Simultaneous saccharification and extractive fermentation for acetone/butanol production from pretreated hardwood

Applied Biochemistry and Biotechnology - An integrated bioreactor-extractor was investigated for applicability in simultaneous saccharification and extractive fermentation (SSEF), and production of...     

Simultaneous saccharification and fermentation of lignocellulose

Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of 80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.     

Simultaneous saccharification and fermentation of pretreated wheat straw to ethanol with selected yeast strains and β-glucosidase supplementation

Previous shake flask and stirred tank evaluations of temperature tolerant (37–43°C) yeasts in simultaneous saccharification and fermentation (SSF) on Sigmacell-50 cellulose substrates to ethanol have identified several good microorganisms for further SSF studies (27). Of these, the glucose fermenting yeastCandida acidothermophilum, C. brassicae, Saccharomyces cerevisiae, S. uvarum, and a mixed culture of the cellobiose fermenting yeastBrettanomyces clausenii withS. cerevisiae as a control were chosen for shake flask SSF screening experiments with pretreated wheat straw. This study indicates that theSaccharomyces strainscerevisiae anduvarum, give very good performance at high cellulase loadings or when supplemented with Novo-188 β-glucosidase. In fact, with the higher enzyme loadings these yeast will give complete conversion of cellulose to ethanol. Yet at the lower, more economical enzyme loadings, the mixed culture ofBrettanomyces clausenii andS. cerevisiae performs better than any single yeast.

     

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.     

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.     

Evaluation of pretreated herbaceous crops for the simultaneous saccharification and fermentation process

Applied Biochemistry and Biotechnology - Three dilute acid pretreated herbaceous crops (Weeping lovegrass,Eragrotis curvula; Sericea lespedeza,Lespedeza cuneata; and switch-grass,Panicum virgatum)...     

High solids simultaneous saccharification and fermentation of pretreated wheat straw to ethanol

Wheat straw was pretreated with dilute (0.5%) sulfuric acid at 140°C for 1 h. Pretreated straw solids were washed with deionized water to neutrality and then stored frozen at –20°C. The approximate composition of the pretreated straw solids was 64% cellulose, 33% lignin, and 2% xylan. The cellulose in the pretreated wheat straw solids was converted to ethanol in batch simultaneous saccharification and fermentation experiments at 37°C using cellulase enzyme fromTrichoderma reesei (Genencor 150 L) with or without supplementation with β–glucosidase fromAspergillus niger (Novozyme 188) to produce glucose sugar and the yeastSaccharomyces cerevisiae to ferment the glucose into ethanol. The initial cellulose concentrations were adjusted to 7.5, 10, 12.5, 15, 17.5, and 20% (w/w). Since wheat straw particles do not form slurries at these concentrations and cannot be mixed with conventional impeller mixers used in laboratory fermenters, a simple rotary fermenter was designed and fabricated for these experiments. The results of the simultaneous saccharification and fermentation (SSF) experiments indicate that the cellulose in pretreated wheat straw can be efficiently fermented into ethanol for up to a 15% cellulose concentration (24.4% straw concentration).     

Simultaneous saccharification and fermentation of cellulosic biomass to acetic acid

Astrain of Clostridium thermoaceticum (ATCC 49707) was evaluated for its homoacetate potential. This thermophilic anaerobe best produces acetate from glucose at pH 6.0 and 59°C with a yield of 83% of theoretical. Enzyme hydrolysis of two substrates, a-cellulose and a pulp mill sludge, yielded 68% and 70% digestion, respectively. The optimum conditions for the simultaneous saccharification and fermentation (SSF) were substrate dependent: 55°C, pH 6.0 for α-cellulose, and 55°C, pH 5.5 for the pulp mill sludge. In the SSF with α-cellulose, the overall yield of acetate was strongly influenced by the enzyme loading. In a fed-batch operation of SSF with α-cellulose, an overall acetic acid yield of 60 wt% was obtained. Among the factors limiting the yields were incomplete digestion by the enzyme and the end-product inhibition. In the SSF of pulp mill sludge, inhibitors present in the sludge severely limited bacterial action. A large accumulation of glucose developed over the entire process, changing the intended SSF operation into a separate hydrolysis and fermentation operation. Despite a long lag phase of microbial growth, a terminal yield of 85% was obtained with this substrate.     

Simultaneous saccharification and cofermentation of dilute-acid pretreated yellow poplar hardwood to ethanol using xylose-fermenting Zymomonas mobilis

Simultaneous saccharification and cofermentation (SSCF) was carried out at approximately 15% total solids using conditioned dilute-acid pretreated yellow poplar feedstock, an adapted variant of National Renewable Energy Laboratory (NREL) xylose-fermenting Zymomonas mobilis and either commercial or NREL-produced cellulase enzyme preparations. In 7 d, at a cellulase loading of 12 filter paper units pergram cellulose (FPU/g), the integrated system produced more than 3% w/v ethanol and achieved 54% conversion of all potentially available biomass sugars (total sugars) entering SSCF. A control SSCF employing Sigmacell cellulose and a commercial cellulase at an enzyme loading of 14 FPU/gachieved 65% conversion of total sugars to ethanol.     

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.     

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.     

Simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid in a two-zone fermentor-extractor system

Simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid was investigated using a two-zone bioreactor. The system is composed of an immobilized cell reactor, a separate column reactor containing the lignocellulosic substrate and a hollow-fiber membrane. It is operated by recirculating the cell free enzyme (cellulase) solution from the immobilized cell reactor to the column reactor through the membrane. The enzyme and microbial reactions thus occur at separate locations, yet simultaneously. This design provides flexibility in reactor operation as it allows easy separation of the solid substrate from the microorganism, in situ removal of the product and, if desired, different temperatures in the two reactor sections. This reactor system was tested using pretreated switchgrass as the substrate. It was operated under a fed-batch mode with continuous removal of lactic acid by solvent extraction. The overall lactic acid yield obtainable from this bioreactor system is 77% of the theoretical.     

Simultaneous saccharification and cofermentation of peracetic acid-pretreated biomass

Previous work in our laboratories has demonstrated the effectiveness of peracetic acid for improving enzymatic digestibility of lignocellulosic materials. The use of dilute alkali solutions as a pre-pretreatment prior to peracetic acid lignin oxidation increased carbohydrate hydrolysis yields in a synergistic as opposed to additive manner. Deacetylation of xylan is easily achieved using dilute alkali solutions under mild conditions. In this article, we evaluate the effectiveness of peracetic acid combined with an alkaline pre-pretreatment through simulataneous saccharification and cofermentation (SSCF) of pretreated hybrid poplar wood and sugar can ebagasse. Respective ethanol yields of 92.8 and 91.9% of theoretical are achieved using 6% NaOH/15% peracetic acid-pretreated substrates and recombinant Zymomonas mobilis CP4/p ZB5. Reduction of acetyl groups of the lignocellulosic materials is demonstrated following alkaline pre-pretreatments. Such processing may be helpful in reducing peracetic acid requirements. The influence of deacetylation is more significant in combined pretreatments using lower peracetic acid loadings.     

Enzymatic saccharification and fermentation of xylose-optimized dilute acid-treated lignocellulosics

The cellulose reactivity of two lignocellulosic feedstocks, switchgrass and poplar, was evaluated under straight saccharification (SS) and simultaneous saccharification and fermentation (SSF) conditions following dilute sulfuric acid pretreatments designed for optimum xylose yields. The optimum pretreatment conditions, within the constraints of the experimental system (Parr batch reactor), were 1.2% acid, 180°C, and 0.5 min for switchgrass and 1% acid, 180°C, and 0.56 min for poplar. The cellulase enzyme preparation was from Trichoderma reesei and fermentations were done with Saccharomyces cerevisiae. Time courses for SS were monitored as the sum of glucose and cellobiose; those for SSF as the sum of glucose, cellobiose, and ethanol. Percentage conversions under SS conditions were 79.1% and 91.4% for the pretreated poplar and switchgrass feedstocks, respectively. Analogous values under SSF conditions were 73.0% and 90.3% for pretreated poplar and switchgrass, respectively.     

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.     

Xylanase supplementation on enzymatic saccharification of dilute acid pretreated poplars at different severities

Three pairs of solid substrates from dilute acid pretreatment of two poplar wood samples were enzymatically hydrolyzed by cellulase preparations supplemented with xylanase. Supplementation of xylanase improved cellulose saccharification perhaps due to improved cellulose accessibility by xylan hydrolysis. Total xylan removal directly affected enzymatic cellulose saccharification. Furthermore, xylan removal by pretreatment and xylanase are indifferent to enzymatic cellulose saccharification. However, more enzymatic xylose and glucose yields were obtained for a substrate with lower xylan content after a severer pretreatment at the same xylanase dosage. The effectiveness of xylanase at increased dosages depended on the substrates structure or accessibility. High xylanase dosages were more effective on well pretreated substrates than on under-pretreated substrates with high xylan content. The application sequence of xylanase and cellulase affected cellulose saccharification. This effect varied with substrate accessibility, perhaps due to competition between xylanase and cellulase binding to the substrate.     

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.     

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.     

Bioconversion of cellulose into ethanol by nonisothermal simultaneous saccharification and fermentation

The kinetic characteristics of cellulase and beta-glucosidase during hydrolysis were determined. The kinetic parameters were found to reproduce experimental data satisfactorily and could be used in a simultaneous saccharification and fermentation (SSF) system by coupling with a fermentation model. The effects of temperature on yeast growth and ethanol production were investigated in batch cultures. In the range of 35-45 degrees C, using a mathematical model and a computer simulation package, the kinetic parameters at each temperature were estimated. The appropriate forms of the model equation for the SSF considering the effects of temperature were developed, and the temperature profile for maximizing the ethanol production was also obtained. Briefly, the optimum temperature profile began at a low temperature of 35 degrees C, which allows the propagation of cells. Up to 10 h, the operating temperature increased rapidly to 39 degrees C, and then decreased slowly to 36 degrees C. In this nonisothermal SSF system with the above temperature profile, a maximum ethanol production of 14.87 g/L was obtained.     

Whole broth cellulase production for use in simultaneous saccharification and fermentation

Applied Biochemistry and Biotechnology - Cellulase, an enzyme that catalyzes the breakdown of cellulose into glucose, is produced inside fungal cells and secreted into the surrounding media....