Bioresource Technology 187 (2015) 288–298

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Optimization of chip size and moisture content to obtain high, combined sugar recovery after sulfur dioxide-catalyzed steam pretreatment of softwood and enzymatic hydrolysis of the cellulosic component Colin Olsen a, Valdeir Arantes b, Jack Saddler c,⇑ a b c

Neucel Specialty Cellulose Ltd, PO Box 2000, 300 Marine Drive, Port Alice, BC V0N 2N0, Canada Lorena School of Engineering, University of São Paulo Estrada Municipal do Campinho s/n, CP 116, 12602-810 Lorena, SP, Brazil Forestry Products Biotechnology/Bioenergy Group, Faculty of Forestry, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada

h i g h l i g h t s  We steam pretreated softwood chips and then used the solids for enzymatic hydrolysis.  We report on the influences of chip size and moisture content on sugar recovery.  Optimizing chip size provides a small increase in sugar recovery after pretreatment.  Elevated chip moisture provides maximum sugar recovery after both process steps.  Elevated chip moisture also promotes good process control of the pretreatment step.

a r t i c l e

i n f o

Article history: Received 10 February 2015 Received in revised form 18 March 2015 Accepted 19 March 2015 Available online 30 March 2015 Keywords: Bioconversion Steam pretreatment Combined sugar recovery Softwood Response surface methodology

a b s t r a c t The influence of chip size and moisture content on the combined sugar recovery after steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis of the cellulosic component were investigated using response surface methodology. Chip size had little influence on sugar recovery after both steam pretreatment and enzymatic hydrolysis. In contrast, the moisture of the chips greatly influenced the relative severity of steam pretreatment and, as a result, the combined sugar recovery from the hemicellulosic and cellulosic fractions. Irrespective of chip size and the pretreatment temperature, time, and SO2 loading that were used, the relative severity of pretreatment was highest at a moisture of 30–40 w/w%. However, the predictive model indicated that an elevated moisture content of roughly 50 w/w% (about the moisture content of a standard softwood mill chip) would result in the highest, combined sugar recovery (80%) over the widest range of steam pretreatment conditions. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Renewable fuels and chemicals can be produced from the sugars derived from the hemicellulosic and cellulosic components of biomass after pretreatment, enzymatic hydrolysis and fermentation. Concerns over volatile oil prices, energy security, the environmental consequences of continued fossil fuel dependence and the limited supplies of sugar and starch rich biomass for both human consumption and conversion to ethanol have encouraged ongoing work into the development ⇑ Corresponding author. Tel.: +1 604 822 9741; fax: +1 604 822 8157. E-mail addresses: [email protected] (C. Olsen), [email protected] (V. Arantes), [email protected] (J. Saddler). http://dx.doi.org/10.1016/j.biortech.2015.03.084 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

of biomass-to-sugars-and-fuels/chemicals processes (NREL, 2008; Representative Rahall NJ II, 2007; USDA, 2011). Agricultural residues (such as corn stover and wheat straw), herbaceous energy crops (such as switchgrass) and forest residues (such as softwood and hardwood chips) have all been investigated as potential feedstocks for bioconversion to fuels and chemicals (Galbe and Zacchi, 2012). A fairly recent Canadian estimate placed the total national available biomass at between 24 and 87 million dry tonnes per year, of which forest residues may contribute as much as 80% (Mabee and Saddler, 2010). A more recent assessment concluded that the availability of forest residues in British Columbia (primarily softwood) would be sufficient to support up to 10 bioconversion facilities for the production of advanced bioethanol (Mabee et al., 2011).

289

C. Olsen et al. / Bioresource Technology 187 (2015) 288–298

Pretreatment is widely recognized as a necessary first step in the bioconversion process, although many different strategies for the pretreatment of lignocellulosic biomass have been proposed (Chandra et al., 2007; Galbe and Zacchi, 2007; Yang and Wyman, 2008). A variation of steam pretreatment is arguably the preferred method of pretreatment as it appears to be the technology of choice for a number of advanced bioethanol facilities in Europe (Inbicon, Chemtex), North America (Abengoa, DSM-POET) and Brazil (Raizen, GranBio). However, all of these facilities primarily utilize herbaceous biomass feedstocks. If softwoods are to be used, earlier work has indicated that an acid catalyst needs to be combined with steam pretreatment to both enhance subsequent enzymatic hydrolysis of the cellulosic component while providing better recovery of the hemicellulose derived sugars (Boussaid et al., 1998; Clark and Mackie, 1987). Although previous work has shown that the addition of SO2 did not result in significant sulfonation or the solubilization of the lignin it did appear to increase the accessibility of the enzymes to the cellulose, possibly through the modification and redistribution of the lignin fraction (Clark et al., 1989; Donaldson et al., 1988). This work also showed that, when using softwoods, more severe conditions are required to produce a cellulosic fraction that can be readily hydrolyzed at low enzyme loadings. However, at these more severe conditions a significant amount of the hemicellulose derived sugars are degraded. Thus, to try to achieve maximum combined sugar recovery (from both the hemicellulose and cellulose components) the steam pretreatment conditions have to be a compromise between maximizing the recovery of hemicellulose-derived sugars and enhancing the enzymatic hydrolysis of the cellulosic component.

Several organic acids, and sulfuric acid (H2SO4) in particular, have been proposed as effective acid catalysts (Lee and Jeffries, 2011; Galbe and Zacchi, 2002). As another acid catalyst, sulfur dioxide (SO2) has been shown to have several advantages such as its rapid penetration into and its uniform distribution throughout wood chips (Mamers and Menz, 1984; Canadian Pulp and Paper Association, 1985; Wayman et al., 1984). Although less attention has been paid to the initial size and moisture content of the lignocellulosic feedstock, these two parameters are known to influence the effectiveness of steam pretreatment (Brownell et al., 1986; Monavari et al., 2009; Ballesteros et al., 2000; Ewanick and Bura, 2011). For example, the relative severity of steam pretreatment has been shown to decrease as the size of the softwood feedstock increases (Cullis et al., 2004). In subsequent work Swedish researchers were able to increase the combined recovery of soluble glucose and mannose after the pretreatment and enzymatic hydrolysis of softwood, from 71% to 73%, by decreasing chip thickness from 5–6 mm to 1–2 mm (Monavari et al., 2009). Earlier work using aspen (Populus tremuloides Michaux) showed that the interior temperature of green chips increased more slowly than did that of air dried chips during steam pretreatment. This difference in temperature profile resulted in the ‘undercooking’ of chip interiors and the ‘overcooking’ of chip exteriors at high reaction temperatures (Brownell et al., 1986). In other work the residual xylan content of the pretreatment-derived solid fraction of switchgrass decreased, from 3.9 to 2.5 w/w% (weight percent), as Table 2 Summary of results for steam pretreated lodgepole pine: water insoluble fraction. Run

Run

Temperature (°C)

Time (min)

SO2 (w/w%)

Chip size (inch)

Moisture content (w/w%)

1a 4 6 7 10 11 13 16 18 19 21 24 25 28 30 31

195 215 215 195 215 195 195 215 215 195 195 215 195 215 215 195

2.75 7.25 2.75 7.25 2.75 7.25 2.75 7.25 2.75 7.25 2.75 7.25 2.75 7.25 2.75 7.25

1.5 1.5 3.5 3.5 1.5 1.5 3.5 3.5 1.5 1.5 3.5 3.5 1.5 1.5 3.5 3.5

3/8 3/8 3/8 3/8 7/8 7/8 7/8 7/8 3/8 3/8 3/8 3/8 7/8 7/8 7/8 7/8

22.5 22.5 22.5 22.5 22.5 22.5 22.5 22.5 47.5 47.5 47.5 47.5 47.5 47.5 47.5 47.5

b

205 205 205 205 205 205

5 5 5 5 5 5

2.5 2.5 2.5 2.5 2.5 2.5

5/8 5/8 5/8 5/8 5/8 5/8

35 35 35 35 35 35

39c 40 41 42 43 44 45 46 47 48

185 225 205 205 205 205 205 205 205 205

5 5 0.5 9.5 5 5 5 5 5 5

2.5 2.5 2.5 2.5 0.5 4.5 2.5 2.5 2.5 2.5

5/8 5/8 5/8 5/8 5/8 5/8 1/8 9/8 5/8 5/8

35 35 35 35 35 35 35 35 10 60

33 34 35 36 37 38

a b c

Samples 1–31 belong to the fractional factorial design. Samples 33–38 are replicates of the center point. Samples 39–48 are axial points.

WIF yield

WIF composition AILa

Table 1 Experimental design for the response surface methodology model of lodgepole pine.

Manc

Xyld

Glucan hydrolysise

(–)

(w/w%)f

(w/w WIF%)g

1 4 6 7 10 11 13 16 18 19 21 24 25 28 30 31

56.9 47.6 47.3 54.3 56.3 57.4 59.7 49.9 52.1 59.9 61.0 44.8 62.0 50.2 49.8 56.5

50.09 72.66 68.17 56.15 48.34 52.50 47.35 85.85 51.31 43.84 40.74 74.23 42.83 62.48 57.11 48.79

49.96 24.03 32.50 42.55 48.63 44.05 51.90 10.98 48.86 55.31 56.98 23.77 57.39 35.03 43.14 50.90

0.51 0.15 0.25 0.29 0.39 0.34 0.45 0.16 0.31 0.32 0.55 0.00 0.49 0.32 0.34 0.26

0.49 0.11 0.08 0.17 0.29 0.26 0.36 0.14 0.26 0.26 0.60 0.00 0.58 0.24 0.24 0.18

38.1 75.1 66.5 49.7 40.3 44.9 32.5 88.1 44.5 27.8 24.3 76.8 22.5 61.7 53.2 37.4

CPh

49.6

60.30

38.38

0.28

0.22

58.4

3.4

6.77

12.13

29.50

34.41

11.1

61.2 41.9 65.8 49.6 59.3 47.7 51.2 49.0 62.9 58.9

42.54 88.40 38.84 61.81 46.36 61.53 57.62 53.19 46.71 45.43

56.68 9.89 58.26 36.55 53.05 35.53 41.32 43.25 52.18 55.46

0.60 0.00 0.92 0.23 0.42 0.18 0.22 0.29 0.54 0.47

0.59 0.00 1.01 0.21 0.31 0.11 0.07 0.12 0.52 0.38

24.5 91.0 16.5 60.5 31.5 63.1 53.9 53.8 28.5 28.8

SE

i

39 40 41 42 43 44 45 46 47 48 a

Glub

(%)

Acid insoluble lignin. Glucan. Mannan. d Xylan. e Calculated as the glucan present in the raw wood minus that remaining in the WIF after steam pretreatment. f Expressed in units of w/w% of raw wood. g Expressed in units of odg w/w% of the water insoluble fraction. h Samples 33–38 are replicates of the center point. i Standard error of the center point. b

c

290

C. Olsen et al. / Bioresource Technology 187 (2015) 288–298

Table 3 Fitted coefficients of the response surface methodology model of steam pretreated lodgepole pine. Independent variables are presented in coded form. Response a

Yield Glucan Mannan Xylan Hemicelluloseg Glucose EH recoveryh Combined recoveryi a b c d e f g h i

R2

Model b

c

d

e

f

2

2

2

49.87  4.51T  2.37t  1.77S  0.95TMc  1.04Si Mc + 1.69t + 0.63S + 2.48Mc 43.07  15.08T  9.48t  5.70S + 3.59Mc  3.41Tt  3.28TS + 3.69t2 + 6.14Mc2 1.23  0.58T  0.65t  0.23S + 0.36t2  0.13Si2 + 0.25Mc2 1.84  1.14T  1.37t  0.45S + 0.50Tt + 0.89t2  0.44Si2 + 0.46Mc2 49.17  13.83T  6.96t  5.91S + 1.94Si + 1.94Mc  6.24Tt  2.65tMc  3.48SSi  3.68SiMc + 2.92t2  1.94Si2 + 4.70Mc2 33.30 + 3.93T + 4.66t  3.10Tt + 2.46TMc  1.78tS  1.91SMc  1.78T2  2.01t2  2.86Mc2 24.79  6.23T  3.29t  2.30S + 1.47Mc  3.55Tt  3.32TS + 1.15tMc  1.13SiMc  1.22T2 + 3.16Mc2 62.32  8.19T  2.48t  3.51S + 2.14Mc  7.69Tt  3.91TS + 1.34TSi + 1.40TMc  2.52tS  2.10T2 + 2.88Mc2

0.88 0.91 0.80 0.82 0.91 0.80 0.92 0.90

Equation created in units of w/w% of raw wood. Temperature. Time. SO2 loading. Chip moisture content. Chip size. Sugars derived from hemicellulose are mannose, xylose, galactose and arabinose. Recovery of all five soluble sugars in the EH. Recovery of all five soluble sugars as both monomers and oligomers after steam pretreatment and enzymatic hydrolysis.

Fig. 1. The effect of chip size on the recovery of hemicellulose-derived sugars obtained in the water soluble fraction after the steam pretreatment of lodgepole pine. SO2 and moisture content are set at 1.5 and 47.5 w/w%, respectively.

C. Olsen et al. / Bioresource Technology 187 (2015) 288–298 Table 4 Summary of results for steam pretreated lodgepole pine: sugar recovery of the water soluble fraction. Run

WSF recoveriesa Hemi (%)

Glucose

Total

(–) 1 4 6 7 10 11 13 16 18 19 21 24 25 28 30 31

53.2 28.2 36.7 54.8 66.6 80.3 59.7 19.4 59.8 73.8 78.5 20.2 81.3 38.4 47.8 59.0

21.0 29.1 32.9 34.2 22.2 28.2 19.3 28.9 31.9 31.0 14.3 25.5 13.1 39.1 35.5 24.1

31.7 28.8 34.2 41.0 36.9 45.5 32.7 25.8 41.1 45.2 35.6 23.7 35.7 38.9 39.6 35.7

CP

45.6

34.8

38.4

SE

10.4

10.5

7.6

39 40 41 42 43 44 45 46 47 48

81.6 27.4 77.8 48.9 66.1 47.9 44.1 43.7 73.8 67.1

17.0 34.4 9.3 40.3 22.8 36.6 33.4 31.1 20.8 22.0

38.5 32.0 32.0 43.2 37.2 40.3 37.0 35.3 38.4 36.9

a All recoveries are calculated as a percentage of the individual sugars or group of sugars (as both monomers and oligomers) in the raw wood subsequently present in the WSF.

a result of increasing the moisture content of the raw material from 12 to 80 w/w% prior to SO2 impregnation (Ewanick and Bura, 2011). It appeared that increasing the moisture content of the raw biomass did not result in a decrease in the relative severity of steam pretreatment. Unfortunately, these researchers were not able to determine if the effectiveness of the SO2 impregnation step was influenced by the moisture content of the biomass. In the work reported here a model of the SO2 catalyzed steam pretreatment and enzymatic hydrolysis of lodgepole pine was developed using response surface methodology (RSM). By using RSM, the goal was to determine if chip size and moisture content influenced the combined recovery of hemicellulose and cellulose derived sugars after steam treatment and subsequent enzymatic hydrolysis of the cellulosic component.

2. Methods 2.1. Experimental design Response surface methodology (RSM) is a statistical technique which allows the simultaneous investigation of multiple parameters on a single process outcome. The five steam pretreatment variables that were studied were: temperature, time, SO2 loading, chip size, and initial chip moisture content. The 32 steam pretreatment experimental conditions that were compared and used for the central composite experimental design (CCD) are listed in Table 1. Note that the half fraction of the 25 factorial design was chosen using the block generator I = 12345 = 1, and that the order of experiments was randomized (Box and Draper, 1987). Chip size was varied from 1/8 in (3 mm) to 1-3/4 in (45 mm) to represent the range of chips sizes encountered in a typical softwood mill setting.

291

The moisture content of the chips was varied from 10 to 60 w/w% (wet weight basis). Each of the five independent variables ranged from 2 to +2 when converted to coded form. 2.2. Raw wood Healthy, freshly-felled lodgepole pine (approximately 75 years old) sourced from British Columbia’s Thompson-Okanagan interior region was cut into three bolts measuring roughly 14 cm in diameter, debarked and chipped. Klason lignin analysis showed that the chips contained: 45.9 ± 0.3 w/w% glucan, 26.9 ± 0.6 w/w% lignin (both acid insoluble and acid soluble lignin), 11.8 ± 0.9 w/w% mannan, 6.3 ± 0.3 w/w% xylan, 2.9 ± 0.2 w/w% galactan, and 1.8 ± 0.1 w/w% arabinan. The ranges listed here are ±one standard deviation. 2.3. Chipping and screening After chipping, a Williams classifier removed the dust retained on a pan, the over-thick chips retained on the bar (TH) 10 mm screen and the over-sized chips retained on the round hole (RH) 45 mm (roughly 1–3/4 in) screen. The remaining chips retained on the RH 1/8 in (3 mm), RH 3/8 in (10 mm), RH 5/8 in (16 mm), RH 7/8 in (22 mm) and RH 9/8 in (29 mm) screens were used for steam pretreatment. Chip thickness was primarily in the 2–6 mm range for the size fractions up to RH 7/8 in and 8–10 mm for the RH 9/8 in fraction (data not shown). In other words, chip thickness was found to increase slightly as chip size increased. The moisture content of the fully classified chip samples were determined (31–39 w/w% on a wet weight basis) by drying to a constant weight at 105 °C before storage in plastic bags in aliquots of 200 odg (oven dry grams). The samples were immediately frozen to 20 °C. 2.4. Adjustment of initial moisture content Those wood chip samples requiring an increase in moisture content had water added via a spray bottle to enhance homogenous uptake. The samples were stored at 4 °C until the excess moisture had been incorporated into the raw wood. The adjusted samples were then frozen again at 20 °C. Those samples requiring a decrease in moisture content were air dried at room temperature and the weight of the samples was monitored until the desired value had been reached. The adjusted samples were then frozen and stored at 20 °C. 2.5. SO2-catalyzed steam pretreatment Thawed wood samples were impregnated with gaseous SO2 (Praxair Canada Inc., Mississauga, Ontario) prior to steam pretreatment. The gaseous SO2 was added to sealed plastic bags containing 200 odg of raw wood with the amount added controlled by monitoring the total weight of the bags. The impregnated samples were left overnight in a fume hood at room temperature. Steam pretreatment was carried out the following day. Steam pretreatment was carried out in batches of 200 odg using a 2 L StakeTech II steam gun (Stake Technologies, Norval, Ontario). At the end of each reaction, the slurry was removed and vacuum filtered in a Buchner funnel, separating the water soluble fraction (WSF) from the water insoluble fraction (WIF). The WIF was then washed in stages with approximately 5 L of water, also using vacuum filtration. Samples of the WSF (including wash water) were then frozen at 20 °C, while WIF samples were refrigerated until the time of analysis. 2.6. Enzymatic hydrolysis The washed, never-dried WIF obtained after steam pretreatment was enzymatically hydrolyzed in sodium acetate buffer

292

C. Olsen et al. / Bioresource Technology 187 (2015) 288–298

Fig. 2. The effect of moisture content on the recovery of hemicellulose-derived sugars obtained in the water soluble fraction after the steam pretreatment of lodgepole pine. SO2 and chip size are set at 1.5 w/w% and 10 mm (3/8 in), respectively.

(50 mM, pH 4.8) at 50 °C in an orbital shaker operating at 150 rpm. Hydrolysis was carried out at a moderate substrate consistency (10 w/w%) using a total weight of 25 g (2.5 odg WIF) in 125 mL Erlenmeyer screw-top flasks. At this consistency, end-product inhibition, limited cellulose accessibility, and non-productive binding of the enzymes to lignin were all expected to influence the extent of hydrolysis. The commercial enzyme preparation CellicÒ CTec2 (Novozymes North America Inc., Franklinton, North Carolina) was used at a loading of 70 mg total protein/g glucan. After 72 h, a 1 mL sample of the hydrolyzate was taken and the reaction was halted by boiling the sample on a hot-plate at 100 °C for 10 min. The boiled sample was then centrifuged for 10 min at 13,000g and 4 °C before freezing the supernatant at 20 °C. Each hydrolysis reaction was conducted in duplicate. 2.7. Analytical methods All analyses were repeated in triplicate. The moisture content of the WIF was determined in the same manner as for the raw wood. The cellulose, hemicellulose, and acid insoluble lignin (AIL) contents of the raw wood and pretreatment-derived solid fractions were

quantified according to the TAPPI standard method T-222-om-06 (TAPPI, 2006). The analysis of the resulting Klason filtrate for acid soluble lignin (ASL) was done by measuring UV absorbance at 205 nm (Dence, 1992). Both the WIF- and WSF-derived soluble monomeric sugars were quantified by high performance liquid chromatography (HPLC) using a Dionex ICS-3000 system (Dionex Corporation, Sunnyvale, California) as previously described (Kumar et al., 2012). Nanopure water at 1.0 mL/min was used as the eluent and 0.2 M NaOH was added post-column. The column was reconditioned between each sample (injection volume 20 lL) with 1.0 M NaOH and all samples were filtered through either a 0.45 or 0.20 lm filter (Chromatographic Specialties, Brockville, Ontario) prior to analysis. Fucose (Sigma Aldrich Canada Ltd., Oakville, Ontario) acted as an internal standard. Post-hydrolysis of a duplicate sample using 4% H2SO4 followed by autoclaving for 1 h at 121 °C allowed for the quantification of sugars in oligomeric form. 2.8. Enzymatic hydrolyzate The soluble monomeric sugars present in the free liquid of the enzymatic hydrolysis reactions (enzymatic hydrolyzate, EH) were

C. Olsen et al. / Bioresource Technology 187 (2015) 288–298 Table 5 Summary of results for steam pretreated and enzymatically hydrolyzed lodgepole pine: combined sugar recovery. All recoveries are calculated as a percentage of the individual sugars or group of sugars (as both monomers and oligomers) in the raw wood subsequently present in the WSF or EH. Run

EH Recoveryb

Combined Recoveryc

(–)

WIF Digestibilitya (%)

1 4 6 7 10 11 13 16 18 19 21 24 25 28 30 31

68.1 81.4 85.9 81.5 88.1 82.2 81.0 62.9 89.4 70.9 73.3 79.4 50.4 86.2 88.1 86.4

28.4 13.6 19.3 27.6 35.3 30.4 36.7 5.0 33.3 34.5 37.2 12.3 31.6 22.2 27.6 36.2

60.0 42.4 53.4 68.6 72.2 75.9 69.5 30.8 74.4 79.6 72.8 36.1 67.3 61.1 67.2 71.9

CP

87.7

24.0

62.4

SE

6.2

8.7

4.3

39 40 41 42 43 44 45 46 47 48

65.8 91.5 50.4 86.4 68.4 70.7 84.3 81.8 78.8 75.7

33.4 5.5 28.6 22.9 31.5 17.5 26.1 25.4 37.8 36.2

71.9 37.6 60.6 66.1 68.6 57.8 63.0 60.6 76.2 73.1

3.1. Composition and yield of the steam pretreatment-derived, cellulose-rich water insoluble fraction

a

Conversion of the glucan available in the WIF to soluble monomeric glucose. Sum of all five sugars in the enzymatic hydrolyzate. Sum of all five sugars recovered after both steam pretreatment and enzymatic hydrolysis. c

quantified using the previously described HPLC method. Sugars present in the CellicÒ CTec2 enzyme preparation were subtracted from those present in the hydrolyzate. During enzymatic hydrolysis conducted at moderate to high substrate consistency, the conversion of insoluble carbohydrate polymers to soluble sugars changes the density, volume, and mass of the hydrolyzate. Thus, the previously determined correction factor (0.92) was applied to avoid an overestimation of cellulose hydrolysis (Kristensen et al., 2009). 2.9. Statistical analysis A second-order approximation was fitted to each set of data. In the generalized equation, the predicted value of an individual response variable is denoted Y, while all five independent variables (k = 5) are present in their coded form (Xi) (Box et al., 2005). The presence of the quadratic terms of the form aiiX2 and the crossproduct terms of the form aijXXj allow for the estimation of curvature in the response: k k1 X k k X X X ai X i þ aij X i  X j þ aii X 2i i¼1

i¼1 j¼1

insignificant (p > 0.10) were removed from the equation and the values of the remaining regression coefficients were determined. The lack-of-fit test ratio (Flof) was used to test each equation, for which a plof value less than 0.05 indicated a lack-of-fit. The results of the extended ANOVA are available in the reference of Olsen (2012).

3. Results and discussion

b

Y ¼ a0 þ

293

ð1Þ

i¼1

Multiple linear regression was used to fit the equation to each set of data and an extended analysis of variance (ANOVA) was performed to determine which terms were significant at a 90% confidence interval. All data analysis was conducted using the commercially available software Statistica 6.0 (StatSoft Inc., Tulsa, Oklahoma). Those terms found to be statistically

It was apparent (Table 2) that nearly all of the combinations of steam pretreatment conditions used were sufficiently severe such that complete hydrolysis of the hemicellulose component of the raw wood could be achieved. Consequently, the resulting water insoluble fractions were composed almost entirely of cellulose and lignin. Previous work on softwoods showed that maximum overall sugar recovery could be achieved when SO2 catalyzed steam pretreatment resulted in solubilization of 10–20% of the original glucan present in the wood chip (Clark and Mackie, 1987; Stenberg et al., 1998) and a water insoluble fraction (WIF) yield of roughly 65 w/w% (Clark and Mackie, 1987; Söderström et al., 2004). Glucan solubilization, which ranged from 17% to 91%, and WIF yield, which ranged from 42 to 66 w/w% (Table 2), indicated that at least a part of this potentially optimal range had been captured. Although chip size had a very limited influence on the composition of the steam pretreatment-derived solid fraction as a whole, in contrast, the data (Table 3) indicated that all of the remaining variables, including moisture content, had a significant effect on the composition of the solid fraction. As well as influencing the composition of the WIF it was apparent that the temperature, time, and SO2 loading used for pretreatment and the moisture content of the chips influenced the final WIF yield. Although earlier work had suggested that the relative severity of steam pretreatment could be reduced as softwood chip size is increased, this work compared a much broader feedstock size range, from less than 1 to 50 mm (Cullis et al., 2004). It was likely that the relatively narrow ranges of chip length (3–45 mm) and thickness (essentially 2–6 mm) used in the current study limited the influence of chip size on the WIF yield. Similar results were obtained after steam pretreating Norway spruce (Picea abies (L.) H. Karst) chips of 1–2 and 5–6 mm thickness, again resulting in no real differences in yield after steam pretreatment (Monavari et al., 2009). It has also been shown that, when SO2 is used as the acid catalyst, the mass transfer of gaseous SO2 to the interior of even relatively thick wood chips is relatively rapid (Wayman et al., 1984; Monavari et al., 2009). In comparison, the mass transfer of charged ionic components such as those present in Kraft and sulfite pulping liquors into the interior of wood chips is much slower and may not be complete by the time the respective pulping reactions commence (Canadian Pulp and Paper Association, 1985, 1989). Thus, it is likely that the combination of an SO2 catalyst and a relatively narrow chip size range resulted in the lack of influence of chip size on WIF yield. In contrast, the initial moisture content of the chips had a significant effect on the steam pretreatment-derived solid fraction yield with highest values achieved when chips had either a low or high moisture content and lowest yields achieved when chips had a moderate moisture content. Steam pretreatment at 205 °C, 5 min, and 2.5 w/w% SO2 (Run 47, Table 1) using 5/8 in (16 mm) chips at 10 w/w% moisture resulted in a WIF yield of 63 w/w%. When the moisture content was increased first to 35 (Runs 33–38) and then to 60 w/w% (Run 48), the yield first decreased to 50 ± 2 w/w% before rising again to 59 w/w%. The resulting model

294

C. Olsen et al. / Bioresource Technology 187 (2015) 288–298

Fig. 3. The effect of moisture content on the recovery of all sugars (primarily glucose) obtained after the enzymatic hydrolysis of the water insoluble fraction of steam pretreated lodgepole pine. SO2 and chip size are set at 1.5 w/w% and 10 mm (3/8 in), respectively.

(which contains a quadratic moisture content term) indicated that the lowest WIF yield, and therefore the greatest gross effect of steam pretreatment on the raw wood, always occurred at moderate moisture contents ranging from 30 to 40 w/w% (Table 3). In addition, the data indicated that the potentially desirable yield of 65 w/w% could be achieved over the full range of moisture contents that was investigated. There are several possible reasons for the observed, non-linear relationship between the moisture content of the chips and the final WIF yield. As the solid fraction yield is primarily determined by the extent of hemicellulose solubilization occurring during steam pretreatment it is possible that, at moisture contents below 30 w/w%, the rate and extent of the hydrolysis is water limited. Earlier work had shown that green wood displayed a greater chemical reactivity than air dry wood towards hydrolysis, despite the fact that the temperature of air dry wood chips was shown to approach the intended temperature of steam pretreatment more rapidly (Brownell et al., 1986). Thus, it is possible that the amount of gaseous SO2 adsorbed to wood chips that had a moisture content of less than 30 w/w% was less than that applied, resulting in lower steam pretreatment severity and, consequently, a higher than

anticipated WIF yield. As mentioned earlier, a chip moisture content higher than 40 w/w% would also be problematic as hemicellulose hydrolysis proceeds at a reduced rate and to a lesser extent due to the increased heat capacity of the moist wood chips. This earlier work showed that the majority of the latent heat released upon steam condensation was used to heat the water present in green wood chips, resulting in less energy being available for the hydrolysis reactions (Brownell et al., 1986). 3.2. Sugar recovery in the steam pretreatment-derived, hemicelluloserich water soluble fraction Although chip size did not influence the amount of glucose released into the water soluble fraction (WSF) the response surfaces (Fig. 1) indicated that chip size did have a small yet statistically significant influence on the recovery of soluble, hemicellulose-derived sugars. For example, 1/8 in (3 mm) chips of 35 w/w% moisture that were steam pretreated at 205 °C, 5 min, and 2.5 w/w% SO2 (Run 45, Table 1) resulted in a 44% recovery of soluble hemicellulose-derived sugars (Table 4). As chip size was increased to 5/8 in (16 mm, Runs 33–38) and then to 9/8 in

C. Olsen et al. / Bioresource Technology 187 (2015) 288–298

295

Fig. 4. The effect of moisture content on the combined sugar recovery obtained after the steam pretreatment and subsequent enzymatic hydrolysis of lodgepole pine. SO2 and chip size are set at 1.5 w/w% and 10 mm (3/8 in), respectively.

(29 mm, Run 46), the recovery first increased slightly to 46 ± 5% before decreasing again to 44%. Thus it appears that a small gain in hemicellulose recovery could be made at each of the moisture contents by optimizing chip size. For steam pretreatment conducted at 205 °C, 5 min, and 2.5 w/w% SO2 using chips with a 22.5 w/w% moisture, a chip size of about 1 in (25 mm) was predicted to lead to the highest recovery of soluble, hemicellulosederived sugars. When the moisture content of the chips was increased to 35 and then to 47.5 w/w%, the optimum chip size decreased to 3/4 in (19 mm) and then to 1/2 in (13 mm). Although this result could encourage the production of more uniform chips, to maximize the recovery of soluble, hemicellulose-derived sugars, these relatively small increases might not merit the costs of implementing an enhanced chipping and screening strategy at a commercial scale (Monavari et al., 2009). As shown by the response surfaces (Fig. 2), a moisture content of 47.5 w/w% for the chips resulted in the good recovery of hemicellulose-derived sugars (above 80%) over wide ranges of steam pretreatment temperatures and time. Over the ranges of steam pretreatment operating conditions used in this study,

chips with a lower moisture content (22.5 w/w%) generally showed a much lower hemicellulose recovery. Previous work found that the relative severity of steam pretreatment (as measured by both WIF yield and the recovery of soluble, hemicellulose-derived sugars in the WSF) was reduced as the moisture content of the chips increased (Cullis et al., 2004). The more recent work reported here found that chips with either low or high moisture contents showed a decrease in the relative severity of steam pretreatment. For example, chips of 5/8 in (16 mm) and 10 w/w% moisture that were steam pretreated at 205 °C, 5 min, and 2.5 w/w% SO2 (Run 47, Table 1) resulted in a WSF hemicellulose-derived sugar recovery of 74% (Table 4). When the moisture content was increased to 35 (Runs 33–38) and then to 60 w/w% (Run 48), the recovery first decreased to 46 ± 5% before rising again to 67%. The corresponding model (which contains a quadratic moisture content term) also showed that chips with a moderate moisture content (30–40 w/w%) resulted in the minimum recovery of soluble, hemicellulosederived sugars at each combination of pretreatment operating conditions and chip sizes.

296

C. Olsen et al. / Bioresource Technology 187 (2015) 288–298

Fig. 5. Combined sugar recovery obtained after the steam pretreatment and subsequent enzymatic hydrolysis of lodgepole pine as a function of (A) the water insoluble fraction yield and (B) the extent of glucan hydrolysis after steam pretreatment. The mean value of the center point is depicted and the error bars represent ±one standard deviation of the mean.

3.3. Sugar recovery after enzymatic hydrolysis of the steam pretreatment-derived, cellulose-rich water insoluble fraction It was apparent that no clear relationship existed between the glucan or lignin contents of the WIF and the ease of enzymatic digestibility of this cellulose rich component of steam pretreated softwoods, as had been previously reported by other workers (Clark and Mackie, 1987; Stenberg et al., 1998). Although chip size was shown to have a very small but statistically significant influence on the recovery of soluble sugars after enzymatic hydrolysis (Table 3), the strong relationship between moisture content and the relative severity of steam pretreatment implied it would likely have a significant influence on the ease of enzymatic hydrolysis of the WIF component. For example, steam pretreated chips (5/8 in (16 mm) and 10 w/w% moisture), at 205 °C, 5 min, and 2.5 w/w% SO2 (Run 47, Table 1) resulted in an enzymatic hydrolysis sugar

recovery of 38% (Table 5). When the moisture content was increased to 35 (Runs 33–38) and to 60 w/w% (Run 48), the recovery decreased to 24 ± 2% before rising again to 36%. Chips with a 47.5 w/w% moisture content resulted in good enzymatic hydrolysis sugar recovery (above 35%) over a wide range of steam pretreatment conditions (Fig. 3) while achieving the same good recovery using chips with a 22.5 w/w% moisture content was limited to a combined range of higher temperatures (225 °C) and shorter residence times (0.50 min). 3.4. Combined sugar recovery after steam pretreatment and enzymatic hydrolysis Although the combined sugar recovery ranged widely, from 31% to 80% (Table 5), the model of combined sugar recovery (Fig. 4) suggested that maximum sugar recovery (hemicellulose and

C. Olsen et al. / Bioresource Technology 187 (2015) 288–298

cellulose derived) would be achieved when pretreatment is carried out using a combination of lower temperatures (185 °C) and longer residence times (9.5 min). The response surfaces in Fig. 4 were set at an SO2 loading and chip size of 1.5 w/w% and 10 mm (3/8 in), respectively, yet the corresponding model (Table 3) suggests that this prediction of maximum sugar recovery using a combination of lower temperatures and longer residence times holds true over the full ranges of SO2 loadings, chip sizes and moisture contents that were investigated. Although it is very unlikely that complete sugar recovery will ever be achieved, the response surfaces did show that the range of thermal severity over which a combined sugar recovery of at least 70% can be achieved, increased with increasing moisture content. Increasing the range over which high sugar recovery might be achieved should be advantageous from a process control perspective as any deviation from the desired pretreatment operating conditions or feedstock characteristics is less likely to result in a large decrease in combined sugar recovery. Although chip size had a very limited influence on the combined sugar recovery achieved after steam pretreatment and enzymatic hydrolysis (Table 3) the moisture content of the chip greatly affected this response variable. Specifically, highest recoveries were achieved at low and high moisture contents while the lowest recoveries were achieved at moderate moisture contents. For example, chips (5/8 in (16 mm) and 10 w/w% moisture) steam pretreated at 205 °C, 5 min, and 2.5 w/w% SO2 (Run 47, Table 1) resulted in a combined sugar recovery of 76% (Table 5). When the moisture content was increased to 35 (Runs 33–38) and then to 60 w/w% (Run 48), the recovery decreased to 62 ± 3% before rising again to 73%. Although the response surfaces (Fig. 4) were set at an SO2 loading and chip size of 1.5 w/w% and 10 mm (3/8 in), respectively, the corresponding model suggested that the combined sugar recovery approached a minimum at a moderate moisture content of roughly 35 w/w% at each combination of pretreatment operating conditions. As mentioned earlier, previous work had shown that maximum combined hemicellulose and cellulose derived sugar recovery from SO2 catalyzed steam pretreated softwoods is typically achieved when pretreatment results in a WIF yield of roughly 65 w/w% (Clark and Mackie, 1987; Stenberg et al., 1998). In these two previous studies a substrate consistency of 2 w/w% and enzyme loadings of 250 and 320 mg protein/g substrate, respectively, were used. In contrast, the work reported here used a higher substrate consistency of 10 w/w% and a much reduced enzyme loading of 7–41 mg protein/g substrate (70 mg protein/g glucan). The highest combined sugar recovery achieved experimentally in the current study (80%) did not correspond to a WIF yield of 65 w/w% after steam pretreatment or to a glucan hydrolysis extent of 10–20%. This was likely due to the fact that enzymatic hydrolysis was conducted under more challenging conditions. The highest combined sugar recovery achieved experimentally in the current study occurred when chips (3/8 in (10 mm)) were steam pretreated at 195 °C, for 7.25 min, using 1.5 w/w% SO2 and a 47.5 w/w% moisture (Run 19, Table 1). Under these conditions, the WIF yield was lower, at 60 w/w% and the extent of glucan hydrolysis was much higher, at nearly 30% (Table 2 and Fig. 5). It is probable that, as a result of the more challenging enzyme hydrolysis conditions (increased substrate consistency, lower enzyme loading), hemicellulose sugar recovery after steam pretreatment was responsible for an increased amount of the maximum combined sugar recovery. 4. Conclusions The moisture content of the softwood chips prior to SO2 catalyzed steam pretreatment was shown to have a significant impact on the combined recovery of hemicellulose and cellulose derived

297

sugars, likely due to it influencing the relative severity of steam pretreatment. A moisture content of roughly 50 w/w% appeared optimal as it allowed for maximum combined sugar recovery over wide ranges of steam pretreatment conditions. In contrast, chip size had a much smaller influence although size optimization could provide a slight increase in the recovery of soluble sugars after pretreatment. Acknowledgements The authors wish to thank the Natural Sciences and Engineering Research Council of Canada (NSERC), Natural Resources Canada (NRCan) and Genome British Columbia for their support of this study. Bolts of lodgepole pine were kindly supplied by Kerry Rouck of Gorman Brothers Lumber Ltd. (Kelowna, BC.). Access to chipping and screening equipment was kindly arranged by Paul Bicho and Bernard Yuen of FPInnovations (Vancouver, BC.) as well as Rodger Beatson of the British Columbia Institute of Technology (BCIT, Burnaby, BC.). Finally, we wish to thank Novozymes (Franklinton, North Carolina) for providing the enzymes used in this study. References Ballesteros, I., Oliva, J., Navarro, A., González, A., Carrasco, J., Ballesteros, M., 2000. Effect of chip size on steam explosion pretreatment of softwood. Appl. Biochem. Biotechnol. 84–86, 97–110. Boussaid, A., Cai, Y., Robinson, J., Gregg, D.J., Nguyen, Q., Saddler, J.N., 1998. Sugar recovery and fermentability of hemicellulose hydrolysates from steamexploded softwoods containing bark. Biotechnol. Prog. 17, 887–892. Box, G.E.P., Draper, N.R., 1987. Blocking and fractionating 2k factorial designs. In: Empirical Model-Building and Response Surfaces. John Wiley & Sons Inc., New York, NY. Box, G.E.P., Hunter, J.S., Hunter, W.G., 2005. Modeling, geometry, and experimental design. In: Balding, D.J. et al. (Eds.), Statistics for Experimenters: Design, Innovation, and Discovery. Wiley-Interscience, Hoboken, NJ. Brownell, H.H., Yu, E.K.C., Saddler, J.N., 1986. Steam-explosion pretreatment of wood: effect of chip size, acid, moisture content and pressure drop. Biotechnol. Bioeng. 28, 792–801. Chandra, R.P., Bura, R., Mabee, W.E., Berlin, A., Pan, X., Saddler, J.N., 2007. Substrate pretreatment: the key to effective enzymatic hydrolysis of lignocellulosics? Adv. Biochem. Eng. Biotechnol. 108, 67–93. Clark, T.A., Mackie, K.L., 1987. Steam explosion of the softwood Pinus radiata with sulphur dioxide addition. I Process optimization. J. Wood Chem. Technol. 7, 373–403. Clark, T.A., Mackie, K.L., Dare, P.H., McDonald, A.G., 1989. Steam explosion of the softwood Pinus radiata with sulphur dioxide addition. II. Process characterisation. J. Wood Chem. Technol. 9, 135–166. Cullis, I.F., Saddler, J.N., Mansfield, S.D., 2004. Effect of initial moisture content and chip size on the bioconversion efficiency of softwood lignocellulosics. Biotechnol. Bioeng. 85, 413–421. Dence, C.W., 1992. The determination of lignin. In: Lin, S.Y., Dence, C.W. (Eds.), Methods in Lignin Chemistry. Springer-Verlag, Berlin, Germany, pp. 33–61. Donaldson, L.A., Wong, K.K.Y., Mackie, K.L., 1988. Ultrastructure of steam-exploded wood. Wood Sci. Technol. 22, 103–114. Ewanick, S., Bura, R., 2011. The effect of biomass moisture content on bioethanol yields from steam pretreated switchgrass and sugarcane bagasse. Bioresour. Technol. 102, 2651–2658. Galbe, M., Zacchi, G., 2012. Pretreatment: the key to efficient utilization of lignocellulosic materials. Biomass Bioenergy 46, 70–78. Galbe, M., Zacchi, G., 2007. Pretreatment of lignocellulosic materials for efficient bioethanol production. Adv. Biochem. Eng. Biotechnol. 108, 41–65. Galbe, M., Zacchi, G., 2002. A review of the production of ethanol from softwood. Appl. Microbiol. Biotechnol. 59, 618–628. Kristensen, J., Felby, C., Jørgensen, H., 2009. Determining yields in high solids enzymatic hydrolysis of biomass. Appl. Biochem. Biotechnol. 156, 127–132. Kumar, L., Arantes, V., Chandra, R.P., Saddler, J.N., 2012. The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility. Bioresour. Technol. 103, 201–208. Lee, J., Jeffries, T.W., 2011. Efficiencies of acid catalysts in the hydrolysis of lignocellulosic biomass over a range of combined severity factors. Bioresour. Technol. 102, 5884–5890. Mabee, W.E., Saddler, J.N., 2010. Bioethanol from lignocellulosics: status and perspectives in Canada. Bioresour. Technol. 101, 4806–4813. Mabee, W.E., McFarlane, P.N., Saddler, J.N., 2011. Biomass availability for lignocellulosic ethanol production. Biomass Bioenergy 35, 4519–4529. Mamers, H., Menz, D.N.J., 1984. Explosion pretreatment of Pinus radiata wood chips for the production of fermentation substrates. APPITA 37, 644–649.

298

C. Olsen et al. / Bioresource Technology 187 (2015) 288–298

Monavari, S., Galbe, M., Zacchi, G., 2009. Impact of impregnation time and chip size on sugar yield in pretreatment of softwood for ethanol production. Bioresour. Technol. 100, 6312–6316. National Renewable Energy Laboratory (NREL), 2008. The Impact of Ethanol Blending on U.S. Gasoline Price. NREL/SR-670-44517. Olsen, C.A., 2012 (unpublished results). Empirical Process Modeling of the Acid Catalyzed Steam Pretreatment of Radiata and Lodgepole Pine (MASc thesis). The University of British Columbia, Vancouver, BC. Representative Rahall NJ II, 2007. Energy Independence and Security Act of 2007. Congress, Washington DC. Söderström, J., Galbe, M., Zacchi, G., 2004. Effect of washing on yield in one- and two-Step steam pretreatment of softwood for production of ethanol. Biotechnol. Prog. 20, 744–749. Stenberg, K., Tengborg, C., Galbe, M., Zacchi, G., 1998. Optimisation of steam pretreatment of SO2-impregnated mixed softwoods for ethanol production. J. Chem. Technol. Biotechnol. 71, 299–308.

TAPPI, 2006. TAPPI Test Method 222 om-06, Acid Insoluble Lignin in Wood and Pulp. In: TAPPI Test Methods. Technical Association of the Pulp and Paper Industry, Norcross, GA. Technical Section: Canadian Pulp and Paper Association, 1989. Volume 5: Alkaline Pulping. In: Grace, T.M., et al. (Eds.), Pulp and Paper Manufacture. McGraw-Hill, Montreal, QC. Technical Section: Canadian Pulp and Paper Association, 1985. Volume 4: Sulfite Science & Technology. In: Ingruber, O.V., et al. (Eds.), Pulp and Paper Manufacture. McGraw-Hill, Montreal, QC. United States Department of Agriculture (USDA), 2011. Why Have Food Commodity Prices Risen Again? WRS-1103. Wayman, M. et al., 1984. Hydrolysis of biomass by sulphur dioxide. Biomass 6, 183– 191. Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioprod. Biorefin. 2, 26–40.

Optimization of chip size and moisture content to obtain high, combined sugar recovery after sulfur dioxide-catalyzed steam pretreatment of softwood and enzymatic hydrolysis of the cellulosic component.

The influence of chip size and moisture content on the combined sugar recovery after steam pretreatment of lodgepole pine and subsequent enzymatic hyd...
2MB Sizes 1 Downloads 8 Views