An analysis of variance (ANOVA) showed that the effect of initial moisture content on the ethanol yields of ensiled alfalfa stems including both unwashed and washed was not significant (Fig. 2A).
In order to exclude the effect of sulfuric acid pretreatment, the effect of initial moisture content on the ethanol yields of ensiled alfalfa stems not subjected to sulfuric acid pretreatment (0 g/ kg DM) was examined alone (Fig. 2B) with ANOVA, and also shown to be not significant, because ensiled alfalfa stems were all freeze dried, which did not result in a significant effect on substrates structure.
The ethanol yields of alfalfa stems after ensiling (Fig. 3, lined bars at acid 0 g/kg DM) were lower than that of air-dried (Fig. 3, solid bar). This may have been due to: (1) some soluble glucan, which could be easily fermented during SSF, were consumed during bacterial metabolism; (2) the ‘‘added ash’’ in the ensiled alfalfa stems could have had an inhibitory effect on SSF, which has been reported previously (Maiorella et al., 1984).