Bioresource Technology 157 (2014) 1–5

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Saccharification behavior of cellulose acetate during enzymatic processing for microbial ethanol production Shinji Hama a, Kohsuke Nakano a, Kaoru Onodera a, Masashi Nakamura a, Hideo Noda a, Akihiko Kondo b,⇑ a b

Bio-energy Corporation, Research and Development Laboratory, 2-9-7 Minaminanamatsu, Amagasaki 660-0053, Japan Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan

h i g h l i g h t s  The potential application of cellulose acetate to enzymatic processing.  Prior to saccharification, cellulose acetate was subjected to deacetylation.  After deacetylation, enzymatic saccharification at 50 °C yielded 88.1–99.1%.  Cellulose acetate exhibited a temperature dependence during saccharification.  Presaccharification prior to SSF was found effective for increasing ethanol yield.

a r t i c l e

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Article history: Received 22 October 2013 Received in revised form 29 December 2013 Accepted 2 January 2014 Available online 10 January 2014 Keywords: Acetyl cellulose Deacetylation Cellulase Biorefinery Microbial fermentation

a b s t r a c t This study was conducted to realize the potential application of cellulose acetate to enzymatic processing, followed by microbial ethanol fermentation. To eliminate the effect of steric hindrance of acetyl groups on the action of cellulase, cellulose acetate was subjected to deacetylation in the presence of 1N sodium hydroxide and a mixture of methanol/acetone, yielding 88.8–98.6% at 5–20% substrate loadings during a 48 h saccharification at 50 °C. Ethanol fermentation using Saccharomyces cerevisiae attained a high yield of 92.3% from the initial glucose concentration of 44.2 g/L; however, a low saccharification yield was obtained at 35 °C, decreasing efficiency during simultaneous saccharification and fermentation (SSF). Presaccharification at 50 °C prior to SSF without increasing the total process time attained the ethanol titers of 19.8 g/L (5% substrate), 38.0 g/L (10% substrate), 55.9 g/L (15% substrate), and 70.9 g/L (20% substrate), which show a 12.0–16.2% improvement in ethanol yield. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Cellulose acetate is one of the important cellulose derivatives for producing various consumer products including textiles, plastic films, and cigarette filters. The properties of cellulose acetate depend on its degree of substitution (DS), and cellulose diacetate with a DS of 2.5 is widely used because of its good solubility in solvents, molecular weight, and melt properties (Puls et al., 2011). Recently, how to reduce the tremendous amount of wastes generated from daily life and industry has become a serious problem of public concern. Since the global production of cellulose-acetate-based materials exceeds 800,000 tons per year (Puls et al., 2011), investigations on the biological utilization of cellulose acetate could prove useful for diminishing the environmental impact of these waste materials. Although there is a question of whether cellulose acetate is susceptible to biological degradation, recent studies have led to a ⇑ Corresponding author. Tel./fax: +81 78 803 6196. E-mail address: [email protected] (A. Kondo). 0960-8524/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2014.01.002

considerable increase in the knowledge of the biodegradability of cellulose acetate. Researchers have thus far isolated bacterial strains capable of degrading cellulose acetate with a DS of 1.7– 2.5 (Moriyoshi et al., 2002; Ishigaki et al., 2000). Previous studies on the effect of DS on the biological utilization of cellulose acetate have been reviewed (Puls et al., 2011). The importance of deacetylation in the efficient biological degradation of cellulose acetate has been elucidated, in which acetyl esterases play a crucial role in the initial step and the resulting cellulose backbone is susceptible to cellulase action (Puls, 2004). Acetyl groups can also be eliminated by chemical hydrolysis using base catalysts. During chemical hydrolysis at room temperature, the acetyl content in cellulose decreases, and the glycosidic linkages are rather stable. The deacetylated cellulose with a DS of less than 0.8 is easily degraded by subsequent cellulase treatment (He et al., 2008). Biorefinery is a concept that describes the production of fuels and chemicals from renewable resources via biological processes. Cellulose acetate is a renewable resource-based biopolymer produced from natural polysaccharides, and shows considerable potential to expand its applications to environmentally friendly

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S. Hama et al. / Bioresource Technology 157 (2014) 1–5

materials (Mohanty et al., 2003). However, compared with the investigation on the degradation behavior of cellulose-acetatebased materials, few investigations have been reported on the production of fuels and chemicals through the biological utilization of cellulose acetate. Microbial ethanol fermentation from saccharified cellulose has been studied extensively and is one of the approaches to assessing the availability of cellulosic materials as potential resources. In general, two ethanol fermentation processes have been proposed: separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). SHF, in which the activities of cellulase and microorganisms are inhibited by a high glucose concentration, requires a long overall process time. In SSF, the glucose produced during microbial fermentation can be converted rapidly into ethanol, thereby reducing process time (Alfani et al., 2000). Inevitably, however, there are also disadvantages of SSF over SHF; the optimum temperature in enzymatic saccharification is often higher than that in fermentation (Olofsson et al., 2008). In this study, the potential use of cellulose acetate for biorefinery purpose was investigated through chemical deacetylation, enzymatic hydrolysis, and microbial ethanol fermentation. Particular attention is paid to the saccharification behavior of cellulose acetate depending on temperature and biomass concentration. The comparison of saccharification behavior and a strategy to increase process performance are also discussed. 2. Methods 2.1. Materials Cellulose acetate was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). The specifications of cellulose acetate given by the supplier are as follows: acetyl content, 38–40%; degree of substitution, 2.4; mean degree of polymerization, 150; and molecular weight, approximately 40,000. The lignocellulosic material used in this study was a water-insoluble fraction of rice straw obtained by hot water treatment, as described previously (Matano et al., 2012). Commercial cellulase, Cellic CTec2 (Novozymes, Bagsvaerd, Denmark) with a filter paper unit (FPU) of 120 per 1 ml (measured in our laboratory), was used for enzymatic saccharification. All other chemicals were of analytical grade. For microbial fermentation, the yeast Saccharomyces cerevisiae NBRC1440 (Matano et al., 2012) was used for ethanol fermentation. 2.2. Deacetylation of cellulose acetate Cellulose acetate (1 g) was immersed in 20 ml of methanol/acetone (1:1, v/v), into which 1 ml of 1 N sodium hydroxide was added. The mixture was then allowed to stand at room temperature for 2 h. The cellulose acetate collected was washed thoroughly with distilled water and 50 mM citric acid buffer (pH 5.0), dried at 70 °C for 24 h, and then ground into powder prior to use. The weight loss of the sample was calculated as the difference in weight before and after deacetylation (He et al., 2008). The glucose content in cellulose acetate was determined by sulfuric acid hydrolysis as described previously (Sluiter et al., 2008). The resultant sugar solutions were analyzed by high-performance liquid chromatography (HPLC) as described in the next section. The acetyl content in cellulose acetate was determined by the Eberstadt method (Genung and Mallatt, 1941). One gram of dry cellulose acetate was added to 40 ml of 75% ethanol, heated at 60 °C for 30 min, and saponified with 40 ml of 0.5N sodium hydroxide by heating at 60 °C for 15 min. The mixture was allowed to stand at room temperature for 48 h. An excess amount of 0.5N

hydrochloric acid was then added to the saponified solution. The excess hydrochloric acid was titrated with 0.5 N sodium hydroxide to pH 7.0. A parallel blank experiment using crystalline cellulose was also carried out and acetyl content was calculated as follows:

Acetyl content ð%Þ ¼ fðA  BÞNb  ðC  DÞNag  4:3=W where A: volume (ml) of 0.5N NaOH added to the sample; B: volume (ml) of 0.5N NaOH added to the blank; C: volume (ml) of 0.5N HCl added to the sample; D: volume (ml) of 0.5N HCl added to the blank; Na: normality of hydrochloric acid; Nb: normality of sodium hydroxide; W: dry weight (g) of the sample. Changes in the chemical bonds and molecular structure of cellulose acetate were analyzed using a Fourier transform infrared (FT-IR) spectrometer (IRAffinity-1, Shimadzu Corp., Kyoto, Japan) equipped with an attenuated total reflection system. Spectra were obtained from an average of 20 scans from 700 to 4000 cm1 with 8 cm1 resolution. 2.3. Enzymatic hydrolysis Cellulose acetate samples (5–20% loadings based on dry weight) were suspended in 50 mM citric acid buffer (pH 5.0). Enzymatic hydrolysis was initiated by adding cellulose to the samples at a dosage of 20 FPU/g cellulose acetate (unless otherwise noted). 50 ml polypropylene tubes (Corning Inc., NY, USA) containing the above solutions were incubated at 35 or 50 °C in a rotator (Thermo Block Rotator SN-06BN; Nissin, Tokyo, Japan). The sugar solutions obtained at specified intervals were analyzed by HPLC using an ULTRON PS-80H column (300  8.0 mm I.D.; Shinwa Chemical Industries Ltd., Kyoto, Japan) maintained at 80 °C. The mobile phase (pH 2.1) was prepared by adding perchloric acid to distilled water, which was then flowed at a rate of 0.7 ml/min. The absorbance of glucose was measured on a refractive index detector. 2.4. Ethanol fermentation Microbial ethanol fermentation was carried out by SHF, SSF, or a combination of presaccharification and SSF. The yeast S. cerevisiae was aerobically cultivated at 30 °C for 24 h using a basal medium containing 10 g/L yeast extract, 20 g/L polypeptone, and 20 g/L dextrose (YPD). The cells were collected by centrifugation for 10 min at 6000g and washed with distilled water. For SHF, 5 ml of YPD containing 1 g-wet weight of S. cerevisiae was added to a 100 ml closed bottle containing 45 ml of the sugar solutions obtained by enzymatic hydrolysis at 50 °C for 48 h. The bottles were equipped with a bubbling CO2 outlet and incubated at 35 °C (within the acceptable limits for ethanol fermentation using the yeast S. cerevisiae NBRC1440) at a stirring rate of 300 rpm. For SSF, cells were added into the mixtures of cellulose acetate in a closed bottle immediately after adding cellulase. Unless otherwise noted, the sugar solutions obtained by presaccharification at 50 °C for 24 h were used for ethanol fermentation. Ethanol and glucose concentrations in the fermentation broth were determined by HPLC under the same conditions as those described above. 3. Results and discussion 3.1. Deacetylation of cellulose acetate Table 1 shows the glucose and acetyl contents in cellulose acetate before and after deacetylation by alkaline treatment. The acetyl content in cellulose acetate decreased significantly after deacetylation, in which the corresponding weight loss of cellulose acetate was observed. Accordingly, the glucose content in the deacetylated cellulose increased to 92.3 wt%. To further confirm

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S. Hama et al. / Bioresource Technology 157 (2014) 1–5 Table 1 Glucose and acetyl contents in cellulose acetate.

a b c

Deacetylationa

Glucoseb (wt%)

Acetyl contentc (%)

 +

58.8 92.3

41.1 3.87

Cellulose acetate was subjected to alkaline treatment (+). Glucose content in dried cellulose acetate. Acetyl content in cellulose acetate determined by the Eberstadt method.

the occurrence of deacetylation, the structural changes of cellulose acetate were investigated by FT-IR spectroscopy (Supplementary data Fig. S1). The spectra of treated cellulose acetate showed a decrease in the absorbance of the bands at 1754 cm1 (C@O stretching of acetyl group), 1245 cm1 (CAO stretching of acetyl group), and 1375 cm1 (CAH blending in the methyl of acetyl group). In treated cellulose acetate, an increase in the absorbance at 3470 cm1, attributed to the OH stretching bond, was also observed. These results clearly show the removal of acetyl groups from cellulose acetate through saponification.

3.2. Enzymatic saccharification of cellulose acetate To examine the role of deacetylation in the saccharification of cellulose acetate, cellulose acetate samples (5% loading to buffer) before and after deacetylation were subjected to enzymatic saccharification at 50 °C using cellulase (Fig. 1). Before deacetylation, the glucose concentration in the saccharification mixtures did not increase; however, after deacetylation, 47.5 g/L glucose was obtained at 48 h, a yield 98.6% of the theoretical value (considering the decrease in the concentration of the exogenous glucose present in commercial cellulase, 2.0 g/L at 0 h). In the case of deacetylated cellulose, increasing the substrate concentration in the saccharification mixtures (10%, 15%, and 20%) during a 48 h reaction at 50 °C reached glucose concentrations of 91.5 g/L (99.1% yield), 134.9 g/L (97.4% yield), and 163.9 g/L (88.8% yield), respectively (data not shown). The above results thus show the importance of the deacetylation of cellulose acetate for obtaining a high glucose yield during enzymatic processing. Therefore, deacetylated cellulose acetate with an average glucose content of 92.3 wt% was used in the subsequent experiments. Cellobiohydrolases (CBHs), the principal enzymes responsible for cellulose hydrolysis, are rather sensitive to cellulose substituents including acetyl groups because the active site of CBHs exists inside a tunnel formed by stable surface loops (Divne et al., 1994).

Compared with that of CBHs, the active site of endoglucanases (EGs) is widely open and placed in a cleft, allowing a random hydrolysis of the cellulose chain (Spezio et al., 1993). Nonetheless, the accessibility of EGs is clearly a function of the DS of cellulose acetate in the range from 0.9 to 1.7 (Puls et al., 2011). Because cellulase-catalyzed saccharification needs the synergistic action of principal enzymes such as CBHs and EGs (Ganner et al., 2012; Kumar and Murthy, 2013), the effect of the steric hindrance of acetyl groups on the action of CBHs and/or EGs may account for the difficulties in the saccharification of cellulose acetate. 3.3. Ethanol fermentation of cellulose acetate Because a high glucose yield was obtained through the enzymatic saccharification of cellulose acetate, ethanol fermentation was carried out using S. cerevisiae and 5% substrate concentration in the saccharification mixtures. Herein, two process configurations were examined; SHF and SSF, during which the temperature was maintained at 35 °C considering the thermal resistance of yeast cells. As shown in Fig. 2, the glucose in the saccharification mixtures produced during SHF was converted into ethanol, yielding 92.3% (20.8 g/L at 22 h from the initial glucose concentration of 44.2 g/L) of the theoretical value. However, SSF resulted in a low yield of 59.4% at 24 h, and this yield did not significantly increase after 48 h. The result appears to be inconsistent with the generality of SSF, i.e., the reduced end-product (e.g., glucose) inhibition of enzymatic hydrolysis, thereby leading to the increased ethanol yield (Olofsson et al., 2008). Previous studies showed the instability of exoglucanases in cellulase components against shear stress induced by agitation (Gunjikar et al., 2001; Taneda et al., 2012; Ye et al., 2012). Although such an instability was considered under vigorous stirring during SSF, decreasing stirring rate had no significant impact on the ethanol yield during SSF (data not shown). 3.4. Saccharification behavior of cellulose acetate Fig. 3 shows a unique saccharification behavior of cellulose acetate depending on temperature and biomass concentration in comparison with that of hydrothermally treated rice straw. In both substrates, a negative impact of increasing biomass concentration on saccharification yield was observed. In a previous paper, the yield-determining factors in high-solid saccharification such as insufficient mixing, product inhibition, and enzyme adsorption have been extensively discussed (Kristensen et al., 2009). Notably,

45

50 Concentration (g/L)

Glucose concentration (g/L)

40

40 30

20

35 30 25 20 15 10 5

10

0 0

0

0

10

20 30 Reaction time (h)

40

50

Fig. 1. Time course of enzymatic saccharification of cellulose acetate. Cellulose acetate with (d) and without (s) alkali treatment was used as the substrate.

10

20 30 Time (h)

40

50

Fig. 2. Ethanol fermentation of cellulose acetate. The vertical axis shows the concentrations of ethanol (ds) and glucose (N4) in the fermentation broth. Closed symbols show the data obtained in separate hydrolysis (50 °C) and fermentation (35 °C), whereas open symbols show those obtained in simultaneous saccharification and fermentation (35 °C).

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Saccharification yield (%)

a

Table 2 Ethanol fermentation of cellulose acetate.

100

80

Temperature

Biomass concentration (%)

Ethanol (g/L)

Yield (%)

35 °C at 0–48 h

5 10 15 20

16.2 ± 0.03 31.5 ± 0.3 44.5 ± 1.6 59.6 ± 2.5

68.7 66.8 63.0 63.3

50 °C at 0–24 h and 35 °C at 24–48 h

5 10 15 20

19.8 ± 0.2 38.0 ± 0.7 55.9 ± 0.5 70.9 ± 2.5

84.0 80.7 79.2 75.3

60 35°C 50°C

40

20

interactions between enzymes and cellulose may significantly hamper enzyme desorption from cellulose acetate.

0 5%

10%

15%

20%

Biomass concentration

Saccharification yield (%)

b

3.5. Ethanol fermentation of cellulose acetate employing presaccharification

100

80

60 35°C 50°C

40

20

0 5%

10%

15%

20%

Biomass concentration Fig. 3. Effect of temperature on enzymatic saccharification. (a) Alkali-treated cellulose acetate and (b) hydrothermally treated rice straw were used as the substrates. Saccharification yields after a 24 h hydrolysis at different biomass concentrations were compared between 35 °C (white bar) and 50 °C (gray bar).

at all the biomass concentrations examined, decreasing temperature from 50 to 35 °C decreased saccharification yield more significantly in cellulose acetate than in hydrothermally treated rice straw. The detailed time profiles of the enzymatic saccharification of cellulose acetate are provided in data (Supplementary data Fig. S2). Interestingly, the glucose concentrations in the saccharification mixtures at 35 °C remained lower than those at 50 °C even after a long reaction time, and increasing cellulase dosage up to 40 FPU/g-biomass did not increase glucose yield (Fig. S2a–d). In the case of hydrothermally treated rice straw, saccharification at 35 °C for 48–72 h produced almost the same glucose concentration as that at 50 °C (Fig. S2e–h). The temperature dependence during enzymatic saccharification of cellulose acetate was similar to that of phosphoric acid swollen cellulose as pure cellulose (data not shown). The enzymatic saccharification of lignocellulose requires cellulase adsorption onto an insoluble substrate, during which a nonproductive binding of enzymes to cellulose or lignin occurs (Várnai et al., 2011). The binding of cellulase onto a cellulosic substrate is reversible, although such reversibility depends on various factors including temperature, pH, and substrate. In principle, the rate of cellulase adsorption/desorption seems to increase with increasing temperature, thus leading to the increased reversibility and recyclability of enzymes (Carrard and Linder, 1999; Tu et al., 2009; Wang et al., 2012). On the basis of the mechanisms underlying the enzymatic action on cellulose, the results obtained using cellulose acetate suggest that the rate of cellulase adsorption/ desorption highly depends on thermodynamics. During the course of enzymatic saccharification at a low temperature, hydrophilic

To eliminate the drawbacks associated with the low temperature during SSF and long process time of SHF, cellulose presaccharification (Souza et al., 2012) was carried out. Table 2 shows the ethanol concentrations and yields at various substrate loadings after a total process time of 48 h with and without a 24 h presaccharification at 50 °C. The ethanol titers of SSF with presaccharification were 19.8 g/L (5% substrate), 38.0 g/L (10% substrate), 55.9 g/L (15% substrate), and 70.9 g/L (20% substrate). The results showed a 12.0–16.2% increase in ethanol yield by employing presaccharification, which further suggests a significant temperature dependence of enzymatic saccharification using cellulose acetate. At all the substrate loadings examined, the glucose concentration in the broth during SSF with presaccharification decreased to zero up to 18 h, and then the ethanol concentration increased gradually with fermentation time. A short-term (2–4 h) presaccharification produced no significant increase in ethanol yield (data not shown). Given the significant temperature dependence during saccharification, the use of thermotolerant yeast strains (Benjaphokee et al., 2012; Pessani et al., 2011) would be another strategy to further increase ethanol yield.

4. Conclusion Cellulose acetate, whose acetyl groups were removed from the cellulose backbone, was efficiently converted into glucose by enzymatic saccharification at 50 °C and high substrate loadings of up to 20%. The resulting glucose can be fermented into ethanol at a high yield of 92.3% during SHF; however, a low saccharification yield was obtained at 35 °C, leading to the reduced fermentation efficiency during SSF. Owing to the significant temperature dependence during saccharification, presaccharification prior to SSF was found effective for increasing ethanol yield from cellulose acetate without changing the total process time in comparison with SHF. Acknowledgement This work was supported by the Japanese Ministry of the Environment for Technical Development of Measures to Prevent Global Warming (2011–2012).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.01. 002.

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Saccharification behavior of cellulose acetate during enzymatic processing for microbial ethanol production.

This study was conducted to realize the potential application of cellulose acetate to enzymatic processing, followed by microbial ethanol fermentation...
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