Liyue
Zhang
,
Yue
Liu
and
Zhiqiang
Li
*
Key Laboratory of National Forestry and Grassland Administration, Beijing Co-built on Bamboo and Rattan Science and Technology, International Centre for Bamboo and Rattan, Beijing 100102, China. E-mail: lizq@icbr.ac.cn
First published on 25th September 2019
In this study, the conditions for the pretreatment of bamboo by ammonium sulfite to achieve high cellulose recovery were investigated and optimized. To obtain higher cellulose recovery under low-severity pretreatment conditions such as ammonia sulfite concentration, pretreatment time and pretreatment temperature, three-factor and three-level experiments were designed by the Box–Behnken design based on response surface methodology. The results showed that the cellulose recovery yield after 48 h enzymatic hydrolysis could reach 58.36–59.87%; moreover, the recovered cellulose was pretreated with 20% ammonium sulfite at 150 °C for 6 h, and the obtained yield was in agreement with the predicted yield (58.87%). It was about 13-fold higher than that of the untreated bamboo (4.41%). Pretreatment temperature and ammonia sulfite concentration are significantly important factors than pretreatment time in the design space for achieving high cellulose recovery. Moreover, SEM analysis of the pretreated bamboo substrate under optimized conditions illustrated that the biomass surface had become more rough and porous after pretreatment.
Bamboo is one of the most abundant renewable lignocellulose resources; it usually contains 40–60% cellulose and 20–32% hemicellulose, which can be converted to fermentable sugars; moreover, it has 20–30% lignin that resists the accessibility of enzymes to cellulose. Therefore, pretreatment is a necessary process in biorefinery to increase the conversion efficiency via the removal of lignin and improvement of enzyme accessibility.5 However, pretreatment is also the most expensive process in the biomass utilization project,6 and the development of low-cost and high value-added pretreatment method is of great importance for the further use of cellulose.
Numerous pretreatment technologies, such as acid-based pretreatments,7 alkali-based pretreatments,8 organosolv pretreatments,9 ionic liquid pretreatments,10 and physically assisted chemical pretreatments,11 have been studied to remove lignin, break the resistance and depolymerize cellulose for fermentable sugar production.
Sulfite pretreatment is a traditional way in the pulping industry for papermaking; it can be conducted in a wide range of pH and temperature, which has been described in a textbook.12 The goal of pulping is to remove lignin as much as possible without the concurrent loss and degradation of hemicellulose and cellulose; this would lead to a pulp with high yield and strength.13 Sulfite process has also been used for pretreating wood chips,12 and it has been first used for softwoods (spruce and red pine) through enzymatic saccharification. The result of the study showed that the sulfite-treated softwood chips could significantly become soft, and the enzymatic cellulose conversion yield of over 90% was achieved.13 Then, alkaline sulfite or acid sulfite has been used for the pretreatment of other lignocellulosic biomass such as bamboo14 and switchgrass.15 In the pretreatment process, the active reagents could be sulfite (SO32−), bisulfite (HSO3−), or a combination of two of the three reagents sulfite (SO32−), bisulfite (HSO3−), and sulfur dioxide (SO2, or H2SO3) depending on the pH value of the pretreatment liquor at pretreatment temperature.16
Ammonium sulfite as a kind of neutral sulfite has been used for papermaking for a long time17 and can be easily decomposed into ammonia and sulfite at about 70 °C. Therefore, the effects of the pretreatment of ammonium sulfite and sodium sulfite can be significantly different. Ammonium sulfite may exhibit the effects of both ammonia and sulfite on the pretreated bamboo. However, only few studies have been reported on the pretreatment of lignocellulosic biomass. It was first used for wheat straw, and the result demonstrated that ammonium sulfite could significantly improve the enzymatic hydrolysis but under a more severe pretreatment condition; since bamboo is more rigid and compact than wheat straw, harsh pretreatment conditions would be needed.18 Hence, considering the pretreatment cost and the final value of the produced glucose, the ammonia sulfite pretreatment conditions of bamboo need to be optimized. The aim of this study was to find low-severity pretreatment parameters (pretreatment temperature, time, and ammonia sulfite concentration) to achieve higher cellulose recovery yield. Milled bamboo was selected as feedstock, and a series of pretreatments were performed based on the Box–Behnken design involving three variables: pretreatment temperature, time, and ammonia sulfite concentration. The total cellulose recovery yield (TCRY) was calculated by enzymatic hydrolysis efficiency obtained after 48 h enzymatic hydrolysis multiplied with the solid recovery rate after the ammonia sulfite pretreatment. Environmental scanning electron microscopy was conducted to compare the structural changes of raw bamboo and the preferred pretreated substrate.
The enzymatic hydrolysis efficiency was calculated by the following equation:
Enzymatic hydrolysis efficiency (%) = (glucose content in the liquid supernatant after enzymatic hydrolysis × 0.9/glucose content in liquid after pretreatment) × 100 |
The monosaccharide concentrations were analyzed by ion chromatography using an amperometric detector (Metrohm Corporation, Switzerland). Detection was performed at 32 °C using the Hamilton RCX-30 column and Metrosep RP2 guard column. The cellulose content was presented by the glucose concentration. Moreover, the hemicellulose content was the combination of the concentrations of xylose, arabinose, galactose and mannose.
The acid-soluble lignin was analyzed at 205 nm via a UV-visible spectrophotometer using 3% sulfuric acid as the control blank. Acid-insoluble lignin of all the samples was determined by the weight of the residues after the two-step sulfuric acid hydrolysis. Fermentation inhibitors, including acetic acid, formic acid, furfural, levulinic acid and 5-hydroxylmethylfurural (HMF), were degraded from cellulose and hemicellulose during the pretreatment process. They were analyzed using a high-performance liquid chromatograph (HPLC) equipped with the Aminex HPX-87H (30 cm × 7.8 mm) column at the temperature of 25 °C and a UV detector at 210 nm. Eluent was 0.1% phosphoric acid at the rate of 0.7 mL min−1.
Coded levels of factors | Factors | ||
---|---|---|---|
Ammonium sulfite concentration (wt%) | Time (h) | Temperature (°C) | |
Low level (−1) | 10 | 3 | 120 |
Central level (0) | 20 | 6 | 150 |
High level (1) | 30 | 9 | 180 |
Run | Factors | Composition (%) | Removal (%), lignin | Solid recovery rate (%) | Enzymatic hydrolysis efficiency (%) | Cellulose recovery yield (%) | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
C | T | H | Glucan | Xylan | Lignin | bExp | cPred | ||||
a CK-control check (untreated raw bamboo). C-concentration (%). T-temperature (°C). H-time (h). b Exp-experimental. c Pred-predicted. | |||||||||||
CK | — | — | — | 42.22 ± 0.23 | 17.51 ± 0.36 | 29.37 ± 0.02 | — | 100 | 4.41 | — | — |
1 | 20 | 6 | 150 | 55.66 ± 1.02 | 17.76 ± 0.12 | 11.58 ± 0.01 | 37.08 | 71.30 | 83.97 | 59.87 | 58.87 |
2 | 20 | 6 | 150 | 57.12 ± 0.84 | 18.21 ± 0.48 | 11.09 ± 0.00 | 37.83 | 70.54 | 82.73 | 58.36 | 58.87 |
3 | 10 | 6 | 120 | 46.43 ± 0.62 | 19.02 ± 0.29 | 25.87 ± 0.01 | 10.49 | 92.56 | 38.22 | 35.38 | 36.39 |
4 | 20 | 9 | 180 | 57.89 ± 0.24 | 15.05 ± 0.35 | 15.99 ± 0.00 | 39.08 | 48.28 | 71.54 | 34.54 | 35.59 |
5 | 20 | 6 | 150 | 56.25 ± 0.48 | 17.64 ± 0.27 | 11.32 ± 0.01 | 37.54 | 71.24 | 81.92 | 58.36 | 58.87 |
6 | 20 | 6 | 150 | 56.95 ± 0.15 | 17.41 ± 1.12 | 11.43 ± 0.01 | 37.68 | 70.71 | 82.80 | 58.55 | 58.87 |
7 | 10 | 3 | 150 | 49.43 ± 0.21 | 19.21 ± 1.14 | 23.81 ± 0.01 | 17.86 | 82.33 | 34.69 | 28.56 | 28.60 |
8 | 30 | 6 | 180 | 48.76 ± 0.37 | 14.97 ± 0.89 | 1.22 ± 0.01 | 64.51 | 49.72 | 63.68 | 31.66 | 30.66 |
9 | 20 | 9 | 120 | 54.50 ± 1.29 | 17.34 ± 0.57 | 16.30 ± 0.00 | 13.74 | 79.34 | 53.47 | 42.42 | 42.05 |
10 | 30 | 9 | 150 | 60.30 ± 0.47 | 20.63 ± 0.31 | 7.82 ± 0.01 | 55.22 | 63.28 | 57.46 | 36.36 | 36.32 |
11 | 20 | 3 | 180 | 64.27 ± 1.23 | 17.36 ± 0.34 | 11.24 ± 0.02 | 46.24 | 56.21 | 50.24 | 28.24 | 28.61 |
12 | 20 | 3 | 120 | 43.32 ± 1.06 | 17.78 ± 0.41 | 25.27 ± 0.00 | 15.51 | 83.69 | 61.23 | 51.24 | 50.19 |
13 | 30 | 3 | 150 | 49.59 ± 0.56 | 19.33 ± 0.51 | 14.60 ± 0.01 | 30.71 | 77.24 | 56.40 | 43.56 | 44.19 |
14 | 30 | 6 | 120 | 47.37 ± 0.40 | 19.05 ± 0.21 | 16.80 ± 0.01 | 18.45 | 78.61 | 60.88 | 47.86 | 48.27 |
15 | 20 | 6 | 150 | 55.98 ± 1.04 | 18.35 ± 0.16 | 11.74 ± 0.01 | 37.92 | 71.04 | 83.35 | 59.21 | 58.87 |
16 | 10 | 6 | 180 | 57.17 ± 1.48 | 2.06 ± 0.20 | 38.78 ± 0.00 | 25.07 | 63.06 | 83.35 | 26.37 | 25.96 |
17 | 10 | 9 | 150 | 44.09 ± 0.65 | 4.52 ± 0.51 | 23.04 ± 0.03 | 28.23 | 65.88 | 54.58 | 35.96 | 35.32 |
Pretreatment | Sum of arabinose and galactose (g L−1) | Xylose (g L−1) | Glucose (g L−1) | Soluble lignin (g L−1) | Formic acid (g L−1) | Acetic acid (g L−1) | Levulinic acid (g L−1) | Furfural (g L−1) | HMF (g L−1) | ||
---|---|---|---|---|---|---|---|---|---|---|---|
C | T | H | |||||||||
20 | 6 | 150 | 0.48 ± 0.05 | 0.83 ± 0.01 | 2.11 ± 0.30 | 10.89 ± 0.01 | 3.99 ± 0.41 | 21.53 ± 1.34 | 17.69 ± 2.33 | 0.25 ± 0.12 | 0.00 ± 0.00 |
20 | 6 | 150 | 0.48 ± 0.08 | 0.79 ± 0.01 | 2.13 ± 0.32 | 11.11 ± 0.02 | 3.79 ± 0.25 | 18.72 ± 2.23 | 8.91 ± 1.05 | 0.18 ± 0.02 | 0.00 ± 0.00 |
10 | 6 | 120 | 0.11 ± 0.02 | 1.80 ± 0.14 | 0.08 ± 0.00 | 3.08 ± 0.01 | 1.42 ± 0.08 | 3.55 ± 0.21 | 1.74 ± 0.65 | 0.07 ± 0.01 | 0.37 ± 0.01 |
20 | 9 | 180 | 0.02 ± 0.00 | 0.06 ± 0.03 | 0.09 ± 0.00 | 11.47 ± 0.03 | 3.18 ± 0.34 | 6.37 ± 0.58 | 1.99 ± 0.26 | 0.07 ± 0.01 | 0.02 ± 0.00 |
20 | 6 | 150 | 0.49 ± 0.01 | 0.75 ± 0.08 | 2.21 ± 0.37 | 10.97 ± 0.01 | 3.38 ± 0.56 | 14.63 ± 0.51 | 6.70 ± 1.02 | 0.23 ± 0.00 | 0.01 ± 0.00 |
20 | 6 | 150 | 0.50 ± 0.01 | 0.62 ± 0.02 | 2.09 ± 0.21 | 10.01 ± 0.01 | 4.01 ± 0.16 | 19.49 ± 1.33 | 13.73 ± 2.04 | 0.18 ± 0.00 | 0.00 ± 0.00 |
10 | 3 | 150 | 0.40 ± 0.02 | 1.18 ± 0.03 | 0.80 ± 0.04 | 5.25 ± 0.05 | 0.47 ± 0.06 | 2.03 ± 0.31 | 15.81 ± 2.19 | 0.05 ± 0.00 | 0.07 ± 0.00 |
30 | 6 | 180 | 0.18 ± 0.03 | 0.28 ± 0.03 | 2.09 ± 0.05 | 18.95 ± 0.04 | 17.00 ± 1.25 | 9.57 ± 0.53 | 35.37 ± 2.67 | 0.05 ± 0.00 | 0.03 ± 0.00 |
20 | 9 | 120 | 0.09 ± 0.00 | 0.99 ± 0.01 | 0.13 ± 0.02 | 4.04 ± 0.06 | 1.97 ± 0.24 | 5.73 ± 0.23 | 14.64 ± 1.35 | 0.02 ± 0.00 | 0.02 ± 0.00 |
30 | 9 | 150 | 0.54 ± 0.02 | 0.53 ± 0.02 | 1.16 ± 0.15 | 16.22 ± 0.04 | 4.63 ± 0.01 | 11.88 ± 1.35 | 26.28 ± 2.35 | 0.02 ± 0.02 | 0.06 ± 0.00 |
20 | 3 | 180 | 0.52 ± 0.04 | 0.53 ± 0.01 | 4.47 ± 0.09 | 13.58 ± 0.02 | 13.67 ± 0.21 | 8.62 ± 0.34 | 26.70 ± 1.57 | 0.10 ± 0.0 | 0.01 ± 0.00 |
20 | 3 | 120 | 0.12 ± 0.00 | 1.83 ± 0.28 | 0.11 ± 0.01 | 4.55 ± 0.15 | 2.01 ± 0.34 | 9.12 ± 1.23 | 6.15 ± 0.95 | 0.08 ± 0.00 | 0.20 ± 0.00 |
30 | 3 | 150 | 0.20 ± 0.00 | 0.75 ± 0.01 | 0.17 ± 0.24 | 8.02 ± 0.04 | 4.02 ± 0.02 | 12.61 ± 1.55 | 8.88 ± 1.25 | 0.11 ± 0.00 | 0.17 ± 0.00 |
30 | 6 | 120 | 0.03 ± 0.00 | 1.19 ± 0.07 | 0.05 ± 0.00 | 5.42 ± 0.09 | 13.02 ± 1.58 | 9.47 ± 0.84 | 0.54 ± 0.11 | 0.28 ± 0.00 | 0.01 ± 0.00 |
20 | 6 | 150 | 0.47 ± 0.06 | 0.82 ± 0.05 | 2.22 ± 0.13 | 11.08 ± 0.06 | 3.27 ± 0.28 | 16.40 ± 2.01 | 13.89 ± 1.82 | 0.20 ± 0.00 | 0.00 ± 0.00 |
10 | 6 | 180 | 0.02 ± 0.00 | 0.26 ± 0.01 | 0.14 ± 0.00 | 7.36 ± 0.24 | 0.58 ± 0.09 | 6.58 ± 0.63 | 4.53 ± 0.56 | 0.03 ± 0.00 | 0.04 ± 0.00 |
10 | 9 | 150 | 0.39 ± 0.01 | 0.52 ± 0.00 | 4.69 ± 0.36 | 8.29 ± 0.02 | 1.35 ± 0.08 | 3.08 ± 0.01 | 8.73 ± 1.62 | 0.01 ± 0.00 | 0.01 ± 0.00 |
The contents of arabinose, galactose, xylose, glucose, soluble lignin and fermentation inhibitor in all the pretreated liquors are listed in Table 3. It was clearly found that the content of carbohydrate was low in the spent liquor, and the soluble lignin content was in accordance with the enzymatic hydrolysis efficiency; this meant that the content of soluble lignin in the spent liquid was increased significantly (from 3.08% to 16.22%) after pretreatment, and the higher lignin removal could result in higher enzymatic hydrolysis efficiency.
Furfural and HMF were obtained from the dehydration of pentoses and hexoses, respectively; acetic acid was mainly obtained from the acetyl groups on hemicelluloses; moreover, levulinic and formic acids were the products of the successive decomposition of HMF.21 The contents of formic acid, acetic acid, and levulinic acid were much higher than those of furfural and HMF; this might be due to the long pretreatment time.
The chemical component in the spent liquor was extremely complicated according to the HPLC analysis. The spent liquor contained different kinds of monosaccharides, fermentation inhibitors, extractives, waxes, lignosulfonates, unreacted ammonium sulfite and so on. The cost of solvent recovery was really high. Normally, the spent liquor is used as a fertilizer.22 To fully utilize the pretreated liquor, it was subjected to pyrolysis with hydrogen to break lignin into flammable gas, whereas ammonium sulfite was decomposed into sulfur dioxide and ammonia.23
Y
TCRY was set as the responsive variable for modelling to reach the highest cellulose recovery yield, and the final quadratic polynomial model is shown by the following equation:
YTCRY = +58.87 + 4.15A − 0.29B − 7.01C − 3.65AB − 1.80AC + 3.78BC − 13.28A2 − 9.48B2 − 10.28C2 |
In the abovementioned equation, positive coefficients indicated a synergistic effect, whereas negative signs suggested an antagonistic effect. The analysis of variance (ANOVA) showed the possible effects of all variables on YTCRY. The fit of the model was evaluated by comparing the R2 and the adjusted Radj2. The statistical significance was determined by an F-test, which should not be less than 0.80, and the P-value, which should be less than 0.05 for a good fit of the developed model.24,25 The ANOVA results of the developed model are listed in Table 4. The model fitted the data with an R2 of 0.9969 for YTCRY, which suggested a strong correlation coefficient between all the data. The model parameters, such as temperature and concentration, showed a high level of significance (p < 0.0001), whereas the time implied a low level of significance (p = 0.4507 > 0.05), and the quadratic terms A2, B2, and C2 and the interactions of AB, BC, and AC were significant with the P-value less than 0.05. The value of the predicted R-squared (Rpre2 = 0.9613) significantly suggested the strong predictive ability of the model. The value of lack of fit (p = 0.0954 > 0.05) further indicated model adequacy. In addition, the experimental values versus the predicted values showed a high quality of fit throughout the design space (Table 2).
Source | Sum of squares | DF | Mean square | F value | P-value prob. > F | Significance |
---|---|---|---|---|---|---|
a DF-degree of freedom. A-concentration. B-time. C-temperature. **-denotes very significance difference at P < 0.01. — denotes not significance at P > 0.05. | ||||||
Model | 2398.84 | 9 | 266.45 | 252.69 | <0.0001 | ** |
A | 137.53 | 1 | 137.53 | 130.38 | <0.0001 | ** |
B | 0.67 | 1 | 0.67 | 0.64 | 0.4507 | — |
C | 393.26 | 1 | 393.26 | 372.82 | <0.0001 | ** |
AB | 53.29 | 1 | 53.29 | 50.52 | 0.0002 | ** |
AC | 12.92 | 1 | 12.92 | 12.25 | 0.0100 | ** |
BC | 57.15 | 1 | 57.15 | 54.18 | 0.0002 | ** |
A 2 | 742.14 | 1 | 742.14 | 703.57 | <0.0001 | ** |
B 2 | 378.70 | 1 | 378.70 | 359.02 | <0.0001 | ** |
C 2 | 444.64 | 1 | 444.64 | 421.53 | <0.0001 | ** |
Residual | 7.38 | 7 | 1.05 | |||
Lack of fit | 5.65 | 3 | 1.88 | 4.33 | 0.0954 | Not significant |
Pure error | 1.74 | 4 | 0.43 | |||
Corrected total | 2406.22 | 16 | ||||
R 2 | 0.9969 | |||||
R adj 2 | 0.9930 | |||||
R pred 2 | 0.9613 |
The slope of the response surface could indicate the extent of the response under various pretreatment conditions, whereas the steep curved surface indicated that the response value was very sensitive to the relevant pretreatment conditions.20Fig. 1(A–C) show the effects of two parameters on the total cellulose recovery yield. The three steep slopes clearly suggested the relatively strong interaction effect between all the pretreatment conditions. Moreover, the shape of the contour plot could reflect the intensity of the interaction effects. In general, an oval shape revealed a strong interaction of the two factors, which was opposite to the case of the circular shape;26 the contour plot showed an apparent oval shape, which was in agreement with the result of the 3-D response surface.
This journal is © The Royal Society of Chemistry 2019 |