Bioresource Technology 176 (2015) 175–180
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Liquid hot water pretreatment on different parts of cotton stalk to facilitate ethanol production Wei Jiang a,b, Senlin Chang a,b, Hongqiang Li a, Piotr Oleskowicz-Popiel c, Jian Xu a,⇑ a
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China c Poznan University of Technology, Faculty of Civil and Environmental Engineering, Institute of Environmental Engineering, Berdychowo 4, 60-965 Poznan, Poland b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Cotton stalk were separated into
stem, branch, boll shell for ethanol production. Hydrothermal pretreatment were optimized for different parts of cotton stalk. Boll shell was found to be more sensitive to pretreatment condition than stem. Ratio of ethanol yield to pretreatment energy for different parts was different.
a r t i c l e
i n f o
Article history: Received 25 September 2014 Received in revised form 6 November 2014 Accepted 8 November 2014 Available online 15 November 2014 Keywords: Cotton stalk Liquid hot water pretreatment Simultaneous saccharification and fermentation Bioethanol Energy input
a b s t r a c t To investigate pretreatment demand for different parts of biomass, cotton stalk was separated into stem, branch and boll shell, which were treated by liquid hot water pretreatment (LHWP) with severity from 2.77 to 4.42. Based on weight loss (WL, w/w) mainly caused by hemicellulose removal, it was found that boll shell (WL, 46.93%) was more sensitive to LHWP than stem (WL, 38.85%). Although ethanol yield of 18.3, 16.27 and 21.08 g/100 g was achieved from stem, branch and boll shell with pretreatment severity at 4.42, ratio of ethanol yield to pretreatment energy input for particular parts was different. For boll shell and branch, the maximum ratio of ethanol yield to energy input were 1.37 and 1.33 g ethanol kJ1 with severity at 4.34, while it was 1.20 for stem at 3.66. This indicates that different pretreatment demands for different parts of plants should be considered in order to save pretreatment energy input. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction As fossil fuel has currently become the major resource to meet the growing energy demand, it has caused economic, environmental and social sustainability problems (Kaygusuz, 2012). Biofuel has been considered as a strategic alternative energy to reduce the dependence on non-renewable resources (Buruiana et al., 2014). Unlike non-renewable energy only from fossil fuels, bioethanol ⇑ Corresponding author. Tel./fax: +86 10 82544852. E-mail addresses: [email protected], [email protected] (J. Xu). http://dx.doi.org/10.1016/j.biortech.2014.11.023 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
can be obtained either through fermentation of sugars from renewable biomass materials or by synthesis (Sun and Cheng, 2002). As one of the renewable resources, biomass has huge potential to meet the energy needs for modern society, especially in industrialized and developing countries (Ong et al., 2011). As a by-product of cotton industry, the annual yield of cotton stalk (CS) is about 40 million tons in China (Du et al., 2013). However, nowadays they are utilized with low efficiency by direct combustion or as livestock feed and compost (Watanabe et al., 2012). In CS, the content of nutrients such as protein is extremely low, while the cellulose content can get to 32–46% (Zhou et al.,
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2010). This makes CS a potential biomass feedstock for bioethanol production (Binod et al., 2012; Kaur et al., 2012). Nonetheless, natural CS is extremely difficult to be utilized due to its complicated structure. To improve the enzymatic digestibility and the subsequent fermentation of CS, pretreatment to break down its structure properly is indispensable (Pienkos and Zhang, 2009). Schell et al. (2003) applied sulfuric acid, hydrochloric acid and nitric acid to pretreat CS and found that acid pretreatment could remove the hemicellulose component significantly. More cellulose was therefore exposed, which contributed to enhancement of the subsequent enzymatic hydrolysis and fermentation. Alkali pretreatment on CS was investigated by Chang and Holtzapple (2000). It was observed that alkali was more effective on herbaceous crops than for hardwood material. Gould (1985) found that hydrogen peroxide pretreatment could reduce cellulose crystallinity by oxidative degradation, which ultimately resulted in improved enzymatic conversion of CS. By adopting ozone to pretreat CS, Neely (1984) found that there were no acid, alkali or toxic compounds formed during the pretreatment process and ozone pretreatment could be widely employed in lignin removal. Although some progress has been made, the same pretreatment conditions were adopted on the whole CS in all the investigations without any exception. The structural differences among different parts of CS were not considered, which led to the ignorance of the various demand for different part of CS. In a previous investigation with Miscanthus as feedstock (Li et al., 2013), it has been illustrated that different parts of Miscanthus had different optimal pretreatment condition. In the present study, CS was separated into three parts: stem, branch and boll shell, which was pretreated respectively. Liquid hot water pretreatment (LHWP) has been widely used due to its unique advantages such as no additive chemicals requirement, no special demand for non-corrosive reactor, and lower toxic compounds formation (Li et al., 2014, 2013; Xu et al., 2010a,b). The aim of this investigation was to optimize pretreatment conditions for different parts of CS in order to improve the utilization efficiency economically which would provide valuable practical experience for bioethanol production from CS.
t1: time when the reactor temperature reaches 100 °C; t2: time when the reactor temperature cooled down to 100 °C; T: reactor temperature (°C). After pretreatment, tap water was used to cool down the reactor to 80 °C. The slurry was then pumped out and filtered into two parts by vacuum filtration: hydrolyzate and solid residue. The pH and volume of hydrolyzate were measured before it was stored at 4 °C. The solid residue was dried at 60 °C and then put into sealed plastic bags for compositional analysis and simultaneous saccharification and fermentation. 2.3. Prehydrolysis and simultaneous saccharification fermentation on solid residue (SSF) Cellulase with filter paper activity (FPA) of 173.9 IU g1 was purchased from the SUKAHAN (WeiFang) Bio-Technology Company. Active dry yeasts (Angel thermal resistant ethanol active dry yeast, Product code: 800000012) were purchased from Angel Yeast Co., Ltd. Prehydrolysis was done with the ratio of liquid to solid at 8% at 50 °C, 120 rpm for 24 h and the cellulase loading was set as 15 IU/g dry matter (DM) (Xu et al., 2011). SSF was then performed at 42 °C and 120 rpm with the supplementation of 20 IU FPA g1 DM, 0.15 g yeast. The fermentation bottles were filled with N2 before fermentation locks prefilled with glycerol were equipped. Weight loss caused by CO2 release was determined at 0, 2, 4, 8, 12, 24, 36, 48 h. The conversion of cellulose to ethanol was calculated according to Eq. (2):
Cellulose conversion ¼
1:045 C L 0:51C M C C 0:9
100
ð2Þ
1.045: coefficient of CO2 conversion to ethanol; 0.51: coefficient of glucose conversion to ethanol; 0.9: coefficient of glucose to cellulose; CL: weight loss caused by CO2 release, g; CM: mass of sample used in SSF, g; CC: mass percentage of cellulose in sample, %. When the fermentation was completed, the fermentation liquor was filtered through 0.45 lm membrane, the final ethanol was determined by HPLC to evaluate the fermentation.
2. Methods
2.4. Toxic test on hydrolyzate
2.1. Feedstock preparation
Toxic tests were carried out with 15 g hydrolyzate obtained from pretreatment or water with the same amount as control, 0.3 g pure glucose, 0.024 g yeast and 30 lL urea (24 w/v). Weight loss caused by CO2 release was determined at 0, 2, 4, 8, 12, 24, 36, and 48 h. The ethanol yield was calculated based on the weight loss by multiplying 1.045, the conversion constant of CO2 to ethanol (Li et al., 2013).
CS was kindly provided by the Institute of Botany, Chinese Academy of Sciences. After harvesting in September, 2012, it was separated into 3 parts by clipper: stem, branch, and boll shell. The dry matter content was 93.60%, 93.11%, and 93.19%, respectively with the proportions of the 3 parts were about 36.16:31.17:32.67 (wt%). They were milled individually into particle size smaller than 2 mm with a laboratory miller (LXJMI-8111, Delixi Co., Ltd.). The milled samples were then stored in sealed barrels for LHWP. 2.2. Liquid hot water pretreatment A laboratory customized high pressure reactor with working volume of 1 L was used for LHWP as described in previous studies (Li et al., 2014, 2013). The solid loading was 40 g and the ratio of solid (milled stem, branch and boll shell) to water was 1:9 (w/v). The LHWP severity (PS) (Overend et al., 1987) was 2.77–4.42 calculated according to the following formula as described in a previous study (Li et al., 2013).
PS ¼
Z
t2
t1
exp
T 100 dt 14:75
ð1Þ
2.5. Analytical methods 2.5.1. Compositional analysis of solid residue The compositional analysis on the treated and untreated samples was carried out with the adapted NREL procedure (Li and Xu, 2013b; Sluiter et al., 2008). Polysaccharides were degraded into sugar monomers by two-step sulfuric acid hydrolysis: samples were treated by 72% H2SO4 in the first step at 30 °C for 60 min, followed by the second step at 121 °C for 1 h by diluting the H2SO4 concentration with Millipore water to 4%. Residual and filtrate were collected separately. The residual was dried at 105 °C overnight and the Klason lignin was calculated by subtracting the ash content from the dried residual (Sluiter et al., 2008). The amounts of sugars in the filtrate were determined by HPLC. Ash content was determined by heating the dried residual to 550 °C for 3 h. All the experiments were repeated twice.
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2.5.2. Oligosaccharides, monosaccharides and its degradation products analysis in hydrolyzate To determine the monosaccharides and its degradation products, an aliquot of the hydrolyzate was filtered through 0.45 lm membrane which was then directly analyzed by HPLC (Agilent 1260 Infinity USA). Hi-Plex H column (300 7.7 mm, Varian, Inc., Shropshire, UK) was used at 65 °C with 5% H2SO4 at a flow rate of 0.6 mL min1. Both refractive index detector (RID) and diode array detector (DAD) were employed for determining monosaccharides, glycolic acid, formic acid, acetic acid, HMF and furfural. Weak acid hydrolysis was carried out on hydrolyzate with 8% H2SO4, at 121 °C for 60 min (Li and Xu, 2013b). The oligosaccharides were quantified by the increased monosaccharides according to Eq. (3):
OS ¼ ðMSA MSB Þ 0:9
ð3Þ
OS: oligosaccharides in the hydrolyzate, mg; MSA: monosaccharides in the hydrolyzate after weak acid hydrolysis, mg; MSB: monosaccharides in the hydrolyzate before weak acid hydrolysis, mg. 3. Results and discussion 3.1. Effect of LHWP on solid recoveries The solid recoveries (w/w, %) on different parts of cotton stalk after pretreatments are presented in Table 1. It can be obviously seen that solid recoveries of stem, branch and boll shell were dramatically impacted by PS. For these three materials, the solid recoveries had a similar variation tendency which decreased as PS gradually increased. When the PS increased from 2.77 to 4.42, the solid recoveries for branch dropped from 79.34% to 56.50%. Similarly, 83.26% of the solid recovery of stem was obtained with PS of 2.77, while it was just 61.15% when the PS at 4.42 was used. For boll shell, the solid recoveries of 71.61–53.07% were observed when the PS changed from 2.27 to 4.42. After LHWP, the maximum weight losses were 43.5%, 38.85% and 46.93%, for branch, stem and boll shell, respectively. Linear relationships between PS and solid recoveries of these three materials were made and are shown in
Fig. 1. The maximum absolute value of straight line slope was observed to be 14.86 for branch, followed by 13.04 and 12.77 for stem and boll shell, respectively. This indicated that the impact of LHWP on the solid recoveries of different materials was different. The structure of boll shell was more sensitive to LHWP, while the stem was more resistant. It might be attributed to the different microstructural characteristics of the three materials. 3.2. Effect of PS on chemical composition of different parts of cotton stalk The effect of PS on the composition of different raw materials is presented in Table 1. Obviously, the reduction of the solid recoveries was mainly caused by hemicellulose degradation. There was no hemicellulose detected in solid residue for branch when the PS was more than 4.34. For stem, the critical PS point to remove all the hemicellulose seemed to be 4.42. Although the hemicellulose in boll shell reached lowest with PS at 4.25, it could not be completely removed for all the pretreatments employed. This illustrated that the LHWP can have a positive effect on these three materials by removing hemicellulose partly or completely. However, the optimal LHWP condition for different materials was obviously different. The optimal PS to pretreat branch might be between 4.25 and 4.34, while it was between 4.34 and 4.42 for stem. The possible optimal PS condition for boll shell was about 4.25. Therefore, the selection of PS condition should be based on the sensitivity of different parts of CS instead of using one severe PS to pretreat the whole CS. This would save energy input for the parts with lower PS requirement. The presence of hemicellulose has been proved to be one of the most important factors in preventing cellulose digestion process (Öhgren et al., 2007). In LHWP process, the acetyl groups on hemicellulose can be released to form acetic acid which can further accelerate the hemicellulose degradation and thus improve the enzymatic hydrolysis (Sun and Cheng, 2002). Compared to hemicellulose, cellulose and Klason lignin were much more resistant to LHWP which can be barely degraded. The recoveries of the main components of three materials are presented in Table 1. Obviously,
Table 1 Composition and main component recoveries of different parts of cotton stalk before/after LHWP. Samples
Branch
Stem
Boll shell
PS
Solid recoveries (%)
Composition, %
Component recoveries, %
Cellulose
Hemi-cellulose
Klason lignin
Ash
14.82 ± 2.77 10.6 ± 1.27 15.31 ± 1.2 17.35 ± 0.22 13.48 ± 0.77 6.9 ± 0.43 0 0
21.73 ± 0.84 26.91 ± 1.06 27.04 ± 0.5 26.45 ± 1.32 33.23 ± 5.6 36.15 ± 1.61 39.22 ± 0.68 39.15 ± .53
3.78 ± 0.23 3.73 ± 0.44 3.23 ± 0.26 2.98 ± 0.17 2.38 ± 0.36 1.47 ± 0.11 1.98 ± 0.1 2.27 ± 0.09
0 2.77 3.07 3.36 3.66 4.25 4.34 4.42
79.34 ± 0.23 77.35 ± 3.27 75.82 ± 0.09 69.78 ± 1.29 60.34 ± 0.59 57.06 ± 0.19 56.50 ± 0.19
28.26 ± 1.88 26.59 ± 2.0 29.54 ± 3.1 33.19 ± 0.35 34.38 ± 1.82 41.25 ± 0.85 47.5 ± 1.46 48.19 ± 0.28
0 2.77 3.07 3.36 3.66 4.25 4.34 4.42
83.26 ± 0.41 79.31 ± 1.80 75.91 ± 3.19 74.64 ± 2.57 63.80 ± 0.29 60.11 ± 1.33 61.15 ± 0.57
35.58 ± 6.01 29.85 ± 4.07 29.22 ± 2.41 27.22 ± 8.56 36.28 ± 7.81 33.73 ± 13.75 44.27 ± 11.16 45.81 ± 10.68
17.59 ± 4.13 12 ± 1.12 11.2 ± 0.92 7.77 ± 2.63 11.22 ± 1.31 5.89 ± 3.28 5.47 ± 1.96 0
24.33 ± 1.06 31.79 ± 1.17 29.3 ± 0.47 34.98 ± 3.05 30.78 ± 2.09 40.46 ± 0.63 38.5 ± 1.64 32.53 ± 8.62
0 2.77 3.07 3.36 3.66 4.25 4.34 4.42
71.61 ± 0.06 70.75 ± 0.78 69.15 ± 1.27 62.89 ± 1.00 55.66 ± 0.91 53.47 ± 0.04 53.07 ± 0.71
37 ± 0.74 39.2 ± 2.76 42.53 ± 0.58 41.61 ± 0.75 43.48 ± 0.58 62.06 ± 4.11 71.73 ± 10.96 69.63 ± 6.85
15.42 ± 1.71 16.17 ± 1.97 16.99 ± 0.49 15 ± 0.65 14 ± 0.63 9.27 ± 1.46 16.97 ± 0.69 12.18 ± 0.46
18.54 ± 0.38 25.28 ± 0.1 26.06 ± 0.33 27.84 ± 0.29 31.21 ± 1.1 38.08 ± 0.3 39.92 ± 1.52 40.08 ± 0.47
Cellulose
Hemi-cellulose
Klason lignin
74.65 84.76 89.05 84.98 88.08 95.91 96.35
56.75 85.15 88.76 64.67 28.09 0 0
98.25 93.5 94.35 94.5 100.38 102.99 101.79
2.36 ± 0.44 1.95 ± 0.04 1.84 ± 0.15 1.74 ± 0.06 1.55 ± 0.14 1.78 ± 0.07 1.98 ± 0.06 0.98 ± 1.14
69.85 65.14 58.06 76.09 60.49 74.79 78.72
56.82 50.52 33.57 47.60 21.35 18.70 0
108.81 95.51 109.17 94.42 106.11 95.13 81.78
5.85 ± 0.17 2.32 ± 0.15 2.2 ± 0.03 2.19 ± 0.06 2.12 ± 0.09 2.23 ± 0.07 2.13 ± 0.16 2.18 ± 0.17
75.87 81.33 78.15 73.37 98.13 108 103.49
75.07 77.95 67.27 57.09 33.46 58.85 41.91
97.63 99.43 103.83 105.85 114.29 115.10 114.70
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90 solid recoveries(1) y = -13.035x + 120.19 R² = 0.8881
85
solid recoveries(2) solid recoveries(3)
solid recoveries (%)
80 75 70 y = -12.774x + 109.82 R² = 0.9518
65 60
y = -14.868x + 123.45 R² = 0.9599
55 50 2.5
3
3.5
4
4.5
5
PS Fig. 1. Relationship between PS and solid recoveries.
most of cellulose and lignin were still remained in the solid residue for all the pretreatments. It should be noted that over 100% recoveries of Klason lignin found for some pretreatments. This might be caused by the formation of pseudo-lignin in the acid pretreatment environment (Sannigrahi et al., 2011). 3.3. Degradation products from different parts of cotton stalk in LHWP The effects of LHWP on the formation of glycolic acid, formic acid, acetic acid from branch, stem and boll shell are shown in Table 2. It can be clearly seen that glycolic acid, formic acid and acetic acid gradually accumulated as PS increased. The most obvious accumulation was found on acetic acid. When the PS was 2.77, there was no detectable acetic acid observed in the hydrolyzate of stem. However, acetic acid reached 1.17 g/L with the PS at 4.42. For boll shell, the acetic acid sharply increased from 0.21 g/L to 1.20 g/L as the PS changed from 2.27 to 4.42. In LHWP at higher PS, accumulation of acetic acid was contributed to the acid-catalyzed hydrolysis of polysaccharide and degradation of monosaccharides. In addition, glycolic acid and furan aldehydes including furfural and 5-hydroxymethlfurfural (HMF) were also formed which have been confirmed to inhibit the growth of yeast, thus reduce the ethanol yield (Liu et al., 2004; Wahlbom and Hahn-Hägerdal, 2002). Formic acid can be produced from degradation of HMF (Ulbricht et al., 1984). The pKa values for formic acid, glycolic acid and acetic acid are 3.75, 3.90, and 4.76, Table 2 Organic acids formed after LHWP (average value ± standard deviation). Samples
PS
Glycolic acid (g/L)
Formic acid (g/L)
Acetic acid (g/L)
Branch
2.77 3.36 4.25 4.42
0.07 ± 0.01 0.04 ± 0.05 0.12 ± 0.01 0.23 ± 0.01
0.1 ± 0.01 0.15 ± 0.00 0.3 ± 0.00 0.38 ± 0.00
0.21 ± 0.04 0.35 ± 0.01 1.08 ± 0.01 1.4 ± 0.03
Stem
2.77 3.36 4.25 4.42
0 0.16 ± 0.04 0.25 ± 0.02 0.32 ± 0.01
0 0.06 ± 0.02 0.11 ± 0.01 0.15 ± 0.08
0 0.35 ± 0.06 1.01 ± 0.02 1.18 ± 0.18
Boll shell
2.77 3.36 4.25 4.42
0.06 ± 0.00 0.06 ± 0.00 0.11 ± 0.01 0.17 ± 0.07
0 0.18 ± 0.01 0.38 ± 0.01 0.42 ± 0.11
0.22 ± 0.06 0.35 ± 0.04 0.98 ± 0.22 1.21 ± 0.09
respectively. Of them, formic acid with the maximum pKa was the most toxic compound on Saccharomyces cerevisiae and acetic acid was the least (Larsson et al., 1999). An investigation made by Larsson et al. (1999) showed that the activity of yeast was completely inhibited when the concentration of aliphatic acids exceeded 100 mM, whereas the ethanol production was improved as the inhibitors were lower than 100 mM. Although effective, the LHWP was detrimental to monomeric sugar recoveries in the hydrolyzate due to higher PS causing degradation of monosaccharides to form inhibitors (Weil et al., 1998). As mentioned above, the composition and structure were different for different parts of CS. If CS were treated entirely, it might increase the risk of forming more inhibitors, for the part which did not require the higher PS as the other parts. Therefore, the pretreatment condition should be optimized for different parts of cotton stalk to avoid more inhibitors formation. 3.4. Toxic tests on hydrolyzate After pretreatment, most of the inhibitors were left in hydrolyzate which could inhibit the yeast activity. To test the toxic effect of the hydrolyzate on the ethanol production, the fermentation trials on hydrolyzate from the LWHP were preformed which are shown in Fig. 2(a)–(c). For branch, the ethanol yield from hydrolyzate obtained with PS at 4.25 or 4.42 was much lower than that from the control. While the ethanol yield was found to be higher with PS lower than 4.25. The ethanol yield for hydrolyzate from stem was far lower than the control, when the PS used was higher than 3.94. However, with PS at 2.77, 3.07 and 3.66, the ethanol yields started to surpass the control after 36 h. When PS at 2.77 and 3.36 were used, the ethanol yields were fairly similar to the control at the end of the fermentation process. Similarly, the ethanol yield from hydrolyzate of boll shell was inhibited with PS higher than 3.94. The final ethanol yield was very close to the control with PS at 3.07 or 3.66. While for PS at 3.36, the ethanol yield was much higher than the control all the time. There were many factors affecting the fermentation. Low concentration of acetic acid presented in hydrolyzate may increase the ethanol yield (Linde et al., 2008; Palmqvist and HahnHägerdal, 2000b). Although furfural at 2 g/L or higher had significantly toxic effect on yeast fermentation (Couallier et al., 2006), HMF and furfural levels below 1.0 g/L can minimize their toxicity
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2.77 4.25
12
3.36 3.66 control (a)
3.07 4.42
control ps3.66
ps3.36 ps4.42 (a)
10
6 3
5
0
(b)
9 6 3 0
(c)
16 12
Ethanol (g/100g DM)
Ethanol (g/L)
ps3.07 ps4.34
15
9
0
(b)
15 10 5 0
(c)
20
8
15
4 0
ps2.77 ps4.25
10
0
10
20
30
40
50
Time (h)
5 0
0
20
Fig. 2. Fermentation on hydrolyzate from (a) branch, (b) stem, (c) boll shell.
to yeasts (Kim et al., 2009). Moreover, the tolerance of yeasts to inhibitors could be also enhanced in the continuous production process due to its adaptation (Palmqvist and Hahn-Hägerdal, 2000a). Since ethanol yield could be influenced by many complicated factors, the optimal pretreatment condition should be obtained according to the characteristic of feedstocks to achieve the best fermentation efficiency. 3.5. Simultaneous saccharification and fermentation Simultaneous saccharification and fermentation (SSF) was conducted to evaluate the convertibility of the solid residue obtained from different parts of CS including branch, stem and boll shell before/after LHWP. The results are shown in Fig. 3(a)–(c). For all the materials, the ethanol yield was improved as PS increased. But when the PS was relatively lower, the ethanol yield approximated to that of the control. Boll shell pretreated with lower PS at 2.77 gave ethanol conversion of 11.41 ± 0.01 g/100 g feedstock, close to that from the untreated which was 10.63 ± 0.07 g/100 g feedstock. For branch, the ethanol conversion was 6.27 ± 0.01 g/ 100 g with PS at 2.77, lower than that from the untreated (7.24 ± 0.07 g/100 g feedstock). Compared to ethanol conversion of 8.02 ± 0.03 g/100 g feedstock from the untreated stem, it was 8.98 ± 0.01 g/100 g feedstock when PS at 3.05 was used. It can be concluded that lower LHWP had no apparent effect on promoting the ethanol conversion. When PS increased to 3.94, the ethanol conversions were greatly improved by 65.3%, 41.30% and 78.68% for branch, stem and boll shell, compared to the untreated materials, respectively. The highest ethanol conversions of 16.81 ± 0.00 g/ 100 g feedstock and 16.29 ± 0.003 g/100 g feedstock were obtained from stem and branch at a PS of 4.34. However when the PS further increased to 4.42, the ethanol conversion was not as high as that
40
60
80
100
120
Time (h) Fig. 3. SSF on solid of pretreated (a) branch, (b) stem, (c) boll shell.
from the pretreatment at 4.34. This might be caused by either more inhibitors formed, or some pseudo-lignin deposited on the surface of cellulose preventing the conversion process (Kumar et al., 2013). For boll shell, the ethanol conversions with PS at 4.34 and 4.42 were not only almost the same, but also fairly higher than that from the other two materials. This further indicated the sensitivity of these three materials to the PS was different. 3.6. Ratio of ethanol yield to energy input To evaluate the optimal pretreatment technology for different parts of CS, the ratio of ethanol yield to pretreatment energy input was used. The energy input can be estimated by the formulas of specific heat (Eq. (4)):
Q ¼ C W M W DT þ C m M m DT
ð4Þ
where Q: energy input, kJ; Cw: specific heat capacity of water, kJ (kg °C)1; Cm: specific heat capacity of materials, kJ (kg °C)1; Mw: mass of water, kg; Mm: mass of materials, kg; DT: the difference between initial temperature and terminated temperature of the reactor, °C. Since biomass contributed much less than water, there were only water heat absorption were considered. Table 3 presents the ratio of ethanol yield to pretreatment energy input of these three materials (branch, stem and boll shell). It can be seen that the maximum ratios for boll shell and branch were 1.37 and 1.33 g ethanol kJ1 with PS at 4.34. While it was 1.20 g ethanol kJ1 for stem obtained as PS of 3.66 was used. This might be caused by the different structure and composition of different parts of CS which
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Table 3 Ratio of ethanol yield to pretreatment energy input for branch, stem and boll shell. PS
2.77 3.07 3.36 3.66 4.25 4.34 4.42
Ratio of ethanol yield to pretreatment energy input (g ethanol kJ1) Branch
Stem
Boll shell
0.94 0.96 0.95 0.93 1.19 1.33 1.25
1.18 1.15 1.03 1.20 1.11 1.14 1.11
1.30 1.32 1.15 1.06 1.31 1.37 1.36
cannot be ignored. The economic utilization of CS should be based on its individual structural and compositional property. 4. Conclusions LHWP was employed to treat different parts of cotton stalk including branch, stem and boll shell. The results showed that different part had different PS requirement for hemicellulose removal. To completely remove hemicellulose for branch and stem, PS at 4.34 and 4.42 was needed respectively, while only 58.09% hemicellulose was removed for boll shell with PS at 4.42. Although the maximum ethanol yield can be obtained at the same PS (4.34), the ratio of ethanol yield to energy input for different part was different. In order to utilize CS economically, pretreatment condition for different parts of CS should be optimized. Acknowledgements The work was financially supported by the National Natural Science Foundation of China (No. 21276259), the National Basic Research Program of China (973 Program: 2011CB707401), National High Technology Research and Development Program of China (863 Program: 2012AA022301), the Chinese Academy of Sciences Fellowship for Young International Scientists (2013Y2GB0004), International (Regional) Cooperation and Exchange Projects Program of NSFC: Research Fund for International Young Scientists (21450110062) and the 100 Talents Program of the Chinese Academy of Sciences. References Binod, P., Kuttiraja, M., Archana, M., Janu, K.U., Sindhu, R., Sukumaran, R.K., Pandey, A., 2012. High temperature pretreatment and hydrolysis of cotton stalk for producing sugars for bioethanol production. Fuel 92 (1), 340–345. Buruiana, C.-T., Vizireanu, C., Garrote, G., Parajó, J.C., 2014. Optimization of corn stover biorefinery for coproduction of oligomers and second generation bioethanol using non-isothermal autohydrolysis. Ind. Crops Prod. 54, 32–39. Chang, V.S., Holtzapple, M.T., 2000. Fundamental factors affecting biomass enzymatic reactivity. In: Twenty-First Symposium on Biotechnology for Fuels and Chemicals. Springer, pp. 5–37. Couallier, E.M., Payot, T., Bertin, A.P., Lameloise, M., 2006. Recycling of distillery effluents in alcoholic fermentation. Appl. Biochem. Biotechnol. 133 (3), 217– 237. Du, S., Zhu, X., Wang, H., Zhou, D., Yang, W., Xu, H., 2013. High pressure assist-alkali pretreatment of cotton stalk and physiochemical characterization of biomass. Bioresour. Technol. 148, 494–500. Gould, J.M., 1985. Studies on the mechanism of alkaline peroxide delignification of agricultural residues. Biotechnol. Bioeng. 27 (3), 225–231. Kaur, U., Oberoi, H.S., Bhargav, V.K., Sharma-Shivappa, R., Dhaliwal, S.S., 2012. Ethanol production from alkali- and ozone-treated cotton stalks using thermotolerant Pichia kudriavzevii HOP-1. Ind. Crops Prod. 37 (1), 219–226.
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Liquid hot water pretreatment on different parts of cotton stalk to facilitate ethanol production Wei Jiang a,b, Senlin Chang a,b, Hongqiang Li a, Piotr Oleskowicz-Popiel c, Jian Xu a,⇑ a
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China c Poznan University of Technology, Faculty of Civil and Environmental Engineering, Institute of Environmental Engineering, Berdychowo 4, 60-965 Poznan, Poland b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Cotton stalk were separated into
stem, branch, boll shell for ethanol production. Hydrothermal pretreatment were optimized for different parts of cotton stalk. Boll shell was found to be more sensitive to pretreatment condition than stem. Ratio of ethanol yield to pretreatment energy for different parts was different.
a r t i c l e
i n f o
Article history: Received 25 September 2014 Received in revised form 6 November 2014 Accepted 8 November 2014 Available online 15 November 2014 Keywords: Cotton stalk Liquid hot water pretreatment Simultaneous saccharification and fermentation Bioethanol Energy input
a b s t r a c t To investigate pretreatment demand for different parts of biomass, cotton stalk was separated into stem, branch and boll shell, which were treated by liquid hot water pretreatment (LHWP) with severity from 2.77 to 4.42. Based on weight loss (WL, w/w) mainly caused by hemicellulose removal, it was found that boll shell (WL, 46.93%) was more sensitive to LHWP than stem (WL, 38.85%). Although ethanol yield of 18.3, 16.27 and 21.08 g/100 g was achieved from stem, branch and boll shell with pretreatment severity at 4.42, ratio of ethanol yield to pretreatment energy input for particular parts was different. For boll shell and branch, the maximum ratio of ethanol yield to energy input were 1.37 and 1.33 g ethanol kJ1 with severity at 4.34, while it was 1.20 for stem at 3.66. This indicates that different pretreatment demands for different parts of plants should be considered in order to save pretreatment energy input. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction As fossil fuel has currently become the major resource to meet the growing energy demand, it has caused economic, environmental and social sustainability problems (Kaygusuz, 2012). Biofuel has been considered as a strategic alternative energy to reduce the dependence on non-renewable resources (Buruiana et al., 2014). Unlike non-renewable energy only from fossil fuels, bioethanol ⇑ Corresponding author. Tel./fax: +86 10 82544852. E-mail addresses: [email protected], [email protected] (J. Xu). http://dx.doi.org/10.1016/j.biortech.2014.11.023 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
can be obtained either through fermentation of sugars from renewable biomass materials or by synthesis (Sun and Cheng, 2002). As one of the renewable resources, biomass has huge potential to meet the energy needs for modern society, especially in industrialized and developing countries (Ong et al., 2011). As a by-product of cotton industry, the annual yield of cotton stalk (CS) is about 40 million tons in China (Du et al., 2013). However, nowadays they are utilized with low efficiency by direct combustion or as livestock feed and compost (Watanabe et al., 2012). In CS, the content of nutrients such as protein is extremely low, while the cellulose content can get to 32–46% (Zhou et al.,
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2010). This makes CS a potential biomass feedstock for bioethanol production (Binod et al., 2012; Kaur et al., 2012). Nonetheless, natural CS is extremely difficult to be utilized due to its complicated structure. To improve the enzymatic digestibility and the subsequent fermentation of CS, pretreatment to break down its structure properly is indispensable (Pienkos and Zhang, 2009). Schell et al. (2003) applied sulfuric acid, hydrochloric acid and nitric acid to pretreat CS and found that acid pretreatment could remove the hemicellulose component significantly. More cellulose was therefore exposed, which contributed to enhancement of the subsequent enzymatic hydrolysis and fermentation. Alkali pretreatment on CS was investigated by Chang and Holtzapple (2000). It was observed that alkali was more effective on herbaceous crops than for hardwood material. Gould (1985) found that hydrogen peroxide pretreatment could reduce cellulose crystallinity by oxidative degradation, which ultimately resulted in improved enzymatic conversion of CS. By adopting ozone to pretreat CS, Neely (1984) found that there were no acid, alkali or toxic compounds formed during the pretreatment process and ozone pretreatment could be widely employed in lignin removal. Although some progress has been made, the same pretreatment conditions were adopted on the whole CS in all the investigations without any exception. The structural differences among different parts of CS were not considered, which led to the ignorance of the various demand for different part of CS. In a previous investigation with Miscanthus as feedstock (Li et al., 2013), it has been illustrated that different parts of Miscanthus had different optimal pretreatment condition. In the present study, CS was separated into three parts: stem, branch and boll shell, which was pretreated respectively. Liquid hot water pretreatment (LHWP) has been widely used due to its unique advantages such as no additive chemicals requirement, no special demand for non-corrosive reactor, and lower toxic compounds formation (Li et al., 2014, 2013; Xu et al., 2010a,b). The aim of this investigation was to optimize pretreatment conditions for different parts of CS in order to improve the utilization efficiency economically which would provide valuable practical experience for bioethanol production from CS.
t1: time when the reactor temperature reaches 100 °C; t2: time when the reactor temperature cooled down to 100 °C; T: reactor temperature (°C). After pretreatment, tap water was used to cool down the reactor to 80 °C. The slurry was then pumped out and filtered into two parts by vacuum filtration: hydrolyzate and solid residue. The pH and volume of hydrolyzate were measured before it was stored at 4 °C. The solid residue was dried at 60 °C and then put into sealed plastic bags for compositional analysis and simultaneous saccharification and fermentation. 2.3. Prehydrolysis and simultaneous saccharification fermentation on solid residue (SSF) Cellulase with filter paper activity (FPA) of 173.9 IU g1 was purchased from the SUKAHAN (WeiFang) Bio-Technology Company. Active dry yeasts (Angel thermal resistant ethanol active dry yeast, Product code: 800000012) were purchased from Angel Yeast Co., Ltd. Prehydrolysis was done with the ratio of liquid to solid at 8% at 50 °C, 120 rpm for 24 h and the cellulase loading was set as 15 IU/g dry matter (DM) (Xu et al., 2011). SSF was then performed at 42 °C and 120 rpm with the supplementation of 20 IU FPA g1 DM, 0.15 g yeast. The fermentation bottles were filled with N2 before fermentation locks prefilled with glycerol were equipped. Weight loss caused by CO2 release was determined at 0, 2, 4, 8, 12, 24, 36, 48 h. The conversion of cellulose to ethanol was calculated according to Eq. (2):
Cellulose conversion ¼
1:045 C L 0:51C M C C 0:9
100
ð2Þ
1.045: coefficient of CO2 conversion to ethanol; 0.51: coefficient of glucose conversion to ethanol; 0.9: coefficient of glucose to cellulose; CL: weight loss caused by CO2 release, g; CM: mass of sample used in SSF, g; CC: mass percentage of cellulose in sample, %. When the fermentation was completed, the fermentation liquor was filtered through 0.45 lm membrane, the final ethanol was determined by HPLC to evaluate the fermentation.
2. Methods
2.4. Toxic test on hydrolyzate
2.1. Feedstock preparation
Toxic tests were carried out with 15 g hydrolyzate obtained from pretreatment or water with the same amount as control, 0.3 g pure glucose, 0.024 g yeast and 30 lL urea (24 w/v). Weight loss caused by CO2 release was determined at 0, 2, 4, 8, 12, 24, 36, and 48 h. The ethanol yield was calculated based on the weight loss by multiplying 1.045, the conversion constant of CO2 to ethanol (Li et al., 2013).
CS was kindly provided by the Institute of Botany, Chinese Academy of Sciences. After harvesting in September, 2012, it was separated into 3 parts by clipper: stem, branch, and boll shell. The dry matter content was 93.60%, 93.11%, and 93.19%, respectively with the proportions of the 3 parts were about 36.16:31.17:32.67 (wt%). They were milled individually into particle size smaller than 2 mm with a laboratory miller (LXJMI-8111, Delixi Co., Ltd.). The milled samples were then stored in sealed barrels for LHWP. 2.2. Liquid hot water pretreatment A laboratory customized high pressure reactor with working volume of 1 L was used for LHWP as described in previous studies (Li et al., 2014, 2013). The solid loading was 40 g and the ratio of solid (milled stem, branch and boll shell) to water was 1:9 (w/v). The LHWP severity (PS) (Overend et al., 1987) was 2.77–4.42 calculated according to the following formula as described in a previous study (Li et al., 2013).
PS ¼
Z
t2
t1
exp
T 100 dt 14:75
ð1Þ
2.5. Analytical methods 2.5.1. Compositional analysis of solid residue The compositional analysis on the treated and untreated samples was carried out with the adapted NREL procedure (Li and Xu, 2013b; Sluiter et al., 2008). Polysaccharides were degraded into sugar monomers by two-step sulfuric acid hydrolysis: samples were treated by 72% H2SO4 in the first step at 30 °C for 60 min, followed by the second step at 121 °C for 1 h by diluting the H2SO4 concentration with Millipore water to 4%. Residual and filtrate were collected separately. The residual was dried at 105 °C overnight and the Klason lignin was calculated by subtracting the ash content from the dried residual (Sluiter et al., 2008). The amounts of sugars in the filtrate were determined by HPLC. Ash content was determined by heating the dried residual to 550 °C for 3 h. All the experiments were repeated twice.
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2.5.2. Oligosaccharides, monosaccharides and its degradation products analysis in hydrolyzate To determine the monosaccharides and its degradation products, an aliquot of the hydrolyzate was filtered through 0.45 lm membrane which was then directly analyzed by HPLC (Agilent 1260 Infinity USA). Hi-Plex H column (300 7.7 mm, Varian, Inc., Shropshire, UK) was used at 65 °C with 5% H2SO4 at a flow rate of 0.6 mL min1. Both refractive index detector (RID) and diode array detector (DAD) were employed for determining monosaccharides, glycolic acid, formic acid, acetic acid, HMF and furfural. Weak acid hydrolysis was carried out on hydrolyzate with 8% H2SO4, at 121 °C for 60 min (Li and Xu, 2013b). The oligosaccharides were quantified by the increased monosaccharides according to Eq. (3):
OS ¼ ðMSA MSB Þ 0:9
ð3Þ
OS: oligosaccharides in the hydrolyzate, mg; MSA: monosaccharides in the hydrolyzate after weak acid hydrolysis, mg; MSB: monosaccharides in the hydrolyzate before weak acid hydrolysis, mg. 3. Results and discussion 3.1. Effect of LHWP on solid recoveries The solid recoveries (w/w, %) on different parts of cotton stalk after pretreatments are presented in Table 1. It can be obviously seen that solid recoveries of stem, branch and boll shell were dramatically impacted by PS. For these three materials, the solid recoveries had a similar variation tendency which decreased as PS gradually increased. When the PS increased from 2.77 to 4.42, the solid recoveries for branch dropped from 79.34% to 56.50%. Similarly, 83.26% of the solid recovery of stem was obtained with PS of 2.77, while it was just 61.15% when the PS at 4.42 was used. For boll shell, the solid recoveries of 71.61–53.07% were observed when the PS changed from 2.27 to 4.42. After LHWP, the maximum weight losses were 43.5%, 38.85% and 46.93%, for branch, stem and boll shell, respectively. Linear relationships between PS and solid recoveries of these three materials were made and are shown in
Fig. 1. The maximum absolute value of straight line slope was observed to be 14.86 for branch, followed by 13.04 and 12.77 for stem and boll shell, respectively. This indicated that the impact of LHWP on the solid recoveries of different materials was different. The structure of boll shell was more sensitive to LHWP, while the stem was more resistant. It might be attributed to the different microstructural characteristics of the three materials. 3.2. Effect of PS on chemical composition of different parts of cotton stalk The effect of PS on the composition of different raw materials is presented in Table 1. Obviously, the reduction of the solid recoveries was mainly caused by hemicellulose degradation. There was no hemicellulose detected in solid residue for branch when the PS was more than 4.34. For stem, the critical PS point to remove all the hemicellulose seemed to be 4.42. Although the hemicellulose in boll shell reached lowest with PS at 4.25, it could not be completely removed for all the pretreatments employed. This illustrated that the LHWP can have a positive effect on these three materials by removing hemicellulose partly or completely. However, the optimal LHWP condition for different materials was obviously different. The optimal PS to pretreat branch might be between 4.25 and 4.34, while it was between 4.34 and 4.42 for stem. The possible optimal PS condition for boll shell was about 4.25. Therefore, the selection of PS condition should be based on the sensitivity of different parts of CS instead of using one severe PS to pretreat the whole CS. This would save energy input for the parts with lower PS requirement. The presence of hemicellulose has been proved to be one of the most important factors in preventing cellulose digestion process (Öhgren et al., 2007). In LHWP process, the acetyl groups on hemicellulose can be released to form acetic acid which can further accelerate the hemicellulose degradation and thus improve the enzymatic hydrolysis (Sun and Cheng, 2002). Compared to hemicellulose, cellulose and Klason lignin were much more resistant to LHWP which can be barely degraded. The recoveries of the main components of three materials are presented in Table 1. Obviously,
Table 1 Composition and main component recoveries of different parts of cotton stalk before/after LHWP. Samples
Branch
Stem
Boll shell
PS
Solid recoveries (%)
Composition, %
Component recoveries, %
Cellulose
Hemi-cellulose
Klason lignin
Ash
14.82 ± 2.77 10.6 ± 1.27 15.31 ± 1.2 17.35 ± 0.22 13.48 ± 0.77 6.9 ± 0.43 0 0
21.73 ± 0.84 26.91 ± 1.06 27.04 ± 0.5 26.45 ± 1.32 33.23 ± 5.6 36.15 ± 1.61 39.22 ± 0.68 39.15 ± .53
3.78 ± 0.23 3.73 ± 0.44 3.23 ± 0.26 2.98 ± 0.17 2.38 ± 0.36 1.47 ± 0.11 1.98 ± 0.1 2.27 ± 0.09
0 2.77 3.07 3.36 3.66 4.25 4.34 4.42
79.34 ± 0.23 77.35 ± 3.27 75.82 ± 0.09 69.78 ± 1.29 60.34 ± 0.59 57.06 ± 0.19 56.50 ± 0.19
28.26 ± 1.88 26.59 ± 2.0 29.54 ± 3.1 33.19 ± 0.35 34.38 ± 1.82 41.25 ± 0.85 47.5 ± 1.46 48.19 ± 0.28
0 2.77 3.07 3.36 3.66 4.25 4.34 4.42
83.26 ± 0.41 79.31 ± 1.80 75.91 ± 3.19 74.64 ± 2.57 63.80 ± 0.29 60.11 ± 1.33 61.15 ± 0.57
35.58 ± 6.01 29.85 ± 4.07 29.22 ± 2.41 27.22 ± 8.56 36.28 ± 7.81 33.73 ± 13.75 44.27 ± 11.16 45.81 ± 10.68
17.59 ± 4.13 12 ± 1.12 11.2 ± 0.92 7.77 ± 2.63 11.22 ± 1.31 5.89 ± 3.28 5.47 ± 1.96 0
24.33 ± 1.06 31.79 ± 1.17 29.3 ± 0.47 34.98 ± 3.05 30.78 ± 2.09 40.46 ± 0.63 38.5 ± 1.64 32.53 ± 8.62
0 2.77 3.07 3.36 3.66 4.25 4.34 4.42
71.61 ± 0.06 70.75 ± 0.78 69.15 ± 1.27 62.89 ± 1.00 55.66 ± 0.91 53.47 ± 0.04 53.07 ± 0.71
37 ± 0.74 39.2 ± 2.76 42.53 ± 0.58 41.61 ± 0.75 43.48 ± 0.58 62.06 ± 4.11 71.73 ± 10.96 69.63 ± 6.85
15.42 ± 1.71 16.17 ± 1.97 16.99 ± 0.49 15 ± 0.65 14 ± 0.63 9.27 ± 1.46 16.97 ± 0.69 12.18 ± 0.46
18.54 ± 0.38 25.28 ± 0.1 26.06 ± 0.33 27.84 ± 0.29 31.21 ± 1.1 38.08 ± 0.3 39.92 ± 1.52 40.08 ± 0.47
Cellulose
Hemi-cellulose
Klason lignin
74.65 84.76 89.05 84.98 88.08 95.91 96.35
56.75 85.15 88.76 64.67 28.09 0 0
98.25 93.5 94.35 94.5 100.38 102.99 101.79
2.36 ± 0.44 1.95 ± 0.04 1.84 ± 0.15 1.74 ± 0.06 1.55 ± 0.14 1.78 ± 0.07 1.98 ± 0.06 0.98 ± 1.14
69.85 65.14 58.06 76.09 60.49 74.79 78.72
56.82 50.52 33.57 47.60 21.35 18.70 0
108.81 95.51 109.17 94.42 106.11 95.13 81.78
5.85 ± 0.17 2.32 ± 0.15 2.2 ± 0.03 2.19 ± 0.06 2.12 ± 0.09 2.23 ± 0.07 2.13 ± 0.16 2.18 ± 0.17
75.87 81.33 78.15 73.37 98.13 108 103.49
75.07 77.95 67.27 57.09 33.46 58.85 41.91
97.63 99.43 103.83 105.85 114.29 115.10 114.70
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90 solid recoveries(1) y = -13.035x + 120.19 R² = 0.8881
85
solid recoveries(2) solid recoveries(3)
solid recoveries (%)
80 75 70 y = -12.774x + 109.82 R² = 0.9518
65 60
y = -14.868x + 123.45 R² = 0.9599
55 50 2.5
3
3.5
4
4.5
5
PS Fig. 1. Relationship between PS and solid recoveries.
most of cellulose and lignin were still remained in the solid residue for all the pretreatments. It should be noted that over 100% recoveries of Klason lignin found for some pretreatments. This might be caused by the formation of pseudo-lignin in the acid pretreatment environment (Sannigrahi et al., 2011). 3.3. Degradation products from different parts of cotton stalk in LHWP The effects of LHWP on the formation of glycolic acid, formic acid, acetic acid from branch, stem and boll shell are shown in Table 2. It can be clearly seen that glycolic acid, formic acid and acetic acid gradually accumulated as PS increased. The most obvious accumulation was found on acetic acid. When the PS was 2.77, there was no detectable acetic acid observed in the hydrolyzate of stem. However, acetic acid reached 1.17 g/L with the PS at 4.42. For boll shell, the acetic acid sharply increased from 0.21 g/L to 1.20 g/L as the PS changed from 2.27 to 4.42. In LHWP at higher PS, accumulation of acetic acid was contributed to the acid-catalyzed hydrolysis of polysaccharide and degradation of monosaccharides. In addition, glycolic acid and furan aldehydes including furfural and 5-hydroxymethlfurfural (HMF) were also formed which have been confirmed to inhibit the growth of yeast, thus reduce the ethanol yield (Liu et al., 2004; Wahlbom and Hahn-Hägerdal, 2002). Formic acid can be produced from degradation of HMF (Ulbricht et al., 1984). The pKa values for formic acid, glycolic acid and acetic acid are 3.75, 3.90, and 4.76, Table 2 Organic acids formed after LHWP (average value ± standard deviation). Samples
PS
Glycolic acid (g/L)
Formic acid (g/L)
Acetic acid (g/L)
Branch
2.77 3.36 4.25 4.42
0.07 ± 0.01 0.04 ± 0.05 0.12 ± 0.01 0.23 ± 0.01
0.1 ± 0.01 0.15 ± 0.00 0.3 ± 0.00 0.38 ± 0.00
0.21 ± 0.04 0.35 ± 0.01 1.08 ± 0.01 1.4 ± 0.03
Stem
2.77 3.36 4.25 4.42
0 0.16 ± 0.04 0.25 ± 0.02 0.32 ± 0.01
0 0.06 ± 0.02 0.11 ± 0.01 0.15 ± 0.08
0 0.35 ± 0.06 1.01 ± 0.02 1.18 ± 0.18
Boll shell
2.77 3.36 4.25 4.42
0.06 ± 0.00 0.06 ± 0.00 0.11 ± 0.01 0.17 ± 0.07
0 0.18 ± 0.01 0.38 ± 0.01 0.42 ± 0.11
0.22 ± 0.06 0.35 ± 0.04 0.98 ± 0.22 1.21 ± 0.09
respectively. Of them, formic acid with the maximum pKa was the most toxic compound on Saccharomyces cerevisiae and acetic acid was the least (Larsson et al., 1999). An investigation made by Larsson et al. (1999) showed that the activity of yeast was completely inhibited when the concentration of aliphatic acids exceeded 100 mM, whereas the ethanol production was improved as the inhibitors were lower than 100 mM. Although effective, the LHWP was detrimental to monomeric sugar recoveries in the hydrolyzate due to higher PS causing degradation of monosaccharides to form inhibitors (Weil et al., 1998). As mentioned above, the composition and structure were different for different parts of CS. If CS were treated entirely, it might increase the risk of forming more inhibitors, for the part which did not require the higher PS as the other parts. Therefore, the pretreatment condition should be optimized for different parts of cotton stalk to avoid more inhibitors formation. 3.4. Toxic tests on hydrolyzate After pretreatment, most of the inhibitors were left in hydrolyzate which could inhibit the yeast activity. To test the toxic effect of the hydrolyzate on the ethanol production, the fermentation trials on hydrolyzate from the LWHP were preformed which are shown in Fig. 2(a)–(c). For branch, the ethanol yield from hydrolyzate obtained with PS at 4.25 or 4.42 was much lower than that from the control. While the ethanol yield was found to be higher with PS lower than 4.25. The ethanol yield for hydrolyzate from stem was far lower than the control, when the PS used was higher than 3.94. However, with PS at 2.77, 3.07 and 3.66, the ethanol yields started to surpass the control after 36 h. When PS at 2.77 and 3.36 were used, the ethanol yields were fairly similar to the control at the end of the fermentation process. Similarly, the ethanol yield from hydrolyzate of boll shell was inhibited with PS higher than 3.94. The final ethanol yield was very close to the control with PS at 3.07 or 3.66. While for PS at 3.36, the ethanol yield was much higher than the control all the time. There were many factors affecting the fermentation. Low concentration of acetic acid presented in hydrolyzate may increase the ethanol yield (Linde et al., 2008; Palmqvist and HahnHägerdal, 2000b). Although furfural at 2 g/L or higher had significantly toxic effect on yeast fermentation (Couallier et al., 2006), HMF and furfural levels below 1.0 g/L can minimize their toxicity
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2.77 4.25
12
3.36 3.66 control (a)
3.07 4.42
control ps3.66
ps3.36 ps4.42 (a)
10
6 3
5
0
(b)
9 6 3 0
(c)
16 12
Ethanol (g/100g DM)
Ethanol (g/L)
ps3.07 ps4.34
15
9
0
(b)
15 10 5 0
(c)
20
8
15
4 0
ps2.77 ps4.25
10
0
10
20
30
40
50
Time (h)
5 0
0
20
Fig. 2. Fermentation on hydrolyzate from (a) branch, (b) stem, (c) boll shell.
to yeasts (Kim et al., 2009). Moreover, the tolerance of yeasts to inhibitors could be also enhanced in the continuous production process due to its adaptation (Palmqvist and Hahn-Hägerdal, 2000a). Since ethanol yield could be influenced by many complicated factors, the optimal pretreatment condition should be obtained according to the characteristic of feedstocks to achieve the best fermentation efficiency. 3.5. Simultaneous saccharification and fermentation Simultaneous saccharification and fermentation (SSF) was conducted to evaluate the convertibility of the solid residue obtained from different parts of CS including branch, stem and boll shell before/after LHWP. The results are shown in Fig. 3(a)–(c). For all the materials, the ethanol yield was improved as PS increased. But when the PS was relatively lower, the ethanol yield approximated to that of the control. Boll shell pretreated with lower PS at 2.77 gave ethanol conversion of 11.41 ± 0.01 g/100 g feedstock, close to that from the untreated which was 10.63 ± 0.07 g/100 g feedstock. For branch, the ethanol conversion was 6.27 ± 0.01 g/ 100 g with PS at 2.77, lower than that from the untreated (7.24 ± 0.07 g/100 g feedstock). Compared to ethanol conversion of 8.02 ± 0.03 g/100 g feedstock from the untreated stem, it was 8.98 ± 0.01 g/100 g feedstock when PS at 3.05 was used. It can be concluded that lower LHWP had no apparent effect on promoting the ethanol conversion. When PS increased to 3.94, the ethanol conversions were greatly improved by 65.3%, 41.30% and 78.68% for branch, stem and boll shell, compared to the untreated materials, respectively. The highest ethanol conversions of 16.81 ± 0.00 g/ 100 g feedstock and 16.29 ± 0.003 g/100 g feedstock were obtained from stem and branch at a PS of 4.34. However when the PS further increased to 4.42, the ethanol conversion was not as high as that
40
60
80
100
120
Time (h) Fig. 3. SSF on solid of pretreated (a) branch, (b) stem, (c) boll shell.
from the pretreatment at 4.34. This might be caused by either more inhibitors formed, or some pseudo-lignin deposited on the surface of cellulose preventing the conversion process (Kumar et al., 2013). For boll shell, the ethanol conversions with PS at 4.34 and 4.42 were not only almost the same, but also fairly higher than that from the other two materials. This further indicated the sensitivity of these three materials to the PS was different. 3.6. Ratio of ethanol yield to energy input To evaluate the optimal pretreatment technology for different parts of CS, the ratio of ethanol yield to pretreatment energy input was used. The energy input can be estimated by the formulas of specific heat (Eq. (4)):
Q ¼ C W M W DT þ C m M m DT
ð4Þ
where Q: energy input, kJ; Cw: specific heat capacity of water, kJ (kg °C)1; Cm: specific heat capacity of materials, kJ (kg °C)1; Mw: mass of water, kg; Mm: mass of materials, kg; DT: the difference between initial temperature and terminated temperature of the reactor, °C. Since biomass contributed much less than water, there were only water heat absorption were considered. Table 3 presents the ratio of ethanol yield to pretreatment energy input of these three materials (branch, stem and boll shell). It can be seen that the maximum ratios for boll shell and branch were 1.37 and 1.33 g ethanol kJ1 with PS at 4.34. While it was 1.20 g ethanol kJ1 for stem obtained as PS of 3.66 was used. This might be caused by the different structure and composition of different parts of CS which
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Table 3 Ratio of ethanol yield to pretreatment energy input for branch, stem and boll shell. PS
2.77 3.07 3.36 3.66 4.25 4.34 4.42
Ratio of ethanol yield to pretreatment energy input (g ethanol kJ1) Branch
Stem
Boll shell
0.94 0.96 0.95 0.93 1.19 1.33 1.25
1.18 1.15 1.03 1.20 1.11 1.14 1.11
1.30 1.32 1.15 1.06 1.31 1.37 1.36
cannot be ignored. The economic utilization of CS should be based on its individual structural and compositional property. 4. Conclusions LHWP was employed to treat different parts of cotton stalk including branch, stem and boll shell. The results showed that different part had different PS requirement for hemicellulose removal. To completely remove hemicellulose for branch and stem, PS at 4.34 and 4.42 was needed respectively, while only 58.09% hemicellulose was removed for boll shell with PS at 4.42. Although the maximum ethanol yield can be obtained at the same PS (4.34), the ratio of ethanol yield to energy input for different part was different. In order to utilize CS economically, pretreatment condition for different parts of CS should be optimized. Acknowledgements The work was financially supported by the National Natural Science Foundation of China (No. 21276259), the National Basic Research Program of China (973 Program: 2011CB707401), National High Technology Research and Development Program of China (863 Program: 2012AA022301), the Chinese Academy of Sciences Fellowship for Young International Scientists (2013Y2GB0004), International (Regional) Cooperation and Exchange Projects Program of NSFC: Research Fund for International Young Scientists (21450110062) and the 100 Talents Program of the Chinese Academy of Sciences. References Binod, P., Kuttiraja, M., Archana, M., Janu, K.U., Sindhu, R., Sukumaran, R.K., Pandey, A., 2012. High temperature pretreatment and hydrolysis of cotton stalk for producing sugars for bioethanol production. Fuel 92 (1), 340–345. Buruiana, C.-T., Vizireanu, C., Garrote, G., Parajó, J.C., 2014. Optimization of corn stover biorefinery for coproduction of oligomers and second generation bioethanol using non-isothermal autohydrolysis. Ind. Crops Prod. 54, 32–39. Chang, V.S., Holtzapple, M.T., 2000. Fundamental factors affecting biomass enzymatic reactivity. In: Twenty-First Symposium on Biotechnology for Fuels and Chemicals. Springer, pp. 5–37. Couallier, E.M., Payot, T., Bertin, A.P., Lameloise, M., 2006. Recycling of distillery effluents in alcoholic fermentation. Appl. Biochem. Biotechnol. 133 (3), 217– 237. Du, S., Zhu, X., Wang, H., Zhou, D., Yang, W., Xu, H., 2013. High pressure assist-alkali pretreatment of cotton stalk and physiochemical characterization of biomass. Bioresour. Technol. 148, 494–500. Gould, J.M., 1985. Studies on the mechanism of alkaline peroxide delignification of agricultural residues. Biotechnol. Bioeng. 27 (3), 225–231. Kaur, U., Oberoi, H.S., Bhargav, V.K., Sharma-Shivappa, R., Dhaliwal, S.S., 2012. Ethanol production from alkali- and ozone-treated cotton stalks using thermotolerant Pichia kudriavzevii HOP-1. Ind. Crops Prod. 37 (1), 219–226.
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