated for maximizing ethanol concentration from cellulose in a single reactor. RESULTS: Ethanol production from cellulose was carried out in one-pot bioethanol production process. A. cellulolyticus C-1 and S. cerevisiae were co-cultured in a single reactor. Cellulase producing-medium supplemented with 2.5 g/l of yeast extract was used for productions of both cellulase and ethanol. Cellulase production was achieved by A. cellulolyticus C-1 using Solka-Floc (SF) as a cellulase-inducing substrate. Subsequently, ethanol was produced with addition of both 10%(v/v) of S. cerevisiae inoculum and SF at the culture time of 60 h. Dissolved oxygen levels were adjusted at higher than 20% during cellulase producing phase and at lower than 10% during ethanol producing phase. Cellulase activity remained 8--12 FPU/ml throughout the one-pot process. When 50--300 g SF/l was used in 500 ml Erlenmeyer flask scale, the ethanol concentration and yield based on initial SF were as 8.7--46.3 g/l and 0.15--0.18 (g ethanol/g SF), respectively. In 3-l fermentor with 50--300 g SF/l, the ethanol concentration and yield were 9.5--35.1 g/l with their yields of 0.12--0.19 (g/g) respectively, demonstrating that the one-pot bioethanol production is a reproducible process in a scale-up bioconversion of cellulose to ethanol. CONCLUSION: A. cellulolyticus cells produce cellulase using SF. Subsequently, the produced cellulase saccharifies the SF, and then liberated reducing sugars are converted to ethanol by S. cerevisiae. These reactions were carried out in the one-pot process with two different microorganisms in a single reactor, which does require neither an addition of extraneous cellulase nor any pretreatment of cellulose. Collectively, the one-pot bioethanol production process with two different microorganisms could be an alternative strategy for a practical bioethanol production using biomass.     

High titer ethanol production from simultaneous enzymatic saccharification and fermentation of aspen at high solids: a comparison between SPORL and dilute acid pretreatments

Native aspen (Populus tremuloides) was pretreated using sulfuric acid and sodium bisulfite (SPORL) and dilute sulfuric acid alone (DA). Simultaneous enzymatic saccharification and fermentation (SSF) was conducted at 18% solids using commercial enzymes with cellulase loadings ranging from 6 to 15 FPU/g glucan and Saccharomyces cerevisiae Y5. Compared with DA pretreatment, the SPORL pretreatment reduced the energy required for wood chip size-reduction, and reduced mixing energy of the resultant substrate for solid liquefaction. Approximately 60% more ethanol was produced from the solid SPORL substrate (211 L/ton wood at 59 g/L with SSF efficiency of 76%) than from the solid DA substrate (133 L/ton wood at 35 g/L with SSF efficiency 47%) at a cellulase loading of 10 FPU/g glucan after 120 h. When the cellulase loading was increased to 15 FPU/g glucan on the DA substrate, the ethanol yield still remained lower than the SPORL substrate at 10 FPU/g glucan.     

Simultaneous saccharification and fermentation of lignocellulosic residues pretreated with phosphoric acid–acetone for bioethanol production

Bermudagrass, reed and rapeseed were pretreated with phosphoric acid–acetone and used for ethanol production by means of simultaneous saccharification and fermentation (SSF) with a batch and fed-batch mode. When the batch SSF experiments were conducted in a 3% low effective cellulose, about 16 g/L of ethanol were obtained after 96 h of fermentation. When batch SSF experiments were conducted with a higher cellulose content (10% effective cellulose for reed and bermudagrass and 5% for rapeseed), higher ethanol concentrations and yields (of more than 93%) were obtained. The fed-batch SSF strategy was adopted to increase the ethanol concentration further. When a higher water-insoluble solid (up to 36%) was applied, the ethanol concentration reached 56 g/L of an inhibitory concentration of the yeast strain used in this study at 38 °C. The results show that the pretreated materials can be used as good feedstocks for bioethanol production, and that the phosphoric acid–acetone pretreatment can effectively yield a higher ethanol concentration.     

Simultaneous improvement of saccharification and ethanol production from crystalline cellulose by alleviation of irreversible adsorption of cellulase with a cell surface-engineered yeast strain

Enzymatic hydrolysis of cellulosic material is an essential step in the bioethanol production process. However, complete cellulose hydrolysis by cellulase is difficult due to the irreversible adsorption of cellulase onto cellulose. Thus, part of the cellulose remains in crystalline form after hydrolysis. In this study, after 96-h hydrolysis of Avicel crystalline cellulose, 47.1 % of the cellulase was adsorbed on the cellulose surface with 10.8 % crystalline cellulose remaining. In simultaneous saccharification and fermentation of 100 g/L Avicel with 1.0 filter paper unit/mL cellulase, a wild-type yeast strain produced 44.7 g/L ethanol after 96 h. The yield of ethanol was 79.7 % of the theoretical yield. On the other hand, a recombinant yeast strain displaying various cellulases, such as β-glucosidase, cellobiohydrolase, and endoglucanase, produced 48.9 g/L ethanol, which corresponds to 87.3 % of the theoretical yield. Higher ethanol production appears to be attributable to higher efficiency of cellulase displayed on the cell surface. These results suggest that cellulases displayed on the yeast cell surface improve hydrolysis of Avicel crystalline cellulose. Indeed, after the 96-h simultaneous saccharification and fermentation using the cellulase-displaying yeast, the amount of residual cellulose was 1.5 g/L, one quarter of the cellulose remaining using the wild-type strain, a result of the alleviation of irreversible adsorption of cellulases on the crystalline cellulose.     

Effect of hydrothermolysis process conditions on pretreated switchgrass composition and ethanol yield by SSF with Kluyveromyces marxianus IMB4

Hot compressed liquid water was used to treat switchgrass in a method called hydrothermolysis to disrupt lignin, dissolve hemicellulose, and increase accessibility of cellulose to cellulase. Three temperatures (190, 200, and 210 °C) and hold times (10, 15, and 20 min) were tested. Switchgrass treated at 190 °C for 10 min had the greatest xylan recovery in the prehydrolyzate. Less than 0.65 g/L glucose were released into the prehydrolyzate for all pretreatment conditions, indicating most glucose was retained as cellulose in the solid substrate. 5-Hydroxymethylfurfural (HMF) and furfural formation in the prehydrolyzate were found to be less than 1 g/L for all treatments. The highest concentration of ethanol, 16.8 g/L (72% of theoretical), was produced from switchgrass pretreated at 210 °C and 15 min using simultaneous saccharification and fermentation (SSF) at 45 °C with the thermotolerant yeast Kluyveromyces marxianus IMB4 and 15 FPU cellulase/g glucan.     

Ethanol production in simultaneous saccharification and fermentation of cellulose with temperature profiling

The effects of temperature on enzymatic saccharification of cellulose and simulataneous saccharification and fermentation (SSF) were investigated with 100 g·l⁻¹ Solka Floc, 5g·l⁻¹Trichoderma reesei cellulase, and Zymomonas mobilis ATCC 29191. The following results were obtained: 1) Ethanol fermentation under glucose dificient conditions can proceed for more than 100 h at 30°C but gradually ceases after 50 h of operation at 40°C. 2) Equivalent glucose yield based on cellulose for SSF operated at its optimum temperature (37°C) is higher than that for enzymatic saccharification of cellulose at the same temperature by 32%. However, the same equivalent glucose yields were obtained for both processes if they were operated at their respective optimum temperature. 3) SSF with temperature cycling increased the ethanol productivity but gave similar ethanol yield to SSF at 37°C. 4) SSF with temperature profiling gave an ethanol yield of 0.32 g·g⁻¹ and cellulose use of 0.86 g·g⁻¹ which were increased by 39% and 34% over SSF with temperature cycling and at 37°C.