Pretreatment of Dried Distiller Grains with Solubles by Soaking in Aqueous Ammonia and Subsequent Enzymatic/Dilute Acid Hydrolysis to Produce Fermentable Sugars.

Dried distillers grains with solubles (DDGS), a co-product of corn ethanol production in the dry-grind process, was pretreated by soaking in aqueous ammonia (SAA) using a 15 % w/w NH4OH solution at a solid/liquid ratio of 1:10. The effect of pretreatment on subsequent enzymatic hydrolysis was studied at two temperatures (40 and 60 °C) and four reaction times (6, 12, 24, and 48 h). Highest glucose yield of 91 % theoretical was obtained for the DDGS pretreated at 60 °C and 24 h. The solubilized hemicellulose in the liquid fraction was further hydrolyzed with dilute H2SO4 to generate fermentable monomeric sugars. The conditions of acid hydrolysis included 1 and 4 wt% acid, 60 and 120 °C, and 0.5 and 1 h. Highest yields of xylose and arabinose were obtained at 4 wt% acid, 120 °C, and 1 h. The fermentability of the hydrolysate obtained by enzymatic hydrolysis of the SAA-pretreated DDGS was demonstrated in ethanol fermentation by Saccharomyces cerevisiae. The fermentability of the hydrolysate obtained by consecutive enzymatic and dilute acid hydrolysis was demonstrated using a succinic acid-producing microorganism, strain Escherichia coli AFP184. Under the fermentation conditions, complete utilization of glucose and arabinose was observed, whereas only 47 % of xylose was used. The succinic acid yield was 0.60 g/g total sugar consumed.

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

BACKGROUND: The 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. RESULTS: Two 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. CONCLUSIONS: This study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.

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 ß-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.

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.

Production of astaxanthin from corn fiber as a value-added co-product of fuel ethanol fermentation.

Five strains of the yeast Phaffia rhodozyma, NRRL Y-17268, NRRL Y-17270, ATCC 96594 (CBS 6938), ATCC 24202 (UCD 67-210), and ATCC 74219 (UBV-AX2) were tested for astaxanthin production using the major sugars derived from corn fiber. The sugars tested included glucose, xylose, and arabinose. All five strains were able to utilize the three sugars for astaxanthin production. Among them, ATCC 74219 was the best astaxanthin producer. Kinetics of sugar utilization of this strain was studied, both with the individual sugars and with their mixtures. Arabinose was found to give the highest astaxanthin yield. It also was observed that glucose at high concentrations suppressed utilization of the other two sugars. Corn fiber hydrolysate obtained by dilute sulfuric acid pretreatment and subsequent enzyme hydrolysis was tested for astaxanthin production by strain ATCC 74219. Dilution of the hydrolysate was necessary to allow growth and astaxanthin production. All the sugars in the hydrolysate diluted with two volumes of water were completely consumed. Astaxanthin yield of 0.82 mg/g total sugars consumed was observed.