**4. Conclusions**

mined in this study, 0.18~0.39 g/g were generally lower than those obtained earlier with the fermentation of the enzymatic hydrolyzates from several other energy crops, such as switch‐ grass, miscanthus and gamagrass, using identical yeast strains (Ge et al. 2011, 2012). This should be attributed to the presence of substantial amount of fermentation inhibitors in the poplar hydrolyzates, such as furfural, acetic acid and formic acid HMF (see discussion below), though the pretreated biomass has been extensively washed with water. It should be noted that enzymatic hydrolyzates prepared from other energy crops largely lacked these inhibitors because these biomass were pretreated with concentrated (84%, w/v) phosphoric acid under moderate reaction conditions (50°C for 45 min) (Ge et al. 2011, 2012). However, this phosphoric acid-based pretreatment approach is regarded as too expensive to be economically feasible.

112 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

Of particular interest is the observation that the self-flocculating SPSC01 yeast always pro‐ duced higher ethanol yields than the ATCC4126 strain from the same enzymatic hydrolyzates (Table 2). While no ethanol was detected from the fermentation of the unwashed hydrolyzates by the ATCC strain, marginal levels of ethanol could be produced by the SPSC01 strain. When the washed hydrolyzates were tested for fermentation, the SPSC01 yeast could produce up to 35% more ethanol then the ATCC strain. The SPSC01 yeast is an industrial strain that has been reported to have high ethanol productivity, high ethanol tolerance and lower capital invest‐ ment required for yeast cell recovery (Bai et al. 2004; Zhao and Bai, 2009; Zhao et al. 2009). It has been successfully used for continuous ethanol fermentation at commercial scales in China (Bai et al. 2008). The results from this study indicated that this self-flocculating strain could also have a higher tolerance to fermentation inhibitors than the non-flocculating yeast, thus being able to produce higher ethanol yields. Fermentation with the self-flocculating yeast may

represent a promising strategy to increase the production of cellulosic ethanol.

Figure 2 presents HPLC chromatograms from washed and non-washed enzymatic hydroly‐ zates; analysis was conducted at 280 (A) and 210 (B) nm. Retention times of furfural, acetic acid and formic acid HMF were 44.4, 14.7, 13.6 minutes, respectively. Peaks at 9 and 12 minutes remain unidentified. Examination of the UV traces showed that, for the most part, washing did not remove any compounds, but decreased peak intensity. Results from Figure 1 demon‐ strate that washing biomass is critical to maximize sugar recovery; however UV traces at 280 and 210 nm are qualitatively similar. Mass spectrometry analysis of the hydrolyzates would have most likely revealed more peaks, aiding in identifying which peaks need to be removed

In related work, aliphatic acid and furans from wet distillers grain (Ximenes et al. 2010), corn stover (Hodge et al. 2008), wheat straw (Panagiotou and Olsson 2007) and poplar wood (Cantarella et al. 2004) wash waters were analyzed. Having detected and quantified com‐ pounds in wash waters, solutions were reconstituted and tested for their effect on saccharifi‐ cation cocktails. Cantarella et al. (2004) pretreated poplar in steam at a severity of 4.13 and tested the effect of washing the pretreated biomass. Cantarella et al. (2004) washed poplarpretreated material with either 12.5 or 66.7 volumes of water to biomass ratio prior to enzy‐ matic hydrolysis and fermentation steps. They reported that using washed biomass resulted

**3.3. Differences between washed and non-washed hydrolyzates**

and/or minimized prior to enzymatic hydrolysis and fermentation.

Dilute acid pretreatment processes resulted in the production of inhibitory byproducts, such as furfural, acetic acid, formic acid, and HMF that hindered both the enzymatic saccharification and fermentation steps. Washing the pretreated biomass with water did not entirely remove the inhibitory compounds, but significantly decreased their concentrations, which resulted in recovery of 5.3 times more glucose and substantially increased ethanol yields. The selfflocculating yeast strain SPSC01 showed higher tolerance to fermentation inhibitors than the non-flocculating ATCC4126 yeast, resulting in up to 35% increase in ethanol yield.

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