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研究生：	童淑珠 Tung, Shu-Chu
論文名稱：	水源中2-MIB與geosmin鑑定與氧化之研究 Identification and oxidation of 2-MIB and geosmin in source water
指導教授：	林財富 Lin, Tsair-Fu
學位類別：	博士 Doctor
系所名稱：	工學院 - 環境工程學系 Department of Environmental Engineering
論文出版年：	2006
畢業學年度：	94
語文別：	英文
論文頁數：	146
中文關鍵詞：	藍綠細菌、放線菌、土霉味物質、前氧化處理
外文關鍵詞：	Actinomyces; geosmin; 2-methylisoborneol; Oscill
相關次數：	點閱：152 下載：2
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本研究主要探討南台灣地區重要的自來水水源澄清湖及鳳山水庫發生的土霉味問題，預期成果則為確認土霉味物質geosmin及2-MIB的產生源，並評估水廠前氧化處理對溶解態土霉味物質及胞內土霉味物質的影響。

本研究藉由澄清湖淨水廠及鳳山淨水廠原水中geosmin及2-MIB濃度的長期監測資料，進行分析，結果發現此兩種臭味物質的濃度變化趨勢與水溫及日均溫變動情形一致，因考量geosmin及2-MIB為水體中微生物的代謝產物，其產臭特性應受水體中的水質參數影響，又鳳山淨水廠一整年皆可監測到2-MIB的存在，濃度也都超過臭味的閾值，最高濃度出現在2003年的7月，濃度高達183 ng/L。而由鳳山淨水廠採回之原水，置於實驗室20C、25C及30C培養，培養8天後，其2-MIB濃度分別增加2.1、4.5及7.4倍。除了溫度影響2-MIB生成，本研究也發現在沒有營養鹽添加的情況下，原水培養之2-MIB的濃度可高達1000-2000 ng/L，兩次採樣及培養的結果發現在氨氮高時需氧化為硝酸鹽才能被產2-MIB的微生物所利用，因此微生物的生長及2-MIB的產生會有1-2天的遲滯期，且於2-MIB快速增長的生長期，僅有30%為溶解態，其他則存於細胞內。

研究中共分離出四株產臭的微生物，其一為產2-MIB藍綠細菌，以光學顯微鏡觀察判定為顫藻屬(Oscillatoria)，另外三株放線菌，以16S rRNA片段基因定序後比對NCBI的基因資料庫，分別判定為產geosmin及2-MIB的Streptomyces malaysiensis ，產geosmin的S. roseoflavus，及產2-MIB的S. caelestis。此四株微生物的產臭特性受培養溫度影響甚大，此與環境觀測結果相一致，進而可判斷南台灣兩大水源geosmin主要來自放線菌的代謝物，而2-MIB則來自藍綠細菌與放線菌的代謝物。

本研究發現不論是環境採樣分析或是純化微生物的培養都發現溫度是影響2-MIB生成量最顯著的因子，為了建立一簡單的預警模式，提供水廠採取適當的應變措施處理突發的臭味問題，本研究也收集來自台灣地區自來水七區管理處之鳳山淨水廠的原水監測資料含水溫共十項，並收集高雄氣象站監測的日均溫等四項參數，利用因數分析、主成分分析及逐步回歸分析等統計技術分析十四項環境因子對2-MIB濃度高低的影響程度，結果僅發現水溫或氣溫為影響2-MIB生成最顯著的因子

由於水溫與氣溫非獨立變數，基於氣溫資料為監測站自動監測紀錄，資料容易取得，因此本研究擬利用氣溫資料建立簡便的2-MIB濃度預測模式，以提供水廠在面臨臭味事件時能即時採取應變措施，如粉狀活性碳或氧化劑劑量的施放。因考量微生物代謝的特性，即當日測得的2-MIB濃度應受採樣前數日的環境特性影響，此研究中計算起始日(含採樣當日及前6日)往前推算共1~14天的平均氣溫與2-MIB濃度進行相關分析，結果發現採樣前1日至前9日共9天的平均氣溫與2-MIB的濃度相關係數最大，並建立預測方程式2-MIB concentration (ng/L) = 0.3406+0.0579×T1-9 (R2=0.90, F=119.174, p<0.001) ，採樣數據為2000年12月至2001年7月。

利用所建立的預測模式預測2001年8月至2003年7月鳳山淨水廠原水中2-MIB的濃度，結果發現70%的觀測值位於95%的預測區間內。其中因兩場暴雨導致2001年9月與10的觀測值遠低於預測值，還有2003年7月23 mm的累積雨量遠低於歷年七月平均雨量400 mm，導致兩測值高出預估值60%-80%，而創歷史新高的2-MIB濃度183 ng/L。因此進行2-MIB濃度預估時，除了以平均氣溫計算外，另需考慮採樣前的雨量變化，以求得較佳的近似值。

氯、高錳酸鉀與臭氧為常施用於自來水系統的前氧化劑，本研究發現批次反應中，氯與高錳酸鉀對水中溶解態的2-MIB幾無去除效果，即使氧化劑劑量已達10mg/L，2-MIB的去除率也僅有10%。在半批次反應系統中，臭氧施放過量時，2-MIB以假一階的反應動力進行降解，臭氧供應量4 mg/L，鳳山淨水廠原水中溶解態的2-MIB去除率可達50%以上。另外當水體中存在產臭的藍綠菌細胞時，此三種氧化劑皆能導致細胞膜受損，致胞內的2-MIB釋出，然而只有臭氧對溶解態的2-MIB有移除效果，因此當水廠發生藻華所引起的臭味事件時，較好的處理對策應先考慮移除細菌或藻類細胞，再行前氧化程序，以避免溶解態的2-MIB或消毒副產物的前驅物濃度提高，致後續的處理成效降低。

以上幾點具體成果希望能提供水廠管理與技術方面的指引，有助於其評選最佳的土臭味問題控制策略。

Musty and earthy odor is present in the source water of Feng-Shen Waterworks (FSW) and Cheng-Chin Lake Waterworks (CCLW) south Taiwan year round. MIB concentrations were between 10ng/L and 200 ng/L, and higher concentrations happened in warm seasons, i.e. between May and October. In cooler seasons, from November to April, the concentrations were much lower. However the concentrations always exceed the odor threshold 2-10 ng/L. Although 2-methylisoborneol (2-MIB) and geosmin are responsible for the musty and earthy odor, respectively, the possible odor producers remained unknown.

In this study four 2-MIB and geomsin producers were isolated for determining their odorant production, and release and destruction of the odorants under oxidation processes. The odorant producers, including a cyanobacterium and two actinomycetes were isolated from two source waters in southern Taiwan, and then identified using molecular biotechnology. Although the cyanobacterium, a 2-MIB producer, was only identified as Oscillatoria sp., the three actinomytecetes were identified as Streptomyces caelestis producing 2-MIB, S. roseoflavus producing geosmin and S. malaysiensis producing 2-MIB and geosmin after comparing the extracted DNA sequence with the NCBI database.

Both the purified cyanobacterium and actinomycetes were incubated at 20, 25 and 30C for the determination of 2-MIB and geosmin production ratios. At 20 and 25C, a substantial proportion (around 80%) of 2-MIB retained intracellularly for the cyanobacterium at the logarithmic growth phase. The intracellular 2-MIB soon released into water once the cyanobacterium reached its stationary growth phase. The 2-MIB production ratio was about 300 ng/ug chl. a at three temperatures. For actinomycetes (except S. roseoflavus), about 50% of either geosmin or 2-MIB were present within cells at the logarithmic growth phase. The production ratio for 2-MIB and geosmin was around 1-5 ng/mg-biomass for the two actinomycetes.

Two statistical methods, including principle component analysis and multiple linear regression technique were employed to correlate the 2-MIB concentrations with all the corresponding water quality and meteorological data collected from December 2000 to June 2001. The results suggested that 2-MIB concentration was highly correlated to water temperature or ambient temperature. Further analysis of ambient temperature with 2-MIB concentration suggested that a good correlation was observed between 2-MIB concentration and T1-9. The correlation equation, with regression coefficient = 0.90, is expressed as

2-MIB concentration (ng/L) = 0.3406+0.0579×T1-9 (R2=0.90, F=119.174, p<0.001)

The equation was then employed to predict the 2-MIB concentrations for the period of July 2001 to July 2003. The data fits the predicted line closely, with about 70% of the observed 2-MIB concentrations falling into the 95% prediction intervals. These high correlations between 2-MIB concentration and environmental temperatures may provide a simple way for the utility to estimate the concentration of odorants, and serve as a means for the selection of control measure

To understand the effect of oxidants used in waterworks on the dissolved and cell-bounded 2-MIB concentration, three oxidants, sodium hypochlorite, potassium permanganate, and ozone were tested. Chlorine and permanganate were much less effective, both removing only about 10% of the 2-MIB within 60 minutes with 10 mg/L addition in batch system. In semi-batch system, the overdose ozone applied, the 2-MIB decayed under 1st order reaction. Oxidation of the Oscillatoria sp. laden water was also conducted with addition of chlorine, permanganate, and ozone. As all the three oxidants might break the cell membrane of O. sp. and caused the release of almost all the MIB from cells into water, only ozone could effectively destroy dissolved MIB to a degree of higher than 50%.

Abstract (in Chinese)	--I
Abstract-------------------------------------------------------------------------------------IV
Figure of Contents	-VIII
Table of Contents	XI

Chapter 1 Introduction	1-1

Chapter 2 Literature Review	2-1
2-1	The off-flavors problem in drinking water	2-1
2-2	Off-flavors producing microorganisms	2-5
2-3	Biosynthesis of geosmin and 2-MIB	2-13
2-4	Effect of environmental factors on odor producing microorganisms	2-17
2-5	Control of odors	2-23

Chapter 3 Materials and Methods		3-1
3-1	Site description	3-1
3-2		The statistical methods	3-6
3-3  Isolation, purification, culture and growth measurement of cyanobacteria and actinomycetes	3-9
3-4	DAN extraction, PCR and Phylogenetic analyses amplification	3-14
3-5  Chlorine, permanganate and ozone oxidation experiments-------------------	3-16
3-6  Analysis of 2-MIB and geosmin------------------------------------------------- 3-19

Chapter 4 Results and Dissussion------------------------------------------------------- 4-1
4-1  Occurrence of 2-MIB and geosmin in two source waters--------------------- 4-1
4-2  The cell growth and 2-MIB production of Oscillatoria sp.--------------------4-10
4-3  The cell growth and odorants production of actinomycetes-------------------4-22
4-4  Prediction of 2-MIB concentration in source water----------------------------4-35
4-5  Oxidation of dissolved 2-MIB and Oscillatoria sp.–laden water------------ 4-45

Chapter 5 Summary and Conclusions-------------------------------------------------- 5-1
Reference 	6-1
 
Contents of Figure

Figure 2-1-1 The taste and odor wheel of drinking water----------------------------	2-2
Figure 2-1-2 Maps of the waterworks with odor problems in Taiwan	2-4
Figure 2-3-1 MVA pathway and MEP pathway	2-16
Figure 2-5-1 Ozone reaction pathway ------------------------------------------------- 2-31
Figure 3-1-1 Schematic diagram of the experimental procedure	3-2
Figure 3-1-2 The advanced treatment process of CCLWTP	3-3
Figure 3-1-3 The conventional treatment process of FSWTP-----------------------	3-4
Figure 3-3-1 The broth culture of actinomycetes	3-11
Figure 3-3-2 The procedure of SEM observation------------------------------------- 3-12
Figure 3-3-3 The photo of S-R cell	3-13
Figure 3-4-1 The procedure of DNA extraction---------------------------------------3-15
Figure 3-5-1 The apparatus of ozonation	3-18
Figure 4-1-1 The concentrations of 2-MIB and geosmin and corresponding temperature in the source water of FSR------------------------------- 4-3
Figure 4-1-2 The trend of 2-MIB concentrations in the source water of Cheng-Chin Lake waterworks--------------------------------------------------------- 4-4
Figure 4-1-3 The correlation of 2-MIB concentrations and daily-mean temperature		--------------------------------------------------------------------------4-4
Figure 4-1-4 The concentrations of MIB of FSW source water cultivated under 20C, 25C and 30C	4-6
Figure 4-1-5(a) The concentration changes of 2-MIB in cultivated experiments (water from dry season)	4-8
Figure 4-1-5(b) The concentration changes of nutrient in cultivated experiments (water from dry season)	4-8
Figure 4-1-6(a) The concentration changes of 2-MIB and optical density at 665nm in cultivated experiments (water from wet season)	4-9
Figure 4-1-6(b) The concentration changes of nutrients and pH in cultivated experiments (water from wet season)	4-9
Figure 4-2-1 The microphoto of Oscillatoria sp. under 400 times of magnifications using optical microscope	4-12
Figure 4-2-2(a) The cell growth and 2-MIB production for Oscillatoria sp. under 25C and 1000 lux of incubation	4-13
Figure 4-2-2(b) The intra- and extra-cellular specific 2-MIB production for Oscillatoria sp. under 25C and 1000 lux of incubation	4-13
Figure 4-2-3(a) The cell growth and 2-MIB production for Oscillatoria sp. under 30C and 1000 lux of incuabtion	4-14
Figure 4-2-3(b) The intra- and extra-cellular specific 2-MIB production for Oscillatoria sp. under 30C and 1000 lux of incuabtion	4-14
Figure 4-2-4 Effects of temperature on 2-MIB and chl. a concentration and specific 2-MIB production for O. sp.	4-19
Figure 4-2-5 Effects of light intensity on 2-MIB and chl. a concentration and specific 2-MIB production for O. sp.	4-20
Figure 4-2-6 Effects of NO3-N on 2-MIB and chl. a concentration and specific 2-MIB production for O. sp.	4-20
Figure 4-2-7 Effects of PO4--P on 2-MIB and chl. a concentration and specific 2-MIB production for O. sp.	4-21
Figure 4-3-1 The SEM microphoto of the spore chain of strain FS1	4-23
Figure 4-3-2 The SEM microphoto of the spore chain of strain FS4	4-24
Figure 4-3-3 The SEM microphoto of the spore chain of strain FS6	4-24
Figure 4-3-4 Effects of temperature on (a) growth and odorants production, and (b) specific odorants production and odorant distribution for S. malaysiensis-	4-30
Figure 4-3-5 Effects of temperature on (a) growth and 2-MIB production, and (b) specific 2-MIB production and odorant distribution for S. caelestis--------	4-31
Figure 4-3-6 Effects of temperature on (a) growth and geosmin production, and (b) specific geosmin production and odorant distribution for S. roseoflavu------------------------------------------------------------------ 4-32
Figure 4-4-1 The correlation between water and ambient temperature	4-39
Figure 4-4-2. The scree plot of principle component analysis-----------------------4-39
Figure 4-4-3 The contours of regression coefficient for 2-MIB concentrations and different time periods of average of mean daily temperatures-	4-43
Figure 4-4-4 2-MIB fitted values with ambient temperature (T1-9)	4-43
Figure 4-4-5 The predicted and observed 2-MIB concentrations for FSR	4-44
Figure 4-5-1 The degradation of 2-MIB under different ozone dosage------------ 4-48
Figure 4-5-2 The degradation of 2-MIB during ozonation under different matrix		4-49
Figure 4-5-3 Percent removal efficiencies of 2-MIB by different oxidant	4-51
Figure 4-5-4 The concentration change of potassium, magnesium, and calcium during ozonation	4-55
Figure 4-5-5 The concentration change of potassium, 2-MIB, and chlorophyll a during ozonation	4-56
Figure 4-5-6 The concentration change of potassium, 2-MIB, and chlorophyll a under different chlorine dosage	4-56
Figure 4-5-7 The concentration change of potassium, 2-MIB, and chlorophyll a under different permanganate dosage	4-57
Figure 4-5-8 The change of O. sp. filament before and after oxidation	4-57






 
Contents of Table
                                                             
Table 2-1-1 The five musty/earthy compounds	2-3 
Table 2-1-2. The location of source water and waterworks	2-5
Table 2-2-1 Geosmin and/or 2-MIB producing actimomycete	2-7
Table 2-2-2 Cyanobacteria for production of geosmin and/or 2-MIB-------------- 2-9
Table 2-4-1 Tthe effect of light intensity on odorants production by cyanobacteria--------------------------------------------------------------- 2-18
Table 2-4-2 The effect of temperature on odorant producing actinomycetes----- 2-19
Table 2-4-3 The effects of temperature on odorant producing cyanobacteria---- 2-19
Table 2-5-1 Concentrations of 2-MIB at different sampling locations in nine waterworks ----------------------------------------------------------------- 2-26
Table 3-3-1 The composition of BG-11medium	3-10
Table 3-3-2 The composition of starch-casein medium	3-11
Table 3-3-3 The composition of M medium	3-11
Table 3-4-1 Primers used in this study	3-17
Table 3-6-1 The conditions of GC/MS	3-20
Table 4-3-1 Growth and cultural characteristic of three pure actinomycetes strains on solid starch-casein agar 		4-23
Table 4-3-2 Sequencing results of the three pure actinomycete strains	4-25
Table 4-3-3 Effects of spore formation on mean biomass dry weight, odorant production, and specific odorant production of three S. spp. at 30C	 		4-29
Table 4-4-1 The meteorological and water quantity/quality parameters monitored at FSWs	4-36
Table 4-4-2 Correlation matrix for environmental parameters		4-40
Table 4-4-3 The result of principle component analysis----------------------------- 4-41
Table 4-4-4. The coefficient of variables with the first regression------------------4-41
Table 4-4-5. The coefficient of variables with the second regression--------------4-42
Table 4-4-6. The coefficient of variables with the third regression-----------------4-42
Table 4-4-7 The results of stepwise regression (independent variable: 2-MIB concentration)--------------------------------------------------------------- 4-42
Table 4-5-1 The water quality of FSR in 2000		4-48
Table 4-5-2 Comparison of reaction coefficients of 2-MIB degradation with different ozonation tests		4-49
Table 4-5-3 Comparison of percent removal of 2-MIB among three different semi-batch ozonation tests	4-50
Table 4-5-4 Comparison of 2-MIB removal efficiencies for different chlorination and permanganation 	4-51

                                    

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