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Optimization of the Thermal Dry Treatment To Enhance the Enzymatic Hydrolysis of a Spent-Sawdust Matrix Used for Grifola frondosa Cultivation† Akihiro Hideno,‡ Hideki Aoyagi,*,‡ James C. Ogbonna,§ and Hideo Tanaka‡ Institute of Life Science and Bioengineering, Graduate School of Life and EnVironmental Science, UniVersity of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan, and Department of Microbiology, UniVersity of Nigeria, Nsukka, Nigeria ReceiVed May 28, 2007. ReVised Manuscript ReceiVed September 10, 2007
Maitake mushroom (Grifola frondosa) is extensively cultivated in Japan using sawdust matrices (SM), mainly made of hard wood chips, as media. This leads to the accumulation of a large amount of spent-sawdust matrix (SSM). For example, one of the maitake mushroom cultivation companies in Japan produces more than 230 tons (wet weight) of SSM every day. There is yet no major utilization method of the SSM, and this has resulted in a high cost of treating the waste material. Our studies have been focused on effective methods for bioconversion of SSM into bioethanol. Given that the moisture content of SSM was about 70% after the maitake cultivation, drying of the SSM is required to prevent decay during storage and to reduce the cost of transportation. In the present study, the effects of drying conditions on the efficiency of grinding and a simultaneous saccharification and fermentation process (SSF) were investigated. Thermal analysis (TG/DTG) of the raw SSM was conducted using a thermo-analysis instrument (EXSTRAR6000 TG/DTA, Seiko Instruments, Inc., Japan). The thermal analysis was also used for determining appropriate thermal dry treatment conditions for saccharification of the SSM. The dry-treated SSM was milled using a beads beater (TOMY Micro smash MS-100) for enzymatic saccharification. The results of thermal analysis of the raw SSM showed that the optimal drying condition for grinding and saccharification was in the temperature range of 25 and 200 °C, at a rate temperature increase of 50 °C/min. To develop a large-scale drying process, effects of ovendrying were also investigated. The results obtained from the oven-drying showed that the optimal condition was 200 °C for 30 min and, thus, conforms with the results of the thermal analysis. The ethanol concentration yield by SSF from the oven-dried SSM (at 200 °C for 30 min) was higher than that of the air-dried SSM at room temperature.
Introduction Many species of mushrooms cultivated on a solid medium (consisting of wood powder) are important agricultural foods in the world. Global production is greater than six million tons and has an approximate value of at least 14 billion U.S. dollars.1 The fruiting body of a white rot fungus Grifola frondosa is known as an edible mushroom, maitake in Japan, China, and the U.S.A.2 The maitake mushroom is now being cultivated extensively in Japan using sawdust matrices (SM). However, the disposal of spent-sawdust matrix (SSM) after cultivation of G. frondosa has being very problematic and expensive.3 For example, over 230 tons (wet weight) of SSM is constantly being † Presented at the International Conference on Bioenergy Outlook 2007, Singapore, April 26–27, 2007. * To whom correspondence should be addressed. Fax: +81-298534605. E-mail:
[email protected]. ‡ University of Tsukuba. § University of Nigeria. (1) Rinker, D. L. Handling and using spent mushroom substrate around the world. In Mushroom Biology and Mushroom Products; Sanchez, J. E., Huerta, G., Montiel, E., Eds.; Impresos Jupiter: Cuernavaca, Mexico, 2002; pp 43–60. (2) Shen, Q.; Royse, D. J. Effects of nutrient supplements on biological efficiency, quality and crop cycle time of maitake (Grifola frondosa). Appl. Microbiol. Biotechnol. 2001, 57, 74–78. (3) Seto, M.; Nishibori, K.; Masai, E.; Fukuda, M.; Ohdaira, Y. Degradation of polychlorinated biphenyls by a “Maitake” mushroom, Grifola frondosa. Biotechnol. Lett. 1999, 21, 27–31.
discharged by a production factory of maitake mushroom in Japan on a daily basis. SSM can be regarded as the lignocellulosic biomass. SSM have important advantages considering their accumulation and constant availability irrespective of the season compared to other lignocellulosic biomass that are scattered and seasonal. We have been working on the development of methods for the bioconversion of SSM into bioethanol. Because the moisture content of SSM was about 70%, drying of the SSM is necessary to prevent decay during storage and to reduce the cost of transportation. In the present study, effects of drying conditions on the efficiency of grinding and simultaneous saccharification and fermentation process (SSF) were investigated by the thermal analysis (TG/DTG) method. Experimental Section Substrates and Enzymes. Figure 1 shows the scheme for making maitake mushroom and SSM. Wood chips of beech wood, SM, and SSM were kindly supplied from Yukiguni Maitake Co., Ltd. Niigata, Japan. Because the SSM had the shape of the block, it was necessary to homogenize SSM as the first step of the pretreatment. The raw sample of SSM was homogenized manually (4) Hideno, A.; Aoyagi, H.; Isobe, S.; Tanaka, H. Utilization of spentsawdust matrix after cultivation of Grifola frondosa as substrate for ethanol production by simultaneous saccharification and fermentation. Food Sci. Technol. Res. 2007, 13, 111–117.
10.1021/ef7002975 CCC: $40.75 2008 American Chemical Society Published on Web 10/23/2007
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Figure 1. Flow diagram for the cultivation of G. frondosa and production of fruiting bodies. (/) WC/CB ) about 3.4–3.7:1 (weight). Figure 2. DTA curves corresponding to percentage weight losses (TG) of the raw SSM in the atmosphere.
Table 1. Various Sugars and Lignin Contents of the SSM Tested component
[% (w/w)]
arabinose rhamnose galactose glucose xylose mannose klason lignin
0.8 0.3 0.9 37.1 14.9 1.5 14.9
for use as the substrate. The constitutional neutral sugars and lignin content of this material are shown in Table 1. Cellulclast 1.5L (cellulase derived from Trichoderma reesei QM9414) and Novozyme 188 (β-glucosidase derived from Aspergillus niger) were kindly supplied from Novozymes Japan Co. Ltd., Chiba, Japan. Thermal Analysis of Raw (Hydroscopic) SSM. Simultaneous thermogravimetry (TG) and differential thermal analysis (DTA) of raw SSM were performed using an EXSTRAR6000 TG/DTA (Seiko Instruments, Inc., Japan) at heating rates of 10 °C/min and the temperature range from 25 to 500 °C in the atmosphere. TG was calculated using eq 1 TG (%) ) (weight loss because of thermal decomposition/original weight) × 100 (1) DTA was simultaneously measured with TG on the TG/DTA instrument, and R-alumina was used as a reference. Optimization of the Drying Condition To Enhance the Enzymatic Hydrolysis of SSM. The raw SSM (approximately 30 mg, wet weight) was ejected from the thermal analysis equipment after its analysis in the temperature range of 25 and 200 or 500 °C at the rate of temperature increase of 10–100 °C/min in the atmosphere; this dry sample was inserted into a micro test tube containing a stainless-steel bead and milled using a beads beater (TOMY Micro smash MS-100) at 4000 rpm for 30 s. The milled SSM was used as a substrate for enzymatic saccharification. In the process, the TG/DTA instrument was used for “thermal analysis” and “thermal dry treatment”. A total of 1 mL of enzyme solution (Celluclast 1.5L, 0.8 FPU/ mL; Novozyme 188, 1.6-cellobiase unit/mL, in 0.05 M citrate buffer at pH 4.8) was added into this micro test tube containing milled SSM. The micro test tube was incubated at 50 °C for 60 min. The sample solution was taken after 60 min of incubation and centrifuged (1100g, 15 min). The amount of reducing sugars in the supernatants was measured. The percentage of saccharification (S) was calculated using eq 2 S ) (B × 0.9/A) × 100
(2)
Here, A is the total reducing sugars in SSM (in milligrams), and B is the reducing sugars acquired by cellulase (in milligrams).
Optimization of the Dry Treatment for Enhanced Enzymatic Hydrolysis of SSM in the Dry Oven. The raw SSM (approximately 10 g, wet weight) was placed in a glass Petri dish to dry in the drying oven at various conditions (at 100–250 °C for 5–720 min). This dried sample was milled in a food processor (BMFT08, ZOJIRUSHI, Osaka, Japan) at about 1200 rpm for 60 s and passed through a 0.5 mm screen. The milled sample (10 mg, dry weight) was put into a 15 mL glass test tube with 1 mL of enzyme solution. The mixture was incubated at 50 °C for 60 min and centrifuged (1100g, 15 min). The amount of reducing sugars in the supernatants was measured. The percentage of saccharification (S) was calculated using eq 2.
Results and Discussion Thermal Analysis of Raw (Hydroscopic) SSM. Thermal analysis is convenient, reproducible, and a useful method for characterizing heterogeneous organic materials. The thermal degradability is affected by the chemical composition of the material because different components of lignocellulosic biomass have different thermal behaviors.5 To understand the thermal properties of SSM, typical TG and DTA curves obtained from raw SSM are shown in Figure 2. TG is the most widely used technique to characterize the thermal decomposition of polymer materials.6 TG is a technique in which the mass of a sample is measured as a function of the temperature. Samples are subjected to a controlled temperature program, which records the TG curves. Thus, the mass of the samples are plotted on the ordinate (which decreased downward), and the temperature is plotted on the abscissa (which decreased from left to right). DTA is the measurement of the difference in temperature between a sample and a reference as heat is applied to the system. The initial weight loss observed at temperatures lower than 100 °C is due to the evaporation of free water. The devolatilization process started at 225 °C, and the maximum weight loss measured occurs at 300–325 °C. At 325 °C, the slope of the TG curve changes and a sharp weight loss continued up to 430 °C. These losses in weights were attributed to pyrolyses of cellulose, hemicellulose, and lignin.5 The endothermal peak of SSM observed at 80 °C was relevant to the weight loss peak in the corresponding TG curve at 80 °C. Two exothermic peaks, (5) Negro, M. J.; Manzanares, P.; Oliva, J. M.; Ballesteros, I.; Ballesteros, M. Changes in various physical/chemaical parameters of Pinus pinaster wood after steam explosion pretreatment. Biomass Bioenergy 2003, 25, 301– 308. (6) Wang, H.; Tao, X.; Newton, E. Thermal degradation kinetics and lifetime prediction of a luminescent conducting polymer. Polym. Int. 2004, 53, 20–26.
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Table 2. Optimization of the Dry Treatment for Enhanced Enzymatic Hydrolysis of SSM by Thermal Analysis condition of thermal analyses temperature range (°C) 25–500 25–500 25–500 25–500 25–200 25–200 50a
measurement value of thermal analysis
rate of temperature increase (°C/min)
heating time (min)
temperature of sample (°C)
TG (%)
DTA (uV)
saccharification rate
10 20 50 100 50 100 0
12.21 ( 0.31 8.85 ( 0.20 5.53 ( 0.18 4.14 ( 0.06 5.41 ( 0.10 4.30 ( 0.12 1 week
98.25 ( 2.72 121.30 ( 4.38 154.88 ( 10.28 203.85 ( 5.51 121.05 ( 1.76 121.85 ( 1.97 ND
52.28 ( 2.63 51.83 ( 2.09 48.76 ( 5.33 48.92 ( 2.23 40.60 ( 1.02 38.11 ( 1.47 ND
56.33 ( 6.96 90.30 ( 8.10 150.80 ( 0.70 239.45 ( 14.97 120.13 ( 5.94 119.95 ( 3.58 ND
12.87 ( 2.16 10.79 ( 1.43 10.83 ( 1.97 6.79 ( 1.15 17.64 ( 1.27 14.62 ( 1.55 5.18 ( 0.25
a The oven dry sample without thermal analysis. Values represent means of four repetitions ( standard deviation (SD). ND ) not detected. The “heating time” showed the time to put the sample in the thermal analysis instrument. The “temperature of sample” showed the temperature at the end time of analyses.
Figure 3. Effect of the heating temperature on enzymatic hydrolysis of SSM dried in the large-scale convectional oven.
observed at 325 and 425 °C, respectively, were due to the pyrolysis of cellulose, hemicellulose, and lignin. Drying by Thermal Analysis for the Enhancement of Enzymatic Hydrolysis of SSM. To determine the effects of drying conditions on the efficiency of grinding and saccharification, the enzymatic hydrolysis of SSM dried by using thermal analysis was carried out. Various conditions and data of thermal analyses of SSM and the saccharification rate of dried SSM using thermal analysis were summarized in Table 2. Table 2 showed that the optimal drying condition for grinding and saccharification was in the temperature range of 25 and 200 °C and at a heating rate of 50 °C/min in the atmosphere; however, their mechanism are not well-understood. Studies on this aspect are currently being investigated. This rate of saccharification was approximately 17.6% (which corresponded to 3-fold) higher than the values obtained with the oven-dried sample at 50 °C for 1 week, To develop a method for a large-scale drying process, the drying treatment in conventional oven was also investigated. The results obtained from the drying process showed that the optimal condition was 200 °C for 30 min (Figure 3) and showed a similar trend as the results obtained with the thermal analysis.
Steam explosion is widely used for the pretreatment of lignocellulosic biomass. The steam explosion is typically initiated at a temperature of 160–260 °C (corresponding pressure of 0.69–4.83 MPa) for several seconds to a few minutes before the material is exposed to atmospheric pressure.7 The process causes hemicellulose degradation and lignin transformation because of the high temperature, thus increasing the potential of cellulose hydrolysis. It was reported that the optimal hemicellulose solubilization and hydrolysis can be achieved by either a high temperature and short residence time (270 °C, 1 min) or a lower temperature and longer residence time (190 °C, 10 min).8 Considering these reports in combination with our results, the process of drying by thermal analysis can be caused by the similar situation of the steam explosion. Namely, the moisture in SSM may be changed to the high-pressure saturated steam by the extreme increase of the temperature in a brief time. Moreover, SSM can be easily caused by this phenomenon, because the hyphae of G. frondosa penetrate SSM and it lead to the moisture in the SSM. However, little difference between the optimal condition and the other conditions in the oven drying process was noted. This could be due to much dispersion of heat in the oven and ineffective heating of SSM. The ethanol concentration yield by SSF with the oven-dried SSM (at 200 °C for 30 min) was higher than the value obtained using air-dried SSM at room temperature (data not shown).4 However, this difference was little. The heating method of SSM in a large scale is an issue, which requires further investigation. Acknowledgment. This work was supported in part by (1) The Biomass Energy Research Project, Ministry of Agriculture, Forestry, and Fisheries, Japan, (2) the 21st Century COE Program (Functional analysis of the communication mechanism in bioconsortia and its agricultural hyper-utilization) from the Ministry of Education, Culture Sports, Science, and Technology, Japan (MEXT), and (3) Grant-in-Aid for Scientific Research A (16208017) from the Japan Society for the Promotion of Science. The authors thank Prof. Shuichi Doi (University of Tsukuba) for his advice during the early days of this study. The authors thank Dr. Kozo Nishibori of Yukiguni Maitake Co. for the gift of fresh mushroom, G. frondosa. The authors are grateful to the Chemical Analysis Center, University of Tsukuba, for thermal analysis data. EF7002975 (7) Sun, Y.; Cheng, J. Hydrolysis of lignocellulosic materials for ethanol production: A review. Bioresour. Technol. 2002, 83, 1–11. (8) Duff, S. J. B.; Murray, W. D. Bioconversion of forest products industry waste cellulosics to fuel ethanol: A review. Bioresour. Technol. 1996, 55, 1–33.
