Optimum Condition of Rice Straw Hydrolysate Detoxification with Charcoal Powder for Cellulosic Ethanol Production by Pichiastipitis TISTR 5806

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1 Journal of Food Science and Engineering 6 (2016) doi: / / D DAVID PUBLISHING Optimum Condition of Rice Straw Hydrolysate Detoxification with Charcoal Powder for Cellulosic Ethanol Production by Pichiastipitis TISTR 5806 Teerapatr Srinorakutara 1, Yuttasak Subkaree 2, Nassapat Boonvitthya 3 and Nantana Bamrungchue 2 1. Thailand Institute of Scientific and Technological Research (TISTR), 35 Mu 3, Technopolis, Khlong 5, KhlongLuang, Pathum Thani 12120, Thailand 2. Department of Energy Technology, Thailand Institute of Scientific and Technological Research (TISTR), 35 Mu 3, Technopolis, Khlong 5, KhlongLuang, Pathum Thani 12120, Thailand 3. PTT Research and Technology Institute, PTT Public Company Limited, 71 M.2 Phahonyothin Rd., Sanubtup, Wangnoi, Ayutthaya 13170, Thailand Abstract: In this study, the rice straw was hydrolysed by using 3.0% (w/v) H 2 SO 4 followed by enzymatic hydrolysis. The rice straw obtained was treated with charcoal powder and the optimal condition of detoxification with charcoal powder was investigated. The results showed that the optimal condition for detoxification was the use of 2.5 grams of sterilized charcoal. The mixture was operated at ph 5.0, 30 C and 160 rpm for 5 min. The detoxified was then used for ethanol production using P. stipitis TISTR The condition of the detoxified fermentation which gave maximum ethanol concentration of 21 g/l was at ph 5.0, 30 C and 160 rpm for 72 h. Without detoxification, the P. stipitis TISTR 5806 could not however utilize the for ethanol production. Key words: Detoxification, charcoal, rice straw, ethanol fermentation, P. stipitis. 1. Introduction Ethanol from lignocelluloses biomass has become an increasingly popular alternative to gasoline as one option to reduce dependence on oil and mitigate global warming [1]. As the first generation materials for ethanol production has been blamed for causing food insecurity [1], the switching to inedible plant material should thus help to reduce pressure on the food crops [2]. In addition, lignocellulose, abundant feedstock, can be regenerated and given a low cost. The lignocellulosic material can provide both C 5 and C 6 sugar especially glucose and xylose, which they can convert to bioethanol by microorganism. Since the pentose sugar, D-xylose comprises approximately one-third of the total carbohydrate sugars Corresponding author: Teerapatr Srinorakutara, Ph.D., research field: biofuel technology. inlignocellulosic biomass [3], therefore the favorable economics for large-scale production of ethanol from lignocellulosic materials should require not only a good conversion of glucose but also an effective conversion of xylose. However, there is only some microorganism which can utilize both glucose and xylose such as P. stipitis, Candida shehatae, Kuyveromycesmaxinus and etc. In terms of wild-type microorganisms, strains of the yeast Pichiastipitis show the most promise in the short term for direct high-yield fermentation of xylose without byproduct formation [3]. However, these microorganisms are sensitive to inhibitors such as acetic, furfural, hydroxymethyl furfural and phenolic compounds leading to low ethanol production rate and yield [4-8]. Ge et. al. [9] also reported that the sugars, furfural, acetic acid and phenolic compounds are

2 76 Optimum Condition of Rice Straw Hydrolysate Detoxi- fication with Charcoal Powder for produced during acid hydrolysis of corn cob. It is known that these compounds affect ethanol fermentation performance and must be removed [9]. Several treatments e.g. ion exchange, overliming, activated charcoal adsorption, laccase oxidation, and bioabatement treatments have been reported for the detoxification of hydrolyzates to improve the fermentability of acid s into ethanol [10, 11]. Larbnongsaeng et. al. [12] reported that charcoal powder can treat rice straw. The detoxified can produce 16 g/l of ethanol by P. stipitis TISTR 5806 as detoxified cannot produce. The charcoal powder detoxification is the best method comparing to coffee seed residues adding, and overliming [12]. In addition, charcoal powder is inexpensive alternative in the detoxification and expected to be cheaper than activated charcoal. The optimal condition powder detoxification of from rice straw will be therefore investigated prior to ethanol production by P. stipitis TISTR 5806 in this research. 2. Materials and Methods 2.1 Rice Straw and Enzymes Rice straw was harvested from Nakhon Pathom province, central region of Thailand. Prior to chemical pretreatment, it was cut into small size (< 2.0 mm) by using wood chopper and kept in plastic bag for this study. The Cellic CTec2 and Cellic HTec2 enzymes (Novozymes) were bought from East Asiatic (Thailand) Co., Ltd. The Cellic CTec2 and Cellic HTec2 enzymes consist of FPU/mL and FPU/mL respectively. They were measured by Filter Paper Activity at ph 4.8 and 50 C [13]. 2.2 Charcoal Powder Charcoal was bought from Thailand grocery. It was crushed to powder with Hammer Mill passed through sieve size of 1.25 mm. The charcoal powder was kept in plastic bag for this study. 2.3 Yeast P. stipitis TISTR 5806 Yeast P. stipitis TISTR 5806 was obtained from TISTR Culture Collection. It was kept on YM slant at 4 C. The yeast was activated by tranfering to other YM slant and incubated at room temperature prior to use. After yeast growing, the yeast colony was transferred to YM broth for preparation the yeast starter. 2.4 Rice Straw Hydrolysate Production The 630 grams of chopped rice straw were treated with 3,600 ml of 3.0% (w/v) H 2 SO 4 at 121 C, 1.5 bar for 15 min in autoclave. The pretreated rice straw was cooled and adjusted ph to 5.0 with NaOH solution. The 45 FPU/g DM of Cellic CTec2 and 15 FPU/g DM of Cellic HTec2 enzymes were then added. The mixture was transfered to 5 liters Fermenter for enzyme hydrolysis. The mixture was operated at ph 5.0, 50 C and 250 rpm for 72 h. After enzyme hydrolysis, the solid residue was separated by vacuum filtration passed through filter paper Whatman No. 1. The rice straw was kept in refrigerator for next experiment (Adapt from Srinorakutara et. al. [14]). 2.5 Charcoal Powder Detoxification The optimal condition of rice straw detoxification with charcoal powder was investigated with various factors. The charcoal powder both sterilized and sterilized of grams was added into 100 ml of rice straw. The mixtures were operated at temperature of C, ph 4-6 and 160 rpm for 5 min to 24 h. The detoxified s were separated by vacuum filtration passed through filter paper Whatman No. 1. The detoxified s were used to rapidly test the ethanol production by P. stipitis TISTR Ethanol Fermentation The nutrients consisting of 1.0 g/l of yeast extract,

3 Optimum Condition of Rice Straw Hydrolysate Detoxi- fication with Charcoal Powder for g/l of (NH 4 ) 2 HPO 4 and g/l of MgSO 4.7H 2 O were added to detoxified. After that the yeast P. stipitis TISTR 5806 starter was then added to detoxified. The initial yeast was cell/ml. The was carried out at ph 5.0, 30 C and 160 rpm for 72 h. The fermented broth was harvested, centrifuged and analyzed for reducing sugar, glucose, xylose and ethanol concentration. 2.7 Analytical Method The concentration of reducing sugar was analyzed by DNS method [15]. The concentration of glucose and xylose was analyzed by YSI glucose analyser. Ethanol concentration was analyzed by Gas chromatography (GC) with HP-INNOWAX N-133 column a length 30 m, outer diameter mm and inner diameter 0.25 µ at column temperature increased with rate of 15 C/min to 120 C, inject temperature 220 C. Helium is carrierr gas at flow rate 50 ml/min and analysed by Flame-Ionized 3.1 Rice Straw Hydrolysate The rice straw was hydrolysedusing 3.0% (w/v) H 2 SO 4 at 121 C, 1.5 bar for 15 minute in autoclave followed by enzymatic hydrolysis. The rice straw composed of g/l of total reducing sugar, g/l of glucose, g/ /L of xylose, and detector (FID). 3. Results and Discussion g/l of furfural (Table 1). This showed the high dosage of glucose and xylose comparing to total reducing sugarwhich is equivalent to a 46-52% (w/w) and 35-38% (w/w) of total reducing sugars respectively. Although the high conversion of both glucose and xylose to ethanol would lead to favorable economics forlarge-scale production of ethanol from lignocellulosic materials, the ethanol production from this hydrlysate could not however occur without detoxification. The P. stipitis TISTR 5806 could not grow and ferment glucose and xylose to ethanol in this [12] because it did not only consist of sugars but also consist of highh inhibitors. The inhibited by inhibitors. Therefore this is a main problem that should be solved. 3.2 Charcoal Powder Detoxification Effect of Sterilized and Non-sterilized Charcoal Powder After detoxification with both sterilized and sterilized charcoal powders, the obtained could produce g/l of ethanol by using P. stipitis TISTR 5806 as shown in 1. These results showed that the sterilized and sterilized charcoal powder could equally removee the amount of inhibitors. The sterilization at 121 C for 15 min could not change the property powder. The temperature of sterilization could not increase the porosity powder. However, the major target of this sterilization increase the porosity powder but it was to investigate on ethanol production by P. stipitis TISTR Table 1 Composition of rice straw. Composition Reducing sugar Glucose Xylose Furfural P. stipitis TISTR 5806 would be Content (g/l) Sterilized Sterilized rice straw was not to the contamination of detoxifiedd Charcoal powder 1 Ethanol concentration obtained from of detoxified by P. stipitis TISTR The was detoxified with 5.0 grams of sterilized and sterilized charcoal powder in 100 ml at ph 5.0, 50 C and 160 rpm for 24 h.

4 78 Optimum Condition of Rice Straw Hydrolysate Detoxi- fication with Charcoal Powder for The results showed that there were no contamination and effect on ethanol fermentation. The after detoxification with sterilized and sterilized charcoal powder could equally produce amount of ethanol. Therefore the charcoal powder did not need to sterilizee before detoxification of because it was easy method and low cost Effect of Charcoal Powder Content The proportions of activated charcoal used to treat the can strongly influence the removal of compounds. The increasing of activated charcoal is increasing of adsorbate. Thereforee high activated charcoal leads to high adsorption of inhibitor [16]. However, the charcoals can also adsorp sugar. The use of high charcoal is a cause of sugar loss. In addition, the use of higher charcoal is also a cause of higher cost. In this study, an increase in level powder from 2.5 grams to 5.0 grams in 100 ml was able to rise ethanol concentration from 21 g/l to 23 g/l after fermentation with P. stipitis TISTR 5806 ( 2). However, ethanol concentration would decrease as charcoal powder was more than 5.0 grams in 100 ml. As a result, the higher concentration powder might adsorp higher sugar. The reducing sugar loss was over 15% (w/w) as charcoal powder of 10 grams in 100 ml used for detoxification. The reducing sugar loss was over 30% (w/w) when charcoal powder of 20 grams in 1000 ml used (data not shown). This agrees with Mussatto and Roberto [16] who found that the sugars reduced 31.3% at 30% used. The results showed that optimal content powder was between 2.5 grams to 5.0 grams in 100 ml. As the 2.5 gram in 100 ml was selected for production because of cost saving. This level agrees with Lee et al. [10] who found that the activated carbon ata 2.5 wt% level on the prehydrolyzate was able to remove 42% of formic acid, 14% of aceticacid, 96% of hydroxymethyl- furfural (HMF) and 93% of the furfural. However, the 8.9% of sugars were also removed [10] Effect of Temperature Mussatto and Roberto [16] explained that the adsorption of compounds increases with higher temperature, because high temperatures provide a faster rate of diffusion of adsorbate molecules from the solution to adsorbent. However,it was found thatt there was no difference in ethanol production from detoxified using P. stipitis TISTR 5806 in this study ( 3). The results showed that there was no effect of temperture on adsorption powder. Charcoal powder (g) in 100 ml 2 Ethanol concentration obtained from detoxified by P. stipitis TISTR The was detoxified with grams at ph 5.0 and 50 C for 24 h. Temperaure ( C) 3 Ethanol concentration obtained from detoxified by P. stipitis TISTR The was detoxified with 2.5 grams at ph 5.0 and C for 24 h.

5 Optimum Condition of Rice Straw Hydrolysate Detoxi- fication with Charcoal Powder for 79 Charcoal powder was able to adsorp the inhibitor at various temperatures from 30 to 50 C. This is an advantage of operation because there is no need to control of temperature for detoxification. The after enzymatic hydrolysis can suddenly detox with charcoal powder without temperature control. However, 30 C is chosen as the optimal temperature for detoxification in this study because this level is close to room temperature (28-31 C) of Thailand. This level of detoxification temperature agrees with Kamal et. al. [17] who used activated charcoal for detoxification at the room temperature as this range was enough to remove the inhibitors [17]. However, this temperature was difference from many researches such as Chaud et. al. [11], Mussatto et. al., [18] and in review of Mussantto and Roberto [16] who used the temperature above 45 C for detoxification with activated charcoal Effect of ph Mussatto and Robertoo [16] explained that the both weak organic acids and bases compounds are most readily adsorbed in the ionized state. The weak organic acids (such as acetic acid, phenol molecules) are most readily adsorbed in the low ph (acids). On other hand, the weakly basic compounds are also most readily adsorbed in high ph (alkaline). As a rule, organic acids are best adsorbed from acidic solution and organic bases adsorbed in basic solution [16]. In this study, the charcoal powder was used to adsorb the inhibitors in acidic solution, low ph of 4-6. The after the detoxification at low ph of 4-5 could produce higher ethanol than after detoxification at ph 5.5 and 6 as shown in 4. This result showed that at the low ph of 4-5 could remove higher inhibitors than at high ph of 5.5 and 6. This may be because at low ph, the organic acids, especially phenolic compounds are most readily adsorbed. These phenolic compounds are knownn as high effect on ethanol fermentation performance. Therefore, the low ph of 4-5 is optimal ph for charcoal powder detoxification in this study. However, 4 Ethanol concentration obtained from detoxified by P. stipitis TISTR The was detoxified with 2.5 grams at ph and 30 C for 24 h. Contact time of detoxification 5 Ethanol concentration obtained from detoxified by P. stipitis TISTR The was detoxified with 2.5 grams at ph 5.0 and 30 C for 5 min-24 h. best optimal ph of this detoxification is 5.0 becausee the initial ph of the rice straw is ph 5 which will help to save the cost for ph control Effect of Contact Time Contact time is an important variable in activated charcoal treatment. During the adsorption process, the charcoal surface is progressively blocked by adsorbate, becoming completely covered after the time passes by. When this happens, the charcoal cannot adsorb any more compounds [16]. In this study, the after detoxification with contact time of 5 minutes to 4 h. showed that there was no differencee of ethanol production after fermentation by P. stipitis TISTR

6 80 Optimum Condition of Rice Straw Hydrolysate Detoxi- fication with Charcoal Powder for This result indicated that the adsorption of charcoal powder may be stopped at only 5 minutes of contact time. The only 5 minutes powder detoxification is very short time comparing with other researchs, which contact time was over 45 min for activated charcoal detoxification [11, 16-18]. This may be because the charcoal powders consist of porosity less than activated charcoal. The surface of charcoal powder may be blocked by adsorbate faster than activated charcoal. This may be a disadvantage of charcoal powder on adsorption. It may be a cause to still remain inhibitors in the. The obtained from hydrolyzing rice straw with 3.0% (w/v) H 2 SO 4 followed by enzymatic hydrolysis could not directly produce ethanol by P. stipitis TISTR5806. The must be detoxified before ethanol fermentation by P. stipitis TISTR 5806 [11]. This study showed the performance powder on detoxification of rice straw. The optimal condition for detoxification was 2.5 gram of sterilized charcoal as the detoxification was operated at ph 5.0, 30 C and 160 rpm for 5 minutes. Although this charcoal powder detoxification was a cause for reducing sugar loss of 5-7% (w/w), the after detoxification with this condition could produce g/l of ethanol by P. stipitis TISTR The detoxified s could not however use to produce ethanol. The P. stipitis TISTR 5806 could completely consume glucose within 72 h of fermentation. After fermentation, the reducing sugar still remained above 40 g/l as almost all of it was xylose ( 6). This indicated that the P. stipitis TISTR 5806 could utilize a little of xylose for ethanol production only. Ethanol concentration obtained from this study was only g/l. However, Rouhollah et. al. [19] reported that ethanol concentration around 30 g/l would inhibit the xylose fermentation process.therefore, limitation of xylose utilization of P. 6 Time course of ethanol production from detoxifiedd byp. stipitis TISTR stipitis TISTR inhibition of ethanol concentration. (seee Table 3 of Chandel et. al. [20]) as the industial scale which need not only high yield but also highh ethanol concentration. The use of xylose-fermenting yeast tolerant to highh initial sugar problem. 4. Conclusion ns Fermentation time (h) 5806 may not be a result from The limitation of xylose utilizing may come from two reasons. The first one may be a high concentration n of initial reducing sugar. The second one may be inhibitors that still remained in rice straw after charcoal detoxification. Many researchess reported that the native P. stipitisgave high conversionn of xylose to ethanol at initial sugar lower than 60 g/l consists of very low level of inhibitor. The initial sugar at the low level is not however suitable for concentrationn may overcome this The charcoal powder was able to remove inhibitor of rice straw. The detoxifiedd could use to produce ethanol by P. stipitis TISTR The optimal condition of detoxification rice straw was 2.5 grams of sterilized charcoal as the detoxification was operated at ph 5.0, 30 C and 160 rpm for 5 min. The detoxified could be fermented to g/l of ethanol by P. stipitis TISTR 5806 at ph 5.0, 30 C and 160 rpm for 72 h. However,

7 Optimum Condition of Rice Straw Hydrolysate Detoxi- fication with Charcoal Powder for 81 the P. stipitis TISTR 5806 could not use the detoxified to produce the ethanol. Acknowledgments This work was financially and facilely supported by the Thai government and Thailand Institute of Scientific and Technological Research (TISTR). References [1] Diep, N. G., Fujimoto, S., Yanagida, T., Minova, T., Sakanishi, K, Nakagoshi, N., and Tran, X. D Comparison of the Potential for Ethanol Production from Rice Straw in Vietnam and Japan via Techno-Economic Evaluation. Int. Energ. J. 13: [2] Sassner, P., Gallbe, M., and Zacchi, G Techno-economic Evaluation of Bioethanol Production from Three Different Lignocellulosic Materials. Biomass Bioenerg 32: [3] McMillan, J. D Xylose Fermentation to Ethanol. A Review. National Renewable Energy Laboratory. [4] Sun, Y., and Cheng, J Hydrolysis of Lignocellulosic Materials for Ethanol Production. A Review. Bioresource Technol 83: [5] Diaz, M. J., Ruiz, E., Romeo, I., Cara, C., Moya, M., and Castro, E Inhibition of Pichiastipitis Fermentation of Hydrolysate from Olive Tree Cutting. World J. Microb. Biot. 25: [6] Bellido, C., Bolado, S., Coca, M., Lucas, S., and Gonzales-Benito, G Effect of Inhibitors Formed during Wheat Straw Pretreatment on Ethanol Fermentation by Pichiastipitis. Bioresource Technol. 102: [7] Yang, X., Zhang, S., Zuo, Z., Men, X., and Tian, S Ethanol Production from the Enzymatic Hydrolysis of Non-detoxified Steam-Exploded Corn Stalk. Bioresource Technol. 102: [8] Chandel, A. K., Kapoor, R. K., Singh, A., and Kuhad, R. C Detoxification of Sugarcane Bagasse Hydrolysate Improves Ethanol Production by Candida Shehatae NCIM Bioresource Technol. 98: [9] Ge, J. P., Cai, B. Y., Liu, G. M., Ling, H. Z., Fang, B. Z., Song, G., and Yang X, F., and Ping, W. X Comparison of Different Detoxification Methods for Corn Cob Hemicelluose Hydrolysate to Improve Ethanol Production by Candida Shehatae ACCC Afr. J. Microbiol Res 5 (10): [10] Lee, J. M., Venditti, R. A., Jameel, H., and Kenealy, W. R Detoxification of Woody Hydrolyzates with Activated Carbon for Bioconversion to Ethanol by the Thermophilic Anaerobic Bacterium Thermoanaero- Bacterium Saccharolyticum. Biomass Bioenerg 35: [11] Chaud, L. C. S., da Silva, D. D. V., de Mattos, R. T., and de Almeida Felipe, M. G Evaluation of Oat Hull Hemicellulosic Hydrolysate Fermentative Employing Pichiastipitis. Braz. Arch. Biol. Technol. 55: [12] Larbnongsaeng, C., Srinorakutara, T., and Pratinthong, N Detoxification of Rice Straw Hydrolysates with Charcoal for Efficient Improvement of Ethanol Fermentation Using Pichiastipitis TISTR In Proceedings of the 4th International Conference on Fermentation Technology Value Added Agriculture Products, Aug. 3-29, 2011, KhonKaen, Thailand, 18. [13] Ghose, T. K Measurement of Cellulase Activity. Pure Appl. Chem. 59: [14] Srinorakutara, T., Subkaree, Y., Bamrungchue, N., Suttikul S., Panphan V., Pripanpong P., and Burapatana V The Effect of High Solid Loading and Sulfuric Acid Pretreatment Following Commercial Cellulose Saccharification in 5L Fermenter on Reducing Sugar Concentration for Cellulosic Ethanol Production. In Proceedingsof the 24th Annual Meeting of the Thai Society for Biotechnology Renewable Energy and Global Care ; Nov , 2012, Ubon Ratchatani, Thailand, P-V-94. [15] Miller, G. L Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal. Chem. 3: [16] Mussatto, S. I, and Roberto, I. C Alternatives for Detoxi- Fication of Dilute-Acid Lignocellulosic Hydrolysate for Use in Fermentative Process. A Review. Bioresource Technol. 93: [17] Kamal, S. M. M., Mohamad, N. L., Abdullah, A. G. L., and Abdullah, N Detoxification of Sago Trunghy drolysate Using Activated Charcoal for Xylitol Production. Procedia Food Sci 1: [18] Mussatto, S. I., Santos, J. C., and Roberto, I. C Effect of ph and Activated Charcoal Adsorption on Hemicellulosichy Drolysate Detoxification for Xylitol Production. J. Chem. Technol. Biotechnol 79: [19] Rouhollah, H., Irag, N., Giti, E., and Sorah, A Mixed Sugar Fermentation by Pichiastipitis, Saccharomycescerevisiae, and Isolated Xylose-Fermenting Klyvero Mycesmarxianus and Their Concultures. Afr. J. Biotechnol. 6 (9): [20] Chandel, A. K., Chandrasekhar, G., Radhika, K., Ravinder, R., and Ravinda, P Bioconversion of Pentose Sugars into Ethanol. A Review and Future Directions. Biotechnol Mol. Biol. Rev. 6 (1):