**3.1. Pretreatment and enzymatic hydrolysis**

Poplar biomass was pretreated at 140 °C for 40 min. This condition corresponded to a combined severity of 1.16 (Abatzoglou et al. 1992). The composition of the hydrolyzate was analyzed by HPLC and calculations were made to express the concentrations in terms of compounds obtained from 100 g of biomass. These pretreatment conditions resulted in the recovery of 12% and 41% of the possible glucose and xylose, respectively; these calculations were based on previously reported high specific gravity compositional analysis (Djioleu et al. 2012). Carbo‐ hydrate recoveries are presented in Table 1. Dilute acid pretreatment resulted in the release of xylose from hemicellulose as compared to that of glucose from cellulose, and results presented in Table 1 reflect this trend. Dilute acid hydrolyzates also contained furfural, acetic acid, formic acid and HMF. By determining HPLC concentrations, liquid volumes and initial feedstock masses, amounts of furfural, acetic acid, formic acid and HMF were calculated as 0.71, 1.56, 2.41 and 0.04 g per 100 g, respectively.

After pretreatment, the biomass was either washed with three volumes of water or used as is (non-washed), and the resulting wash waters were analyzed by HPLC. Table 1 presents the compositional analysis of the resulting wash waters; furfural, acetic acid, formic acid and HMF were 0.14, 0.31, 0.41 and 0.01 g per 100 g, respectively. Of the inhibitory compounds monitored, formic acid was generated in the highest concentration. In contrast to dilute acid hydrolyzates, wash waters contained similar proportions of glucose and xylose. Furfural, acetic acid, formic acid and HMF concentrations in the wash waters were at most 18% of those present in dilute acid hydrolyzates,indicatingthatinhibitoryproductscouldremainboundtothepretreatedbiomass.

The washed and non-washed pretreated pellets were subjected to enzymatic hydrolysis. The resultsarepresentedinFigure1.Washingthepretreatedpellethadasignificanteffectonglucose recovery,whereglucoseconcentrationsinthewashedconditionwere5.3timeshigherthanthose from the non-washed samples. As expected, concentrations of furfural, acetic acid, formic acid and HMF were significantly higher in the enzymatic hydrolyzates of non-washed samples.


**Table 1.** Composition of pretreatment hydrolyzate and wash water of high specific gravity poplar pretreated in dilute acid (0.98 % v/v) at 140 °C for 40 min.

#### **3.2. Ethanol production from washed and non-washed hydrolyzates**

The fermentability of the enzymatic hydrolysates was evaluated using two yeast strains, selfflocculating yeast SPSC01 and conventional *Saccharomyces cerevisiae* ATCC4126. Both yeast strains solely metabolize glucose and not xylose. A total of four hydrolysate samples, two from

**Figure 1.** Carbohydrate, furan and aliphatic acid yields of washed and non-washed enzymatically hydrolysed dilute acid pretreated poplar.

washed pretreatments and two from non-washed pretreatments were directly used for the fermentation, and ethanol yields based on glucose (*Y*E/G) were determined. Since the initial glucose concentrations in the hydrolyzates were low (less than 4.0 g/L) due to inefficient enzymatic saccharification (Table 2), all the fermentations were completed within 6 hours, as indicated by pre-experiments (data not shown).


a : *Y*E/G refers to ethanol yields based on the glucose contained in hydrolysates

c : Not accurately detected because of out of the detection limit of GC

d: Not calculated due to inaccurately determined ethanol concentration by GC

**Table 2.** Ethanol yields of the fermentation of four different enzymatic hydrolyzates with two yeast strains ATCC4126 and SPSC01. Of the four hydrolyzate samples, two were prepared from non-washed pretreated biomass and two from washed pretreated biomass. The pretreatments were conducted at 140 °C (A) and 160 °C (B), respectively.

It is shown in Table 2 that trace amounts of ethanol were detected in the fermentation broth of the non-washed enzymatic hydrolysates. In contrast, significant amounts of ethanol were generated from the washed enzymatic hydrolyzates, in particular from the hydrolyzate sample that contained a glucose concentration of 3.76 g/L, producing 1.08 g/L and 1.46 g/L of ethanol by the ATCC4126 and SPSC01 strains, respectively. However, the ethanol yields *Y*E/G deter‐

**Figure 2.** Chromatogram of enzymeatic hydrolyzate of washed and unwashed biomass after dilute acid pretreatment. Compounds detected at A) 280 nm and B) 210nm. Compounds are 1) Furfural, retention time = 44.35 min; 2) Formica‐

0 
 10 
 20 
 30 
 40 
 50 
 60 

**Retention 
 Time 
 (minute)** 

Figure 
 2: 
 Chromatogram 
 of 
 enzymatic 
 hydrolyzate 
 of 
 washed 
 and 
 unwashed 
 biomass 
 after 
 dilute 
 acid 
 pretreatment. 
 Compounds 
 detected 
 at 
 A) 
 280 
 nm 
 and 
 B) 210 nm. 

 Compounds 
 are 
 1) 
 Furfural, 
 retention 
 time 
 = 
 44.35 
 min; 
 2) 
 Formic 
 acid,

retention 
 time 
 = 
 13.6 
 min; 
 3) 
 Acetic 
 acid, 
 retention 
 time 
 = 
 14.7 
 min

cid, retention time = 13.6 min; 3) Acetic acid, retention time = 14.7min

0 
 10 
 20 
 30 
 40 
 50 
 60 

**A** 

Washed 
 Unwashed 

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111

Washed 
 Unwashed 

**B** 

1 

The Effect of Washing Dilute Acid Pretreated Poplar Biomass on Ethanol Yields

**Retention 
 Time 
 (minute)** 

2 

3 

 0.000 
 0.005 
 0.010 
 0.015 
 0.020 
 0.025 
 0.030 
 0.035 
 0.040 
 0.045 


0.000 

0.001 

0.002 

0.003 

0.004 

**Absorbance 
 (MV) 
 at 
 210 
 nm** 

0.005 

0.006 

0.007 

0.008 

0.009 

**Absorbance 
 (MV) 
 at 
 280 
 nm** 

washed pretreatments and two from non-washed pretreatments were directly used for the fermentation, and ethanol yields based on glucose (*Y*E/G) were determined. Since the initial glucose concentrations in the hydrolyzates were low (less than 4.0 g/L) due to inefficient enzymatic saccharification (Table 2), all the fermentations were completed within 6 hours, as

**Figure 1.** Carbohydrate, furan and aliphatic acid yields of washed and non-washed enzymatically hydrolysed dilute

**Glucose 
 Xylose 
 Furfural 
 Ace0c 
 Acid 
 Formic 
 Acid 
 HMF** 

\* 
 \* 

\* 

\* 

No 
 Wash 
 Wash 

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

**ATCC4126 SPSC01**

**Table 2.** Ethanol yields of the fermentation of four different enzymatic hydrolyzates with two yeast strains ATCC4126 and SPSC01. Of the four hydrolyzate samples, two were prepared from non-washed pretreated biomass and two from

washed pretreated biomass. The pretreatments were conducted at 140 °C (A) and 160 °C (B), respectively.

**g/100 g Ethanol (g/l)** *Y***E/Ga**

**(g/g)**

**g/100 g**

**(g/g)**

Non-washed-A 0.20±0.00 0 0 0 0.08±0.01b --c --c Non-washed-B 0.19±0.01 0 0 0 0.07±0.01b --c --c Washed-A 2.32±0.06 0.41±0.03 0.18 0.90 0.48±0.05 0.21 1.05 Washed-B 3.76±0.11 1.08±0.09 0.29 5.75 1.46±0.15 0.39 7.74

**Ethanol (g/l)** *Y***E/Ga**

: *Y*E/G refers to ethanol yields based on the glucose contained in hydrolysates

d: Not calculated due to inaccurately determined ethanol concentration by GC

: Not accurately detected because of out of the detection limit of GC

indicated by pre-experiments (data not shown).

**content (g/l)**

**Samples Glucose**

0 

acid pretreated poplar.

1 

2 

3 

4 

**Yield 
 (g/100 
 g 
 of 
 natural 
 biomass)** 

5 

6 

\* 

7 

a

c

Figure 
 2: 
 Chromatogram 
 of 
 enzymatic 
 hydrolyzate 
 of 
 washed 
 and 
 unwashed 
 biomass 
 after 
 dilute 
 acid 
 pretreatment. 
 Compounds 
 detected 
 at 
 A) 
 280 
 nm 
 and 
 B) 210 nm. 

 Compounds 
 are 
 1) 
 Furfural, 
 retention 
 time 
 = 
 44.35 
 min; 
 2) 
 Formic 
 acid, retention 
 time 
 = 
 13.6 
 min; 
 3) 
 Acetic 
 acid, 
 retention 
 time 
 = 
 14.7 
 min **Figure 2.** Chromatogram of enzymeatic hydrolyzate of washed and unwashed biomass after dilute acid pretreatment. Compounds detected at A) 280 nm and B) 210nm. Compounds are 1) Furfural, retention time = 44.35 min; 2) Formica‐ cid, retention time = 13.6 min; 3) Acetic acid, retention time = 14.7min

It is shown in Table 2 that trace amounts of ethanol were detected in the fermentation broth of the non-washed enzymatic hydrolysates. In contrast, significant amounts of ethanol were generated from the washed enzymatic hydrolyzates, in particular from the hydrolyzate sample that contained a glucose concentration of 3.76 g/L, producing 1.08 g/L and 1.46 g/L of ethanol by the ATCC4126 and SPSC01 strains, respectively. However, the ethanol yields *Y*E/G deter‐ 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.

in the production of at least 20 g/L of ethanol, while the use of the non-washed control produced

The Effect of Washing Dilute Acid Pretreated Poplar Biomass on Ethanol Yields

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113

Cantarella et al. (2004) showed that increasing formic acid concentrations of washed steam pretreated poplar from 3. 7 to 11.5 mg/ml decreased sugar recovery from enzymatic hydrolysis by 60%. Formic acid was also shown to inhibit enzymatic hydrolysis by Arora et al. (2012). They showed that adding 5 or 10 mg/ml formic acid to washed dilute acid pretreated poplar biomass resulted in recovery of 47% and 14%, respectively, of potential sugars. Using steam explosion-pretreated wheat straw as their system, Panagitou and Olsson (2007) reported the effects of adding 4 and 15 mg/ml of formic acid to their hydrolyzate; the higher concentration

In work reported by Moreno et al. (2012), wheat straw was pretreated by steam explosion; the pretreated slurry was incubated with *Pycnoporus cinnabarinus* or *Trametes villosa* laccases prior to fermentation with *Kluyveromyces marxianus*. Biomass loadings of 5, 6 and 7% were tested. No differences in ethanol yields at 5% and 6% were observed; however, loadings at 7% resulted in an 86% reduction in ethanol yields compared to the control, which was not prior incubated with laccases. These results indicate that inhibitory byproducts are present in the pretreatment hydrolyzates. Incubation with *T. villosa* laccases removed almost 100 % of vanillin, syringal‐ dehyde, p-coumaric acid and ferulic acid from pretreated hydrolyzates, enabling ethanol to

Although this report is centered on the effects of aliphatic acids and furans on enzymatic hydrolysis and fermentation, it is important to note that other generated products may play key roles in inhibiting enzymatic hydrolysis and fermentation (Palmqvist and Hahn-Hägerdal 1999; Moreno et al 2012). Lignin derivatives can result in nonproductive binding of the saccharification cocktail with lignin derivatives (Berlin et al. 2006); and released sugars and their degradation compounds can deactivate or obstruct enzyme active sites (Kumar and Wyman 2008). It is critical to establish a better understanding of pretreatment chemistry in terms of generated degradation products. By understanding which compound plays a critical role in inhibiting enzymatic hydrolysis and fermentation, attempts can be made to minimize their generation, thereby improving processing yields. Pretreatments at 0.98% (w/v) dilute acid, 140 °C for 40 min resulted in the recovery of 12% and 41% of possible glucose and xylose, respectively. The authors recognize that these were low carbohydrate yields. Pretreatment were re-conducted at 0.98% (w/v) dilute acid, 160 °C for 40 min. Glucose recovery from nonwashed and washed biomass was 0.92 and 19.85 g/100g, respectively, indicating that a 20 °C increase in temperature significantly augmented sugar recovery. Conversely, formic acid contents were 0.65 and 0.04 g/100 g non-washed and washed biomass, respectively; higher content was determined in non-washed biomass as for the 140 °C pretreatment conditions.

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

no ethanol.

annihilated sugar recovery.

glucose yields greater than 0.33 g/g.

**4. Conclusions**

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.

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

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 and/or minimized prior to enzymatic hydrolysis and fermentation.

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 in the production of at least 20 g/L of ethanol, while the use of the non-washed control produced no ethanol.

Cantarella et al. (2004) showed that increasing formic acid concentrations of washed steam pretreated poplar from 3. 7 to 11.5 mg/ml decreased sugar recovery from enzymatic hydrolysis by 60%. Formic acid was also shown to inhibit enzymatic hydrolysis by Arora et al. (2012). They showed that adding 5 or 10 mg/ml formic acid to washed dilute acid pretreated poplar biomass resulted in recovery of 47% and 14%, respectively, of potential sugars. Using steam explosion-pretreated wheat straw as their system, Panagitou and Olsson (2007) reported the effects of adding 4 and 15 mg/ml of formic acid to their hydrolyzate; the higher concentration annihilated sugar recovery.

In work reported by Moreno et al. (2012), wheat straw was pretreated by steam explosion; the pretreated slurry was incubated with *Pycnoporus cinnabarinus* or *Trametes villosa* laccases prior to fermentation with *Kluyveromyces marxianus*. Biomass loadings of 5, 6 and 7% were tested. No differences in ethanol yields at 5% and 6% were observed; however, loadings at 7% resulted in an 86% reduction in ethanol yields compared to the control, which was not prior incubated with laccases. These results indicate that inhibitory byproducts are present in the pretreatment hydrolyzates. Incubation with *T. villosa* laccases removed almost 100 % of vanillin, syringal‐ dehyde, p-coumaric acid and ferulic acid from pretreated hydrolyzates, enabling ethanol to glucose yields greater than 0.33 g/g.

Although this report is centered on the effects of aliphatic acids and furans on enzymatic hydrolysis and fermentation, it is important to note that other generated products may play key roles in inhibiting enzymatic hydrolysis and fermentation (Palmqvist and Hahn-Hägerdal 1999; Moreno et al 2012). Lignin derivatives can result in nonproductive binding of the saccharification cocktail with lignin derivatives (Berlin et al. 2006); and released sugars and their degradation compounds can deactivate or obstruct enzyme active sites (Kumar and Wyman 2008). It is critical to establish a better understanding of pretreatment chemistry in terms of generated degradation products. By understanding which compound plays a critical role in inhibiting enzymatic hydrolysis and fermentation, attempts can be made to minimize their generation, thereby improving processing yields. Pretreatments at 0.98% (w/v) dilute acid, 140 °C for 40 min resulted in the recovery of 12% and 41% of possible glucose and xylose, respectively. The authors recognize that these were low carbohydrate yields. Pretreatment were re-conducted at 0.98% (w/v) dilute acid, 160 °C for 40 min. Glucose recovery from nonwashed and washed biomass was 0.92 and 19.85 g/100g, respectively, indicating that a 20 °C increase in temperature significantly augmented sugar recovery. Conversely, formic acid contents were 0.65 and 0.04 g/100 g non-washed and washed biomass, respectively; higher content was determined in non-washed biomass as for the 140 °C pretreatment conditions.
