The fungal plant pathogen F. graminearum produces trichothecene mycotoxins that may remain as a contaminant in barley DDGS after fuel ethanol production . New cost-effective and commercially viable methods to reduce mycotoxin contamination in barley DDGS need to be developed and implemented. Our work has a direct relevance to commercial barley ethanol plants in the USA (such as the ethanol plant in Hopewell, Virginia) and in Europe. Preparation of the barley-mash dilutes mycotoxin levels from the ground grain through the addition of DI water or 10% galactose solution (Figure 2). Mycotoxin levels are then concentrated during the formation of DDGS (Table 2). DON is soluble in water , and therefore we would expect a mycotoxin dilution of approximately fourfold in the mash compared with the dry grain (all mashes in this study were 20% solids). However, not all DON may dissolve in water , and therefore increases in ground grain taken from the mash during subsampling may explain the smaller dilutions when the concentration of dry ground grain is compared with levels in the mash at the start of fermentation (Figure 2).
We found large reductions in DON via conversion (52.4% to 58.1%) during fermentation of the hulless barley line VA06H-25, which contained the highest levels of DON in its starting ground grain (Figure 4). This alone demonstrates the tremendous potential for commercial ethanol yeasts to be engineered to express enzymes that modify mycotoxins (such as trichothecene 3-O-acetyltransferases) during fermentation. In a recent study, seven different trichothecene 3-O-acetyltransferases transformed into the yeast strain RW2802 were analyzed for their ability to modify DON into 3ADON during a series of feeding assays ; conversion levels ranged from 50.5% to 100%, depending on the source of the acetyltransferase . In our study, the enzyme FgTRI101 resulted in a 55.3% mean conversion of DON for the VA06H-25 (hulless barley line), but previous feeding assays with the same enzyme reported a reduction of 92.6% in yeast cultures . There may be several reasons for the different levels of conversion in our barley ethanol fermentations compared with the previously published feeding assays. It is possible that 'pure' yeast cultures allow higher acetylation rates because of the greater accessibility to DON by the acetyltransferases. The complex matrix of proteins and sugars in barley mashes  might impede the ability of the acetyltransferases to interact with DON. The starting concentration of yeast might also play a role in determining DON acetylation rates; the OD600 of yeast inoculum for our hulled line (VA04B-125) was approximately half that of the inoculum for the hulless line (VA06H-25), and might have contributed to the differences in acetylation rates during fermentation between these two lines.
We compared the acetylation levels of two different acetyltransferases (FgTRI101 and FfTRI201) during fermentation, using ground grain from VA04B-125 (hulled barley). Previous work has shown that the enzyme FgTRI101 has a catalytic efficiency towards DON that is 9.2 times greater than that of FfTRI201, but FfTRI201 results in higher DON conversion levels than FgTRI101 likely because of its higher protein expression in yeast . In our study, FfTRI201 converted more DON to 3ADON during fermentation than did FgTRI101 (Figure 4), and this was confirmed in the corresponding DDGS (data not shown). Western blot analyses of mashes containing VA04B-125 detected FfTRI201 in all three mashes tested, but FgTRI101 was not detected. Previous studies have reported that FfTRI201 is expressed at higher levels than FgTRI101 in yeast , which might explain why the FgTRI101 levels in the VA04B-125 mashes were below the limit of detection in our western blot.
In our fermentation assays, it is likely that glucose (repression) and galactose (induction) were competing for control of the GAL1 promoter (Figure 1), responsible for FgTRI101 and FfTRI201 expression, and therefore the expression of the acetyltransferases may not have been optimal in the fermentations. Alternative methods to induce protein expression (for example, using inducers other than galactose) may yield larger reductions in DON, especially in grain containing reduced amounts of DON (the substrate). Future studies could use promoters such as CUP1  induced by copper (100 μmol/l Cu2+) [35, 36]. The effect of copper on fermentation and DDGS production is unknown; however, addition of copper (30 mg/kg dry mass) to animal feed has been reported to suppress bacterial infections in the gut of swine . Alternatively, for constitutive expression, the phosphoglycerate kinase promoter (PGK1) can be used, and requires no additional components .
Previous reports have indicated a threefold increase in the concentration of DON in DDGS relative to starting material . In our study, DON concentrations in DDGS from Ethanol Red fermentations were about 1.6 to 8.2 times higher than in the starting ground grain (Table 2). Unexpectedly, ground grain from resistant genotypes (e.g., Eve), containing a low DON concentration, resulted in the corresponding DDGS having DON levels that were concentrated more than those in DDGS derived from ground grain with high DON levels (e.g., VA06H-25) (Table 2). It is possible that resistant genotypes harbor more masked DON (DON glucosides), through expression of a UDP-glucosyltransferase, [40, 41] than do susceptible genotypes (which accumulate high levels of DON), which may be subsequently hydrolyzed by the yeast, causing DON to be released during fermentation . This may help explain our results (Table 2) showing DON concentrating in DDGS relative to the ground grain, but this was not investigated further in the present study, and we were unable to calculate a proper mass balance to compare the masses of DON because of the subsampling of mashes during the course of the fermentation.
The reduction in total solid mass during fermentation (in which glucose is converted to ethanol and carbon dioxide), together with the loss of moisture during drying of the DDGS, increases the concentration of mycotoxins in DDGS. Because the laboratory yeast strain RW2802 does not consume galactose, the components (including DON) of its corresponding DDGS were diluted. Mycotoxin dilutions caused by galactose and other residuals (such as unreacted starch, oligosaccharides, maltose and glucose) remaining because of incomplete fermentation, made calculating the concentration of mycotoxins in the DDGS unreliable, and therefore a mass balance was used (Table 3). Fermentations containing yeast transformed with FgTRI101 or FfTRI201 reduced the mass of DON and increased the mass of 3ADON in all DDGS samples (Table 3). These enzymes are probably inactive in the DDGS because the thermostability values of these enzymes  are approximately 15°C lower than the temperature at which the DDGS was prepared.
Ethanol yields were greatest in mashes containing Ethanol Red and galactose. This industrial yeast strain was developed for fuel ethanol production and has the unique ability to utilize both galactose and glucose. In most yeast strains, galactose utilization is about one-third that of glucose . The model (laboratory) yeast strain RW2802 does not have the ability to utilize galactose efficiently, thus in our experiments, ethanol yields for RW2802 were significantly lower in the presence of galactose. This is perhaps due to the energy cost on the yeast cells to synthesize enzymes in the Leloir pathway, which make up approximately 5% of all total cellular enzymes . DON is a known protein synthesis inhibitor , but ethanol yields were not affected by DON in our fermentations.
Another approach to reduce DON in DDGS might be to add an exogenous trichothecene 3-O-acetyltransferase preparation to the mash at the start of fermentation. However, the amount of enzyme needed for this approach to be successful is presently unknown. Moreover, the enzyme stability may limit the effectiveness of this strategy , and no such preparation is commercially available at this time. Washing the grain  before fermentation can be implemented in order to reduce DON levels before mash preparation, in addition to DON modificiation during fermentation. Reduction of mycotoxins in fermentation mashes does not have to be limited to barley. This strategy could also be applied to other fuel ethanol crops such as corn, wheat and sugarcane. For example, in addition to deoxynivalenol, the mycotoxin zearalenone is another common contaminant of corn ethanol co-products , and a lactonohydrolase has been shown to decrease levels of zearalenone in spiked cultures of Schizosaccharomyces pombe and E. coli .
The EDGE process was developed as a new method for increasing ethanol yields from barley in a commercial setting to advance biofuels made from non-food feedstocks . Employing yeast to express mycotoxin-detoxification genes represents a potential strategy to reduce mycotoxin levels in fuel ethanol co-products. However, a number of issues must be addressed before this process is commercialized. First, integrating a transgene into the yeast genome would be preferred over maintaining the gene on a plasmid (which generally requires selective conditions for plasmid propagation). Second, the composition of DDGS in future work using transformed yeast would need to be evaluated. Analysis of DDGS composition in this study showed that DDGS produced by transformed yeast was similar to DDGS produced by commercial yeast, except for the change in the concentration of components due to added galactose and residual sugars that were not utilized to completion. Third, the use of a transgenic yeast strain for fuel ethanol production will need to be accepted by policy makers and ethanol production facilities in order to be implemented on a commercial scale.