**2.1. Biomass**

into targeted products (Lynd et al. 2008). The cell wall is designed by nature as an elegant interwoven hemicellulose, cellulose and lignin tapestry that maintains its integrity, resulting in wood products that can sustain daily use for hundreds of years. To release the coveted carbohydrates from plant cell walls, the tapestry must be subjected to some form of pretreat‐ ment, ensuring the exposure of sugar polymers, which can subsequently be hydrolyzed.

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

There are a number of available pretreatment technologies (Tao et al. 2011). However, dilute acid pretreatment, though it contains many drawbacks, is most likely to be adopted at the deployment scale due to its relatively low cost and ease of use (Sannigrahi et al. 2011). Regrettably, dilute acid pretreatment results in the production of inhibitory compounds that inhibit downstream biochemical conversion processing steps. These inhibitory compounds are formed from the degradation of hemicellulose into furfural, acetic acid and formic acid; or lignin-derived phenolic compounds, oligomers and re-polymerized furans named humins (van Dam et al. 1986). Such compounds can inhibit enzymatic hydrolysis by at least 50% (Cantarella et al. 2004). In a sense, the dilute acid-based biochemical platform is caught in a chicken and egg situation: pretreatment is essential to loosen the sugar polymer tapestry, but pretreating biomass causes the formation of inhibitory products that hinder subsequent downstream processing steps. In other words, without pretreatment, the expensive processing enzymes cannot access the complex carbohydrates to release the coveted monomeric sugars,

To circumvent the negative effects of dilute acid pretreatment, namely the production of inhibitory products, pretreated biomass is washed prior to enzymatic hydrolysis. Successive washes remove inhibitory products, resulting in biomass amenable to subsequent enzymatic hydrolysis. At the bench scale, inhibitory compounds are removed by washing with up to 30 volumes of water (Djioleu et al. 2012). At the pilot scale, inhibitory compounds are removed from pretreated biomass by washing with at least three volumes of water (Hodge et al. 2008). Washing pretreated biomass will be difficult to replicate at the deployment scale due to the daunting amount of water that will be required. Another approach consists of enhancing our understanding of which compounds critically impede enzymatic hydrolysis, and how to

The conversion of cellulosic biomass into fuels and biochemicals can be conducted with a range of feedstocks. Cellulosic biomass can be sourced from various streams: forestry products and residues, agricultural byproducts, dedicated energy crops, food processing and municipal solid wastes. In particular, wood energy crops, such as hybrid poplars (*Populus deltoides*), are hardwoods that can find use as biorefinery feedstock. *P. deltoides* is being increasingly planted and managed in the United States as short-rotation plantations for timber, pulp and renewable energy (Studer et al. 2011). The use of *P. deltoides* as a feedstock and its response to various pretreatment technologies combined with enzymatic hydrolysis was reported by the Consor‐ tium for Applied Fundamentals and Innovation (CAFI), where the technologies were com‐ pared with identical characterized poplar feedstock (Kim et al. 2009). The series of papers were reported in one single 2009 issue of *Biotechnology Progress*. *P. deltoides*is an interesting feedstock that can be deconstructed into fermentable sugars. The production of a fermentable sugar stream was examined by our group (Martin et al. 2011; Djioleu et al. 2012), where high and

which will be fermented into fuels or bioproducts.

minimize their generation during pretreatment.

High-density poplar was secured from University of Arkansas Pine Tree Branch Station. The material was identical to what was studied by Djioleu et al. (2012) and Martin et al. (2011). The biomass was transformed into chips, which were then ground to 10 mesh using a Wiley Mini Mill (Thomas Scientific, Swedesboro, NJ) as described by Torget et al. (1988). The moisture content was determined with an Ohaus MB45 Moisture Analyzer (Pine Brook, NJ). The poplar used in this study was reported to have a specific gravity of 0.48, as reported by Martin et al. (2011).

#### **2.2. Pretreatment**

Twenty-five grams of biomass were weighed and mixed with 250 ml of 0.98% (w/v) sulfuric acid (EMD, Gibstown, NJ), resulting in a solids concentration of 10%. The reaction mixture was placed in a 1 L Parr (Moline, IL) 4525 reaction vessel. The reaction temperature used in these experiments was 140 °C. Reaction time was set as the time when the reactor reached 140 °C. After 40 min, heating was halted and the reactor was cooled under a stream of cold tap water. Temperature decreased from 140 °C down to 100 °C in about four min. When the mixture inside the reactor reached a temperature lower than 60 °C, the contents were retrieved. On average, the cool down period lasted approximately 10 min. The mixture was filtered with a Buchner apparatus fitted with Whatman filter paper. The remainder of the reaction solids were removed from the vessel and likewise filtered through a Buchner apparatus. The volume of the hydrolyzate was recorded and saved for further testing. The mass of filtered solids was recorded and its moisture content determined, using Ohaus MB45 Moisture Analyzer. The filtered solids were either used as is (referred to throughout the work as non-washed) or washed (referred to throughout the work as washed) with three volumes of Millipore water as suggested by Hodge et al. (2008). The wash liquid was saved and kept at 4 °C for further testing.

#### **2.3. Enzyme hydrolysis**

The hydrolysis was essentially conducted as in Djioleu et al. (2012), but carried out in a 600 ml Parr reactor described by Martin et al. (2010). Forty grams of washed or non-washed pretreated biomass were placed in the Parr reactor with 20 ml of Accellerase ®1500 (Genencor), 200 ml of pH 4.9 sodium citrate buffer, and 180 ml of Millipore filtered water. The reactor was stirred continuously at a slow speed as reported by Martin et al. (2010) and maintained at 50 °C for 24 hours. The entire sample was collected at the end of the run and stored at 4 °C.

**3. Results and discussion**

**3.1. Pretreatment and enzymatic hydrolysis**

2.41 and 0.04 g per 100 g, respectively.

acid (0.98 % v/v) at 140 °C for 40 min.

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,

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

http://dx.doi.org/10.5772/56129

109

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.

**g/100 g glucose xylose furfural acetic acid formic acid HMF**

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

Hydrolyzate 0.828 ± 0.030 4.420 ± 0.103 0.710 ± 0.028 1.560 ± 0.323 2.410 ± 0.231 0.037 ±0.003 Wash water 0.111 ± 0.077 0.103 ± 0.006 0.137 ± 0.023 0.311 ± 0.034 0.412 ± 0.126 0.007 ±0.002

**Table 1.** Composition of pretreatment hydrolyzate and wash water of high specific gravity poplar pretreated in dilute

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

## **2.4. HPLC analysis**

Aliquots from pretreatment hydrolyzates, wash waters and enzyme hydrolyzates were analyzed by high-pressure liquid chromatography (HPLC) for carbohydrates and inhibitory byproducts. Two instruments were used to conduct these analyses. Carbohydrates were analyzed with Waters 2695 Separations module (Milford, MA) equipped with Shodex (Waters, Milford, MA) precolumn (SP-G, 8 µm, 6 x 50 mm) and Shodex column (SP0810, 8 µm x 300 mm). Millipore filtered water (0.2 mL/min) was the mobile phase and the column was heated to 85 ˚C with an external heater. Carbohydrates were detected with a Waters 2414 Refractive Index Detector (Milford, MA) as described by Djioleu et al. (2012). Inhibitory byproducts were analyzed on a Waters 2695 Separations module equipped with a Bio-Rad (Hercules, CA) Aminex HPX-87H Ion Exclusion 7.8 mm X 30 mm column, heated to 55˚C. The mobile phase was 0.005 M H2SO4, flowing at 0.6 ml/min. Compounds were detected with a UV index using the Waters 2996 Photodiode Array detector. Furfural and hydroxymethylfurfural (HMF) were detected at 280 nm; whereas, formic acid and acetic acid were detected at 210 nm.

#### **2.5. Fermentation**

Fermentation was carried out in 50 ml shake flasks with two strains of yeast, self-flocculating SPSC01 and ATCC4126. The SPSC01 strain was provided by Dalian University of Technology, China (Bai et al. 2004). Preculture of both yeast strains was carried out in medium consisting of 30 g/L glucose, 5 g/L yeast extract and 5 g/L peptone. The overnight grown yeasts were harvested by centrifugation at 4,100 *g* for 30 min. The pellets of yeast cells were washed twice with de-ionized water, and then re-suspended in 50 mM sodium citrate buffer (pH 4.8) to reach a cell concentration of 2 to 4×109 /ml. The re-suspended yeast cells were inoculated into 10 ml of each hydrolysate to reach a yeast cell concentration of 8×107 /ml. Ethanol fermentations were performed at 30°C on a rotary shaker at 150 rpm for 8 hours. Glucose content of the samples was assayed using a glucose colorimetric assay kit (Cayman Chemical, MI). Produced ethanol was quantified by gas chromatography (GC) on the Shimadzu GC-2010 equipped with a flame ionization detector (FID) and a Stabilwax®-DA column (cross-bond polyethylene glycol, 0.25 mm ×0.25 µm ×30 m), as described early by Ge et al. (2011). Before injection into the GC, 50 µl of fermentation broth was diluted 10 times with de-ionized water and supplemented with 50 µl of 0.1 mg/ml n-butanol as an internal standard.

#### **2.6. Statistical analysis**

Experiments were conducted in duplicate (pretreatment and enzymatic saccharification) or triplicate (fermentation). Calculations of carbohydrate and degradation compounds, including HMF, furfural, formic acid, and acetic acid, were calculated using Microsoft Office Excel 2007. Analysis of the variance (ANOVA) was determined using JMP 9.0, LSMeans Differences Student's t, with α= 0.10.
