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Hydrolysis of Chlorella biomass for fermentable sugars in the presence of HCl and MgCl2
Bioresource Technology 102 (2011) 10158–10161
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Hydrolysis of Chlorella biomass for fermentable sugars in the presence of HCl and MgCl2 Na Zhou a,b,c, Yimin Zhang a, Xiaobin Wu a, Xiaowu Gong c, Qinhong Wang b,⇑ a
Key Laboratory for Green Chemical Technology of State Education Ministry, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China c Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, China b
a r t i c l e
i n f o
Article history: Received 3 June 2011 Received in revised form 5 August 2011 Accepted 11 August 2011 Available online 19 August 2011 Keywords: Chlorella biomass Fermentable sugar Hydrolysis Synergic effect Bioethanol
a b s t r a c t When Chlorella biomass was hydrolyzed in the presence of 2% HCl and 2.5% MgCl2, a sugar concentration of nearly 12%, and a sugar recovery of about 83% was obtained. Fermentation experiments demonstrated that glucose in the Chlorella biomass hydrolysates was converted into ethanol by Saccharomyces cerevisiae with a yield of 0.47 g g1, which is 91% of the theoretical yield. This chemical hydrolysis approach is thus a novel route for the hydrolysis of biomass to generate fermentable sugars. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The utilization of algae as biomass feedstock has several advantages over terrestrial biomass because of their high productivities (estimates 5000–7000 gallons/acre/year), short life cycle, use of marginal or non-arable land, and avoidance of feedstock and food conflict. Chlorella biomass grows very fast with an extrapolated productivity of 25 g m2 per day, and accounts for about 30% of the production of algal biomass. Chlorella biomass consists of about 40–70% carbohydrate, 10–20% protein and residual low-molecularweight compounds such as fatty acids, free amino acids, and amines. The high content of carbohydrate makes Chlorella biomass a potential biomass feedstock (Brennan and Owende, 2010). As with many other biomass feedstocks, one of the major obstacles in the utilization of algal biomass is its hydrolysis to simple soluble sugars, and the concentration of total soluble sugar is usually lower than 5% (Nguyen et al., 2009; Wang et al., 2011). To produce bioethanol and many other biobased products from biomass materials, the cost of sugar formation would be >30% of total production cost based on a conservative estimation (Wyman, 2007). Therefore, several methods, which include concentrated acid, dilute acid and enzymatic hydrolysis, have been commonly used to hydrolyze feedstock into simple sugars (Sun and Cheng, 2002). ⇑ Corresponding author. Address: Tianjin Institute of Industrial Biotechnology, CAS, 32 XiQiDao, Tianjin Airport Economic Area, 300308 Tianjin, China. Tel./fax: +86 22 84861950. E-mail address: [email protected] (Q. Wang). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.08.051
The concentrated acid hydrolysis is usually carried out by first adding 70–77% sulfuric acid (H2SO4) to destroy the connection of intra- and inter-chain hydrogen bonds of biomass, followed by adding water to dilute the acid to 20–30% for releasing fermentable sugars (Xiang et al., 2003; Harmer et al., 2009). Although the sugar recovery of this method was as high as 90%, the hazards of handling concentrated acids and the complexities of recycling them have limited its application. Dilute acid hydrolysis has the advantage of not requiring acid recovery, but the hydrolysis process occurs in two stages to accommodate the differences in the structure between hemicellulose and cellulose, and the conversion efficiency is as low as 50– 70% (Yang and Wyman, 2009; Lee and Jeffries, 2011) Enzymatic hydrolysis in combination with thermochemical pretreatment can give a high yield (75–95%) of soluble sugars from feedstocks, but the high cost of the enzymes and long reaction time (2–3 days) have hampered its application (Zhang et al., 2006; Kumar and Wyman, 2009). Most importantly, these three approaches are nearly fully developed with little room for further cost savings (Gray et al., 2006). Therefore, alternative methods are desirable (Mosier et al., 2005). Some investigators have proposed chloride salt (MgCl2) as Lewis acid to hydrolyze biomass. Zhang et al. (2011) found that approximately 35% of total xylan of cattails was directly hydrolyzed to the xylose when cattails were treated with 0.4 mol L1 MgCl2 above 180 °C. Although MgCl2 is neutral at room temperature, it is a mild acid (pH 4.3–5.0) at 165–230 °C (Fig. S1). Moreover, MgCl2 ($75 per ton) is cheaper than HCl or H2SO4 (more than $100 per ton), and generates less chemical discharge, which is essential for developing the cost-effective processes of biomass hydrolysis. In this
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work, a chemical hydrolysis technology to directly converse hemicelluloses, cellulose and other polysaccharides in Chlorella biomass to glucose, xylose, arabinose and other soluble monosaccharides using a mixture of dilute HCl and MgCl2 was investigated.
tions of different sugars and ethanol were quantified with calibration curves and used to calculate the sugar yield.
2. Methods
3.1. Hydrolysis of Chlorella biomass with different catalysts
2.1. Materials
Chlorella biomass was hydrolyzed in the presence of HCl (Fig. 1, curves B and D; Fig. 2). Two hydrolysis conditions (180 °C for 10 min; 120 °C for 60 min) were selected for the hydrolytic experiments. When the hydrolysis process occurred at 180 °C for 10 min, only 6.77% of total sugar (glucose: 6.22 g L1, 4.40%; xylose and arabinose: 3.35 g L1, 2.37%) was released in the presence of 0.5% HCl. As the concentration of HCl was increased to 2%, total sugar recovery reached 43.78% (glucose: 46.17 g L1, 32.67%; xylose and arabinose: 15.70 g L1, 11.11%). To obtain a total sugar yield of >70%, more than 4% of HCl must be used; no significant effect and even a decreased yield was observed when higher concentrations of HCl were introduced (Fig. 1, curve B). In addition, some degradation products were formed at high temperatures (Fig. 3).
2.2. Composition determination and chemical hydrolysis of Chlorella biomass NREL methods (NREL, 2010) were used to determine solid content (96%), composition and the total sugar content of Chlorella biomass. Cellulose, hemicelluloses and starch accounted for 35.28%, 10.19%, and 20.52% of dry Chlorella biomass, respectively, and the total sugar amounted to 73.58% (62.00% for glucose; 11.58% for xylose and arabinose). For a typical hydrolysis experiment, the reaction was performed in a stainless steel cylindrical reactor with a total volume of 15 mL. After the Chlorella biomass (a loading of 20% of 4 mL, for all reactions) and the catalyst were loaded, the reactor was heated to 120–180 °C at a rate 4–6 °C min1. When the set temperature was reached, the reaction time was recorded. After the reaction was over, the reactor was cooled to below 50 °C with room-temperature water, and the reaction solution was centrifuged at 7000 g for 10 min and the supernatant was collected.
100
80
Total sugar yield (%)
All chemicals were of analytical purity. The standard samples used for HPLC quantification were from Sigma and of chromatographic purity. Chlorella sp. TIB-A01 (which was a kind gift from Dr. Shulin Chen of Washington State University, USA) was grown in Basal medium containing the following components (mg L1): Glucose 40000, KNO3 7000, KH2PO4 1250, MgSO47H2O 1000, FeSO47H2O 49.8, Co(NO3)26H2O 4.9, CuSO45H2O 15.7, MoO3 7.1, H3BO3 114.2, EDTA 500, MnCl24H2O 14.2, CaCl22H2O 111, ZnSO47H2O 88.2. Cell incubation was carried out at 30 °C in a 7.5 L bioreactor at 300 rpm. The pH of the medium was automatically controlled at 6.0. Finally, the Chlorella biomass was harvested at OD600 = 43.65 and dried at 60 °C for composition determination and subsequent hydrolysis after centrifugation at 7000 g for 10 min and washing with distilled water twice.
3. Results and discussion
(A) (C) 60
(B) (D)
40
20
0 0
1
2
3
4
5
6
7
HCl (%) Fig. 1. Total sugar recovery from Chlorella biomass hydrolyzed with HCl or HCl/ MgCl2. (A) and (C) were with HCl plus 2.5% MgCl2; (B) and (D) were with HCl. (A) and (B) were operated at 180 °C for 10 min; (C) and (D) were at 120 °C for 60 min.
2.3. Microbial fermentation of Chlorella biomass hydrolysate 100 Ò
2% HCl + 2.5% MgCl2 80
Total sugar yield (%)
Saccharomyces cerevisiae Y01 (Ethanol Red yeast of Lesaffre group, France, which is industrial yeast for conventional ethanol production from corn) was used for ethanologenic fermentation of Chlorella hydrolysates. First, S. cerevisiae Y01 was incubated at 30 °C in YP medium (10.0 g L1yeast extract and 20.0 g L1peptone) containing 2% glucose at 200 rpm for 12 h. The cells in a 1mL aliquot of the culture were collected by centrifugation at 7000 g for 2 min, resuspended in 50-mL YNB medium (6.7 g L1, without amino acids) containing the Chlorella biomass hydrolysate with a pH meter adjusted to pH 5.5 with NaOH. Samples were collected at intervals for OD600 determination and HPLC analysis.
60
2% HCl 40
20
2.4. Analysis methods Sugars and ethanol were analyzed with an Agilent 1200 HPLC apparatus (Agilent Technologies, Santa Clara, California, USA) equipped with a HPX-87H ion exchange column (Biorad Laboratories Inc., Hercules, California, USA) at 63 °C. H2SO4 (5 mM) was used as a mobile phase at a flow of 0.6 mL min1. The concentra-
2% H2SO4 2.5% MgCl2
0
Different catalysts Fig. 2. Comparison of total sugar yield released from Chlorella biomass in the presence of different catalysts (hydrolysis condition: 180 °C, 10 min). Q: glucose; P: xylose and arabinose.
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4
40
2
20
0
0 1
2
3
4
CSF
40
(A) (C)
30
(B)
20
2
OD at 600 nm
60
4
Concentration / g L-1
80
Concentration of degradation products / g L-1
Total sugar recovery (%)
50
10
0
0 0
10
20
30
40
50
time / h
Fig. 3. Total sugar yield (j) of Chlorella biomass and concentration of degradation products (s) as a function of combined severity factor (CSF).
Fig. 4. (A) Glucose consumption, (B) ethanol production and (C) grow of S. cerevisiae Y01 in Chlorella biomass hydrolysates under oxygen-limited conditions.
Similar results were observed when Chlorella biomass was hydrolyzed with HCl at low temperature for a long time, but the yields were not as high as those at high temperature (Fig. 1). H2SO4 was used to release sugars from Chlorella biomass as a comparison to that of HCl. Though it is commonly used as acid catalyst to convert many feedstocks to fermentable sugars (Lee and Jeffries, 2011; Lu et al., 2009), it released less sugar than HCl (Fig. 2). Although the underlying mechanism should be further explored, this result was similar to that of Kim et al. (2011) and Israilides et al. (1978). Hydrolysis of Chlorella biomass in the presence of MgCl2 yielded no more than 8.62% of total sugar (glucose: 9.37 g L1, 6.63%; xylose and arabinose: 2.81 g L1, 1.99%) (Table S1). However, when a mixture of MgCl2 and HCl was used to as acidic catalyst, the exciting results were observed (Figs. 1 and 2).
3.3. Overall evaluation of combined severity factor on the hydrolysis
3.2. Synergic hydrolysis of Chlorella biomass with HCl and MgCl2 In the presence of 2% HCl and 2.5% MgCl2 the sugar yield was 83.47% (glucose: 90.74 g L1, 64.21%; xylose and arabinose: 27.21 g L1, 19.26%), which was almost two fold the sum of the sugar recovery when 2% HCl or 2.5% MgCl2 were used individually (Fig. 2). The addition of 2.5% MgCl2 and 2% HCl released more sugar than the addition of 4% HCl (Fig. 1). The ratio of MgCl2 and HCl was important for maximum sugar yield (Fig. S2 and Fig. 1), as was temperature and time (Fig. S3). These results indicated that adding moderate amount of inorganic MgCl2 into low concentration of HCl would have the similar or better effect on sugar release from Chlorella biomass as those at high concentration of HCl, and MgCl2 could have some synergic effect with HCl for Chlorella biomass decomposition. However, adding MgCl2 into much lower or higher concentration of HCl did not show apparent synergic effect (Fig. S2 and Fig. 1). Furthermore, the total sugar yield slightly decreased when higher concentration of MgCl2 was introduced, perhaps because the released sugars would be further decomposed at those conditions with higher MgCl2 concentrations (Fig. S2). These results suggested that sugar yield should be affected by the concentration of HCl and MgCl2. In addition, the experiment results showed that reaction temperature and time would also dramatically affect the sugar recovery (Fig. S3). Therefore, sugar recovery should be dependent on the triangle relation of catalyst concentration, reaction time and temperature, and optimal combination of these factors would give optimal sugar recovery.
Combined severity factor (CSF, Eq. (1)) has been reported to predict the hydrolysis of polysaccharides in hydrothermal processes and to explain results by including the effects of three major operational variables (catalyst concentration, reaction time and temperature) in a single parameter (Lee and Jeffries, 2011). The release of total sugars as a function of the CSF is summarized in Fig. 3. The total sugar conversion reached a plateau at CSF = 1.7–2.6. As was expected, a low sugar yield which was obtained at low temperature (120 °C) or at low concentration of HCl (0.5% HCl) corresponded to a CSF value <1.7. When the CSF was more than 2.6, the total sugar yield declined, likely because of the decomposition of glucose, xylose and arabinose into 5-hydroxymethyl-2-furfural (HMF) and furfural aldehyde at these harsh conditions (180 °C in the presence of 6%HCl, or in mixed HCl/MgCl2 for a long time) (Fig. 3).
CSF ¼ log10 ðt eðT100Þ=14:75 Þ pH
ð1Þ
where t is the reaction time (min), e is the natural logarithm, T is the reaction temperature (°C).
3.4. Microbial fermentation of Chlorella biomass hydrolysate S. cerevisiae Y01 produced 22.60 g L1 ethanol after 48 h and the yield was 0.47 g g1, which was 91% of the theoretical yield (Fig. 4). Xylose (19.46 g L1) in the hydrolysate was not consumed (data not shown). The conversion of glucose to ethanol was very similar to that of pure glucose or enzyme hydrolysis-derived sugars (Bai et al., 2007; Yuan et al., 2011), demonstrating the hydrolysis technology did not introduce inhibitors that interfered with ethanol production.
4. Conclusions Chemical hydrolysis of Chlorella biomass with a release of more than 83% of total sugars was achieved in the presence of HCl and MgCl2. Since MgCl2 is cheap ($75 per ton) and less chemical discharge is generated, this technology would be a cost-effective route for the pretreatment of biomass to generate fermentable sugars.
N. Zhou et al. / Bioresource Technology 102 (2011) 10158–10161
Acknowledgements The work was supported by the National Knowledge Innovation Project of the Chinese Academy of Sciences (KSCX1-YW-11E) and the National Basic Research Program (973 Program, 2011CB2009 02 and 2011CB200906). Qinhong Wang is supported by the 100 Talents Program of the Chinese Academy of Sciences. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.08.051. References Bai, F.W., Anderson, W.A., Moo-Young, M., 2007. Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnol. Adv. 26 (1), 89–105. Brennan, L., Owende, P., 2010. Biofuels from microalgae – A review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energ. Rev. 14 (2), 557–577. Gray, K.A., Zhao, L., Emptage, M., 2006. Bioethanol. Curr. Opin. Chem. Biol. 10 (2), 141–146. Harmer, M.A., Fan, A., Liauw, A., Kumar, R.K., 2009. A new route to high yield sugars from biomass: phosphorica-sulfuric acid. Chem. Commun. (43), 6610–6612. Israilides, C.J., Grant, G.A., Han, Y.W., 1978. Sugar level, fermentability, and acceptability of straw treated with different acids. Appl. Environ. Microb. 36 (1), 43–46. Kim, N.-J., Li, H., Jung, K., Chang, H.N., Lee, P.C., 2011. Ethanol production from marine algal hydrolysates using Escherichia coli KO11. Bioresour. Technol. 102, 7466–7469. Kumar, R., Wyman, C.E., 2009. Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies. Biotechnol. Bioeng. 102 (2), 457–467.
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Lee, J.W., Jeffries, T.W., 2011. Efficiencies of acid catalysts in the hydrolysis of lignocellulosic biomass over a range of combined severity factors. Bioresour. Technol. 102 (10), 5884–5890. Lu, X., Zhang, Y., Angelidaki, I., 2009. Optimization of H2SO4-catalyzed hydrothermal pretreatment of rapeseed straw for bioconversion to ethanol: focusing on pretreatment at high solids content. Bioresour. Technol. 100 (12), 3048–3053. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M., 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96 (6), 673–686. Nguyen, M.T., Choi, S.P., Lee, J., Lee, J., Sim, S.J., 2009. Hydrothermal acid pretreatment of Chlamydomonas reinhardtii biomass for ethanol production. J. Microbiol. Biotechnol. 19, 161–166. NREL, 2010. Chemical analysis and testing standard procedure, no. 001-014, National Renewable Energy Labs., Golden, CO.. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83 (1), 1–11. Wang, X., Liu, X., Wang, G., 2011. Two-stage hydrolysis of invasive algal feedstock for ethanol fermentation. J. Integr. Plant Biol. 53 (3), 246–252. Wyman, C.E., 2007. What is (and is not) vital to advancing cellulosic ethanol. Trends Biotechnol. 25 (4), 153–157. Xiang, Q., Lee, Y.Y., Pettersson, P.O., Torget, R.W., 2003. Heterogeneous aspects of acid hydrolysis of alpha-cellulose. Appl. Biochem. Biotechnol. 105–108, 505– 514. Yang, B., Wyman, C.E., 2009. Dilute acid and autohydrolysis pretreatment. Methods Mol. Biol. 581, 103–114. Yuan, D., Rao, K., Relue, P., Varanasi, S., 2011. Fermentation of biomass sugars to ethanol using native industrial yeast strains. Bioresour. Technol. 102 (3), 3246– 3253. Zhang, B., Shahbazi, A., Wang, L., Diallo, O., Whitmore, A., 2011. Magnesium chloride pretreatment for enzymatic hydrolysis of cattails. 33rd Symposium on Biotechnology for Fuels and Chemicals, Sheraton Seattle, WA, USA. Zhang, Y.H.P., Cui, J., Lynd, L.R., Kuang, L.R., 2006. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 7 (2), 644–648.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Hydrolysis of Chlorella biomass for fermentable sugars in the presence of HCl and MgCl2 Na Zhou a,b,c, Yimin Zhang a, Xiaobin Wu a, Xiaowu Gong c, Qinhong Wang b,⇑ a
Key Laboratory for Green Chemical Technology of State Education Ministry, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China c Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, China b
a r t i c l e
i n f o
Article history: Received 3 June 2011 Received in revised form 5 August 2011 Accepted 11 August 2011 Available online 19 August 2011 Keywords: Chlorella biomass Fermentable sugar Hydrolysis Synergic effect Bioethanol
a b s t r a c t When Chlorella biomass was hydrolyzed in the presence of 2% HCl and 2.5% MgCl2, a sugar concentration of nearly 12%, and a sugar recovery of about 83% was obtained. Fermentation experiments demonstrated that glucose in the Chlorella biomass hydrolysates was converted into ethanol by Saccharomyces cerevisiae with a yield of 0.47 g g1, which is 91% of the theoretical yield. This chemical hydrolysis approach is thus a novel route for the hydrolysis of biomass to generate fermentable sugars. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The utilization of algae as biomass feedstock has several advantages over terrestrial biomass because of their high productivities (estimates 5000–7000 gallons/acre/year), short life cycle, use of marginal or non-arable land, and avoidance of feedstock and food conflict. Chlorella biomass grows very fast with an extrapolated productivity of 25 g m2 per day, and accounts for about 30% of the production of algal biomass. Chlorella biomass consists of about 40–70% carbohydrate, 10–20% protein and residual low-molecularweight compounds such as fatty acids, free amino acids, and amines. The high content of carbohydrate makes Chlorella biomass a potential biomass feedstock (Brennan and Owende, 2010). As with many other biomass feedstocks, one of the major obstacles in the utilization of algal biomass is its hydrolysis to simple soluble sugars, and the concentration of total soluble sugar is usually lower than 5% (Nguyen et al., 2009; Wang et al., 2011). To produce bioethanol and many other biobased products from biomass materials, the cost of sugar formation would be >30% of total production cost based on a conservative estimation (Wyman, 2007). Therefore, several methods, which include concentrated acid, dilute acid and enzymatic hydrolysis, have been commonly used to hydrolyze feedstock into simple sugars (Sun and Cheng, 2002). ⇑ Corresponding author. Address: Tianjin Institute of Industrial Biotechnology, CAS, 32 XiQiDao, Tianjin Airport Economic Area, 300308 Tianjin, China. Tel./fax: +86 22 84861950. E-mail address: [email protected] (Q. Wang). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.08.051
The concentrated acid hydrolysis is usually carried out by first adding 70–77% sulfuric acid (H2SO4) to destroy the connection of intra- and inter-chain hydrogen bonds of biomass, followed by adding water to dilute the acid to 20–30% for releasing fermentable sugars (Xiang et al., 2003; Harmer et al., 2009). Although the sugar recovery of this method was as high as 90%, the hazards of handling concentrated acids and the complexities of recycling them have limited its application. Dilute acid hydrolysis has the advantage of not requiring acid recovery, but the hydrolysis process occurs in two stages to accommodate the differences in the structure between hemicellulose and cellulose, and the conversion efficiency is as low as 50– 70% (Yang and Wyman, 2009; Lee and Jeffries, 2011) Enzymatic hydrolysis in combination with thermochemical pretreatment can give a high yield (75–95%) of soluble sugars from feedstocks, but the high cost of the enzymes and long reaction time (2–3 days) have hampered its application (Zhang et al., 2006; Kumar and Wyman, 2009). Most importantly, these three approaches are nearly fully developed with little room for further cost savings (Gray et al., 2006). Therefore, alternative methods are desirable (Mosier et al., 2005). Some investigators have proposed chloride salt (MgCl2) as Lewis acid to hydrolyze biomass. Zhang et al. (2011) found that approximately 35% of total xylan of cattails was directly hydrolyzed to the xylose when cattails were treated with 0.4 mol L1 MgCl2 above 180 °C. Although MgCl2 is neutral at room temperature, it is a mild acid (pH 4.3–5.0) at 165–230 °C (Fig. S1). Moreover, MgCl2 ($75 per ton) is cheaper than HCl or H2SO4 (more than $100 per ton), and generates less chemical discharge, which is essential for developing the cost-effective processes of biomass hydrolysis. In this
10159
N. Zhou et al. / Bioresource Technology 102 (2011) 10158–10161
work, a chemical hydrolysis technology to directly converse hemicelluloses, cellulose and other polysaccharides in Chlorella biomass to glucose, xylose, arabinose and other soluble monosaccharides using a mixture of dilute HCl and MgCl2 was investigated.
tions of different sugars and ethanol were quantified with calibration curves and used to calculate the sugar yield.
2. Methods
3.1. Hydrolysis of Chlorella biomass with different catalysts
2.1. Materials
Chlorella biomass was hydrolyzed in the presence of HCl (Fig. 1, curves B and D; Fig. 2). Two hydrolysis conditions (180 °C for 10 min; 120 °C for 60 min) were selected for the hydrolytic experiments. When the hydrolysis process occurred at 180 °C for 10 min, only 6.77% of total sugar (glucose: 6.22 g L1, 4.40%; xylose and arabinose: 3.35 g L1, 2.37%) was released in the presence of 0.5% HCl. As the concentration of HCl was increased to 2%, total sugar recovery reached 43.78% (glucose: 46.17 g L1, 32.67%; xylose and arabinose: 15.70 g L1, 11.11%). To obtain a total sugar yield of >70%, more than 4% of HCl must be used; no significant effect and even a decreased yield was observed when higher concentrations of HCl were introduced (Fig. 1, curve B). In addition, some degradation products were formed at high temperatures (Fig. 3).
2.2. Composition determination and chemical hydrolysis of Chlorella biomass NREL methods (NREL, 2010) were used to determine solid content (96%), composition and the total sugar content of Chlorella biomass. Cellulose, hemicelluloses and starch accounted for 35.28%, 10.19%, and 20.52% of dry Chlorella biomass, respectively, and the total sugar amounted to 73.58% (62.00% for glucose; 11.58% for xylose and arabinose). For a typical hydrolysis experiment, the reaction was performed in a stainless steel cylindrical reactor with a total volume of 15 mL. After the Chlorella biomass (a loading of 20% of 4 mL, for all reactions) and the catalyst were loaded, the reactor was heated to 120–180 °C at a rate 4–6 °C min1. When the set temperature was reached, the reaction time was recorded. After the reaction was over, the reactor was cooled to below 50 °C with room-temperature water, and the reaction solution was centrifuged at 7000 g for 10 min and the supernatant was collected.
100
80
Total sugar yield (%)
All chemicals were of analytical purity. The standard samples used for HPLC quantification were from Sigma and of chromatographic purity. Chlorella sp. TIB-A01 (which was a kind gift from Dr. Shulin Chen of Washington State University, USA) was grown in Basal medium containing the following components (mg L1): Glucose 40000, KNO3 7000, KH2PO4 1250, MgSO47H2O 1000, FeSO47H2O 49.8, Co(NO3)26H2O 4.9, CuSO45H2O 15.7, MoO3 7.1, H3BO3 114.2, EDTA 500, MnCl24H2O 14.2, CaCl22H2O 111, ZnSO47H2O 88.2. Cell incubation was carried out at 30 °C in a 7.5 L bioreactor at 300 rpm. The pH of the medium was automatically controlled at 6.0. Finally, the Chlorella biomass was harvested at OD600 = 43.65 and dried at 60 °C for composition determination and subsequent hydrolysis after centrifugation at 7000 g for 10 min and washing with distilled water twice.
3. Results and discussion
(A) (C) 60
(B) (D)
40
20
0 0
1
2
3
4
5
6
7
HCl (%) Fig. 1. Total sugar recovery from Chlorella biomass hydrolyzed with HCl or HCl/ MgCl2. (A) and (C) were with HCl plus 2.5% MgCl2; (B) and (D) were with HCl. (A) and (B) were operated at 180 °C for 10 min; (C) and (D) were at 120 °C for 60 min.
2.3. Microbial fermentation of Chlorella biomass hydrolysate 100 Ò
2% HCl + 2.5% MgCl2 80
Total sugar yield (%)
Saccharomyces cerevisiae Y01 (Ethanol Red yeast of Lesaffre group, France, which is industrial yeast for conventional ethanol production from corn) was used for ethanologenic fermentation of Chlorella hydrolysates. First, S. cerevisiae Y01 was incubated at 30 °C in YP medium (10.0 g L1yeast extract and 20.0 g L1peptone) containing 2% glucose at 200 rpm for 12 h. The cells in a 1mL aliquot of the culture were collected by centrifugation at 7000 g for 2 min, resuspended in 50-mL YNB medium (6.7 g L1, without amino acids) containing the Chlorella biomass hydrolysate with a pH meter adjusted to pH 5.5 with NaOH. Samples were collected at intervals for OD600 determination and HPLC analysis.
60
2% HCl 40
20
2.4. Analysis methods Sugars and ethanol were analyzed with an Agilent 1200 HPLC apparatus (Agilent Technologies, Santa Clara, California, USA) equipped with a HPX-87H ion exchange column (Biorad Laboratories Inc., Hercules, California, USA) at 63 °C. H2SO4 (5 mM) was used as a mobile phase at a flow of 0.6 mL min1. The concentra-
2% H2SO4 2.5% MgCl2
0
Different catalysts Fig. 2. Comparison of total sugar yield released from Chlorella biomass in the presence of different catalysts (hydrolysis condition: 180 °C, 10 min). Q: glucose; P: xylose and arabinose.
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100
4
40
2
20
0
0 1
2
3
4
CSF
40
(A) (C)
30
(B)
20
2
OD at 600 nm
60
4
Concentration / g L-1
80
Concentration of degradation products / g L-1
Total sugar recovery (%)
50
10
0
0 0
10
20
30
40
50
time / h
Fig. 3. Total sugar yield (j) of Chlorella biomass and concentration of degradation products (s) as a function of combined severity factor (CSF).
Fig. 4. (A) Glucose consumption, (B) ethanol production and (C) grow of S. cerevisiae Y01 in Chlorella biomass hydrolysates under oxygen-limited conditions.
Similar results were observed when Chlorella biomass was hydrolyzed with HCl at low temperature for a long time, but the yields were not as high as those at high temperature (Fig. 1). H2SO4 was used to release sugars from Chlorella biomass as a comparison to that of HCl. Though it is commonly used as acid catalyst to convert many feedstocks to fermentable sugars (Lee and Jeffries, 2011; Lu et al., 2009), it released less sugar than HCl (Fig. 2). Although the underlying mechanism should be further explored, this result was similar to that of Kim et al. (2011) and Israilides et al. (1978). Hydrolysis of Chlorella biomass in the presence of MgCl2 yielded no more than 8.62% of total sugar (glucose: 9.37 g L1, 6.63%; xylose and arabinose: 2.81 g L1, 1.99%) (Table S1). However, when a mixture of MgCl2 and HCl was used to as acidic catalyst, the exciting results were observed (Figs. 1 and 2).
3.3. Overall evaluation of combined severity factor on the hydrolysis
3.2. Synergic hydrolysis of Chlorella biomass with HCl and MgCl2 In the presence of 2% HCl and 2.5% MgCl2 the sugar yield was 83.47% (glucose: 90.74 g L1, 64.21%; xylose and arabinose: 27.21 g L1, 19.26%), which was almost two fold the sum of the sugar recovery when 2% HCl or 2.5% MgCl2 were used individually (Fig. 2). The addition of 2.5% MgCl2 and 2% HCl released more sugar than the addition of 4% HCl (Fig. 1). The ratio of MgCl2 and HCl was important for maximum sugar yield (Fig. S2 and Fig. 1), as was temperature and time (Fig. S3). These results indicated that adding moderate amount of inorganic MgCl2 into low concentration of HCl would have the similar or better effect on sugar release from Chlorella biomass as those at high concentration of HCl, and MgCl2 could have some synergic effect with HCl for Chlorella biomass decomposition. However, adding MgCl2 into much lower or higher concentration of HCl did not show apparent synergic effect (Fig. S2 and Fig. 1). Furthermore, the total sugar yield slightly decreased when higher concentration of MgCl2 was introduced, perhaps because the released sugars would be further decomposed at those conditions with higher MgCl2 concentrations (Fig. S2). These results suggested that sugar yield should be affected by the concentration of HCl and MgCl2. In addition, the experiment results showed that reaction temperature and time would also dramatically affect the sugar recovery (Fig. S3). Therefore, sugar recovery should be dependent on the triangle relation of catalyst concentration, reaction time and temperature, and optimal combination of these factors would give optimal sugar recovery.
Combined severity factor (CSF, Eq. (1)) has been reported to predict the hydrolysis of polysaccharides in hydrothermal processes and to explain results by including the effects of three major operational variables (catalyst concentration, reaction time and temperature) in a single parameter (Lee and Jeffries, 2011). The release of total sugars as a function of the CSF is summarized in Fig. 3. The total sugar conversion reached a plateau at CSF = 1.7–2.6. As was expected, a low sugar yield which was obtained at low temperature (120 °C) or at low concentration of HCl (0.5% HCl) corresponded to a CSF value <1.7. When the CSF was more than 2.6, the total sugar yield declined, likely because of the decomposition of glucose, xylose and arabinose into 5-hydroxymethyl-2-furfural (HMF) and furfural aldehyde at these harsh conditions (180 °C in the presence of 6%HCl, or in mixed HCl/MgCl2 for a long time) (Fig. 3).
CSF ¼ log10 ðt eðT100Þ=14:75 Þ pH
ð1Þ
where t is the reaction time (min), e is the natural logarithm, T is the reaction temperature (°C).
3.4. Microbial fermentation of Chlorella biomass hydrolysate S. cerevisiae Y01 produced 22.60 g L1 ethanol after 48 h and the yield was 0.47 g g1, which was 91% of the theoretical yield (Fig. 4). Xylose (19.46 g L1) in the hydrolysate was not consumed (data not shown). The conversion of glucose to ethanol was very similar to that of pure glucose or enzyme hydrolysis-derived sugars (Bai et al., 2007; Yuan et al., 2011), demonstrating the hydrolysis technology did not introduce inhibitors that interfered with ethanol production.
4. Conclusions Chemical hydrolysis of Chlorella biomass with a release of more than 83% of total sugars was achieved in the presence of HCl and MgCl2. Since MgCl2 is cheap ($75 per ton) and less chemical discharge is generated, this technology would be a cost-effective route for the pretreatment of biomass to generate fermentable sugars.
N. Zhou et al. / Bioresource Technology 102 (2011) 10158–10161
Acknowledgements The work was supported by the National Knowledge Innovation Project of the Chinese Academy of Sciences (KSCX1-YW-11E) and the National Basic Research Program (973 Program, 2011CB2009 02 and 2011CB200906). Qinhong Wang is supported by the 100 Talents Program of the Chinese Academy of Sciences. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.08.051. References Bai, F.W., Anderson, W.A., Moo-Young, M., 2007. Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnol. Adv. 26 (1), 89–105. Brennan, L., Owende, P., 2010. Biofuels from microalgae – A review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energ. Rev. 14 (2), 557–577. Gray, K.A., Zhao, L., Emptage, M., 2006. Bioethanol. Curr. Opin. Chem. Biol. 10 (2), 141–146. Harmer, M.A., Fan, A., Liauw, A., Kumar, R.K., 2009. A new route to high yield sugars from biomass: phosphorica-sulfuric acid. Chem. Commun. (43), 6610–6612. Israilides, C.J., Grant, G.A., Han, Y.W., 1978. Sugar level, fermentability, and acceptability of straw treated with different acids. Appl. Environ. Microb. 36 (1), 43–46. Kim, N.-J., Li, H., Jung, K., Chang, H.N., Lee, P.C., 2011. Ethanol production from marine algal hydrolysates using Escherichia coli KO11. Bioresour. Technol. 102, 7466–7469. Kumar, R., Wyman, C.E., 2009. Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies. Biotechnol. Bioeng. 102 (2), 457–467.
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