**1. Introduction**

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Ethanol sold in the US or Brazil is produced from feedstocks that contain starch or sucrose: corn starch in the US and sugar cane juice in Brazil. The use of these readily available fer‐ mentable sugar sources rouses societal discussions that are anchored on debates involving the use of food commodities for energy production (Wallington et al. 2012). From a sustainability perspective, conversion of cellulosic biomass to ethanol produces less greenhouse gases and particulate matter with a diameter less than 2.5 µm. Furthermore, the cost in dollars per liter in gas equivalent of using corn and corn stover as feedstock are 0.9 and 0.3, respectively (Hills et al. 2009). The production of fuels and biochemicals from cellulosic feedstock is desirable from both societal and environmental perspectives.

Although appealing, the deconstruction of cellulosic biomass into fermentable sugars is problematic. Cellulosic biomass conversion to industrial chemicals and fuels is performed via thermochemical, biochemical or a combination of these platforms. Unfortunately there is no clear technology winner and both conversion platforms have tradeoffs. The thermochemical platform is robust in terms of feedstock processing, but somewhat complicated in terms of the resulting product portfolio (Sharara et al. 2012). On the other hand, the biochemical platform can successfully yield industrial chemicals or fuels, but is delicate in terms of feedstock deconstruction into monomeric sugars (Lynd et al. 2008). This chapter is centered on biomass deconstruction using the biochemical platform.

In the biochemical platform, unfortunately, the deconstruction of plant cell wall into useable and fermentable carbohydrates remains challenging. Feedstock must be reduced in size, pretreated, and hydrolyzed with enzymes to produce a sugar stream that can be fermented

© 2013 Frederick et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Frederick et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

low specific gravity poplar was pretreated in 1% (v/v) dilute acid in non-agitated batch reactors

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

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

107

In this work, high specific gravity poplar was pretreated in 0.98% (w/v) dilute acid at 140 °C in a 1 L stirred reactor and the hydrolyzates were fermented with two ethanol producing strains. This work examined the side-by-side effect of washing and not washing the pretreated

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

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

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

and hydrolyzed using Accelerase ® 1500 enzymes.

**2. Materials and methods**

**2.1. Biomass**

Martin et al. (2011).

**2.2. Pretreatment**

testing.

**2.3. Enzyme hydrolysis**

biomass on sugar yields and its effect on fermentation to ethanol.

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, which will be fermented into fuels or bioproducts.

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 minimize their generation during pretreatment.

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 low specific gravity poplar was pretreated in 1% (v/v) dilute acid in non-agitated batch reactors and hydrolyzed using Accelerase ® 1500 enzymes.

In this work, high specific gravity poplar was pretreated in 0.98% (w/v) dilute acid at 140 °C in a 1 L stirred reactor and the hydrolyzates were fermented with two ethanol producing strains. This work examined the side-by-side effect of washing and not washing the pretreated biomass on sugar yields and its effect on fermentation to ethanol.
