Nhuan P. Nghiem

Bioethanol production from corn stover using aqueous ammonia pretreatment and two-phase simultaneous saccharification and fermentation (TPSSF)

An integrated bioconversion process was developed to convert corn stover derived pentose and hexose to ethanol effectively. In this study, corn stover was pretreated by soaking in aqueous ammonia (SAA), which retained glucan ( approximately 100%) and xylan (>80%) in the solids. The pretreated carbohydrates-rich corn stover was converted to ethanol via two-phase simultaneous saccharification and fermentation (TPSSF). This single-reactor process employed sequential simultaneous saccharification and fermentation (SSF), i.e. pentose conversion using recombinant Escherichia coli KO11 in the first phase, followed by hexose conversion with Saccharomyces cerevisiae D5A in the second phase. In the first phase, 88% of xylan digestibility was achieved through the synergistic action of xylanase and endo-glucanase with minimal glucan hydrolysis (10.5%). Overall, the TPSSF using 12-h SAA-treated corn stover resulted in the highest ethanol concentration (22.3g/L), which was equivalent to 84% of the theoretical ethanol yield based on the total carbohydrates (glucan+xylan) in the untreated corn stover.

Pretreatment of corn stover using low-moisture anhydrous ammonia (LMAA) process.

A simple pretreatment method using anhydrous ammonia was developed to minimize water and ammonia inputs for cellulosic ethanol production, termed the low moisture anhydrous ammonia (LMAA) pretreatment. In this method, corn stover with 30-70% moisture was contacted with anhydrous ammonia in a reactor under nearly ambient conditions. After the ammoniation step, biomass was subjected to a simple pretreatment step at moderate temperatures (40-120°C) for 48-144 h. Pretreated biomass was saccharified and fermented without an additional washing step. With 3% glucan loading of LMAA-treated corn stover under best treatment conditions (0.1g-ammonia+1.0 g-water per g biomass, 80°C, and 84 h), simultaneous saccharification and cofermentation test resulted in 24.9 g/l (89% of theoretical ethanol yield based on glucan+xylan in corn stover).

Production of ethanol from winter barley by the EDGE (enhanced dry grind enzymatic) process

BackgroundUS legislation requires the use of advanced biofuels to be made from non-food feedstocks. However, commercialization of lignocellulosic ethanol technology is more complex than expected and is therefore running behind schedule. This is creating a demand for non-food, but more easily converted, starch-based feedstocks other than corn that can fill the gap until the second generation technologies are commercially viable. Winter barley is such a feedstock but its mash has very high viscosity due to its high content of β-glucans. This fact, along with a lower starch content than corn, makes ethanol production at the commercial scale a real challenge.ResultsA new fermentation process for ethanol production from Thoroughbred, a winter barley variety with a high starch content, was developed. The new process was designated the EDGE (enhanced dry grind enzymatic) process. In this process, in addition to the normal starch-converting enzymes, two accessory enzymes were used to solve the β-glucan problem. First, β-glucanases were used to hydrolyze the β-glucans to oligomeric fractions, thus significantly reducing the viscosity to allow good mixing for the distribution of the yeast and nutrients. Next, β-glucosidase was used to complete the β-glucan hydrolysis and to generate glucose, which was subsequently fermented in order to produce additional ethanol. While β-glucanases have been previously used to improve barley ethanol production by lowering viscosity, this is the first full report on the benefits of adding β-glucosidases to increase the ethanol yield.ConclusionsIn the EDGE process, 30% of total dry solids could be used to produce 15% v/v ethanol. Under optimum conditions an ethanol yield of 402 L/MT (dry basis) or 2.17 gallons/53 lb bushel of barley with 15% moisture was achieved. The distillers dried grains with solubles (DDGS) co-product had extremely low β-glucan (below 0.2%) making it suitable for use in both ruminant and mono-gastric animal feeds.

Pretreatment and Fractionation of Corn Stover by Soaking In Ethanol and Aqueous Ammonia

A new process for pretreatment of lignocellulosic biomass, designated the soaking in ethanol and aqueous ammonia (SEAA) process, was developed to improve hemicellulose preservation in solid form. In the SEAA process, an aqueous ammonia solution containing ethanol is used. Corn stover was treated with 15 wt.% ammonia at 1:9 solid–liquid ratio (by weight) at 60 °C for 24 h with ethanol added at 1, 5, 20, and 49 wt.% (balance was water). The extents by which xylan was solubilized with no ethanol and with ethanol added at 1, 5, 20, and 49 wt.% of the total liquid were 17.2%, 16.7%, 14.5%, 10.4%, and 6.3% of the original xylan, respectively. Thus, at the highest ethanol concentration used the loss of hemicellulose to the liquid phase was reduced by 63%. The digestibility of glucan and xylan in the pretreated corn stover samples by cellulase was not affected by ethanol addition of up to 20 wt.%. The enzymatic digestibility of the corn stover treated with 49 wt.% ethanol added was lower than the digestibility of the sample treated with no ethanol addition. Thus, based on these results, 20 wt.% was found to be the optimum ethanol concentration for use in the SEAA process for pretreatment of corn stover.

Integration of succinic acid and ethanol production with potential application in a corn or barley biorefinery.

Production of succinic acid from glucose by Escherichia coli strain AFP184 was studied in a batch fermentor. The bases used for pH control included NaOH, KOH, NH4OH, and Na2CO3. The yield of succinic acid without and with carbon dioxide supplied by an adjacent ethanol fermentor using either corn or barley as feedstock was examined. The carbon dioxide gas from the ethanol fermentor was sparged directly into the liquid media in the succinic acid fermentor without any pretreatment. Without the CO2 supplement, the highest succinic acid yield was observed with Na2CO3, followed by NH4OH, and lowest with the other two bases. When the CO2 produced in the ethanol fermentation was sparged into the media in the succinic acid fermentor, no improvement of succinic acid yield was observed with Na2CO3. However, several-fold increases in succinic acid yield were observed with the other bases, with NH4OH giving the highest yield increase. The yield of succinic acid with CO2 supplement from the ethanol fermentor when NH4OH was used for pH control was equal to that obtained when Na2CO3 was used, with or without CO2 supplementation. The benefit of sparging CO2 from ethanol fermentation on the yield of succinic acid demonstrated the feasibility of integration of succinic acid fermentation with ethanol fermentation in a biorefinery for production of fuels and industrial chemicals.

Economic analysis of fuel ethanol production from winter hulled barley by the EDGE (Enhanced Dry Grind Enzymatic) process.

A process and cost model was developed for fuel ethanol production from winter barley based on the EDGE (Enhanced Dry Grind Enzymatic) process. In this process, in addition to β-glucanases, which are added to reduce the viscosity of the mash, β-glucosidase is also added to completely hydrolyze the oligomers obtained during the hydrolysis of β-glucans to glucose. The model allows determination of capital costs, operating costs, and ethanol production cost for a plant producing 40 million gallons of denatured fuel ethanol annually. A sensitivity study was also performed to examine the effects of β-glucosidase and barley costs on the final ethanol production cost. The results of this study clearly demonstrate the economic benefit of adding β-glucosidase. Lower ethanol production cost was obtained compared to that obtained without β-glucosidase addition in all cases except one where highest β-glucosidase cost allowance and lowest barley cost were used.

Fractionation of Corn Fiber Treated by Soaking in Aqueous Ammonia (SAA) for Isolation of Hemicellulose B and Production of C5 Sugars by Enzyme Hydrolysis

A process was developed to fractionate and isolate the hemicellulose B component of corn fiber generated by corn wet milling. The process consisted of pretreatment by soaking in aqueous ammonia followed by enzymatic cellulose hydrolysis, during which the hemicellulose B was solubilized by cleavage into xylo-oligosaccharides and subsequently recovered by precipitation with ethanol. The pretreatment step resulted in high retention of major sugars and improvement of subsequent enzymatic hydrolysis. The recovered hemicellulose B was hydrolyzed by a cocktail of enzymes that consisted of β-glucosidase, pectinase, xylanase, and ferulic acid esterase (FAE). Xylanase alone was ineffective, demonstrating yields of less than 2% of xylose and arabinose. The greatest xylose and arabinose yields, 44% and 53%, respectively, were obtained by the combination of pectinase and FAE. A mass balance accounted for 87% of the initially present glucan, 91% of the xylan, and 90% of the arabinan. The developed process offered a means for production of corn fiber gum as a value-added co-product and C5 sugars, which could be converted to other valuable co-products through fermentation in a corn wet-milling biorefinery.

Conversion of deoxynivalenol to 3- acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases

BackgroundThe trichothecene mycotoxin deoxynivalenol (DON) may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation) when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm) in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O- acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON), and may reduce its toxicity in plants and animals.ResultsTwo Fusarium trichothecene 3-O- acetyltransferases (FgTRI101 and FfTRI201) were cloned and expressed in yeast (Saccharomyces cerevisiae) during a series of small-scale ethanol fermentations using barley (Hordeum vulgare). DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red®, which also consumed galactose when present in the mash.ConclusionsThis study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.

Production of Astaxanthin from Cellulosic Biomass Sugars by Mutants of the Yeast Phaffia rhodozyma

Astaxanthin is a potential high-value coproduct in an ethanol biorefinery. Three mutant strains of the astaxanthin-producing yeast Phaffia rhodozyma, which were derived from the parent strain ATCC 24202 (UCD 67-210) and designated JTM166, JTM185, and SSM19, were tested for their capability of utilizing the major sugars that can be generated from cellulosic biomass, including glucose, xylose, and arabinose, for astaxanthin production. While all three strains were capable of metabolizing these sugars, individually and in mixtures, JTM185 demonstrated the greatest sugar utilization and astaxanthin production. Astaxanthin yield by this strain (milligrams astaxanthin per gram of sugar consumed) was highest for xylose, followed by arabinose and then glucose. The kinetics of sugar utilization by strain JTM185 was studied in fermenters using mixtures of glucose, xylose, and arabinose at varied concentrations. It was found that glucose was utilized preferentially, followed by xylose, and lastly, arabinose. Astaxanthin yield was significantly affected by sugar concentrations. Highest yields were observed with sugar mixtures containing the highest concentrations of xylose and arabinose. Hydrolysates produced from sugarcane bagasse and barley straw pretreated by the soaking in aqueous ammonia method and hydrolyzed with the commercial cellulase preparation, Accellerase™ 1000, were used for astaxanthin production by the mutant strain JTM185. The organism was capable of metabolizing all of the sugars present in the hydrolysates from both biomass sources and produced similar amounts of astaxanthin from both hydrolysates, although these amounts were lower when compared to yields obtained with reagent grade sugars.

Scale-up of ethanol production from winter barley by the EDGE (enhanced dry grind enzymatic) process in fermentors up to 300 l.

A fermentation process, which was designated the enhanced dry grind enzymatic (EDGE) process, has recently been developed for barley ethanol production. In the EDGE process, in addition to the enzymes normally required for starch hydrolysis, commercial β-glucanases were used to hydrolyze (1,3)(1,4)-β-d-glucans to smaller molecules, thus reducing the viscosity of the mash to levels sufficiently low to allow transport and mixing in commercial equipment. Another enzyme, a developmental β-glucosidase, then was used to hydrolyze the resulting oligomers to glucose, which subsequently was fermented to produce additional ethanol. The EDGE process was developed with Thoroughbred, a winter hulled barley, using a shake flask model. To move toward commercialization, it is necessary to prove that the developed process would be applicable to other barley varieties and also to demonstrate its scalability. Experiments were performed in 7.5, 70, and 300-l fermentors using Thoroughbred and Eve, a winter hull-less barley. It was shown that the process was scalable for both barley varieties. Low levels of glucose throughout the course of the fermentations demonstrated the high efficiency of the simultaneous saccharification and fermentation process. Final ethanol concentrations of 14% (v/v) were achieved for initial total solids of 28.5–30% (w/w), which gave an ethanol yield of 83–87% of the theoretical values. The distillers dried grains with solubles co-products contained very low levels of β-glucans and thus were suitable for use in feed formulations for all animal species.