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Improved efficiency of butanol production by absorbed lignocellulose fermentation
Journal of Bioscience and Bioengineering VOL. 115 No. 3, 298e302, 2013 www.elsevier.com/locate/jbiosc
Improved efficiency of butanol production by absorbed lignocellulose fermentation Qin He and Hongzhang Chen* National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China Received 23 May 2012; accepted 22 September 2012 Available online 22 October 2012
Alkali-treated steam-exploded corn stover (SECSAT) was used as solid substrate for acetoneebutanoleethanol (ABE) production by absorbed lignocellulose fermentation (ALF) using Clostridium acetobutylicum ATCC 824. The ABE concentration in ALF culture had increased by 47% compared with that in submerged culture. More surprisingly, the acetone production was promoted and ethanol production was lower in the presence of SECSAT than that in its absence. ALF was also successfully in cofermentation of glucose and xylose, although decreased fermentability with an increase in the proportion of xylose. An invariable chemical composition and dry weight of SECSAT was found in ALF. Partial simultaneous saccharification and fermentation of SECSAT using a certain amount of cellulase could not only enhance the ABE concentration by 71%, but also significantly increase the area proportion of fiber cells in SECSAT from 53% to 90%, which would be an excellent paper making material. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Acetoneebutanoleethanol (ABE) fermentation; Steam explosion; Absorbed lignocellulose fermentation; Alkaline treatment; Corn stover; Fiber cells]
The interests in production of butanol as a chemical and also as an alternative liquid fuel have renewed due to the depletion of fossil fuels and deterioration of global environment situation in recent years. However, compared with petrochemical-derived butanol, biobutanol production is still not in economically competitive, because of its major drawbacks: high cost of feedstocks, low butanol concentration in fermentation broth and coproduction of low-value byproducts of acetone and ethanol. Although great progress has been made on genetics, upstream processing, fermentation and downstream processing recently, the acetoneebutanoleethanol (ABE) fermentation is yet to reach a sustainable and economically viable process. Biobutanol is now usually produced industrially by submerged fermentation (SmF) in which the microorganism and nutrient source are normally suspended or dissolved in a liquid medium and the growth takes place as a dispersed cell suspension. Solid-state fermentation (SSF) systems exhibit several advantages over SmF, including improved product characteristics, higher product yields, easier product recovery and reduced energy requirements despite heat and mass transfer problems and growth limited by low water content. However, Clostridium acetobutylicum was unable to grow on solid cellulosic substrates (1), in this case, an efficient process for fermentation, absorbed substrate fermentation (ASF) might be an attractive alternative to SSF for the production of butanol due to the * Corresponding author. Tel.: þ86 10 82544982; fax: þ86 10 28627071. E-mail address: [email protected] (H. Chen).
fact that medium composition can be designed as it is done in submerged culture. Meanwhile, the substrate used in the ASF process is similar to SSF, it behaves the nature of high porosity, good mechanical resistance and better water absorbency. In the former studies, researchers evaluated the potential of bagasse to be used as inert substrate (2). Bagasse was impregnated with a liquid medium containing glucose and calcium carbonate for lactic acid production. An increased yield and productivity of lactic acid were found in ASF. The solid support used in ASF includes: hemp, perlite, polyurethane foam, sugarcane bagasse and vermiculite (3). The authors considered lignocellulosic biomass as an effective potential substrate in the sense of porosity and absorb ability. Porous lignocellulosic carrier almost behaves all the characteristics of the ideal cell supports, in particular, could solve problems of cell injury caused by the immobilized cells and shear stress, could improve the cell density to facilitate large-scale cell culture, at the same time could greatly reduce inhibition on microorganisms because the concentration gradients of substrate and product between the internal and surface of substrate. However, more work should be done to improve the porosity of lignocellulosic biomass. As a pretreatment method for increasing porosity of lignocellulose and making lignocellulose more accessible to microorganisms and nutrients, steam explosion is an effective technique (4). Unfortunately, a wide range of compounds, some of which are toxic to butanol production is generated during pretreatment of lignocellulose (5). Previous works have proved that phenolic compounds (6) from lignin degradation are more
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.09.017
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inhibitory than weak acids, furan derivatives, in either cell growth or ABE production. Thus, the removal of inhibitors prior to fermentation is essential for successful butanol production from absorbed lignocellulose fermentation (ALF). Corresponding methods include water washing (7), delignification (8), etc. After detoxification, increase of porosity and surface area is found in the steam-exploded lignocellulose. The objective of this work is to evaluate the applicability and performance of using the absorbed lignocellulose fermentation technique for butanol production by C. acetobutylicum ATCC 824 growing on treated corn stover impregnated with different liquid medium. The characteristics of the solid substrate are also investigated to increase the fermentability and economic competitiveness of butanol production.
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Rad Laboratories Inc.) and a refractive index detector. The mobile phase was 0.5 mM H2SO4 at a flow rate of 0.5 mL/min at 65 C. Sugar utilization was calculated as utilized sugars (g/l) divided by added sugars (g/l) and was expressed in percentage. ABE and acid (acetic and butyric) were measured using a gas chromatograph (7890A, Agilent Technologies) equipped with a flame ionization detector (FID) and an HP-Innowax capillary column (30 m 0.32 mm). Oven temperature was held at 85 C for 4.5 min, programmed at increments of 20 C per min until 170 C, the final temperature was held for 2.5 min. Both injector and detector temperatures were set at 250 C. Nitrogen was used as the carrier gas and isobutanol was used as the internal standard. Productivity was calculated as the maximum ABE concentration achieved (g/l) divided by the fermentation time and is expressed as g/(l h). Yield was calculated as the total amount of solvents produced divided by the amount of fermentable sugar utilized and was expressed as g/g. The concentration of C. acetobutylicum in the fermentation broth was measured as OD 600 nm using an ultravioletevisible spectrophotometer (UV-1240, Shimadzu Co., Kyoto, Japan). The measured OD was correlated with dry weight using an established proportional constant (13). Pulp properties of residual solid substrate after partial simultaneous saccharification and fermentation After partial simultaneous saccharification and fermentation, the carrier was collected using a plastic 60-mL syringe, at the front end of which several holes of 1 mm diameter were punched. Then the carrier was dried after water washed. Fiber cell content was observed and determined through a light microscope after the carrier was separated in the acetic acid and hydrogen peroxide (30%) solution (1:1, v/v) for about 48 h at 60 C. The UV-G microscopic particle size analysis software was used to measure the length and diameter of each cell. Fiber cell content was expressed by area proportion, and fiber content of the carrier before fermentation was performed as control (14). The pulp was achieved by the followed process: fermented SECSAT were pretreated with 2% NaOH solution (w:v) at 115 C for 1 h with the solideliquid ratio maintained at 1:10 (w:v). The residual solid was rinsed to neutral pH with distilled water and bleached by 1% NaOH (w/v) and 4% H2O2 (w/v) at 60 C for 2 h. The pulp characteristics of this material were then studied. The pulps, with the beating degree of 38 SR and the grammage of 60 g/m2, were kept and tested under controlled environment (temperature of 23 1 C and RH of 50 1%). The tensile index was measured by the tensile testing machine (DLD-1, Beijing Guance Testing Machine Ltd., China) according to China national standard no. GB/T 453-2002.
MATERIALS AND METHODS Corn stover pretreatment The corn stover was harvested from the suburb of Beijing, China. After air-dried, the stover was chopped into 2e4 cm in length. About 200 g air-dried chipped corn stover was soaked in 200 mL distilled water for 15 min. Steam explosion pretreatment was carried out in a 7.5 L batch reactor (Weihai Automatic Control Reactor Ltd., China) as described in a previous work (9). The chipped stalk was fed into the reactor and the pretreatment was operated at 1.1 MPa for 4 min. After steam explosion, the material was washed with 1 L of 80 C water and filtered by nylon cloth (200 mesh). The washed steam-exploded corn stover (SECS) was dried at 60 C until constant weight. SECS was soaked in distilled water containing 1% NaOH (w/v) for 24 h at room temperature for alkaline treatment, the solid substrate concentration was 10% (w/v). Solid residues were thoroughly washed with water until neuter pH was achieved, and then filtered by nylon cloth (200 mesh). Alkali-treated SECS (SECSAT) was dried at 60 C oven to constant weight and stored at room temperature. Properties of solid substrate The chemical composition of raw and pretreated corn stover was determined according to the methods previously described (10). The porosity was measured by mercury porosimetry method using a mercury porosimeter (Micromeritics Autopore IV, Micromeritics, USA). The specific surface area was obtained by the standard BrunauereEmmetteTeller (BET) method using Beishide 3H-2000A apparatus (Beishide instrument-S&T Co., Ltd., Beijing).
RESULTS AND DISCUSSION Applicability of butanol production by absorbed lignocellulose fermentation The applicability of butanol production by ALF was tested firstly and the results were shown in Table 1. After 72-h anaerobic fermentation, nearly 99% of sugar was consumed when 60 g/l glucose was used as carbon source and the solideliquid ratio was 1:10 (w/v) in the fermentation. A total of 18.54 g/l ABE were produced and of which 10.07 g/l were butanol. An ABE yield of 0.31 g/g and a productivity of 0.26 g/(l h) were obtained. Cells which were grown in 60 g/l glucose in submerged fermentation produced 12.63 g/l ABE. The ABE concentration had increased by 47% compared with control exercise. Performance of ABE production using 54 g/l xylose was also investigated in this paper. ABE (14.55 g/l) were produced and of which 8.44 g/l were butanol, an ABE yield of 0.27 g/g and a productivity of 0.20 g/(l h) were reached eventually. Water washed SECS was also used as absorbed substrate for butanol fermentation. The efficiency of fermentation using this carrier was significantly lower than that of SECSAT added. This may be due to inhibitors absorbed on the steam-exploded straw (15), so pretreatment and detoxification of this material were essential.
Microorganism and butanol fermentation C. acetobutylicum ATCC 824 was maintained as a spore suspension in 6% (w/v) corn mash at 4 C. The preculture medium contained the following components per liter of distilled water: 30 g glucose, 4.3 g CH3COONH4, 1.768 g KH2PO4, 2.938 g K2HPO4, 10 mg p-aminobenzoic acid, 10 mg biotin, and 1 mL mineral salts solution (11). The initial pH of the medium was adjusted to 6.5 0.2 with 1 M NaOH or 1 M H2SO4. The medium was sterilized at 115 C for 15 min. Cells were grown anaerobically at 37 C for 20e36 h without agitation before being transferred into the fermentation medium. Batch fermentation experiments were carried out in a 100-mL serum bottle with a 60 mL working volume and 6 g air-dried SECSAT. The fermentation medium was similar to the seed culture medium except carbon sources. The pH of the medium was adjusted to 6.5 0.2 before autoclaving at 115 C for 15 min. After cooling to room temperature, the fermentation media were inoculated with 6 mL inoculum, and then infused with filtered oxygen-free nitrogen gas to maintain strict anaerobic conditions. Cultures were incubated at 37 C for 72 h without agitation. Samples were withdrawn at intervals for ABE, acids, and sugar analyses. All experiments were carried out at least twice to ensure reproducibility. Cellulase was added in the partial simultaneous saccharification and fermentation (PSSF) at 24 h after inoculation and at a loading of 5 FPA/g dry substrate. The filter paper activity of cellulase was measured by DNS method (12). Analytical procedures for butanol fermentation Glucose and xylose were determined by high-performance liquid chromatography (Agilent 1200 HPLC, Agilent Technologies, USA) with an Aminex HPX-87H column (300 mm 7.8 mm, Bio-
TABLE 1. Improved efficiency of ABE production and sugar utilization in absorbed lignocellulose fermentation. Carbon sources
Total sugar (g/L)
Controla Glucoseb Glucosec Xylosed a b c d
Using Using Using Using
60 60 60 54 60 60 60 54
g/l g/l g/l g/l
Consumption ratio of sugar (%) 97.00 88.75 98.73 98.43
0.83 1.00 0.67 2.79
Ethanol (g/L) 1.05 0.67 1.00 0.40
0.02 0.01 0.01 0.01
Acetone (g/L) 3.97 5.19 7.47 5.71
0.03 0.05 0.04 0.03
glucose as carbon source (CS), submerged fermentation, 72 h. glucose as CS, SECS as substrate, absorbed lignocellulose fermentation, 72 h. glucose as CS, SECSAT as substrate, absorbed lignocellulose fermentation, 72 h. xylose as CS, SECS as substrate, absorbed lignocellulose fermentation, 72 h.
Butanol (g/L) 7.61 7.17 10.07 8.44
0.03 0.03 0.04 0.02
Total acids (g/L)
Total solvent (g/L)
Yield of ABE (g/g)
12.63 13.03 18.54 14.55
0.22 0.24 0.31 0.27
3.11 2.28 2.02 5.33
0.17 0.07 0.12 0.30
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40
3
2
1
30
8
25 6
20 15
4
10 2
5 0
0
Products concentration (g/L)
35 4
Sugar concentration (g/L)
Biomass concentration (g/L)
10
0 0
12
24
36
48
60
72
Time (h) FIG. 1. Time course of sugar uptake and ABE production by C. acetobutylicum ATCC 824 on complex medium containing of 36 g/l glucose and 18 g/l xylose in ALF. Symbols: open squares, glucose concentration in broth; open triangles, xylose concentration in broth; open obtriangular, acetic acid concentration in broth; open circles, butyric acid concentration in broth; open obtriangular, biomass concentration in broth; closed squares, acetone concentration in broth; closed triangles, butanol concentration in broth; closed obtriangular, ethanol concentration in broth.
Since lignin could be considerably extractable with dilute alkali (16), the pretreatment of steam explosion coupling with alkali treated was adopted to remove lignin from SECS. Surprisingly, changed ABE ratio could also be found in ALF, it was approximately 1:7.5:10.1 (ethanol:acetone:butanol) when glucose was used as substrate, whereas for xylose, the ratio was 1:14.2:21.1. It changed largely compared to 1:3:6 in submerged fermentation. The content of ethanol decreased significantly, especially in xylose fermentation, it was less than 3% (w/w) of the total solvent. On the contrary, the relative content of acetone in the solvent increased in both of glucose and xylose fermentation, the acetone ratio went up to 40%. However, the relative amount of butanol had no obvious changes. The reasons for this phenomenon were still under investigation, but it was clear that this changed solvent composition will raise economics of the ABE fermentation. Compared with other former research reports on ABE production by this strain from glucose or xylose (13,17), ALF had obvious advantages especially in xylose fermentation. It was found that (17) a level of fermented xylose limited to 47 g/l because an initial slower growth rate and a longer metabolic transition resulted in higher cellular and acids concentration. Previous researchers (13) also reported that 7.91 g/l butanol was produced using 60 g/l xylose as the substrate, and the butanol yield on xylose reached 0.19 g/g. Analyzed the reasons for this difference, it may be that in the ALF, Clostridium could attach to the surface of SECSAT and grow on the porous surface, thus greatly increased the contact surface of the substrate and microorganisms and improved the cell culture density (the growth curve was shown in Fig. 1, the free cell concentration in ALF fermentation was a little larger than that in submerged fermentation, the absorbed cell concentration in ALF fermentation was not measured in this paper). At the same time, the concentration gradients of substrate and product between internal and surface of the substrate and the buffering effect
weakened the inhibition and poisoning on C. acetobutylicum ATCC 824, and thus greatly improved the fermentation efficiency. The detailed reasons for these phenomenons of increased butanol concentration and changed solvent ratios in ALF will be reported in more detail elsewhere. ABE production by cofermentation of glucose and xylose Owing to the removal of hemicellulose sugars by steam explosion, the water extract of SECS contained a mixture of hexoses and pentoses, always a certain proportion of glucose and xylose as the major carbohydrates, so it would be very important to convert all resulting sugars into solvents to lower the cost of fermentation. Of the strict anaerobic bacterium, C. acetobutylicum was one of the few strains able to ferment xylose and other pentoses (18), but in glucose/xylose mixtures, xylose was often left over at the end of fermentation. Glucose-mediated catabolic repression and butanol inhibition of xylose transport and metabolism were the factors that limit xylose bioconversion by C. acetobutylicum (2). Here we utilized different proportions of mixtures to study the performance of C. acetobutylicum in ALF. Table 2 shows the fermentation efficiency of three different proportions of sugar mixture. It could be seen that an increased proportion of xylose affected fermentation adversely. A former study (19) evaluated and compared the fermentability of separated versus mixed sugars. The researchers performed separate fermentations of glucose, xylose, and glucose/xylose (1:1) sugars to determine the ABE production period. During the course of fermentation, the ABE concentration increased until 60 h and then remained almost constant, possible due to end product inhibition on the microorganisms. The final ABE concentration in the mixed glucose/ xylose culture was lower than the average ABE concentration obtained in separate glucose and xylose cultures, indicating that glucose partially inhibited xylose utilization in glucose/xylose mixtures. It was coincident with the results of this paper. Another report (20) also investigated batch fermentation of glucose/xylose mixture by C. acetobutylicum ATCC 824. They found that using xylose-pregrown cells and pH control, an important amount of xylose was left over at the end of the fermentation when the glucose concentration was higher than that of xylose whereas addition of 10 g/l CaCO3 (to prevent the pH dropping below 4.8) increased xylose uptake. Total solvent of 15.8 g/l was achieved by ABE fermentation using a proportion of 30 g/l glucose and 30 g/l xylose with 10 g/l CaCO3 adding and xylose-pregrown cells used as inoculum. The concentration was obviously lower than a total solvent of 17.52 g/l obtained from the ALF in this paper using the same substrate. Fig. 1 shows the time course of sugar uptake and ABE production by C. acetobutylicum ATCC 824 on complex medium containing of 36 g/l glucose and 18 g/l xylose in ALF. From the results, we can observe that only 0.52 g/l glucose was left at 48 h, and 97.78% of xylose was utilized at 72 h, the ABE was accumulated by 16.95 g/l, and the ABE yield reached 0.32 g/g. Although the inoculum was without xylose-pregrown, xylose and glucose were used simultaneously in the fermentation process, yet a significantly lower consumption rate in xylose than glucose because of the glucosemediated catabolic repression. Composition of SECS and SECSAT before and after fermentation To remove the fermentation inhibitors in butanol production, alkaline treatment was used in this paper. First
TABLE 2. ABE production and sugar utilization using mixture of glucose and xylose with SECSAT as the absorbed substrate at 72 h. Carbon sources
Total sugar (g/L)
Consumption rate of sugar (%)
Ethanol (g/L)
Acetone (g/L)
Butanol (g/L)
Total acids (g/L)
Total solvent (g/L)
Yield of ABE (g/g)
Glucose/xylose Glucose/xylose Glucose/xylose
30/30 36/18 18/36
97.38 0.07 98.81 0.03 98.11 0.05
0.92 0.02 0.72 0.02 0.53 0.05
6.97 0.03 6.59 0.03 5.84 0.01
9.63 0.03 9.64 0.02 8.66 0.02
5.03 0.67 5.06 0.29 4.55 0.33
17.52 16.95 15.03
0.30 0.32 0.28
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TABLE 3. Chemical compositions and pore characteristics of SECS and SECSAT before and after fermentation. Sample SECS SECSAT Fermented SECSAT
Hemicellulose (%)
Cellulose (%)
Klason lignin (%)
Ash (%)
Porosity (%)
Specific surface area (m2/g)
27.15 0.64 23.73 1.27 21.75 2.46
30.33 0.85 56.78 1.22 59.21 0.85
23.01 3.31 9.06 2.13 8.73 1.62
4.15 0.42 4.47 0.26 3.65 0.72
70.75 2.13 82.61 1.78 83.03 1.64
1.067 0.15 1.686 0.22 1.693 0.27
we analyzed the chemical composition of SECS and SECSAT to prove the usefulness of alkaline treatment in removal of inhibitors such as phenolic compounds. As shown in Table 3, the lignin content in SECSAT was decreased sharply to 9.06% compared with 23.01% of that without alkaline treatment. The removal of lignin could not only remove fermentation inhibitors (6), but also increase the porosity and specific surface of the carrier. All of these made SECSAT more suitable for using as the absorbed carrier for microorganism, meanwhile the soluble lignin in alkaline liquid could be separated to produce high-value lignin-derived products, and alkali could also be recovered and reused for raw materials treatment. The compared fermentation results between SECS and SECSAT shown in Table 1 had proved the improvement effect of removal of lignin in ALF. More important, the chemical composition of SECSAT before and after fermentation varied in a small range, and the recycled substrate had an unchanged mass. This showed that in the butanol production process, the SECSAT was used as a support but not as a nutrient carrier in fermentation. This was mainly because that without cellulase activity, C. acetobutylicum ATCC 824 could not hydrolyze the glycoside linkage to get fermentable carbon sources. Thus, we tried the methods of enzymatic hydrolysis for getting more free sugars and lowering the cost of fermentation.
As shown in Fig. 2, after enzyme adding, a slight increase in glucose content was found in the fermentation broth at 36 h. This phenomenon was mainly due to the cellulase degradation of miscellaneous cells to generate some glucose, and the enzymatic rate was slightly higher than the consumption rate of glucose. Since then, the glucose content was in sharp decline, and the glucose was completely consumed at 72 h. The final butanol concentration reached 12.27 g/l, the total solvent was 21.59 g/l, compared with ALF without enzyme adding, it was increased by 22% and 16%, respectively, and compared with submerged fermentation, it enhanced the ABE concentration by 71%. This was mainly due to the partial enzyme hydrolysis of SECSAT. Microscopic images of SECSAT before and after PSSF are shown in Fig. 3. Compared with the two materials, area percent of fiber cells of the SECSAT after PSSF was significantly increased, it was 90% versus 53% that before PSSF. As we know, corn stover was not a material commonly used in the paper making mainly because of high levels of miscellaneous cells. However, this method actualized the separation of fiber cells and miscellaneous cells.
60
12
50
10
40
8
30
6
20
4 2
10 0
Products concentration (g/L)
Sugar concentration (g/L)
Partial simultaneous saccharification and fermentation of SECSAT for butanol production Fiber cells and parenchyma cells were the main parts of the fiber structure of SECSAT. Because the parenchyma cells were more susceptible to enzymatic hydrolysis to prepare fermentable monosaccharide (14), and the fiber cell-based fraction was more suitable as a paper making raw material. Thus, it would be appropriate to add a certain amount of cellulase to achieve partial saccharification and fermentation (PSSF) during the fermentation process. We took 60 g/l glucose as the fermentation substrate, and added cellulase at 24 h in ALF with the enzyme loading of 5 FPA/g dry SECSAT.
0 0
12
24
36
48
60
72
Time (h) FIG. 2. Time course of sugar uptake and ABE production by C. acetobutylicum ATCC 824 on glucose (60 g/L) with adding 5 FPA/g substrate at 24 h in ALF. Symbols: open squares, glucose concentration in broth; open obtriangular, acetic acid concentration in broth; open circles, butyric acid concentration in broth; closed squares, acetone concentration in broth; closed triangles, butanol concentration in broth; closed obtriangular, ethanol concentration in broth.
FIG. 3. Photomicrograph of SECSAT before and after PSSF (100). (A) SECSAT before fermentation. (B) SECSAT after PSSF.
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Corn stover steam explosion Steam-exploded corn stover alkai treatment Liquid medium
Alkali-treated steamexploded corn stover
mixed Butanol fermentation
ABE solvent
enzymatic hydrolysis
Cellulase
Fermented solid carrier paper making Paper
FIG. 4. The flow diagram of butanol fermentation.
Additionally, the tensile index of this sheet reached 41.68 N m/g indicating adequate strength properties for paper making. Fig. 4 shows the detailed flow diagram of butanol fermentation by ALF to explain the novel technology clearly. Conclusions A novel technology for butanol production by ALF was studied. This technology could not only increase the ABE yield, but also change the solvent ratio. Mixed pentose and glucose fermentation also showed higher fermentability although lower than the average level of separated sugar fermentation. Furthermore, PSSF of SECSAT for butanol production could increase fermentability and prepare an excellent paper making material. All of these results would economize industrial production of ABE. ACKNOWLEDGMENTS Financial support to this study was provided by the National Basic Research Program of China (973 Project, No. 2011CB707401), the National High Technology Research and Development Program of China (863 Program, SS2012AA022502), and the National Key Project of Scientific and Technical Supporting Program of China (No. 2011BAD22B02).
1. Sabathé, F. and Soucaille, P.: Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum, FEMS Microbiol. Lett., 217, 15e22 (2002). 2. Ounine, K., Petitdemange, H., Raval, G., and Gay, R.: Regulation and butanol inhibition of D-xylose and D-glucose uptake in Clostridium acetobutylicum, Appl. Environ. Microbiol., 49, 874e878 (1985). 3. Ooijkaas, L. P., Weber, F. J., Buitelaar, R. M., Tramper, J., and Rinzema, A.: Defined media and inert supports: their potential as solid-state fermentation production systems, Trends Biotechnol., 18, 356e360 (2000). 4. Chen, H. Z. and Liu, L. Y.: Unpolluted fractionation of wheat straw by steam explosion and ethanol extraction, Bioresour. Technol., 98, 666e676 (2007). 5. Ramos, L. P.: The chemistry involved in the steam treatment of lignocellulosic materials, Quim. Nova, 26, 863e871 (2003). 6. Wang, L. and Chen, H. Z.: Increased fermentability of enzymatically hydrolyzed steam-exploded corn stover for butanol production by removal of fermentation inhibitors, Process Biochem., 46, 604e607 (2011). 7. Koukiekolo, R., Cho, H. Y., Kosugi, A., Inui, M., Yukawa, H., and Doi, R. H.: Degradation of corn fiber by Clostridium cellulovorans cellulases and hemicellulases and contribution of scaffolding protein CbpA, Appl. Environ. Microbiol., 71, 3504e3511 (2005). 8. Sánchez, O., Sierra, R., and Alméciga-Díaz, C. J.: Delignification process of agro-industrial wastes an alternative to obtain fermentable carbohydrates for producing fuel, pp. 111e154, in: Manzanera, M. (Ed.), Alternative fuel. InTech (2011). 9. Li, H. Q. and Chen, H. Z.: Detoxification of steam-exploded corn straw produced by an industrial-scale reactor, Proc. Biochem., 43, 1447e1451 (2008). 10. Goering, H. and Van Soest, P. J.: Forage fiber analysis: apparatus, reagents, procedures, and some applications, vol. 379. US Department of Agriculture, Washington, DC, USA (1970). 11. George, H., Johnson, J., Moore, W., Holdeman, L., and Chen, J.: Acetone, isopropanol, and butanol production by Clostridium beijerinckii (syn. Clostridium butylicum) and Clostridium aurantibutyricum, Appl. Environ. Microbiol., 45, 1160e1163 (1987). 12. Ghose, T.: Measurement of cellulase activities, Pure Appl. Chem., 59, 257e268 (1987). 13. Sun, Z. J. and Liu, S. J.: Production of n-butanol from concentrated sugar maple hemicellulosic hydrolysate by Clostridia acetobutylicum ATCC 824, Biomass Bioenergy, 39, 39e47 (2012). 14. Chen, H. Z., Li, H. Q., and Liu, L. Y.: The inhomogeneity of corn stover and its effects on bioconversion, Biomass Bioenergy, 35, 1940e1945 (2011). 15. Ezeji, T., Qureshi, N., and Blaschek, H. P.: Butanol production from agricultural residues: impact of degradation products on Clostridium beijerinckii growth and butanol fermentation, Biotechnol. Bioeng., 97, 1460e1469 (2007). 16. Chen, H. Z., Han, Y. J., and Xu, J.: Simultaneous saccharification and fermentation of steam exploded wheat straw pretreated with alkaline peroxide, Proc. Biochem., 43, 1462e1466 (2008). 17. Fond, O., Engasser, J. M., Matta-El-Amouri, G., and Petitdemange, H.: The acetone butanol fermentation on glucose and xylose. I. Regulation and kinetics in batch cultures, Biotechnol. Bioeng., 28, 160e166 (1986). 18. Ounine, K., Petitdemange, H., Raval, G., and Gay, R.: Acetoneebutanol production from pentoses by Clostridium acetobutylicum, Biotechnol. Lett., 5, 605e610 (1986). 19. Mu, X. D., Sun, W., Liu, C., and Wang, H. S.: Improved efficiency of separate hexose and pentose fermentation from steam-exploded corn stalk for butanol production using Clostridium beijerinckii, Biotechnol. Lett., 33, 1587e1591 (2011). 20. Kanouni, A. E., Zerdani, I., Zaafa, S., Znassni, M., Loutfi, M., and Boudouma, M.: The improvement of glucose/xylose fermentation by Clostridium acetobutylicum using calcium carbonate, World J. Microbiol. Biotechnol., 14, 431e435 (1998).
Improved efficiency of butanol production by absorbed lignocellulose fermentation Qin He and Hongzhang Chen* National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China Received 23 May 2012; accepted 22 September 2012 Available online 22 October 2012
Alkali-treated steam-exploded corn stover (SECSAT) was used as solid substrate for acetoneebutanoleethanol (ABE) production by absorbed lignocellulose fermentation (ALF) using Clostridium acetobutylicum ATCC 824. The ABE concentration in ALF culture had increased by 47% compared with that in submerged culture. More surprisingly, the acetone production was promoted and ethanol production was lower in the presence of SECSAT than that in its absence. ALF was also successfully in cofermentation of glucose and xylose, although decreased fermentability with an increase in the proportion of xylose. An invariable chemical composition and dry weight of SECSAT was found in ALF. Partial simultaneous saccharification and fermentation of SECSAT using a certain amount of cellulase could not only enhance the ABE concentration by 71%, but also significantly increase the area proportion of fiber cells in SECSAT from 53% to 90%, which would be an excellent paper making material. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Acetoneebutanoleethanol (ABE) fermentation; Steam explosion; Absorbed lignocellulose fermentation; Alkaline treatment; Corn stover; Fiber cells]
The interests in production of butanol as a chemical and also as an alternative liquid fuel have renewed due to the depletion of fossil fuels and deterioration of global environment situation in recent years. However, compared with petrochemical-derived butanol, biobutanol production is still not in economically competitive, because of its major drawbacks: high cost of feedstocks, low butanol concentration in fermentation broth and coproduction of low-value byproducts of acetone and ethanol. Although great progress has been made on genetics, upstream processing, fermentation and downstream processing recently, the acetoneebutanoleethanol (ABE) fermentation is yet to reach a sustainable and economically viable process. Biobutanol is now usually produced industrially by submerged fermentation (SmF) in which the microorganism and nutrient source are normally suspended or dissolved in a liquid medium and the growth takes place as a dispersed cell suspension. Solid-state fermentation (SSF) systems exhibit several advantages over SmF, including improved product characteristics, higher product yields, easier product recovery and reduced energy requirements despite heat and mass transfer problems and growth limited by low water content. However, Clostridium acetobutylicum was unable to grow on solid cellulosic substrates (1), in this case, an efficient process for fermentation, absorbed substrate fermentation (ASF) might be an attractive alternative to SSF for the production of butanol due to the * Corresponding author. Tel.: þ86 10 82544982; fax: þ86 10 28627071. E-mail address: [email protected] (H. Chen).
fact that medium composition can be designed as it is done in submerged culture. Meanwhile, the substrate used in the ASF process is similar to SSF, it behaves the nature of high porosity, good mechanical resistance and better water absorbency. In the former studies, researchers evaluated the potential of bagasse to be used as inert substrate (2). Bagasse was impregnated with a liquid medium containing glucose and calcium carbonate for lactic acid production. An increased yield and productivity of lactic acid were found in ASF. The solid support used in ASF includes: hemp, perlite, polyurethane foam, sugarcane bagasse and vermiculite (3). The authors considered lignocellulosic biomass as an effective potential substrate in the sense of porosity and absorb ability. Porous lignocellulosic carrier almost behaves all the characteristics of the ideal cell supports, in particular, could solve problems of cell injury caused by the immobilized cells and shear stress, could improve the cell density to facilitate large-scale cell culture, at the same time could greatly reduce inhibition on microorganisms because the concentration gradients of substrate and product between the internal and surface of substrate. However, more work should be done to improve the porosity of lignocellulosic biomass. As a pretreatment method for increasing porosity of lignocellulose and making lignocellulose more accessible to microorganisms and nutrients, steam explosion is an effective technique (4). Unfortunately, a wide range of compounds, some of which are toxic to butanol production is generated during pretreatment of lignocellulose (5). Previous works have proved that phenolic compounds (6) from lignin degradation are more
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.09.017
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inhibitory than weak acids, furan derivatives, in either cell growth or ABE production. Thus, the removal of inhibitors prior to fermentation is essential for successful butanol production from absorbed lignocellulose fermentation (ALF). Corresponding methods include water washing (7), delignification (8), etc. After detoxification, increase of porosity and surface area is found in the steam-exploded lignocellulose. The objective of this work is to evaluate the applicability and performance of using the absorbed lignocellulose fermentation technique for butanol production by C. acetobutylicum ATCC 824 growing on treated corn stover impregnated with different liquid medium. The characteristics of the solid substrate are also investigated to increase the fermentability and economic competitiveness of butanol production.
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Rad Laboratories Inc.) and a refractive index detector. The mobile phase was 0.5 mM H2SO4 at a flow rate of 0.5 mL/min at 65 C. Sugar utilization was calculated as utilized sugars (g/l) divided by added sugars (g/l) and was expressed in percentage. ABE and acid (acetic and butyric) were measured using a gas chromatograph (7890A, Agilent Technologies) equipped with a flame ionization detector (FID) and an HP-Innowax capillary column (30 m 0.32 mm). Oven temperature was held at 85 C for 4.5 min, programmed at increments of 20 C per min until 170 C, the final temperature was held for 2.5 min. Both injector and detector temperatures were set at 250 C. Nitrogen was used as the carrier gas and isobutanol was used as the internal standard. Productivity was calculated as the maximum ABE concentration achieved (g/l) divided by the fermentation time and is expressed as g/(l h). Yield was calculated as the total amount of solvents produced divided by the amount of fermentable sugar utilized and was expressed as g/g. The concentration of C. acetobutylicum in the fermentation broth was measured as OD 600 nm using an ultravioletevisible spectrophotometer (UV-1240, Shimadzu Co., Kyoto, Japan). The measured OD was correlated with dry weight using an established proportional constant (13). Pulp properties of residual solid substrate after partial simultaneous saccharification and fermentation After partial simultaneous saccharification and fermentation, the carrier was collected using a plastic 60-mL syringe, at the front end of which several holes of 1 mm diameter were punched. Then the carrier was dried after water washed. Fiber cell content was observed and determined through a light microscope after the carrier was separated in the acetic acid and hydrogen peroxide (30%) solution (1:1, v/v) for about 48 h at 60 C. The UV-G microscopic particle size analysis software was used to measure the length and diameter of each cell. Fiber cell content was expressed by area proportion, and fiber content of the carrier before fermentation was performed as control (14). The pulp was achieved by the followed process: fermented SECSAT were pretreated with 2% NaOH solution (w:v) at 115 C for 1 h with the solideliquid ratio maintained at 1:10 (w:v). The residual solid was rinsed to neutral pH with distilled water and bleached by 1% NaOH (w/v) and 4% H2O2 (w/v) at 60 C for 2 h. The pulp characteristics of this material were then studied. The pulps, with the beating degree of 38 SR and the grammage of 60 g/m2, were kept and tested under controlled environment (temperature of 23 1 C and RH of 50 1%). The tensile index was measured by the tensile testing machine (DLD-1, Beijing Guance Testing Machine Ltd., China) according to China national standard no. GB/T 453-2002.
MATERIALS AND METHODS Corn stover pretreatment The corn stover was harvested from the suburb of Beijing, China. After air-dried, the stover was chopped into 2e4 cm in length. About 200 g air-dried chipped corn stover was soaked in 200 mL distilled water for 15 min. Steam explosion pretreatment was carried out in a 7.5 L batch reactor (Weihai Automatic Control Reactor Ltd., China) as described in a previous work (9). The chipped stalk was fed into the reactor and the pretreatment was operated at 1.1 MPa for 4 min. After steam explosion, the material was washed with 1 L of 80 C water and filtered by nylon cloth (200 mesh). The washed steam-exploded corn stover (SECS) was dried at 60 C until constant weight. SECS was soaked in distilled water containing 1% NaOH (w/v) for 24 h at room temperature for alkaline treatment, the solid substrate concentration was 10% (w/v). Solid residues were thoroughly washed with water until neuter pH was achieved, and then filtered by nylon cloth (200 mesh). Alkali-treated SECS (SECSAT) was dried at 60 C oven to constant weight and stored at room temperature. Properties of solid substrate The chemical composition of raw and pretreated corn stover was determined according to the methods previously described (10). The porosity was measured by mercury porosimetry method using a mercury porosimeter (Micromeritics Autopore IV, Micromeritics, USA). The specific surface area was obtained by the standard BrunauereEmmetteTeller (BET) method using Beishide 3H-2000A apparatus (Beishide instrument-S&T Co., Ltd., Beijing).
RESULTS AND DISCUSSION Applicability of butanol production by absorbed lignocellulose fermentation The applicability of butanol production by ALF was tested firstly and the results were shown in Table 1. After 72-h anaerobic fermentation, nearly 99% of sugar was consumed when 60 g/l glucose was used as carbon source and the solideliquid ratio was 1:10 (w/v) in the fermentation. A total of 18.54 g/l ABE were produced and of which 10.07 g/l were butanol. An ABE yield of 0.31 g/g and a productivity of 0.26 g/(l h) were obtained. Cells which were grown in 60 g/l glucose in submerged fermentation produced 12.63 g/l ABE. The ABE concentration had increased by 47% compared with control exercise. Performance of ABE production using 54 g/l xylose was also investigated in this paper. ABE (14.55 g/l) were produced and of which 8.44 g/l were butanol, an ABE yield of 0.27 g/g and a productivity of 0.20 g/(l h) were reached eventually. Water washed SECS was also used as absorbed substrate for butanol fermentation. The efficiency of fermentation using this carrier was significantly lower than that of SECSAT added. This may be due to inhibitors absorbed on the steam-exploded straw (15), so pretreatment and detoxification of this material were essential.
Microorganism and butanol fermentation C. acetobutylicum ATCC 824 was maintained as a spore suspension in 6% (w/v) corn mash at 4 C. The preculture medium contained the following components per liter of distilled water: 30 g glucose, 4.3 g CH3COONH4, 1.768 g KH2PO4, 2.938 g K2HPO4, 10 mg p-aminobenzoic acid, 10 mg biotin, and 1 mL mineral salts solution (11). The initial pH of the medium was adjusted to 6.5 0.2 with 1 M NaOH or 1 M H2SO4. The medium was sterilized at 115 C for 15 min. Cells were grown anaerobically at 37 C for 20e36 h without agitation before being transferred into the fermentation medium. Batch fermentation experiments were carried out in a 100-mL serum bottle with a 60 mL working volume and 6 g air-dried SECSAT. The fermentation medium was similar to the seed culture medium except carbon sources. The pH of the medium was adjusted to 6.5 0.2 before autoclaving at 115 C for 15 min. After cooling to room temperature, the fermentation media were inoculated with 6 mL inoculum, and then infused with filtered oxygen-free nitrogen gas to maintain strict anaerobic conditions. Cultures were incubated at 37 C for 72 h without agitation. Samples were withdrawn at intervals for ABE, acids, and sugar analyses. All experiments were carried out at least twice to ensure reproducibility. Cellulase was added in the partial simultaneous saccharification and fermentation (PSSF) at 24 h after inoculation and at a loading of 5 FPA/g dry substrate. The filter paper activity of cellulase was measured by DNS method (12). Analytical procedures for butanol fermentation Glucose and xylose were determined by high-performance liquid chromatography (Agilent 1200 HPLC, Agilent Technologies, USA) with an Aminex HPX-87H column (300 mm 7.8 mm, Bio-
TABLE 1. Improved efficiency of ABE production and sugar utilization in absorbed lignocellulose fermentation. Carbon sources
Total sugar (g/L)
Controla Glucoseb Glucosec Xylosed a b c d
Using Using Using Using
60 60 60 54 60 60 60 54
g/l g/l g/l g/l
Consumption ratio of sugar (%) 97.00 88.75 98.73 98.43
0.83 1.00 0.67 2.79
Ethanol (g/L) 1.05 0.67 1.00 0.40
0.02 0.01 0.01 0.01
Acetone (g/L) 3.97 5.19 7.47 5.71
0.03 0.05 0.04 0.03
glucose as carbon source (CS), submerged fermentation, 72 h. glucose as CS, SECS as substrate, absorbed lignocellulose fermentation, 72 h. glucose as CS, SECSAT as substrate, absorbed lignocellulose fermentation, 72 h. xylose as CS, SECS as substrate, absorbed lignocellulose fermentation, 72 h.
Butanol (g/L) 7.61 7.17 10.07 8.44
0.03 0.03 0.04 0.02
Total acids (g/L)
Total solvent (g/L)
Yield of ABE (g/g)
12.63 13.03 18.54 14.55
0.22 0.24 0.31 0.27
3.11 2.28 2.02 5.33
0.17 0.07 0.12 0.30
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40
3
2
1
30
8
25 6
20 15
4
10 2
5 0
0
Products concentration (g/L)
35 4
Sugar concentration (g/L)
Biomass concentration (g/L)
10
0 0
12
24
36
48
60
72
Time (h) FIG. 1. Time course of sugar uptake and ABE production by C. acetobutylicum ATCC 824 on complex medium containing of 36 g/l glucose and 18 g/l xylose in ALF. Symbols: open squares, glucose concentration in broth; open triangles, xylose concentration in broth; open obtriangular, acetic acid concentration in broth; open circles, butyric acid concentration in broth; open obtriangular, biomass concentration in broth; closed squares, acetone concentration in broth; closed triangles, butanol concentration in broth; closed obtriangular, ethanol concentration in broth.
Since lignin could be considerably extractable with dilute alkali (16), the pretreatment of steam explosion coupling with alkali treated was adopted to remove lignin from SECS. Surprisingly, changed ABE ratio could also be found in ALF, it was approximately 1:7.5:10.1 (ethanol:acetone:butanol) when glucose was used as substrate, whereas for xylose, the ratio was 1:14.2:21.1. It changed largely compared to 1:3:6 in submerged fermentation. The content of ethanol decreased significantly, especially in xylose fermentation, it was less than 3% (w/w) of the total solvent. On the contrary, the relative content of acetone in the solvent increased in both of glucose and xylose fermentation, the acetone ratio went up to 40%. However, the relative amount of butanol had no obvious changes. The reasons for this phenomenon were still under investigation, but it was clear that this changed solvent composition will raise economics of the ABE fermentation. Compared with other former research reports on ABE production by this strain from glucose or xylose (13,17), ALF had obvious advantages especially in xylose fermentation. It was found that (17) a level of fermented xylose limited to 47 g/l because an initial slower growth rate and a longer metabolic transition resulted in higher cellular and acids concentration. Previous researchers (13) also reported that 7.91 g/l butanol was produced using 60 g/l xylose as the substrate, and the butanol yield on xylose reached 0.19 g/g. Analyzed the reasons for this difference, it may be that in the ALF, Clostridium could attach to the surface of SECSAT and grow on the porous surface, thus greatly increased the contact surface of the substrate and microorganisms and improved the cell culture density (the growth curve was shown in Fig. 1, the free cell concentration in ALF fermentation was a little larger than that in submerged fermentation, the absorbed cell concentration in ALF fermentation was not measured in this paper). At the same time, the concentration gradients of substrate and product between internal and surface of the substrate and the buffering effect
weakened the inhibition and poisoning on C. acetobutylicum ATCC 824, and thus greatly improved the fermentation efficiency. The detailed reasons for these phenomenons of increased butanol concentration and changed solvent ratios in ALF will be reported in more detail elsewhere. ABE production by cofermentation of glucose and xylose Owing to the removal of hemicellulose sugars by steam explosion, the water extract of SECS contained a mixture of hexoses and pentoses, always a certain proportion of glucose and xylose as the major carbohydrates, so it would be very important to convert all resulting sugars into solvents to lower the cost of fermentation. Of the strict anaerobic bacterium, C. acetobutylicum was one of the few strains able to ferment xylose and other pentoses (18), but in glucose/xylose mixtures, xylose was often left over at the end of fermentation. Glucose-mediated catabolic repression and butanol inhibition of xylose transport and metabolism were the factors that limit xylose bioconversion by C. acetobutylicum (2). Here we utilized different proportions of mixtures to study the performance of C. acetobutylicum in ALF. Table 2 shows the fermentation efficiency of three different proportions of sugar mixture. It could be seen that an increased proportion of xylose affected fermentation adversely. A former study (19) evaluated and compared the fermentability of separated versus mixed sugars. The researchers performed separate fermentations of glucose, xylose, and glucose/xylose (1:1) sugars to determine the ABE production period. During the course of fermentation, the ABE concentration increased until 60 h and then remained almost constant, possible due to end product inhibition on the microorganisms. The final ABE concentration in the mixed glucose/ xylose culture was lower than the average ABE concentration obtained in separate glucose and xylose cultures, indicating that glucose partially inhibited xylose utilization in glucose/xylose mixtures. It was coincident with the results of this paper. Another report (20) also investigated batch fermentation of glucose/xylose mixture by C. acetobutylicum ATCC 824. They found that using xylose-pregrown cells and pH control, an important amount of xylose was left over at the end of the fermentation when the glucose concentration was higher than that of xylose whereas addition of 10 g/l CaCO3 (to prevent the pH dropping below 4.8) increased xylose uptake. Total solvent of 15.8 g/l was achieved by ABE fermentation using a proportion of 30 g/l glucose and 30 g/l xylose with 10 g/l CaCO3 adding and xylose-pregrown cells used as inoculum. The concentration was obviously lower than a total solvent of 17.52 g/l obtained from the ALF in this paper using the same substrate. Fig. 1 shows the time course of sugar uptake and ABE production by C. acetobutylicum ATCC 824 on complex medium containing of 36 g/l glucose and 18 g/l xylose in ALF. From the results, we can observe that only 0.52 g/l glucose was left at 48 h, and 97.78% of xylose was utilized at 72 h, the ABE was accumulated by 16.95 g/l, and the ABE yield reached 0.32 g/g. Although the inoculum was without xylose-pregrown, xylose and glucose were used simultaneously in the fermentation process, yet a significantly lower consumption rate in xylose than glucose because of the glucosemediated catabolic repression. Composition of SECS and SECSAT before and after fermentation To remove the fermentation inhibitors in butanol production, alkaline treatment was used in this paper. First
TABLE 2. ABE production and sugar utilization using mixture of glucose and xylose with SECSAT as the absorbed substrate at 72 h. Carbon sources
Total sugar (g/L)
Consumption rate of sugar (%)
Ethanol (g/L)
Acetone (g/L)
Butanol (g/L)
Total acids (g/L)
Total solvent (g/L)
Yield of ABE (g/g)
Glucose/xylose Glucose/xylose Glucose/xylose
30/30 36/18 18/36
97.38 0.07 98.81 0.03 98.11 0.05
0.92 0.02 0.72 0.02 0.53 0.05
6.97 0.03 6.59 0.03 5.84 0.01
9.63 0.03 9.64 0.02 8.66 0.02
5.03 0.67 5.06 0.29 4.55 0.33
17.52 16.95 15.03
0.30 0.32 0.28
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TABLE 3. Chemical compositions and pore characteristics of SECS and SECSAT before and after fermentation. Sample SECS SECSAT Fermented SECSAT
Hemicellulose (%)
Cellulose (%)
Klason lignin (%)
Ash (%)
Porosity (%)
Specific surface area (m2/g)
27.15 0.64 23.73 1.27 21.75 2.46
30.33 0.85 56.78 1.22 59.21 0.85
23.01 3.31 9.06 2.13 8.73 1.62
4.15 0.42 4.47 0.26 3.65 0.72
70.75 2.13 82.61 1.78 83.03 1.64
1.067 0.15 1.686 0.22 1.693 0.27
we analyzed the chemical composition of SECS and SECSAT to prove the usefulness of alkaline treatment in removal of inhibitors such as phenolic compounds. As shown in Table 3, the lignin content in SECSAT was decreased sharply to 9.06% compared with 23.01% of that without alkaline treatment. The removal of lignin could not only remove fermentation inhibitors (6), but also increase the porosity and specific surface of the carrier. All of these made SECSAT more suitable for using as the absorbed carrier for microorganism, meanwhile the soluble lignin in alkaline liquid could be separated to produce high-value lignin-derived products, and alkali could also be recovered and reused for raw materials treatment. The compared fermentation results between SECS and SECSAT shown in Table 1 had proved the improvement effect of removal of lignin in ALF. More important, the chemical composition of SECSAT before and after fermentation varied in a small range, and the recycled substrate had an unchanged mass. This showed that in the butanol production process, the SECSAT was used as a support but not as a nutrient carrier in fermentation. This was mainly because that without cellulase activity, C. acetobutylicum ATCC 824 could not hydrolyze the glycoside linkage to get fermentable carbon sources. Thus, we tried the methods of enzymatic hydrolysis for getting more free sugars and lowering the cost of fermentation.
As shown in Fig. 2, after enzyme adding, a slight increase in glucose content was found in the fermentation broth at 36 h. This phenomenon was mainly due to the cellulase degradation of miscellaneous cells to generate some glucose, and the enzymatic rate was slightly higher than the consumption rate of glucose. Since then, the glucose content was in sharp decline, and the glucose was completely consumed at 72 h. The final butanol concentration reached 12.27 g/l, the total solvent was 21.59 g/l, compared with ALF without enzyme adding, it was increased by 22% and 16%, respectively, and compared with submerged fermentation, it enhanced the ABE concentration by 71%. This was mainly due to the partial enzyme hydrolysis of SECSAT. Microscopic images of SECSAT before and after PSSF are shown in Fig. 3. Compared with the two materials, area percent of fiber cells of the SECSAT after PSSF was significantly increased, it was 90% versus 53% that before PSSF. As we know, corn stover was not a material commonly used in the paper making mainly because of high levels of miscellaneous cells. However, this method actualized the separation of fiber cells and miscellaneous cells.
60
12
50
10
40
8
30
6
20
4 2
10 0
Products concentration (g/L)
Sugar concentration (g/L)
Partial simultaneous saccharification and fermentation of SECSAT for butanol production Fiber cells and parenchyma cells were the main parts of the fiber structure of SECSAT. Because the parenchyma cells were more susceptible to enzymatic hydrolysis to prepare fermentable monosaccharide (14), and the fiber cell-based fraction was more suitable as a paper making raw material. Thus, it would be appropriate to add a certain amount of cellulase to achieve partial saccharification and fermentation (PSSF) during the fermentation process. We took 60 g/l glucose as the fermentation substrate, and added cellulase at 24 h in ALF with the enzyme loading of 5 FPA/g dry SECSAT.
0 0
12
24
36
48
60
72
Time (h) FIG. 2. Time course of sugar uptake and ABE production by C. acetobutylicum ATCC 824 on glucose (60 g/L) with adding 5 FPA/g substrate at 24 h in ALF. Symbols: open squares, glucose concentration in broth; open obtriangular, acetic acid concentration in broth; open circles, butyric acid concentration in broth; closed squares, acetone concentration in broth; closed triangles, butanol concentration in broth; closed obtriangular, ethanol concentration in broth.
FIG. 3. Photomicrograph of SECSAT before and after PSSF (100). (A) SECSAT before fermentation. (B) SECSAT after PSSF.
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Corn stover steam explosion Steam-exploded corn stover alkai treatment Liquid medium
Alkali-treated steamexploded corn stover
mixed Butanol fermentation
ABE solvent
enzymatic hydrolysis
Cellulase
Fermented solid carrier paper making Paper
FIG. 4. The flow diagram of butanol fermentation.
Additionally, the tensile index of this sheet reached 41.68 N m/g indicating adequate strength properties for paper making. Fig. 4 shows the detailed flow diagram of butanol fermentation by ALF to explain the novel technology clearly. Conclusions A novel technology for butanol production by ALF was studied. This technology could not only increase the ABE yield, but also change the solvent ratio. Mixed pentose and glucose fermentation also showed higher fermentability although lower than the average level of separated sugar fermentation. Furthermore, PSSF of SECSAT for butanol production could increase fermentability and prepare an excellent paper making material. All of these results would economize industrial production of ABE. ACKNOWLEDGMENTS Financial support to this study was provided by the National Basic Research Program of China (973 Project, No. 2011CB707401), the National High Technology Research and Development Program of China (863 Program, SS2012AA022502), and the National Key Project of Scientific and Technical Supporting Program of China (No. 2011BAD22B02).
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