Reed is a widespread-growing, inexpensive, and readily available lignocellulosic material source in northeast China. The objective of this study is to evaluate the liquid hot water (LHW) pretreatment efficiency of reed based on the enzymatic digestibility and ethanol fermentability of water-insoluble solids (WISs) from reed after the LHW pretreatment. Several variables in the LHW pretreatment and enzymatic hydrolysis process were optimized. The conversion of glucan to glucose and glucose concentrations are considered as response variables in different conditions. The optimum conditions for the LHW pretreatment of reed area temperature of 180°C for 20min and a solid-to-liquid ratio of 1 : 10. These optimum conditions for the LHW pretreatment of reed resulted in a cellulose conversion rate of 82.59% in the subsequent enzymatic hydrolysis at 50°C for 72 h with a cellulase loading of 30 filter paper unit per gram of oven-dried WIS. Increasing the pretreatment temperature resulted in a higher enzymatic digestibility of the WIS from reed. Separate hydrolysis and fermentation of WIS showed that the conversion of glucan to ethanol reached 99.5% of the theoretical yield. The LHW pretreatment of reed is a suitable method to acquire a high recovery of fermentable sugars and high ethanol conversion yield.
Lignocellulosic material (LCM) is an abundant, natural, and renewable carbon source for biofuel production. For a long time, studies have been performed to enhance the LCM enzymatic hydrolysis for the efficient conversion of cellulose to ethanol [
Reed is an abundant and inexpensive lignocellulosic raw material that can be found throughout northeastern China. According to statistics, the reed output all over the world was about >70 million tons, especially in Asia and Europe. In China, the planting areas for reed are over 10 million mu, and the reed output reaches 3 million tons. The region with the greatest reed output is Panjin, Liaoning province, China. The Panjin reed field, which covers an area of 1.2 million mu, is the largest reed-producing region in the world today. Five hundred thousand tons of reed are produced in the fields every year. Reed has been used in the papermaking industry for years as a good raw material because of its high cellulose content and good fiber properties. Nevertheless, studies on bioethanol production are few. Reed may be used as an alternative raw material for ethanol production. In this study, the LHW pretreatment of reed and the enzymatic hydrolysis of pretreated reed were investigated to determine its potential application for ethanol production by a bioconversion process. The pretreatment and enzymatic hydrolysis conditions were optimized to obtain a high conversion of cellulose to ethanol, and to enhance the enzymatic digestibility of reed, to obtain a high glucose yield. Ethanol fermentation was also conducted by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes using the pretreated reed as a glucose source to determine the feasibility of using reed in bioethanol production.
Reed, which has a moisture content of 11.67%, was provided by the Yingkou papermaking mill, Yingkou, Liaoning province, China. The reed was milled to a particle size of 40 mesh to 60 mesh by using a laboratory ball mill (Taijihuan Nanometer Limited Company, Qinhuangdao, China) and was stored in a plastic bag until it was used in the experiments. Before the LHW pretreatment, the chemical compositions of reed were determined.
The commercial cellulase used in the enzymatic hydrolysis was purchased from the Imperial JADE Biotechnology Co., Ltd., Ningxia, China. The cellulase derived from
The yeast
The LHW pretreatment was conducted in a 15 L digester with four small tanks (mechanical mill of the Shanxi University of Science and Technology, China). About 40 g of reed and a given volume deionized water were loaded in the small tanks. The pretreatment temperature was controlled at 170°C, 180°C, 190°C, 200°C, and 210°C. The pretreatment time was set at either 20 min or 40 min. After pretreatment, the water-insoluble solids (WISs) and the reed prehydrolyzates were separated by filtration with the Büchner funnel. The WISs were washed with deionized water to obtain a pH of approximately 7. The WISs were used for subsequent enzymatic hydrolysis and ethanol fermentation.
Enzymatic hydrolysis of the washed WISs was performed at 36°C or 50°C for 72 h in 100 mL Erlenmeyer flasks. Each flask contained 20 mL to 50 mL of 0.05 M sodium citrate buffer (pH 4.8) and had a solid-to-liquid ratio of 1 : 50 weight per volume (W/V) of WIS. The enzyme loading was 10 to 30 filter paper unit (FPU) per gram of oven-dried WIS. The samples were collected at 1, 5, 9, 12, 24, 36, 48, and 72 h for glucose concentration determination. All enzymatic hydrolysis experiments were performed in duplicates, and the average results were determined.
Separate hydrolysis and fermentation (SHF) was performed to check the fermentability of pretreated reed. The WIS from the pretreatment experiments (at 180°C and 210°C for 20 min each) was used as the substrate. About 100 mL of the enzymatic hydrolysis liquor and some nutrients, namely, 3 g/L yeast extract, 5 g/L peptone, 25 g/L KH2PO4, 0.3 g/L MgCl2, and 0.25 g/L CaCl2, were added into 250 mL Erlenmeyer flasks. The total liquid volume was 100 mL. The solid concentration was 6% (by WIS weight) during the hydrolysis. The resulting slurry was inoculated with 1 mL of activated yeast. The flasks were autoclaved at 121°C for 20 min. The experiments were performed in duplicates in a constant-temperature incubator at 36°C for 72 h. The flasks were sealed with rubber stoppers and equipped with cannulas to remove the generated carbon dioxide. The cannulas were inserted into a container filled with water. The simultaneous saccharification and fermentation (SSF) experiment was conducted based on the SSF protocol by the National Renewable Energy Laboratory (NREL) LAP-008 [
All experiments were performed in duplicates in the same conditions, and the average values were reported.
The contents of xylan, Klason lignin, ash, and benzene-alcohol (2 : 1) extractives were determined using the Chinese National Standard methods, namely, the GB/T2677.9-1994, GB/T2677.8-1994, GB/T2677.3-1993, and GB/T2677.6-1994, respectively. The acid-soluble lignin content was determined using the method described in GB/T10337-1989. The glucan content was determined according to NREL methods [
The conversion of cellulose to glucose in the enzymatic hydrolysis was determined by the ratio of the glucose concentration that was released during enzymatic hydrolysis to the total glucose in the substrate and was calculated using formula:
For SHF and SSF of WIS, the conversion of cellulose to ethanol was calculated using formula (
The chemical compositions of reed used in this study were determined and shown in Table
Chemical compositions of reed and other biomass sources (%, by dry weight).
Compositions | Reed | Rice Straw | Corn stover | Wheat straw |
---|---|---|---|---|
Glucan | 40.5 | 34.6 | 36.1 | 37.8 |
Xylan | 25.9 | 21.3 | 21.4 | 22.8 |
Klason lignin | 16.2 | 9.6 | 17.2 | 16.3 |
Acid-soluble lignin | 2.0 | 3.4 | — | 1.8 |
Ash | 3.6 | 14.5 | 7.1 | 6.3 |
The pretreatment conditions were selected based on whether they could modify the structural and chemical characteristics of the biomass that limited the enzyme availability to cellulose in cell-wall microfibrils [
Glucan contents of WIS in different pretreatment temperature and time (%, by weight of WIS).
Temperature (°C) | Pretreatment time | |
---|---|---|
20 min | 40 min | |
170 | 45.47 | 51.16 |
180 | 53.98 | 55.55 |
190 | 54.31 | 57.46 |
200 | 54.29 | 56.81 |
210 | 56.42 | 57.82 |
Conversion of cellulose to glucose in the enzymatic hydrolysis of WIS from LHW-pretreated reed, with different pretreatment temperatures and times. *Enzymatic hydrolysis conditions: 15 FPU/g oven-dried WIS, pH 4.8, solid-to-liquid ratio 1 : 50 (w/v), and 50°C for 72 h.
Pretreatment time of 20 min
Pretreatment time of 40 min
The solid-to-liquid ratio is the ratio between the oven-dried WIS quality and the entire liquid volume in the LHW pretreatment. When the reed quantity was kept constant, a higher solid-to-liquid ratio led to a lower substrate concentration, which further decreased the end-product inhibition. Reducing the solid-to-liquid ratio would decrease process cost by lowering the reactor size and the amount of heat requirement during the pretreatment. When the solid-to-liquid ratio was increased from 1 : 10 to 1 : 25 (w/v) in the LHW pretreatment at 180°C, the glucose release slightly increases in the later enzymatic hydrolysis period. For example, the conversion of cellulose to glucose at 72 h of enzymatic hydrolysis increased from 75.97% to 83.39% with only a difference of 7.42% after 48 h (Figure
Effect of solid-to-liquid ratio on enzymatic hydrolysis of the WIS from the pretreated reed. *LHW pretreatment: 180°C for 20 min. **Enzymatic hydrolysis: 30 FPU/g oven-dried WIS, pH 4.8, at 50°C.
Glucose release from WIS in the enzymatic hydrolysis
Conversion of cellulose of WIS
A suitable pH in the enzymatic hydrolysis system is beneficial to the cellulase function on the substrate. The systemic pH has to be adjusted to improve cellulase effectiveness in hydrolysis. In this study, two different methods were used for adjusting and controlling the pH. One method involves the use of sodium citrate buffer (pH 4.8, 0.05 M), and the other method involves the use of H2SO4. Figure
Enzymatic hydrolysis of WIS from LWH-pretreated reed at 180°C and 210°C by using two types of pH adjustment in the hydrolysis system. *Enzymatic hydrolysis: 30 FPU/g oven-dried WIS at 36°C.
LHW pretreatment at180°C
LHW pretreatment at 210°C
The enzymatic temperature affects cellulase activity and effectiveness in enzymatic hydrolysis. The temperature in the enzymatic hydrolysis is generally kept at 50°C, which is a suitable temperature for the cellulase activity. However, the yeast fermentation temperature is 36°C. Two different enzymatic temperatures were used for the enzymatic hydrolysis of WIS to compare the effects of different enzymatic temperatures on WIS enzymatic digestibility. Figure
Temperature effects on the WIS conversion from reed in the LHW pretreatment at 180°C and 210°C for 20 min. *Enzymatic hydrolysis: 30 FPU/g oven-dried WIS at pH 4.8.
Conversion of WIS from reed pretreated at 180°C
Conversion of WIS from reed pretreated at 210°C
Aside from the enzymatic temperature and pH, cellulase loading is also an important factor for the enzymatic hydrolysis of cellulosic substrates. Increasing the cellulase loading generally resulted in an increase in glucose release and cellulose conversion. The enzymatic hydrolysis curves of the WIS in reed after the LHW pretreatment with respect to several cellulase loadings are shown in Figure
Enzymatic hydrolysis of WIS from reed in the LHW pretreatment at 180°C and 210°C with respect to different cellulase loadings.
Conversion of WIS from reed pretreated at 180°C
Conversion of WIS from reed pretreated at 210°C
A high glucose concentration is necessary to obtain high ethanol concentration by fermentation, which results in a decrease in the production cost, such as ethanol distillation cost. Decreasing the solid-to-liquid ratios during the enzymatic hydrolysis of a cellulosic substrate may increase glucose consistency. Nevertheless, very low solid-to-liquid ratio indicates a very high initial solid concentration, which causes difficulty in uniformly mixing the enzyme liquid and the WIS because the amount of liquid in the system is reduced. The trends of change in the cellulose conversion are illustrated in Figure
Enzymatic hydrolysis of WIS from reed pretreated at 180°C and 210°C with respect to different solid-to-liquid ratios in enzymatic hydrolysis.
Glucose release of WIS from reed pretreated at 180°C
Cnversion of WIS from reed pretreated at 180°C
Glucose release of WIS from reed pretreated at 210°C
Conversion of WIS from reed pretreated at 210°C
Ethanol production depends on the sugar yield and the mixture fermentability. The WIS obtained from different pretreatment conditions, namely, 180°C at 20 min and 210°C at 20 min, were fermented by SHF and SSF methods by using special ethanol yeast to investigate the fermentability of the WIS from LWH-pretreated reed. Table
Ethanol fermentation of WIS after LHW pretreatment.
Substrates | SHF | SSF | ||||
---|---|---|---|---|---|---|
Ethanol concentrations |
Residual glucose |
Conversion of cellulose to ethanol (%) | Ethanol concentrations |
Residual glucose |
Conversion of cellulose to ethanol (%) | |
Reed | 5.07 | 0.06 | 36.9 | 4.30 | 0.39 | 23.4 |
Reed at 180°C for 20 min | 17.69 | 0.06 | 96.6 | 17.60 | 0.34 | 72.1 |
Reed at 210°C for 20 min | 18.99 | 0.06 | 99.5 | 21.75 | 0.36 | 85.5 |
Reed can be used as a substrate for ethanol production in northeast China. The LHW pretreatment significantly enhanced the enzymatic digestibility of reed. The optimum conditions of LHW pretreatment for reed were a temperature of 180°C for 20 min and a solid-to-liquid ratio of 1 : 10, which resulted in a cellulose conversion rate of 82.59% in the subsequent enzymatic hydrolysis at 50°C for 72 h with a cellulase loading of 30 FPU/g oven-dried WIS. After the LHW pretreatment, the conversion of glucan to ethanol in WIS from LWH-pretreated reed reached 99.5% of theoretical yield by the SHF process.
This study was financially supported by the National Key Basic Research Development Program (2011CB707401), the National Natural Science Foundation of China (nos. 31100440, 21276143), and the International Science & Technology Cooperation Program of China (2010DFA32560).