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

<p>Abstract</p> <p>Background</p> <p>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-<it>O-</it>acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON), and may reduce its toxicity in plants and animals.</p> <p>Results</p> <p>Two <it>Fusarium </it>trichothecene 3-<it>O-</it>acetyltransferases (FgTRI101 and FfTRI201) were cloned and expressed in yeast (<it>Saccharomyces cerevisiae</it>) during a series of small-scale ethanol fermentations using barley (<it>Hordeum vulgare</it>). 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.</p> <p>Conclusions</p> <p>This study demonstrates the potential of using yeast expressing a trichothecene 3-<it>O</it>-acetyltransferase to modify DON during commercial fuel ethanol fermentation.</p

Pretreatment of Sweet Sorghum Bagasse for Ethanol Production Using Na2CO3 Obtained by NaOH Absorption of CO2 Generated in Sweet Sorghum Juice Ethanol Fermentation

(1) Background: Commercial production of fuel ethanol currently uses sugarcane and corn as feedstocks. Attempts to develop other renewable feedstocks that are more abundant have led to lignocellulosic biomass, which requires pretreatment prior to enzymatic hydrolysis to generate fermentable sugars. One of the largest cost components of pretreatment is chemical cost. Ethanol fermentation also produces large quantities of CO2 as a co-product contributing to global warming. (2) Methods: Sweet sorghum has emerged as a potential new feedstock for ethanol production. In the present study, the CO2 produced in sweet sorghum juice (SSJ) fermentation was captured by absorption in 5 M NaOH. The resultant Na2CO3 solution was used for pretreatment of sweet sorghum bagasse (SSB), which is the solid residue in SSJ extraction. The pretreated SSB was fermented in SSJ to produce additional ethanol. (3) Results: CO2 absorption efficiency of 92.0% was observed. Pretreatment of SSB by the obtained Na2CO3 solution resulted in no loss of glucan and only 8.1 wt% loss of xylan. Ethanol yield from glucan in the pretreated SSB was 81.7% theoretical. (4) Conclusions: CO2 from SSJ fermentation captured as Na2CO3 could be used for efficient SSB pretreatment. Further study focusing on pretreatment process optimization is needed

Pretreatment and Fractionation of Wheat Straw for Production of Fuel Ethanol and Value-added Co-products in a Biorefinery

An integrated process has been developed for a wheat straw biorefinery. In this process, wheat straw was pretreated by soaking in aqueous ammonia (SAA), which extensively removed lignin but preserved high percentages of the carbohydrate fractions for subsequent bioconversion. The pretreatment conditions included 15 wt% NH4OH, 1:10 solid:liquid ratio, 65 oC and 15 hours. Under these conditions, 48% of the original lignin was removed, whereas 98%, 83% and 78% of the original glucan, xylan, and arabinan, respectively, were preserved. The pretreated material was subsequently hydrolyzed with a commercial hemicellulase to produce a solution rich in xylose and low in glucose plus a cellulose-enriched solid residue. The xylose-rich solution then was used for production of value-added products. Xylitol and astaxanthin were selected to demonstrate the fermentability of the xylose-rich hydrolysate. Candida mogii and Phaffia rhodozyma were used for xylitol and astaxanthin fermentation, respectively. The cellulose-enriched residue obtained after the enzymatic hydrolysis of the pretreated straw was used for ethanol production in a fed-batch simultaneous saccharification and fermentation (SSF) process. In this process, a commercial cellulase was used for hydrolysis of the glucan in the residue and Saccharomyces cerevisiae, which is the most efficient commercial ethanol-producing organism, was used for ethanol production. Final ethanol concentration of 57 g/l was obtained at 27 wt% total solid loading

Soaking in Aqueous Ammonia (SAA) Pretreatment of Whole Corn Kernels for Cellulosic Ethanol Production from the Fiber Fractions

Corn fiber is a co-product of commercial ethanol dry-grind plants, which is processed into distillers dried grains with solubles (DDGS) and used as animal feed, yet it holds high potential to be used as feedstock for additional ethanol production. Due to the tight structural make-up of corn fiber, a pretreatment step is necessary to make the cellulose and hemicellulose polymers in the solid fibrous matrix more accessible to the hydrolytic enzymes. A pretreatment process was developed in which whole corn kernels were soaked in aqueous solutions of 2.5, 5.0, 7.5, and 10.0 wt% ammonia at 105 &deg;C for 24 h. The pretreated corn then was subjected to a conventional mashing procedure and subsequent ethanol fermentation using a commercial strain of natural Saccharomyces cerevisiae with addition of a commercial cellulase. Pretreatment of the corn with 7.5 wt% ammonia solution plus cellulase addition gave the highest ethanol production, which improved the yield in fermentation using 25 wt% solid from 334 g ethanol/kg corn obtained in the control (no pretreatment and no cellulase addition) to 379 g ethanol/kg corn (a 14% increase). The process developed can potentially be implemented in existing dry-grind ethanol facilities as a &ldquo;bolt-on&rdquo; process for additional ethanol production from corn fiber, and this additional ethanol can then qualify as &ldquo;cellulosic ethanol&rdquo; by the Environmental Protection Agency&rsquo;s (EPA&rsquo;s) Renewable Fuels Standard and thereby receive RINs (Renewable Identification Numbers)

Sorghum as a renewable feedstock for production of fuels and industrial chemicals

Considerable efforts have been made in the USA and other countries to develop renewable feedstocks for production of fuels and chemicals. Among these, sorghum has attracted strong interest because of its many good characteristics such as rapid growth and high sugar accumulation, high biomass production potential, excellent nitrogen usage efficiency, wide adaptability, drought resistance, and water lodging tolerance and salinity resistance. The ability to withstand severe drought conditions and its high water usage efficiency make sorghum a good renewable feedstock suitable for cultivation in arid regions, such as the southern US and many areas in Africa and Asia. Sorghum varieties include grain sorghum, sweet sorghum, and biomass sorghum. Grain sorghum, having starch content equivalent to corn, has been considered as a feedstock for ethanol production. Its tannin content, however, may cause problems during enzyme hydrolysis. Sweet sorghum juice contains sucrose, glucose and fructose, which are readily fermentable by Saccharomyces cerevisiae and hence is a good substrate for ethanol fermentation. The enzyme invertase, however, needs to be added to convert sucrose to glucose and fructose if the juice is used for production of industrial chemicals in fermentation processes that employ microorganisms incapable of metabolizing sucrose. Biomass sorghum requires pretreatment prior to enzymatic hydrolysis to generate fermentable sugars to be used in the subsequent fermentation process. This report reviews the current knowledge on bioconversion of sorghum to fuels and chemicals and identifies areas that deserve further studies

Integrated Process for Extraction of Wax as a Value-Added Co-Product and Improved Ethanol Production by Converting Both Starch and Cellulosic Components in Sorghum Grains

Grain sorghum is a potential feedstock for fuel ethanol production due to its high starch content, which is equivalent to that of corn, and has been successfully used in several commercial corn ethanol plants in the United States. Some sorghum grain varieties contain significant levels of surface wax, which may interact with enzymes and make them less efficient toward starch hydrolysis. On the other hand, wax can be recovered as a valuable co-product and as such may help improve the overall process economics. Sorghum grains also contain lignocellulosic materials in the hulls, which can be converted to additional ethanol. An integrated process was developed, consisting of the following steps: 1. Extraction of wax with boiling ethanol, which is the final product of the proposed process; 2. Pretreatment of the dewaxed grains with dilute sulfuric acid; 3. Mashing and fermenting of the pretreated grains to produce ethanol. During the fermentation, commercial cellulase was also added to release fermentable sugars from the hulls, which then were converted to additional ethanol. The advantages of the developed process were illustrated with the following results: (1) Wax extracted (determined by weight loss): ~0.3 wt % of total mass. (2) Final ethanol concentration at 25 wt % solid using raw grains: 86.1 g/L. (3) Final ethanol concentration at 25 wt % solid using dewaxed grains: 106.2 g/L (23.3% improvement). (4) Final ethanol concentration at 25 wt % solid using dewaxed and acid-treated grains (1 wt % H2SO4) plus cellulase (CTec2): 117.8 g/L (36.8% improvement)

The effects of ethanol on hydrolysis of cellulose and pretreated barley straw by some commercial cellulolytic enzyme products

The effect of ethanol at levels ranging from 2.5% v/v to 15% v/v on the activities of two recently developed commercial cellulosic biomass hydrolytic enzyme products, Accellerase&reg; 1500 and Accellerase&reg; XY, was investigated. The substrates used for study of the effect of ethanol on Accellerase&reg; 1500 included α-cellulose, cellobiose and barley straw pretreated by the soaking in aqueous ammonia method. The initial rates of glucose production and final glucose concentrations obtained at 48 h of hydrolysis were used as the response factors. To study the effect of ethanol on Accellerase&reg; XY, beechwood xylan and the pretreated barley straw were used as substrates. The response factors included the initial rates of xylose production and final xylose concentrations obtained at 48 h of hydrolysis. It was found that ethanol acted as either activator or inhibitor depending on its concentration and the type of substrate used. At 10% v/v, which is expected to be the final concentration of ethanol in a commercial cellulosic biomass-based process, ethanol acted as an inhibitor in all cases. However, even at this ethanol level, both commercial enzymes still retained at least about 50% of their activities

Utilization of Sweet Sorghum Juice for the Production of Astaxanthin as a Biorefinery Co-Product by <i>Phaffia rhodozyma</i>

This work investigates cultivating the red-pigmented yeast Phaffia rhodozyma in sweet sorghum juice (SSJ) to assess the production of astaxanthin as a potential biorefinery co-product. Shake flask cultures on defined sugar medium indicated that all three sugars (sucrose, glucose, and fructose) could be consumed with adequate nitrogen and nutrient supplementation. Only modest biomass growth and astaxanthin production could be achieved in SSJ without nitrogen supplementation; however, combining nitrogen supplementation with yeast extract in diluted SSJ could metabolize all sugars present in 168 h. A 2 L bioreactor trial with full strength (i.e., undiluted) SSJ produced up to 29 g/L of biomass, 65.4 mg/L of astaxanthin, an overall cell astaxanthin content of 2.49 mg astaxanthin/g dry cell mass, and a volumetric astaxanthin productivity of 0.389 mg/L/h after 168 h of cultivation. Further process optimization is needed since glucose metabolism was incomplete in undiluted SSJ