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.     

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.     

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.     

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.     

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.     

Continuous fermentation of cellulosic biomass to ethanol

Experimental results are presented for continuous conversion of pretreated hardwood flour to ethanol. A simultaneous saccharification and fermentation (SSF) system comprised ofTrichoderma reesei cellulase supplemented with additional β-glucosidase and fermentation bySaccharomyces cerevisiae was used for most experiments, with data also presented for a direct microbial conversion (DMC) system comprised ofClostridium thermocellum. Using a batch SSF system, dilute acid pretreatment of mixed hardwood at short residence time(10 s, 220°C, 1% H₂SO₄) was compared to poplar wood pretreated at longer residence time (20 min, 160°C, 0.45% H₂SO₄). The short residence time pretreatment resulted in a somewhat (10–20%) more reactive substrate, with the reactivity difference particularly notable at low enzyme loadings and/or low agitation. Based on a preliminary screening, inhibition of SSF by byproducts of short residence time pretreatment was measurable, but minor. Both SSF and DMC were carried out successfully in well-mixed continuous systems, with steady-state data obtained at residence times of 0.58–3 d for SSF as well as 0.5 and 0.75 d for DMC. The SSF system achieved substrate conversions varying from 31% at a 0.58-d residence time to 86% at a 2-d residence time. At comparable substrate concentrations (4–5 g/l) and residence times (0.5–0.58 d), substrate conversion in the DMC system (77%) was significantly higher than that in the SSF system (31%). Our results suggest that the substrate conversion in SSF carried out in CSTR is relatively insensitive to enzyme loading in the range 7–25 U/g cellulose and to substrate concentration in the range of 5–60 g/L cellulose in the feed.     

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.     

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.     

Fuel ethanol production from corn fiber current status and technical prospects

Corn fiber, which consists of about 20% starch, 14% cellulose, and 35% hemicellulose, has the potential to serve as a low cost feedstock for production of fuel ethanol. Currently, the use of corn fiber to produce fuel ethanol faces significant technical and economic challenges. Its success depends largely on the development of environmentally friendly pretreatment procedures, highly effective enzyme systems for conversion of pretreated corn fiber to fermentable sugars, and efficient microorganisms to convert multiple sugars to ethanol. Several promising pretreatment and enzymatic processes for conversion of corn fiber cellulose, hemicellulose, and remaining starch to fermentable sugars were evaluated. These hydrolyzates were then examined for ethanol production in bioreactors, using genetically modified bacteria and yeast. Several novel enzymes were also developed for use in pretreated corn fiber saccharification. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Optimization of sucrose ethanol fermentation for K,Na, Ca,and cu metal contents

The effect of the trace metals Cu, K, Na, and Ca, separately or in mixture, on fermentation time, ethanol production rate, and cell growth in the fermentation of synthetic media containing sucrose is discussed. The results are related to the range of contents found in raw materials, molasses and raisins, in order to determine their optimum concentrations for alcohol production.     

Preliminary studies on the processing sequence for southern red oak and municipal solid waste using a hybrid dilute acid/ enzymatic hydrolysis process for ethanol production

Based on this preliminary study, a metric ton of dry southern red oak chips subjected to a first-stage dilute sulfuric acid hydrolysis would yield 132 kg of xylose and 40 kg of glucose and mannose. A second-stage dilute sulfuric acid hydrolysis on the first-stage residue would yield only 128 kg of additional glucose, but a second-stage cellulytic enzyme hydrolysis on the first-stage residue would yield an additional 265 kg of glucose. Fermentation of these hydrolyzates would show that the hybrid process would yield over 50% more ethanol. Results on other biomass are also included.     

Effect of pretreatment on simultaneous saccharification and fermentation of hardwood into Acetone/Butanol

Aspergillus niger NCIM 1207 produces high levels of extracellular beta-glucosidase and xylanase activities in submerged fermentation. Among the nitrogen sources, ammonium sulfate, ammonium dihydrogen orthophosphate, and corn-steep liquor were the best for the production of cellulolytic enzymes by A. niger. The optimum pH and temperature for cellulase production were 3.0-5.5 and 28 degrees C, respectively. The cellulase complex of this strain was found to undergo catabolite repression in the presence of high concentrations of glucose. Glycerol at all concentrations caused catabolite repression of cellulase production. The addition of glucose (up to 1% concentration) enhanced the production of cellulolytic enzymes, but a higher concentration of glucose effected the pronounced repression of enzymes. Generally the growth on glucose- or glycerol-containing medium was accompanied by a sudden drop in the pH of the fermentation medium to 2.0.     

Factors that affect the biosynthesis of xylitol by xylose-fermenting yeasts

Xylitol is a sweetener with important technological properties like anticariogenicity, low caloric value, and negative dissolution heat. Because it can be used successfully in food formulations and pharmaceutical industries, its production is in great demand. Xylitol can be obtained by microbiological process, since many yeasts and filamentous fungi synthesize the xylose reductase enzyme, which catalyses the xylose reduction into xylitol as the first step in the xylose metabolism. The xylitol production by biotechnological means has several economic advantages in comparison with the conventional process based on the chemical reduction of xylose. The efficiency and the productivity of this fermentation chiefly depends upon the microorganism and the process conditions employed. In this mini-review, the most significant upstream parameters on xylitol production by biotechnological process are described.     

An integrated bioconversion process for production of l-lactic acid from starchy potato feedstocks

The potential market for lactic acid as the feedstock for biodegradable polymers, oxygenated chemicals, and specialty chemicals is significant. L-lactic acid is often the desired enantiomer for such applications. However, stereospecific lactobacilli do not metabolize starch efficiently. In this work, Argonne researchers have developed a process to convert starchy feedstocks into L-lactic acid. The processing steps include starch recovery, continuous liquefaction, and simultaneous saccharification and fermentation. Over 100 g/L of lactic acid was produced in less than 48 h. The optical purity of the product was greater than 95%. This process has potential economical advantages over the conventional process.     

Continuous attrition bioreactor with enzyme recycling for the bioconversion of cellulose

A new type of reactor, the attrition bioreactor, has been developed to increase the rate of the enzymatic hydrolysis of cellulose and also to cut pretreatment costs. It was found that the attrition bioreactor could be operated continuously or semicontinuously in conjunction with a membrane filter to produce a high cellulose conversion rate and low enzyme consumption. The membrane filter served to contain the enzyme and cellulose within the reactor while allowing sugar to permeate as a product.     

Characterization of an immobilized yeast cells fluidized-bed bioreactor for ethanol fermentation

The operational characterization of a fluidized-bed bioreactor for ethanol fermentation using Ca-alginate immobilized yeast cells is described. An additional air stream is supplied to the fermenter to ensure and maintain satisfactory fluidization behavior of beads and to avoid slug formation. The influence of physical properties such as bead density and liquid density on the fluidization quality and stability are discussed.     

Production of ethanol from starch by co-immobilizedZymomonas mobilis-glucoamylase in a fluidized-bed reactor

The production of ethanol from starch was studied in a fluidized-bed reactor (FBR) using co-immobilizedZymomonas mobilis and glucoamylase. The FBR was a glass column of 2.54 cm in diameter and 120 cm in length. TheZ. mobilis and glucoamylase were co-immobilized within small uniform beads (1.2-2.5 mm diameter) of κ-carrageenan. The substrate for ethanol production was a soluble starch. Light steep water was used as the complex nutrient source. The experiments were performed at 35κC and pH range of 4.0-5.5. The substrate concentrations ranged from 40 to 185 g/L, and the feed rates from 10 to 37 mL/min. Under relaxed sterility conditions, the FBR was successfully operated for a period of 22 d, during which no contamination or structural failure of the biocatalyst beads was observed. Volumetric productivity as high as 38 g ethanol/(Lh), which was 74% of the maximum expected value, was obtained. Typical ethanol volumetric productivity was in the range of 15-20 g/(Lh). The average yield was 0.49 g ethanol/g substrate consumed, which was 90% of the theoretical yield. Very low levels of glucose were observed in the reactor, indicating that starch hydrolysis was the rate-limiting step.     

Ethanol production from cellulose by coupled saccharification/fermentation usingSaccharomyces cerevisiae and cellulase complex fromSclerotiun rolfsii UV-8 mutant

Using cellulase/hemicellulase complex of Sclerotium rolfsii UV-8 mutant and Saccharomyces cerevisiae for fermentation, the coupled saccharification/fermentation (CSF) of 15% AT-rice straw was carried out at 40 degrees C, pH 4.5 for the first 24 h and further incubation was performed at 30 degrees C for 72 h. Increasing the amount of cellulase activity from 3-12 IU FPA/g of substrate resulted in increased yields of ethanol from 1.5-3.6% in 96 h. It has been observed that the coupled system was advantageous over the two stage (separate hydrolysis/fermentation) system as it produced higher amounts of ethanol from cellulose (3.6% as compared to 2.3% ethanol from rice straw).     

Xylose fermentation

The economic impact of conversion of xylose to ethanol for a wood-to-ethanol plant was examined, and the maximum potential reduction in the price of ethanol from utilization of xylose is estimated to be 0.42 per gallon from a base case price of0.42 per gallon from a base case price of 1.65. The sensitivity of the price of ethanol to the yield, ethanol concentration and rate of the xylose fermentation was also examined, and the price of ethanol is most affected by changes in yield and ethanol concentration, with rate of lesser importance. Current performances of various xylose conversion biocatalysts were analyzed, andC. shehatae andP. stipitis appear to be the best yeasts.