Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52108
標題: 本土木質纖維分解菌之篩選與其在經超音波前處理稻殼之發酵研究
Isolation of Lignocellulolytic Microorganisms from Soil and its Fermentation with Rice Hull by Ultrasonic Pretreatment
作者: 楊珺堯
Yang, Chun-Yao
關鍵字: Rice hull;稻殼;Ultrasonic pretreatment;Lignocellulose;Xylooligosaccharide;超音波前處理;木質纖維素;木寡醣
出版社: 食品暨應用生物科技學系所
引用: [1] AACC. 1983. Approved methods of the American Association of Cereal Chemists (8th ed.): AACC method 32-10. AACC. St. Paul, Minn. USA. [2] Alvira, P., E. Tomás-Pejó, M. Ballesteros, and M.J. Negro. 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review. Bioresource Technol. 101: 4851-4861. [3] Balata, M., H. Balata, and C. Őz. 2008. Progress in bioethanol processing. Prog. Energy Combust. Sci. 34: 551-573. [4] Béguin, P., and J.P. Aubert. 1994. The biological degradation of cellulose. Fems Microbiol. Rev. 13: 25-58. [5]Cann, I.O., S. Kocherginskaya, M.R. King, B.A. White, and R.I. Mackie. 1999. Molecular Cloning, Sequencing, and Expression of a Novel Multidomain Mannanase Gene from Thermoanaerobacterium polysaccharolyticum. J. Bacteriol. 181: 1643-1651. [6] Duff, S.J.B., W.D. Murray. 1996. Bioconversion of forest products industry waste cellulosics to fuel ethanol: A review. Bioresource Technol. 55: 1-33. [7] Ebringerová, A and Z. Hromádková. 1997. The effect of ultrasound on the structure and properties of the water-soluble corn hull heteroxylan. Ultrason. Sonochem. 4: 305-309. [8] Economou, C.N., A. Makri, G. Aggelis, S. Pavlou, and D.V. Vayenas. 2010. Semi-solid state fermentation of sweet sorghum for the biotechnological production of single cell oil. Bioresource Technol. 101: 1385-1388. [9] Fang, H.Y., S.M. Chang, C.H. Lan, and T.J. Fang. 2008. Purification and characterization of a xylanase from Aspergillus carneus M34 and its potential use in photoprotectant preparation. Process Biochem. 43: 49-55. [10] Fang, H.Y., S.M. Chang, M.C Hsieh, and T.J. Fang. 2007. Production, optimization growth conditions and properties of the xylanase from Aspergillus carneus M34. J. Mol. Catal. B-Enzym. 49: 36-42. [11] Gírio F.M., C. Fonseca, F. Carvalheiro, L.C. Duarte, S. Marques, and R. Bogel-Łukasik. 2010. Hemicelluloses for fuel ethanol: A review. Bioresource Technol. 101: 4775-4800. [12] Gullón, P., M.J. González-Muñoz, M.P. Van Gool, H.A. Schols, J.Hirsch, A. Ebringerová, and J.C. Parajó. 2010. Production, refining, structural characterization and fermentability of rice husk xylooligosaccharides. J Agric Food Chem. 58: 3632–3641. [13] Gullón, P., P. Moura, M.P. Esteves, F.M. Girio, H. Domínguez, and J.C. Parajó. 2008. Assessment on the fermentability of xylooligosaccharides from rice husks by probiotic bacteria. J Agric Food Chem. 56: 7482-7487. [14] Haltrich D., B. Nidetzky, K.D. Kulbe, W. Steiner, and S. Župančič. 1996. Production of fungal xylanases. Bioresource Technol. 58: 137-161. [15] Hayashi, N., T. Sakaki, and K. Doi. 2005. Water-soluble saccharides useful as health foods and their manufacture by hydrolysis of hemicellulose-containing plants with pressurized hot water. Japan Patent. JP 2005-023041. [16] Hendriks, A.T.W.M., and G. Zeeman. 2009. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresource Technol. 100: 10-18. [17] Higuchi, T. 1985. Biosynthesis and biodegradation of wood components. Academic Press, Inc., N.Y. [18] Hromádková, Z., and A. Ebringerová. 2003. Ultrasonic extraction of plant materials––investigation of hemicellulose release from buckwheat hulls. Ultrason. Sonochem. 10: 127-133. [19] Huang, G., J.X. Shi, and T.A.G. Langrish. 2007. NH4OH–KOH pulping mechanisms and kinetics of rice straw. Bioresource Technol. 98: 1218–1223. [20] Isogai, A., and R.H. Atalla. 1998. Dissolution of cellulose in aqueous NaOH solutions. Cellulose. 5: 309-319. [21] Jeya, M.,Y.W. Zhang, I.W. Kim, and J.K. Lee. 2009. Enhanced saccharification of alkali-treated rice straw by cellulase from Trametes hirsuta and statistical optimization of hydrolysis conditions by RSM. Bioresource Technol. 100: 5155–5161. [22] Juliano, B.O. 1985. Rice: chemistry and technology. American Association of Cereal Chemists, Inc. USA. [23] Kim, C.H. 1995. Characterization and substrate specificity of an Endo-β-1,4-D-Glucanase I (Avicelase I) from an extracellular multienzyme complex of Bacillus circulans. Appl. Environ. Microbiol. 61: 959-965. [24] Kim, T.H. and Y.Y. Lee, 2005. Pretreatment and fractionation of corn stover by ammonia recycle percolation process. Bioresource Technol. 96: 2007–2013. [25] Kim, T.H. and Y.Y. Lee. 2006. Fractionation of corn stover by hot-water and aqueous ammonia treatment. Bioresource Technol. 97: 224–232. [26] Kim, T.H., F. Taylor, and K.B. Hicks. 2008. Bioethanol production from barley hull using SAA (soaking in aqueous ammonia) pretreatment. Bioresource Technol. 99: 5694-5702. [27] Ko, J.K., J.S. Bak, M.W. Jung, H.J. Lee, I.G. Choi, T.H. Kim, and K.H. Kim. 2009. Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes. Bioresource Technol. 100: 4374–4380. [28] Kulkarni, N., A. Shendye, and M. Rao.1999. Molecular and biotechnological aspects of xylanases. Fems Microbiol. Rev. 23: 411-456. [29] Kumagai, S., N. Hayashi, T. Sakaki, M. Nakada, and M. Shibata. 2004. Fractionation and saccharification of cellulose and hemicellulose in rice hull by hotcompressed-water treatment with two-step heating. J Jpn Inst Energy. 83: 776-781. [30] Kumar, P., D.M. Barrett, M.J. Delwiche, and P. Stroeve. 2009. Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production. Ind. Eng. Chem. Res. 48: 3713-3729. [31] Lee, Y.J., B.K. Kim, B.H. Lee, K.I. Jo, N.K. Lee, C.H. Chung, Y.C. Lee, and J.W. Lee. 2008. Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresource Technol. 99: 378–386. [32] Luche, J.L. 1998. Synthetic organic sonochemistry, Plenum Press, New York. USA. [33] Ma, H., W.W. Liu, X. Chen, Y.J. Wu, and Z.L Yu. 2009. Enhanced enzymatic saccharification of rice straw by microwave pretreatment. Bioresource Technol. 100: 1279–1284. [34] Mäkeläinen, H., M. Juntunen and O. Hasselwander. 2009. Prebiotic Potential of xylo-oligosaccharides. Springer Science. [35] Manisseri, C., and M. Gudipati. 2010. Bioactive xylo-oligosaccharides from wheat bran soluble polysaccharides. LWT-Food Sci. Technol. 43: 421-430. [36] Miller, G.L. 1959. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal. Chem. 31: 426-428. [37] Mosier, N., C. Wyman, B. Dale, R. Elander, Y.Y. Lee, M. Holtzapple, and M. Ladisch. 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technol. 96: 673-686. [38] Moure, A., P. Gullón, H. Domínguez, and J.C. Parajo. 2006. Advances in the manufacture, purification and applications of xylo-oligosaccharides as food additives and nutraceuticals. Process Biochem. 41: 1913-1923. [39] Ohta, K., D.S. Beall, J.P. Mejia, K.T. Shanmugam, and L.O. Ingram. 1991. Genetic improvement of escherichia coli for ethanol production: chromosomal integration of zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase Ⅱ. Appl. Environ. Microbiol. 57: 893-900. [40] Parajó, J.C., G. Garrote, J.M. Cruz, and H. Domínguez. 2004. Production of xylooligosaccharides by autohydrolysis of lignocellulosic materials. Trends Food Sci Technol. 15: 115-120. [41] Persson, T., J.L. Ren, E. Joelsson, and A.S. Jönsson. 2009. Fractionation of wheat and barley straw to access high-molecular-mass hemicelluloses prior to ethanol production. Bioresource Technol. 100: 3905-3913. [42] Saha, B.C., L.B. Iten, M.A. Cotta, and Y.V. Wu. 2005. Dilute Acid Pretreatment, Enzymatic Saccharification, and Fermentation of Rice Hulls to Ethanol. Biotechnol. Prog. 21: 816-822. [43] Sasson, Y. and R. Neumann. 1997. Handbook of Phase Transfer Catalysis. Blackie Academic & Professional. London. [44] Selvendran, R.R. 1985. Developments in the chemistry and biochemistry of pectic and hemicellulose polymers. J. Cell. Sci. Suppl. 2: 51-88. [45] Shallom, D. and Y. Shoham. 2003. Microbial hemicellulases. Curr. Opin. Microbiol. 6: 219-228. [46] Shao, X., L. Lynd, C.Wyman, and A. Bakker. 2009. Kinetic modeling of cellulosic biomass to ethanol via simultaneous saccharification and fermentation: Part I. Accommodation of intermittent feeding and analysis of staged reactors. Biotechnol. Bioeng. 102: 59-65. [47] Shrestha, P., M. Rasmussen, S.K. Khanal, A.L. Pometto III, and J.H. Van Leeuwen. 2008. Solid-Substrate Fermentation of Corn Fiber by Phanerochaete chrysosporium and Subsequent Fermentation of Hydrolysate into Ethanol. J Agric Food Chem. 56: 3918–3924. [48] Sun, R.C., X.F. Sun, and X.H. Ma. 2002. Effect of ultrasound on the structural and physiochemical properties of organosolv soluble hemicelluloses from wheat straw. Ultrason. Sonochem. 9: 95-101. [49] Sun, Y. and J. Cheng. 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technol. 83: 1–11. [50] Suslick, K.S. and E. Britannica. 1994. The chemistry of ultrasound. Yearbook of Science& the Future, Chicago, 138-155. [51] Tuomela, M., M. Vikman, A. Hatakka, and M. Itävaara. 2000. Biodegradation of lignin in a compost environment: a review. Bioresource Technol. 72: 169-183. [52] Vadiveloo, J., B. Nurfariza, and J.G. Fadel. 2009. Nutritional improvement of rice husks. Anim. Feed Sci. Technol. 151: 299–305. [53] Van. Laere, K.M.J., R. Hartemink, M. Bosveld, H.A. Schols, and A.G.J. Voragen. 2000. Fermentation of plant cell wall derived polysaccharides and their corresponding oligosaccharides by intestinal bacteria. J Agric Food Chem. 48: 1644-1652. [54] Vázquez, M.J., J.L. Alonso, H. Domínguez and J.C. Parajó. 2000. Xylooligosaccharides: manufacture and applications. Trends Food Sci. Technol. 11: 387-393. [55] Vegas, R., A. Moure, H. Domínguez, J.C. Parajó, J.R. Álvarez, and S. Luque. 2006. Purification of oligosaccharides from rice husk autohydrolysis liquors by ultra- and nano-filtration. Desalination. 199: 541–543. [56] Vegas, R., J.L. Alonso, H. Domínguez, and J.C. Parajó. 2004. Processing of rice husk autohydrolysis liquors for obtaining food ingredients. J Agric Food Chem. 52: 7311-7317. [57] Vila, C., G. Garrote, H. Domínguez, and J.C. Parajó. 2002. Hydrolytic processing of rice husks in aqueous media: a kinetic assessment. Collect. Czech. Chem. Commun. 67: 509-530. [58] Vilkhu, K., R. Mawson, L. Simons, and D. Bates. 2008. Applications and opportunities for ultrasound assisted extraction in the food industry — A review. Innov Food Sci Emerg. 9: 161-169. [59] Wakiyama, M., K. Yoshihara, S. Hayashi, and K. Ohta. 2008. Purification and Properties of an Extracellular β-Xylosidase from Aspergillus japonicus and Sequence Analysis of the Encoding Gene. J. Biosci. Bioeng. 106: 398-404. [60] Wakiyama, M., K. Yoshihara, S. Hayashi, and K. Ohta. 2010. An extracellular endo-1,4-β-xylanase from Aspergillus japonicus: Purification, properties, and characterization of the encoding gene. J. Biosci. Bioeng. 109: 227-229. [61] Wikipedia, the free encyclopedia. 2007. Ultrasound. Available from: http://en.wikipedia.org/wiki/Ultrasound. Accessed 21 September 2009. [62] Yachmenev, V., B. Condon, T. Klasson, and A. Lambert. 2009. Acceleration of the enzymatic hydrolysis of corn stover and sugar cane bagasse celluloses by low intensity uniform ultrasound. J. Biobased Mater. Bioenergy. 3: 25–31. [63] Yang, F., L. Li , Q. Li, W. Tan, W. Liu, and M. Xian. 2010. Enhancement of enzymatic in situ saccharification of cellulose in aqueous-ionic liquid media by ultrasonic intensification. Carbohydr. Polym. 81: 311-316. [64] Yu, J., J. Zhang, J. He, Z. Liu, and Z. Yu. 2009. Combinations of mild physical or chemical pretreatment with biological pretreatment for enzymatic hydrolysis of rice hull. Bioresource Technol. 100: 903-908. [65] Zhang, L., Y. Mao, J. Zhou, and J. Cai. 2005. Effects of coagulation conditions on the properties of regenerated cellulose films prepared in NaOH/urea aqueous solution. Ind. Eng. Chem. Res. 44: 522-529. [66] Zhang, Z., and Z.K. Zhao. 2010. Microwave-assisted conversion of lignocellulosic biomass into furans in ionic liquid. Bioresource Technol. 101: 1111-1114. [67] 中華民國國家標準(CNS)。2009。食品中粗灰分之檢驗方法。經濟部標準檢驗局。台北市。 [68] 中華民國國家標準(CNS)。2009。食品中粗脂肪之檢驗方法。經濟部標準檢驗局。台北市。 [69] 中華民國國家標準(CNS)。2009。食品中粗蛋白之檢驗方法。經濟部標準檢驗局。台北市。 [70] 尤智立。2003。嗜高溫纖維分解菌纖維分解酵素的探討。國立中山大學生物科學研究所碩士論文。 [71] 田以正。2005。提升寡木醣生產效率及建立連續分離系統之研究。靜宜大學食品營養學系碩士論文。 [72] 行政院農委會農糧署。2007。稻穀收購數量與價格。96年農業統計年報。 [73] 施照輝。2003。白腐菌前處理對硫酸鹽紙漿漂白促進效果之研究。國立台灣大學森林學研究所博士論文。 [74] 胡俊榮。2002。玉米聚木醣分子性質對其聚木醣酶降解性及寡醣組成之影響。靜宜大學食品營養學系碩士論文。 [75] 郁儀豪。2003。蔗渣堆肥中嗜高溫菌之分離與應用。國立台灣大學環境工程學研究所碩士論文。 [76] 食品微生物之檢驗方法。2003。黴菌及酵母菌數之檢驗。署受食字第0929210167公告。行政院衛生署食品藥物管理局。台北市。 [77] 倪禮豐。2007。稻殼再利用技術。花蓮區農業專訊。61: 19-20。 [78] 張志豪。2000。耐高溫及中溫菌的分離及其纖維素分解酵素之研究。國立台灣大學農業化學系碩士論文。 [79] 張為憲、呂政義、張永和。1995。食品化學: 榖類。華香園出版社。台北市。 [80] 張為憲、林宏基、甘子能、陳昭義、蕭寧馨。2003。高等食品化學: 醣類。華香園出版社。台北市。 [81] 陳文恆、郭家倫、黃文松、王嘉寶。2007。纖維酒精技術之發展。農業生技產業季刊。9: 62-69。 [82] 陳維倫。2004。具分泌木聚醣酶漂前黑液菌種型態鑑定及生化特性分析。國立台灣大學森林學系碩士論文。 [83] 楊杰修。2009。Aspergillus carneus M34聚木糖酶特性分析、最適化固態發酵生產條件與其應用於木寡醣生產之探討。國立中興大學食品暨應用生物科技學系碩士論文。 [84] 葉丁源。1996。嗜高溫放線菌纖維素分解酵素之探討。國立台灣大學農業化學系碩士論文。 [85] 廖佳瑋。2000。Aspergillus carneus M34生產聚木糖酶最適化條件之探討。國立中興大學食品科學系碩士論文。 [86] 齊倍慶。2001。從堆肥中篩選纖維素分解酵素生產菌及其酵素性質研究。國立清華大學生命科學系碩士論文。 [87] 蕭淞云。2006。自稻桿與蔗渣生產木寡醣之研究。國立台灣大學微生物與生化研究所碩士論文。 [88] 謝松源。2007。進口食材污染真菌之檢測及鑑定。植物重要防疫檢疫病害診斷鑑定技術研習會專刊。6: 24-38。 [89] 蘇遠志、黃世佑。1975。微生物化學工程學。天然書社。台北市。
摘要: 
稻殼是台灣常見且大宗的農業廢棄物,由於其含有豐富的木質纖維素,可作為微生物的培養基質,因此在生物資源應用的領域上是有發展的必要性。本研究探討具木質纖維分解能力菌種之篩選與超音波放射技術在稻殼前處理上之應用,並測試分離菌株在不同培養時間與不同前處理之培養基質的酵素活性與發酵液產物之組成變化。

本研究自土樣中篩選出32株可利用稻殼木質纖維素之菌株,並利用不同酵素活性的測試挑選出活性較高之菌株CY6-1進行後續實驗,此分離菌株CY6-1的四種酵素活性(CMCase, β-glucosidase, Avicelase, Xylanase) 活性皆優於對照組Phanerochaete chrysosporium BCRC36201。此分離菌株CY6-1經鑑定結果歸屬於Aspergillus japonicus var. japonicus。

本研究藉由超音波放射的方式來作為稻殼前處理之步驟,不僅可萃取稻殼中可用之纖維素與半纖維素,也可利用超音波所產生的噴流與局部熱點來震碎及破壞附著於稻殼表面的二氧化矽,此前處理可將稻殼前處理的時間由24小時縮短至1.5小時外,並提升1.4倍之半纖維素產率。所使用的超音波前處理條件為300W、28 kHz於80℃作用1.5小時,並以場發射掃描式電子顯微鏡觀察未處理與經超音波處理之稻殼的微結構變化,發現到經過超音波處理之稻殼表面較未處理之表面較光滑,但若超音波加上12% NaOH處理之稻殼表面則會有皺縮的現象產生。

在酵素活性測試方面,發現在不同稻殼克數、不同超音波前處理方式與不同時間的培養下,酵素活性皆以4 g /50 mL比例之超音波處理的稻殼(USRHM)培養時最佳,不僅有助於延長酵素活性的穩定性,且更可有效地提升β-glucosidase之活性,於培養第28天後可達5.62 U/mL,而xylanase的活性則於培養第10天達到7.37 U/mL。

以稻殼為基質之發酵液在經過28天的培養,其發酵液之組成主要以木四糖、木六糖與一些分子量較大之低聚木糖,其木寡醣產率可以達到1.17 mg/ mL(木四糖與木六糖)。

Rice hull is a common and enormous amount of agricultural waste in Taiwan. It contains an abundant lignocellulosic biomass and can be used as a substrate for microorganism. Developing the methods to utilize rice hull is essential in the field of bio-resources. The aim of this thesis is to explore the methodology of utilizing rice hull by the isolation of cellulolytic and xylanolytic microorganism, pretreatment of rice hull with ultrasonic irradiation, testing the enzyme activities, and analyzing the composition of hydrolysis products of the fermentation broth with the isolate on different culture times for various pretreatment methods.

Microorganism being able to hydrolyze the lignocellulose of rice hull was isolated from soil, and the isolate CY6-1 was selected from 32 strains by enzyme activity tests. Those enzyme activities of the isolate CY6-1 were better than Phanerochaete chrysosporium BCRC36201, including CMCase, β-glucosidase, avicelase, and xylanase. The isolate CY6-1 is identified as Aspergillus japonicus var. japonicus.

Using ultrasonic irradiation as pretreatment, it has been employed to not only extract cellulose and hemicellulose from rice hull, but also remove silica on the surface via liquid jet and hot spots. The pretreatment can shorten the treated time from 24 hours to 1.5 hours, and increase 1.4 fold in yield of hemicellulose. In this work, rice hull was treated at 80℃ for 1.5 hours in 300W and 28 kHz ultrasonic system. And field emission scanning electron microscope (FESEM) was used to observe the structural change of the rice hull before and after the pretreatments. The structure of rice hull treated with ultrasound was smoother than untreated rice hull, and the structure was corroded when using ultrasonic pretreatment in combination with 12% NaOH.

For enzyme activity test, the highest enzyme activity of isolate CY6-1 was observed at using 4 g USRHM/50 mL in different grams of rice hull and culture times by various pretreatments. In this culture condition, it could not only extend the stability of enzyme activity, but also increase β-glucosidase activity better than in other culture conditions. After 28- day culture by isolate CY6-1, β-glucosidase activity could achieve 5.62 U/mL, and the highest xylanase activity was 7.37 U/mL after 10 days of culture.

The fermentation broth of rice hull was cultured with isolate CY6-1in 28 days, and compositions of the broth were xylotetraose, xylohexaose, and higher molecular weight xylooligosaccharide, and the yield of xylotetraose and xylohexaose were 1.17 mg/mL.
URI: http://hdl.handle.net/11455/52108
Appears in Collections:食品暨應用生物科技學系

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