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Mass balance and transformation of corn stover by pretreatment with different dilute organic acids
Bioresource Technology 112 (2012) 319–326
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Mass balance and transformation of corn stover by pretreatment with different dilute organic acids Lei Qin a, Zhi-Hua Liu a, Bing-Zhi Li a, Bruce E. Dale b, Ying-Jin Yuan a,⇑ a Key Laboratory of Systems Bioengineering, Ministry of Education Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, P.O. Box 6888, Tianjin 300072, PR China b Biomass Conversion Research Lab, Department of Chemical Engineering and Materials Science, Michigan State University, 3815 Technology Boulevard, Lansing, MI 48910, USA
a r t i c l e
i n f o
Article history: Received 17 December 2011 Received in revised form 25 February 2012 Accepted 28 February 2012 Available online 6 March 2012 Keywords: Corn stover Pretreatment Organic acid Mass balance Combined severity
a b s t r a c t Previous studies indicated high xylose yield could be achieved after pretreatment using organic acids, but it is necessary to systematically investigate the effects of different parameters during organic acid pretreatments. Corn stover was pretreated with sulfuric, oxalic, citric, tartaric and acetic acid at 50 and 90 mM from 130 to 190 °C. The xylan balance for each different acid was distinct, but all balances were very close to 100% by determining xylan recovery, xylooligomer yield, xylose yield and furfural yield. The effects of combined severity on the recovery or yields of these components were also studied. The acid pKa value affected the proportion of xylan degradation products. The maximum value of xylose and xylooligomer yield for specific acid pretreatment was also determined by pKa value. The maximum xylose yield was obtained after pretreatment with sulfuric and oxalic acid, but more xylooligomers were obtained after pretreatment with weaker acids. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Cellulosic ethanol, as modern adjunct to fossil fuels, has attracted more attention because lignocelluloses is renewable and in large quantity (Zhong et al., 2010). In cellulosic ethanol production, an appropriate pretreatment is crucial for high efficiency in subsequent enzymatic hydrolysis and fermentation processes (da Costa Sousa et al., 2009). Physical or/and chemical pretreatments disrupt lignocellulosic structures, and thereby increasing the access to polymeric sugars for enzymes catalyzing the conversion of polymeric sugars to fermentable sugars. A desirable pretreatment not only provides higher sugar yields and limited amounts of degradation byproducts which inhibit enzymatic hydrolysis or fermentation, but also cost less on equipment, energy and catalyst (Mosier et al., 2005; Wyman et al., 2005; Kootstra et al., 2009a). Dilute acid pretreatment is regarded as a promising method for industry. During dilute acid pretreatment, hemicellulose is removed from the biomass and degraded to xylose and other products, but lignin is reabsorbed on cellulose (Lee et al., 1999; Mosier et al., 2005; Garlock et al., 2011). Dilute sulfuric acid are commonly applied in pretreatment (Lloyd and Wyman, 2005; Saha et al., 2005; Cara et al., 2008; Castro et al., 2010). Pretreatment using nitric acid (Zhang et al., 2011), phosphoric acid (Geddes et al., 2010) and ⇑ Corresponding author. Tel./fax: +86 22 27403888. E-mail address: [email protected] (Y.-J. Yuan). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.134
hydrochloric acid (Goldstein et al., 1983) have also been studied. Organic acids were often employed at a high concentration, which is defined as an organosolv system. Such organosolv pretreatments increase the dosages of acid during pretreatment and therefore need more alkali for subsequent neutralization or water for washing. However, dilute organic acid pretreatment has received less attention. Only oxalic acid (Scordia et al., 2011; Lee et al., 2010), maleic acid (Lu and Mosier, 2007) and fumaric acid (Kootstra et al., 2009b) have apparently been investigated. Dilute organic acid pretreatment has some desirable characteristics compared with dilute inorganic acid, including effective hydrolysis, less degradation products and more oligomeric sugars (Kootstra et al., 2009a). Mass balances for the transformation of compounds during the entire pretreatment and hydrolysis processes are an important means of comparing different pretreatments (Li et al., 2010; Lau and Dale, 2009). One important mass balance is the carbon balance for glucan and xylan, due to their high content in biomass. It is difficult to close the mass balances accurately because of the complexity of the degradation products (Garlock et al., 2011). In addition, combined severity (CS), a function of reaction time, temperature and pH, is usually applied to evaluate dilute sulfuric acid pretreatment (Lloyd and Wyman, 2005; Kabel et al., 2007). The combined severity was defined as (Overend and Chornet, 1987; Chum et al., 1990):
CS ¼ log R0 pH; R0 ¼ t exp½ðT H T R Þ=x
ð1Þ ð2Þ
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In which t is reaction time in minutes, TH is the hydrolysis temperature in °C, and TR is a base temperature, usually set at 100 °C, and x is a fitting parameter, which in most studies is assigned the value of 14.75. Eqs. (1) and (2) were also modified for use with sodium hydroxide pretreatments (Silverstein et al., 2007). It is worth considering whether the above equations adequately describe pretreatment using different organic acids. In the present study, four organic acids, including acetic acid, oxalic acid, tartaric acid and citric acid, were used in pretreatment, in consideration of their low price and different acidities. Mass balances and the combined severity metric were used to compare the effects of different dilute organic acids in pretreatment. 2. Methods 2.1. Raw materials Corn stover was from the suburbs of Tianjin City, China. The corn stover was stripped from the leaves, air-dried and milled. The portion of particles smaller than 0.84 mm was separated by screening. The resulting ground biomass was air-dried until the moisture content was between 5% and 10% based on the total weight and then stored in sealed bags at ambient room temperature until further use. 2.2. Pretreatment with different dilute acids Pretreatment was performed with five different acids, including sulfuric acid, acetic acid, oxalic acid, tartaric acid and citric acid. In order to compare these pretreatments with different acids, uniform molar concentrations of 50 and 90 mM were used, which were also within the range usually used for dilute sulfuric acid (Lloyd and Wyman, 2005; Castro et al., 2010). Milled corn stover (15 g dry matter) was mixed with the dilute acid solution to a final weight of 150 g, and the resulting acid concentration was 50 and 90 mM (The volume of mixture was taken to be 150 mL). The mixture was then transferred to a 316 stainless steel reactor (inner height diameter is 220.0 35.0 mm, 5.0 mm wall) capped on both ends with 316 stainless steel caps. Reactors were heated in an oil bath filled with dimethyl silicon oil. Pretreatment was performed at 130, 150, 170 and 190 °C. Reactors were plunged into the preheated oil at the desired temperature. The heating-up process in these reactors was monitored by thermocouples. After 30 min, the reactors were cooled to room temperature by quenching in ice water. The pretreatments were performed in duplicates. The pretreated slurry was pressed through eight-layer filter cloth to separate free liquid from hydrolyzed solids. The pH of the resulting liquid hydrolysate was determined at room temperature. Then the hydrolysate was collected and stored at 20 °C for component analysis. The solid fraction was washed with about 750 mL deionized water. Washed stream from solid fraction was also separated by filtration. The component of washed stream was also determined. The solid fraction was dried at room temperature until the moisture content (based on the total weight) was not more than 10%. 2.3. Analytical methods The National Renewable Energy Laboratory (NREL) laboratory analysis protocol (LAP) was followed to determine the composition of untreated and pretreated biomass (NREL, 2004). The untreated corn stover raw material consisted of glucan 36.1%, xylan 20.7%, arabinan 2.8%, acid insoluble lignin 18.6% and extractives 18.3%. Glucose, xylose, acetic acid, hydroxymethylfurfural (HMF) and furfural in the hydrolysate were analyzed by high performance
liquid chromatography (HPLC) following LAP-015 from NREL. The HPLC system consists of an Aminex HPX-87H organic acid column (Bio-rad, Hercules, CA), a Waters (Milford, MA) 1515 pump, a Waters column heater module and a Waters 2414 refractive index detector. The mobile phase was 5 mM sulfuric acid, deionized water filtered through 0.22 lm filters. Operating conditions for the HPLC column were 60 °C with a mobile phase flow rate of 0.6 mL/min. For sample analysis, 10 lL of sample was injected and complete sample elution could be accomplished within 48 min. An external standard consisting of glucose, xylose, acetic acid, HMF and furfural was used for quantification. Oligomeric sugars in the hydrolysate and washed solids stream were acid hydrolyzed to analyze these oligomeric sugars, following LAP-014 from NREL. Standards were prepared to calculate the percent recovery of monosaccharides. 2.4. Calculation Solids recovery was calculated as a percentage of the total solids recovered after pretreatment based on the initial sample (dry weight). The closures of mass balance for glucan and xylan undergoing dilute acid pretreatment were determined. Polymers, oligomers, monomers and degradation products should provide close to 100% mass balance closure according to the degradation pathways. However, not all degradation products were measured, only HMF and furfural. The equations for xylan mass balance were expressed as:
Xylan recovery ð%Þ ¼
Xylan in pretreated solid ðgÞ 100% Xylan in untreated solid ðgÞ
Xylooligomer yield ð%Þ ¼
Xylooligomer in hydrolysate ðgÞ Xylan in untreated solid ðgÞ 100%
Xylose yield ð%Þ ¼
ð3Þ
ð4Þ
Xylose in hydrolysate ðgÞ 132 Xylan in untreated solid ðgÞ 150 100%
Furfural yield ð%Þ ¼
ð5Þ
Furfural in hydrolysate ðgÞ 132 Xylan in untreated solid ðgÞ 96 100%
ð6Þ
The xylose and furfural are converted to xylan equivalent by multiplying the ratio of the molecular weights of xylose to xylan (132/150) and furfural to xylan (132/96). Xylooligomer is reported in xylan equivalents. The equations for glucan were similar to xylan except the ratios for glucose to glucan and HMF to glucan are (162/180) and (162/126), respectively. 3. Results and discussion 3.1. Composition of the pretreated solids and hydrolysates Pretreated solid recovery, solid compositions and hydrolysate components were variable between different acid pretreatments (Table 1). Solid recoveries are different for different acids. The lowest and highest solid recovery was obtained after sulfuric and acetic acid pretreatment at any condition, respectively. For every acid, increased temperature and increased acid concentration decreased the solid recovery, indicating that harsh conditions increased the biomass conversion into dissoluble compounds in pretreatment. The xylan content obviously decreased along with increased temperature and acid concentration. This result is similar to previous
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Table 1 Compositions of solid fractions and components of hydrolysates for different dilute acid pretreatments. All data are mean values of two separate experiments. Standard deviations are given between brackets. Factors
Xylan
Lignin
Glucose Xylose
Acetic acida HMF
Furfural
pHb
130/50
82.2 86.7 86.3 88.3 88.7 67.3 71.5 79.0 80.1 86.4 60.3 61.8 65.8 65.7 77.3 54.0 57.1 57.8 58.2 61.9 70.6 75.2 86.1 86.3 88.7 59.5 61.9 71.6 74.0 83.8 54.9 57.0 60.6 60.7 74.0 49.7 54.7 52.8 55.8 60.5
20.9 (0.7) 23.2 (0.7) 22.4 (1.9) 22.9 (0.5) 23.0 (0.7) 14.9 (0.9) 16.5 (1.6) 21.0 (0) 21.8 (0.6) 22.9 (0.7) 9.2 (2.1) 10.1 (1.8) 13.1 (0.8) 14.4 (0.3) 22.1 (1.5) 5.0 (0.4) 6.8 (2.2) 8.4 (0.5) 9.2 (0.8) 17.5 (0.4) 16.6 (0.4) 17.7 (1.8) 22.7 (0.6) 22.1 (0.2) 22.8 (0.5) 8.2 (1.4) 10.9 (1.1) 18.4 (0.2) 19.5 (1.4) 23.2 (1.4) 5.1 (0.6) 4.9 (0.5) 6.3 (1.8) 8.5 (1.9) 20.3 (1.3) 0 (0) 0 (0) 0.6 (0.4) 1.0 (0.3) 15.6 (0)
23.6 (0.5) 22.8 (0.1) 21.1 (0.5) 22.3 (0.8) 21.1 (1.1) 26.9 (0.1) 26.3 (2.0) 24.3 (1.1) 23.9 (0.8) 21.5 (0.3) 32.1 (0.2) 26.8 (1.7) 30.5 (0.2) 25.1 (0.6) 24.2 (0.6) 31.7 (0.9) 29.5 (1.2) 29.8 (1.4) 28.9 (0.2) 26.9 (1.0) 25.3 (2.1) 23.2 (0.8) 21.8 (0.7) 22.0 (0.5) 20.1 (1.8) 29.5 (1.7) 26.9 (0) 23.1 (0.4) 23.7 (1.1) 21.6 (2.4) 32.3 (1.2) 29.5 (1.2) 27.8 (0.5) 29.0 (0.2) 23.2 (2.0) 33.2 (0.3) 31.7 (1.2) 30.3 (1.1) 30.9 (1.4) 27.4 (1.3)
1.2 (0.2) 1.1 (0) 1.0 (0) 1.1 (0.1) 1.1 (0.1) 1.8 (0.4) 1.5 (0.3) 1.2 (0.1) 1.2 (0.1) 1.3 (0.1) 2.6 (0) 2.1 (0.1) 1.1 (0.2) 1.2 (0.2) 0.9 (0.2) 4.0 (1.1) 2.3 (0.8) 2.1 (0.2) 2.2 (0.1) 0.3 (0.4) 1.4 (0.1) 1.2 (0.2) 1.1 (0) 1.2 (0.2) 1.1 (0.2) 2.7 (0) 2.2 (0.3) 1.3 (0.1) 1.2 (0.1) 1.1 (0) 4.1 (0.1) 3.0 (0.2) 1.4 (0.1) 1.4 (0.2) 0.8 (0.1) 5.9 (0.1) 3.0 (0.3) 2.4 (0.3) 1.8 (0.2) 0.1 (0.1)
0.5 0.3 0.3 0.2 3.3 1.6 1.1 0.5 0.3 3.6 3.3 2.3 1.3 0.9 4.0 4.3 3.7 3.1 3.3 5.5 1.7 0.9 0.4 0.3 5.9 3.5 2.6 0.8 0.4 6.5 3.0 3.0 1.8 1.2 6.1 5.5 4.2 4.2 4.7 7.6
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0.2 (0.1) 0.1 (0) 0 (0) 0 (0) 0 (0) 1.6 (0.5) 1.1 (0.4) 0.7 (0.1) 0.7 (0.5) 0.1 (0) 6.7 (1.1) 5.4 (0.6) 5.9 (1.3) 5.3 (0.5) 1.5 (0.3) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0.7 (0.1) 0.3 (0.1) 0.1 (0) 0.1 (0) 0 (0) 3.8 (0.9) 1.8 (0.4) 1.2 (0) 1.4 (0.5) 0.1 (0) 11.5 (0.5) 8.4 (0) 9.2 (1.0) 8.9 (0.3) 3.0 (0.1)
1.5 2.2 3.1 3.0 4.3 1.5 2.2 3.2 3.0 4.3 1.5 2.4 3.1 2.9 4.1 1.5 2.8 3.0 2.9 3.7 1.2 1.6 2.8 2.6 4.1 1.2 1.6 2.8 2.6 4.1 1.3 1.9 2.8 2.6 4.0 1.2 2.3 2.8 2.3 3.6
170/50
190/50
130/90
150/90
170/90
190/90
a
Components of hydrolysates (g/L)
Solid recovery Glucan
150/50
b
Compositions of solid fractions (%)
Temperature (°C)/acid concentration (mM) Acid type Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
(2.2) (3.6) (2.4) (3.3) (2.3) (0.4) (0.2) (3.5) (3.0) (2.2) (4.1) (3.7) (2.5) (4.7) (4.1) (2.0) (2.6) (2.5) (1.8) (2.1) (0.7) (1.3) (1.7) (1.4) (0.4) (0.9) (0.9) (1.3) (1.1) (0.3) (0.3) (0.9) (0.2) (0.7) (0.6) (2.7) (0.8) (1.6) (1.4) (0.5)
42.9 (0.7) 42.2 (1.6) 41.1 (0.1) 40.8 (0.6) 41.0 (0.3) 52.6 (1.5) 49.3 (1.0) 44.1 (1.9) 43.0 (1.2) 39.6 (0.8) 55.0 (5.9) 53.2 (3.2) 53.5 (0.4) 52.2 (0.8) 43.9 (1.9) 58.1 (3.1) 56.4 (2.6) 57.3 (1.5) 57.2 (1.6) 53.6 (4.7) 48.6 (1.0) 43.9 (3.7) 40.0 (0.7) 40.9 (0.7) 39.4 (1.8) 55.8 (0.1) 55.2 (2.6) 48.4 (1.2) 46.8 (1.1) 40.8 (0.6) 57.3 (4.0) 54.9 (1.7) 54.5 (1.8) 56.3 (1.6) 45.7 (1.4) 56.9 (1.9) 56.9 (0.2) 58.6 (0.5) 58.5 (0.3) 54.6 (1.5)
3.0 (0.7) 1.9 (0.4) 2.3 (0.1) 1.7 (0.2) 1.5 (0) 11.5 (1.5) 7.9 (1.1) 5.8 (1.8) 2.1 (0.1) 1.6 (0) 16.9 (0.1) 14.2 (1.2) 7.6 (0.5) 6.7 (2.3) 1.2 (0) 9.6 (0.1) 10.4 (1.0) 8.2 (0.8) 7.9 (0.9) 1.4 (0.5) 10.0 (0.4) 5.4 (0.9) 1.4 (0.2) 1.7 (0) 1.5 (0.1) 18.9 (0.5) 16.0 (1.5) 2.9 (0.2) 3.0 (0.5) 1.5 (0) 14.2 (0.8) 18.7 (0.4) 12.1 (1.2) 12.1 (1.3) 1.2 (0.2) 4.1 (0.3) 11.6 (1.2) 7.3 (0.5) 9.2 (0.6) 1.9 (0.1)
(0.2) (0.2) (0.2) (0.1) (0.4) (0.2) (0.3) (0.4) (0.2) (0.5) (0.3) (0.5) (0.4) (0.2) (0.2) (0.8) (0.4) (0.4) (0.6) (0.1) (0.2) (0.2) (0.3) (0.1) (0.3) (0) (0.3) (0.4) (0.1) (0.3) (0.4) (0.3) (1.0) (0.1) (1.0) (0.5) (0.6) (0.2) (0.2) (0.4)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0.3 (0.1) 0.2 (0) 0.1 (0) 0.2 (0) 0.1 (0) 0.9 (0.4) 0.3 (0.1) 0.4 (0.1) 0.4 (0) 0.1 (0) 1.2 (0.2) 1.1 (0.3) 1.1 (0.2) 1.2 (0) 0.4 (0.2) 0.2 (0) 0 (0.1) 0 (0) 0 (0) 0 (0) 0.6 (0.1) 0.4 (0.1) 0.2 (0) 0.2 (0) 0.1 (0) 0.7 (0) 0.7 (0.1) 0.5 (0) 0.6 (0.1) 0.1 (0) 2.7 (0.3) 1.5 (0.1) 1.5 (0.1) 1.5 (0.2) 0.2 (0)
(0) (0.1) (0.2) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (0) (0.1) (0) (0) (0) (0.1) (0.1) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0.1) (0) (0.1) (0.2) (0) (0) (0.1) (0) (0) (0)
For acetic acid pretreatment, the acetic acid concentrations are determined as the total acetic acid including initial part and production part. The pH values of hydrolysates were measured at room temperature.
studies (Cara et al., 2008). The residual xylan content was less than 10% when temperatures exceeded 170 °C (down from 20.7% of the raw material), and even approached zero at 190 °C/90 mM after sulfuric and oxalic acid pretreatment. For acetic acid pretreatment, xylan content scarcely changed, and was still 15.6% at 190 °C/ 90 mM. Pretreatments with organic acid resulted in lower glucan content and higher xylan content in pretreated solids than sulfuric acid at the same condition. The lignin contents in pretreated solid increased somewhat with the increased temperature and acid concentration due to lost xylan. In pretreatment hydrolysates, monosaccharide, oligosaccharide and degradation product concentrations were determined (Table 1). The glucose concentrations were almost constant, around 2 g/ L for citric, tartaric and acetic acid pretreatment, but a little higher for sulfuric and oxalic acid pretreatment. Xylose concentrations ranged from 1.2 to 18.9 g/L, and it was higher after sulfuric and oxalic acid pretreatment than that after other pretreatments. The above observations indicate that pretreatments with organic acid were less efficient in releasing glucose and xylose due to lower concentration of H+ ions formation in liquor, because the release of glucose and xylose is catalyzed by acid. Acetic acid was produced during degradation of hemicellulose and lignin (Palmqvist and Hahn-Hagerdal, 2000), and its content in hydrolysates increased with increased temperature and acid
concentration (Table 1). Acetic acid pretreatment led to a higher acetic acid concentration including the acid added to initiate pretreatment. The acetic acid concentrations in hydrolysates except acetic acid pretreatment hardly inhibit fermentation using tolerant yeast (Ding et al., 2011). HMF rarely appeared below 170 °C for all the acidic pretreatments, and its concentration was always less than 1.0 g/L. When the pretreatment temperature was 190 °C, HMF concentration exceeded 1.0 g/L. The trend of furfural generation during pretreatment was similar to that of HMF, and the maximum concentration of furfural reached 11.5 g/L after sulfuric acid pretreatment at 190 °C. It is reported that the formation of HMF and furfural is promoted by temperature and H+ ion (Mamman et al., 2008). As a result, organic acid pretreatment led to lower HMF and furfural concentration than sulfuric acid at the same condition (Table 1). Furfural and HMF concentration exceeding 1.3 g/L were unexpected (Ding et al., 2011), due to the significant inhibition to fermentation and the loss of xylose. It implies that pretreatment with organic acid perhaps led to better fermentability due to lower inhibitor concentration. 3.2. Mass balance closures for glucan, xylan and lignin The glucan and xylan degradation process has been mentioned in several reports (Palmqvist and Hahn-Hagerdal, 2000; Karimi
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et al., 2006). Hence, the mass balance for glucan and xylan from beginning to end was available and significant to evaluate the pretreatment effect. Glucan recovery, glucooligomer yield, glucose yield and HMF yield after different dilute acid pretreatments were determined for glucan balance closure (Table 2). Glucan balance closure for all conditions was close to 100%, confirming this method’s reliability. Glucan recoveries were above 90% under most conditions due to the low solubility of glucan, except for some harsher conditions, such as 190 °C/90 mM. Glucooligomer and glucose yields were less than 10% under most conditions. HMF yields were also very low. Glucose and HMF yields were 13.1% and 7.8% by sulfuric acid pretreatment at 190 °C/90 mM, which were the highest achieved; about twofold more than achieved by oxalic acid pretreatment. It is reported that the degradations of glucose and xylose with organic acid pretreatment were less than sulfuric acid due to the higher activation energy for degradations with organic acid (Lu and Mosier, 2007). However, the degradations of glucan and xylan with organic acid seemed less as well. Xylan recovery and xylan-products yield were highly variable between the different pretreatment conditions (Fig. 1). In contrast to cellulose, hemicellulose was apt to be hydrolyzed during acid pretreatment. Xylan recovery after any acid pretreatment decreased along with increased temperature and acid concentration.
Xylan recoveries with different acid pretreatments under the same condition increased according to the order of sulfuric < oxalic < citric < tartaric < acetic acid. Xylan mainly remained in the solid fraction after acetic acid pretreatment, indicating dilute acetic acid is too weak to degrade xylan to xylose. Removal of hemicellulose increased the mean pore size of the biomass, therefore increased the accessibility of enzyme to glucan, which was an important feature and the mechanism of action in acid pretreatment (Alvira et al., 2010). Excess xylan remaining in solid indicates the lack of pretreatment strength which led to a low enzymatic efficiency in enzymatic hydrolysis (Cara et al., 2008). At the same condition, organic acid pretreatment induced to lower furfural yield than sulfuric acid pretreatment, which agreed with previous report (Kootstra et al., 2009b). Other component yields varied in more complicated patterns than did xylan. For 50 mM acid at 130 °C, xylan recovery after sulfuric acid pretreatment was 83.2%, lower than that following pretreatment with organic acids, which was more than 90%. At 150 °C, the xylose yield was 43.4% after sulfuric acid pretreatment, and after other acid pretreatments, decreased according to the order of oxalic, citric, tartaric and acetic acid. At 170 °C, xylooligomer yields decreased after sulfuric and oxalic acid pretreatment, and increased after citric, tartaric and acetic acid pretreatment than that at 150 °C; xylose
Table 2 Glucan recovery, glucooligomer yield, glucose yield and HMF yield for different dilute acid pretreatments. All data are mean values of two separate experiments. Standard deviations are given between brackets. Temperature (°C)/acid concentration (mM)
Acid type
Glucan recovery (%)
Glucooligomer yield (%)
Glucose yield (%)
HMF yield (%)
Total closure (%)
130/50
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
97.8 (4.3) 101.5 (0.4) 98.3 (3.1) 99.8 (5.2) 100.9 (3.4)
1.6 0.3 0.2 0.6 0.1
(1.5) (0.5) (0.3) (0.8) (0.2)
2.2 1.8 2.0 1.7 1.9
(0.6) (0.2) (0.1) (0.6) (0.4)
0.1 (0.1) 0 (0) 0 (0) 0 (0) 0 (0)
101.7 103.7 100.5 102.1 102.9
150/50
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
98.3 97.7 96.6 95.5 94.8
(3.4) (2.3) (0.1) (1.0) (4.4)
1.1 1.5 3.6 1.8 0.9
(1.2) (1.0) (3.5) (0.8) (0.8)
3.6 3.0 2.2 2.3 2.4
(1.0) (0.6) (0.2) (0.2) (0.4)
0.7 0.3 0.2 0.3 0.1
(0.3) (0) (0) (0) (0)
103.8 (3.8) 102.5 (2.5) 102.6 (3.5) 99.9 (1.3) 98.1 (4.5)
170/50
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
92.0 91.2 97.7 95.1 94.1
(3.2) (0.4) (3.0) (8.4) (1.2)
0.9 2.3 1.1 1.6 2.1
(0) (0.7) (0.7) (1.0) (0.3)
5.9 4.4 2.5 2.5 1.8
(0.4) (0.3) (0.6) (0.6) (0.7)
2.4 1.0 1.2 1.3 0.2
(0.6) (0.1) (0.1) (0.1) (0)
101.2 (3.3) 98.8 (0.8) 102.4 (3.1) 100.5 (8.4) 98.3 (1.4)
190/50
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
87.0 89.3 91.9 92.4 92.0
(1.1) (0.1) (6.4) (5.4) (4.8)
0.6 3.1 1.2 0.5 1.6
(0.9) (1.6) (1.0) (0.7) (1.2)
8.2 4.6 5.0 4.9 0.7
(2.1) (1.7) (0) (0.9) (0.8)
3.1 2.8 3.0 3.1 0.8
(0.4) (0.5) (0.1) (0) (0.4)
98.9 (2.5) 99.9 (2.4) 101.0 (6.5) 100.9 (5.5) 95.1 (5.0)
130/90
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
95.1 91.5 95.6 97.8 96.8
(1.0) (9.3) (3.5) (0.2) (4.9)
1.0 1.5 0.2 1.0 0.5
(1.0) (0.8) (0.2) (0.4) (0.6)
2.9 2.4 2.2 2.2 2.2
(0.4) (0.3) (0.4) (0.4) (0.4)
0.4 (0.1) 0.1 (0.1) 0 (0) 0 (0) 0 (0)
99.4 (1.5) 95.4 (9.4) 97.9 (3.6) 101.1 (0.6) 99.4 (5.0)
150/90
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
92.0 94.6 96.1 96.1 94.8
(1.3) (3.2) (4.1) (3.7) (1.8)
0.2 0.6 0.6 1.8 0.7
(0.2) (0.4) (0.8) (0.1) (0.5)
5.8 4.5 2.6 2.3 2.1
(0.1) (0.8) (0.5) (0.3) (0.1)
1.9 1.0 0.4 0.4 0.1
(0.2) (0.2) (0) (0.2) (0)
99.9 (1.4) 100.7 (3.3) 99.7 (4.2) 100.7 (3.7) 97.7 (1.9)
170/90
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
87.3 86.7 91.5 94.8 93.8
(7.1) (1.3) (2.7) (3.8) (3.6)
0 (0) 0 (0) 0.9 (1.0) 1.5 (0.5) 1.7 (0.3)
8.9 6.5 2.8 2.9 1.5
(0.3) (0.6) (0.1) (0.4) (0.2)
2.2 1.9 1.6 1.8 0.3
(0) (0.2) (0) (0.1) (0)
98.3 (7.1) 95.1 (1.5) 96.8 (2.9) 101.0 (3.8) 97.3 (3.6)
190/90
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
78.4 86.5 85.8 90.5 91.7
(4.6) (1.6) (3.4) (2.6) (3.3)
0 (0) 0 (0) 0 (0) 0 (0) 1.0 (0.1)
13.1 (0.2) 6.2 (0.6) 5.1 (0.5) 4.5 (0.3) 0.2 (0.1)
7.8 3.8 3.6 3.7 0.7
(0.7) (0.2) (0.3) (0.4) (0)
99.2 96.4 94.6 98.6 93.6
(4.5) (0.7) (3.1) (5.3) (3.4)
(4.7) (1.7) (3.4) (2.7) (3.3)
L. Qin et al. / Bioresource Technology 112 (2012) 319–326
323
Fig. 1. Xylan balance for different dilute acid pretreatments. Xylan recovery, xylooligomer yield, xylose yield and furfural yield are presented together to close to 100%, for the calculation bases of which are all initial xylan. Error bar represents the standard deviation of duplicate experiments. XO: xylooligomer, SA: sulfuric acid, OA: oxalic acid, CA: citric acid, TA: tartaric acid, AA: acetic acid.
yields increased about 20% after sulfuric, oxalic and tartaric acid pretreatment than that at 150 °C. At 190 °C, xylose yields decreased by 26.9% and 14.2% after sulfuric and oxalic acid pretreatment and increased a little after citric and tartaric acid pretreatment from 170 °C; furfural yields accounted for 30–40% after most acid pretreatments except acetic acid pretreatment. For 90 mM acid, xylose yields went up to 71.3% and 61.0% after sulfuric and oxalic acid pretreatment at 150 °C, respectively. Xylooligomer yields came up to 27.3% and 16.8% after citric and tartaric acid pretreatment, but reduced to zero and 4.8% after sulfuric and oxalic acid pretreatment from 130 °C, respectively. Similar trend was observed when the condition was changed from 150 to 170 °C at 50 mM acid, which indicated that a similar yield can be achieved at low temperature-high acid concentration or high temperature-low acid concentration (Lloyd and Wyman, 2005). At 170 °C, xylose yield came down to 53.5% after sulfuric acid pretreatment, and went up to 71.3%, 48.6% and 47.0% after oxalic, citric and tartaric acid pretreatment, respectively; xylooligomer yield after acetic acid pretreatment increased to 20.2%. At 190 °C, furfural yield was 73.1% after sulfuric acid pretreatment and around 50% after oxalic, citric and tartaric acid pretreatment; xylose yield obviously decreased after any pretreatment. It is observed that the maximum xylose yield was obtained at 150 °C/90 mM with sulfuric acid and 170 °C/90 mM with oxalic acid. However, the total yield of xylose and xylooligomer with different acids was equivalent, which can reach 72.3%, 71.3%, 69.2% and 63.1% at 170 °C/90 mM with oxalic, sulfuric, citric and tartaric acid, respectively. Xylooligomer production was taking into account, because it can be hydrolyzed to xylose in subsequent enzymatic hydrolysis. The relationship between the yield of xylose plus xylooligomer and the xylan recovery was linear dependence in the xylan recovery range of 30–100% after any acid pretreatment (Fig. 2), which indicated that no furfural or other degradation product generated within this range. The optimal pretreatment condition should enable the xylan recovery around 20%. At this critical point, the total soluble sugar yield reached the maximum and the degradation product just produced (Fig. 2). In addition, these linear relationships had the similar function with different acids, which indicated that the release of total soluble sugar was not affected by acid species. But the proportion of xylose and
Fig. 2. Total yield of xylose and xylooligomer as a function of xylan recovery for different acid pretreatments at 50/90 mM acid concentration and 130–190 °C.
xylooligomer in total soluble sugar was affected by acid species mainly due to the H+ ion concentration. In general, organic acid pretreatment obtained an equivalent xylan recovery and soluble sugar yield to sulfuric acid, and produced more xylooligomer and less furfural except acetic acid pretreatment in this study when temperature and acid concentration was over 170 °C and 90 mM. It is noticeable that the xylan balance closures were not close to 100% at 190 °C under both 50 and 90 mM acid concentration, which indicated some degradation products not determined generated from furfural. Formic acid and furfural resinification may generate from furfural in pretreatment, which was reported previously (Palmqvist and Hahn-Hagerdal, 2000; Karimi et al., 2006). Acid insoluble lignin recoveries for different pretreatments scarcely changed, which were around 100% (Fig. 3). Acid soluble lignin was ignored in this study due to its small amount. This result agreed with many reports that lignin can melt in acid pretreatment due to high temperatures and redeposit as spherical droplets on the residual surfaces unless there is post-washing with hot water
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Fig. 3. Acid insoluble lignin recovery for different dilute acid pretreatments. Error bar represents the standard deviation of duplicate experiments. SA: sulfuric acid, OA: oxalic acid, CA: citric acid, TA: tartaric acid, AA: acetic acid.
or organic solvent (Selig et al., 2007; Garlock et al., 2011). In this study, pretreated solids were washed with cold water, which was not helpful for lignin removal. 3.3. Effects of combined severity on different acid pretreatments In order to investigate the effects of pretreatment parameters on the component conversion, the combined severity based on Eqs. (1) and (2) was employed to describe the pretreatment condition. The combined severity factor has been plotted against the xylan recovery, xylooligomer yield, xylose yield and furfural yield after pretreatment (Fig. 4). The trends of xylan recovery, xylose yield and furfural yield versus combined severity were consistent with previous reports (Larsson et al., 1999; Kabel et al., 2007). The trend of xylooligomer yield has seldom been reported. The function of the recovery or yield with different acid pretreatments versus combined severity had the same form (e.g. monotonic for xylan recovery and furfural yield, non-monotonic for xylooligomer yield and xylose yield), but the parameters in the function of different acid pretreatments were not the same. Namely, the recovery or yield trends for different acid pretreatments didn’t coincide. This result seems reasonable, because combined severity is a parameter comprised of temperature, pH and time, but not acid species. The difference of the functions among different acid pretreatments was from the acid dissociation constant. The effects of temperature on pH are negligible for strong acid (i.e. sulfuric acid) due to complete ionization at any temperature. In contrast, the pH of a weak acid obviously decreases along the increased temperature due to the heat-needed ionization process (Henderson–Hasselbalch equation; Palmqvist and Hahn-Hagerdal, 2000). The pH of organic acid is much lower at higher temperature than that determined at room temperature. Therefore, a higher pH than the real pH during the pretreatment reaction was used to calculate the combined severity of weak acid. As a result, the combined severity without modified by acid dissociation constant was applied before a novel parameter was invented. The drift degree of combined severities along x-axis in Fig.4 for different acid pretreatments was in accordance with the pKa value, the negative logarithm of dissociation constant (these five acid pKa values at ordinary temperature is sulfuric acid-3, oxalic acid 1.27, tartaric acid 2.98, citric acid 3.13, acetic acid 4.76).
An evident peak value existed in the function of xylooligomer and xylose yield versus combined severity after pretreatment with specific acid. The peak values of xylooligomer yields for different acid pretreatments were distinct and in the order of acetic acid > citric acid > tartaric acid > oxalic acid > sulfuric acid (Fig. 4B). In contrast, the peak values of xylose yield were in the opposite order as: sulfuric and oxalic acid > tartaric and citric acid > acetic acid, which paralleled the order of pKa of each acid as well (Fig. 4C). These regular variations indicated that the maximum value of xylose yield and xylooligomer yield for a specific acid pretreatment was determined by acid pKa value. Furfural was generated at a specific combined severity and sharply rose for each acid pretreatment (Fig. 4D). Furfural yield also should have peak value at a harsher combined severity than that used in this study, which was not desired during pretreatment (Larsson et al., 1999). Another significant point in Fig. 4B and C was the intersection point of the trends of any two acid pretreatments, which represented the pretreatments with the same combined severity and the same xylooligomer or xylose yield. When the combined severity was higher than this point, the xylose yield of pretreatment with stronger acid was higher than that with weaker acid, and vice versa. Xylooligomer yield ran contrary pattern to xylose yield as expected. This model indicated that a weaker acid pretreatment can never result a high xylose yield even if pretreatment temperature or acid concentration was increased. Pretreatment with maleic and fumaric acid was also investigated in previous study (Kootstra et al., 2009b). Maximum xylose yields were about 70% and 45% after pretreatment with maleic and fumaric acid at 50 mM and 170 °C, respectively. The pKa value of maleic and fumaric acid is 1.92 and 3.03, which was agreed with the relationship between pKa and xylose yield observed in this study. According to the result in previous and current study, high xylose yield would be obtained using the acid with pKa < 2.0 after pretreatment, such as oxalic acid and maleic acid. In contrast, using organic acids with a higher pKa value can never reach a high xylose yield. In previous study, sulfuric acid and acetic acid were both added to pH = 1.0 during biomass pretreatment, but xylose yield after acetic acid pretreatment was nearly half of that after sulfuric acid pretreatment (Pedersen et al., 2010).
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Fig. 4. Combined severity versus xylan recovery (A), xylooligomer yield (B), xylose yield (C) and furfural yield (D). Combined severity (CS) was defined by Eqs. (1) and (2). The pH of each acid was measured at ambient temperature. The two duplicate pretreatment data were both shown in the figure, and were not replaced by an average.
4. Conclusion In conclusion, organic acid pretreatment was milder than sulfuric acid due to the sustained release of H+ ions. Organic acid pretreatment resulted in higher xylan content in pretreated solids and lower xylose concentration in pretreated hydrolysate than sulfuric acid at the same condition. However, organic acid pretreatment produced more xylooligomer and less furfural. The dissociation constant decided the maximum xylooligomer and xylose yield. Although a high pKa acid pretreatment can never lead high xylose yield, a roughly equivalent soluble sugar yield was obtained.
Acknowledgements The authors are grateful for the financial support by Major International Joint Research Project (21020102040) from the National Natural Science Foundation of China, the National Basic Research Program of China (‘‘973’’ Program: 2011CBA00802, 2007CB714301) from Ministry of Science and Technology of China, Key Program (20736006) from the National Natural Science Foundation of China.
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Mass balance and transformation of corn stover by pretreatment with different dilute organic acids Lei Qin a, Zhi-Hua Liu a, Bing-Zhi Li a, Bruce E. Dale b, Ying-Jin Yuan a,⇑ a Key Laboratory of Systems Bioengineering, Ministry of Education Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, P.O. Box 6888, Tianjin 300072, PR China b Biomass Conversion Research Lab, Department of Chemical Engineering and Materials Science, Michigan State University, 3815 Technology Boulevard, Lansing, MI 48910, USA
a r t i c l e
i n f o
Article history: Received 17 December 2011 Received in revised form 25 February 2012 Accepted 28 February 2012 Available online 6 March 2012 Keywords: Corn stover Pretreatment Organic acid Mass balance Combined severity
a b s t r a c t Previous studies indicated high xylose yield could be achieved after pretreatment using organic acids, but it is necessary to systematically investigate the effects of different parameters during organic acid pretreatments. Corn stover was pretreated with sulfuric, oxalic, citric, tartaric and acetic acid at 50 and 90 mM from 130 to 190 °C. The xylan balance for each different acid was distinct, but all balances were very close to 100% by determining xylan recovery, xylooligomer yield, xylose yield and furfural yield. The effects of combined severity on the recovery or yields of these components were also studied. The acid pKa value affected the proportion of xylan degradation products. The maximum value of xylose and xylooligomer yield for specific acid pretreatment was also determined by pKa value. The maximum xylose yield was obtained after pretreatment with sulfuric and oxalic acid, but more xylooligomers were obtained after pretreatment with weaker acids. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Cellulosic ethanol, as modern adjunct to fossil fuels, has attracted more attention because lignocelluloses is renewable and in large quantity (Zhong et al., 2010). In cellulosic ethanol production, an appropriate pretreatment is crucial for high efficiency in subsequent enzymatic hydrolysis and fermentation processes (da Costa Sousa et al., 2009). Physical or/and chemical pretreatments disrupt lignocellulosic structures, and thereby increasing the access to polymeric sugars for enzymes catalyzing the conversion of polymeric sugars to fermentable sugars. A desirable pretreatment not only provides higher sugar yields and limited amounts of degradation byproducts which inhibit enzymatic hydrolysis or fermentation, but also cost less on equipment, energy and catalyst (Mosier et al., 2005; Wyman et al., 2005; Kootstra et al., 2009a). Dilute acid pretreatment is regarded as a promising method for industry. During dilute acid pretreatment, hemicellulose is removed from the biomass and degraded to xylose and other products, but lignin is reabsorbed on cellulose (Lee et al., 1999; Mosier et al., 2005; Garlock et al., 2011). Dilute sulfuric acid are commonly applied in pretreatment (Lloyd and Wyman, 2005; Saha et al., 2005; Cara et al., 2008; Castro et al., 2010). Pretreatment using nitric acid (Zhang et al., 2011), phosphoric acid (Geddes et al., 2010) and ⇑ Corresponding author. Tel./fax: +86 22 27403888. E-mail address: [email protected] (Y.-J. Yuan). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.134
hydrochloric acid (Goldstein et al., 1983) have also been studied. Organic acids were often employed at a high concentration, which is defined as an organosolv system. Such organosolv pretreatments increase the dosages of acid during pretreatment and therefore need more alkali for subsequent neutralization or water for washing. However, dilute organic acid pretreatment has received less attention. Only oxalic acid (Scordia et al., 2011; Lee et al., 2010), maleic acid (Lu and Mosier, 2007) and fumaric acid (Kootstra et al., 2009b) have apparently been investigated. Dilute organic acid pretreatment has some desirable characteristics compared with dilute inorganic acid, including effective hydrolysis, less degradation products and more oligomeric sugars (Kootstra et al., 2009a). Mass balances for the transformation of compounds during the entire pretreatment and hydrolysis processes are an important means of comparing different pretreatments (Li et al., 2010; Lau and Dale, 2009). One important mass balance is the carbon balance for glucan and xylan, due to their high content in biomass. It is difficult to close the mass balances accurately because of the complexity of the degradation products (Garlock et al., 2011). In addition, combined severity (CS), a function of reaction time, temperature and pH, is usually applied to evaluate dilute sulfuric acid pretreatment (Lloyd and Wyman, 2005; Kabel et al., 2007). The combined severity was defined as (Overend and Chornet, 1987; Chum et al., 1990):
CS ¼ log R0 pH; R0 ¼ t exp½ðT H T R Þ=x
ð1Þ ð2Þ
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In which t is reaction time in minutes, TH is the hydrolysis temperature in °C, and TR is a base temperature, usually set at 100 °C, and x is a fitting parameter, which in most studies is assigned the value of 14.75. Eqs. (1) and (2) were also modified for use with sodium hydroxide pretreatments (Silverstein et al., 2007). It is worth considering whether the above equations adequately describe pretreatment using different organic acids. In the present study, four organic acids, including acetic acid, oxalic acid, tartaric acid and citric acid, were used in pretreatment, in consideration of their low price and different acidities. Mass balances and the combined severity metric were used to compare the effects of different dilute organic acids in pretreatment. 2. Methods 2.1. Raw materials Corn stover was from the suburbs of Tianjin City, China. The corn stover was stripped from the leaves, air-dried and milled. The portion of particles smaller than 0.84 mm was separated by screening. The resulting ground biomass was air-dried until the moisture content was between 5% and 10% based on the total weight and then stored in sealed bags at ambient room temperature until further use. 2.2. Pretreatment with different dilute acids Pretreatment was performed with five different acids, including sulfuric acid, acetic acid, oxalic acid, tartaric acid and citric acid. In order to compare these pretreatments with different acids, uniform molar concentrations of 50 and 90 mM were used, which were also within the range usually used for dilute sulfuric acid (Lloyd and Wyman, 2005; Castro et al., 2010). Milled corn stover (15 g dry matter) was mixed with the dilute acid solution to a final weight of 150 g, and the resulting acid concentration was 50 and 90 mM (The volume of mixture was taken to be 150 mL). The mixture was then transferred to a 316 stainless steel reactor (inner height diameter is 220.0 35.0 mm, 5.0 mm wall) capped on both ends with 316 stainless steel caps. Reactors were heated in an oil bath filled with dimethyl silicon oil. Pretreatment was performed at 130, 150, 170 and 190 °C. Reactors were plunged into the preheated oil at the desired temperature. The heating-up process in these reactors was monitored by thermocouples. After 30 min, the reactors were cooled to room temperature by quenching in ice water. The pretreatments were performed in duplicates. The pretreated slurry was pressed through eight-layer filter cloth to separate free liquid from hydrolyzed solids. The pH of the resulting liquid hydrolysate was determined at room temperature. Then the hydrolysate was collected and stored at 20 °C for component analysis. The solid fraction was washed with about 750 mL deionized water. Washed stream from solid fraction was also separated by filtration. The component of washed stream was also determined. The solid fraction was dried at room temperature until the moisture content (based on the total weight) was not more than 10%. 2.3. Analytical methods The National Renewable Energy Laboratory (NREL) laboratory analysis protocol (LAP) was followed to determine the composition of untreated and pretreated biomass (NREL, 2004). The untreated corn stover raw material consisted of glucan 36.1%, xylan 20.7%, arabinan 2.8%, acid insoluble lignin 18.6% and extractives 18.3%. Glucose, xylose, acetic acid, hydroxymethylfurfural (HMF) and furfural in the hydrolysate were analyzed by high performance
liquid chromatography (HPLC) following LAP-015 from NREL. The HPLC system consists of an Aminex HPX-87H organic acid column (Bio-rad, Hercules, CA), a Waters (Milford, MA) 1515 pump, a Waters column heater module and a Waters 2414 refractive index detector. The mobile phase was 5 mM sulfuric acid, deionized water filtered through 0.22 lm filters. Operating conditions for the HPLC column were 60 °C with a mobile phase flow rate of 0.6 mL/min. For sample analysis, 10 lL of sample was injected and complete sample elution could be accomplished within 48 min. An external standard consisting of glucose, xylose, acetic acid, HMF and furfural was used for quantification. Oligomeric sugars in the hydrolysate and washed solids stream were acid hydrolyzed to analyze these oligomeric sugars, following LAP-014 from NREL. Standards were prepared to calculate the percent recovery of monosaccharides. 2.4. Calculation Solids recovery was calculated as a percentage of the total solids recovered after pretreatment based on the initial sample (dry weight). The closures of mass balance for glucan and xylan undergoing dilute acid pretreatment were determined. Polymers, oligomers, monomers and degradation products should provide close to 100% mass balance closure according to the degradation pathways. However, not all degradation products were measured, only HMF and furfural. The equations for xylan mass balance were expressed as:
Xylan recovery ð%Þ ¼
Xylan in pretreated solid ðgÞ 100% Xylan in untreated solid ðgÞ
Xylooligomer yield ð%Þ ¼
Xylooligomer in hydrolysate ðgÞ Xylan in untreated solid ðgÞ 100%
Xylose yield ð%Þ ¼
ð3Þ
ð4Þ
Xylose in hydrolysate ðgÞ 132 Xylan in untreated solid ðgÞ 150 100%
Furfural yield ð%Þ ¼
ð5Þ
Furfural in hydrolysate ðgÞ 132 Xylan in untreated solid ðgÞ 96 100%
ð6Þ
The xylose and furfural are converted to xylan equivalent by multiplying the ratio of the molecular weights of xylose to xylan (132/150) and furfural to xylan (132/96). Xylooligomer is reported in xylan equivalents. The equations for glucan were similar to xylan except the ratios for glucose to glucan and HMF to glucan are (162/180) and (162/126), respectively. 3. Results and discussion 3.1. Composition of the pretreated solids and hydrolysates Pretreated solid recovery, solid compositions and hydrolysate components were variable between different acid pretreatments (Table 1). Solid recoveries are different for different acids. The lowest and highest solid recovery was obtained after sulfuric and acetic acid pretreatment at any condition, respectively. For every acid, increased temperature and increased acid concentration decreased the solid recovery, indicating that harsh conditions increased the biomass conversion into dissoluble compounds in pretreatment. The xylan content obviously decreased along with increased temperature and acid concentration. This result is similar to previous
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Table 1 Compositions of solid fractions and components of hydrolysates for different dilute acid pretreatments. All data are mean values of two separate experiments. Standard deviations are given between brackets. Factors
Xylan
Lignin
Glucose Xylose
Acetic acida HMF
Furfural
pHb
130/50
82.2 86.7 86.3 88.3 88.7 67.3 71.5 79.0 80.1 86.4 60.3 61.8 65.8 65.7 77.3 54.0 57.1 57.8 58.2 61.9 70.6 75.2 86.1 86.3 88.7 59.5 61.9 71.6 74.0 83.8 54.9 57.0 60.6 60.7 74.0 49.7 54.7 52.8 55.8 60.5
20.9 (0.7) 23.2 (0.7) 22.4 (1.9) 22.9 (0.5) 23.0 (0.7) 14.9 (0.9) 16.5 (1.6) 21.0 (0) 21.8 (0.6) 22.9 (0.7) 9.2 (2.1) 10.1 (1.8) 13.1 (0.8) 14.4 (0.3) 22.1 (1.5) 5.0 (0.4) 6.8 (2.2) 8.4 (0.5) 9.2 (0.8) 17.5 (0.4) 16.6 (0.4) 17.7 (1.8) 22.7 (0.6) 22.1 (0.2) 22.8 (0.5) 8.2 (1.4) 10.9 (1.1) 18.4 (0.2) 19.5 (1.4) 23.2 (1.4) 5.1 (0.6) 4.9 (0.5) 6.3 (1.8) 8.5 (1.9) 20.3 (1.3) 0 (0) 0 (0) 0.6 (0.4) 1.0 (0.3) 15.6 (0)
23.6 (0.5) 22.8 (0.1) 21.1 (0.5) 22.3 (0.8) 21.1 (1.1) 26.9 (0.1) 26.3 (2.0) 24.3 (1.1) 23.9 (0.8) 21.5 (0.3) 32.1 (0.2) 26.8 (1.7) 30.5 (0.2) 25.1 (0.6) 24.2 (0.6) 31.7 (0.9) 29.5 (1.2) 29.8 (1.4) 28.9 (0.2) 26.9 (1.0) 25.3 (2.1) 23.2 (0.8) 21.8 (0.7) 22.0 (0.5) 20.1 (1.8) 29.5 (1.7) 26.9 (0) 23.1 (0.4) 23.7 (1.1) 21.6 (2.4) 32.3 (1.2) 29.5 (1.2) 27.8 (0.5) 29.0 (0.2) 23.2 (2.0) 33.2 (0.3) 31.7 (1.2) 30.3 (1.1) 30.9 (1.4) 27.4 (1.3)
1.2 (0.2) 1.1 (0) 1.0 (0) 1.1 (0.1) 1.1 (0.1) 1.8 (0.4) 1.5 (0.3) 1.2 (0.1) 1.2 (0.1) 1.3 (0.1) 2.6 (0) 2.1 (0.1) 1.1 (0.2) 1.2 (0.2) 0.9 (0.2) 4.0 (1.1) 2.3 (0.8) 2.1 (0.2) 2.2 (0.1) 0.3 (0.4) 1.4 (0.1) 1.2 (0.2) 1.1 (0) 1.2 (0.2) 1.1 (0.2) 2.7 (0) 2.2 (0.3) 1.3 (0.1) 1.2 (0.1) 1.1 (0) 4.1 (0.1) 3.0 (0.2) 1.4 (0.1) 1.4 (0.2) 0.8 (0.1) 5.9 (0.1) 3.0 (0.3) 2.4 (0.3) 1.8 (0.2) 0.1 (0.1)
0.5 0.3 0.3 0.2 3.3 1.6 1.1 0.5 0.3 3.6 3.3 2.3 1.3 0.9 4.0 4.3 3.7 3.1 3.3 5.5 1.7 0.9 0.4 0.3 5.9 3.5 2.6 0.8 0.4 6.5 3.0 3.0 1.8 1.2 6.1 5.5 4.2 4.2 4.7 7.6
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0.2 (0.1) 0.1 (0) 0 (0) 0 (0) 0 (0) 1.6 (0.5) 1.1 (0.4) 0.7 (0.1) 0.7 (0.5) 0.1 (0) 6.7 (1.1) 5.4 (0.6) 5.9 (1.3) 5.3 (0.5) 1.5 (0.3) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0.7 (0.1) 0.3 (0.1) 0.1 (0) 0.1 (0) 0 (0) 3.8 (0.9) 1.8 (0.4) 1.2 (0) 1.4 (0.5) 0.1 (0) 11.5 (0.5) 8.4 (0) 9.2 (1.0) 8.9 (0.3) 3.0 (0.1)
1.5 2.2 3.1 3.0 4.3 1.5 2.2 3.2 3.0 4.3 1.5 2.4 3.1 2.9 4.1 1.5 2.8 3.0 2.9 3.7 1.2 1.6 2.8 2.6 4.1 1.2 1.6 2.8 2.6 4.1 1.3 1.9 2.8 2.6 4.0 1.2 2.3 2.8 2.3 3.6
170/50
190/50
130/90
150/90
170/90
190/90
a
Components of hydrolysates (g/L)
Solid recovery Glucan
150/50
b
Compositions of solid fractions (%)
Temperature (°C)/acid concentration (mM) Acid type Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
(2.2) (3.6) (2.4) (3.3) (2.3) (0.4) (0.2) (3.5) (3.0) (2.2) (4.1) (3.7) (2.5) (4.7) (4.1) (2.0) (2.6) (2.5) (1.8) (2.1) (0.7) (1.3) (1.7) (1.4) (0.4) (0.9) (0.9) (1.3) (1.1) (0.3) (0.3) (0.9) (0.2) (0.7) (0.6) (2.7) (0.8) (1.6) (1.4) (0.5)
42.9 (0.7) 42.2 (1.6) 41.1 (0.1) 40.8 (0.6) 41.0 (0.3) 52.6 (1.5) 49.3 (1.0) 44.1 (1.9) 43.0 (1.2) 39.6 (0.8) 55.0 (5.9) 53.2 (3.2) 53.5 (0.4) 52.2 (0.8) 43.9 (1.9) 58.1 (3.1) 56.4 (2.6) 57.3 (1.5) 57.2 (1.6) 53.6 (4.7) 48.6 (1.0) 43.9 (3.7) 40.0 (0.7) 40.9 (0.7) 39.4 (1.8) 55.8 (0.1) 55.2 (2.6) 48.4 (1.2) 46.8 (1.1) 40.8 (0.6) 57.3 (4.0) 54.9 (1.7) 54.5 (1.8) 56.3 (1.6) 45.7 (1.4) 56.9 (1.9) 56.9 (0.2) 58.6 (0.5) 58.5 (0.3) 54.6 (1.5)
3.0 (0.7) 1.9 (0.4) 2.3 (0.1) 1.7 (0.2) 1.5 (0) 11.5 (1.5) 7.9 (1.1) 5.8 (1.8) 2.1 (0.1) 1.6 (0) 16.9 (0.1) 14.2 (1.2) 7.6 (0.5) 6.7 (2.3) 1.2 (0) 9.6 (0.1) 10.4 (1.0) 8.2 (0.8) 7.9 (0.9) 1.4 (0.5) 10.0 (0.4) 5.4 (0.9) 1.4 (0.2) 1.7 (0) 1.5 (0.1) 18.9 (0.5) 16.0 (1.5) 2.9 (0.2) 3.0 (0.5) 1.5 (0) 14.2 (0.8) 18.7 (0.4) 12.1 (1.2) 12.1 (1.3) 1.2 (0.2) 4.1 (0.3) 11.6 (1.2) 7.3 (0.5) 9.2 (0.6) 1.9 (0.1)
(0.2) (0.2) (0.2) (0.1) (0.4) (0.2) (0.3) (0.4) (0.2) (0.5) (0.3) (0.5) (0.4) (0.2) (0.2) (0.8) (0.4) (0.4) (0.6) (0.1) (0.2) (0.2) (0.3) (0.1) (0.3) (0) (0.3) (0.4) (0.1) (0.3) (0.4) (0.3) (1.0) (0.1) (1.0) (0.5) (0.6) (0.2) (0.2) (0.4)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0.3 (0.1) 0.2 (0) 0.1 (0) 0.2 (0) 0.1 (0) 0.9 (0.4) 0.3 (0.1) 0.4 (0.1) 0.4 (0) 0.1 (0) 1.2 (0.2) 1.1 (0.3) 1.1 (0.2) 1.2 (0) 0.4 (0.2) 0.2 (0) 0 (0.1) 0 (0) 0 (0) 0 (0) 0.6 (0.1) 0.4 (0.1) 0.2 (0) 0.2 (0) 0.1 (0) 0.7 (0) 0.7 (0.1) 0.5 (0) 0.6 (0.1) 0.1 (0) 2.7 (0.3) 1.5 (0.1) 1.5 (0.1) 1.5 (0.2) 0.2 (0)
(0) (0.1) (0.2) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (0) (0.1) (0) (0) (0) (0.1) (0.1) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0.1) (0) (0.1) (0.2) (0) (0) (0.1) (0) (0) (0)
For acetic acid pretreatment, the acetic acid concentrations are determined as the total acetic acid including initial part and production part. The pH values of hydrolysates were measured at room temperature.
studies (Cara et al., 2008). The residual xylan content was less than 10% when temperatures exceeded 170 °C (down from 20.7% of the raw material), and even approached zero at 190 °C/90 mM after sulfuric and oxalic acid pretreatment. For acetic acid pretreatment, xylan content scarcely changed, and was still 15.6% at 190 °C/ 90 mM. Pretreatments with organic acid resulted in lower glucan content and higher xylan content in pretreated solids than sulfuric acid at the same condition. The lignin contents in pretreated solid increased somewhat with the increased temperature and acid concentration due to lost xylan. In pretreatment hydrolysates, monosaccharide, oligosaccharide and degradation product concentrations were determined (Table 1). The glucose concentrations were almost constant, around 2 g/ L for citric, tartaric and acetic acid pretreatment, but a little higher for sulfuric and oxalic acid pretreatment. Xylose concentrations ranged from 1.2 to 18.9 g/L, and it was higher after sulfuric and oxalic acid pretreatment than that after other pretreatments. The above observations indicate that pretreatments with organic acid were less efficient in releasing glucose and xylose due to lower concentration of H+ ions formation in liquor, because the release of glucose and xylose is catalyzed by acid. Acetic acid was produced during degradation of hemicellulose and lignin (Palmqvist and Hahn-Hagerdal, 2000), and its content in hydrolysates increased with increased temperature and acid
concentration (Table 1). Acetic acid pretreatment led to a higher acetic acid concentration including the acid added to initiate pretreatment. The acetic acid concentrations in hydrolysates except acetic acid pretreatment hardly inhibit fermentation using tolerant yeast (Ding et al., 2011). HMF rarely appeared below 170 °C for all the acidic pretreatments, and its concentration was always less than 1.0 g/L. When the pretreatment temperature was 190 °C, HMF concentration exceeded 1.0 g/L. The trend of furfural generation during pretreatment was similar to that of HMF, and the maximum concentration of furfural reached 11.5 g/L after sulfuric acid pretreatment at 190 °C. It is reported that the formation of HMF and furfural is promoted by temperature and H+ ion (Mamman et al., 2008). As a result, organic acid pretreatment led to lower HMF and furfural concentration than sulfuric acid at the same condition (Table 1). Furfural and HMF concentration exceeding 1.3 g/L were unexpected (Ding et al., 2011), due to the significant inhibition to fermentation and the loss of xylose. It implies that pretreatment with organic acid perhaps led to better fermentability due to lower inhibitor concentration. 3.2. Mass balance closures for glucan, xylan and lignin The glucan and xylan degradation process has been mentioned in several reports (Palmqvist and Hahn-Hagerdal, 2000; Karimi
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et al., 2006). Hence, the mass balance for glucan and xylan from beginning to end was available and significant to evaluate the pretreatment effect. Glucan recovery, glucooligomer yield, glucose yield and HMF yield after different dilute acid pretreatments were determined for glucan balance closure (Table 2). Glucan balance closure for all conditions was close to 100%, confirming this method’s reliability. Glucan recoveries were above 90% under most conditions due to the low solubility of glucan, except for some harsher conditions, such as 190 °C/90 mM. Glucooligomer and glucose yields were less than 10% under most conditions. HMF yields were also very low. Glucose and HMF yields were 13.1% and 7.8% by sulfuric acid pretreatment at 190 °C/90 mM, which were the highest achieved; about twofold more than achieved by oxalic acid pretreatment. It is reported that the degradations of glucose and xylose with organic acid pretreatment were less than sulfuric acid due to the higher activation energy for degradations with organic acid (Lu and Mosier, 2007). However, the degradations of glucan and xylan with organic acid seemed less as well. Xylan recovery and xylan-products yield were highly variable between the different pretreatment conditions (Fig. 1). In contrast to cellulose, hemicellulose was apt to be hydrolyzed during acid pretreatment. Xylan recovery after any acid pretreatment decreased along with increased temperature and acid concentration.
Xylan recoveries with different acid pretreatments under the same condition increased according to the order of sulfuric < oxalic < citric < tartaric < acetic acid. Xylan mainly remained in the solid fraction after acetic acid pretreatment, indicating dilute acetic acid is too weak to degrade xylan to xylose. Removal of hemicellulose increased the mean pore size of the biomass, therefore increased the accessibility of enzyme to glucan, which was an important feature and the mechanism of action in acid pretreatment (Alvira et al., 2010). Excess xylan remaining in solid indicates the lack of pretreatment strength which led to a low enzymatic efficiency in enzymatic hydrolysis (Cara et al., 2008). At the same condition, organic acid pretreatment induced to lower furfural yield than sulfuric acid pretreatment, which agreed with previous report (Kootstra et al., 2009b). Other component yields varied in more complicated patterns than did xylan. For 50 mM acid at 130 °C, xylan recovery after sulfuric acid pretreatment was 83.2%, lower than that following pretreatment with organic acids, which was more than 90%. At 150 °C, the xylose yield was 43.4% after sulfuric acid pretreatment, and after other acid pretreatments, decreased according to the order of oxalic, citric, tartaric and acetic acid. At 170 °C, xylooligomer yields decreased after sulfuric and oxalic acid pretreatment, and increased after citric, tartaric and acetic acid pretreatment than that at 150 °C; xylose
Table 2 Glucan recovery, glucooligomer yield, glucose yield and HMF yield for different dilute acid pretreatments. All data are mean values of two separate experiments. Standard deviations are given between brackets. Temperature (°C)/acid concentration (mM)
Acid type
Glucan recovery (%)
Glucooligomer yield (%)
Glucose yield (%)
HMF yield (%)
Total closure (%)
130/50
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
97.8 (4.3) 101.5 (0.4) 98.3 (3.1) 99.8 (5.2) 100.9 (3.4)
1.6 0.3 0.2 0.6 0.1
(1.5) (0.5) (0.3) (0.8) (0.2)
2.2 1.8 2.0 1.7 1.9
(0.6) (0.2) (0.1) (0.6) (0.4)
0.1 (0.1) 0 (0) 0 (0) 0 (0) 0 (0)
101.7 103.7 100.5 102.1 102.9
150/50
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
98.3 97.7 96.6 95.5 94.8
(3.4) (2.3) (0.1) (1.0) (4.4)
1.1 1.5 3.6 1.8 0.9
(1.2) (1.0) (3.5) (0.8) (0.8)
3.6 3.0 2.2 2.3 2.4
(1.0) (0.6) (0.2) (0.2) (0.4)
0.7 0.3 0.2 0.3 0.1
(0.3) (0) (0) (0) (0)
103.8 (3.8) 102.5 (2.5) 102.6 (3.5) 99.9 (1.3) 98.1 (4.5)
170/50
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
92.0 91.2 97.7 95.1 94.1
(3.2) (0.4) (3.0) (8.4) (1.2)
0.9 2.3 1.1 1.6 2.1
(0) (0.7) (0.7) (1.0) (0.3)
5.9 4.4 2.5 2.5 1.8
(0.4) (0.3) (0.6) (0.6) (0.7)
2.4 1.0 1.2 1.3 0.2
(0.6) (0.1) (0.1) (0.1) (0)
101.2 (3.3) 98.8 (0.8) 102.4 (3.1) 100.5 (8.4) 98.3 (1.4)
190/50
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
87.0 89.3 91.9 92.4 92.0
(1.1) (0.1) (6.4) (5.4) (4.8)
0.6 3.1 1.2 0.5 1.6
(0.9) (1.6) (1.0) (0.7) (1.2)
8.2 4.6 5.0 4.9 0.7
(2.1) (1.7) (0) (0.9) (0.8)
3.1 2.8 3.0 3.1 0.8
(0.4) (0.5) (0.1) (0) (0.4)
98.9 (2.5) 99.9 (2.4) 101.0 (6.5) 100.9 (5.5) 95.1 (5.0)
130/90
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
95.1 91.5 95.6 97.8 96.8
(1.0) (9.3) (3.5) (0.2) (4.9)
1.0 1.5 0.2 1.0 0.5
(1.0) (0.8) (0.2) (0.4) (0.6)
2.9 2.4 2.2 2.2 2.2
(0.4) (0.3) (0.4) (0.4) (0.4)
0.4 (0.1) 0.1 (0.1) 0 (0) 0 (0) 0 (0)
99.4 (1.5) 95.4 (9.4) 97.9 (3.6) 101.1 (0.6) 99.4 (5.0)
150/90
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
92.0 94.6 96.1 96.1 94.8
(1.3) (3.2) (4.1) (3.7) (1.8)
0.2 0.6 0.6 1.8 0.7
(0.2) (0.4) (0.8) (0.1) (0.5)
5.8 4.5 2.6 2.3 2.1
(0.1) (0.8) (0.5) (0.3) (0.1)
1.9 1.0 0.4 0.4 0.1
(0.2) (0.2) (0) (0.2) (0)
99.9 (1.4) 100.7 (3.3) 99.7 (4.2) 100.7 (3.7) 97.7 (1.9)
170/90
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
87.3 86.7 91.5 94.8 93.8
(7.1) (1.3) (2.7) (3.8) (3.6)
0 (0) 0 (0) 0.9 (1.0) 1.5 (0.5) 1.7 (0.3)
8.9 6.5 2.8 2.9 1.5
(0.3) (0.6) (0.1) (0.4) (0.2)
2.2 1.9 1.6 1.8 0.3
(0) (0.2) (0) (0.1) (0)
98.3 (7.1) 95.1 (1.5) 96.8 (2.9) 101.0 (3.8) 97.3 (3.6)
190/90
Sulfuric acid Oxalic acid Citric acid Tartaric acid Acetic acid
78.4 86.5 85.8 90.5 91.7
(4.6) (1.6) (3.4) (2.6) (3.3)
0 (0) 0 (0) 0 (0) 0 (0) 1.0 (0.1)
13.1 (0.2) 6.2 (0.6) 5.1 (0.5) 4.5 (0.3) 0.2 (0.1)
7.8 3.8 3.6 3.7 0.7
(0.7) (0.2) (0.3) (0.4) (0)
99.2 96.4 94.6 98.6 93.6
(4.5) (0.7) (3.1) (5.3) (3.4)
(4.7) (1.7) (3.4) (2.7) (3.3)
L. Qin et al. / Bioresource Technology 112 (2012) 319–326
323
Fig. 1. Xylan balance for different dilute acid pretreatments. Xylan recovery, xylooligomer yield, xylose yield and furfural yield are presented together to close to 100%, for the calculation bases of which are all initial xylan. Error bar represents the standard deviation of duplicate experiments. XO: xylooligomer, SA: sulfuric acid, OA: oxalic acid, CA: citric acid, TA: tartaric acid, AA: acetic acid.
yields increased about 20% after sulfuric, oxalic and tartaric acid pretreatment than that at 150 °C. At 190 °C, xylose yields decreased by 26.9% and 14.2% after sulfuric and oxalic acid pretreatment and increased a little after citric and tartaric acid pretreatment from 170 °C; furfural yields accounted for 30–40% after most acid pretreatments except acetic acid pretreatment. For 90 mM acid, xylose yields went up to 71.3% and 61.0% after sulfuric and oxalic acid pretreatment at 150 °C, respectively. Xylooligomer yields came up to 27.3% and 16.8% after citric and tartaric acid pretreatment, but reduced to zero and 4.8% after sulfuric and oxalic acid pretreatment from 130 °C, respectively. Similar trend was observed when the condition was changed from 150 to 170 °C at 50 mM acid, which indicated that a similar yield can be achieved at low temperature-high acid concentration or high temperature-low acid concentration (Lloyd and Wyman, 2005). At 170 °C, xylose yield came down to 53.5% after sulfuric acid pretreatment, and went up to 71.3%, 48.6% and 47.0% after oxalic, citric and tartaric acid pretreatment, respectively; xylooligomer yield after acetic acid pretreatment increased to 20.2%. At 190 °C, furfural yield was 73.1% after sulfuric acid pretreatment and around 50% after oxalic, citric and tartaric acid pretreatment; xylose yield obviously decreased after any pretreatment. It is observed that the maximum xylose yield was obtained at 150 °C/90 mM with sulfuric acid and 170 °C/90 mM with oxalic acid. However, the total yield of xylose and xylooligomer with different acids was equivalent, which can reach 72.3%, 71.3%, 69.2% and 63.1% at 170 °C/90 mM with oxalic, sulfuric, citric and tartaric acid, respectively. Xylooligomer production was taking into account, because it can be hydrolyzed to xylose in subsequent enzymatic hydrolysis. The relationship between the yield of xylose plus xylooligomer and the xylan recovery was linear dependence in the xylan recovery range of 30–100% after any acid pretreatment (Fig. 2), which indicated that no furfural or other degradation product generated within this range. The optimal pretreatment condition should enable the xylan recovery around 20%. At this critical point, the total soluble sugar yield reached the maximum and the degradation product just produced (Fig. 2). In addition, these linear relationships had the similar function with different acids, which indicated that the release of total soluble sugar was not affected by acid species. But the proportion of xylose and
Fig. 2. Total yield of xylose and xylooligomer as a function of xylan recovery for different acid pretreatments at 50/90 mM acid concentration and 130–190 °C.
xylooligomer in total soluble sugar was affected by acid species mainly due to the H+ ion concentration. In general, organic acid pretreatment obtained an equivalent xylan recovery and soluble sugar yield to sulfuric acid, and produced more xylooligomer and less furfural except acetic acid pretreatment in this study when temperature and acid concentration was over 170 °C and 90 mM. It is noticeable that the xylan balance closures were not close to 100% at 190 °C under both 50 and 90 mM acid concentration, which indicated some degradation products not determined generated from furfural. Formic acid and furfural resinification may generate from furfural in pretreatment, which was reported previously (Palmqvist and Hahn-Hagerdal, 2000; Karimi et al., 2006). Acid insoluble lignin recoveries for different pretreatments scarcely changed, which were around 100% (Fig. 3). Acid soluble lignin was ignored in this study due to its small amount. This result agreed with many reports that lignin can melt in acid pretreatment due to high temperatures and redeposit as spherical droplets on the residual surfaces unless there is post-washing with hot water
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Fig. 3. Acid insoluble lignin recovery for different dilute acid pretreatments. Error bar represents the standard deviation of duplicate experiments. SA: sulfuric acid, OA: oxalic acid, CA: citric acid, TA: tartaric acid, AA: acetic acid.
or organic solvent (Selig et al., 2007; Garlock et al., 2011). In this study, pretreated solids were washed with cold water, which was not helpful for lignin removal. 3.3. Effects of combined severity on different acid pretreatments In order to investigate the effects of pretreatment parameters on the component conversion, the combined severity based on Eqs. (1) and (2) was employed to describe the pretreatment condition. The combined severity factor has been plotted against the xylan recovery, xylooligomer yield, xylose yield and furfural yield after pretreatment (Fig. 4). The trends of xylan recovery, xylose yield and furfural yield versus combined severity were consistent with previous reports (Larsson et al., 1999; Kabel et al., 2007). The trend of xylooligomer yield has seldom been reported. The function of the recovery or yield with different acid pretreatments versus combined severity had the same form (e.g. monotonic for xylan recovery and furfural yield, non-monotonic for xylooligomer yield and xylose yield), but the parameters in the function of different acid pretreatments were not the same. Namely, the recovery or yield trends for different acid pretreatments didn’t coincide. This result seems reasonable, because combined severity is a parameter comprised of temperature, pH and time, but not acid species. The difference of the functions among different acid pretreatments was from the acid dissociation constant. The effects of temperature on pH are negligible for strong acid (i.e. sulfuric acid) due to complete ionization at any temperature. In contrast, the pH of a weak acid obviously decreases along the increased temperature due to the heat-needed ionization process (Henderson–Hasselbalch equation; Palmqvist and Hahn-Hagerdal, 2000). The pH of organic acid is much lower at higher temperature than that determined at room temperature. Therefore, a higher pH than the real pH during the pretreatment reaction was used to calculate the combined severity of weak acid. As a result, the combined severity without modified by acid dissociation constant was applied before a novel parameter was invented. The drift degree of combined severities along x-axis in Fig.4 for different acid pretreatments was in accordance with the pKa value, the negative logarithm of dissociation constant (these five acid pKa values at ordinary temperature is sulfuric acid-3, oxalic acid 1.27, tartaric acid 2.98, citric acid 3.13, acetic acid 4.76).
An evident peak value existed in the function of xylooligomer and xylose yield versus combined severity after pretreatment with specific acid. The peak values of xylooligomer yields for different acid pretreatments were distinct and in the order of acetic acid > citric acid > tartaric acid > oxalic acid > sulfuric acid (Fig. 4B). In contrast, the peak values of xylose yield were in the opposite order as: sulfuric and oxalic acid > tartaric and citric acid > acetic acid, which paralleled the order of pKa of each acid as well (Fig. 4C). These regular variations indicated that the maximum value of xylose yield and xylooligomer yield for a specific acid pretreatment was determined by acid pKa value. Furfural was generated at a specific combined severity and sharply rose for each acid pretreatment (Fig. 4D). Furfural yield also should have peak value at a harsher combined severity than that used in this study, which was not desired during pretreatment (Larsson et al., 1999). Another significant point in Fig. 4B and C was the intersection point of the trends of any two acid pretreatments, which represented the pretreatments with the same combined severity and the same xylooligomer or xylose yield. When the combined severity was higher than this point, the xylose yield of pretreatment with stronger acid was higher than that with weaker acid, and vice versa. Xylooligomer yield ran contrary pattern to xylose yield as expected. This model indicated that a weaker acid pretreatment can never result a high xylose yield even if pretreatment temperature or acid concentration was increased. Pretreatment with maleic and fumaric acid was also investigated in previous study (Kootstra et al., 2009b). Maximum xylose yields were about 70% and 45% after pretreatment with maleic and fumaric acid at 50 mM and 170 °C, respectively. The pKa value of maleic and fumaric acid is 1.92 and 3.03, which was agreed with the relationship between pKa and xylose yield observed in this study. According to the result in previous and current study, high xylose yield would be obtained using the acid with pKa < 2.0 after pretreatment, such as oxalic acid and maleic acid. In contrast, using organic acids with a higher pKa value can never reach a high xylose yield. In previous study, sulfuric acid and acetic acid were both added to pH = 1.0 during biomass pretreatment, but xylose yield after acetic acid pretreatment was nearly half of that after sulfuric acid pretreatment (Pedersen et al., 2010).
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Fig. 4. Combined severity versus xylan recovery (A), xylooligomer yield (B), xylose yield (C) and furfural yield (D). Combined severity (CS) was defined by Eqs. (1) and (2). The pH of each acid was measured at ambient temperature. The two duplicate pretreatment data were both shown in the figure, and were not replaced by an average.
4. Conclusion In conclusion, organic acid pretreatment was milder than sulfuric acid due to the sustained release of H+ ions. Organic acid pretreatment resulted in higher xylan content in pretreated solids and lower xylose concentration in pretreated hydrolysate than sulfuric acid at the same condition. However, organic acid pretreatment produced more xylooligomer and less furfural. The dissociation constant decided the maximum xylooligomer and xylose yield. Although a high pKa acid pretreatment can never lead high xylose yield, a roughly equivalent soluble sugar yield was obtained.
Acknowledgements The authors are grateful for the financial support by Major International Joint Research Project (21020102040) from the National Natural Science Foundation of China, the National Basic Research Program of China (‘‘973’’ Program: 2011CBA00802, 2007CB714301) from Ministry of Science and Technology of China, Key Program (20736006) from the National Natural Science Foundation of China.
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