Modeling simultaneous saccharification and fermentation of softwood

Simultaneous saccharification and fermentation (SSF) of wood has been modeled for the past 15–20 years, but the substrates used for model evaluation have so far not included pretreated softwood. In the present study, data from lab-scale batch SSF of SO₂-impregnated, steam-pretreated spruce chips were used to evaluate a model found in the literature. The model, which was somewhat modified, consists of a number of nonlinear, coupled ordinary differential equations, which were solved numerically. Some parameter values were fitted to data by use of least-squares minimization. A difficulty in parameter estimation was the lack of cellobiose measurements, something that was relieved by adding assumptions about parameter relations. The simulated concentration profiles agreed well with the measured concentrations of glucose and ethanol. It is therefore concluded that the basic model features apply to softwood SSF. The model predicts rate saturation with respect to enzyme concentration at concentrations above 60 FPU/g cellulose, although this was not observed in the experimental data, which only comprised enzyme concentrations up to 32 FPU/g cellulose.     

Recirculation of process streams in fuel ethanol production from softwood based on simultaneous saccharification and fermentation

The effect of process stream recirculation on ethanol production from steam- pretreated softwood based on simultaneous saccharification and fermentation (SSF) was investigated for two process configurations. In the first configuration, a part of the stillage stream after distillation was recycled and, in the second configuration, the liquid after SSF was recycled. The aim was to minimize the energy consumption in the distillation of the fermentation broth and in the evaporation of the stillage, as well as the use of fresh water. However, recirculation leads to an increased concentration of nonvolatiles in the first configuration, and of both volatiles and nonvolatiles in the second configuration. These substances might be inhibitory to the enzymes and the yeast in SSF. When 60% of the fresh water was replaced by stillage, the ethanol yield and the productivity were the same as for the configuration without recirculation. The ethanol production cost was reduced by 17%. In the second configuration, up to 40% of the fresh water could be replaced without affecting the final ethanol yield, although the initial ethanol productivity decreased. The ethanol production cost was reduced by 12%. At higher degrees of recirculation, fermentation was clearly inhibited, resulting in a decrease in ethanol yield while hydrolysis seemed unaffected.     

Thermotolerant yeast for simultaneous saccharification and fermentation of cellulose to ethanol

Ten promising microbial strains were screened for glucose fermentation over the temperature range of 37–47°C, and five temperature-tolerant yeasts (Saccharomyces cerevisiae SERI strain (D₅A),S. uvarum, andCandida generaacidothermophilium, brassicae, andlusitaniae), were chosen for SSF evaluation on Sigmacell-50 cellulose with Genencor 150 L cellulase enzyme.Brettanomyces clausenii (Y-1414) was included for comparison to previous studies both by itself and in mixed culture withS. cerevisiae (D₅A). Good conversion rates were achieved at temperatures as high as 43°C withC. brassicae andS. uvarum; mixed cultures of either of these yeasts with the thermotolerant cellobiose fermenting yeastC. lusitaniae achieved higher rates and yields than any of the three yeasts alone. However, the mixed culture ofB. clausenii andS. cerevisiae at 37°C achieved as high conversion rates and higher yields than any of the other yeasts tested.     

纤维素酶在食品发酵工业中的应用及其前景

本文论述了纤维素与纤维素酶的研究进展。着重阐述了纤维素酶在食品与发酵工业中的应用及其木质纤维类废物利用等最新动态、同时阐明了纤维素与纤维素酶在食品及发酵工业应用中存在的问题。     

Optimization of the simultaneous saccharification and fermentation process using thermotolerant yeasts

Different treatments to improve the thermotolerance of fermenting yeasts for simultaneous ethanol saccharification and fermentation process of cellulosic materials have been examined. Yeasts of the generaSaccharomyces andKluyveromyces were tested for growth and fermentation at progressively higher temperatures in the range of 42–47°C. The best results were obtained withK. marxianus LG, which was then submitted to different treatments in order to achieve thermotolerant clones. A total of 35 new clones were obtained that dramatically improved the SSF of 10% Solka-floc substrate at 45°C when compared to the original strain, some with ethanol concentrations as high as 33 g/L.     

Simultaneous saccharification and fermentation of steam-pretreated spruce to ethanol

Ethanol production was studied in simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce at 42°C, using a thermotolerant yeast. Three yeast strains of Kluyveromyces marxianus were compared in test fermentations. SSF experiments were performed with the best of these on 5% (w/w) of substrate at a cellulase loading of 37 filter paper units/g of cellulose, and a β-glucosidase loading of 38 IU/gof cellulose. The detoxification of the substrate and the lack of pH control in the experiments increased the final ethanol concentration. The final ethanol yield was 15% lower compared to SSF with Saccharomyces cerevisiae at 37°C, owing to the cessation of ethanol fermentation after the first 10 h.     

Simultaneous saccharification and fermentation of cassava bagasse for l-(+)-lactic acid production using Lactobacilli

Saccharification and fermentation of cassava (Manihot esculenta) bagasse was carried out in a single step for the production of L-(+)-lactic acid by Lactobacillus casei and Lactobacillus delbrueckii. Using 15.5% w/v of cassava bagasse as the raw material, a maximum starch to lactic acid conversion of 96% was obtained with L. casei with a productivity rate of 1.40mg/mL·h and maximum yield of 83.8 mg/mL. It was 94% with L. delbrueckii with a productivity rate of 1.36 mg/mL·h. and maximum yeild of 81.9 mg/mL. Supplementation of bagasse with 0.01% w/v MnCl₂ showed positive influence on the lactic acid production by L. casei.     

Ammonia fiber explosion treatment of corn stover

Optimizing process conditions and parameters such as ammonia loading, moisture content of biomass, temperature, and residence time is necessary for maximum effectiveness of the ammonia fiber explosion process. Approximate optimal pretreatment conditions for corn stover were found to be temperature of 90°C, ammonia: dry corn stover mass ratio of 1∶1, moisture content of corn stover of 60% (dry weight basis), and residence time (holding at target temperature), of 5 min. Approximately 98% of the theoretical glucose yield was obtained during enzymatic hydrolysis of the optimal treated corn stover using 60 filter paper units (FPU) of cellulase enzyme/g of glucan (equal to 22 FPU/g of dry corn stover). The ethanol yield from this sample was increased up to 2.2 times over that of untreated sample. Lowering enzyme loading to 15 and 7.5 FPU/g of glucan did not significantly affect the glucose yield compared with 60 FPU, and any differences between effects at different enzyme levels decreased as the treatment temperature increased.     

Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol

A total of 27 yeast strains belonging to the groupsCandida, Saccharomyces, andKluyveromyces were screened for their ability to grow and ferment glucose at temperatures ranging 32-45°C. K. marxianus andK. fragilis were found to be the best ethanol producing organisms at the higher temperature tested and, so, were selected for subsequent simultaneous saccharification and fermentation (SSF) studies.     

Pretreatment of switchgrass by ammonia fiber explosion (AFEX)

The effects of ammonia fiber explosion (AFEX) pretreatment of switch grass using its major process variables are reported. The optimal pretreatment conditions for switchgrass were found to be near 100°C reactor temperature, and ammonia loading of 1:1 kg of ammonia: kg of dry matter with 80% moisture content (dry weight basis [dwb]) at 5 min residence time. Hydrolysis results of AFEX-treated and untreated samples showed 93% vs 16% glucan conversion, respectively. The ethanol yield of optimized AFEX-treated switchgrass was measured to be about 0.2 g ethanol/g dry biomass, which is 2.5 times more than that of the untreated sample.     

The simultaneous saccharification and fermentation of pretreated woody crops to ethanol

Four promising woody crops (Populusmaximowiczii x nigra (NE388), P.trichocarpa x deltoides (Nll), P.tremuloides, and SweetgumLiquidambar styraciflua) were pretreated by dilute sulfuric acid and evaluated in the simultaneous saccharification and fermentation (SSF) process for ethanol production. The yeastSaccharomyces cerevisiae was used in the fermentations alone, and in mixed cultures with β -glucosidase producingBrettanomyces dausenii. Commercial Genencor 150L cellulase enyme was either employed alone or supplemented with β- glucosidase. All SSFs were run at 37 …C for 8 d and compared to saccharifications at 45…C under the same enzyme loadings.S. cerevisiae alone achieved the highest ethanol yields and rates of hydrolysis at the higher enzyme loadings, whereas the mixed culture performed better at the lower enzyme loadings without β -glucosidase supplementation. The best overall rates of fermentation (3 d) and final theoretical ethanol yields (86–90%) were achieved with P.maximowiczii x nigra (NE388) and SweetgumLiquidambar styraciflua, followed by P.tremuloides and P.trichocarpa xdeltoides (N1l) with slightly slower rates and lower yields. Although there were some differences in SSF performance, all these pretreated woody crops show promise as substrates for ethanol production.     

Analysis of biomass cellulose in simultaneous saccharification and fermentation processes

A direct method for determining the cellulose content of biomass residues resulting from simultaneous saccharifiaction and fermentation (SSF) experiment has been developed and evaluated. The method improves on classical cellulose assays by incorporating the enzymatic removal of yeast glucans from the biomass residue prior to acid hydrolysis and subsequent quantification of cellulose-derived glucose. An appropriate cellulasefree, commercially available, yeast-lysing enzyme preparation fromCytophaga was identified. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzymatic lysis. The method was applied to the analysis of cellulose and yeast-associated glucans in SSF residues from three pretreated feedstocks; hybrid poplar, switchgrass, and cornstover. Cellulose assays employing the lysing-enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucans were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to subsequent chemical and physical analysis of SSF residues. Oregon State University Agricultural Experiment Station Technical Publication Number 10977.     

Fourier transform infrared imaging and microscopy studies of Pinus radiata pulps regarding the simultaneous saccharification and fermentation process

The distribution and chemical patterns of lignocellulosic components at microscopic scale and their effect on the simultaneous saccharification and fermentation process (SSF) in the production of bioethanol from Pinus radiata pulps were analyzed by the application of diverse microscopical techniques, including scanning electronic microscopy (SEM), confocal laser scanning microscopy (CLSM) and attenuated total reflectance (ATR) – Fourier transform infrared microspectroscopy. This last technique was accompanied with multivariate methods, including principal component analysis (PCA) and multivariate curve resolution with alternating least squares (MCR-ALS) to evaluate the distribution patterns and to generate pure spectra of the lignocellulosic components of fibers. The results indicate that the information obtained by the techniques is complementary (ultrastructure, confocality and chemical characterization) and that the distribution of components affects the SSF yield, identifying lignin coalescence droplets as a characteristic factor to increase the SSF yield. Therefore, multivariate analysis of the infrared spectra enabled the in situ identification of the cellulose, lignin and lignin-carbohydrates arrangements. These techniques could be used to investigate the lignocellulosic components distribution and consequently their recalcitrance in many applications where minimal sample manipulation and microscale chemical information is required.     

Process improvement in acetone-butanol production from hardwood by simultaneous saccharification and extractive fermentation

The acetone-butanol production by simultaneous saccharification and extractive fermentation (SSEF) was investigated. In the SSEF employing cellulase enzymes andClostridium acetobutylicum, both glucan and xylan fractions of pretreated aspen are concurrently converted into acetone and butanol. Continuous removal of the fermentation products from the bioreactor by extraction was an important factor that allowed long-term fed-batch operation. The use of membrane extraction prevented the problems of phase separation and extractant loss. Increase in substrate feeding as well as reduction of nutrient supply was found to be beneficial in suppressing the acid production, thereby improving the solvent yield. Because of prolonged low growth conditions prevalent in the fed-batch operation, the butanol-to-acetone ratio in the product was significantly higher at 2.6–2.8 compared to the typical value of two.     

Pretreatment of corn stover by soaking in aqueous ammonia

Soaking in aqueous ammonia (SAA) was investigated as a pretreatment method for corn stover. In this method, the feedstock was soaked in aqueous ammonia over an extended period (10–60 d) at room temperature. It was done without agitation at atmospheric pressure. SAA treatment removed 55–74% of the lignin, but retained nearly 100% of the glucan and 85% of the xylan. The xylan remaining in the corn stover after SAA treatment was hydrolyzed along with the glucan by xylanase present in the Spezyme CP enzyme. In the simultaneous saccharification and fermentation (SSF) test of SAA-treated corn stover, using S. cerevisiae (D₅A), an ethanol yield of 73% of theoretical maximum was obtained on the basis of the glucan content in the treated corn stover. The accumulation of xylose in the SSF appears to inhibit the cellulase activity on glucan hydrolysis, which limits the yield of ethanol. In the simultaneous saccharification and co-fermentation (SSCF) test, using recombinant E. coli (KO11), both the glucan and xylose were effectively utilized, resulting in on overall ethanol yield of 77% based on the glucan and xylan content of the substrate. When the SSCF process is used, the fact that the xylan fraction is retained during pretreatment is a desirable feature since the overall bioconversion can be carried out in a single step without separate recovery of xylose from the pretreatment liquid.     

Nonisothermal simultaneous saccharification and fermentation for direct conversion of lignocellulosic biomass to ethanol

The enzymatic reaction in the simultaneous saccharification and fermentation (SSF) is operated at a temperature much lower than its optimum level. This forces the enzyme activity to be far below its potential, consequently raising the enzyme requirement. To alleviate this problem, a nonisothermal simultaneous saccharification and fermentation process (NSSF) was investigated. The NSSF is devised so that saccharification and fermentation occur simultaneously, yet in two separate reactors that are maintained at different temperatures. Lignocellulosic biomass is retained inside a column reactor and hydrolyzed at the optimum temperature for the enzymatic reaction (50°C). The effluent from the column reactor is recirculated through a fermenter, which runs at its optimum temperature (20-30°C). The cellulase enzyme activity is increased by a factor of 2-3 when the hydrolysis temperature is raised from 30 to 50°C. The NSSF process has improved the enzymatic reaction in the SSF to the extent that it reduces the overall enzyme requirement by 30-40%. The effect of temperature on β-glucosidase activity was the most significant among the individual cellulase compounds. Both ethanol yield and productivity in the NSSF are substantially higher than those in the SSF at the enzyme loading of 5 IFPU/g glucan. With 10 IFPU/g glucan, improvement in productivity was more discernible for the NSSF. The terminal yield attainable in 4 d with the SSF was reachable in 40 h with the NSSF.     

Optimization of Bioethanol Production from Enzymatic Treatment of Argan Pulp Feedstock

Argan pulp is an abundant byproduct from the argan oil process. It was investigated to study the feasibility of second-generation bioethanol production using, for the first time, enzymatic hydrolysis pretreatment. Argan pulp was subjected to an industrial grinding process before enzymatic hydrolysis using Viscozyme L and Celluclast 1.5 L, followed by fermentation of the resulting sugar solution by Saccharomyces cerevisiae. The argan pulp, as a biomass rich on carbohydrates, presented high saccharification yields (up to 91% and 88%) and an optimal ethanol bioconversion of 44.82% and 47.16% using 30 FBGU/g and 30 U/g of Viscozyme L and Celluclast 1.5 L, respectively, at 10%_w/v of argan biomass.     

Large-scale ethanol fermentation through pipeline delivery of biomass

Issues of traffic congestion and community acceptance limit the size of biomass-processing plants based on truck delivery to about 2 million (M) dry t/yr or less. In this study, the cost of ethanol from an ethanol fermentation plant processing 2 M dry t/yr of corn stover supplied by truck is compared with that of larger plants in the range of 4–38 M dry t/yr supplied by a combination of trucks plus pipelines. For corn stover, a biomass source with a low yield per gross hectare, the cost of ethanol from larger plants is always higher. For wood chips from the boreal forest, a biomass source with a relatively high yield per gross hectare, a plant processing 14–38 M dry t/yr produces ethanol at a 13% reduction in cost compared with a plant producing 2 M dry t/yr supplied by truck. Processing of value-added products, such as chemicals from lignin, would be enabled by larger-scale plants.     

Two-step steam pretreatment of softwood with SO2 impregnation for ethanol production

Two-step steam pretreatment of softwood was investigated with the aim of improving the enzymatic digestibility for ethanol production. In the first step, softwood was impregnated with SO₂ and steam pretreated at different severities. The first step was performed at low severity to hydrolyze the hemicellulose and release the sugars into the solution. The combination of time and temperature that yielded the highest amount of hemicellulosic sugars in the solution was determined. In the second step, the washed solid material from the optimized first step was impregnated once more with SO₂ and steam pretreated under more severe conditions to enhance the enzymatic digestibility. The investigated temperature range was between 180 and 220°C, and the residence times were 2, 5 and 10 min. The effectiveness of pretreatment was assessed by both enzymatic hydrolysis of the solids and simultaneous saccharification and fermentation (SSF) of the whole slurry after the second pretreatment step, in the presence of antibiotics. For each pretreatment combination, the liquid fraction was fermented to determine any inhibiting effects. At low severity in the second pretreatment step, a high conversion of cellulose was obtained in the enzymatic hydrolysis step, and at a high severity a high conversion of cellulose was obtained in the second pretreatment step. This resulted in an overall yield of sugars that was nearly constant over a wide range of severity. Compared with the one-step steam pretreatment, the two-step steam pretreatment resulted in a higher yield of sugar and in a slightly higher yield of ethanol. The overall sugar yield, when assessed by enzymatic hydrolysis, reached 80%. In the SSF configuration, an overall ethanol yield of 69% was attained.     

Effect of surfactants and zeolites on simultaneous saccharification and fermentation of steam-exploded poplar biomass to ethanol

In this work, the effect of the addition of different concentrations of Tween-80 and three different zeolite-like products on enzymatic hydrolysis, ethanol fermentation, and simultaneous saccharification and fermentation (SSF) process has been investigated. The ability of these products to enhance the effectiveness of the SSF process to ethanol of steam-exploded poplar biomass using the thermotolerant strainKluyveromyces marxianus EMS-26 has been tested. Tween-80 (0.4 g/L) increased enzymatic hydrolysis yield by 20% when compared to results obtained in hydrolysis in absence of the additive. Zeolite-like products (ZESEP-56 and ZECER-56) (2.5 g/L) improved rates of conversion and ethanol yields in the fermentation of liquid fraction recovered from steam-exploded poplar. The periods required for the completion of fermentation were approx 10 h in the presence of zeolite-like products and 24 h in the absence of additives. The probable mode of action is through lowered levels of inhibitory substances because of adsorption by the additive.