Production of 2,3-butanediol from cellulose and Jatropha hulls after ionic liquid pretreatment and dilute-acid hydrolysis
© Jiang et al.; licensee Springer. 2013
Received: 13 August 2013
Accepted: 14 August 2013
Published: 20 August 2013
Abundant Jatropha waste is a promising renewable feedstock for the production of sugars and 2,3-butanediol fermentation. To obtain high yield of water-soluble products and high concentration of reducing-sugars, ionic liquid (IL) pretreatment and dilute acid hydrolysis at 150°C were combined in this work. The destruction of crystalline structure and increase surface area of biomasses after IL-pretreatment, made their hydrolysis more efficient. Compared with original cellulose, after IL-pretreatment, both the yield and concentration of reducing-sugars increased by 139%, and the water-soluble products yield increased by 128% after hydrolysis. Compared with water-washed Jatropha hulls, after IL-pretreatment, the yield and concentration of reducing-sugars increased by 80% and 76%, respectively, and the water-soluble products yield increased by 70% after hydrolysis. IL-pretreatment benefited the fermentation of Jatropha hull hydrolysate with 66.58% diol yield and its productivity increased from 0.35 to 0.40 g/(L · h).
KeywordsIonic liquid Hydrolysis 2,3-Butanediol Fermentation Klebsiella oxytoca Jatropha hulls
2,3-Butanediol is a promising liquid fuel and bulk chemical for comprehensive industry applications (Garg and Jain 1995; Syu 2001). Biological production of 2,3-butanediol on an industrial-scale is still in its early stage but with strong prospects of growth. However, the substrates account for more than half of the total production cost, and strongly influence the economy of its production (Celińska and Grajek 2009; Wang et al. 2010; Ji et al. 2011). As the most abundant, cheap and renewable source of sugars substrate, lignocellulose is a promising feedstock for biorefinery. Jatropha hulls are wastes from Jatropha seeds for biodiesel synthesis. One tonne of Jatropha seeds provides about 350-L crude oil for biodiesel production leaving 2.4 tonne hulls as waste (Sharma et al. 2009). As Jatropha hulls are rich in carbohydrates, studies are required to efficiently utilize these polysaccharides to produce sugars for fermentation.
Fermentable sugars can be produced by pretreatment and subsequent enzymatic hydrolysis. In previous study (Marasabessy et al. 2012), a dilute acid pretreatment of Jatropha hulls at optimum conditions (0.9% sulfuric acid, 30 min, 178°C) for enzymatic hydrolysis and ethanol fermentation resulted in 29% pentose degradation into furfural and 5% hexose degradation into 5-hydroxymethylfurfural (5-HMF). The degradation of sugars could not only result in low yield and high cost of sugars derived from biomass, but also lead to the formation of toxic by-products for fermentation. In addition, slow hydrolysis rate, high cost of enzyme and sensitivity to contaminants originated from other biomass components restrict its economical feasibility (Brinder et al. 2010). Two-step dilute sulfuric acid hydrolysis was used to effectively hydrolyze Jatropha hulls at 150°C for 2,3-butanediol fermentation in our previous work (Jiang et al. 2012). Hemicellulose and amorphous cellulose of Jatropha hulls can be easily hydrolyzed without any pretreatment but crystalline cellulose is more difficult to be hydrolyzed, thus harsh conditions are needed for it. However, severe conditions also accelerate the secondary decomposition of sugars. In addition, low concentration of fermentable sugars in the hydrolysate is one of the critical issues in the utilization of lignocellulose for biofuels and bio-based chemicals production. Cellulose crystallinity is a negative factor that affects biomass hydrolysis. Ionic liquids (ILs) have excellent properties to treat lignocellulose and make the crystallinity remarkable decrease and structure essentially amorphous and porous for efficient hydrolysis (Tian et al. 2011; Li et al. 2013). ILs are recyclable and IL-pretreatment is considered as an environmentally-friendly alternative to conventional pretreatment methods (Liu et al. 2012; Shafiei et al. 2013). As IL-pretreatment can effectively decrystallize cellulose, both hemicellulose and cellulose components can be hydrolyzed simultaneously at relatively moderate conditions after IL-pretreated. This strategy seems a promising route to make full utilization of raw material and achieve high concentration of sugars in the hydrolysate of Jatropha hulls.
In this study, the feasibility of combination of IL-pretreatment with dilute acid-hydrolysis of Jatropha hulls for fermentable sugars to produce 2,3-butanediol was evaluated. Microcrystalline cellulose and Jatropha hulls were pretreated by IL1-butyl-3-methylimidazoliuma chloride ([BMIM]Cl) before their subsequent dilute sulfuric acid hydrolysis. Untreated cellulose and water-washed Jatropha hulls were employed as control samples for comparison. The hydrolysates were further fermented to 2,3-butanediol with Klebsiella oxytoca.
Materials and methods
Original Jatropha hulls (OJH) were obtained from Xishuangbanna Tropical Botanical Garden in Yunnan, China. They were air-dried at 70°C for 24 h, milled then passed through 80–150 meshes. Structural carbohydrates and lignin in Jatropha hulls were determined according to the National Renewable Energy Laboratory (NREL) procedure (Sluiter et al. 2008). The elemental composition of Jatropha hulls were analyzed by an organic elemental analyzer (Vario EL III, Hanau, Germany). Microcrystalline cellulose (Cat. No. 6288) was purchased from Sigma (Shanghai), milled to the size of less than 150 meshes and dried in an oven at 60°C for 24 h before use. [BMIM]Cl (purity 99%) was from Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (Lanzhou, China). The strain used for 2,3-butanediol production was Klebsiella oxytoca (CICC 22912) from China Center of Industrial Culture Collection (Beijing).
where I002 was the highest peak intensity at an angle of diffraction of 2θ = 22.5°, whereas Iam was the peak for the amorphous cellulose at 2θ = 18°.
Measurement of specific surface area
Specific surface area of samples was determined by Bruner Emmett and Teller (BET) method (Tristar II 3020, Micromeritics Instrument Co., Ltd, Northcross, GA). Samples were degassed at 100°C for 3 h and nitrogen with relative pressure of 0.05-0.985 was applied in the analyses.
Dilute sulfuric acid hydrolysis
The hydrolysates were neutralized with calcium hydroxide to pH 7.0 and concentrated under vacuum at 60–70°C to achieve approximately 100 g/L sugar concentration. The concentrated hydrolysate solutions (100 mL) were detoxicated in a 250-mL flask containing 2-g activated charcoal (Baoman Biochemical Co. Ltd., Shanghai). The flask was placed in a shaker at 50°C and 200 rpm for 2 h. Finally, after removing the charcoal by filtration, the solutions were ready for fermentation.
Biomass recovery and chemical composition before and after pretreatment
Chemical and elemental compositions of Jatropha hulls before and after pretreatment
12.9 ± 0.7
14.6 ± 0.2
12.9 ± 0.1
34.3 ± 0.6
48.5 ± 0.4
48.8 ± 0.4
22.4 ± 0.1
25.0 ± 0.7
29.9 ± 0.7
The chemical components and elemental composition of untreated and pretreated Jatropha hulls were analyzed and listed in Table 1. The content of cellulose in ILJH (48.8%) was nearly equivalent that in WJH (48.5%), but the content of hemicellulose in ILJH (12.9%) was slightly lower than that in WJH (14.6%). The composition of C, H and N in Jatropha hulls changed little (C: 39.2-40.0%; H: 5.5-5.9%; N: 1.1-1.3%) before and after IL-pretreatment.
Structural characteristics of the recovered biomass
Crystallinity index and BET surface area of biomass
Crystallinity index (%)
BET surface area ( m 2/g )
Dilute sulfuric acid-hydrolysis
The yields of water-soluble products, reducing-sugars, 5-HMF and furfural as well as concentration of reducing-sugars
Yield of reducing-sugars (%)**
Concentration of reducing-sugars (g/L)
Yield of water-soluble products (%)
Yield of 5-HMF (%)**
Yield of furfural (%)**
Furfural and 5-HMF were inhibitors in fermentation, while their formation also meant the loss of fermentable sugars. So, furfural and 5-HMF should be avoided. In this work, the increase of the total yield of furfurals (5-HMF and furfural) after IL-pretreatment meant more secondary degradation (from 2.1% for WJH to 3.0% for ILJH). The by-products can be reduced by using more moderate reaction conditions (such as, lower H2SO4 concentration, shorter reaction time and lower temperature).
2,3-Butanediol fermentation by Klebsiella oxytoca for 60 h using different substrates in flasks
Diol (2,3-butanediol + acetoin)
Productivity g/(L · h)
Hydrolysate from cellulose
Hydrolysate from ILC
Hydrolysate from WJH
Hydrolysate from ILJH
Original Jatropha hulls hydrolysate from the first-step hydrolysis
Jiang et al. (2012)
First-step hydrolysate from pretreated Jatropha hulls
Jiang et al. (2012)
Second-step hydrolysate from the solid residue of the first step
Jiang et al. (2012)
Ji et al. (2009)
Corncob acid hydrolysate
Cheng et al. (2010)
Wood acid hydrolysate
Grover et al. (1990)
Figure 5 showed fermentation results of hydrolysates from WJH and ILJH in flasks, with initial reducing-sugars concentrations of 77.41 g/L and 76.91 g/L, respectively. For the fermentation of WJH hydrolysate, 3.58 g/L DCW and 20.70 g/L diol were produced after 60 h, giving diol yield of 28.60% (equivalent to 57.20% of the theoretical value) and diol productivity of 0.35 g/(L · h). For the fermentation of ILJH hydrolysate, products contained 4.61 g/L DCW and 24.13 g/L diol, with diol yield of 33.29% (equivalent to 66.58% of the theoretical value) and diol productivity of 0.40 g/(L · h), which were higher than those of WJH hydrolysate.
In the case of bulk chemicals and biofuels, the cost of the raw material mostly affected the price of the final products. The efficient utilization of biomass was essential for the economical production of 2,3-butanediol. In the previous two-step dilute sulfuric acid hydrolysis of Jatropha hulls, total water-soluble products yield was 64%, which was higher than that (37%) from the first-step hydrolysis (Jiang et al. 2012). In this work, the yield of water-soluble products of ILJH reached similar total value (60.2%) by just using the first-step hydrolysis conditions. The yield of water-soluble products for ILJH was greatly enhanced (60.2% vs. 37%). In the combination of IL-pretreatment and enzyme hydrolysis of cellulose (Tian et al. 2011), glucose yield was 59% after 72 h hydrolysis time as compared with 80.2% yield of reducing-sugars for ILC in this work. Therefore, this hydrolysis work reached relatively high yield of water-soluble products for IL-pretreated biomass samples under milder conditions.
Separation of 2,3-butanediol from fermentation media is one of economic barriers for the commercial production of microbial 2,3-butanediol (Ji et al. 2011). High concentration of 2,3-butanediol can cut the cost of downstream separation. So, high concentration of initial fermentable sugars is required for practical applications. In the previous study, for example, concentration of glucose (200 g/L) was used and relative high concentration of 2,3-butanediol (95.5 g/L) was achieved (Ji et al. 2009). However, the concentration of total reducing-sugars obtained from lignocellulose hydrolysates was about 20–30 g/L (Guo et al. 2008; Cheng et al. 2010; Jiang et al. 2012). So, increasing the reducing-sugars concentration in lignocellulose hydrolysates is one of the key problems for the high efficient production of biomass-derived 2,3-butanediol. In this work, after IL-pretreatment, the concentration of reducing-sugars increased to 53.4 g/L from 21.5 g/L for cellulose, and the concentration of reducing-sugars increased to 39.7 g/L from 22.6 for lignocellulose.
In the previous work (Table 4), some chemicals after hydrolysis had significantly unfavorable influence on the 2,3-butanediol metabolic pathway and biological activities (Jiang et al. 2012). After fermentation of OJH hydrolysate (obtained from the first-step hydrolysis) for 60 h, only 5.5% diol yield was achieved. However, after washed with neutral detergent to remove extractives (e.g., proteins, lipids, pectins and nonfibrous carbohydrates) and two-step hydrolysis, diol yields reached 35.6% and 41.4% from the hydrolysates of the first- and second-step hydrolysis, respectively. Compared with OJH hydrolysate, the fermentation efficiency of WJH and ILJH hydrolysates were much higher. The reason was that most of fermentation inhibitors produced from the extractives during hydrolysis of OJH were removed by water-washed and IL-pretreatment. IL-pretreatment benefited the fermentation of Jatropha hulls hydrolysate more than water-washed pretreatment might due to the more effective removal of extractives. On the other hand, the yields of diol from WJH (28.60%) and ILJH (33.29%) hydrolysates in this work were slightly lower than that from the first-step hydrolysate from neutral detergent pretreated Jatropha hulls (35.6%) due to minor extractives still remaining in WJH and ILJH. Glucose had higher efficiency for fermentation than other sugars (Wang et al. 2010). Therefore, the diol yield from the cellulose hydrolysate solution (41.6%) was higher than that from the hydrolysate of ILJH (33.29%), and was close to that of second-step hydrolysate (41.4%) from the solid residue (mainly cellulose) of the first-step hydrolysis of Jatropha hulls. Corncob acid hydrolysates were used as feedstocks for fermentation and after 60 h of fed-batch fermentation, a maximal 35.7 g/L 2,3-butanediol was obtained, giving a productivity of 0.59 g/(L · h) and a highest diol yield of 50% reported so far (Cheng et al. 2010). In the work of Grover et al. (1990), wood acid hydrolysate neutralized with Ca(OH)2 had been used for 2,3-butanediol production, obtaining 13.3 g/L 2,3-butanediol with a yield of 29% and a productivity of 0.28 g/L h.
In conclusion, in this study, IL-pretreatment and dilute acid-hydrolysis were performed to produce fermentable sugars from cellulose and Jatropha hulls. The yield of water-soluble products increased to 90.0% for IL-pretreated cellulose from 39.5% for original cellulose. For Jatropha hulls, after IL-pretreatment, the yield of water-soluble products rose to 60.2% from 35.5% for water-washed Jatropha hulls. IL-pretreatment also benefited the fermentation of Jatropha hulls hydrolysate due to the removal of extractives, with diol productivity increased to 0.40 from 0.35 g/(L · h) for water-washed Jatropha hulls. The techniques developed in this paper may be applied to other similar industrial microorganisms for the production of biofuels from biomass wastes.
This work was supported by the Chinese Academy of Sciences [100 talents program, CAS 135 program (XTBG-T02) and equipment R&D grant (No.YZ201260)], Yunnan 100 overseas talents program and the Natural Science Foundation of China (No. 21076220).
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