1a. Objectives (from AD-416)
Develop new hulled and hulless barley lines having traits desirable to enhance production of fuel ethanol and add value to the animal feed coproduct, DDGS; engineer commercial ethanol yeasts to detoxify mycotoxins such as DON that are sometimes found on barley kernels in wet growing years and that would be detrimental to animals who ate the resulting ethanol coproduct, DDGS, derived from that grain and; evaluate hull removal strategies to reduce DON levels in raw barley kernels prior to fuel ethanol production as another way to reduce levels of DON in DDGS.
1b. Approach (from AD-416)
Virginia Tech breeders will send new barley varieties to us for evaluation. Varieties containing high levels of starch, beta-glucan, and protein will be fermented to ethanol and the coproduct, DDGS. Varieties producing the highest ethanol yields and DDGS with highest protein will be selected as top candidates. Virginia Tech researchers will transform commercial ethanol yeasts with a 3-O-acetyltransferase (TRI101) enzyme, which catalyzes the acetylation of DON at the third carbon, reducing the toxicity of DON by two orders of magnitude. We will test the ability of these yeasts to detoxify DON during a series of small-scale ethanol fermentations using raw barley grain containing different levels of DON. We expect the enzyme to detoxify DON during simultaneous saccharification and fermentation. It is expected that the use of these transgenic yeasts during fermentation will add additional value by the production of barley DDGS with low DON levels. ERRC researchers will remove barley hulls using several different types of mills (pearling, roller milling, precision impact mill) to determine if removal of hull will result in barley kernels with lower DON levels.
3. Progress Report
Levels of the mycotoxin deoxynivalenol (DON) were determined for harvested grain from 18 selected barley varieties and elite experimental lines grown and harvested in 2010. Each barley variety or line was replicated twice. DON concentrations ranged from 3.3 to 99.3 ppm. Hulled and hulless varieties with high and low DON were processed at the USDA ARS Sustainable Biofuels and CoProducts Research Lab in Wyndmoor, PA to track the fate of DON during ethanol fermentation. These varieties were fermented using commercial ethanol yeast known as Ethanol Red, and DON concentrations were measured throughout the fermentation process. Results from these experiments indicated that DON was concentrated 1.6 to 8.2 times in DDGS compared to the starting ground grain (i.e., the DDGS contained much higher concentrations of DON than raw grain). This study is the first to confirm that DON is concentrated in DDGS during fuel ethanol production from barley as a source. This result underscores the importance of developing strategies to reduce DON during the fermentation process. A series of novel DON detoxification genes were assessed for their ability to detoxify DON during barley ethanol fermentations using yeast expressing one of these detoxification genes. The results showed that DON could be significantly reduced during fuel ethanol fermentation ((9.2% to 55.3% of DON was converted to a less toxic derivative of DON). In a set of experiments to lower the levels of DON in barley grain, hulls were removed from a set of hulled barley grain lots using scarification, precision dehulling, and improved roller milling methods. DON is currently being quantified in the resulting fractions (untreated kernels with hulls, kernels without hulls, and hulls without kernels). The results will determine if this process significantly lowers the amount of DON in the fermentable parts of the barley kernel. The research progress was monitored by conference calls, e-mail, and by 3 one-week visits by Virginia Tech employees to ERRC to conduct research in our laboratories.