酚化合物為許多工廠會使用到的原料之一，其排放的廢水中含有高濃度酚，具有高毒性及難分解性，可用物理、化學及生物等方式處理。但物理化學方法成本高，容易產生二次污染物，以生物處理最具有經濟性，更無有害副產物產生。而現今的污水處理方式也以生物處理中的活性污泥法為主，但在廢污水處理過程中會產生大量CO2、且國內污泥產量日益增加而最終處置場地有限。若以微藻做為處理方式，其具有處理污水中碳、氮、磷的去除以及降解廢水中有機污染物的能力，處理後的微藻更可作為燃料等其他用途。
本研究將小球藻於含酚培養基中進行培養，酚濃度為0~1000 mg/L，觀察藻體生長情形以及酚濃度變化，進而探討小球藻對酚的耐受程度、降解能力以及利用GC/MS檢測酚降解後所產生的中間產物，再利用生長動力學模式探討小球藻之比生長速率與基質間之關係。 
研究結果發現，在酚濃度200 mg/L時可使小球藻達到最高比生長速率，而600 mg/L以上則開始對小球藻產生明顯抑制現象，當濃度為1000 mg/L時小球藻生物質量完全沒有增加，同時酚也幾乎沒有減少，而酚濃度100~300 mg/L可在48 ~ 84小時內完全去除，同時總有機碳去除率也有96~100 %。由GC/MS檢測結果顯示，推測酚經由間位裂解後進入三羧酸循環中。本研究利用Monod動力學模擬在100~400以及600~1000 mg/L時分為低濃度及高濃度兩段行計算，其R2值分別為0.8435及0.638，顯示在高濃度基質下會抑制小球藻生長，不適合使用Monod模式；而利用Haldane模式求得之R2值則分別為0.9527及0.9892，明顯優於Monod模式，較適合於本研究中模擬使用。

Phenolic compounds are commonly used in many industries, which discharge into wastewater with the characteristics of toxic and low biodegradability. Physical or chemical treatments are costly and easily generate secondary pollutants, while biological methods produce large amounts of waste sludge, which cost much during the final disposal. Microalgae have been proven to have the ability to remove the organic carbon, nitrogen, and phosphorous in the wastewater, and produce bioenergy simultaneously, nevertheless, the degradation ability and phenomenon on the phenolic compounds still not clear. 
In this study, chlorella sp. was cultured in up to 1000 mg phenol /L contained culture media; subsequently, the growth rates of chlorella and residual phenol concentration were examined, and the extent of chlorella toleration was investigated,  GC/MS analysis was executed to exam  the intermediate products during the phenolic degradation. Finally, growth kinetic model was simulated to investigate the growth rate of chlorella. 
The results indicated that 200 mg phenol /L initial concentration has the maximum chlorella growth rate, and 100–300 mg/L of phenol can be removed completely within 48–84 hours, the removal rate of total organic carbon was 96%–100%. Whereas exceeded 600 mg phenol/L, the growth of chlorella was inhibited, and at  1000 mg phenol /L of initial concentration, the growth of chlorella biomass and  phenol degradation were stope. On the basis of the GC/MS results, phenol is inferred to have entered the tricarboxylic acid cycle following the meta-cleavage process. The simulation with Monod equation at 100–400 and 600–1000 mg phenol/L, the R2 were 0.84 and 0.64 respectively, it showed that, high concentration of phenol inhibits chlorella growth so the Monod equation was not applicable in this study. In contrast, the Haldane equation yielded R2 of 0.95 and 0.99 respectively, indicating that this equation was superior to the Monod equation and thus it can be applied in the simulation of this study.

目錄
第一章	研究背景與目的	1
1-1	研究背景	1
1-2	研究目的	2
第二章	文獻回顧	3
2-1	藻類特性	3
2-2	小球藻(Chella sp.)	3
2-3	微藻培養方式	4
2-4	光合作用	8
2-5	生長曲線與測定	11
2-6	影響微藻生長因子	13
2-6-1 光照強度和週期影響	13
2-6-2 溫度的影響	14
2-6-3  pH值的影響	14
2-6-4 碳源的影響	15
2-6-5 氮源的影響	15
2-6-6 磷源的影響	16
2-7	酚類化合物	17
2-7-1.	背景來源	17
2-7-2.	毒性	18
2-7-3.	處理技術	19
2-7-4.	代謝途徑	25
2-8	微生物動力學模式	28
2-8-1.	基質生長動力學模式	28
2-8-2.	基質抑制動力學模式	30
第三章	研究方法	32
3-1	實驗流程	32
3-2	實驗材料	35
3-2-1	藻種來源及培養基成分	35
3-2-2	酚	35
3-3	實驗方法	36
3-3-1	小球藻前期培養	36
3-3-2	酚濃度空白試驗	37
3-3-3	小球藻對酚之耐受程度	38
3-3-4	小球藻細胞表面吸附酚含量	39
3-3-5	小球藻對酚之降解產物	40
3-4	分析方法	41
3-4-1	藻體生長濃度(OD660)測定	41
3-4-2	藻體生物質量的測定(biomass)	41
3-4-3	總有機碳濃度測定	41
3-4-4	酚濃度測定	41
3-4-5	有機化合物定性分析	41
3-4-6	藻體比生長速率計算	42
3-4-7	生長動力學式計算	43
3-5	實驗儀器與設備	44
第四章	結果與討論	45
4-1	小球藻對酚之耐受程度	45
4-1-1	酚濃度空白試驗	45
4-1-2	小球藻生物質量與酚濃度變化	47
4-1-3	總有機碳濃度變化	53
4-2	小球藻細胞表面吸附酚之能力	56
4-3	小球藻對酚之降解產物	58
4-4	小球藻生長動力學探討	62
第五章	結論與建議	67
5-1	結論	67
5-2	建議	68
參考文獻	69

 
圖目錄
圖 2- 1Chlorella sp.圖片	4
圖 2- 2光合作用光反應流程圖	9
圖 2- 3光合作用暗反應流程圖	10
圖 2- 4綠藻之生長曲線	12
圖 2- 5酚結構式	17
圖 2- 6酚降解途徑	27
圖 2- 7基質濃度與比生長速率圖	29
圖 3- 1實驗流程圖	32
圖 4- 1空白實驗之培養基中含酚濃度變化	46
圖 4- 2小球藻於酚濃度0~1000 mg/L之生長質量濃度變化	48
圖 4- 3不同初始酚濃度在培養期間其濃度變化	50
圖 4- 4小球藻降解酚之速率	50
圖 4- 5不同酚濃度與生物質量變化比較	52
圖 4- 6不同初始酚濃度培養之總有機碳濃度	54
圖 4- 7總有機碳濃度去除率	54
圖 4- 8小球藻降解酚之中間產物GC/MS檢測結果	60
圖 4- 9  3-Methylcatechol MS圖譜	60
圖 4- 10 Monod生長動力模式	64
圖 4- 11 Haland生長動力模式	65
圖 4- 12降解酚之實驗值與模式預估比較圖	66


 
	
表目錄
表 2- 1光合作用比較表	11
表 2- 2生物降解酚化合物之效率與操作條件	24
表 2- 3基質抑制模式	30
表 3- 1培養基成分表	35
表 3- 2藻類培養設備	44
表 3- 3分析儀器與設備	44
表4- 1 小球藻植種前後培養基中含酚濃度	57
表4- 2 MS檢測結果之化合物	61

 

參考文獻
A.C. Sofia, Lima, Paula M.L. Castro Rui Morais (2003). "Biodegradation of p-nitrophenol by microalgae." Journal of Applied Phycology 15: 2137-142.
Agarry, S. E., Durojaiye, A. O., and Solomon, B. O. (2008) "Microbial degradation of phenols: A review. " Int. J. Environ. Pollut. 32:12–28.
Bared Razika. Boucheta Abbes. Chaib Messaoud. Kacimi soufi.(2010). "Phenol and Benzoic Acid Degradation by Pseudomomons aeruginosa ." Journal of Water Resource and Protection 2: 799-791.
Barker E.L., Peter E. B., Petrecia H. F., Grant S. K.(1978)."Phenol poisoning due to contaminated drinking water. "Arch. Environ. Health 33:89-94
Bajaj, M., Gallert, C., and Winter, J. (2008) "Biodegradation of high phenol containing synthetic wastewater by an aerobic fixed bed reactor. " Bioresour.Technol Vol. 99: 8376–8381
Becker, E. W. (1993). "Microalgae: biotechnology and microbiology, Cambridge University Press. " Bioengineering 81, 470-472. 
Bhaskar, Das. Tapas, K. Mandal, Sanjukta, Patra. (2015). "A Comprehensive Study on Chlorella pyrenoidosa for Phenol Degradation and its Potential Applicability as Biodiesel Feedstock and Animal Feed." Applied Biochemistry and Biotechnology 176 (5):1382-401
Bridle, T. R., and A. K. Sachdev. (1984). " Thermal destruction of Camiro-Vargas, T. K., Hernández-Ayón, J. M., Valenzuela-Espinoza, E., Delgadillo-Hinojosa, F., Cajal-Medrano, R. (2005). "Dissolved inorganic carbon uptake by Rhodomonas sp. and Isochrysis aff. galbana determined by a potentiometric technique." Aquacultural engineering 33(2): 83-95.
Camiro-Vargas, T. K.,Hernández-Ayón, J. M.,Valenzuela-Espinoza, E.,Delgadillo-Hinojosa, F. and Cajal-Medrano, R. (2005). "Dissolved inorganic carbon uptake by Rhodomonas sp. and Isochrysis aff. galbana determined by a potentiometric technique." Aquacultural engineering 33(2): 83-95.
Chen, F. (1996). "High cell density culture of microalgae in heterotrophic growth." Trends in biotechnology 14(11): 421-426.
Chisti, Y. (2007). "Biodiesel from microalgae." Biotechnology advances 25(3): 294-306.
Christen, P., Vega, A., Casalot, L., Simon, G. and Auria. R., (2012)"Kinetics of aerobic phenol biodegradation by the acidophilic andhyperthermophilic archaeon Sulfolobus solfataricus 98/2." Biochemical Engineering Journal62:56-61 
Chojnacka, K., Noworyta, A. (2004). "Evaluation of Spirulina sp. growth in photoautotrophic, heterotrophic and mixotrophic cultures." Enzyme and Microbial Technology 34(5): 461-465.
Conway, H. (1977). "Interactions of inorganic nitrogen in the uptake and assimilation by marine phytoplankton." Marine biology 39(3): 221-232.
De la Noue, J.De Pauw, N. (1988). "The potential of microalgal biotechnology: A review of production and uses of microalgae." Biotechnology advances 6(4): 725-770.
Edwards, V.H., (1970) " The influence of high substrate concentration on microbial kinetics. " Biotechnol. Bioeng. 12: 679–712.
El-Naas, M., Al-Zuhair, S., and Makhlouf, S. (2010)."Batch degradation of phenol in a spouted bed bioreactor system." J. Ind. Eng. Chem 16:267–272.
Fujita, K. (1959). "The metabolism of acetate in Chlorella cells." Journal of Biochemistry 46(3): 253-268.
Gabriele Pinto, Antonino Pollio, Lucio Previtera and Fabio Temussi (2002). "Biodegradation of phenols by microalgae. " Biotechnology Letters 24:2047-2051.
Gao, K. Yu, A. (2000). "Influence of CO2, light and watering on growth of Nostoc flagelliforme mats." Journal of Applied Phycology 12(2): 185-189.
Gao, Q.T., Wong, Y.S., Tam, N.F.Y. (2011). "Removal and biodegradation of nonylphenol by immobilized Chlorella vulgaris." Bioresource Technology 102: 10230-10238.
Gennaro P.D., Rescalli E., Galli E.(2001) "Characterization of Rhodococcus opacus R7, a strain able to degrade naphthalene and oxylene isolated from a polycyclic aromatic hydrocarbon contaminated soil . " Research Microbiol. 152(7):641-651
Gogate, P.R., and Pandit, A., (2004) "A review of imperative technologies for wastewater treatment I: oxidation technologies at ambient conditions. " Advances in Environmental ResearchVol. 8:501-551 
Gurujeyalekshmi, G., Oreil, P. (1988). "Isolation of phenol degrading Bacillus stearothermophilus and partial characterization of the phenol hydroxylase. " Environ. Microbiol 55(2): 500-502.
H. M. Ehrhardt and H. J. Reh,(1985) "Phenol degradation by microorganisms adsorbed on activated carbon. " Appl.Microbial Biotechnol 21:32～36.
H. Bettmann and H. J. Rehm, (1984). "Degradation of phenol by polymer entrapped microorganisms." Appl Microbiol Biotechnology 20: 285-290.
Hill, J., Nelson, E., Tilman, D., Polasky, S. and Tiffany, D. (2006). "Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels." Proceedings of the National Academy of Sciences of the United States of America 103(30): 11206-11210.
Hill, G.A., Robinson, C.W. (1975). "Substrate inhibition kinetics: phenol     degradation by Pseudomonas putida. " Biotechnol Bioeng 17:1599–1615
Hirata, S., Hayashitani, M., Taya, M., Tone S.(1996). "Carbon dioxide
Hu, Q. (2004). "5 Environmental Effects on Cell Composition." Handbook of microalgal culture: Biotechnology and applied Phycology: 83.
Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M. and Darzins, A. (2008). "Microalgal triacylglycerols as feedstocks for biofuel production: Perspectives and advances." Plant Journal 54(4): 621-639.
Huertas, E., Montero, O., Lubián, L. M. (2000). "Effects of dissolved inorganic carbon availability on growth, nutrient uptake and chlorophyll fluorescence of two species of marine microalgae." Aquacultural engineering 22(3): 181-197.
Hulzebos, E.M., Adema, D.M.M., Dirven-van Breemen, E.M., Henzen, L. van Dis, W.A., Herbold, H.A., Hoekstra, J.A., Baerselman, R., van Gestel, C.A.M., (1993). "Phytotoxicity studies with Lactuca sativa in soil and nutrient solution Environ." Environmental Toxicology and Chemistry 12:1079–1094
Jácome-Pilcoa, C.R., Cristiani-Urbinab, E., Flores-Coteraa, L.B., Velasco-Garcíac, R., Ponce-Noyolaa, T., and Cañizares-Villanuevaa., R.O., (2009)"Continuous Cr(VI) removal by Scenedesmus incrassatulus in an airlift photobioreactor" Bioresource Technology100:2388-2391 
Kahru, A., Reiman, R., and Ratsep, A., (1998)"The efficiency of different phenol-degrading bacteria, and activatd sludges in detoxification of phenolic leachates. "ChemosphereVol. 37:301–318 
Khazi Mahammedilyas Basha, Aravindan Rajendran, Viruthagiri Thangavelu (2010). "Recnt advances in the Biodegradation of henol: Areview" Society of Applied Sciences 1(2)；219-234.
Komarkova, E., Klapkova, J. P., Stiborova, M., and Soccol, C. R. (2003). "Physiological changes of Candida tropicalis population degrading phenol in fed batch reactor. " Braz. Arch. Biol. Technol 46:537–543.
Kumar, A., Kumar, S., and Kumar, S. (2005). "Biodegradation kinetics of phenol and catechol using Pseudomonas putida MTCC 1194. " Biochem. Eng. J 22:151–159.
Kumaran, P., Paruchuri, Y.L.(1997). "Kinetics of phenol biotransformation. "Water ResearchVolume 31(1): 11-22
Lee, K., and C.G. Lee.(2001). "Effect of light/dark cycles on wasterwater treatments by microalgae."Biotechnology and Bioprocess Engineering 6(3):194-199
Li, Y., Horsman, M., Wang, B., Wu, N. and Lan, C. Q. (2008). "Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans." Applied Microbiology and Biotechnology 81(4): 629-636.
Li, Y., Li, J., Wang, C., and Wang. P., (2010)"Growth kinetics and phenol biodegradation of psychrotrophic Pseudomonas putida LY1" Bioresource Technology(101)6740-6744.
Lika, K., and Papadakis, I. A. (2009). Modeling the biodegradation of phenoliccompounds by microalgae. J. Sea Res. 62, 135–146.
Liu, W., Au, D. W. T., Anderson, D. M., Lam, P. K. S. and Wu, R. S. S. (2007). "Effects of nutrients, salinity, pH and light:dark cycle on the production of reactive oxygen species in the alga Chattonella marina." Journal of experimental marine biology and ecology 346(1-2): 76-86.
Liu, Y. J., Zhang, A. N., and Wang, X. C. (2009)."Biodegradation of phenol by using free and immobilized cells of Acinetobacter sp. XA05 and Sphingomonas sp. FG03." Biochem. Eng. J 44:187–192.
Lukáš Jelínek, Gita Procházková, Cristina Quintelas, Eliška Beldíková, Tomáš Brányik.(2015)."Chlorella vulgaris biomass enriched by biosorption of polyphenols. "Algal Research10：1–7
Minowa, T., Yokoyama, S. y., Kishimoto, M. and Okakura, T. (1995). "Oil production from algal cells of Dunaliella tertiolecta by direct thermochemical liquefaction." Fuel 74(12): 1735-1738.
Minii Bajaj, Claudia Gallert, and Josef Winter. (2006). "Phenol degradation kinetics of an aerobic mixed culture." Biochemical Engineering Journal 46: 205-209.
Nagase, H., Yoshihaea, Kl., Eguchi, K., Hirata, K., Matsui, R., and Hirata, K., (1997)"Characteristics of biological NOx removal form flue gas in a Dunaliella tertiolecta culture system." Journal of Fermentation and Bioengineering 83: 461-465
Nuhoglu, A., and Yalcin, B. (2005). "Modelling of phenol removal in a batch reactor. " Process Biochem 40: 1233–1239.
Ohlrogge J., Browse J.( 1995) " Lipid biosynthesis" Plant Cell 7: 957-970.
Oliver J. Hao, Michael H. Kim, Eric A. Seagren , Hyunook Kim (2002) "Kinetics of phenol and chlorophenol utilization by Acinetobacter species. " Chemosphere 31:11-22.
P. Kumaran and Y. L. Paruchuri (1997) "Kinetics of phenol biotransformation. " Pergamon 96:797-807.
Perez-Garcia, O., Escalante, F. M. E., De-Bashan, L. E. and Bashan, Y. (2011). "Heterotrophic cultures of microalgae: Metabolism and potential products." Water Research 45(1): 11-36.
Pierre Christen, Armando Vega, Laurence Casalot , Gwenola Simon, and Richard Auria. (2012). " Kinetics of aerobic phenol biodegradation by the acidophilic andhyperthermophilic archaeon Sulfolobus solfataricus 98/2." Biochemical Engineering Journal 62: 56-61.
Pulz, O. (2001). "Photobioreactors: production systems for phototrophic microorganisms." Applied Microbiology and Biotechnology 57(3): 287-293.
Rai, L. C.,Gaur, J. P.Kumar, H. D. (1981). "Phycology and heavy-metal pollution." Biological Reviews of the Cambridge Philosophical Society 56(2): 99-151.
Sceagg, A.H., (2006). " The effect of phenol on the growth of Chlorella vulgaris and Chlorella VT-1" Enzyme and Microbial Technology 39: 796-799.
Sceagg, A.H., Spiller, L., Morrison, J., (2003). " The effect of 2,4-dichlorophenol on the microalga Chlorella VT-1" Enzyme and Microbial Technology 32: 616-622.
Schafer, T.E. Lapp, C.A. Hanes, C.M. Lewis, J.B. Wataha, J.C. Schuster G.S. (1999) "Estrogenicity of bisphenol A and bisphenol A dimethacrylate in vitro." Journal of Biomedical Materials Research 45:192–197
Semple, K.T., Cain, R.B., Schmidt, S., (1999). " Biodegradation of aromatic compounds by microalgae" FEMS Microbiology Letters 170: 291-300.
Semple, K. T. (1997). "Biodegradation of phenols by a eukaryotic alga. " Res. Microbiol 148:365–367.
Shi, X. M., Liu, H. J., Zhang, X. W. and Chen, F. (1999). "Production of biomass and lutein by Chlorella protothecoides at various glucose concentrations in heterotrophic cultures." Process Biochemistry 34(4): 341-347.
Spolaore, P.,Joannis-Cassan, C.,Duran, E. and Isambert, A. (2006). "Commercial applications of microalgae." Journal of Bioscience and Bioengineering 101(2): 87-96.
Staples, C.A., Dorn, P.B., Klecka, G.M., O'Block, S.T., Harris, L.R., (1998) "A review of the environmental fate, effects, and exposures of bisphenol A" Chemosphere 36: 2149–2173
Stoilova, I., Krastanov, A., Stanchev, V., Daniel, D., Gerginova, M., Alexieva,Z. (2006). "Biodegradation of high amounts of phenol, catechol, 2,4- dichlorophenol and 2,6-dimethoxyphenol by Aspergillus awamori cells. " Enzyme Microb. Technol 39:1036–1041.
Sukenik, A., Carmeli, Y.Berner, T. (1989). "Regulation of fatty acid composition by irradiance level in the eustigmatophyte Nannochloropsis sp. 1." Journal of Phycology 25(4): 686-692.
Tanaka, T, Yamada, K, Tonosaki, T, Konishi, T, Goto, H, Taniguchi, M. (2000). "Enzymatic degradation of alkylphenols, bisphenol A, synthetic estrogen and phthalic ester. " Water Sci. Technol 42:89-95
Taghreed Al-Khalid, and Muftah H. El-Naas,. (2012) "Aerobic Biodegradation of Phenols:A Comprehensive Review." Environmental Science and Technology Vol.42:1631–1690
Terry, K.L., Raymond, L. P. (1985). "System design for the autotrophic production of microalgae." Enzyme and Microbial Technology 7(10): 474-487.
V. Arutchelvan, V. Kanakasabai,R. Elangovan, S. Nagarajan, V.Muralikrishnan (2006). "Kinetics of high strength phenol degradation using Bacillus brevis." Journal of Hazardous Materials 129: 216-222.
Van der Heide, T.,Roijackers, R. M.,Van Nes, E. H. and Peeters, E. T. (2006). "A simple equation for describing the temperature dependent growth of free-floating macrophytes." Aquatic Botany 84(2): 171-175.
Verma, N. M., Mehrotra, S.,Shukla, A. and Mishra, B. N. (2010). "Prospective of biodiesel production utilizing microalgae as the cell factories: A comprehensive discussion."African Journal of Biotechnology 9(10): 1402-1411.
Wang, Y., Tian, Y., Han, B., Zhaw, H. B., Bi, J. N., and Cai, B. L. (2007). "Biodegradation of phenol by free and immobilized Acinetobacter sp. Strain PD12." J. Environ. Sci 19:222–225.
Yang, R.D., Humphry, A.E. (1975). "Dynamic steady state studies of phenol biodegradation in pure and mixed cultures."Biotech. Bioengg 17: 1211–1235
Yang, C.,Hua, Q.Shimizu, K. (2000). "Energetics and carbon metabolism during growth of microalgal cells under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions." Biochemical Engineering Journal 6(2): 87-102.
藍大鈞(2001) “藻類固定二氧化碳與藻體的利用研究”長庚大學化學工程研究所碩士論文。
王藝蓉(2003) “以光生化反應器培養微藻生產脂肪酸”大葉大學       食品工學系碩士論文。
潘忠政(2001) “整合鹼液吸收及光合作用以固定二氧化碳”大葉大學環境工程研究所碩士論文。
陳國誠、詹益亮(1990) “含酚廢水的生物處理法”科學月刊，0246 期。