臺灣博碩士論文加值系統

本研究旨在探討使用微生物降解羽毛粉取代動物飼糧中之高價動物性蛋白質－魚粉之可行性。試驗主要分為兩大部分，分別為發酵羽毛粉的製備與發酵羽毛粉之動物試驗。發酵羽毛粉製備部分，以米麴菌及枯草芽孢桿菌做為發酵菌株，試驗使用10 g 羽毛分別以不同之發酵時間、培養溫度、接種菌量、溶液 pH 值與含水量進行初步發酵條件測試，之後添加不同碳氮源以及其他營養成分，以刺激微生物的生長及其角蛋白酶活性之提升。爾後，再進行發酵條件優化試驗，以初步結果得知其發酵時間及含水量對於酵素活性較有影響，且羽毛粉加工法部分亦會先使用高溫高壓部分破壞後再進行處理，因此增加其發酵時間、含水量與增加其滅菌時間進行試驗。其結果顯示米麴菌之最適發酵條件為：發酵時間 10 日、含水量 350％、溶液 pH 8、接種菌量 1 mL/10 g 羽毛、培養溫度為 37℃ 與滅菌時間為 40 分鐘；枯草芽孢桿菌之最適發酵條件為：發酵時間 10 日、含水量 350％、溶液 pH 8、接種菌量 2 mL/10 g 羽毛、培養溫度為 37℃ 與滅菌時間為30分鐘，兩者均可使處理後的羽毛粉之胃蛋白酶消化率達到 60％ 以上。第二部分之動物試驗，以土番鴨為試驗動物，並進行飼糧中魚粉 0％、25％、50％、75％ 及 100％之取代。其結果顯示，枯草芽孢桿菌發酵羽毛粉可完全取代飼糧中之魚粉，其生長性能及血液性狀無不良影響。

This study was conducted to investigate the fermented feather meal to replace the high price feed Ingredient of animal diets - fish meal . The experiment is divided into two parts, the manufacture of fermented feather meal and the animal experiments of using fermented feather meal respectively. Feather meal was fermented by Aspergillus oryzae and Bacillus subtilis as fermentation microorganisms. There was 10 g of feather meal to test using different fermentation period, temperature, inoculums volume of microorganism, pH of the solution and moisture percent in initial fermentation conditions test. After that, adding different carbon and nitrogen sources and other nutrient source to stimulate the microorganism growth and enhance the activity of the microbial keratinase. According to the results, the incubate period and the moisture are most effective of enzyme activity of the feather meal fermentation. And there was some feather processing going to use high temperature high pressure to be treated in the beginning. So we increase the incubate period, moisture content and sterilizing time for testing. The results show that the optimum fermentation conditions of Aspergillus oryzae was: incubate period of 10 days, the moisture content of 350%, the solution pH 8, inoculums volume of microorganism 1 mL/10 g feather meal, the culture temperature is 37℃ and sterilized for 40 minutes. The optimum fermentation conditions of Bacillus subtilis was : incubate period of 10 days, the moisture content of 350%, the solution pH 8, inoculums volume of microorganism 2 mL/10 g feather meal, the culture temperature is 37℃ and sterilization time of 30 minutes. The pepsin digestibility of both two microorganism degraded feather meal are more than 60%. The second part of the test animals was chosen the mule duck as experimental animals. And using the Bacillus subtilis fermented feather meal to replace 0%, 25%, 50%, 75% and 100% fish meal of the diet. The results showed that Bacillus subtilis fermented feather meal can be completely replaced all fishmeal of the diet. There's no significantly adverse effect on the growth performance and parameters of blood characters of mule duck.

中文摘要 ..................................................................................................................................... I
英文摘要 ................................................................................................................................... II
謝誌 .......................................................................................................................................... IV
總目錄 ....................................................................................................................................... V
表目錄 .................................................................................................................................... VII
圖目錄 ................................................................................................................................... VIII
壹、前言 .................................................................................................................................... 1
貳、文獻探討 ............................................................................................................................ 2
一、羽毛的簡介 .................................................................................................................... 2
（一）家禽羽毛 ................................................................................................................. 2
（二）羽毛的分類與功能 ................................................................................................. 2
（三）羽毛的構造與顏色 ................................................................................................. 3
二、羽毛角蛋白的簡介 ........................................................................................................ 6
三、角蛋白酶的簡介 ............................................................................................................ 8
（一）角蛋白酶 ................................................................................................................. 8
（二）碳源、氮源及其它營養源的影響 ......................................................................... 8
（三）角蛋白酶對於角蛋白之作用 ................................................................................. 9
四、微生物的固態發酵 ........................................................................................................ 9
五、羽毛粉的加工方法 ...................................................................................................... 10
（一）高溫高壓水解法 ................................................................................................... 10
（二）酸、鹼化學水解法 ............................................................................................... 10
（三）膨化處理法 ........................................................................................................... 11
（四）酵素處理法 ........................................................................................................... 11
（五）微生物法 ............................................................................................................... 12
六、蛋白質飼料 .................................................................................................................. 13
七、羽毛粉於飼料之應用 .................................................................................................. 13
八、鴨隻安全之血液評估指標 .......................................................................................... 14
（一）鹼性磷酸酶 ........................................................................................................... 14
（二）丙胺酸轉胺酶 ....................................................................................................... 14
（三）天門冬胺酸轉胺酶 ............................................................................................... 14
（四）肌酸激酶 ............................................................................................................... 15
（五）乳酸脫氫酶 ........................................................................................................... 15
参、材料與方法 ...................................................................................................................... 16
一、試驗一：發酵條件、碳氮源及其他營養源對不同菌株發酵羽毛之探討 .............................................................................................................................................. 16
（一）試驗目的 ............................................................................................................... 16
（二）試驗方法 ............................................................................................................... 16
（三）各試驗測定項目之分析 ....................................................................................... 22
（四）統計分析 ............................................................................................................... 24
二、試驗二：發酵時間、含水量及滅菌時間對不同菌株發酵羽毛粉之影響 ........... 25
（一）試驗目的 ............................................................................................................... 25
（二）試驗方法 ............................................................................................................... 25
（三）統計分析 ............................................................................................................... 26
三、試驗三：發酵羽毛粉取代魚粉對土番鴨飼糧生長性能之影響 ............ 27
（一）試驗目的 ............................................................................................................... 27
（二）試驗動物及飼養管理 ........................................................................................... 27
（三）測定項目及分析 ................................................................................................... 28
（四）統計分析 ............................................................................................................... 28
肆、結果 .................................................................................................................................. 29
一、試驗一：發酵條件、碳氮源及其他營養源對不同菌株發酵羽毛之探討 .............................................................................................................................................. 29
（一）不同菌株發酵羽毛粉之最適化發酵條件結果 .................................... 29
（二）不同菌株發酵羽毛粉之最適碳、氮源及其他營養源影響 ............. 29
二、試驗二：發酵時間、含水量及滅菌時間對不同菌株發酵羽毛粉之影響 .............................................................................................................................................. 36
（一）發酵時間及含水量對不同菌株發酵羽毛粉之結果 ........................... 36
（二）滅菌時間對不同菌株發酵羽毛粉之結果.............................................. 36
三、試驗三：發酵羽毛粉取代魚粉對土番鴨飼糧生長性能之影響 ............ 40
（一）枯草芽孢桿菌發酵羽毛粉之營養成分 ............................................................. 40
（二）生長性狀 ............................................................................................................. 40
（三）血液性狀 ............................................................................................................. 40
伍、討論 .................................................................................................................................. 47
一、試驗一：發酵條件、碳氮源及其他營養源對不同菌株發酵羽毛粉之影響 .......................................................................................................................................... 47
二、試驗二：發酵時間、含水量及滅菌時間對不同菌株發酵羽毛粉之影響 .............................................................................................................................................. 49
三、試驗三：發酵羽毛粉取代魚粉對土番鴨飼糧生長性狀之影響 ............ 51
（一）發酵羽毛粉成分之影響 .............................................................................. 51
（二）發酵羽毛粉胺基酸之影響.......................................................................... 51
（三）生長性狀之影響 ............................................................................................ 52
陸、結論 .................................................................................................................................. 53
柒、參考文獻 .......................................................................................................................... 54

尹國強、崔英德、黎新明、賈振宇、廖列文。2005。鹼法水解羽毛蛋白的工藝條件研究。食品科學。26:347-349。
白火城、黃森源、林仁壽。1996。家畜臨床血液生化學。立宇出版社，台中。
沈添富。1988。鴨隻營養分需要量手册。行政院農委會。台北。
李青、葉滬生、張慧穎。1998。畜禽飼料的調制。中國農業出版社。北京。
杜連祥、路福平。2005。微生物學實驗技術。中國輕工業出版社。北京。
祁偉廉。2006。鳥羽：台灣野鳥羽毛圖鍵，商周出版社。台北。
林誠一、黃振芳、賴銘葵、林榮新、陳保基、魏恆巍。2005。土番鴨對國產羽毛粉利用效率之評估。畜產研究。38:125-135.
林韓希。2011。飼糧中添加商業酵素對土番鴨生長性能與能量利用之影響。碩士論文。國立宜蘭大學動物科技學系。
徐墨蓮、殷秋妙。1994。提高羽毛利用率的方法初探。飼料研究。5:21-23。
栗曉霞、高建新。2006。酶解羽毛粉對蛋雞生產性能的影響。科學試驗與研究。河北。
馬春祥。1980。家禽學，國立編譯館。台北。
高瑋。1995。鳥類分類學。中台科學技術出版社。台中。
高銘木、賴幸堯。1995。雞毛分解菌篩選之研究。中國環境工程學刊。5:37-43。
崔福齋、馮慶玲。2004。生物材料學。清華大學出版社。北京。
常家傳、馬金生、魯長虎。1995。鳥類學，中台科學技術出版社。台中。
張春生、陳凱祥、田仲源。2011。以固態培養 Aspergillus 屬真菌生產酸性蛋白酶之研究。南台學報。36：91-103
許元昆。2006。家禽電宰廠副產物（羽毛）之回收利用。飼料營養雜誌。66:65-68。
陳國誠。1999。微生物酵素工程學。再版。藝軒出版社，台北。
雷大德、陳瑞祥、陳國隆。2013。篩選羽毛分解菌進行二階段混合型發酵羽毛粉促進肉雞生長之探討。碩士論文。國立嘉義大學動物科學系。
管斌。2010。發酵實驗技術與方案。化學工業出版社。北京。
趙改名。2009。禽產品加工利用。化學工業出版社。北京。
劉海棲。2002。圖解鳥類的奧秘，晨曦出版社。台北。
鄭永祥、詹德芳。2002。細菌性羽毛發酵產物－羽毛溶成物做為飼料蛋白質的評估。飼料營養雜誌。10:10-17。
鄭裕國。2007。生物工程設備。化學工業出版社。北京。
賴智仁。2004。羽毛粉之加工與利用。畜牧半月刊。63:93-96。
謝明國。2003。以 Bacillus licheniformis THSC-1 進行雞毛與猪毛分解效果之研究。碩士論文。東海大學畜產學系。
Adler-Nissen J. 1986. Enzymic hydrolysis of food proteins. Elsevier Applied Science, New York.
Ai, Q., K. Mai, B. Tan, W. Xu, Q. Duan, H. Ma and L. Zhang. 2006. Replacement of fish meal by meat and bone meal in diets for large yellow croaker, Pseudosciaena crocea. Aquaculture, 260: pp. 255–263
Ali, T., N. H. Ali and L. A. Mohamed. 2011. Production, purification and some properties of Extracellular keratinase from feathers-degradation by Aspergillus oryzae NRRL-447. J. Appl. Sci. Environ. Sanit. 6:123-136.
Anbu, P., S. C. B. Gopinatha, A. Hildaa, T. Lakshmi priyab and G. Annaduraic. 2005. Purification of keratinase from poultry farm isolate—Scopulariopsis brevicaulis and statistical optimization of enzyme activity. Enzyme Microb. Technol. 36:639-647.
AOAC International. 2005. Official methods of analysis of AOAC International. AOAC International.
Apple, J. K., C. B. Boger, D. C. Brown, C. V. Maxwell, K. G. Froesen, W. J. Roberts and Z. B. Johnson. 2003. Effect of feather meal on live animal performance and carcass quality and composition of growing-finishing swine. J. Anim. Sci. 81:172-181.
Bagnara, J. T., J. Matsumoto, W. Ferris, S. K. Frost, W. A. Turner Jr., T.T. Tchen and J.D Taylor. 1979. Common origin of pigment cells. Sci. 203:410–415.
Bernal, C., J. Cairo and N. Coello. 2006. Purification and characterization of a novel exocellular keratinase from Kocuria rosea. Enzyme. Microb. Technol. 38:49-54.
Bertsch, A. and N. Coello. 2005. A biotechnological process for treatment and recycling poultry feathers as a feed ingredient. Bioresour. Technol. 96:1703-1780.
Bhargava, K. K., J. B. O’Neil. 1975. Composition and utilization of poultry by-product and hydrolyzed feather meal in broiler diets. Poult. Sci. 54:1511-1518.
Bishop C. D., R. A. Angus and S. A. Watts. 1995. The use of feather meal as a replacement for fish meal in the diet of Oreochromis niloticus fry. Bioresour. Technol. 54:291-295.
Bressollier, P., F. Letourneau, M. Urdaci and B. Verneuil. 1999. Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus. Appl. Environ. Microbiol. 65:2570–2576.
Clarkson, M. J. and T. G. Richards. 1971 The liver with special reference to bile formation. In : Bell DJ, Freeman BM, eds. Physiology and biochemistry of the domestic fowl. New York. Academic Press. 1085-114.
Cohlberg, J. A. 1993. The structure of α- keratin. Trends Biochem. Sci. 18:360-362.
Cruz-Suárez, L.E., M. Nieto-López, C. Guajardo-Barbosa, M. Tapia-Salazar, U. Scholz and D. Ricque-Marie. 2007. Replacement of fish meal with poultry by-product meal in practical diets for Litopenaeus vannamei, and digestibility of the tested ingredients and diets. Aquaculture. 272:466-476.
Das, K. and A. K. Mukherjee. 2007. Comparison of lipopeptide biosurfactants production by Bacillus subtilis strains in submerged and solid state fermentation systems using a cheap carbon source: Some industrial applications of biosurfactants. Pro. Biochem. 42:1191–1199.
Duitman, E. H., L. W. Hamoen, M. Rembold, G. Venema, H. Seitz, W. Saenger, F. Bernhard, R. Reinhardt, M. Schmidt, C. Ullrich, T. Stein, F. Leenders and J. Vater . 1999. The mycosubtilin synthetase of Bacillus subtilis ATCC 6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc. Natl. Acad. Sci. USA 96:13294-13299.
El-Boushy, A. R. and A. F. B. Van der Poel. 1990. Feather meal–a biological waste: its processing and utilization as a feedstuff for poultry. Biol. Wastes 32:39-74.
El-Refai, H. A., M. A. Abdel Naby, A. Gaballa, M. H. El-Araby and A. F. Abdel Fattah. 2005. Improvement of the newly isolated Bacillus pumilis FH9 keratinolytic activity. Process Biochem. 40:2325–2332.
Farag, A. M. and M. A. Hassan. 2004. Purification, characterization and immobilization of a keratinase from Aspergillus oryzae. Enzyme Microbiol. Technol. 34: 85-93.
Fargallo J. A., Velando A., López-Rull I., Gañán N., Lifshitz N., Wakamatsu K. and Torres R. 2014. Sex-specific phenotypic integration: endocrine profiles, coloration, and behavior in fledgling boobies. Behav. Ecol. 25:76–87.
Franson, J. C. 1982. Enzyme activities in plasma, liver and kidney of black ducks and mallards. J. Wildl. Dis. 18:481-485.
Garrett, R., H. and Grisham, C., M. 2002. Principles of biochemistry with a human focus. Harcourt, Inc. Unitede States of America p128-129.
Gradisar, H., J. Friedrich, I. Krizaj, R. Jerala. 2005. Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. Appl. Environ. Microbiol. 71:3420–3426.
Gradisar, H., Kern, S., Friedrich, J., 2000. Keratinase of Doratomyces microsporus. Appl. Microbiol. Biotechnol. 53:196–200.
Grazziotin, A., F. A. Pimentel, E. V. De Jong and A. Brandelli. 2006. Nutritional improvement of feather protein by treatment with microbial keratinase. Anim. Feed Sci. Technol. 126:135-144.
Gupta, R. and P. Ramnani. 2006. Microbial keratinases and their prospective applications: An overview. Appl. Microbiol. Biotechnol. 70:21-33.
Hartley B.S. 1960. Proteolytic enzymes. Annu. Rev. Biochem. 29:45–72.
Haque, A.K.M.A., J. J. Lyons and J. M. Vandepopuliere, 1991. Extrusion processing of broiler starter diets containing ground whole hens, poultry by-product meal, feather meal or ground feathers. Poul. Sci. 70:234-240.
Iovieno P. and E. Baath. 2008. Effect of drying and rewetting on bacterial growth rates in soils. Microb. Ecol. 65:400-407.
Karthikeyan, R., S. Balaji and P. K. Sehgal. 2007. Industrial applications of keratins–A review. J. Sci. Int. Res. 66:710-715.
Kim, J. M., W. J. Lim and H. J. Suh. 2001. Feather-degrading Bacillus species from poultry waste. Process Biochem. 37: 287-291.
Klein, C. and K. D. 1994. Entian. Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633. Appl. Environ. Microbiol. 60:2793–2801.
Kumar, R., S. Balaji, T. S. Uma, A. B. Mandal and P. K. Sehgal. 2010. Optimization of influential parameters for extracellular keratinase production by Bacillus subtilis (MTCC9102) in solid state fermentation using horn meal—A biowaste management. Appl. Biochem. Biotechnol. 160:30-39.
Kunert, J. 1989. Biochemical mechanism of keratin degradation by the actinomyces Streptomyces fradiae and the fungus Microsporum gypseum: a comparison. J. Basic Microbiol. 29:597-604.
Lehninger, A. L., D. L. Nelson and M. M. Cox. 1993. The three-dimensional structure of proteins. In: Principles of Biochemistry. 2nd ed. Worth Publishers, New York.
Lovell, R.T., 1989. Use of soybean products in diets for aquaculture species: revised. Proc. Soybean Utilization Alternatives. The Center for Alternative Crops and Products, Univ. Minnesota, Feb. 16–18, 1988, pp. 235–265.
Lumeij, J. T., and I. Westerhof. 1987. Blood chemistry for the diagnosis of hepatobiliary disease in birds. A review. The Vet. Q. 9:3.
Mathison, G. E. 1964. The microbiological decomposition of keratin. Ann. Soc. Belges. Med. Trop. Parasitol. Mycol. 44:767-791.
Mazotto, A. M. R. R. R. C., M. L. C. Sabrina, F. de L. Marcos, C. Sonia, P. de S. Edilma and B. V. Alane. 2011. Keratinase production by three Bacillus spp. using feather meal and whole feather as substrate in a submerged fermentation. Enzyme Res. 1–7.
Moritz, J. S. and J. D. Latshaw. 2001. Indicators of nutritional value of hydrolyzed feather meal. Poul. Sci. 80:79-86.
Morris W. C. and S. L. Balloun. 1973. Effect of processing methods on utilization of feather meal by broiler chick. Poul. Sci. 52:858-866.
Mousavi, S., M. Salouti, R. Shapoury and Z. Heidari. 2013. Optimization of keratinase production for feather degradation by Bacillus Subtilis. Jundishapur J. Microbiol. 6:e7160.
Mukherjee, A. K., H. Adhikari and S. K. Rai. 2008. Production of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindrical grass and potato peel as low-cost medium: characterization and application of enzyme in detergent formulation. Biochem. Eng. J. 39:353–361.
Nagal, S. and P. C. Jain. 2010. Feather degradation by strains of Bacillus isolation from decomposing feathers. Braz. J. Microbiol. 41:196-200.
NRC, National Research Council. 1984. Nutrient Requirements of Poultry. National Academy Press, Washington, D. C.
NRC, National Research Council. 1993. Nutrient Requirements of Fish. National Academy Press, Washington, D. C.
Pandey, A. 2003. Solid-state fermentation. Biochem. Eng. J. 13:81-84.
Papadopoulos, M. C., A. R. El Boushy and E. H. Ketelaars. 1984. Effects of different processing conditions on amino acid digestibility of feather meal determined by chick assay. Poult. Sci., 64:1729–1741
Papadopoulos M. C., A. R. El Boushy, A. E. Roodbeen and E. H. Ketelaars. 1986. Effects of processing time and moisture-content on amino-acid- composition and nitrogen characteristics of feather meal. Anim. Feed Sci. Technol. 14:279-90.
Parradoa J., B. Rodriguez-Morgadoa, M. Tejadab, T. Hernandezc and C. Garciaca. 2014. Proteomic analysis of enzyme production by Bacillus licheniformisusing different feather wastes as the sole fermentation media. Enzyme Microb. Technol. 57:1-7.
Patel, B. K. and M. V. Jagannadham. 2003. A High Cysteine Containing Thiol Proteinase from the Latex of Ervatamia heyneana: Purification and Comparison with Ervatamin B and C from Ervatamia coronaria. J. Agric. Food Chem. 51:6326-6334.
Rai, S. K., R. Konwarh and A. K. Mukherjee. 2009. Purification, characterization and biotechnological application of an alkaline β-keratinase produced by Bacillus subtilis RM-01 in solid-state fermentation using chicken-feather as substrate. Biochem. Eng. J. 45:218-225.
Reddy, N. and Y. Yang. 2007. Structure and Properties of Chicken Feather Barbs as Natural Protein Fibers. J. Polym. Environ. 15:81-87.
Riffel, A. and A. Brandelli. 2002. Isolation and characterization of a feather-degrading bacterium from the poultry processing industry. J. Ind. Microbiol. Biot. 29:255-258.
Robinson, T., D. and S. P. Nigam. 2001. Solid-state fermentation: a promising microbial technology for secondary metabolite production. Appl. Micro. Biotech. 55:284–289.
Rozs, M., L. Mamczimger, Cs. Vaguolgyi and F. Kevei. 2001. Secretion of a trypsin-like thiol protease by a new keratinolytic strain of Bacillus licheniformis. FEMS Microbio lett. 205:221-224.
Sandhya, C., A. Sumantha, G. Szakacs and A. Pandey. 2005. Comparative evaluation of neutral protease production by Aspergillus oryzae in submerged and solid-state fermentation. Process Biochem. 40:2689-2694.
Silva, L. A. D., A. J. Macedo and C. Termignoni. 2014. Production of keratinase by Bacillus subtilis S14. Annals of Microbio. 64 :1725–1733.
Steinert, P. M. 1993. Structure, function, and dynamics of keratin intermediate filaments. J. Invest. Dermatol. 100:729-734.
Subramaniyam, R. and R. Vimala. 2012. Solid state and submerged fermentation for the Production of bioactive substances: a comparative study. Int. J. Sci. 3:480-486
Sumantha, A., C. Larroche and A. Pandey. 2006. Microbiology and industrial biotechnology of food-grade protease: a perspective. Food Technol. Biotechnol. 44:211-220.
Syed, D. G., J. C. Lee, W. J. Li, C. J. Kim and D. Agasar. 2009. Production, characterization and application of keratinase from Streptomyces Gulbargensis. Bioresour. Technol. 100:1868-1871.
Tacon, A. G. J. 1998. Global trends in aquaculture production with particular reference to low income food deficit countries. FAO Technical Paper No. 12. Rome, Italy.
Takahashi, K., Yamamoto, H., Yoshiko, Y. and Hattori M. 2004. Thermal behavior of fowl feather keratin. Biosci. Biotechnol. Biochem. 68:1875-1881.
Wang, X. and C. M. Parsons. 1997. Effect of processing systems on protein quality of feather meals and hog hair meals. Poul. Sci. 76:491-496.
Wang, Y., J. Guo and D. P. Bureau. 2006. Replacement of fish meal with rendered animal ingredients in feeds for cuneate drum, Nibea miichthioides. Aquaculture. 252:476–483.
Williams1, C. M. , C. S. Richter, J. M. Mackenzie Jr. and Jason C. H. Shih. 1990. Isolation, Identification, and Characterization of a Feather-Degrading Bacterium. Appl. Environ. Microbiol. 56:1509-1515.
Zhang, X., 2011. Study on the optimum process condition of feather fermentation using Bacillus Subtilis mutation strains. Procedia Environ. Sci. 8, 661-668.