Bioprocess Biosyst Eng DOI 10.1007/s00449-013-1115-z

ORIGINAL PAPER

The impact of particle size and initial solid loading on thermochemical pretreatment of wheat straw for improving sugar recovery Oscar A. Rojas-Rejo´n • Arturo Sa´nchez

Received: 30 April 2013 / Accepted: 15 December 2013 Ó Springer-Verlag Berlin Heidelberg 2013

Abstract This work studies the effect of initial solid load (4–32 %; w/v, DS) and particle size (0.41–50 mm) on monosaccharide yield of wheat straw subjected to dilute H2SO4 (0.75 %, v/v) pretreatment and enzymatic saccharification. Response surface methodology (RSM) based on a full factorial design (FFD) was used for the statistical analysis of pretreatment and enzymatic hydrolysis. The highest xylose yield obtained during pretreatment (ca. 86 %; of theoretical) was achieved at 4 % (w/v, DS) and 25 mm. The solid fraction obtained from the first set of experiments was subjected to enzymatic hydrolysis at constant enzyme dosage (17 FPU/g); statistical analysis revealed that glucose yield was favored with solids pretreated at low initial solid loads and small particle sizes. Dynamic experiments showed that glucose yield did not increase after 48 h of enzymatic hydrolysis. Once established pretreatment conditions, experiments were carried out with several initial solid loading (4–24 %; w/v, DS) and enzyme dosages (5–50 FPU/g). Two straw sizes (0.41 and 50 mm) were used for verification purposes. The highest glucose yield (ca. 55 %; of theoretical) was achieved at 4 % (w/v, DS), 0.41 mm and 50 FPU/g. Statistical analysis of experiments showed that at low enzyme dosage, particle size had a remarkable effect over glucose yield and initial solid load was the main factor for glucose yield. Keywords Thermochemical pretreatment  Wheat straw  Enzymatic saccharification  Cellulosic ethanol

O. A. Rojas-Rejo´n (&)  A. Sa´nchez Centro de Investigacio´n y de Estudios Avanzados del, Instituto Polite´cnico Nacional Unidad Guadalajara de Ingenierı´a Avanzada, Control Automa´tico, Guadalajara, Mexico e-mail: [email protected]

Introduction Lignocellulosic biomass represents the largest source of materials currently available for production of alternative biofuels and high value-added bioproducts. Among agricultural residues, wheat straw is particularly attractive because of its high content of polysaccharides [ca. 67 % Dry Basis, (DB)] [23]. Development of efficient strategies of depolymerization through thermochemical pretreatment and enzymatic saccharification is still in progress to increase global yields at low cost [1]. The recalcitrant nature of agro-industrial residues has been pointed out as the main technological impediment for efficient cellulose utilization as raw material [7]. The structural arrangement of fibers into lignocellulosic matter prevents the entrance of water molecules and hence enzyme access [10, 18]. Alteration of lignocellulose structure, changes in composition, and formation of pores may increase amorphous regions and to expose packed cellulose fibers to enzymatic attack [2, 31]. Enzymatic saccharification of lignocellulosic materials using low-protein loadings has proven to be one of the major technical and economic bottlenecks in the overall bioconversion process of lignocellulose to biofuels [2]. The effectiveness of enzymatic hydrolysis of agricultural wastes and residues depends on properties of the substrate (composition, crystallinity, degree of polymerization, etc.), enzyme synergy (origin, composition, specific activities, etc.), mass transfer (substrate adsorption, bulk and pore diffusion, etc.) and intrinsic kinetics [31]. The complete saccharification of lignocellulosic biomass with low quantities of cellulase and hemicellulase continues to be a wide area of research to establish a commercial process for low-cost sugars production from lignocellulose. An effective pretreatment should be inexpensive, use simple equipment, increase the accessibility of fibers, and

123

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avoid loss of carbohydrates and the formation of inhibitory by-products [13, 14]. Dilute acid pretreatment (DAP) has demonstrated to be a promising pretreatment for fuel ethanol production from wheat straw [3, 24]. The main processes of straw defragmentation considered as key stages are pretreatment (size reduction and thermochemical treatment) and enzymatic saccharification. Since size reduction and pretreatment are usually the two first steps of biomass depolymerization in biochemical platforms, exploring the influence of variables such as particle size (PZ) and initial solid loading (ISL) on the performance of enzymatic hydrolysis is a key issue for the industrial deployments of these processes. These two variables greatly influence equipment size and yields, and thus total capital investment and process operation costs [15, 25]. However, limited research has been carried out to investigate the effect of PZ and ISL [11, 16] over xylose yield during thermochemical pretreatment. Some works can be found in the literature dealing with the effect of PZ during enzymatic hydrolysis [4, 20, 31]. Pretreatment has been optimized for efficient enzymatic hydrolysis [1, 2, 21, 34] leaving aside an optimization of pretreatment conditions for xylose production and efficient saccharification of the remaining cellulose in pretreated solids, simultaneously. Table 4 shows a compilation of related works, indicating PZ and ISL employed as well as pretreatment and hydrolysis conditions and yields for a wide variety of materials. The aim of this work was to evaluate the effect of PZ and ISL of wheat straw on the performance of dilute acid pretreatment for maximum monosaccharide yield after enzymatic hydrolysis for values that may be of interest in an industrial setting. The maximum and minimum PZ were chosen to match with previously published results for boundary sizes (i.e., [11, 28] for the PZ tested in pilot plant, and [3, 31] for the smallest PZ tested at laboratory scale). Values of ISL were chosen to match with previously published work of horizontal saccharification systems (i.e., [11, 16]).

Materials and methods Raw material Wheat straw from La Barca (Jalisco, MX) was milled with a hammer mil (Azteca 301012), classified with a vibratory sieve (Alcon, Gdl, MX) (sieves of # 8, # 16, and # 40) and stored at room temperature. Straw composition was determined at CUCBA (University of Guadalajara, Jalisco, M0 exico) according to AOAC International methods (AOAC Official Method 4.6.03 and 4.6.04) [32] with 50.83 ± 2.35, 15.93 ± 4.20, 5.6 ± 0.57 and 12.45 ± 1.03 of cellulose, hemicellulose, lignin and ash, respectively, on

123

a dry weight basis. The PZ used in pretreatment experiments were 50, 25, 12.5, 2.38, 1.19 and 0.41 mm (from 50 to 12.5 mm hand-cut at the length of the straw and from 2.38 to 0.41 mm collected from the sieving stage). Dilute acid pretreatment of straw Straw pretreatment was carried out according to the method proposed by Saha et al. [24] with modifications in the ISL. Wheat straw was slurried in dilute H2SO4 (0.75 % v/v) at 4, 8, 16 and 32 % (w/v; dry solid basis, DS) and pretreated at 121 °C for 1 h in a steam sterilizer with heating and cooling ramps of 30 min each. Pretreated solids were adjusted to pH = 4.6 with NaOH (10 M) before enzyme hydrolysis experiments. Liquid fraction was recovered and samples were taken, centrifuged at 10,000 rpm and supernatants were stored at -15 °C for further analysis. Experimental designs The dependence of ISL (4, 8, 16 and 32 %; w/v, DS) and PZ (0.41–50 mm) on sugar yields from pretreatment and enzyme hydrolysis with a constant enzyme dosage was studied with a full factorial design 4 9 6 (FFD). To establish the impact of pretreatment conditions over yields, enzymatic hydrolysis (as indirect response) was performed under conditions without mass transfer limitations (4 %; w/v, DS). Calculations of improved pretreatment conditions (with respect to PZ and ISL) were made for maximizing xylose and glucose yields for pretreatment and saccharification, respectively. Calculations were carried out to maximize xylose and glucose yields simultaneously at the pretreatment stage. All weights were considered equal to 1. Once pretreatment conditions were established (with respect to PZ and ISL) to maximize glucose yield at enzymatic saccharification, different enzyme dosage [5–50 FPU/g, dry initial cellulose (DIC)] at several ISL (4–24 %; w/v, DS) were analyzed with a full factorial design 2 9 3 9 4 (FFD). To confirm the effect of particle size on enzymatic hydrolysis, experiments with two PZ (0.41 and 50 mm) were carried out. Design-Expert software, ver. 8.0.7.1 (Stat-Easy Inc., Minneapolis, MN, USA) was used for the analysis of variance (ANOVA) of each design. Enzymatic saccharification of straw Experiments were carried out in 250 ml flasks containing 8.41 g of thermochemical pretreated solids. Solids were conditioned for enzymatic saccharification, and re-suspended with distilled water as the liquid phase at 4 % (w/v; DS) (unless otherwise stated). The initial pH of the system was adjusted to 4.6 and during saccharification it was not

Bioprocess Biosyst Eng

controlled. Enzymatic hydrolyses were performed with Accellerase 1500 complex (kindly provided by Genencor) exhibiting exocellulase (56.06 FPU/ml) endoglucanase (2,200–2,800 CMC U/g) and b-glucosidase (525–775 pNPG U/g). Enzyme dosage was adjusted to 17 FPU/g (DIC) for ‘‘pretreatment experiments’’ whilst enzyme dosage and ISL changed for the second part of experimentation stated at 2.3. Flasks with solids, diluent, and enzymes were incubated at 50 °C and 200 rpm for 48 h in an orbital shaker (Thermo Scientific, MaxQ 7000 Benchtop; Marietta, OH, USA). At the end of enzymatic hydrolysis, liquid samples were taken and centrifuged at 10,000 rpm; supernatants were stored at -15 °C for further analysis. Analysis of liquid samples Liquid samples taken from pretreatment and saccharification processes were analyzed for glucose and xylose with an YSI biochemistry analyzer (YSI SELECT 2700/115V Dual Channel).

Results and discussion Thermochemical pretreatment of straw Two main streams were generated from DAP: A liquid fraction with high content of xylose (acid hydrolysates) and a solid fraction enriched of cellulose (pretreated solids). Statistical software Design-Expert (8.0.7.1) was used to select a suitable response surface model and for ANOVA analysis of xylose yields in acid hydrolysates. The F value of the quadratic model chosen was 81.25. ISL was significant for hemicellulose bioconversion to xylose (values of Prob [ F less than 0.0500 indicate that model terms are significant) whilst PZ was not, as shown in Table 1. However, the quadratic factor of PZ shows a modest contribution. A quadratic model, shown in Eq. 1, of xylose yield (Yx) (R2 = 0.8496) was generated as a function of the variables (A-PZ and B-ISL).

Yx ¼ 0:96063 þ 7:25  103 ð AÞ  4:45  102 ðBÞ 5:75
 105 ð AÞðBÞ 1:26  104 A2 þ 6:31
 105 B2 ð1Þ . Figure 1 confirms the contribution of ISL and the quadratic factor of PZ for xylose yield in pretreatment. A significant amount of hemicellulose was converted to xylose at low ISL (ca. 4 %; w/v, DS), whereas with an increase in ISL low yields were obtained, as shown in Fig. 1. The maximum xylose yield 0.97 (g/g) (86.24 % of the theoretical) was obtained in experimental units with ISL of 4 % (w/v, DW) and PZ of 25 mm. This relatively high conversion yield was reported by other authors with wheat straw [3, 24, 28], olive tree biomass [5], rice straw [9], eucalyptus chips [29] and corn stover [30], as shown in Table 4. Weiss et al. [30] reported dilute acid pretreatment of two different sizes (6 and 18 mm) of corn stover at high ISL with severity parameters (R0) ranging from 2.5–3.2 and found no significant differences in xylose yields between PZs studied. This observation differs from the results obtained in this work with a relatively low R0 of 2.39 (determined as [30]), this could be due to nature, structural arrangement, composition of lignocellulose biomass and ISL used ([45 %). The solid fraction of DAP was recovered and the content of structural polysaccharides was determined. From experimental units with an ISL of 4 % (w/v, DS) an increase in cellulose content after DAP was

Table 1 Statistical parameters of xylose yield Factor Intercept

Coefficient 0.44

Std. Error 0.031

A-size

-0.0034

0.018

B-ISL

-0.32

0.018

F value – 0.038 319.16

P value – 0.8460 \0.0001

AB

-0.020

0.022

0.83

0.3660

A2

-0.077

0.031

6.38

0.0140

B2

0.12

0.030

17.15

0.0001

Fig. 1 Response surface of xylose yield at several PZ and ISL of straw; red dots experimental data

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Bioprocess Biosyst Eng Table 2 Statistical parameters of glucose yield Factor Intercept

Coefficient

Std. Error

F value

P value

0.25

0.00343

A-Size

-0.011

0.00192

33.24

–

\0.0001

–

B-ISL

-0.021

0.00198

111.19

\ 0.0001

AB

0.006616

0.002403

7.58

0.0076

A2

-0.0006269

0.003362

3.48

0.0667

B2

-0.007954

0.003280

5.88

0.0180

Fig. 2 Average composition of pretreated solids after DAP obtained with several PZ

observed (Fig. 2). As the system became more concentrated, cellulose and lignin concentrations decreased regardless straw size used, as shown in Fig. 2. Unlike cellulose and lignin, hemicellulose increased in experimental units with high ISL. Therefore, a minimum amount of water (ca. 10 % w/v) may be required to completely hydrolyze hemicellulose fraction. ISL and pretreatment severity are two of the most important metrics of hemicellulose hydrolysis performance [22, 30]. It has been reported that lignin detaches from lignocellulose biomatrix and relocates in amorphous regions during acid pretreatments [1, 6, 17]. Sannigrahi et al. [27] reported an increase in the degree of polymerization of lignin due to dilute acid pretreatments. Presence of lignin causes low yields in saccharification stages [33]. Enzymatic saccharification of pretreated solids Conditioned pretreated solids were subjected to enzymatic hydrolysis under the same process conditions (pH = 7, 50 °C, 200 rpm, and 48 h); to analyze the effect of changing variables (PZ and ISL) at pretreatment stage, ISL of experimental units was adjusted at the same dilution (4 %; w/v, DS) despite loading during pretreatment. It is important to emphasize that enzymatic hydrolysis was performed at low enzyme dosage (17 FPU/g, DIC) to evaluate the effectiveness of changing PZ and ISL at DAP, and to establish through this indirect variable the best pretreatment conditions for high monosaccharide (xylose and glucose) yields. Statistical software Design Expert (8.0.7.1) was used for ANOVA analysis. The F value of the model obtained was significant (51.62) and from Table 2 significant factors such as PZ and ISL (pretreatment conditions) were strongly significant (Values of Prob [ F less than 0.0500 indicate that model terms are significant) to

123

Fig. 3 Response surface of glucose yield at several PZ and ISL of straw; red dots experimental data

achieve high yields. The developed quadratic model (R2 = 0.7964) for the enzymatic hydrolysis in terms of actual pretreatment variables (PZ and ISL) for glucose yield (Yg) is given below by Eq. 2 Yg ¼ 0:27904  2:76  104 ð AÞ  5:18  104 ðBÞ
þ 1:91  105 ð AÞðBÞ 1:02  105 A2 4:06
 105 B2

ð2Þ

. PZ and ISL at pretreatment stage were significant for glucose yield in enzymatic hydrolysis, as shown in Fig. 3. Experimental units with small PZ (0.41 mm) and low ISL (4 %; w/v, DS) achieved the highest glucose yield observed (ca. 30 % g/g). Unlike results of xylose yield at pretreatment stage, PZ affects glucose yield after enzymatic hydrolysis. At high ISL (ca. 25 %; w/v, DS), glucose yield decreased. These observations are consistent with those reported by Hodge et al. [8] and Kristensen et al. [16]. Low glucose yield at high ISL could be due to residual hemicellulose in

Bioprocess Biosyst Eng

pretreated solids (Fig. 2) and to the presence and relocation of lignin [33]. As PZ decreases, glucose yield and production rate increased [31]. There are reports that indicate an important increase in saccharification yields with the reduction of PZ of raw material [1, 20, 31]. Calculation of pretreatment and saccharification process conditions with respect to PZ and ISL

the saccharification step. Statistical software Design Expert (8.0.7.1) was used for ANOVA analysis. The F value of the model obtained was significant (68.27). PZ, ISL and enzyme dosage were found strongly significant for glucose yield. The developed two factor interaction (2FI) model (R2 = 0.9196) generated during enzymatic hydrolysis in terms of saccharification variables (A-ISL, B-enzyme dosage and C-PZ) for glucose yield (Yg’) is given below by Eq. 3. 0

The main purpose of pretreatment is to break down blocking components such as hemicellulose and lignin and to disrupt the crystalline structure of cellulose to enhance enzyme accessibility to the cellulose biomatrix during enzymatic saccharification. Calculations of pretreatment and saccharification conditions for optimal yields were carried out using Design-Expert software. As shown in Table 3, 27.84 mm and 4 % (w/v, DS) were the optimal values for maximizing xylose yield during pretreatment. The contribution of the quadratic term of PZ gave rise to a shift to a larger size value as compared to the other cases. Maximal yield of glucose during enzymatic hydrolysis was favored with the smallest PZ (0.41 mm) and the lowest ISL (4 %; w/v, DS). Dilute systems (4 %; w/v, DS) and small PZ (9.55 mm) produce the maximal value of the sum of yields of xylose and glucose during pretreatment and saccharification, respectively. These results show that more glucan degradation was obtained than the one reported by Kabel et al. [12]. To confirm the effect of PZ over enzymatic hydrolysis yields observed, independent experiments were conducted with the lowest and highest straw sizes (0.41 and 50 mm) pretreated at the same conditions (4 %; w/v, DS). Experimental units were ‘‘inoculated’’ with 5 FPU/g to avoid the linear dependence of reaction rate with enzyme concentration and to observe the actual effect of PZ subjected to a specific pretreatment. Figure 4 shows the dynamic trajectories of glucose yield. After 48 h, the hydrolysis reaches the steady state in both cases.

Yg ¼ 32:451:048ð AÞ þ 0:594ðBÞ  0:2238ðCÞ 2:97  103 ð AÞðBÞ þ 4:49  103 ð AÞðC Þ þ 2:08  103 ðBÞðC Þ ð3Þ

Fig. 4 Time course glucose trajectories at 5 FPU/g with two PZ

Effect of ISL and enzyme dosage over enzymatic hydrolysis of pretreated straw Experiments were conducted to establish whether enzyme dosage improves glucose yields with respect to PZ and ISL at Table 3 Optimum values for pretreatment parameters and their responses for xylose and glucose yields Process

Particle size (mm)

Straw [% (w/v)]

Yx (g/g)

DAP

Yg (g/g)

27.84

4.0

0.89

–

EH

0.41

4.0

–

0.28

DAP ? EH

9.55

4.0

0.85

0.27

DAP dilute acid pretreatment, EH enzymatic hydrolysis

Fig. 5 Response surface of glucose yield at several ISL and enzyme dosage: color surface (0.41 mm; red dots experimental data) and gray scale surface (50 mm; black dots experimental data)

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Bioprocess Biosyst Eng Table 4 Pretreatments and hydrolysis yields Substrate

Pretreatment

Type

Size (mm)

Type

Load [% (w/v)]

Wheat straw Wheat straw

0.41–50

DAP

1–2

DAP

Wheat straw

1.27

DAP

Saccharification

References

Ro

Yield (%)

Enzyme

Dosage (FPU/g BGU/g)

Load [% (w/ v)]

Yield (%)

4–32

2.39

86.0

Cellulase

50

4–24

55

10

1.59–3.24

91.0

Cellulase

40

3

37.0

[3]

7.83

2.35–3.53

92.0

Cellulase

58.97

7.83

47.0

[24]

This work

Wheat straw

50

DAP

NA

4.12

4.1

Cellulase

20

13

65.0

[27]

Wheat straw

60–100

AH

18

3.57

NA

Cellulase

13.3

20

88.0

[11]

Wheat straw

23.5

AH

1.38

3.9–4.02

30.0

Cellulase

17.2

0.86

25.0

[6]

Microcrystalline cotton cellulose

0.78–25.52 lm

Milling

3 and 7

NA

ND

Cellulase

480

0.25–1

90.0

[30]

Barley straw

2

DAP

NA

3.24–3.84

ND

Cellulase

15

NA

50.0

[21]

H. B. carniata

2–12

SE

NA

3.25–4.14

70.0

Cellulase Glucosidase

15, 12.6

2

100.0

[4]

Eucalyptus chips

5

DAP

5

1.88–3.36

90.0

Cellulase

20

2

76.0

[28]

Olive tree pruning

10

DAP

20

3.06–4.23

83.0

Cellulase Glucosidase

15, 15

5

76.5

[5]

Corn stover

NA

DAP

5

3.06

36.0

Cellulase

60

3

53.3

[19]

Corn stover

NA

DAP

20

2.39–3.52

77.5

Cellulase

25

4–35

80.0

[8]

DAP diluted acid pretreatment, AH autohydrolysis, SE steam explosion Table 5 Experimental and predicted values of xylose yield at pretreatment stage and glucose yield at enzymatic saccharification stage Conditions STD (order)

Xylose yield PZ (mm)

ISL (%)

Glucose yield

Experimental and adjusted data Actual

Predicted

Actual

Predicted

1

0.41

4

0.56

0.80

0.27

0.28

2

0.41

4

0.53

0.80

0.27

0.28

3 4

0.41 1.19

4 4

0.55 0.91

0.80 0.80

0.27 0.28

0.28 0.28

5

1.19

4

0.79

0.80

0.28

0.28

6

1.19

4

0.91

0.80

0.29

0.28

7

2.38

4

0.78

0.81

0.25

0.28

8

2.38

4

0.86

0.81

0.25

0.28

9

2.38

4

0.80

0.81

0.27

0.28

10

12.5

4

0.84

0.86

0.30

0.27

11

12.5

4

0.77

0.86

0.30

0.27

12

12.5

4

0.84

0.86

0.30

0.27

13

25

4

0.97

0.89

0.27

0.26

14

25

4

0.91

0.89

0.25

0.26

15

25

4

0.98

0.89

0.27

0.26

16

50

4

0.85

0.83

0.24

0.24

17

50

4

0.83

0.83

0.24

0.24

18 19

50 0.41

4 8

0.79 0.51

0.83 0.65

0.24 0.29

0.24 0.27

20

0.41

8

0.51

0.65

0.29

0.27

21

0.41

8

0.50

0.65

0.28

0.27

22

1.19

8

0.81

0.65

0.26

0.27

23

1.19

8

0.89

0.65

0.26

0.27

123

Bioprocess Biosyst Eng Table 5 continued Conditions STD (order)

Xylose yield PZ (mm)

ISL (%)

Glucose yield

Experimental and adjusted data Actual

Predicted

Actual

Predicted

24

1.19

8

0.83

0.65

0.26

0.27

25

2.38

8

0.71

0.66

0.27

0.27

26

2.38

8

0.75

0.66

0.28

0.27

27 28

2.38 12.5

8 8

0.82 0.83

0.66 0.71

0.28 0.26

0.27 0.27

29

12.5

8

0.84

0.71

0.26

0.27

30

12.5

8

0.80

0.71

0.26

0.27

31

25

8

0.78

0.74

0.27

0.26

32

25

8

0.70

0.74

0.27

0.26

33

25

8

0.71

0.74

0.28

0.26

34

50

8

0.81

0.67

0.24

0.24

35

50

8

0.60

0.67

0.24

0.24

36

50

8

0.65

0.67

0.23

0.24

37

0.41

16

0.35

0.41

0.28

0.26

38

0.41

16

0.35

0.41

0.27

0.26

39

0.41

16

0.36

0.41

0.28

0.26

40

1.19

16

0.44

0.42

0.26

0.26

41

1.19

16

0.41

0.42

0.26

0.26

42

1.19

16

0.43

0.42

0.25

0.26

43 44

2.38 2.38

16 16

0.53 0.53

0.43 0.43

0.26 0.26

0.26 0.26

45

2.38

16

0.41

0.43

0.26

0.26

46

12.5

16

0.49

0.47

0.25

0.26

47

12.5

16

0.42

0.47

0.24

0.26

48

12.5

16

0.36

0.47

0.25

0.26

49

25

16

0.49

0.49

0.25

0.25

50

25

16

0.52

0.49

0.25

0.25

51

25

16

0.43

0.49

0.25

0.25

52

50

16

0.35

0.41

0.23

0.24

53

50

16

0.33

0.41

0.25

0.24

54

50

16

0.28

0.41

0.24

0.24

55

0.41

32

0.16

0.19

0.23

0.22

56

0.41

32

0.16

0.19

0.23

0.22

57

0.41

32

0.18

0.19

0.21

0.22

58 59

1.19 1.19

32 32

0.25 0.28

0.19 0.19

0.22 0.22

0.22 0.22

60

1.19

32

0.22

0.19

0.22

0.22

61

2.38

32

0.21

0.20

0.21

0.22

62

2.38

32

0.24

0.20

0.23

0.22

63

2.38

32

0.23

0.20

0.23

0.22

64

12.5

32

0.16

0.23

0.22

0.22

65

12.5

32

0.17

0.23

0.23

0.22

66

12.5

32

0.15

0.23

0.22

0.22

67

25

32

0.18

0.24

0.22

0.22

68

25

32

0.22

0.24

0.22

0.22

69

25

32

0.13

0.24

0.22

0.22

123

Bioprocess Biosyst Eng Table 5 continued Conditions

Xylose yield

STD (order)

PZ (mm)

ISL (%)

Glucose yield

Experimental and adjusted data Actual

Predicted

Actual

Predicted

70

50

32

0.23

0.14

0.22

0.21

71

50

32

0.17

0.14

0.22

0.21

72

50

32

0.28

0.14

0.22

0.21

Table 6 Experimental and predicted values of glucose yield with optimized pretreatment conditions at two different straw sizes STD (order) 1

ISL (%) 4

Enzyme dosage (FPU/g DIC) 5

Straw size (mm) 0.41

Glucose yield Actual

Predicted

33

30

2

8

5

0.41

20

26

3

16

5

0.41

14

18

4

24

5

0.41

11

9

5

4

17

0.41

44

37

6

8

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The highest glucose yield experimentally obtained was 55 % of the theoretical with 0.41 mm PZ, 4 % (w/v, DS) ISL and 50 FPU/g (DIC) as seen in Fig. 5. It can also be observed that at low enzyme dosage and ISL, PZ had a remarkable effect over glucose yield. As ISL increases (at low enzyme dosage) straw with PZ of 50 mm showed a greater drop in yields than the one presented with PZ of 0.41 mm. It is important to further optimize thermochemical pretreatment to increase yields at low enzyme dosage (\5 FPU/g DIC) since the cost of the enzymes may represent up

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to 16 % of the total cellulosic ethanol production cost [26]. Straw with PZ of 0.41 mm showed a lower decrease in glucose yield changing from 55 at 4 % (w/v, DS) to 44 % at 24 (w/v, DS). Hodge et al. [8] reported cellulose conversions greater than 70 % at 26 % of initial insoluble pretreated solids. However, this result was achieved with washed pretreated solid at high enzyme dosage (yield of 74 % at 24 % ISL and 25 FPU/g). The enzyme dosage used in this work was not supplemented with b-glucosidase, thus producing glucose yields lower than those obtained by Alvira et al. [1] and Cara et al. [5]. Nevertheless, glucose yield has demonstrated to be highly dependent of PZ, ISL, type of lignocellulosic biomass, and enzyme dosage. Yields shown in Table 4 are highly variable and some of them are obtained with mixtures of enzymes. Experimental and adjusted data from pretreatment and saccharification experiments are shown in Tables 5 and 6. Conclusions DAP is an attractive strategy to obtain high quantities of xylose and pretreated solids suitable for enzymatic hydrolysis. Experiments were carried out under similar conditions found in pilot processes for second generation (2G) biofuel production (e.g., without the addition of pH buffer as diluent, unwashed pretreated solids, sterilizing, and avoiding a dry step of pretreated straw). ISL was the main factor affecting sugar yields in both, pretreatment and saccharification processes. PZ significantly affected enzymatic hydrolysis. Thermochemical pretreatment of wheat straw required a minimum of water to disrupt fibers and hydrolyze hemicellulose. Enzymatic hydrolysis was performed to evaluate optimal pretreatment conditions for maximizing sugar recovery. The maximum xylose yield achieved after pretreatment (86.24 %) was obtained with the lowest ISL and small PZ (4 % 25 mm). Enzymatic hydrolysis of pretreated straw at low ISL, small PZ and high enzyme dosage (4 %, 0.41 mm and 50 FPU/g) achieved the highest glucose yield of 55 %. These results show that the role of PZ and ISL must be taken into consideration at the design stage of production plants for 2G biofuels that may use these types of

Bioprocess Biosyst Eng

feedstock. The impact of both PZ and ISL on operation and capital costs may be of great relevance as in the total production costs. Acknowledgments Partial financial support from Secretary of Energy (SENER), Me´xico (Project SENER 2009-150001) is acknowledged.

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