Bioresource Technology 175 (2015) 529–536

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Comparison of aqueous ammonia and dilute acid pretreatment of bamboo fractions: Structure properties and enzymatic hydrolysis Donglin Xin a, Zhong Yang b, Feng Liu a, Xueru Xu a, Junhua Zhang a,⇑ a b

College of Forestry, Northwest A&F University, 3 Taicheng Road, Yangling 712100, China Research Institute of Wood Industry, Chinese Academy of Forestry, Beijing 100091, China

h i g h l i g h t s  Structure and hydrolysability of SAA and DA pretreated bamboo were compared.  Different bamboo fractions had different structure properties and hydrolysabilities.  SAA was more efficient than DA pretreatment in the hydrolysis of bamboo.  Supplementation of xylanase was more effective than increase of cellulases loading.  SAA pretreatment with XYL addition was promising method for bamboo bioconversion.

a r t i c l e

i n f o

Article history: Received 28 September 2014 Received in revised form 29 October 2014 Accepted 30 October 2014 Available online 6 November 2014 Keywords: Bamboo fractions Aqueous ammonia pretreatment Dilute acid pretreatment Structure properties Enzymatic hydrolysis

a b s t r a c t The effect of two pretreatments methods, aqueous ammonia (SAA) and dilute acid (DA), on the chemical compositions, cellulose crystallinity, morphologic change, and enzymatic hydrolysis of bamboo fractions (bamboo yellow, timber, green, and knot) was compared. Bamboo fractions with SAA pretreatment had better hydrolysability than those with DA pretreatment. High crystallinity index resulted in low hydrolysis yield in the conversion of SAA pretreated bamboo fractions, not DA pretreated fractions. The increase of cellulase loading had modestly positive effect in the hydrolysis of both SAA and DA pretreated bamboo fractions, while supplement of xylanase significantly increased the hydrolysis of the pretreated bamboo fractions, especially after SAA pretreatment. The results indicated that SAA pretreatment was more effective than DA pretreatment in conversion of bamboo fractions, and supplementation of xylanase was necessary in effective conversion of the SAA pretreated fractions into fermentable sugars. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction With the increasing in global energy demand and global warming concerns caused by traditional fossil fuels, the use of diverse biomass to produce renewable energy, such as biofuels is of particular interest (Berndes et al., 2003; Klass, 1998). Biomass, such as agricultural residue, grasses, and forestry wastes, are considered to be a viable and sustainable energy source for biofuels production because of its renewability, wide distribution, and abundance (Kuhad and Singh, 1993; Rubin, 2008). Bamboo, a perennial

Abbreviations: CEL, cellulases; CI, crystallinity index; DA, dilute acid pretreatment; DM, dry matter; HPLC, high-performance liquid chromatograph; SEM, scanning electron microscopy; SAA, aqueous ammonia pretreatment process; XRD, X-ray diffraction; XYL, xylanase. ⇑ Corresponding author. Tel.: +86 13892883052; fax: +86 29 87082892. E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.biortech.2014.10.160 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

woody grass in East Asia and South East, has been widely used as a raw material for paper, textiles, food, construction and reinforcing fibers. Recently, bamboo is found to be a promising material for bioethanol production due to its extraordinary growth rate and high contents of carbohydrates that could be converted to fermentable sugars (Kobayashi et al., 2004; Shimokawa et al., 2009; Tsuda et al., 1998). In China, there are more than 200,000 ha of bamboo, and the yields per hectare may reach up to 30 tonnes a year. According to Chand et al. (2006), bamboo stem is composed of bamboo skin, timber, and pith. There no vascular bundles exist in bamboo skin (the outer part) and pith (the inner part). Bamboo timber is the part between skin and pith, where vascular bundles are present. The bamboo timber can be further divided into three parts: bamboo green, timber, and yellow based on the density of vascular bundles. The vascular bundles in bamboo green (outer layer) are dense, while in bamboo yellow (inner layer) are rare. Bamboo timber is the part between bamboo green and yellow. In

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this work, the term ‘‘bamboo timber’’ is not the meaning of the layer between bamboo skin and pith, but the layer between bamboo green and yellow. In bamboo processing industry, bamboo timber is fully utilized while bamboo green and yellow are removed and wasted. However, the wasted bamboo fractions contain high amount of cellulose and hemicelluloses, the bioresources of fermentable sugars production. Therefore, the wasted bamboo fractions are potential materials for biofuels production. Previously, the behaviors of bamboo green, timber, and yellow in sulfite (2%, 4%, and 8% Na2SO3, 180 °C for 30 min), dilute sulfuric acid (2% H2SO4, 180 °C for 30 min), and NaOH (6% and 12% NaOH, 180 °C for 30 min) pretreatments were investigated and the pretreated bamboo fractions were hydrolyzed by cellulases (15 FPU/ g cellulose cellulase and 30 IU/g cellulose b-glucosidase) (Li et al., 2014). It was observed that bamboo timber exhibited higher sugars content and better enzymatic digestibility, while the highest glucose yield of the pretreated bamboo timber was merely 60%. The relatively low hydrolysis yield is mainly due to the high content of lignin, high density and hardness of bamboo. As in other lignocellulosic materials, high degree of lignifications and density of the vascular bundles, heterogeneous and complex structure of cell–well constituents make it difficult for enzymes to access the surface of polysaccharides fibers during enzymatic hydrolysis process (Berlin et al., 2005; Himmel et al., 2007). In order to efficiently convert bamboo to fermentable sugars, it is necessary to remove part of lignin and destroy the complex structure. Among a considerable amount of pretreatment technologies, alkaline pretreatment, such as aqueous ammonia exhibits strong selectivity in lignin removal by degrading the ester bonds those between p-coumaric acid and lignin or between ferulic acid and hemicelluloses and has an ability to swell biomass solids by converting cellulose I to cellulose III (Kim and Lee, 2005; Wada et al., 2004). Removing lignin and swelling cellulose with aqueous ammonia could effectively increase the accessible surface of carbohydrates (cellulose and hemicelluloses) with enzymes, and hence enhance the conversion of carbohydrates to fermentable sugars. It was reported that 73.5% of the lignin in corn stover was removed by 29.5 wt.% aqueous ammonia at room temperature and 1:12 of solid-to-liquid ratio for 60 d, and high glucose yield (92%) and xylose yield (85%) were obtained (Kim and Lee, 2005). However, to our knowledge, less attention has been focused on the aqueous ammonia pretreatment of bamboo. Therefore, in this work, bamboo green, timber, yellow, and another wasted bamboo fractions in bamboo processing industry, bamboo knot, were pretreated by aqueous ammonia. Additionally, dilute acid pretreatment, the most efficient pretreatment method in the work of Li et al. (2014), was performed on the four bamboo fractions as a comparative pretreatment method. The structural characteristics of raw and pretreated bamboo fractions were assessed by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The enzymatic hydrolysis of the pretreated bamboo fractions by different dosages of cellulase was evaluated. Synergistic action of cellulase and xylanase in the hydrolysis of pretreated bamboo fractions was carried out. 2. Methods 2.1. Materials Bamboo (4 years old) was collected from Hangzhou city, Zhejiang Province, China. Air-dried bamboo was fractionated to four parts: bamboo yellow, bamboo timber, bamboo green, and bamboo knot. The four fractions of the bamboo were milled and sieved through a 60 mesh screen scale. The milled fractions (60.3 mm) were then pretreated by aqueous ammonia and dilute acid, as described below.

2.2. Aqueous ammonia and dilute acid pretreatments The bamboo fractions were pretreated with 25 wt.% of aqueous ammonia at 70 °C for 72 h (SAA) and 1 wt.% of H2SO4 at 121 °C for 1 h (DA) in screw-capped bottles at a solid:liquid ratio of 1:10. The pretreated bamboo fractions were washed with distilled water until the pH of the washing to neutral. After that, the solids were air-dried and stored at 20 °C for composition analysis and enzymatic hydrolysis. 2.3. Enzymes The commercial enzyme preparations Celluclast 1.5L and Novozyme 188 (Novo Nordisk A/S, Bagsværd, Denmark) were used as cellulase preparation. Pentopan Mono BG (Novo Nordisk A/S, Bagsværd, Denmark) was used as xylanase (XYL) preparation. Celluclast 1.5L had an activity of 74.7 FPU/ml measured according to IUPAC standard assay (Ghose, 1987). The activity of Novozyme 188 was determined to be 8451 nkat/ml of bG as described by Bailey and Nevalainen (1981). Protein was quantified by the Lowry method, using bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) as standard (Lowry et al., 1951). 2.4. Enzymatic hydrolysis The hydrolysis of the SAA and DA pretreated bamboo fractions by cellulase preparation was performed in tubes with a working volume of 3 ml in 50 mM sodium citrate buffer (pH 5.0) at 50 °C. The hydrolysis was conducted in a shaking incubator, and the shaking speed in the incubator was 200 rpm. The dry matter (DM) content of substrate was 2%. 0.02% NaN3 was added to the hydrolysis broth to prevent bacterial infection. The cellulase preparation contained both Celluclast 1.5L and Novozyme 188 preparations, which were dosed in the range from 10 to 50 FPU/g DM and 500 nkat/g DM, respectively. Samples were withdrawn at 6, 24, and 48 h and boiled for 10 min to stop the enzymatic hydrolysis. After cooling, the samples were centrifuged at 10,000g for 10 min and the supernatants were analyzed for glucose and xylose with a high-performance liquid chromatography (HPLC). The synergy between CEL and XYL in the hydrolysis of the SAA and DA pretreated bamboos was investigated as described above. CEL was dosed at 20 FPU Celluclast 1.5L per gram DM, and 500 nkat Novozyme 188 per gram DM. The dosage of XYL was 0.5 and 2 mg protein per gram DM. Samples were withdrawn at 48 h and boiled for 10 min to stop the enzymatic hydrolysis. After cooling, the samples were centrifuged and the supernatants were analyzed for glucose and xylose by HPLC. Two replicate tests were carried out in all hydrolysis experiments and average values were presented. 2.5. XRD analysis The cellulose crystallinity index (CI) of nonpretreated and pretreated samples was measured by XRD using a Rigaku D/max-3C generator (Rigaku Corporation, Japan). The dried samples were scanned in 2h range from 5° to 50° using the steps of 0.02° in width, and using Cu/Ka radiation (1.54 Å) generated at 35 kV and 35 mA. The CI of cellulose was calculated from the XRD spectra according to the method of Segal et al. (1954):

CrI ¼

I002  Iam  100 I002

In which I002 is the maximum intensity of the (0 0 2) lattice diffraction, and Iam is the peak of the amorphous portion evaluated as the minimum intensity between the (1 0 1) and (0 0 2) lattice planes.

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2.6. Analytical methods Composition of the nonpretreated and pretreated bamboo fractions was determined by National Renewable Energy Laboratory Analytical Procedure: Determination of Structural Carbohydrates and Lignin in Biomass (Sluiter et al., 2008). The amount of glucose and xylose was determined using a HPLC system (Hitachi L-2000, Hitachi Corp., Japan). The system equipped with a refractive index detector (Hitachi Corp., Japan) and an autosampler (Hitachi Corp., Japan). Ion moderated partition chromatography column (Aminex column HPX-87H) with Cation H micro-guard cartridge was used. The Aminex HPX-87H column was maintained at 45 °C with 5 mM H2SO4 as the eluent at a flow rate of 0.5 ml/min. Before injection, samples were filtered through 0.22 lm MicroPES filters, and a volume of 20 ll was injected. Peaks were detected by refractive index and were indentified and quantified by comparison to retention times of authentic standards (D-glucose and D-xylose). The conversion of cellulose to glucose and xylan to xylose was calculated by the following equation:

Table 1 Chemical compositions of bamboo fractions with nonpretreatment, aqueous ammonia pretreatment (SAA), and dilute acid pretreatment (DA). Sample

Pretreatment

Cellulose (%)

Xylan (%)

Lignin (%)

Bamboo yellow

Nonpretreatment SAAa DAb

40.54 ± 2.59 54.47 ± 0.78 44.52 ± 0.1

14.06 ± 0.25 25.67 ± 0.24 8.92 ± 0.45

20.83 ± 0.22 14.42 ± 0.13 37.37 ± 1.42

Bamboo timber

Nonpretreatment SAAa DAb

39.36 ± 0.76 52.43 ± 0.81 43.17 ± 0.43

18.01 ± 0.06 24.72 ± 0.40 7.54 ± 0.07

24.08 ± 0.13 16.82 ± 0.02 36.87 ± 3.76

Bamboo green

Nonpretreatment SAAa DAb

41.78 ± 1.16 49.34 ± 1.57 41.28 ± 0.01

14.67 ± 0.05 22.61 ± 0.76 7.72 ± 0.16

28.66 ± 0.27 23.13 ± 0.07 38.49 ± 0.76

Bamboo knot

Nonpretreatment SAAa DAb

39.90 ± 0.51 51.10 ± 1.02 38.43 ± 0.05

18.42 ± 0.55 25.59 ± 0.49 7.61 ± 0.01

21.90 ± 0.70 17.33 ± 0.06 36.53 ± 0.33

a SAA pretreatment conditions: 21 wt.% of aqueous ammonia, 70 °C, 72 h, S/L ratio 1:10. b DA pretreatment conditions: 1 wt.% of sulfuric acid, 121 °C, 1 h, S/L ratio 1:10.

Cellulose to glucose conversion ð%Þ ¼

Glucose released  0:9  100 Theoretical amount of cellulose in Substrates

Xylan to xylose conversion ð%Þ ¼

Xylose released  0:9 Theoretical amount of xylan in substrates  100

The following equation was used to calculate the synergy factors for formation of glucose in the hydrolysis of pretreated bamboos by CEL and XYL (Kumar and Wyman, 2009):

Synergy factor for formation of glucose ¼

Amount of glucose released by CEL and XYL Sum of amount of glucose released by CEL and XYL alone

The following equation was used to calculate the synergy factors for formation of xylose in the hydrolysis of pretreated bamboos by CEL and XYL:

Synergy factor for formation of xylose ¼

Amount of xylose released by CEL and XYL Sum of amount of xylose released by CEL and XYL alone

3. Results and discussion 3.1. Composition of bamboo green, timber, yellow, and knot The composition of nonpretreated bamboo and pretreated bamboo fractions was shown in Table 1. Nonpretreated bamboo fractions contained 39.4–41.8% cellulose and 14.1–18.4% xylan, high content of carbohydrates made it a promising lignocellulosic substrate for fermentable sugars production. Considerable amount of lignin was observed in the substrates. It was also noticed that the amount of lignin in bamboo green (28.6%) was higher than that in bamboo yellow (20.8%), timber (24.1%), and knot (21.9%). After SAA pretreatment, the amount lignin in bamboo yellow, timber, green, and knot decreased to 14.4%, 16.8%, 23.1%, and 17.3%, respectively, indicating the strong delignification capacity of aqueous ammonia pretreatment. The cellulose and xylan contents of the aqueous ammonia pretreated bamboos increased notably as compared with nonpretreated bamboos due to the solubilization of lignin. It was also noticed that the order of carbohydrates (cellulose and xylan) in the SAA pretreated bamboo fractions was bamboo yellow (80.2%) > bamboo timber (77.2%) > bamboo knot (76.6%) > bamboo green (71.9%). In order to compare

different pretreatment methods on composition and digestibility of bamboo, DA pretreatment was performed. As expected, approximate half of xylan was solubilized in the pretreatment process, indicating that dilute acid exhibited strong ability in removing xylan. The removal of xylan in turn increased the lignin content. It was observed that the lignin contents in the DA pretreated bamboo yellow, timber, green and knot increased to 37.4%, 36.9%, 38.5%, and 36.5%. The results here were in good agreement with the previous results that 40.9%, 37.4%, and 40.2% lignin existed in 2% dilute acid pretreated bamboo yellow, timber, and green (Li et al., 2014).

3.2. XRD analysis of bamboo substrates To investigate the physical structure changes in the pretreated process, the crystallinity of nonpretreated, SAA and DA pretreated bamboo yellow, timber, green, and knot were determined by XRD (Fig. S1). The data of CI was calculated based on the peak height and width between the crystalline peaks (1 0 1–0 0 2) (Table 2). The CIs of nonpretreated bamboo yellow, timber, green, and knot were 43.1%, 52.1%, 55.8%, and 52.3%, respectively. After being pretreated by dilute acid and aqueous ammonia, the CI values increased by 1.5–28.8%. The results here were in good agreement with previous results that the CI of biomass increased after being pretreated by aqueous ammonia, dilute acid and steam explosion (Kim et al., 2003; Kumar et al., 2009; Pang et al., 2013). The phenomenon may be caused by the removal of part of hemicelluloses, lignin (as shown in Table 1), and amorphous cellulose (Mittal et al., 2011; Park et al., 2010) during pretreatment process. It was also observed that the CI values of SAA pretreated bamboo fractions (bamboo yellow, timber, green, and knot) were higher than those of DA pretreated bamboo fractions. The results indicated that much

Table 2 Crystallinity index (%) from XRD analysis of bamboo fractions with nonpretreatment, aqueous ammonia pretreatment (SAA), and dilute acid pretreatment (DA). Substrate

Bamboo yellow

Bamboo timber

Bamboo green

Bamboo knot

Nonpretreatment SAA DA

43.1 55.5 55.0

52.1 63.2 52.9

55.8 65.2 64.7

52.3 62.8 61.5

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more amorphous contents in bamboo fractions were dissolved during SAA pretreatment than DA pretreatment process. 3.3. SEM analysis of bamboo substrates As the data in Table 1, part of hemicelluloses and lignin were solubilized during aqueous ammonia and dilute acid pretreatment processes, the removal of xylan and lignin would result in some physical changes in the bamboo fractions. Hence, the SEM images of the nonpretreated, SAA, and DA pretreated bamboo fractions were captured and it was noticed that nonpretreated bamboo fractions had smooth and well-ordered fibers (Fig. S2). However, the fibers in SAA and DA pretreated substrates were coarse and disordered. The results here clearly indicated that the removal of hemicelluloses and lignin from bamboo fractions by aqueous ammonia and dilute acid significantly changed the morphology of substrates. Previously, it was reported that aqueous ammonia and dilute acid pretreatment significantly altered the structure of barley hull and corn stover (Kim et al., 2008; Sun et al., 2013), which was in good agreement with the results here. The rough and disordered fibers in SAA and DA pretreated fractions could increase the pore volume and the external surface area accessible for enzymes, and therefore enhance the digestibility of cellulose. 3.4. Effect of reaction times on bamboo fractions hydrolysis The hydrolysis of SAA and DA pretreated bamboo fractions was carried out with cellulase (10 FPU/g DM Celluclast 1.5L and 500 nkat/g DM Novozyme 188) at a range of 6–48 h (Fig. 1). The hydrolysis yields of cellulose in SAA pretreated bamboo yellow, timber, green, and knot were as low as 28.9%, 18.9%, 12.9%, and 23.8%, respectively, after 6 h enzymatic hydrolysis (Fig. 1A). When the hydrolysis time increased to 48 h, the hydrolysis yields of cellulose in the four parts of bamboo fractions clearly increased to 42.3%, 31.1%, 17.4%, and 29.3%, respectively. It was observed that

60

(A) SAA

(B) SAA

40

Xylose yield (%)

Glucose yield (%)

50

the order of cellulose digestibility was bamboo yellow > bamboo timber > bamboo knot > bamboo green. As the data in Table 1, SAA pretreated bamboo yellow had more cellulose content and less lignin content than bamboo knot, timber, and green, which made cellulose more accessible to cellulase and hence obtained high cellulose digestibility. However, in the work of Li et al. (2014), pretreated bamboo timber exhibited better enzymatic digestibility as compared with bamboo yellow or green. The possible reason for the distinction was the difference of chemical composition, due to higher content of cellulose and lower content of lignin was present in the pretreated bamboo timber in the work of Li et al. (2014). It was observed that bamboo green exhibited the worst cellulose digestibility as compared with bamboo timber and yellow both in the results here and Li’s. Higher density and lignin content of bamboo green could be the possible reason for the phenomenon. Additionally, it was found that the hydrolysis yields of cellulose in the SAA pretreated bamboo fractions decreased with the increase of the CI values and the extent of relationship was high with the statistical coefficient of determination (R2) being 0.846 (data not shown). The results indicated that lower crystallinity resulted in a larger accessibility surface area of carbohydrate with enzymes, as reported previously (Kumar et al., 2009; Li et al., 2010). A clear increase of xylose yields was observed with the increase of enzymatic hydrolysis time (Fig. 1B). The xylose yields of the SAA pretreated bamboo yellow, timber, green, and knot were 53.7%, 50.1%, 31.4%, and 42.8%, respectively, after 48 h, indicating that significant amount of xylan hydrolytic activities were present in the cellulase preparations. Somewhat surprising, extremely low glucose yields were obtained from DA pretreated bamboo fractions (Fig. 1C). The maximal hydrolysis yield of 11% was achieved from cellulose in DA pretreated bamboo knot after 48 h hydrolysis, However, previously, the hydrolysis yields of cellulose in dilute acid pretreated bamboo fractions were 30–60% (Li et al., 2014), which were higher than the yields in this work. The strong dilute acid pretreatment

30 20 10

40 30 20 10

0

0 0

14

12

24 Time (h)

36

48

0

12

24 Time (h)

36

48

12

24 Time (h)

36

48

25

(C) DA

(D) DA

Xylose yield (%)

12 Glucose yield (%)

50

10 8 6 4 2 0

20 15 10 5 0

0

12

24 Time (h)

36

48

0

Fig. 1. Hydrolysis of 2% bamboo fractions pretreated by aqueous ammonia (SAA) and dilute acid (DA) by Celluclast 1.5L (10 FPU/g DM) and Novozyme 188 (500 nkat/g DM) at 50 °C and pH 5.0 for 6, 24, 48 h. Note: } bamboo yellow; s bamboo knot; h bamboo timber; 4 bamboo green. The error bars represent the standard errors of two independent experiments.

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conditions (2% dilute acid for 30 min at 180 °C) performed in the work of Li et al. was the possible reason for the difference. High pretreatment temperature resulted in serious cellulose cleavage such as the decrease of polymerization degree (Ma et al., 2013), which increased the accessibility cellulose surface area with enzymes. The xylose yields of the DA pretreated bamboo fractions were shown in Fig. 1D. As expected, the xylose yields increased with increasing hydrolysis time. The xylose yields of the DA pretreated bamboo yellow, timber, green, and knot increased to 17.6%, 18.0%, 16.0%, and 20.3%, respectively, at the end of 48 h of enzymatic hydrolysis. Low xylan content in the DA pretreated bamboo fractions and the steric hindrance of high lignin (36.5– 38.5%) could be the possible reason for the low xylose yields. It was observed that both glucose and xylose yields of SAA pretreated bamboo fractions were higher than those of DA pretreated bamboo fractions in this work, indicating that, under the investigated conditions, alkali pretreatment, such as aqueous ammonia pretreatment, was more suitable than dilute acid pretreatment for effective hydrolysis of bamboo fractions. In SAA pretreated process, part of lignin was removed, meanwhile, most of xylan was remained. The removal of part of the lignin increased the accessible surface area of cellulose, additionally, part of the remained xylan in the pretreated bamboo fractions was hydrolyzed by the xylan hydrolytic enzyme in the cellulase preparation, which further increased the accessible surface area of cellulose. However, in the DA pretreated process, most of the lignin was remained and half amount of xylan was solubilized. The presence of large amount of lignin closely interacted with cellulose and showed a negative effect in cellulose hydrolysis. In addition, the remained xylan was covered by lignin and the hydrolysis of xylan was limited, therefore, modest increase of accessible surface area of cellulose by xylan solubilization was obtained in the DA pretreated bamboo fractions hydrolysis. The different effect in the cellulose accessible area with enzymes could be the possible reason for the difference in hydrolysis yields of SAA

and DA pretreated bamboo fractions. The results indicated that the removal of lignin was more necessary than xylan solubilization in the pretreatment process for the efficient hydrolysis of bamboo fractions. 3.5. Effect of cellulase loading on biomass fractions hydrolysis The effect of cellulase loading on the hydrolysis of SAA and DA pretreated bamboo fractions was evaluated and the results were presented in Fig. 2. The data indicated that the glucose and xylose yields of SAA pretreated fractions increased slightly when the cellulase loading was increased from 20 to 50 FPU/g DM (Fig. 2A and B). The hydrolysis yields of cellulose in bamboo yellow, timber, green, and knot increased from 50.7%, 36.4%, 20.7%, and 35.4% to 57.8%, 48.1%, 28.5%, and 47.4%, the xylose yields of the four bamboo fractions increased from 33.7–56.9% to 42.8–65.4%, respectively. The results in Fig. 2C and D indicated that the increase of cellulase loading exhibited feeble effect in the improvement of the glucose and xylose yields of DA pretreated bamboo fractions. It was observed that the maximal glucose yield (11.4%) and xylose yield (22.9%) were obtained from bamboo knot when the cellulase was dosed at 20 FPU/g DM, which approximated the yields obtained at 10 FPU cellulase/g DM (Fig. 1C and D). The results here indicated that the increase of cellulase loading had a modestly positive effect in the hydrolysis of SAA pretreated bamboo fractions, however, negligible effect on DA pretreated bamboo fractions hydrolysis. 3.6. Effect of cellulase and xylanase on bamboo fractions hydrolysis In order to further investigate the effect of lignin removal and xylan solubilization on bamboo fractions hydrolysis, SAA pretreated bamboo fractions with relatively low lignin content and DA pretreated bamboo fractions with low amount of xylan were hydrolyzed by CEL and/or XYL (Fig. 3). Somewhat surprising,

70

70

(A) SAA

(B) SAA

60 Xylose yield (%)

Glucose yield (%)

60 50 40 30 20

50 40 30 20 10

10

0

0 0

10 20 30 40 Cellulase loadings (FPU/g DM)

0

50

10 20 30 40 Cellulase loadings (FPU/g DM)

50

14

(C) DA

25 Xylose yield (%)

Glucose yield (%)

12 10 8 6 4 2

(D) DA

20 15 10 5 0

0 0

10 20 30 40 50 Cellulase loadings (FPU/g DM)

0

10 20 30 40 50 Cellulase loadings (FPU/g DM)

Fig. 2. Hydrolysis of 2% bamboo fractions pretreated with aqueous ammonia (SAA) and dilute acid (DA) by Celluclast 1.5L (20, 30, 40, 50 FPU/g DM) and Novozyme 188 (500 nkat/g DM) at 50 °C and pH 5.0 for 48 h. Note: } bamboo yellow; s bamboo knot; h bamboo timber; 4 bamboo green. The error bars represent the standard errors of two independent experiments.

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100

100

(B) SAA

80

Xylose yield (%)

Glucose yield (%)

(A) SAA

60 40 20 0 0.5 XYL

2 XYL

CEL

80 60 40 20 0

CEL+0.5 XYL CEL+2 XYL

0.5 XYL

(C) DA

2 XYL

CEL

CEL+0.5 XYL CEL+2 XYL

Xylose yield (%)

Glucose yield (%)

(D) DA

0.5XYL

2XYL

CEL

Bamboo green

0.5XYL

CEL+0.5XYL CEL+2XYL

Bamboo timber

2XYL

Bamboo knot

CEL

CEL+0.5XYL CEL+2XYL

Bamboo yellow

some cellulose was hydrolyzed by XYL alone and the hydrolysis yields of cellulose increased with the increase of the XYL dosages. It was observed that the hydrolysis yields of cellulose in SAA and DA pretreated bamboo fractions by 2 mg protein/g DM XYL were 9.0–17.3% and 7.5–10.2%, indicating that some cellulolytic activities were present in the commercial XYL preparation. After supplementation of 0.5 mg protein/g DM XYL to CEL, the hydrolysis yields of cellulose in SAA and DA pretreated bamboo fractions were increased from 23.6–54.1% and 6.8–10.8% to 38.0–70.1% and 13.2– 15.0%, respectively. When employing 2 mg protein/g DM XYL with CEL, the hydrolysis yields of cellulose in SAA and DA pretreated bamboo fractions further increased to 52.6–83.6% and 25.5– 31.8%, which were much higher than those obtained by 50 FPU/g DM cellulases. The results indicated that XYL supplementation was more effective than the increase of cellulases loadings in the hydrolysis of pretreated bamboo fractions. The XYL preparation hydrolyzed residual xylan in the pretreated bamboo fractions and therefore increased the accessibility of cellulase to cellulose. As the data in Fig. 4, the hydrolysis yields of cellulose in the SAA pretreated bamboo fractions were increased nearly linearly with the solubilization of xylan. Previously, the synergy between CEL and XYL on the hydrolysis of steam pretreated corn stover, steam-exploded pretreated barley straw, and hydrothermally pretreated wheat straw was reported (García-Aparicio et al., 2007; Öhgren et al., 2007; Zhang et al., 2011). The corresponding synergy factors for the formation of glucose released from SAA and DA pretreated bamboo fractions were calculated and the results were shown in Table 3. High XYL loadings resulted in higher synergy factors. The synergy factors for formation of glucose by CEL and 2 mg protein/g DM XYL from SAA and DA pretreated bamboo fractions were 1.1–1.5 and 1.4–2.2. Higher synergistic action between CEL and XYL was obtained in the hydrolysis of DA pretreated bamboo fractions, which indicated that the residual xylan in DA pretreated bamboo fractions seemed to evenly cover the cellulose surfaces

Hydrolysis yield of cellulose (%)

Fig. 3. Hydrolysis of 2% bamboo fractions pretreated with aqueous ammonia (SAA) and dilute acid (DA) by CEL (20 FPU/g DM Celluclast 1.5L and 500 nkat/g DM Novozyme 188) and/or XYL (0.5, 2 mg protein/g DM) at 50 °C and pH 5.0 for 48 h. The error bars represent the standard errors of two independent experiments.

Bamboo yellow Bamboo knot Bamboo timber Bamboo green

80 60

R² = 0.9872

R² = 0.9758 R² = 0.9799

40 R² = 0.9469

20 0 0

20

40 60 80 Hydrolysis yield of xylan (%)

100

Fig. 4. Relationship between xylan removal and cellulose hydrolysis in the hydrolysis of 2% bamboo fractions pretreated by aqueous ammonia (SAA) with CEL (20 FPU/g DM Celluclast 1.5L and 500 nkat/g DM Novozyme 188) and/or XYL (0.5, 2 mg protein/g DM) at 50 °C and pH 5.0 for 48 h.

and the removal of xylan by XYL significantly increased the hydrolysis yields of cellulose in DA pretreated bamboo fractions. Our previous results indicated that xylan content affected the synergy between CEL and XYL and high amount of xylan resulted in a stronger synergistic effect in the hydrolysis of hydrothermally pretreated corn stover (Zhang and Viikari, 2014). However, in this work, no relationship between xylan content and synergy factors for formation of glucose was observed, which could be explained by the special characteristics of bamboo fractions, such as high lignin content, density, and hardness. When employing XYL alone, about 10% of the xylan in the SAA pretreated bamboo fractions was converted to xylose because of the XYL preparation was a type of endoxylanase and the hydrolysate was mainly composed of xylo-oligosaccharides. However, the amount of xylose released from xylan in DA pretreated bamboo

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Table 3 Synergy factors for formation of glucose and xylose by CEL (20 FPU/g DM Celluclast 1.5L and 500 nkat/g Novozyme 188) with XYL (0.5, 2 mg protein/g DM) in the hydrolysis of bamboo fractions pretreated with aqueous ammonia (SAA) and dilute acid (DA) for 48 h at pH 5.0 and 50 °C. SAA

DA

Bamboo yellow

Bamboo timber

Bamboo green

Bamboo knot

Bamboo yellow

Bamboo timber

Bamboo green

Bamboo knot

Glucose

0.5 XYL 2 XYL

1.1 1.2

1.2 1.4

1.5 1.6

1.3 1.5

1.2 1.8

1.3 1.5

1.5 2.2

1.1 1.4

Xylose

0.5 XYL 2 XYL

1.2 1.2

1.3 1.3

1.4 1.4

1.4 1.5

1.3 1.4

1.1 1.6

1.1 1.5

1.0 1.0

fractions was below the detection line (Fig. 3D), indicating that xylan in DA pretreated bamboo fractions was tightly covered by cellulose and lignin, and no accessibility of xylan to xylanolytic was present unless part of the cellulose and lignin were removed. The xylose yields of SAA and DA pretreated bamboo fractions slightly increased after the addition of low dosage of XYL (0.5 mg protein/g DM) to CEL. However, when supplementation of 2 mg protein/g DM XYL to CEL, the xylose yields of the SAA and DA pretreated bamboo fractions significantly increased from 37.6–59.5% and 15.1–19.1% to 64.3–87.0% and 19.7–28.8%, respectively. The synergy factors for formation of xylose from SAA and DA pretreated bamboo fractions by CEL and 2 mg protein/g DM XYL were 1.2–1.4 and 1.0–1.6, respectively (Table 3). In this work, the results indicated that significant synergy between the action of cellulase and xylanase in the hydrolysis of both aqueous ammonia and dilute acid pretreated bamboo fractions was observed. Xylan solubilization enhanced the accessibility of cellulase to cellulose and consequently the accessibility of xylanase to xylan was increased due to the solubilization of cellulose, therefore, the hydrolysis yields of both cellulose and xylan were clearly increased. 4. Conclusions Under the investigated conditions, aqueous ammonia pretreatment was shown to be a more effective pretreatment for removing lignin, altering crystallinity index, and enhancing enzymatic hydrolysis of bamboo fractions than dilute acid pretreatment. Supplementation of xylanase was more effective than increase of cellulase loading in saccharification of the pretreated bamboo fractions, especially after aqueous ammonia pretreatment, indicating that aqueous ammonia pretreatment and xylanase addition were a promising combination for the production of fermentable sugars from bamboo. Acknowledgements This work was supported by the Natural Science Foundation of China (No. 31270622). The authors are grateful to Prof. Jinzhong Xie (Research Institute of Subtropical Fores try, Chinese Academy of Forestry, China) for assistance in the collection of bamboos. 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.2014.10. 160. References Bailey, M., Nevalainen, K.M.H., 1981. Induction, isolation and testing of stable Trichoderma reesei mutant with improved production of solubilizing cellulase. Enzyme Microb. Technol. 3, 153–157. Berlin, A., Gilkes, N., Kurabi, A., Bura, R., Tu, M., Kilburn, D., Saddler, J., 2005. Weak lignin-binding enzymes. Appl. Biochem. Biotechnol. Humana Press., 163–170.

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