A comparison of the production of ethanol between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using unpretreated cassava pulp and enzyme cocktail

The processes of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were employed using Saccharomyces cerevisiae for the production of ethanol from cassava pulp without any pretreatment. A combination of amylase, cellulase, cellobiase, and glucoamylase produced the highest levels of ethanol production in both the SHF and the SSF method. A temperature of 37 °C, a pH of 5.0, and an inoculum size of 6% were the optimum conditions for SSF. For the batch process at a pulp concentration of 20%, ethanol production levels from SHF and SSF were the highest, at 23.51 and 34.67 g L(-1) respectively, but in the fed-batch process, the levels of ethanol production from SHF and SSF rose to 29.39 and 43.25 g L(-1) respectively, which were 25% and 24.7% higher than those of the batch process. Thus SSF using the fed-batch provided a more efficient method for the utilization of cassava pulp.     

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.     

Pipeline transport and simultaneous saccharification of corn stover

Pipeline transport of corn stover delivered by truck from the field is evaluated against a range of truck transport costs. Corn stover transported by pipeline at 20% solids concentration (wet basis) or higher could directly enter an ethanol fermentation plant, and hence the investment in the pipeline inlet end processing facilities displaces comparable investment in the plant. At 20% solids, pipeline transport of corn stover costs less than trucking at capacities in excess of 1.4 M drytonnes/yr when compared to a mid range of truck transport cost (excluding any credit for economies of scale achieved in the ethanol fermentation plant from larger scale due to multiple pipelines). Pipelining of corn stover gives the opportunity to conduct simultaneous transport and saccharification (STS). If current enzymes are used, this would require elevated temperature. Heating of the slurry for STS, which in a fermentation plant is achieved from waste heat, is a significant cost element (more than 5 cents/l of ethanol) if done at the pipeline inlet unless waste heat is available, for example from an electric power plant located adjacent to the pipeline inlet. Heat loss in a 1.26 m pipeline carrying 2 M drytonnes/yr is about 5 degrees C at a distance of 400 km in typical prairie clay soils, and would not likely require insulation; smaller pipelines or different soil conditions might require insulation for STS. Saccharification in the pipeline would reduce the need for investment in the fermentation plant, saving about 0.2 cents/l of ethanol. Transport of corn stover in multiple pipelines offers the opportunity to develop a large ethanol fermentation plant, avoiding some of the diseconomies of scale that arise from smaller plants whose capacities are limited by issues of truck congestion.     

Characterization of beta-glucosidase from corn stover and its application in simultaneous saccharification and fermentation

Purification and characterization of beta-glucosidase from corn stover was performed and the enzyme was tried in SSF to evaluate the suitability of plant glycosyl hydrolases in lignocellulose conversion. A beta-glucosidase with M(w) of 62.4 kDa was purified to homogeneity from post-harvest corn stover. The following physicochemical and kinetic parameters of the beta-glucosidase were studied respectively: optimum temperature, thermal stability, optimum pH, pH stability, K(m), V(max), V(i), cellobiose inhibition, tryptic peptide mass spectrometry and effect of metal ions and other reagents on the activity. The beta-glucosidase activity on salicin was optimal at pH 4.8 and 37 degrees C. The unique property of optimum temperature makes the beta-glucosidase potentially useful in SSF. In SSF of steam explosion pretreated corn stover, the supplementation of the purified beta-glucosidase was more effective than Aspergillus niger beta-glucosidase.     

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.     

Effect of surfactants on separate hydrolysis fermentation and simultaneous saccharification fermentation of pretreated lodgepole pine

The effects of surfactants addition on enzymatic hydrolysis and subsequent fermentation of steam exploded lodgepole pine (SELP) and ethanol pretreated lodgepole pine (EPLP) were investigated in this study. Supplementing Tween 80 during cellulase hydrolysis of SELP resulted in a 32% increase in the cellulose‐to‐glucose yield. However, little improvement was obtained from hydrolyzing EPLP in the presence of the same amount of surfactant. The positive effect of surfactants on SELP hydrolysis led to an increase in final ethanol yield after the fermentation. It was found that the addition of surfactant led to a substantial increase in the amount of free enzymes in the 48 h hydrolysates derived from both substrates. The effect of surfactant addition on final ethanol yield of simultaneous saccharification and fermentation (SSF) was also investigated by using SELP in the presence of additional furfural and hydroxymethylfurfural (HMF). The results showed that the surfactants slightly increased the conversion rates of furfural and HMF during SSF process by Saccharomyces cerevisiae. The presence of furfural and HMF at the experimental concentrations did not affect the final ethanol concentration either. The strategy of applying surfactants in cellulase recycling to reduce enzyme cost is presented. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Bioconversion of corn stover derived pentose and hexose to ethanol using cascade simultaneous saccharification and fermentation (CSSF)

A cascade type of fermentation, designated the cascade simultaneous saccharification and fermentation (CSSF), was studied to convert corn stover derived pentose and hexose to ethanol with reduced enzyme input. In detail, each step of CSSF utilizes two sequential SSF phases operating on pentose and hexose, i.e., pentose conversion using xylanase, endo-glucanase, and recombinant Escherichia coli (KO11) with minimal glucose conversion in the first phase SSF, and hexose conversion in the second phase SSF using cellulase, β-glucosidase, and Saccharomyces cerevisiae (D(5)A). In this cascade scheme, multiple stages of 1st and 2nd phase SSF were performed in series; enzymes are recycled from the fermentation broth of the last stage for the use of the next stage. This bioconversion process yielded up to 60% of the theoretical maximum ethanol yield based on the total sugars in untreated corn stover, while enzyme loadings were reduced by 50% (v/v) and the final ethanol concentration reached 27 g/l.     

响应面法优化玉米秸秆同步酶解发酵产乙醇条件

对汽爆玉米秸秆同步酶解发酵生产乙醇的条件进行优化。首先利用Fractional Factorial设计法对影响乙醇产量的7个因素进行评价,筛选出具有显著效应的3个因素,即反应温度、酶添加量、总反应时间,再以Box—Behnken设计法及响应面分析法确定主要因素的最佳水平,即反应温度37℃,每g纤维素添加纤维素酶32u,反应时间87h,此时乙醇体积分数达到3．69％。新工艺条件实验结果表明,乙醇体积分数在87h可达到3．76％,和原工艺相比,反应时间缩短了9h,乙醇体积分数提高了13％。     

Enzymatic hydrolysis and simultaneous saccharification and fermentation of steam-pretreated spruce using crude Trichoderma reesei and Trichoderma atroviride enzymes

The aim of this study was to compare the performance of the enzymes produced by Trichoderma reesei Rut C30 and the good extracellular β-glucosidase-producing mutant Trichoderma atroviride TUB F-1663 to that of commercial preparations in the enzymatic hydrolysis and the simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce (SPS).The concentrated TUB F-1663 enzyme was found to be the most efficient in the hydrolysis of washed SPS at 50 g/L water-insoluble solids (WIS) in terms of the glucose produced (18.5 g/L), even in comparison with commercial cellulases (14.1–16.7 g/L). The enzyme preparations were studied at low enzyme loadings (5 FPU/g WIS) in SSF to produce ethanol from SPS. The enzyme supernatant and whole fermentation broth of T. atroviride as well as the whole broth of T. reesei proved to be as efficient in SSF as the commercial cellulase mixtures (ethanol yields of 61–76% of the theoretical were achieved), while low ethanol yields (<40%) were obtained with the β-glucosidase-deficient T. reesei supernatant.Therefore, it seems, that instead of using commercial cellulases, the TUB F-1663 enzymes and the whole broth of Rut C30 may be produced on-site, using a process stream as carbon source, and employed directly in the biomass-to-bioethanol process.     

Kinetic studies of corn stover saccharification using sulphuric acid

The kinetics of crystalline cellulose and hemicellulose hydrolysis in corn stover were studied with a nonisothermal technique. Reactions were arrested at temperatures between 160 and 240 degrees C and product sugars were analyzed using a Bio-Rad HPX-85 liquid chromatographic column. A simple first-order series reaction model was used for both cellulose and hemicellulose hydrolysis reactions. Kinetic parameters were obtained for three different sulphuric acid concentrations (0.49, 0.92, and 1.47 wt %). Activation energies remained constant over this acid concentration range but the preexponential factors showed an increase with acid concentration. Relationships were obtained between the preexponential factors and acid concentrations. Cellulose hydrolysis and glucose degradation reactions were observed to be of higher order with respect to acid concentration in comparison with the previous studies with other raw materials.     

Ethanol and furfural production from corn stover using a hybrid fractionation process with zinc chloride and simultaneous saccharification and fermentation (SSF)

A two-stage hybrid fractionation process was investigated to produce cellulosic ethanol and furfural from corn stover. In the first stage, zinc chloride (ZnCl₂) was used to selectively solubilize hemicellulose. During the second stage, the remaining treated solids were converted into ethanol using commercial cellulase and Saccharomyces cerevisiae or recombinant Escherichia coli, KO11. This hybrid fractionation process recovered 93.8% of glucan, 89.7% of xylan, 71.1% of arabinan, and 74.9% of lignin under optimal reaction conditions (1st stage: 5% acidified ZnCl₂, 7.5 ml/min, 150 °C (10 min) and 170 °C (10 min); 2nd stage: simultaneous saccharification and fermentation (SSF) using S. cerevisiae). The furfural yield from the hemicellulose hydrolysates was 58%. The SSF of the treated solids resulted in 69–98% of the theoretical maximum ethanol yields based on the glucan content in the treated solids. After fermentation, the solid residues contained primarily lignin. Based on the total lignin in untreated corn stover, the lignin recovery yield was 74.9%.     

Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400

The two main sugars in the agricultural by-product corn stover are glucose and xylose. Co-fermentation of glucose and xylose at high content of water-insoluble solids (WIS) without detoxification is a prerequisite to obtain high ethanol concentration and to reduce production costs. A recombinant strain of Saccharomyces cerevisiae, TMB3400, was used in simultaneous saccharification and fermentation (SSF) of whole pretreated slurry of corn stover at high WIS. TMB3400 co-fermented glucose and xylose with relatively high ethanol yields giving high final ethanol concentration. The ethanol productivity increased with increasing concentration of pretreatment hydrolysate in the yeast production medium and when SSF was performed in a fed-batch mode.     

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.     

Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature

Background

Simultaneous saccharification and fermentation (SSF) is a promising process for bioconversion of lignocellulosic biomass. High glucan loading for hydrolysis and fermentation is an efficient approach to reduce the capital costs for bio-based products production. The SSF of steam-exploded corn stover (SECS) for ethanol production at high glucan loading and high temperature was investigated in this study.

Results

Glucan conversion of corn stover biomass pretreated by steam explosion was maintained at approximately 71 to 79% at an enzyme loading of 30 filter paper units (FPU)/g glucan, and 74 to 82% at an enzyme loading of 60 FPU/g glucan, with glucan loading varying from 3 to 12%. Glucan conversion decreased obviously with glucan loading beyond 15%. The results indicated that the mixture was most efficient in enzymatic hydrolysis of SECS at 3 to 12% glucan loading. The optimal SSF conditions of SECS using a novel Saccharomyces cerevisiae were inoculation optical density (OD)₆₀₀?=?4.0, initial pH 4.8, 50% nutrients added, 36 hours pre-hydrolysis time, 39°C, and 12% glucan loading (20% solid loading). With the addition of 2% Tween 20, glucan conversion, ethanol yield, final ethanol concentration reached 78.6%, 77.2%, and 59.8 g/L, respectively, under the optimal conditions. The results suggested that the solid and degradation products’ inhibitory effect on the hydrolysis and fermentation of SECS were also not obvious at high glucan loading. Additionally, glucan conversion and final ethanol concentration in SSF of SECS increased by 13.6% and 18.7%, respectively, compared with separate hydrolysis and fermentation (SHF).

Conclusions

Our research suggested that high glucan loading (6 to 12% glucan loading) and high temperature (39°C) significantly improved the SSF performance of SECS using a thermal- and ethanol-tolerant strain of S. cerevisiae due to the removal of degradation products, sugar feedback, and solid’s inhibitory effects. Furthermore, the surfactant addition obviously increased ethanol yield in SSF process of SECS.

     

Acidogenic fermentation of corn stover

Corn stover was fermented by anaerobic acidogenic bacteria to produce volatile (C₂–C₆) organic acids. Mild pretreatment with dilute alkali solutions produced a two-fold increase in fermentability. A mixture of lime and sodium carbonate was found to be a better pretreatment agent than sodium hydroxide. Methane generation was inhibited by low temperature (? 25°C) and high solids [≥ 2.5% (w/v)] fermentation. Volatile acid yields of 0.5–0.55 g acetic acid equiv/g dry ash-free (DAF) stover could be obtained in batch fermentations. Several extractants and extraction solvents for organic acids were found to be nontoxic to acidogenic fermentation. The data show that acidogenic fermentation can produce useful volatile fatty acids in high yields from a complex lignocellulosic feedstock. These fermentations are nonsterile, need no stirring, and are easy to run. Moreover, cellulose, pentosans, and other carbohydrates are directly utilized by acidogenic bacteria. Hence, acidogenic fermentation could be useful in converting biomass to chemical feedstocks and fuel.     

Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor

The higher ethanol titer inevitably requires higher solids loading during the simultaneous enzymatic saccharification and fermentation (SSF) using lignocellulose as the feedstock. The mixing between the solid lignocellulose and the liquid enzyme is crucially important. In this study, a bioreactor with a novel helical impeller was designed and applied to the SSF operation of the steam explosion pretreated corn stover under different solids loadings and different enzyme dosages. The performances using the helical impeller and the common Rushton impeller were compared and analyzed by measuring rheological properties and the mixing energy consumption. The results showed that the new designed stirring system had better performances in the saccharification yield, ethanol titer, and energy cost than those of the Rushton impeller stirring. The mixing energy consumption under different solids loadings and enzyme dosages during SSF operation were analyzed and compared to the thermal energy in the ethanol produced. A balance for achieving the optimal energy cost between the increased mixing energy cost and the reduced distillation energy cost at the high solids loading should be made. The potentials of the new bioreactor were tested under various SSF conditions for obtaining optimal ethanol yield and titer. Biotechnol. Bioeng. 2010. 105: 718–728. © 2009 Wiley Periodicals, Inc.     

Continuous simultaneous saccharification and fermentation of starch using Zymomonas mobilis

A continuous process involving simultaneous saccharification and fermentation of liquefied starch has been developed using Zymomonas mobilis. Amyloglucosidase retention and cell recycle have been effected by using an Amicon hollow-fiber membrane system with a MW cutoff of 5000. Relatively high productivities of up to 60 g L(-1) h(-1) have been achieved at ethanol concentrations of 60-65 g/L. The system also offers the potential for reduced enzyme requirements for saccharification.     

Simultaneous saccharification and fermentation of steam-pretreated bagasse using Saccharomyces cerevisiae TMB3400 and Pichia stipitis CBS6054

Sugarcane bagasse--a residue from sugar and ethanol production from sugar cane--is a potential raw material for lignocellulosic ethanol production. This material is high in xylan content. A prerequisite for bioethanol production from bagasse is therefore that xylose is efficiently fermented to ethanol. In the current study, ethanolic fermentation of steam-pretreated sugarcane bagasse was assessed in a simultaneous saccharification and fermentation (SSF) set-up using either Saccharomyces cerevisiae TMB3400, a recombinant xylose utilizing yeast strain, or Pichia stipitis CBS6054, a naturally xylose utilizing yeast strain. Commercial cellulolytic enzymes were used and the content of water insoluble solids (WIS) was 5% or 7.5%. S. cerevisiae TMB3400 consumed all glucose and large fraction of the xylose in SSF. Almost complete xylose conversion could be achieved at 5% WIS and 32 degrees C. Fermentation did not occur with P. stipitis CBS6054 at pH 5.0. However, at pH 6.0, complete glucose conversion and high xylose conversion (>70%) was obtained. Microaeration was required for P. stipitis CBS6054. This was not necessary for S. cerevisiae TMB3400.     

Wet oxidation pretreatment, enzymatic hydrolysis and simultaneous saccharification and fermentation of clover-ryegrass mixtures

The potential of clover (Trifolium repens) and ryegrass (Lolium perenne) mixtures as raw materials for ethanol production was investigated. Wet oxidation, at 175, 185 or 195 degrees C during 10min at two different oxygen pressures and with either addition or no addition of sodium carbonate, was evaluated as pretreatment method for clover-ryegrass mixtures. The enzymatic hydrolysis of cellulose was significantly improved after pretreatment. The highest conversion efficiency, 93.6%, was achieved for the sample pretreated at 195 degrees C, 10min, 1.2MPa and no addition of Na(2)CO(3). For that sample, the overall glucose yield after pretreatment and hydrolysis was 75.5%. No inhibition of cellulose enzymatic conversion by the filtrates was observed. The simultaneous saccharification and fermentation of the pretreated material yielded cellulose conversions of 87.5 and 86.6%, respectively, with Saccharomyces cerevisiae and the filamentous fungus Mucor indicus, and revealed that no addition of nutrients is needed for the fermentation of clover-ryegrass hydrolysates.