Appl Biochem Biotechnol DOI 10.1007/s12010-013-0721-1

Impact of Pretreatment with Dilute Sulfuric Acid Under Moderate Temperature on Hydrolysis of Corn Stover with Two Enzyme Systems Chao Tai & Deepak Keshwani

Received: 31 October 2013 / Accepted: 29 December 2013 # Springer Science+Business Media New York 2014

Abstract Pretreatment of corn stover with dilute sulfuric acid at moderate temperature was investigated, and glucan digestibility by Cellic CTec2 and Celluclast on the pretreated biomass was compared. Pretreatments were carried out from 60 to 180 min at the temperature from 105 to 135 °C, with acid concentrations ranging from 0.5 to 2 % (w/v). Significant portion of xylan was removed during pretreatment, and the glucan digestibility by CTec2 was significantly better than that by Celluclast in all cases. Analysis showed that glucan digestibility by both two enzymes correlated directly with the extent of xylan removal in pretreatment. Confidence interval was built to give a more precise range of glucan conversion and to test the significant difference among pretreatment conditions. Response surface model was built to obtain the optimal pretreatment condition to achieve high glucan conversion after enzymatic hydrolysis. Considering the cost and energy savings, the optimal pretreatment condition of 1.75 % acid for 160 min at 135 °C was determined, and glucan conversion can achieve the range from 72.86 to 76.69 % at 95 % confidence level after enzymatic hydrolysis, making total glucan recovery up to the range from 89.42 to 93.25 %. Keywords Dilute sulfuric acid pretreatment . Lignocellulose . CTec2 . Celluclast . Confidence interval . Response surface model

Introduction Lignocellulosic materials are the most abundant and low-cost biomass available to the world [1]. Bioconversion of lignocellulosic biomass to ethanol is one technology currently being assessed for its potential to supplement the use of current fossil fuel-derived gasoline/petrol [2]. Lignocellulosic biomass is mainly made up of cellulose, hemicellulose, and lignin. The natural structures of the biomass make it hard for microorganism to utilize them directly to produce ethanol. Therefore, saccharification technologies are a crucial step to liberate fermentable sugars before ethanol fermentation [3]. It is acknowledged that the nature of the substrate and pretreatment method used continue to influence the effectiveness of the enzyme access and C. Tai : D. Keshwani (*) Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE 68583, USA e-mail: [email protected]

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attack of the substrate [4, 5]. For a pretreatment to be economically feasible, efficient sugar recovery and high enzyme digestibility must be achieved [6]. To date, dilute acid pretreatment combined with enzymatic hydrolysis has been considered to be a promising procedure for lignocellulosic ethanol production [7–9]. It is particularly important to minimize sugar decomposition in dilute acid pretreatment. It not only improves the sugar yield but also gives a beneficial effect on the subsequent fermentation process by reduction of toxic components. It is well known that sugar decomposition generates various inhibitors including hydroxymethylfurfural (HMF), furfural, and acetic acid, and it is strongly correlated to higher pretreatment temperature and acid concentration [10–12]. Therefore, moderate temperature and lower acid concentration were applied to pretreatment in this study, and the effects were investigated. Many studies have been carried out on the comparison of different pretreatment methods’ effects on subsequent enzymatic hydrolysis efficiency [13–15], but few have been done on comparison of different enzymes’ digestibilities on the same pretreated biomass, especially CTec2 and Celluclast, two of the most promising enzymes in biomass conversion [16–18]. This will provide a better vision at the performance of glucan digestion by these two enzymes. In most of the time, glucan conversion is calculated as the average of corresponding experiment values with standard errors; it works fine for the analysis of difference and comparison of performance, but when it comes to prediction, the result is not so satisfying that experiment values like to fall out of the range of mean with standard errors because (1) sample size in experiment is usually too small to form a good statistical estimate, and (2) the homogeneity of raw and pretreated biomass is challenged which brings more variation. Better statistical calculation way is needed to provide better vision at these variations. The main purpose of this study was to investigate the pretreatment of corn stover with dilute sulfuric acid at moderate temperature and to compare the glucan digestibility of CTec2 and Celluclast on the pretreated biomass. Confidence interval was built to give a more precise range of glucan conversion and to test the significant difference among pretreatment conditions. At last, optimal pretreatment condition for glucan conversion after enzymatic hydrolysis was determined through response surface model.

Materials and Methods Materials Corn stover was collected from Rogers Memorial Farm (Lincoln, NE, USA) in 2012, and it was air dried, milled, screened through a 2.36-mm sieve, and homogenized in a single lot. The enzyme preparations used in this work were Cellic CTec2 which was kindly provided by Novozymes North American, Inc., and a combination of Celluclast and Novozyme 188 which were purchased from Sigma-Aldrich. Pretreatment Experiment Corn stover samples were pretreated with 0.5, 1, and 2 % (w/v) sulfuric acid in sealed flasks in an autoclave at 105, 120, and 135 °C for 60, 120, and 180 min. Solid to liquid ratio is 1:10. The pretreated biomass recovered by filtration through a porcelain Büchner funnel was washed with distilled water until pH was 7. The wet solids were completely transferred to a preweighed plastic bag and then weighed and stored sealed at 4 °C for the enzymatic hydrolysis

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later. A small portion of the wet pretreated biomass was weighed and dried for composition analysis. Enzymatic Hydrolysis To evaluate the sugar recovery from the pretreated biomass and to compare the glucan digestibility of CTec2 and Celluclast, the wet pretreated biomass were hydrolyzed with CTec2 (60 filter paper units (FPU)/g glucan) and a combination of Celluclast (60 FPU/g glucan) and Novozyme 188 (105 CBU/g glucan), respectively. Enzyme activities were determined using the standard analytical procedure developed by the National Renewable Energy Laboratory (NREL) [19]. The solid loading was 5 % (w/v), and 0.05 M sodium citrate buffer was used to maintain pH values 5.0 and 4.8 for CTec2 and Celluclast hydrolysis, respectively. Tetracycline (0.004 %, w/v) and cycloheximide (0.003 %, w/v) were added to the hydrolysis mixture to prevent microbial growth. The hydrolysis was carried out at 50 °C and 150 rpm for 72 h in a controlled environmental incubator shaker (Model I26, New Brunswick Scientific). After the hydrolysis was done, the hydrolysate was centrifuged at 4 °C and 5,000 rpm for 2 min, and the supernatant was used for sugar analysis. Hydrolysis of untreated biomass was conducted with each cellulase as a control. Analytic Methods Chemical composition of raw and pretreated corn stover was analyzed using standard analytical procedures developed by NREL [20, 21]. Monosaccharides in the hydrolysate were measured in a HPLC system (Model Ultimate 3000, Dionex) with a Bio-Rad Aminex HPX87P column (300 mm×7.8 mm), a Bio-Rad De-Ashing guard column, and a refractive index detector. The mobile phase was HPLC grade water at a flow rate of 0.6 mL/min, and the column temperature was 85 °C. Statistical Analysis Statistical analysis was conducted in SAS (version Enterprise 4.3, SAS Institute, USA) with two models: classification model which was used to build a confidence interval and to test a significant difference and response surface model which was used to obtain the optimal condition of pretreatment. For classification model, it was a three-factor, three-level design. For response surface model, the quadratic model for describing the treatment effects was expressed as Eq. (1): yi ¼ β 0 þ

X

β i xi þ

X

β ii x2i þ

X

β ij xi x j

i ¼ 1; 2; …; k;

j ¼ 1; 2; …; k

ð1Þ

where yi was the estimate response, β0 was the overall mean, βixi was the linear effect, βiix2i was the quadratic effect, and βijxixj was the interaction effect.

Results and Discussion Sugar Recovery in Pretreatment Pretreatment of corn stover with dilute sulfuric acid can achieve high digestibility and efficient recovery of hemicellulose sugars with high yield and concentration [6, 22, 23]. After

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pretreatment, considerable portion of xylan has been removed and dissolved into the hydrolysates, as listed in Table 1. Pretreatment of 60 min at 105 °C was sufficient to achieve a significant amount of xylan removal (57.03 %) with 2 % sulfuric acid. On the other hand, decreasing acid concentration from 2 to 0.5 % significantly reduced the xylan removal when time and temperature were at the same level, but there was no obvious trend for glucan removal. It was noticed that up to 99.4 % of xylan can be removed with 2 % sulfuric acid at 135 °C for 180 min, making glucan the largest part (55.03 %) in pretreated corn stover. It was also indicated that with the extension of pretreatment time and increase of temperature, weight loss has been increasing. The greatest weight loss came with the pretreatment of 1 % acid at 135 °C for 120 min, while 93.64 % of xylan and 16.07 % of glucan have been removed into Table 1 Composition of dilute sulfuric acid pretreated corn stover Composition of solids fractionsa

Pretreatment conditions Acid conc. Time (%) (min) Raw corn stover

Temperature (°C)

Glucan (%)

Xylan (%)

ASL (%)

AIL (%)

Weight loss (%)

38.41

15.98

1.95

19.53

0.5

60

105

41.50 (8.76)

16.90 (10.70)

1.32

23.04

15.55

1

60

105

43.63 (10.10)

13.70 (32.10)

1.25

25.60

20.80

2

60

105

49.02 (14.82)

10.29 (57.05)

1.22

26.78

33.27

0.5

120

105

44.05 (13.15)

15.96 (24.33)

1.32

24.03

24.23

1

120

105

47.97 (9.22)

11.72 (46.73)

1.20

26.48

27.31

2

120

105

51.69 (5.39)

7.83 (65.53)

1.18

28.51

29.73

0.5 1

180 180

105 105

43.47 (12.72) 47.72 (14.60)

14.61 (29.49) 9.91 (57.38)

1.17 1.10

24.92 27.48

22.91 31.27

2

180

105

50.05 (17.58)

6.41 (74.68)

1.06

31.06

36.86

0.5

60

120

45.15 (15.04)

13.04 (41.02)

1.38

25.48

27.72

1

60

120

49.33 (16.74)

8.03 (67.39)

1.16

27.42

35.16

2

60

120

52.82 (22.05)

4.69 (83.37)

1.14

29.54

43.32

0.5

120

120

47.75 (13.93)

11.19 (51.52)

1.13

27.25

30.77

1

120

120

52.35 (11.33)

6.13 (75.03)

1.06

30.43

34.92

2 0.5

120 180

120 120

54.58 (13.90) 48.17 (15.60)

2.85 (89.21) 9.33 (60.70)

1.02 1.17

31.35 28.18

39.40 32.70

1

180

120

50.35 (16.43)

4.62 (81.57)

1.05

31.54

36.22

2

180

120

54.81 (16.07)

1.94 (92.85)

1.05

36.92

41.14

0.5

60

135

51.88 (13.97)

8.73 (65.22)

1.29

29.22

36.28

1

60

135

57.11 (7.95)

3.60 (86.06)

1.19

30.84

38.07

2

60

135

58.47 (11.14)

1.18 (95.67)

1.19

32.52

41.62

0.5

120

135

52.18 (11.40)

5.53 (77.45)

1.14

29.19

34.83

1 2

120 120

135 135

57.52 (16.07) 58.08 (11.30)

1.81 (93.64) 0.40 (98.53)

1.20 1.22

32.78 34.33

43.93 41.33

0.5

180

135

52.85 (15.30)

4.44 (82.91)

1.16

28.20

38.44

1

180

135

52.00 (20.80)

1.01 (96.29)

1.18

31.20

41.55

2

180

135

55.03 (16.59)

0.17 (99.40)

1.19

34.13

41.75

Data in parentheses next to the composition number are the percent amount of each component removed during pretreatment a Data are means of three replicates

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hydrolysate, which can be a good source for fermentation with mixed sugars, and inhibitors such as HMF and furfural would not be a problem because of the lower temperature (≤135 °C) [24, 25]. Glucan removal in acid pretreatment at moderate temperature was usually higher than at high temperature since it required longer pretreatment time and, sometimes, elevation of acid concentration [26, 27], and it needed to be balanced by considering both generation of inhibitors and sugar decomposition. Enzymatic Hydrolysis of Pretreated Biomass Pretreated biomass were treated at a high enzyme loading of 60 FPU/g glucan to obtain the maximum possible yields of sugars through digestion by CTec2 and Celluclast according to NREL procedure. Glucan and xylan conversion after 72 h of enzymatic hydrolysis of biomass pretreated at each combination of sulfuric acid concentration, time, and temperature were shown in Fig. 1. It can be seen that hydrolysis by these two enzymes have the same trend for glucan conversion and that at the same levels of pretreatment time and temperature, glucan conversion was increasing with the elevation of acid concentration. It also showed that the digestibility of CTec2 was much greater than that of Celluclast at each the same level of pretreatment, proving that CTec2 is more suitable to hydrolyze biomass pretreated at lower temperatures with sulfuric acid. The highest

Conversion percentage (%)

A 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Glucan conversion (%) Xylan conversion (%)

Conversion percentage (%)

B 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Glucan conversion (%) Xylan conversion (%)

Fig. 1 Glucan and xylan conversion after 72 h of enzymatic hydrolysis of dilute sulfuric acid-pretreated corn stover by CTec2 (a) and Celluclast (b). Glucan/xylan conversion was based on glucan/xylan amount in raw biomass

Appl Biochem Biotechnol

glucan conversion for both CTec2 and Celluclast came with the same biomass pretreated with 2 % acid for 180 min at 135 °C, which made the total glucan recovery in pretreatment and enzymatic hydrolysis up to 92.6 and 76.61 %, respectively. The highest xylan conversion for CTec2 came at the condition of 0.5 % acid for 120 min at 105 °C, while the one for Celluclast came with 0.5 % acid for 60 min at 105 °C, but they did not account much because most of the xylan has been removed in pretreatment. In fact, the success of acid pretreatment is based on the concept of hemicellulose removal in order to maximize glucose conversion after enzymatic hydrolysis [28]. In our case, the glucan digestibility of both two enzymes appeared to have a quadratic relationship with xylan removal (Fig. 2). The removal of xylan improved the exposure of glucan to enzyme attack and, consequently, the digestibility [6, 15]. Although lignin was considered as an important factor hindering the access of enzyme to cellulose [29, 30], high digestibilities of glucan despite high lignin content in some of our pretreated biomass samples have been observed; actually, the highest glucan digestion of 91.21 % came with the pretreated biomass containing 35.32 % of lignin. This implied that xylan and lignin seem to be parallel factors affecting the enzymatic hydrolysis. The removal of either xylan or lignin would make biomass amenable for the enzymatic digestion.

A 100%

Glucan disgestibility (%)

90% 80% 70% 60% 50% 40% 30% 20% 10%

y = 0.6953x2 - 0.1608x + 0.3579 R² = 0.9802

0% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Xylan removal in pretreatment (%)

B

100%

Glucan digestibility (%)

90% 80%

y = 0.6738x2 - 0.3167x + 0.2838 R² = 0.9409

70% 60% 50% 40% 30% 20% 10% 0% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Xylan removal in pretreatment (%) Fig. 2 Correlation between xylan removal in pretreatment and 72 h of glucan digestibility by CTec2 (a) and Celluclast (b). Glucan digestibility was based on glucan amount in pretreated biomass

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Classification Model for Enzymatic Hydrolysis To find out whether there is a leading factor in pretreatment conditions and to give a better prediction of the variance of glucan conversion after 72 h of enzymatic hydrolysis, a threefactor, three-level classification model was built for CTec2 and Celluclast hydrolysis, respectively, with pretreatment conditions as effect items and glucan conversion as response. All the interaction and single effects were significant except the interaction effect of acid concentration and time in CTec2 hydrolysis and the interaction effect of acid concentration, time, and temperature in Celluclast hydrolysis (data not shown). It can be noticed that the first six Table 2 Analysis of variance in classification model for glucan conversion after 72 h of enzymatic hydrolysis of pretreated biomass by CTec2 Estimatea

Effect item

DF

t value

Acid (%)

Time (min)

Temp (°C)

2

180

135

76.0200

54

38.21

2

120

135

75.1533

54

48.24

2 1

60 180

135 135

72.6333 71.3833

54 54

68.76 39.28

Pr>|t|

Confidence intervalb

Letterc

Lower

Upper

Impact of pretreatment with dilute sulfuric acid under moderate temperature on hydrolysis of corn stover with two enzyme systems.

Pretreatment of corn stover with dilute sulfuric acid at moderate temperature was investigated, and glucan digestibility by Cellic CTec2 and Celluclas...
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