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Characterization of a Microbial Culture Capable of Removing Taste- and Odor-Causing 2-Methylisoborneol from Water

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PAGE 1

CHARACTERIZATION OF A MICROBIAL CULTURE CAPABLE OF REMOVING TASTEAND ODOR-CAUSING 2-METHYLISOBORNEOL FROM WATER By CHANCE VENABLE LAUDERDALE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ENGINEERING UNIVERSITY OF FLORIDA 2004

PAGE 2

Copyright 2004 by Chance Venable Lauderdale

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This thesis is dedicated to Marley Lauderdale.

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ACKNOWLEDGMENTS I would like to thank Dr. Paul Chadik, Dr. David Mazyck, Dr. Angela Lindner and Dr. Nancy Szabo for the time and assistance they have offered to me throughout this study and for serving on my graduate advisory committee. I would especially like to thank Dr. Angela Lindner for her patience, her mentoring, and for challenging me to improve the quality of my work. I also thank Matt Booth, Rick Loftis, Adriana Pacheco and Jessica Strait for the training and assistance they provided me in the laboratory. Finally, I would like to thank my parents and my brother, Greg, for giving their support, without which, none of this would have been possible. iv

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................3 Introduction to Taste and Odors in Drinking Water: Description, Causes, and Brief History......................................................................................................................3 Methylisoborneol: Isolation, Formation, Properties, Conventional Treatment and Analysis....................................................................................................................4 Initial Isolation of Methylisoborneol.....................................................................4 Natural Formation of Methylisoborneol................................................................5 Physical and Chemical Characteristics of Methylisoborneol................................6 MIB Detection and Analysis Methods..................................................................8 Conventional Water Treatment for MIB Removal................................................9 Bacterial Transformation of MIB...............................................................................11 Isolation and Identification of Potential MIB-Degrading Bacteria.....................11 Characteristics of MIB-Degrading Bacteria........................................................12 Applications of Technology Using Biological Transformation for MIB Removal in Water Treatment....................................................................................................17 Introduction to the Use of Bioremediation in Water Treatment..........................17 Biological Filtration.............................................................................................18 The Effect of Ozonation the Biological Transformation of MIB........................20 Conclusions.................................................................................................................22 3 CHARACTERIZATION OF A MICROBIAL CULTURE CAPABLE OF REMOVING TASTEAND ODOR-CAUSING 2-METHYLISOBORNEOL FROM WATER: A MANUSCRIPT TO BE SUBMITTED TO WATER RESEARCH.........23 Introduction.................................................................................................................23 v

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Materials and Methods...............................................................................................25 Enrichment and Isolation of MIB Transforming Bacteria..................................25 Source water used as initial inoculum..........................................................25 Enrichment procedures.................................................................................25 Characterization of the Isolated Mixed Culture..................................................27 Cellular morphology....................................................................................27 Genotypic characterization...........................................................................28 Growth characterization of the isolated culture...........................................29 MIB Depletion Potential of the Isolated Culture.................................................29 Oxygen uptake studies.................................................................................29 MIB depletion studies..................................................................................31 Results and Discussion...............................................................................................32 Characterization of the Isolated Mixed Culture..................................................32 Cellular and colony morphology..................................................................32 Genotypic characterization...........................................................................34 Growth characterization...............................................................................35 MIB Depletion Potential of the Isolated Culture.................................................36 Oxygen uptake studies.................................................................................36 MIB depletion studies..................................................................................37 Conclusions.................................................................................................................39 4 CONCLUSIONS........................................................................................................41 LIST OF REFERENCES...................................................................................................44 BIOGRAPHICAL SKETCH.............................................................................................48 vi

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LIST OF TABLES Table page 2-1: Odor threshold concentrations for 5 earthy-musty odor compounds (Lalezary et al., 1986; Persson, 1980)..................................................................................................4 2-2: Physical-chemical characteristics of MIB (Pirbazari et al., 1992) compared to high and low values as described by Ney (1990)...............................................................8 2-3: Conventional treatment methods and powdered activated carbon (PAC) average MIB removal efficiencies (Ashitani et al., 1988).............................................................10 2-4: MIB depletion potential for the identified species isolated from a biological treatment filter (Egashira et al., 1992)......................................................................17 2-5: Column parameters for the biological filtration study conducted by Yagi et al. (1988)...19 2-6: Results for the biological filtration study conducted by Yagi et al. (1988)...............19 3-1: Components of mineral salts media (Izaguirre et al., 1988).....................................26 3-2: Colony and cellular characteristics of the dominant strain in the isolated culture.....33 3-3: Summary of the growth kinetics for the isolated culture grown at 5, 10, and 20 mg/l MIB..................................................................................................................36 vii

PAGE 8

LIST OF FIGURES Figure page 2-1: 2-Methylisoborneol, structure identified by Medskar et al. (1969)..............................5 2-2: Plot depicting the linear relationship between MIB concentration and the filament concentration of Oscillatoria sp.3 (adapted from Hosaka et al., 1995).....................6 2-3: Depletion by Bacillus HI-5 at 2 different initial concentrations of MIB (adapted from Ishida and Miyaji, 1992)..........................................................................................13 2-4: Growth curves for Bacillus HI-5 at 2 different initial MIB concentrations (adapted from Ishida and Miyaji, 1992)..................................................................................14 2-5: Effect of pH variation on the MIB depletion potential of a biologically active column (adapted from Egashira et al., 1992)........................................................................15 2-6: The logarithmic relationship between temperature and the MIB depletion potential of a biologically active column (adapted from Egashira et al., 1992)..........................15 2-7: Kanamachi treatment line (adapted from Muramoto et al., 1995).............................21 2-8: MIB concentrations in raw and processed waters (adapted from Muramoto et al., 1995).........................................................................................................................21 3-1: A schematic of the anthracite column apparatus used to isolate a culture capable of depleting MIB..........................................................................................................26 3-2: A schematic of the reactor used for the oxygen uptake experiments.........................31 3-3: Phase contrast photograph (A) and transmission electron photograph (B) of the MIB-degrading isolated culture when grown on solid agar. Flagella and sporulation denoted with arrows.................................................................................................34 3-4: The neighbor joining phylogenetic tree of the isolated culture constructed from the MicroSeq alignment report. The top 10 closest genetic matches of the isolated culture are presented with their percent genetic difference. A lower % difference indicates a closer match............................................................................................35 viii

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3-5: Growth curves of isolated culture at initial MIB concentrations of 5 mg/l (), 10 mg/l (), and 20 mg/l (). All absorbance measurements were taken at a wavelength of 600 nm..............................................................................................36 3-6: Oxygen uptake rates for the isolated culture over a range of MIB concentrations....37 3-7: MIB depletion percent removal curves for isolated culture microcosms grown with initial MIB concentrations of 25 ng/l () and 5 mg/l ().......................................38 3-8: MIB depletion curves for isolated culture microcosms grown with initial MIB concentrations of 25 ng/l () and 5 mg/l ()..........................................................39 ix

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Engineering CHARACTERIZATION OF A MICROBIAL CULTURE CAPABLE OF REMOVING TASTEAND ODOR-CAUSING 2-METHYLISOBORNEOL FROM WATER By Chance Venable Lauderdale August 2004 Chair: Angela Lindner Major Department: Environmental Engineering Sciences A common blue-green algae metabolite, 2-methylisoborneol (MIB), is responsible for unpalatable drinking water in Asia, Australia, North American and Europe. Current water treatment technologies, are ineffective in removing MIB from potable water. The focus of this project was to examine the potential for microbial transformation of MIB by a culture isolated from a drinking water reservoir, Lake Manatee, in Manatee County, Florida. This culture was characterized using phenotypic and genotypic methods as well as by assessing its growth and MIB-depletion potentials using growth and oxygen uptake experiments and microcosms coupled with solid-phase microextraction (SPME) and gas chromatography/mass spectrophotometry (GC/MS). The predominant strain in the isolated culture was bacillus in shape and possessed spore and flagella, and 16S rRNA analysis determined that this isolated culture is most similar to Bacillus sphaericus (99% match). The ability of this culture to transform MIB was examined by running oxygen uptake measurements and by conducting depletion studies using SPME coupled with

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GC/MS for MIB analysis. The results obtained from these studies demonstrated the isolated culture was capable of using MIB as its sole source of carbon and depleting MIB to below its odor threshold concentration (OTC) of 10 ng/l. Implications of these results are that microbial populations can be isolated from natural water sources for the removal of MIB and potentially other tasteand odor-causing compounds to concentrations that render aesthetically acceptable drinking water.

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CHAPTER 1 INTRODUCTION 2-Methylisoborneol, a common blue-green algae metabolite released in surface waters typically from late spring to early fall, has been a cause of unpalatable drinking water in Asia, Australia, North American and Europe. Conventional water treatment technologies, consisting of breakpoint pre-chlorination, coagulation, sedimentation, and post chlorination, are not effective in removing MIB from potable water to below its odor threshold of 10 ng/l (Lalezary et al., 1986; Ashitani et al., 1988). As a supplement o water treatment, powdered activated carbon (PAC) is often added to water to remove MIB. However, PAC addition is not cost effective at higher MIB concentrations (Herzing et al. 1977). Seeking a possible solution, recent studies have begun to examine biological treatment as an alternative treatment method for MIB removal. To date, several strains of bacteria have been isolated from natural waters that are capable of using MIB as a growth substrate (Ishida and Miyaji, 1992; Egashira et al., 1992). Although these studies have shown MIB removal by these cultures, the potential for microbial communities to deplete MIB to below the odor threshold concentration (OTC) has not been shown. Also, the effect of MIB concentration on microbial growth and metabolic activity has not been fully explored. The focus of this research was to examine the potential for microbial transformation of MIB by a culture isolated from water collected from Lake Manatee in Manatee County, Florida. The hypothesis of this project was that, because of the MIB 1

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2 outbreaks experienced in this reservoir on a seasonal basis, bacterial populations capable of using MIB as a growth substrate could be isolated. The scope of this work was composed of 4 major objectives. The first included a literature review covering the current knowledge of the origins and properties of MIB as well as current technologies involving biological treatment. The next objective was to isolate a bacterial culture capable of growth on MIB from a water sample obtained from Lake Manatee. Subsequently, the isolated culture was to be characterized by using growth kinetics to determine specific growth rates, light and TEM microscopy to examine fine cell structures, and 16S rRNA phylogenetic analysis to assess the closest match of the unknown isolate(s) to known bacterial strains. The final objective was to assess the MIB transformation potential of the microbial community by using oxygen uptake methods and batch microcosm experiments combined with solid phase microextraction analysis to determine the ability of the culture to oxidize and degrade MIB at different initial concentrations and to deplete MIB to below the OTC. The results from this project promise to benefit water treatment facilities and their customers by providing applied results directed towards the remediation of MIB contamination. Additionally, this research will add to the continuously growing body of knowledge addressing the use of biological treatment for improved potable water quality.

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CHAPTER 2 LITERATURE REVIEW Introduction to Taste and Odors in Drinking Water: Description, Causes, and Brief History Tastes and odors are considered by most consumers to be significant factors when determining potable water quality. In 1973, a Gallup Poll indicated that most consumer complaints concerning drinking water involved tastes and odors. As far back as 1957, surveys given to both water utilities and consumers have consistently reported similar problems (Suffet et al., 1996). The occurrence of taste and odor problems is widespread and has been reported in Argentina, Australia, Canada, Denmark, England, Finland, Germany, Israel, Japan, The Netherlands, Norway, Poland, Sweden, U.S.A and U.S.S.R. (Ashitani et al., 1988; Juttner, 1995; Persson, 1983; Suffet et al., 1996; Zimmerman et al., 1995). Some of the more prominent tasteand odor-causing substances are naturally occurring organic compounds that produce an earthy-musty odor in drinking water (Rashash et al., 1997). Examples of these compounds include 2-isopropyl-3-methoxypyrazine (IPMP), 2,3,6-trichloranisole (TCA), 2-isobutyl-3-methoxypyrazine (IBMP), trans-1,10-dimethyl-trans-9-decalol (geosmin) and 1,2,7,7-tetramethylbicyclo[2.2.1]heptan-2-ol, also known as 2-methylisoborneol (MIB). Although these compounds are not deleterious to human health, they can cause malodorous drinking water at extremely low concentrations (10 ng/l) (Persson et al., 1980; Lalezary et al., 1986; Rashash et al., 1997). The odor threshold concentrations (OTC) of these compounds, defined as the concentration at or above which odor can be detected, are provided in Table 2-1 (Lalezary et al., 1986; Persson, 1980). 3

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4 Table 2-1: Odor threshold concentrations for 5 earthy-musty odor compounds (Lalezary et al., 1986; Persson, 1980) Common Name Chemical Formula OTC (ng/l) Geosmin C 12 H 22 O 4 IPMP C 8 H 12 ON 2 2 IBMP C 9 H 14 ON 2 2 TCA C 7 H 5 OCl 3 7 MIB C 11 H 20 O 10 *Odor threshold concentrations determined by an odor panel Water utilities typically rely on conventional treatment methods, supplemented with powder activated carbon (PAC), to remove tasteand odor-causing compounds. These methods can effectively decrease IPMP, TCA, IBMP and geosmin concentrations to below their odor thresholds; however, these processes have been shown to be unsuccessful in removing MIB to below its OTC (Ashitani et al., 1988). Today, some alternative methods for MIB treatment, including biological transformation, granular activated carbon, and ozonation, are being examined. This literature review summarizes previous studies reporting the origins and properties of MIB as well as current technologies involving biological treatment. Methylisoborneol: Isolation, Formation, Properties, Conventional Treatment and Analysis Initial Isolation of Methylisoborneol In 1969, Medsker et al. first reported successful isolation and identification of MIB from actinomycetes, a group of terrestrial gram-positive bacteria that form branching filaments. They described the compound as camphor-smelling and the major odorous constituent in 3 out of 28 species of actinomycetes surveyed. The empirical formula (Table 2-1) and structure (Fig. 2-1) were identified by mass spectrometry.

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5 CH3CH3CH3CH3OH Figure 2-1: 2-Methylisoborneol, structure identified by Medskar et al. (1969) Natural Formation of Methylisoborneol Cyanobacteria are prokaryotic oxygenic phototrophs that are found in a variety of ecological settings, including terrestrial, freshwater and marine habitats (Madigan et al. 2000). In freshwater lakes, especially those that are eutrophic, cyanobacteria can develop massive accumulations, known as blooms. Cyanobacteria and their resulting blooms are responsible for the production of many odor-causing compounds, including MIB in natural waters. Numerous species of MIB-producing cyanobacteria have been isolated and classified, and these include Oscillatoria sp., Anabaena sp. and Phormidium sp. (Hosaka et al., 1995; Juttner et al., 1995; Zimmerman et al., 1995). These microorganisms synthesize MIB during normal growth. MIB is believed to be a methylation product of an unknown monoterpene formed from acetate and mevalonate (Juttner et al., 1995). The function of MIB in the cell has yet to be understood completely, and the compound may simply be a by-product of the photosynthetic pathway. After MIB is synthesized, it is found either bound to thylakoid membranes and cytoplasmic proteins or excreted by the cell. The production of MIB varies by cyanobacteria strain. Some species, such as Oscillatoria sp. 3 and Phormidium tenue, demonstrate a proportionate relationship between the number of filaments, or chains of cells, and MIB concentration. A single filament of Oscillatoria sp. 3 produces an average of 19.5 picograms of MIB (Hosaka et al., 1995). The graph shown in Figure 2-2, compiled from data collected from the Ooba River in Japan,

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6 further illustrates the effect of filament/ml (Oscillatoria sp. 3) on MIB concentration (Hosaka et al., 1995). 01002003000246810121416Number of filaments per mlMIB concentration (ng/l) Figure 2-2: Plot depicting the linear relationship between MIB concentration and the filament concentration of Oscillatoria sp.3 (adapted from Hosaka et al., 1995) These data imply that during a period of algal bloom, typically occurring from late spring to early fall, the MIB concentration in surface water supplies will increase. If eutrophication increases in the freshwater bodies used for drinking water, the potential for increased algal concentrations and, thus, MIB releases is suggested by these findings. Physical and Chemical Characteristics of Methylisoborneol The physical and chemical characteristics of MIB dictate its behavior in the environment and in water treatment plants. The fate of MIB in a treatment process may be predicted and explained, at least in part, by examining its following properties: density, aqueous solubility, octanol-water partition coefficient, and Henrys constant (Ney, 1990). Density is a valuable characteristic when assessing the physical separation potential of a compound in the aqueous phase. The density of MIB is approximately 0.9288 g/cm 3 (Pirbazari et al., 1992). This value is similar to that of water, indicating MIB is unlikely to pool at either the surface or bottom of the water column.

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7 A compounds aqueous solubility is a measurement of its affinity for water. Compounds with high aqueous solubility (>1000 mg/l) are hydrophilic, whereas compounds with low aqueous solubility (<10 mg/l) are hydrophobic. Compounds with a high aqueous solubility are typically more mobile and bioavailable, thus have a higher potential to be biodegraded (Ney, 1990). MIB has an aqueous solubility of 194.5 mg/l (Pirbazari et al., 1992), which is close to the low range, and would, therefore, be expected to be less mobile in the aqueous environment, less biodegradable, and more tending to sorb to sediments. The octanol-water partition coefficient (K ow ) indicates a chemicals tendency to sorb to soils and sediments, bioconcentrate in aquatic organisms and accumulate in the soil. A K ow value greater than 1000 suggests that a chemical has an affinity for bioaccumulation in the food chain, has a low aqueous solubility and has a low mobility in the soil and aqueous phases, whereas a chemical with K ow value of less than 500 would be more bioavailable, soluble, and mobile (Ney, 1990). MIB has an octanol-water partition coefficient of approximately 1349 (Pirbazari et al., 1992), indicating it is lipophilic and thus has a tendency to partition out of the aqueous phase. Henrys Constant (K H ) demonstrates the ability of a chemical to partition between the aqueous phase and the atmosphere and it can be estimated by ratioing the vapor pressure and the aqueous solubility. A chemical with a high K H value (>0.4 l-atm/mol) is more likely to escape the from aqueous phase to the vapor phase, whereas a chemical low K H value (<0.004 l-atm/mol) would most likely remain in the aqueous phase (Ney, 1990). The K H value for MIB is 5 x 10 -8 l-atm/mol (Pirbazari et al., 1992), indicating that MIB will not readily escape from the aqueous phase. Table 2-2 summarizes the physical and chemical characteristics of MIB.

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8 Table 2-2: Physical-chemical characteristics of MIB (Pirbazari et al., 1992) compared to high and low values as described by Ney (1990) Characteristic Observed Value for MIB Low Value High Value Density 0.9288 g/cm 3 Aqueous Solubility 194.5 mg/l <10 mg/l >1000 mg/l Octanol/Water Coefficient 1349 <500 >1000 Henry's Law Constant 5.76 x 10 -8 l -atm/mol <0.004 l-atm/mol >0.4 latm/mol In summary, MIB is expected to have moderately low motility in the aqueous environment, with a large portion partitioned out of the aqueous phase via bioaccumulation and/or sorbtion onto sediments. The low Henrys constant indicates MIB is unlikely to escape into the atmosphere. MIB Detection and Analysis Methods The detection and quantitative analysis of MIB requires the use of technically complex and time-consuming analytical methods, as it is commonly found in natural waters at ultra-trace concentrations (Watson et al., 2000). Because MIB is found at such low natural concentrations, traditional analysis of MIB typically relies on concentrating large sample volumes (100-1000 ml). The preparation and analysis methods used, including liquid-liquid extraction, purge and trap, closed-loop stripping analysis and simultaneous distillation-extraction, are time-intensive and/or require high-resolution mass spectrophotometers (Lloyd et al., 1998). As a means of avoiding the constraints of time and cost involved in using the large sample volumes required in these concentration methods, solid phase microextraction (SPME) methods coupled with a gas chromatograph-mass spectrophotometer (GC/MS) system have been recently developed By combining SPME and GC/MS, detection of MIB at ng/l levels is possible without the large sample volumes. Typically, a sample volume of 25

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9 ml is sufficient. SPME-GC/MS is an inexpensive and rapid method for the analysis of volatile and semi-volatile compounds occurring in the headspace of water matrices (Eisert and Levsen ,1996). Techniques using SPME-GC/MS were originally applied by the Des Moines Water Works for taste and odor analysis and have recently been employed by the City of Tampa for the analysis of MIB and geosmin (Brand, 1995). Currently, a SPME-GC/MS method is undergoing balloting for consideration as Standard Method 6040D (APHA, 2001). This SPME method is based on the adsorption of MIB on a fiber coated with divinylbenzene-carboxen-polydimethyloxane cross-link. This fiber is placed in the headspace of a sealed vial and allowed to equilibrate with an aqueous sample. After equilibrium is reached (typically 30-35 minutes), the fiber is removed and injected into the port of a GC/MS system, where it is heated allowing the analytes to be desorbed for analysis. The minimum detectable concentration of MIB analyzed by these methods < 5 ng/l and the recovery of the laboratory control standard of 20 ng/l is 95% depending on the laboratory (APHA, 2001). Although, this standard method appears to be a good alternative to other forms of MIB analysis, it has not been validated in for many matrices, therefore, prior to sample analysis, recoveries using these matrices with known additions of MIB should be examined (APHA, 2001). Conventional Water Treatment for MIB Removal As stated previously, municipal water utilities have confronted problems with MIB for many years. Typical water treatment consists of breakpoint pre-chlorination, coagulation, sedimentation, rapid sand filtration and post-chlorination. Many studies have reported that current conventional treatment methods do not sufficiently remove MIB (Ashitani et al., 1988; Rashash et al., 1997; Suffet et al., 1996). Powder activated carbon (PAC) is often added as a supplement to a treatment line to decrease the MIB concentration; however, this

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10 practice is cost-effective only for low dosing (Herzing et al., 1977). A study conducted by Ashitani et al. (1988) examined several possible water treatment scenarios, including coagulation alone, pre-chlorination followed by coagulation/sedimentation, pre-chlorination followed by coagulation/sedimentation then PAC, and full conventional treatment (including PAC) (Ashitani et al., 1988). These results (Table 2-3) show MIB removal by coagulation alone was greater than that of prechlorination with coagulation. The authors explanation is that a significant part of the MIB in the raw water was present in the responsible microorganism. These microorganisms are typically removed during coagulation and sedimentation; however, when chlorination was applied prior to coagulation, the cells lysed, releasing intracellular material that contained MIB. Although the PAC (10 ppm)/pre-chlorination/coagulation method was able to remove an average of 42% of the influent MIB, none of the treatment methods studied were capable of decreasing MIB concentrations to below the OTC; thus, alternative technologies must be found. Recently, such water treatment methods have been devised and are showing preliminary success in the removal of MIB. As the focus of this work is microbial transformation of MIB, the remainder of this discussion examines the literature reporting success with biological treatment of MIB. Table 2-3: Conventional treatment methods and powdered activated carbon (PAC) average MIB removal efficiencies (Ashitani et al., 1988) Treatment Method Raw Water MIB (ng/l) Treated Water MIB (ng/l) Removal (%) Coagulation 53 34 36 Pre-Chlorination/Coagulation 53 39 26 PAC (10ppm)/ Pre-Chlorination/Coagulation 53 31 42

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11 Bacterial Transformation of MIB Isolation and Identification of Potential MIB-Degrading Bacteria Most work reporting the identification of microorganisms that are capable of degrading MIB involved the use of a MIB-contaminated raw water as the inoculum source for a liquid culture. The bacterial strains were isolated from the resulting liquid cultures by colony morphology using spread plate methods. Each study then identified isolated strains by using biochemical test kits. These kits are commonly used for many groups of bacteria to determine phenotypic activity (oxidase, catalase, nitrate reduction, amino acid-degrading enzymes, fermentation or utilization of carbohydrates). The tests are typically conducted by adding a small amount of culture to a multi-welled plate containing various biochemical reagents. The reaction in each well is then observed and compared to a database containing phenotypic characteristics of known microorganisms (Chester and Cleary, 1980). Biochemical test kits are commonly used in the identification of gram type negative, nonfermentative bacteria, such as Pseudomonas, Acinetobacter, Flavobacterium, Moraxella and fermentative bacteria not belonging to the Enterobacteriaceae, such as Vibrio and Aeromonas. There are limitations with these kits; however, such as previously unknown, rare, or newly described strains that are not in the database (Chester and Cleary, 1980). This can often lead to strains being unidentified or misidentified when their results are compared to only known species. The two testing kits employed by the studies discussed below for characterization of isolated MIB-degrading microorganism are the Rapid Nonfermentor Test (NFT) and the Minitek Test Kit. Izaguire et al. (1998) reported use of a common technique for the isolation of a pure MIB-degrading culture. In this method, water and sediment samples were collected from contaminated lakes and used as inoculum for a MIB-spiked mineral salts medium. The MIB

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12 degrading bacteria were identified as soon as the cultures showed a decrease in MIB concentration, typically after incubation periods of 3-20 days. Purified isolates were identified from the cultures using the NFT kit, described above. Examples of some of the isolated MIB-degrading bacteria included Pseudomonas aeruginosa, Pseudomonas pseudoalcaligenes, Pseudomonas paucimobilis, and Pseudomonas mendocina. Other publications claiming successful isolation of MIB degraders from natural waters used similar techniques; however, different strains were identified, including Bacillus subtilis and Flavobacterium multivorum (Ishida and Miyaji, 1992; Egashira et al., 1992). Another means of identifying these bacteria would be to compare each gene sequence in a given strain with the gene sequences other known species. The ribosomal ribonucleic acid (rRNA) of one organism can be compared with that of any other organism by a method called 16s rRNA gene sequencing. The results of gene sequencing provides an estimate of the percentage of divergence within sequences that are related but not identical and provides a higher level of accuracy compared to phenotypically based methods (Chester and Cleary, 1980). This information could then be used to construct a phylogenetic tree comparing the isolated culture to known species. Characteristics of MIB-Degrading Bacteria Biological treatment systems are most effective when the conditions for microbial growth are met, including adequate pH and temperature (Egashira et al.,1992). Unfortunately, while some of the microorganisms found capable of MIB transformation have been partially characterized (Ishida and Miyaji, 1992; Egashira et al., 1992), the growth characteristics and environmental requirements of many MIB degraders have not been clearly defined.

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13 Ishida and Miyaji (1992) investigated the kinetics of MIB degradation for a strain these authors isolated, named Bacillus sp. HI-5. This pure strain was isolated by inoculating a minimal salts medium spiked with 100 g/l of MIB with backwash water obtained from a rapid sand filter. The culture was incubated for 18 days while MIB removal was continuously observed. This experiment showed that 30 g/l MIB was removed after the first 70 hours, and, after 7 days all of the MIB had been removed below the minimum analytical detection limit, or MDL, of 20 ng/l. Batch experiments, conducted with Bacillus sp. HI-5, yielded maximum specific growth rates ( max ) of 0.10 hr -1 and 0.03 hr -1 in the presence of 8 and 0.1 mg MIB/l, respectively. A saturation constant (the concentration of substrate where the growth rate is equal to 1/2 max ) was found to be 205 g/l for the culture grown in 8 mg MIB/l. This study also reported corresponding observed MIB depletion rates (Figure 2-3) of 7.7 g/l/hr for the culture grown at an initial concentration of 8 mg/l MIB and 0.5 g/l/hr for the culture grown at the initial concentration of 0.1 mg/l MIB. 0.0010.010.11100406080110120125130140175Time (Hr)MIB (mg/l) MIB 8 mg/l Initial MIB 0.1 mg/l Initial Figure 2-3: Depletion by Bacillus HI-5 at 2 different initial concentrations of MIB (adapted from Ishida and Miyaji, 1992)

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14 The results of the growth study, as shown in Figure 2-4, show the lag time for each culture to be approximately 40 hours. The stationary growth phase for the 0.1 mg/l and 8 mg/l cultures was reached at approximately 130 hours, and the maximum number of microorganisms grown at each MIB concentration were 5.0 x 10 5 CFU/ml and 3.5 x 10 7 CFU/ml, respectively. However, the authors do not report use of a chemical control containing MIB and no cells or a killed control with autoclaved cells. Thus, it is implied that these investigators did not account for volatilization or cell sorption losses of MIB, causing an overestimation of MIB depletion. 1.E+041.E+051.E+061.E+071.E+080406080110120125130140Time (Hr)CFU (N/ml) 8 mg/l MIBInitial 0.1 mg/l MIBinitial Control Figure 2-4: Growth curves for Bacillus HI-5 at 2 different initial MIB concentrations (adapted from Ishida and Miyaji, 1992) Egashira et al. (1992) reported the effects of water temperature and pH on the MIB depletion potential in a pilot plant study with a biological filtration system (packed-column). The microorganisms that were responsible for the depletion in the column were isolated and identified, and their individual depletion potentials were studied. In the biological filter study, a constant concentration of MIB (0.2 g/l) was added to the natural water fed into the

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15 column. The pH and temperature were then systematically adjusted, and the changes in MIB depletion were then measured by GC/MS and recorded. The results for these experiments are shown in Figures 2-5 and 2-6. 7891011121314567891pHMIB Degradation Potential of the Column (g/hr/l) 0 Figure 2-5: Effect of pH variation on the MIB depletion potential of a biologically active column (adapted from Egashira et al., 1992) 1101003.253.33.353.43.451/T (10-3K-1)MIB Degradation Potential of the Packed Column (g/hr/l) Figure 2-6: The logarithmic relationship between temperature and the MIB depletion potential of a biologically active column (adapted from Egashira et al., 1992)

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16 Although no error is reported, the trends in Figures 2-5 and 2-6 suggest that changes in temperature and pH can have a significant influence on the MIB depletion potential of the microbial populations residing in the biological filter used in this study. The maximum MIB depletion potential of the column was observed at a temperature of 30 C and a pH of 8. This correlation may be useful in determining appropriate operating conditions for biological treatment systems designed to remove MIB. After the completion of the biological filtration study, the granular ceramic medium used in the column was treated by ultrasonication. The resulting biological sludge suspension was used as an inoculum in a tripticase soy culture agar that was then incubated for 3 days at 25 C. Microorganisms were then isolated from the agar based on colony morphology. The MIB depletion potential of each isolated bacterium was determined using batch experiments. The isolate bacteria were used to inoculate a minimal mineral medium that was incubated for 7 days with an initial MIB concentration of 20 g/l, under carefully controlled temperature and pH, ranging from 26 to 29 C and 7.3 to 7.6 respectively. MIB depletion potential was determined by the difference between the initial and final MIB concentrations, measured by purge and trap and GC/MS. The isolated species were then characterized using a Minitek identification kit. The results of the MIB depletion potential study for cultures isolated from the biological filter are presented in Table 2-4.

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17 Table 2-4: MIB depletion potential for the identified species isolated from a biological treatment filter (Egashira et al., 1992) Strain Pseudomonas sp. P. aeruginosa Flavobacterium sp. F. multivorum Initial MIB Concentration 20 ug/l 20 ug/l 20 ug/l 20 ug/l Final MIB Concentration 15.7 ug/l 14.82 ug/l 14.0 u/l 16.7 ug/l Incubation Time 24 hr 24 hr 24 hr 24 hr MIB Depletion Rate 0.18 ug/l/hr 0.21 ug/l/hr 0.25 ug/l/hr 0.14 ug/l/hr Total MIB Depletion % 21.6 25.9 29.7 16.7 MIB Depletion %/hr 0.9 1.08 1.24 0.70 As shown in Table 2-4, several species of bacteria that can potentially transform MIB were isolated in this study. This study did not provide; however, the cumulative depletion rate of the original mixed culture composed of these isolated strains. It is unknown whether the depletion rate of the mixed culture differs significantly from those reported. The inclusion of controls to measure the effects of volatilization or cell sorption on MIB depletion was not reported in this study. Applications of Technology Using Biological Transformation for MIB Removal in Water Treatment Introduction to the Use of Bioremediation in Water Treatment Bioremediation is the use of microorganisms to eliminate or detoxify toxic or unwanted chemicals, and can be employed in water treatment to eliminate natural and anthropogenic chemicals from raw water (Characklis and Marshall, 1990). One of the most common bioremediation applications in water treatment is biological filtration. Microorganisms that are present in filtration media oxidize biodegradable organic matter and nitrify ammonium compounds. Recently, laboratoryand pilotscale experiments have shown that some microorganisms have the potential to transform MIB (Izaguirre et al., 1988; Oikawa et al., 1995; Namkung and Rittmann, 1987; Tanaka et al., 1996). Important factors that may dictate the effectiveness of a biological treatment method for

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18 MIB removal include microbial diversity, transformation pathways (intermediate formation), growth characteristics and biofilm development. Biological Filtration Microbial cells often attach firmly to submerged surfaces in aquatic environments. These immobilized cells grow, reproduce, and produce extracellular polymers that can form a tangled matrix of fibers. A biofilm is the assemblage of these fibers (Characklis and Marshall, 1990). Biofilm-based technologies are currently implemented in the treatment of air, water and wastewater. Packed-bed reactors, in which biofilms accumulate on solid substrata or granular media packed within a tower or bed, are often used in water treatment. One example of a packed-bed reactor is a trickling filter where the influent liquid is spread over the top of a granular media by a sprinkler system and allowed to flow through the bed in a thin water layer over the biofilm (Viessman and Hammer, 1998). Oxygen, a required gas for aerobic processes, is drawn up the bed by natural convection. Since biofilm development depends on the adsorbable surface area of the media, a high surface-to-volume ratio, such as small and/or porous particles, is optimal. As mentioned earlier, sand is a typical example of medium used in conventional trickling filters. These trickling filters have shown little success for the removal of MIB from drinking water; thus, alternative media types must be explored (Ashitani et al, 1988). Yagi et al. (1988) examined one example of a biologically active filter for MIB reduction. The filter media for this study included activated carbon (coconut shell, 10x32 mesh), zeolite, and sand, and Bacillus subtilis IAM 12118 was used as the inoculum. The dimensions of the filter are provided in Table 2-5. Each filter was fed 0.9 liters of raw water, which was supplemented with additional MIB to maintain a constant concentration

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19 for each run (1.5 1.6 mg/l). The microbial transformation was estimated by subtracting the total MIB influent by the amount of MIB adsorbed and MIB extracted from the filtrate of a control, consisting of autoclaved medium to eliminating biological activity. The study also examined the amount of MIB adsorbed by the filter medium and the biological cells. The concentration of MIB was determined in these tests by GC/MS, with a minimum detection level of 0.1 mg/l. The results from this study can be found in Table 2-6. Table 2-5: Column parameters for the biological filtration study conducted by Yagi et al. (1988) Internal Diameter 1.5cm Length 20 cm Media volume 35 cm 3 Empty Bed Contact Time 2.4 min Hydraulic Loading 8.3 cm/min Table 2-6: Results for the biological filtration study conducted by Yagi et al. (1988) Parameter Carbon Medium Sand Medium Zeolite Medium Biological Activity Bio-Active Control Bio-Active Control Bio-Active Control MIB in raw water (mg/l) 1.6 1.5 1.5 1.5 1.5 1.6 Volume of filtrate (l) 0.9 0.9 0.9 0.9 0.9 0.9 Loading of MIB (mg/l) 1.4 1.4 1.4 1.4 1.4 1.4 MIB in Filtrate (mg/l) 0.2 <0.01 1.7 1.5 1.7 1.5 MIB in filtrate (mg) 0.18 <0.009 1.5 1.4 1.5 1.4 Estimated Adsorbed (mg) 1.2 1.4 -0.1 0 -0.1 0 Amount Extracted (mg) 0.53 1.33 0.02 0.01 0.06 0.2 Estimated Transformation (mg) 0.7 0.07 0 0 0 0 Estimated Depletion % 58 5 0 0 0 0 *These values, determined by GC/MS, represent the MIB removal characteristics of each column type. These data show that the activated carbon control removed MIB to the lowest effluent concentration. The effectiveness the activated carbon filter to remove MIB was diminished by biological activity; however, it appears that the bacteria present in the in

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20 this filter were responsible for the removal of some MIB via transformation. The data provided also shows that the sand and zeolite filters were not capable of removing MIB regardless of biological activity. It is important to note; however, that no information is provided quantifying the biomass concentration on the filter, allowing the possibility that there was insufficient inoculum present for significant MIB depletion. Also, the raw water fed into the column may have lacked the necessary nutrient concentrations for MIB transformation. The Effect of Ozonation the Biological Transformation of MIB Ozonation is becoming a popular drinking water disinfection method in the United States (Bitton, 1999). This is primarily because of its ability to kill microorganisms without producing trihalomethanes or other halogenated disinfection byproducts. Another consequence of ozonation is the oxidation of large organic compounds (i.e., humics and fulvics) into smaller compounds, such as ketones, ketoaldehydes, alkanes, and alcohols, that may be more easily biodegraded. The reduction of large organics in the influent may also increase the life of a carbon adsorber by saving pore space. Thus, the application of ozonation before a biological system may increase its effectiveness (Muramoto et al., 1995). Muramoto et al. (1995) reported the results of a full-scale application of ozonating water immediately before biological treatment. These researchers examined part of the treatment line in the Kanamachi Purification Plant in Tokyo, Japan (Fig 2-7). The MIB concentrations were analyzed by GC/MS after each stage to determine the removal efficiencies of each process. Figure 2-8 presents the average MIB concentrations that Muramoto et al. (1995) observed at each collection point over 3 months. Regardless of the month of sampling, approximately 20 percent of the influent MIB was removed by

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21 coagulation-sedimentation, and the remainder was removed by the combined ozonation biological treatment system. Chlorine Coagulant Sedimentation Ozonation Biofilter Filtration Effluent Influent Figure 2-7: Kanamachi treatment line (adapted from Muramoto et al., 1995) CoagulationSedimentation Ozonation BAC Raw W ater Figure 2-8: MIB concentrations in raw and processed waters (adapted from Muramoto et al., 1995) As shown in Figure 2-8, the Kanamachi treatment plant was capable of removing MIB to below the OTC, regardless of the MIB loading. Although coagulation and sedimentation processes removed a portion of the MIB, ozonation and BAC treatment effectively removed all of remaining MIB. Although the BAC system shows promise for the removal of MIB, it remains unclear whether the MIB was depleted through bacterial degradation or by adsorption onto the activated carbon.

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22 Conclusions Most consumer complaints regarding drinking water quality are due to malodors and tastes. Often, the odors are described as earthy-musty. Once an earthy-musty odor event occurs, water utilities can typically remove the responsible compounds using conventional water treatment with PAC. However, one chemical responsible for the earthy-musty odor, MIB (a cyanobacteria metabolite), is not effectively removed by these methods. This literature review examined previous studies of isolation and identification of microorganisms and microbial systems capable of removing MIB. Included in this review are papers covering the characteristics and origins of MIB, the isolation of microbial species capable of depleting MIB, the characterization and transformation kinetics of MIB-degrading bacteria, and selected water treatment technologies currently incorporating microbial degradation for MIB removal. Many of the studies mentioned reported MIB removal to below analytical detection limits; however, these studies did not clearly report the final concentration of MIB at the ng/l levels commonly found in natural waters or to a concentration that did not confer odor. To improve upon these methods, additional studies are needed to further assess the MIB transformation potential of microbial communities, particularly, the ability of cultures to deplete MIB at different initial concentrations, including the ng/l levels commonly found in natural waters. Also, improved methods of identification of MIB-degraders that involve genetics-based techniques are desirable because of their increased accuracy. By better understanding MIB concentration effects on the activity of MIB-degrading bacteria and the phylogeny of these microorganisms, drinking water facilities will be better able to design more effective biologically based systems that cater to these specific requirements.

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CHAPTER 3 CHARACTERIZATION OF A MICROBIAL CULTURE CAPABLE OF REMOVING TASTEAND ODOR-CAUSING 2-METHYLISOBORNEOL FROM WATER: A MANUSCRIPT TO BE SUBMITTED TO WATER RESEARCH Introduction Tastes and odors are considered by most consumers to be significant factors when determining potable water quality. The occurrence of taste and odor problems is widespread and has been reported in Asia, Austrailia, North America and Europe (Ashitani et al., 1988; Persson, 1983; Suffet et al., 1996; Zimmerman et al., 1995). Some of the more prominent tasteand odor-causing substances are naturally occurring organic compounds that produce an earthy-musty odor in drinking water (Rashash et al., 1997). Many of these organics have been identified and include 2-isopropyl-3-methyoxy pyrazine (IPMP), 2,3,6-trichloranisole (TCA), 2-isobutyl-3-methyoxy pyrazine (IBMP), trans-1, 10-dimethyl-trans-9-decahol (geosmin) and 2-methylisoborneol (MIB). These compounds are released by blue-green algae, typically from late spring through early fall, in concentrations reported to be as high as 100 ng/l (Tenauchi et al., 1995); however concentrations ranging from 10 ng/l can cause malodorous drinking water (Persson, 1980). While not a concern in terms of health impacts, the offensive odor and taste of MIB may lead to psychosomatic effects, such as headaches, stress, or stomach upsets (Young et al., 1996). Water utilities typically rely on conventional treatment methods to remove tasteand odor-causing compounds. These methods can effectively decrease IPMP, TCA, IBMP and geosmin concentrations to below their odor thresholds; however, they are 23

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24 typically unsuccessful in removing MIB to below its commonly accepted odor threshold concentration (OTC) of 10 ng/l (Ashitani et al., 1988). Recent studies have begun to examine biological treatment as an alternative method for MIB removal. Ishida et al. (1992) and Egashira et al. (1992) isolated several strains of bacteria from natural waters that are capable of using MIB as a growth substrate, including Pseudomonas aeruginosa, Pseudomonas pseudoalcaligenes, Bacillus sp., and Flavobacterium multivorum. Ishida et al. (1992) conducted batch and continuous feed experiments studying the removal of MIB by a bacterium isolated from Lake Kasumigaura in Japan. The results for the batch experiments showed the ability of the bacterium to deplete MIB at the mg/l and g/l levels, while the continuous feed study showed the isolated strain was capable of reducing influent MIB concentrations of 600 ng/l to approximately 60 ng/l. The researchers identified the isolated strain as a Bacillus sp. using phenotypic identification tests. Egashira et al. (1992) examined the removal of g/l concentrations of MIB in drinking water by a biological filter inoculated with surface water from Lake Biwa, Japan. This study also reported the effects of temperature and pH on MIB depletion in a packed column and identified isolated cultures from the filter media using a biochemical test kit. No error was reported in the studies performed by either Egashira et al. (1992) or Ishida et al. (1992); however, the trends of their results support the potential of MIB depletion by bacteria. Although many reports show positive findings on microbial depletion of MIB, the potential for microbial communities to deplete MIB to below the OTC has not been shown. Also, the effect of MIB concentration on microbial growth and metabolic activity has not been fully explored. The purpose of this study was to further assess the MIB

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25 transformation potential of microbial communities, particularly, the ability of cultures to deplete MIB at different initial concentrations including the ng/l levels commonly found in natural waters. The effect of varying MIB concentration on microbial growth and oxidation potential was also examined. The characterization and identification of the isolated culture was conducted by using transmission electron and light microscopy to examine fine cell structures and 16S rRNA gene sequencing to construct alignment profiles and a neighbor joining phylogenetic tree. Materials and Methods Enrichment and Isolation of MIB Transforming Bacteria Source water used as initial inoculum The source of inoculum used in this study was Lake Manatee in Manatee County, FL. Lake Manatee currently feeds the Manatee County Water Treatment Facility to the supply of potable water for Manatee County. This reservoir experiences periods of extensive algal blooms from late spring through early fall, resulting in average MIB concentrations in the raw water of approximately 25 ng/l. Water samples were collected in July 2000 in sterile glass bottles from the raw water source, which were then stored at 4 C until their use in the batch enrichment experiments. Enrichment procedures The enrichment for potential MIB-degrading bacteria was conducted by pumping 1 l of feed solution of buffered mineral salts medium (MSM) (Table 3-1) (Izaguirre et al., 1988) inoculated with 200 ml lake water and spiked with 6 mg/l MIB (Wako Pure Chemicals, LTD., Osaka, Japan) through anthracite-packed glass columns (Figure 3-1). The feed solution was circulated through the columns at a flow rate of 0.5 ml/min by a peristaltic pump for 5 days, at which time biological growth was visible on the anthracite.

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26 Table 3-1: Components of mineral salts media (Izaguirre et al., 1988) Species Concentration (mg/l) NH 4 Cl 50 K 2 HPO 4 100 MgSO 4 50 CaCl 2 20 FeCl 3 1 A control column was also run without added MIB. Further enrichment was then achieved in batch enrichments with flasks containing 10 ml MSM, 6 mg/l MIB, and 0.5 grams of anthracite that was removed from the biologically active columns. These flasks were then incubated in an incubator shaker at 30 C and 300 rpm until turbidity was observed after approximately 3 days. Repeated transfers to fresh MIB-enriched medium were performed before characterization was conducted. Effluent circulated back to pump I noculum with MIB medium Anthraci te Control (MSM Only) Column Parameters Length 9.7 cm Bed Volume 76.3 ml Flow Rate 0.5 ml/min Figure 3-1: A schematic of the anthracite column apparatus used to isolate a culture capable of depleting MIB.

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27 Solid culturing of the MIB-degrading culture was performed by streaking from the liquid cultures onto agar plates composed of MSM medium and 1.5% (w:v) agar Plates were subsequently placed in a sealed 4-liter dessicator equipped with an uncovered beaker containing 10 ml of 100 mg MIB/l of deionized water and incubated at 30 C. Characterization of the Isolated Mixed Culture Cellular morphology The cellular morphologies of the strains present in the isolated mixed culture were identified using light microscopy and transmission electron microscopy (TEM). The liquid cultures were sampled during the stationary phase in order to check for the presence of spores. For light microscopy observations, a pipet tip was touched to a colony and then inserted into a drop of deionized water on a glass slide. The glass slide was then examined and photographed using a Nikon Optiphot-2 microscope (Nikon, Tokyo, Japan) with either differential interference contrast or phase contrast optics. Liquid and solid cultures examined using TEM methods were prepared by first placing samples on a formvar-coated 300 mesh copper grid and treating with a negative stain. The liquid culture was prepared by placing 1 drop of the culture on the grid with an equal amount of 1% aqueous uranyl acetate. The liquid was wicked off the grid with filter paper after 2 minutes and rinsed once with deionized water. For the solid culture, a pipet tip was touched to a colony, inserted into a drop of deionized water mixed with an equal amount of 1% uranyl acetate on the grid, and wicked off the grid after 2 minutes as previously described. The negatively stained sample grids were then observed and photographed at 100 kv on a Zeiss EM10 electron microscope (Zeiss, Oberkochen, Germany).

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28 Genotypic characterization 16S rRNA genetic analysis of the dominant species in the isolated culture was performed by MIDI Labs Inc., (Newark, DE, USA) by using MicroSeq 500 16S ribosomal RNA (rRNA)-based bacterial identification system (Applied Biosystems, Foster City, CA). The 16S rRNA gene was PCR amplified from genomic DNA isolated from the isolated culture. The PCR primers used for the amplification corresponded to E. coli positions 005 and 1540 (full length packages) and 005 and 531 (500 base pair packages). Amplification products were purified from excess primers and deoxy nucleotide triphosphates (dNTPs) using Microcon 100 (Millipore, Billerica, MA, USA) molecular weight cut-off membranes and checked for quality and quantity by running a portion of the products on agarose gel. PCR cycling parameters used were 95 o C and 10 min for initial incubation, 95 o C and 30 seconds for melting, 72 o C and 45 seconds for chain extension and 60 o C and 30 seconds for annealing. Cycle sequencing of the 16S rRNA amplification products was carried out using AmpliTaq FS DNA polymerase and dRhodamine dye terminators (Roche Molecular Systems, Inc., Pleasanton, CA, USA). Excess dye-labeled terminators were removed from the sequencing reactions using a Sephadex G-50 spin column (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The products were collected by centrifuge, dried under vacuum and frozen at C until they were ready to load. Samples were then resuspended in a solution of formamide/blue dextran/EDTA and denatured prior to loading. The samples were electrophoresed on an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The results were analyzed using Applied Biosystems DNA editing and assembly software (Applied Biosystems, Foster City, CA, USA).

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29 Sequences were compared with previously identified sequences in the Microseq database using Microseq software (Applied Biosystems, Foster City, CA, USA) and in the National Center for Biotechnology Information (NCBI) GenBank database using BLAST (Altschul et al. 1990). Isolated culture sequences were aligned with closely related sequences from the Microseq database based on the percent genetic difference of the unknown culture strains with those of known strains. This alignment match was then used to construct the neighbor joining phylogenetic tree. Growth characterization of the isolated culture Specific growth rates and generation times of the isolated culture were determined from turbidity measurements using a UV-VIS spectrophotometer (Milton Roy Company, Ivyland, PA, USA) at a 600 nm wavelength. To assess the effect of MIB concentration on growth, initial MIB concentrations of 5, 10, and 20 mg/l were prepared in triplicate in MSM medium with 20% inoculum (v:v). These concentrations were chosen based on previous publications reporting observed microbial growth on MIB in the given range (Ishida and Miyaji, 1992; Izaguirre et. al, 1988). Chemical (MIB only) and biological (cells only) controls were also included with each set of concentrations. The specific growth rates were calculated using linear fits to the slope of the exponential growth phase of each growth curve obtained. MIB Depletion Potential of the Isolated Culture Oxygen uptake studies Oxidation has been reported as one significant microbial pathway for MIB transformation (Tanaka et. al, 1996; Oikawa et. al, 1995). In order to assess the MIB oxidation potential of the isolated culture, oxygen uptake analysis was first performed. Cell suspensions of the isolated culture were prepared for the oxygen uptake experiments

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30 by harvesting cultures at -log phase by centrifugation at 1.94 g for 25 minutes in a J2-HS Beckman centrifuge (Beckman Coulter, Fullerton, CA, USA). Cells were washed once in MSM to remove residual MIB and resuspended to a wet cell weight concentration of 0.2 g/ml. The final cell suspension was stored in an ice bath during the oxygen uptake experiments. A 1.9-ml glass, water-jacketed reactor (Figure 3-2) was used at a constant temperature of 30 o C to measure the rates of oxygen consumption at various initial substrate concentrations as described by Lindner et al. (2000). An electrolyte and membrane-covered Clarke-type electrode (Instech Laboratories, Plymouth, MA, USA) was inserted into the reactor using a ground-glass port with two rubber o-rings and was connected to a biological oxygen monitor (Yellow Springs, Yellow Springs, OH, USA). Monitor output was sent to an A/D converter board (DAS08-PGL, Computer Boards, Mansfield, MA, USA) for data collection using Labtech Notebook software (Wilmington, MA, USA). In all assays, the reaction chamber was filled with MSM before the addition of cells or substrate. The electrode was calibrated daily (following manufacturers instructions) after application of fresh electrolyte and membrane. Runs for each concentration were conducted in triplicate. Oxygen uptake rates were calculated following the methods presented by Hitchman (1978). To verify oxygen uptake by the isolated culture, controls included runs with MIB only and cells only, and all rates were corrected for endogenous metabolism.

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31 To DO Meterand DAQ system H2O InH2O Out Cells + MSM + MIB Figure 3-2: A schematic of the reactor used for the oxygen uptake experiments MIB depletion studies The protocol for the analysis of MIB depletion was developed using Solid Phase Micro Extraction (SPME) procedures described in Standard Method 6040D (APHA, 2001). The MIB depletion potential of the isolated culture was examined at two MIB concentrations, 5 mg/l and 25 ng/l. These concentrations were chosen because the highest rate of oxygen uptake was observed at 5 mg/l, and 25 ng/l represents average environmental concentrations observed in Lake Manatee during periods of high algal bloom. Microcosms were prepared in triplicate by addition of a stock solution of 100 mg/l MIB to flasks containing a mixture of 50 ml MSM and inoculum added to yield an optical density of 0.5 ( = 600 nm). Controls included with each set of experiments were a substrate control (MIB only) and a killed control with autoclaved cells. All microcosms were incubated in an incubator shaker at 30 C and 300 rpm and sampled at regular time intervals for analysis. The 5 mg/l and 25 ng/l cultures were incubated until no change in MIB concentrations were observed. Samples were collected from the 25 ng/l cultures by transferring 10 ml of the culture to a 20 ml scintillation vial containing 3 g of NaCl, which was then immediately capped. Samples were taken from the 5 mg/l MIB cultures and then serially diluted in deionized water to ng/l MIB concentrations, and these diluted solutions were transferred to 20 ml scintillation vials as previously described. Sample

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32 vials were stored at 4 C prior to analysis. All samples were analyzed by a Varian 3900 gas chromatograph (Varian, Inc., Palo Alto, CA, USA) with an ion trap ion detector, an Equity 5 fused silica capillary column (Supelco, Bellefonte, PA, USA), and a Saturn 2100 mass spectrophotometer (Varian, Inc., Palo Alto, CA, USA) with an auto-sampling system (CTC Analytics, Zweigen, Switzerland) fitted with a SPME fiber (Supelco, Bellefonte, PA, USA). The fiber, coated with divinyl-carboxen-polydimethylsiloxane cross-link, was injected into the headspace of each sample and allowed to equilibrate with the aqueous solution for 30 minutes. The fiber was then removed and inserted into the injection port of the GC/MS system where it was allowed to desorb for 5 minutes. Saturn Workstation version 5.52 data acquisition software (Varian, Inc., Palo Alto, CA, USA) was used for data analysis. Duplicates of each culture sample, a set of MIB standards, and a blank were run during the depletion studies to regularly calibrate the system. Results and Discussion Characterization of the Isolated Mixed Culture Cellular and colony morphology Homogenous, cream-colored, opaque colonies were observed on the agar 36 hours after inoculation, and full growth development was reached after an additional 60 hours of incubation. As summarized in Table 3-2, the colonies, approximately 2 mm in diameter, were round with a glassy surface, minimal elevation, and smooth edges.

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33 Table 3-2. Colony and cellular characteristics of the dominant strain in the isolated culture Characteristics Isolated Culture Colony Morphology Size 2 mm Shape Round Color Cream Topography Glassy Full Development 96 hours Elevation Minimal Edge Smooth Phase contrast light microscopy and transmission electron microscopy were used to elucidate cellular morphologies and fine structure of the strain(s) present in the isolated culture. Both techniques revealed that the isolated culture was composed of predominantly bacillus-shaped bacteria (Figure 3-3). The average cell size was approximately 5 m in length and 1 m in width. The presence of opaque structures, denoted by arrows in Figures 3-3A and 3-3B, indicates morphological evidence of sporulation. When observed under light microscopy the cells were highly motile, and flagella were observed in the TEM micrographs (Figure 3-3B). Flagellation and sporulation are cellular responses that are indicative of nutrient deprivation and allow the cells to move towards nutrient-rich regions or to remain dormant in periods of nutrient deprivation, respectively (Harwood, 1989). Both characteristics of these cellsflagellation and sporulation provide insight into the modes of survival that these cells possess in their natural reservoir environment that undergoes seasonal changes in MIB concentrations.

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34 10 m 1 m Figure 3-3: Phase contrast photograph (A) and transmission electron photograph (B) of the MIB-degrading isolated culture when grown on solid agar. Flagella and sporulation denoted with arrows. Genotypic characterization The 16S rRNA phylogeny results (Figure 3-4) from the MicroSeq alignment confirms placement of the isolated culture the Bacillaceae family of bacteria within a branch dominated by Bacillus strains. The isolated culture showed a 1.68% genetic difference (%GD) with its nearest relative Bacillus fusiformis (accession # AF2132169). Although this is not sufficient enough to place the unknown to the species level, it does match the isolated strain to the Bacillus genus level. It is important to note; however, that results from the BLAST search of the GenBank database showed that the closet match of the MIB unknown was with Bacillus Sphaericus, at a 1% GD. Bacillus sphaericus and its sub-species Bacillus fusiformis are both strictly aerobic, mesophilic bacilli that are capable of forming flagella and spherical endospores in order to adapt to nutrient deprivation. Bacillus sphaericus is currently applied in the remediation of other contaminants, including: urea herbicides (Doi and McGloughlin, 1992).

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35 MIB Unknown Figure 3-4: The neighbor joining phylogenetic tree of the isolated culture constructed from the MicroSeq alignment report. The top 10 closest genetic matches of the isolated culture are presented with their percent genetic difference. A lower % difference indicates a closer match. Growth characterization The effects of varying initial MIB concentration on specific growth rates were determined using the spectrophotometric methods as previously described. Figure 3-5 shows the growth curves of the cultures grown with initial MIB concentrations of 5, 10, and 20 mg/l. The highest specific growth rate calculated from each curve corresponded to an initial MIB concentration of 10 mg/l (0.067 + 0.015 hr -1 ). A summary of the specific growth rates and generation times calculated for each concentration is provided in Table 3-3.

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36 0.50.550.60.650.70.750.805101520Time (hr)Absorbance Figure 3-5: Growth curves of isolated culture at initial MIB concentrations of 5 mg/l (), 10 mg/l (), and 20 mg/l (). All absorbance measurements were taken at a wavelength of 600 nm. Table 3-3: A summary of the growth kinetics for the isolated culture grown at 5, 10, and 20 mg/l MIB. MIB ConcentrationSpecific Growth Rate ()Generation Time5 mg/l0.045 + 0.005 hr-1 15.64 + 1.73 hr 10 mg/l0.067 + 0.0015 hr-110.74 + 2.67 hr20 mg/l0.034 + 0.004 hr-120.56 + 2.42 hr MIB Depletion Potential of the Isolated Culture Oxygen uptake studies Figure 3-6 presents the oxygen uptake rates observed over a range of MIB concentrations from 2.5 mg/l to 20 mg/l. A maximum rate of 0.04 + 0.004 moles O 2 /s/ml was observed at 5 mg/l followed by a dramatic decrease in rates at higher MIB concentrations tested. As no effects on the probe were observed in controls with only MIB, this behavior, reported in other oxygen uptake studies (Lindner et al., 2000, 2003),

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37 suggests a toxicity effect on the cells. Whether this is caused by excessively high MIB concentrations or formation of toxic intermediates is not known. 00.0050.010.0150.020.0250.030.0350.040.0450.050510152025MIB Concentration (mg/l)Oxidation Potential Rates (micromole O2/s/ml cells) Figure 3-6: Oxygen uptake rates for the isolated culture over a range of MIB concentrations. MIB depletion studies The ability of the isolated culture to remove MIB at mg/l and ng/l concentrations was examined (Figure 3-7 and Figure 3-8). Five mg/l MIB, representing the high range of concentrations, was chosen because this was the concentration where the maximum rate of oxygen uptake was observed, as discussed previously. To represent the low and perhaps more environmentally relevant range of MIB concentrations, 25 ng/l was selected.

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38 0102030405060708002040608010Time (hr)% MIB Depleted 0 Figure 3-7: MIB depletion percent removal curves for isolated culture microcosms grown with initial MIB concentrations of 25 ng/l () and 5 mg/l (). As shown in Figure 3-7, each culture was capable of decreasing the concentration of MIB by over 50% in less than 60 hours. There was also no noticeable lag time experienced by either culture before depletion occurred. This is most likely due to each cultures acclimation to the initial concentrations through routine transfers conducted prior to the depletion studies. The culture grown with an initial MIB concentration of 5 mg/l (4.2 mg/l measured) depleted MIB at an average rate of 0.35 + 0.004 mg/l/hr, removing nearly 66% of the MIB to yield a final concentration of 1.8 + 0.02 mg/l MIB after 72 hours of incubation. The culture grown with an initial concentration of 25 ng/l (28.5 ng/l measured) removed MIB at a rate of 0.20 + 0.05 ng/l/hr and, after 96 hours of incubation, had reduced the concentration of MIB by approximately 58% to 9.6 + 0.3 ng/l. While the isolated culture was capable of depleting MIB at both concentration levels, it is interesting to note that it was able to remove MIB to below the OTC of 10 ng/l in the 25 ng/l microcosms and no odor was observed in the culture at the end of the

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39 study. The controls used in this study did not show any significant cell or substrate effects on MIB depletion and/or analytical recovery. 05101520253035020406080100120Time (hr)MIB (ng/l)012345MIB (mg/l) Figure 3-8: MIB depletion curves for isolated culture microcosms grown with initial MIB concentrations of 25 ng/l () and 5 mg/l (). Conclusions Microorganisms indigenous to aqueous surface-water environments that experience seasonal blue-green algae outbreaks may play an important role in cycling tasteand odor-causing compounds released by these microorganisms. Degradation of these compounds, such as MIB, may be a result of pure-culture or mixed-culture activity. Furthermore, opportunity may exist in using these bacteria for enhanced biodegradation for either in situ or ex situ applications. We obtained samples from a drinking water reservoir servicing Manatee County, FL, as it was our hypothesis that, because of the MIB outbreaks experienced in this reservoir on a seasonal basis, bacterial populations capable of using MIB as a growth substrate could be isolated. We report in this study successful isolation of a culture that is capable of degrading MIB to below odor threshold

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40 levels. The predominant strain in this culture was matched to the Bacillus genus level and most closely related by 16S rRNA analysis to Bacillus sphaericus. While previous studies that focused on isolation of MIB-degrading bacteria have also identified Bacillus strains (Ishida and Miyaji, 1992), these results showed the influence of MIB concentration on microbial activity. The maximum growth rates were observed at 10 mg/l MIB, whereas observed oxygen uptake rates were the highest at 5 mg/l MIB. Depletion of MIB was shown at 5 mg/l and 25 ng/l initial concentrations, and final concentrations of MIB below the OTC of 10 ng/l were observed in the latter case. The implications of these results are that microbial populations can be derived from natural water sources for removal of MIB and possibly other tasteand odor-causing compounds to concentrations that render drinking water as wholesome, defined by Young et al. (1996) as both toxicologically and aesthetically acceptable. The culture isolated in this study was capable of using MIB as a growth substrate at relatively high and low concentrations of substrate, thus suggesting the ability of this culture to remove MIB throughout the phases of a seasonal outbreak of blue-green algae. Future work on this study should include further examination of the effectiveness of biological removal of MIB from natural surface waters under water treatment operational conditions that include continuous flow and variation in water characteristics.

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CHAPTER 4 CONCLUSIONS The focus of this project was to examine the potential for microbial transformation of MIB by a culture isolated from water collected from Lake Manatee in Manatee County, Florida. This study was part of a larger project that included an investigation on tailoring granular activated carbon specifically for MIB removal. This work included a literature review and laboratory studies focusing on the isolation and characterization of microbial systems capable of removing MIB. The hypothesis that drove the laboratory phase of this project was that, because of the MIB outbreaks experienced in this reservoir on a seasonal basis, bacterial populations capable of using MIB as a growth substrate could be isolated. In this project, a MIB-degrading bacterial culture was isolated from a Lake Manatee water sample. The sample was used as an inoculum in a feed solution, composed of MSM and 6 mg/l MIB, which was passed through an anthracite column until growth was observed. Subsequently, the isolated culture was characterized by using growth kinetics to determine specific growth rates, light and TEM microscopy to examine fine cell structures, and 16S rRNA phylogenetic analysis to assess the closest match of the unknown isolate(s) to known bacterial strains. Finally, the MIB transformation potential of the microbial community was assessed by using oxygen uptake methods and batch microcosm experiments combined with SPME coupled with 41

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42 GC/MS to determine the ability of the culture to oxidize and degrade MIB at different initial concentrations and to deplete MIB to below the OTC. The results of this study supported the hypothesis by showing that a bacterial population was isolated from Lake Manatee that is capable of using MIB as a growth substrate. The predominant strain in this culture was matched to the Bacillus genus level and most closely related by 16S rRNA analysis to Bacillus sphaericus. While previous studies that focused on isolation of MIB-degrading bacteria have also identified Bacillus strains (Ishida et al., 1992), these results showed the influence of MIB concentration on microbial activity. Maximum growth rates were observed at 10 mg/l MIB, whereas observed oxygen uptake rates were the highest at 5 mg/l MIB. Depletion of MIB was shown at 5 mg/l and 25 ng/l initial concentrations, and final concentrations of MIB below the OTC of 10 ng/l were observed after 96 hr in the latter case. These results imply that microbial populations can be derived from natural water sources for removal of MIB and possibly other tasteand odor-causing compounds to concentrations that render aesthetically acceptable drinking water. The culture isolated in this study was capable of using MIB as a growth substrate at over a relatively large range of concentrations of substrate, thus suggesting the ability of this culture to remove MIB throughout the phases of a seasonal outbreak of blue-green algae. Currently, most drinking water facilities are equipped with powdered activated carbon and oxidation processes, such as chlorination, to remove tasteand odor-causing compounds. However, these technologies are insufficient in removing MIB. Alternatively, biological treatment systems should be considered for the removal of this

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43 compound. One possible biological system application is a biologically active granular activated carbon filter to be used as a polishing step of the treatment process. This system would have several advantages over conventional treatment, including the removal of MIB through carbon adsorption and biodegradation, the production of biologically stable water, the removal of trihalomethane precursors, and the extension of activated carbon bedlife (Bitton, 1999). Future work on this study should address questions concerning the effectiveness of biological removal of MIB from natural surface waters. A closer study of the biodegradation pathway(s) followed and toxicity mechanism(s) should be pursued to elucidate whether intermediates formed render odor and/or toxicity effects. Also, the effect of water treatment operational conditions, including continuous flow and variation in water characteristics, should be determined before a pilot-scale system is designed. Finally, filtration media of different physical and chemical characteristics should be assessed for optimal microbial growth. Answers to these questions will ensure that the ultimate design of a large-scale biological system is the most effective in terms of economics and performance.

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LIST OF REFERENCES Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) Basic local alignment search tool. Journal or Molecular Biology 215, 403-410. APHA, AWWA (2001) Standard Methods for the Examination of Water and Wastewater 20 th Edition. American Public Health Association, Washington, DC. Ashitani, K., Hishida Y., and Fujiwara K. (1988) Behavior of musty odorous compounds during the process of water treatment. Water Science and Technology 20, 261-267. Brand, G. (1995) Evaluation of SPME Technology for the applicability of drinking water analysis. Proceedings Water Quality Technology Conference, San Francisco, CA Nov 6-10, 1994 1, 273. Bitton, G. (1999) Wastewater Microbiology. John Wiley and Sons, Inc., New York, NY. Characklis, W. and Marshall, K. (1990) Biofilms. John Wiley and Sons, Inc., New York, NY. Chester, B. and Cleary,T. (1980) Evaluation of the Minitek System for identification of nonfermentative and nonenteric fermentative gram-negative bacteria. Journal of Clinical Microbiology 12, 509-516. Doi, R. H. and McGloughlin M. (1992) Biology of Bacilli: Applications to Industry. Butterworth-Heinemann, Boston, MA. Egashira, K., Ito, K. and Yoshiy, Y. (1992) Removal of musty odor compound in drinking water by biological filter. Water Science and Technology 25, 307-314. Eisert, R. and Levsen, K. (1996) Solid-phase microextraction coupled to gas chromatography: a new method for the analysis of organics in water. Journal of Chromotography 733, 143-157. Harwood, C. (1989) Bacillus. Plenum Press, New York, NY. 44

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45 Herzing, D., Snoeyink,V. and Wood N. (1977) Activated carbon adsorption of the odorous compounds 2-methylisoborneol and geosmin. Journal of the American Water Works Association 69, 223-228. Hitchman, M. (1978) Measurement of Dissolved Oxygen. John Wiley and Sons, Inc., New York, NY. Hosaka, M., Murata K., Iikura, Y., Oshimi A. and Udagawa, T. (1995) Off-flavor problem in drinking water of Tokyo arising from the occurrence of musty odor in a downstream tributary. Water science and Technology 31, 29-34. Ishida, H. and Y. Miyaji. (1992) Biodegradation of 2-methylisoborneol by oligotrophic bacterium isolated from a eutrophied lake. Water Science and Technology 25, 269-276. Izaguirre, R., Wolfe, L., and Means, E.G. (1988) Bacterial Degradation of 2Methylisoborneol. Water Science and Technology 20, 205-210. Juttner, F. (1995) Physiology and biochemistry of odorous compounds from freshwater cyanobacteria and algae. Water Science and Technology 31, 69-78. Lalezary, S., Pirbazari, M., and McGuire, M. (1986) Oxidation of 5 earthy musty taste and odor compounds. Journal of the American Water Works Association 78, 62-69. Lindner, A.S., Adriaens, P., and Semrau, J.D. (2000) Transformation of ortho-substituted biphenyls by Methylosinus trichosporium OB3b: substituent effects on oxidation kinetics and product formation. Archives of Microbiology 174, 35-41. Lindner, A.S., Whitfield, C., Chen, N., Semrau, J.D., and Adriaens, P. (2003) Quantitative structure-biodegradation relationships for Ortho-substituted biphenyl compounds oxidized by Methylosinus trichosporium OB3b. Environmental Toxicology Chemistry 22, 2251-2257. Llyod, S., Lea, J., Zimba, P., and Grimm, C. (1998) Rapid analysis of Geosmin and 2-methylisoborneol in water using solid phase micro extraction procedures. Water Research 32, 2140-2146. Madigan, M., Martinko, J., and Parker, J. (2000) Brock Biology of Microorganisms. Prentice-Hall, Inc., Upper Saddle River, NJ. Medsker, L., Jenkins, J., Thomas, J., and Koch, C. (1969) 2-Exo-hydroxy-2-methylborane, the major odorous compound produced by several actinomycetes. Environmental Science and Technology 3, 461-464.

PAGE 57

46 Muramoto, S., Udagawa, T., and Okamura, T. (1995) Effective removal of musty odor in the Kanamachi Purification Plant. Water Science and Technology 31, 219-222. Ney, Robert (1990) Where did that Chemical Go? A Practical Guide to Chemical Fate and Transport in the Environment. Van Nostrand Reinhold, New York, NY. Namkung, E. and Rittmann, B. (1987) Removal of taste and odor causing compounds by biofilms grown on humic substances. Journal of the American Water Works Association 79, 107-112. Oikawa E., Ishibashi A., and Ishibashi, Y. (1995) 2-methylisoborneol degradation by the cam operon from Pseudomonas putida. Water Science and Technology 31, 79-86. Persson, P. (1983) Sensory properties and analysis of two muddy odor compounds Geosmin and 2-methylisoborneol in water and fish. Water Research 4, 1113-1118. Pirbazari, M., Borow, H., Craig, S., Ravindran, V., and McGuire, M.J. (1992) Physical chemical characterization of five earthy musty smelling compounds. Water Science & Technology 25, 81-88. Priest, F., Goodfellow, M., and Todd, C. (1988) A numerical Classification of the Genus Bacillus. Journal of General Microbiology 134, 1847-1882. Rashash, D., Dietrich, A., and Hoehn, R. (1997) FPA of selected odorous compounds. Journal of the American Water Works Association 89, 131-141. Suffet, I.H, Corado, A., Chou, D., Maguire, M., and Butterworth, S. (1996) AWWA taste and odor survey. Journal of the American Water Works Association 88, 166-180. Tanaka. A., Oritani, T., Uehara, F., Saito, A., Kishita, H., Niizeki, Y., Yokota, H., and Fuchigami, F. (1996) Biodegradation of a musty odour component, 2-methylisoborneol. Water Research 30, 759-761. Tenauchi, N., Ohtani, T., Yamanaka, K., Tsuji, T., Sudou, T., and Ito, K. (1995) Studies on a biological filter for musty odor removal in drinking water process. Water Science and Technology 31, 229-235. Veissmann, W. and Hammer, M. (1998) Water Supply and Pollution Control. Addison-Wesley Menlo Park, CA. Watson, S., Brownlee, B., Satchwill, T., and Erika E. Hargesheimer. (2000)

PAGE 58

47 Quantitative analysis of trace levels of geosmin and MIB in source and drinking water using headspace SPME. Water Research 34, 2818-2828 Yagi, M., Nakashima, S., and Muramoto, S. (1988) Biological degradation of musty odor compounds, 2-methylisoborneol and geosmin in a bio-activated carbon filter. Water Science and Technology 20, 255-260. Young, W.F., Horth, H., Crane, R., Ogden, T., and Arnott, M. (1996) Taste and odour threshold concentrations of potential potable water contaminants. Water Research 30, 331-340. Zimmerman, W. J., Soliman, C.M., and Rosen, B.H. (1995) Growth and 2methylisoborneol production by the cyanobacterium Phorium LM 689. Water Science and Technology 31, 181-186.

PAGE 59

BIOGRAPHICAL SKETCH I began studying at the University of Florida in August 1997. The multidisciplinary approach to solving problems for the health and well being of society drew me to the field of environmental engineering. As I progressed through my B.S. degree in environmental engineering, I found particular interest in classes covering the biological treatment of contaminants. When I completed my B.S. degree in 2001, I knew I wanted to further explore these subjects; therefore, I decided to attend graduate school for a masters degree in environmental engineering with a focus on biological remediation. The challenge of pursuing this degree has been unparalleled; however, the skills and experience I have obtained along the way, I know, will benefit me for the rest of my life. I look forward to continuing my education in environmental engineering when I begin work as a potable water engineer this June. 48


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CHARACTERIZATION OF A MICROBIAL CULTURE CAPABLE OF
REMOVING TASTE- AND ODOR-CAUSING
2-METHYLISOBORNEOL FROM WATER













By

CHANCE VENABLE LAUDERDALE


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF ENGINEERING

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Chance Venable Lauderdale
































This thesis is dedicated to Marley Lauderdale.















ACKNOWLEDGMENTS

I would like to thank Dr. Paul Chadik, Dr. David Mazyck, Dr. Angela Lindner and

Dr. Nancy Szabo for the time and assistance they have offered to me throughout this

study and for serving on my graduate advisory committee. I would especially like to

thank Dr. Angela Lindner for her patience, her mentoring, and for challenging me to

improve the quality of my work. I also thank Matt Booth, Rick Loftis, Adriana Pacheco

and Jessica Strait for the training and assistance they provided me in the laboratory.

Finally, I would like to thank my parents and my brother, Greg, for giving their support,

without which, none of this would have been possible.
















TABLE OF CONTENTS

Page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ...... ..... ................ ... ...................... .... vii

LIST OF FIGURES .................. ....................... ................. viii

A B ST R A C T .......... ..... ...................................................................................... x

CHAPTER

1 INTRODUCTION ............... ................. ........... ................. ... ..... 1

2 LITER A TU RE REV IEW .................................................. ............................... 3

Introduction to Taste and Odors in Drinking Water: Description, Causes, and Brief
H isto ry ...................................... ................................................... ... 3
Methylisoborneol: Isolation, Formation, Properties, Conventional Treatment and
A n a ly sis ................................ .............................................................................. .. 4
Initial Isolation of M ethylisoborneol .............. ..............................................4
Natural Formation of M ethylisoborneol..................... ......................................5
Physical and Chemical Characteristics of Methylisoborneol.............................6.
M IB D election and A analysis M ethods ........................................ .....................8
Conventional Water Treatment for MIB Removal..............................................9
B acterial Transform ation of M IB ....................................................... ................ ... 11
Isolation and Identification of Potential MIB-Degrading Bacteria....... ........... 11
Characteristics of M IB-Degrading Bacteria................................ ... ................ 12
Applications of Technology Using Biological Transformation for MIB Removal in
W after T reatm ent ........................ .................... .. ..................... .... ............... 17
Introduction to the Use of Bioremediation in Water Treatment..........................17
B biological Filtration.......... .................. .... ..... .. ......... .. ............... 18
The Effect of Ozonation the Biological Transformation of MIB.....................20
C o n clu sio n s..................................................... ................ 2 2

3 CHARACTERIZATION OF A MICROBIAL CULTURE CAPABLE OF
REMOVING TASTE- AND ODOR-CAUSING 2-METHYLISOBORNEOL FROM
WATER: A MANUSCRIPT TO BE SUBMITTED TO WATER RESEARCH.........23

Introduction ................... .... .......... ............. ............ ............ 23









M materials and M methods ..................................... .............................................. 25
Enrichment and Isolation of MIB Transforming Bacteria ......... ...........25
Source w ater used as initial inoculum .................................. ... ..................25
Enrichm ent procedures............................................... ......... ................... 25
Characterization of the Isolated Mixed Culture ..................... ...............27
Cellular m orphology ............................................................................. 27
Genotypic characterization..................... ....... ....................... 28
Growth characterization of the isolated culture ............... ............. .....29
MIB Depletion Potential of the Isolated Culture..............................................29
O xygen uptake studies ........................................ ........................... 29
M IB depletion studies ............................................................................ 31
R results and D discussion ......................... .......... ..... .. ........ .. .......... 32
Characterization of the Isolated Mixed Culture ...............................................32
Cellular and colony morphology........................................... .............32
Genotypic characterization................... ....... ........................... 34
Grow th characterization .................................................... ...... ......... 35
MIB Depletion Potential of the Isolated Culture.............................................. 36
O xygen uptake studies ........................................ ........................... 36
M IB depletion studies ............................................................................ 37
C o n c lu sio n s........................................................................................................... 3 9

4 CON CLU SION S .................................. .. .......... .. .............41

L IST O F R EFE R E N C E S ............................................................................. ............. 44

B IO G R A PH IC A L SK E TCH ..................................................................... ..................48
















LIST OF TABLES


Table pge

2-1: Odor threshold concentrations for 5 earthy-musty odor compounds (Lalezary et al.,
1986; Persson, 1980) ............... ............. ............................... 4

2-2: Physical-chemical characteristics of MIB (Pirbazari et al., 1992) compared to high
and low values as described by Ney (1990)............... ...........................................8

2-3: Conventional treatment methods and powdered activated carbon (PAC) average MIB
rem oval efficiencies (A shitani et al., 1988) .................................. ............... 10

2-4: MIB depletion potential for the identified species isolated from a biological
treatment filter (Egashira et al., 1992)................................ ...............17

2-5: Column parameters for the biological filtration study conducted by Yagi et al.
( 1 9 8 8 ) ........... ................ ..... ....................... ............ 1 9

2-6: Results for the biological filtration study conducted by Yagi et al. (1988) ..............19

3-1: Components of mineral salts media (Izaguirre et al., 1988).............. .......... 26

3-2: Colony and cellular characteristics of the dominant strain in the isolated culture.....33

3-3: Summary of the growth kinetics for the isolated culture grown at 5, 10, and 20
m g/1 M IB ......... .. ...... .................. .. ............ 36















LIST OF FIGURES


Figurege

2-1: 2-Methylisobomeol, structure identified by Medskar et al. (1969).............................5

2-2: Plot depicting the linear relationship between MIB concentration and the filament
concentration of Oscillatoria sp.3 (adapted from Hosaka et al., 1995) .................6

2-3: Depletion by Bacillus HI-5 at 2 different initial concentrations of MIB (adapted from
Ishida and M iyaji, 1992) ...................... .............. ............... ......... 13

2-4: Growth curves for Bacillus HI-5 at 2 different initial MIB concentrations (adapted
from Ishida and M iyaji, 1992).......................................... ............................ 14

2-5: Effect of pH variation on the MIB depletion potential of a biologically active column
(adapted from Egashira et al., 1992) ........ ................................................... ................ 15

2-6: The logarithmic relationship between temperature and the MIB depletion potential of
a biologically active column (adapted from Egashira et al., 1992).........................15

2-7: Kanamachi treatment line (adapted from Muramoto et al., 1995) ...........................21

2-8: MIB concentrations in raw and processed waters (adapted from Muramoto et al.,
1 9 9 5 ) .................................................................................. . 2 1

3-1: A schematic of the anthracite column apparatus used to isolate a culture capable of
depleting M IB ..................................................... ................. 26

3-2: A schematic of the reactor used for the oxygen uptake experiments......................... 31

3-3: Phase contrast photograph (A) and transmission electron photograph (B) of the MIB-
degrading isolated culture when grown on solid agar. Flagella and sporulation
denoted w ith arrow s. ............................ ........... ...... ...... ...... ...... 34

3-4: The neighbor joining phylogenetic tree of the isolated culture constructed from the
MicroSeq alignment report. The top 10 closest genetic matches of the isolated
culture are presented with their percent genetic difference. A lower % difference
indicates a closer m atch ............. .... ...................................................... ...... ........ 35









3-5: Growth curves of isolated culture at initial MIB concentrations of 5 mg/1 (*), 10
mg/1 (0), and 20 mg/1 (A). All absorbance measurements were taken at a
w avelength of 600 nm .......................... ........................ ......... ........... 36

3-6: Oxygen uptake rates for the isolated culture over a range of MIB concentrations. ...37

3-7: MIB depletion percent removal curves for isolated culture microcosms grown with
initial MIB concentrations of 25 ng/1 (*) and 5 mg/1 (*).....................................38

3-8: MIB depletion curves for isolated culture microcosms grown with initial MIB
concentrations of 25 ng/1 (*) and 5 mg/1 (* )............... ................. ...................39















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Engineering

CHARACTERIZATION OF A MICROBIAL CULTURE CAPABLE OF REMOVING
TASTE- AND ODOR-CAUSING 2-METHYLISOBORNEOL FROM WATER
By

Chance Venable Lauderdale

August 2004

Chair: Angela Lindner
Major Department: Environmental Engineering Sciences

A common blue-green algae metabolite, 2-methylisobomeol (MIB), is responsible

for unpalatable drinking water in Asia, Australia, North American and Europe. Current

water treatment technologies, are ineffective in removing MIB from potable water. The

focus of this project was to examine the potential for microbial transformation of MIB by

a culture isolated from a drinking water reservoir, Lake Manatee, in Manatee County,

Florida. This culture was characterized using phenotypic and genotypic methods as well

as by assessing its growth and MIB-depletion potentials using growth and oxygen uptake

experiments and microcosms coupled with solid-phase microextraction (SPME) and gas

chromatography/mass spectrophotometry (GC/MS). The predominant strain in the

isolated culture was bacillus in shape and possessed spore and flagella, and 16S rRNA

analysis determined that this isolated culture is most similar to Bacillus sphaericus (99%

match). The ability of this culture to transform MIB was examined by running oxygen

uptake measurements and by conducting depletion studies using SPME coupled with









GC/MS for MIB analysis. The results obtained from these studies demonstrated the

isolated culture was capable of using MIB as its sole source of carbon and depleting MIB

to below its odor threshold concentration (OTC) of 10 ng/1. Implications of these results

are that microbial populations can be isolated from natural water sources for the removal

of MIB and potentially other taste- and odor-causing compounds to concentrations that

render aesthetically acceptable drinking water.














CHAPTER 1
INTRODUCTION

2-Methylisoborneol, a common blue-green algae metabolite released in surface

waters typically from late spring to early fall, has been a cause of unpalatable drinking

water in Asia, Australia, North American and Europe. Conventional water treatment

technologies, consisting of breakpoint pre-chlorination, coagulation, sedimentation, and

post chlorination, are not effective in removing MIB from potable water to below its odor

threshold of 10 ng/1 (Lalezary et al., 1986; Ashitani et al., 1988). As a supplement o

water treatment, powdered activated carbon (PAC) is often added to water to remove

MIB. However, PAC addition is not cost effective at higher MIB concentrations (Herzing

et al. 1977). Seeking a possible solution, recent studies have begun to examine biological

treatment as an alternative treatment method for MIB removal. To date, several strains

of bacteria have been isolated from natural waters that are capable of using MIB as a

growth substrate (Ishida and Miyaji, 1992; Egashira et al., 1992). Although these studies

have shown MIB removal by these cultures, the potential for microbial communities to

deplete MIB to below the odor threshold concentration (OTC) has not been shown. Also,

the effect of MIB concentration on microbial growth and metabolic activity has not been

fully explored.

The focus of this research was to examine the potential for microbial

transformation of MIB by a culture isolated from water collected from Lake Manatee in

Manatee County, Florida. The hypothesis of this project was that, because of the MIB









outbreaks experienced in this reservoir on a seasonal basis, bacterial populations capable

of using MIB as a growth substrate could be isolated. The scope of this work was

composed of 4 major objectives. The first included a literature review covering the

current knowledge of the origins and properties of MIB as well as current technologies

involving biological treatment. The next objective was to isolate a bacterial culture

capable of growth on MIB from a water sample obtained from Lake Manatee.

Subsequently, the isolated culture was to be characterized by using growth kinetics to

determine specific growth rates, light and TEM microscopy to examine fine cell

structures, and 16S rRNA phylogenetic analysis to assess the closest match of the

unknown isolate(s) to known bacterial strains. The final objective was to assess the MIB

transformation potential of the microbial community by using oxygen uptake methods

and batch microcosm experiments combined with solid phase microextraction analysis to

determine the ability of the culture to oxidize and degrade MIB at different initial

concentrations and to deplete MIB to below the OTC.

The results from this project promise to benefit water treatment facilities and their

customers by providing applied results directed towards the remediation of MIB

contamination. Additionally, this research will add to the continuously growing body of

knowledge addressing the use of biological treatment for improved potable water quality.














CHAPTER 2
LITERATURE REVIEW

Introduction to Taste and Odors in Drinking Water: Description, Causes, and Brief
History

Tastes and odors are considered by most consumers to be significant factors when

determining potable water quality. In 1973, a Gallup Poll indicated that most consumer

complaints concerning drinking water involved tastes and odors. As far back as 1957,

surveys given to both water utilities and consumers have consistently reported similar

problems (Suffet et al., 1996). The occurrence of taste and odor problems is widespread and

has been reported in Argentina, Australia, Canada, Denmark, England, Finland, Germany,

Israel, Japan, The Netherlands, Norway, Poland, Sweden, U.S.A and U.S.S.R. (Ashitani et

al., 1988; Juttner, 1995; Persson, 1983; Suffet et al., 1996; Zimmerman et al., 1995).

Some of the more prominent taste- and odor-causing substances are naturally occurring

organic compounds that produce an earthy-musty odor in drinking water (Rashash et al.,

1997). Examples of these compounds include 2-isopropyl-3-methoxypyrazine (IPMP), 2,3,6-

trichloranisole (TCA), 2-isobutyl-3-methoxypyrazine (IBMP), trans-1,10-dimethyl-trans-9-

decalol (geosmin) and 1,2,7,7-tetramethylbicyclo[2.2.1]heptan-2-ol, also known as

2-methylisoborneol (MIB). Although these compounds are not deleterious to human health,

they can cause malodorous drinking water at extremely low concentrations (<10 ng/1)

(Persson et al., 1980; Lalezary et al., 1986; Rashash et al., 1997). The odor threshold

concentrations (OTC) of these compounds, defined as the concentration at or above which

odor can be detected, are provided in Table 2-1 (Lalezary et al., 1986; Persson, 1980).









Table 2-1: Odor threshold concentrations for 5 earthy-musty odor compounds (Lalezary et
al., 1986; Persson, 1980)
Common Name Chemical Formula OTC (ng/1)
Geosmin C12H220 4
IPMP CsH120N2 2
IBMP C9H140N2 2
TCA C7H5OC13 7
MIB C11H200 10
*Odor threshold concentrations determined by an odor panel

Water utilities typically rely on conventional treatment methods, supplemented with

powder activated carbon (PAC), to remove taste- and odor-causing compounds. These

methods can effectively decrease IPMP, TCA, IBMP and geosmin concentrations to below

their odor thresholds; however, these processes have been shown to be unsuccessful in

removing MIB to below its OTC (Ashitani et al., 1988). Today, some alternative methods for

MIB treatment, including biological transformation, granular activated carbon, and

ozonation, are being examined. This literature review summarizes previous studies reporting

the origins and properties of MIB as well as current technologies involving biological

treatment.



Methylisoborneol: Isolation, Formation, Properties, Conventional Treatment and
Analysis

Initial Isolation of Methylisoborneol

In 1969, Medsker et al. first reported successful isolation and identification of MIB

from actinomycetes, a group of terrestrial gram-positive bacteria that form branching

filaments. They described the compound as "camphor-smelling" and the major odorous

constituent in 3 out of 28 species of actinomycetes surveyed. The empirical formula (Table

2-1) and structure (Fig. 2-1) were identified by mass spectrometry.









CH3

H3C CH3
OH

CH3
Figure 2-1: 2-Methylisobomeol, structure identified by Medskar et al. (1969)

Natural Formation of Methylisoborneol

Cyanobacteria are prokaryotic oxygenic phototrophs that are found in a variety of

ecological settings, including terrestrial, freshwater and marine habitats (Madigan et al.

2000). In freshwater lakes, especially those that are eutrophic, cyanobacteria can develop

massive accumulations, known as blooms. Cyanobacteria and their resulting blooms are

responsible for the production of many odor-causing compounds, including MIB in natural

waters.

Numerous species of MIB-producing cyanobacteria have been isolated and classified,

and these include Oscillatoria sp., Anabaena sp. and Phormidium sp. (Hosaka et al., 1995;

Juttner et al., 1995; Zimmerman et al., 1995). These microorganisms synthesize MIB during

normal growth. MIB is believed to be a methylation product of an unknown monoterpene

formed from acetate and mevalonate (Juttner et al., 1995). The function of MIB in the cell

has yet to be understood completely, and the compound may simply be a by-product of the

photosynthetic pathway. After MIB is synthesized, it is found either bound to thylakoid

membranes and cytoplasmic proteins or excreted by the cell.

The production of MIB varies by cyanobacteria strain. Some species, such as

Oscillatoria sp. 3 and Phormidium tenue, demonstrate a proportionate relationship between

the number of filaments, or chains of cells, and MIB concentration. A single filament of

Oscillatoria sp. 3 produces an average of 19.5 picograms ofMIB (Hosaka et al., 1995). The

graph shown in Figure 2-2, compiled from data collected from the Ooba River in Japan,









further illustrates the effect of filament/ml (Oscillatoria sp. 3) on MIB concentration (Hosaka

et al., 1995).


c 300

200

0 100
0

0 2 4 6 8 10 12 14 16
Number of filaments per ml

Figure 2-2: Plot depicting the linear relationship between MIB concentration and the filament
concentration of Oscillatoria sp.3 (adapted from Hosaka et al., 1995)

These data imply that during a period of algal bloom, typically occurring from late

spring to early fall, the MIB concentration in surface water supplies will increase. If

eutrophication increases in the freshwater bodies used for drinking water, the potential for

increased algal concentrations and, thus, MIB releases is suggested by these findings.

Physical and Chemical Characteristics of Methylisoborneol

The physical and chemical characteristics of MIB dictate its behavior in the

environment and in water treatment plants. The fate of MIB in a treatment process may be

predicted and explained, at least in part, by examining its following properties: density,

aqueous solubility, octanol-water partition coefficient, and Henry's constant (Ney, 1990).

Density is a valuable characteristic when assessing the physical separation potential of

a compound in the aqueous phase. The density of MIB is approximately 0.9288 g/cm3

(Pirbazari et al., 1992). This value is similar to that of water, indicating MIB is unlikely to

pool at either the surface or bottom of the water column.









A compound's aqueous solubility is a measurement of its affinity for water.

Compounds with high aqueous solubility (>1000 mg/1) are hydrophilic, whereas compounds

with low aqueous solubility (<10 mg/1) are hydrophobic. Compounds with a high aqueous

solubility are typically more mobile and bioavailable, thus have a higher potential to be

biodegraded (Ney, 1990). MIB has an aqueous solubility of 194.5 mg/1 (Pirbazari et al.,

1992), which is close to the low range, and would, therefore, be expected to be less mobile in

the aqueous environment, less biodegradable, and more tending to sorb to sediments.

The octanol-water partition coefficient (Kow) indicates a chemical's tendency to sorb to

soils and sediments, bioconcentrate in aquatic organisms and accumulate in the soil. A Kow

value greater than 1000 suggests that a chemical has an affinity for bioaccumulation in the

food chain, has a low aqueous solubility and has a low mobility in the soil and aqueous

phases, whereas a chemical with Kow value of less than 500 would be more bioavailable,

soluble, and mobile (Ney, 1990). MIB has an octanol-water partition coefficient of

approximately 1349 (Pirbazari et al., 1992), indicating it is lipophilic and thus has a tendency

to partition out of the aqueous phase.

Henry's Constant (KH) demonstrates the ability of a chemical to partition between the

aqueous phase and the atmosphere and it can be estimated by ratioing the vapor pressure and

the aqueous solubility. A chemical with a high KH value (>0.4 1-atm/mol) is more likely to

escape the from aqueous phase to the vapor phase, whereas a chemical low KH value (<0.004

1-atm/mol) would most likely remain in the aqueous phase (Ney, 1990). The KH value for

MIB is 5 x 10 81-atm/mol (Pirbazari et al., 1992), indicating that MIB will not readily escape

from the aqueous phase. Table 2-2 summarizes the physical and chemical characteristics

of MB.









Table 2-2: Physical-chemical characteristics of MIB (Pirbazari et al., 1992) compared to high
and low values as described by Ney (1990)
Observed Value
Characteristic erd V e Low Value High Value
for MIB
Density 0.9288 g/cm3
Aqueous Solubility 194.5 mg/1 <10 mg/1 >1000 mg/1
Octanol/Water Coefficient 1349 <500 >1000
5.76 x 10-
Henry's Law Constant 5-atm/l <0.004 1-atm/mol >0.4 1-atm/mol
1 -atm/mol

In summary, MIB is expected to have moderately low motility in the aqueous

environment, with a large portion partitioned out of the aqueous phase via bioaccumulation

and/or sorbtion onto sediments. The low Henry's constant indicates MIB is unlikely to

escape into the atmosphere.

MIB Detection and Analysis Methods

The detection and quantitative analysis of MIB requires the use of technically complex

and time-consuming analytical methods, as it is commonly found in natural waters at ultra-

trace concentrations (Watson et al., 2000). Because MIB is found at such low natural

concentrations, traditional analysis of MIB typically relies on concentrating large sample

volumes (100-1000 ml). The preparation and analysis methods used, including liquid-liquid

extraction, purge and trap, closed-loop stripping analysis and simultaneous distillation-

extraction, are time-intensive and/or require high-resolution mass spectrophotometers (Lloyd

et al., 1998).

As a means of avoiding the constraints of time and cost involved in using the large

sample volumes required in these concentration methods, solid phase microextraction

(SPME) methods coupled with a gas chromatograph-mass spectrophotometer (GC/MS)

system have been recently developed By combining SPME and GC/MS, detection of MIB

at ng/1 levels is possible without the large sample volumes. Typically, a sample volume of 25









ml is sufficient. SPME-GC/MS is an inexpensive and rapid method for the analysis of

volatile and semi-volatile compounds occurring in the headspace of water matrices (Eisert

and Levsen ,1996). Techniques using SPME-GC/MS were originally applied by the Des

Moines Water Works for taste and odor analysis and have recently been employed by the

City of Tampa for the analysis of MIB and geosmin (Brand, 1995). Currently, a SPME-

GC/MS method is undergoing balloting for consideration as Standard Method 6040D

(APHA, 2001). This SPME method is based on the adsorption of MIB on a fiber coated

with divinylbenzene-carboxen-polydimethyloxane cross-link. This fiber is placed in the

headspace of a sealed vial and allowed to equilibrate with an aqueous sample. After

equilibrium is reached (typically 30-35 minutes), the fiber is removed and injected into the

port of a GC/MS system, where it is heated allowing the analytes to be desorbed for analysis.

The minimum detectable concentration of MIB analyzed by these methods < 5 ng/1 and the

recovery of the laboratory control standard of 20 ng/1 is 95+10% depending on the laboratory

(APHA, 2001). Although, this standard method appears to be a good alternative to other

forms of MIB analysis, it has not been validated in for many matrices, therefore, prior to

sample analysis, recoveries using these matrices with known additions of MIB should be

examined (APHA, 2001).

Conventional Water Treatment for MIB Removal

As stated previously, municipal water utilities have confronted problems with MIB for

many years. Typical water treatment consists of breakpoint pre-chlorination, coagulation,

sedimentation, rapid sand filtration and post-chlorination. Many studies have reported that

current conventional treatment methods do not sufficiently remove MIB (Ashitani et al.,

1988; Rashash et al., 1997; Suffet et al., 1996). Powder activated carbon (PAC) is often

added as a supplement to a treatment line to decrease the MIB concentration; however, this









practice is cost-effective only for low dosing (Herzing et al., 1977). A study conducted by

Ashitani et al. (1988) examined several possible water treatment scenarios, including

coagulation alone, pre-chlorination followed by coagulation/sedimentation, pre-chlorination

followed by coagulation/sedimentation then PAC, and full conventional treatment (including

PAC) (Ashitani et al., 1988). These results (Table 2-3) show MIB removal by coagulation

alone was greater than that of prechlorination with coagulation. The authors' explanation is

that a significant part of the MIB in the raw water was present in the responsible

microorganism. These microorganisms are typically removed during coagulation and

sedimentation; however, when chlorination was applied prior to coagulation, the cells lysed,

releasing intracellular material that contained MIB. Although the PAC (10 ppm)/pre-

chlorination/coagulation method was able to remove an average of 42% of the influent MIB,

none of the treatment methods studied were capable of decreasing MIB concentrations to

below the OTC; thus, alternative technologies must be found. Recently, such water treatment

methods have been devised and are showing preliminary success in the removal of MIB. As

the focus of this work is microbial transformation of MIB, the remainder of this discussion

examines the literature reporting success with biological treatment of MIB.

Table 2-3: Conventional treatment methods and powdered activated carbon (PAC) average
MIB removal efficiencies (Ashitani et al., 1988)
Treatment Raw Water MIB Treated Water MIB Removal
Method (ng/1) (ng/1) (%)
Coagulation 53 34 36
Pre-Chlorination/Coagulation 53 39 26
PAC (10ppm)/
Pre-Chlorination/Coagulation 53 31 42









Bacterial Transformation of MIB

Isolation and Identification of Potential MIB-Degrading Bacteria

Most work reporting the identification of microorganisms that are capable of degrading

MIB involved the use of a MIB-contaminated raw water as the inoculum source for a liquid

culture. The bacterial strains were isolated from the resulting liquid cultures by colony

morphology using spread plate methods. Each study then identified isolated strains by using

biochemical test kits. These kits are commonly used for many groups of bacteria to

determine phenotypic activity oxidasee, catalase, nitrate reduction, amino acid-degrading

enzymes, fermentation or utilization of carbohydrates). The tests are typically conducted by

adding a small amount of culture to a multi-welled plate containing various biochemical

reagents. The reaction in each well is then observed and compared to a database containing

phenotypic characteristics of known microorganisms (Chester and Cleary, 1980).

Biochemical test kits are commonly used in the identification of gram type negative,

nonfermentative bacteria, such as Pseudomonas, Acinetobacter, Flavobacterium, Moraxella

and fermentative bacteria not belonging to the Enterobacteriaceae, such as Vibrio and

Aeromonas. There are limitations with these kits; however, such as previously unknown,

rare, or newly described strains that are not in the database (Chester and Cleary, 1980). This

can often lead to strains being unidentified or misidentified when their results are compared

to only known species. The two testing kits employed by the studies discussed below for

characterization of isolated MIB-degrading microorganism are the Rapid Nonfermentor Test

(NFT) and the Minitek Test Kit.

Izaguire et al. (1998) reported use of a common technique for the isolation of a pure

MIB-degrading culture. In this method, water and sediment samples were collected from

contaminated lakes and used as inoculum for a MIB-spiked mineral salts medium. The MIB-









degrading bacteria were identified as soon as the cultures showed a decrease in MIB

concentration, typically after incubation periods of 3-20 days. Purified isolates were

identified from the cultures using the NFT kit, described above. Examples of some of the

isolated MIB-degrading bacteria included Pseudomonas aeruginosa, Pseudomonas

pseudoalcaligenes, Pseudomonas paucimobilis, and Pseudomonas mendocina. Other

publications claiming successful isolation of MIB degraders from natural waters used similar

techniques; however, different strains were identified, including Bacillus subtilis and

Flavobacterium multivorum (Ishida and Miyaji, 1992; Egashira et al., 1992).

Another means of identifying these bacteria would be to compare each gene sequence

in a given strain with the gene sequences other known species. The ribosomal ribonucleic

acid (rRNA) of one organism can be compared with that of any other organism by a method

called 16s rRNA gene sequencing. The results of gene sequencing provides an estimate of

the percentage of divergence within sequences that are related but not identical and provides

a higher level of accuracy compared to phenotypically based methods (Chester and Cleary,

1980). This information could then be used to construct a phylogenetic tree comparing the

isolated culture to known species.

Characteristics of MIB-Degrading Bacteria

Biological treatment systems are most effective when the conditions for microbial

growth are met, including adequate pH and temperature (Egashira et al.,1992).

Unfortunately, while some of the microorganisms found capable of MIB transformation have

been partially characterized (Ishida and Miyaji, 1992; Egashira et al., 1992), the growth

characteristics and environmental requirements of many MIB degraders have not been

clearly defined.









Ishida and Miyaji (1992) investigated the kinetics of MIB degradation for a strain these

authors isolated, named Bacillus sp. HI-5. This pure strain was isolated by inoculating a

minimal salts medium spiked with 100 [tg/l of MIB with backwash water obtained from a

rapid sand filter. The culture was incubated for 18 days while MIB removal was

continuously observed. This experiment showed that 30 [tg/1 MIB was removed after the

first 70 hours, and, after 7 days all of the MIB had been removed below the minimum

analytical detection limit, or MDL, of 20 ng/1. Batch experiments, conducted with Bacillus

sp. HI-5, yielded maximum specific growth rates ([,max) of 0.10 hr1 and 0.03 hr-1 in the

presence of 8 and 0.1 mg MIB/1, respectively. A saturation constant (the concentration of

substrate where the growth rate t is equal to 1/2 [,max) was found to be 205 [tg/l for the

culture grown in 8 mg MIB/1. This study also reported corresponding observed MIB

depletion rates (Figure 2-3) of 7.7 [tg/l/hr for the culture grown at an initial concentration of 8

mg/1 MIB and 0.5 [tg/l/hr for the culture grown at the initial concentration of 0.1 mg/1 MIB.


10





0.1


0.01 --- -MIB 8 mg/l Initial
M- MIB 0.1 mg/I Initial

0.001
o3 NT NO "K #K

Time (Hr)

Figure 2-3: Depletion by Bacillus HI-5 at 2 different initial concentrations of MIB (adapted
from Ishida and Miyaji, 1992)









The results of the growth study, as shown in Figure 2-4, show the lag time for each

culture to be approximately 40 hours. The stationary growth phase for the 0.1 mg/1 and 8

mg/1 cultures was reached at approximately 130 hours, and the maximum number of

microorganisms grown at each MIB concentration were 5.0 x 105 CFU/ml and 3.5 x 107

CFU/ml, respectively. However, the authors do not report use of a chemical control

containing MIB and no cells or a killed control with autoclaved cells. Thus, it is implied that

these investigators did not account for volatilization or cell sorption losses of MIB, causing

an overestimation of MIB depletion.


1.E+08
-4--8 mg/I MIB
Initial
1.E+07 ----0.1 mg/I MIB
initial
-A- Control

S1.E+06
UL

1.E+05


1.E+04
0 40 60 80 110 120 125 130 140
Time (Hr)

Figure 2-4: Growth curves for Bacillus HI-5 at 2 different initial MIB concentrations
(adapted from Ishida and Miyaji, 1992)

Egashira et al. (1992) reported the effects of water temperature and pH on the MIB

depletion potential in a pilot plant study with a biological filtration system (packed-column).

The microorganisms that were responsible for the depletion in the column were isolated and

identified, and their individual depletion potentials were studied. In the biological filter

study, a constant concentration of MIB (0.2 [tg/l) was added to the natural water fed into the









column. The pH and temperature were then systematically adjusted, and the changes in MIB

depletion were then measured by GC/MS and recorded. The results for these experiments are

shown in Figures 2-5 and 2-6.


pH


Figure 2-5: Effect of pH variation on the MIB depletion potential of a biologically active
column (adapted from Egashira et al., 1992)


100


3.25 3.3 3.35 3.4 3.45


1/T (10-3K1)


Figure 2-6: The logarithmic relationship between temperature and the MIB depletion
potential of a biologically active column (adapted from Egashira et al., 1992)











Although no error is reported, the trends in Figures 2-5 and 2-6 suggest that

changes in temperature and pH can have a significant influence on the MIB depletion

potential of the microbial populations residing in the biological filter used in this study.

The maximum MIB depletion potential of the column was observed at a temperature of

30 C and a pH of 8. This correlation may be useful in determining appropriate operating

conditions for biological treatment systems designed to remove MIB.

After the completion of the biological filtration study, the granular ceramic

medium used in the column was treated by ultrasonication. The resulting biological

sludge suspension was used as an inoculum in a tripticase soy culture agar that was then

incubated for 3 days at 25 C. Microorganisms were then isolated from the agar based on

colony morphology. The MIB depletion potential of each isolated bacterium was

determined using batch experiments. The isolate bacteria were used to inoculate a

minimal mineral medium that was incubated for 7 days with an initial MIB concentration

of 20 |tg/l, under carefully controlled temperature and pH, ranging from 26 to 29 OC and

7.3 to 7.6 respectively. MIB depletion potential was determined by the difference

between the initial and final MIB concentrations, measured by purge and trap and

GC/MS. The isolated species were then characterized using a Minitek identification kit.

The results of the MIB depletion potential study for cultures isolated from the biological

filter are presented in Table 2-4.









Table 2-4: MIB depletion potential for the identified species isolated from a biological
treatment filter (Egashira et al., 1992)
Strain Pseudomonas sp. P. aeruginosa Flavobacterium sp. F. multivorum
Initial MIB Concentration 20 ug/1 20 ug/1 20 ug/1 20 ug/1
Final MIB Concentration 15.7 ug/1 14.82 ug/1 14.0 u/1 16.7 ug/1
Incubation Time 24 hr 24 hr 24 hr 24 hr
MIB Depletion Rate 0.18 ug/l/hr 0.21 ug/l/hr 0.25 ug/l/hr 0.14 ug/l/hr
Total MIB Depletion % 21.6 25.9 29.7 16.7
MIB Depletion %/hr 0.9 1.08 1.24 0.70

As shown in Table 2-4, several species of bacteria that can potentially transform

MIB were isolated in this study. This study did not provide; however, the cumulative

depletion rate of the original mixed culture composed of these isolated strains. It is

unknown whether the depletion rate of the mixed culture differs significantly from those

reported. The inclusion of controls to measure the effects of volatilization or cell

sorption on MIB depletion was not reported in this study.

Applications of Technology Using Biological Transformation for MIB Removal in
Water Treatment

Introduction to the Use of Bioremediation in Water Treatment

Bioremediation is the use of microorganisms to eliminate or detoxify toxic or

unwanted chemicals, and can be employed in water treatment to eliminate natural and

anthropogenic chemicals from raw water (Characklis and Marshall, 1990). One of the

most common bioremediation applications in water treatment is biological filtration.

Microorganisms that are present in filtration media oxidize biodegradable organic matter

and nitrify ammonium compounds. Recently, laboratory- and pilot- scale experiments

have shown that some microorganisms have the potential to transform MIB (Izaguirre et

al., 1988; Oikawa et al., 1995; Namkung and Rittmann, 1987; Tanaka et al., 1996).

Important factors that may dictate the effectiveness of a biological treatment method for









MIB removal include microbial diversity, transformation pathways (intermediate

formation), growth characteristics and biofilm development.

Biological Filtration

Microbial cells often attach firmly to submerged surfaces in aquatic environments.

These immobilized cells grow, reproduce, and produce extracellular polymers that can

form a tangled matrix of fibers. A biofilm is the assemblage of these fibers (Characklis

and Marshall, 1990). Biofilm-based technologies are currently implemented in the

treatment of air, water and wastewater. Packed-bed reactors, in which biofilms

accumulate on solid substrata or granular media packed within a tower or bed, are often

used in water treatment. One example of a packed-bed reactor is a trickling filter where

the influent liquid is spread over the top of a granular media by a sprinkler system and

allowed to flow through the bed in a thin water layer over the biofilm (Viessman and

Hammer, 1998). Oxygen, a required gas for aerobic processes, is drawn up the bed by

natural convection. Since biofilm development depends on the adsorbable surface area of

the media, a high surface-to-volume ratio, such as small and/or porous particles, is

optimal. As mentioned earlier, sand is a typical example of medium used in conventional

trickling filters. These trickling filters have shown little success for the removal of MIB

from drinking water; thus, alternative media types must be explored (Ashitani et al,

1988).

Yagi et al. (1988) examined one example of a biologically active filter for MIB

reduction. The filter media for this study included activated carbon (coconut shell, 10x32

mesh), zeolite, and sand, and Bacillus subtilis IAM 12118 was used as the inoculum. The

dimensions of the filter are provided in Table 2-5. Each filter was fed 0.9 liters of raw

water, which was supplemented with additional MIB to maintain a constant concentration









for each run (1.5 1.6 mg/1). The microbial transformation was estimated by subtracting

the total MIB influent by the amount of MIB adsorbed and MIB extracted from the

filtrate of a control, consisting of autoclaved medium to eliminating biological activity.

The study also examined the amount of MIB adsorbed by the filter medium and the

biological cells. The concentration of MIB was determined in these tests by GC/MS,

with a minimum detection level of 0.1 mg/1. The results from this study can be found in

Table 2-6.

Table 2-5: Column parameters for the biological filtration study conducted by Yagi et al.
(1988)
Internal Diameter 1.5cm
Length 20 cm
Media volume 35 cm3
Empty Bed Contact Time 2.4 min
Hydraulic Loading 8.3 cm/min


Table 2-6: Results for the
Parameter


Biological Activity
MIB in raw water (mg/1)
Volume of filtrate (1)
Loading of MIB (mg/1)
MB in Filtrate (mg/1)
MIB in filtrate (mg)
Estimated Adsorbed
(mg)
Amount Extracted (mg)
Estimated
Transformation (mg)
Estimated Depletion %


biological filtration study conducted by Yagi et al. (1988)


*These values, determined by GC/MS, represent the MIB removal characteristics of each
column type.

These data show that the activated carbon control removed MIB to the lowest

effluent concentration. The effectiveness the activated carbon filter to remove MIB was

diminished by biological activity; however, it appears that the bacteria present in the in


Carbon Medium Sand Medium Zeolite Medium
Bio-Active Control Bio-Active Control Bio-Active Control
1.6 1.5 1.5 1.5 1.5 1.6
0.9 0.9 0.9 0.9 0.9 0.9
1.4 1.4 1.4 1.4 1.4 1.4
0.2 <0.01 1.7 1.5 1.7 1.5
0.18 <0.009 1.5 1.4 1.5 1.4

1.2 1.4 -0.1 0 -0.1 0
0.53 1.33 0.02 0.01 0.06 0.2

0.7 0.07 0 0 0 0









this filter were responsible for the removal of some MIB via transformation. The data

provided also shows that the sand and zeolite filters were not capable of removing MIB

regardless of biological activity. It is important to note; however, that no information is

provided quantifying the biomass concentration on the filter, allowing the possibility that

there was insufficient inoculum present for significant MIB depletion. Also, the raw

water fed into the column may have lacked the necessary nutrient concentrations for MIB

transformation.

The Effect of Ozonation the Biological Transformation of MIB

Ozonation is becoming a popular drinking water disinfection method in the United

States (Bitton, 1999). This is primarily because of its ability to kill microorganisms

without producing trihalomethanes or other halogenated disinfection byproducts.

Another consequence of ozonation is the oxidation of large organic compounds (i.e.,

humics and fulvics) into smaller compounds, such as ketones, ketoaldehydes, alkanes,

and alcohols, that may be more easily biodegraded. The reduction of large organic in the

influent may also increase the life of a carbon adsorber by saving pore space. Thus, the

application of ozonation before a biological system may increase its effectiveness

(Muramoto et al., 1995).

Muramoto et al. (1995) reported the results of a full-scale application of ozonating

water immediately before biological treatment. These researchers examined part of the

treatment line in the Kanamachi Purification Plant in Tokyo, Japan (Fig 2-7). The MIB

concentrations were analyzed by GC/MS after each stage to determine the removal

efficiencies of each process. Figure 2-8 presents the average MIB concentrations that

Muramoto et al. (1995) observed at each collection point over 3 months. Regardless of

the month of sampling, approximately 20 percent of the influent MIB was removed by









coagulation-sedimentation, and the remainder was removed by the combined ozonation

biological treatment system.


Influent


Effluent


tuaLuimaLt Chlorine

Figure 2-7: Kanamachi treatment line (adapted from Muramoto et al., 1995)


Coagulation-
Ra Water edimentation


-- July Avg. MIB

S-- August Avg.
MIB
a- -A- September
SAvg. MIB


Ozonation BAC


Figure 2-8: MIB concentrations in raw and processed waters (adapted from Muramoto et
al., 1995)

As shown in Figure 2-8, the Kanamachi treatment plant was capable of removing

MIB to below the OTC, regardless of the MIB loading. Although coagulation and

sedimentation processes removed a portion of the MIB, ozonation and BAC treatment

effectively removed all of remaining MIB. Although the BAC system shows promise for

the removal of MIB, it remains unclear whether the MIB was depleted through bacterial

degradation or by adsorption onto the activated carbon.









Conclusions

Most consumer complaints regarding drinking water quality are due to malodors

and tastes. Often, the odors are described as earthy-musty. Once an earthy-musty odor

event occurs, water utilities can typically remove the responsible compounds using

conventional water treatment with PAC. However, one chemical responsible for the

earthy-musty odor, MIB (a cyanobacteria metabolite), is not effectively removed by these

methods. This literature review examined previous studies of isolation and identification

of microorganisms and microbial systems capable of removing MIB. Included in this

review are papers covering the characteristics and origins of MIB, the isolation of

microbial species capable of depleting MIB, the characterization and transformation

kinetics of MIB-degrading bacteria, and selected water treatment technologies currently

incorporating microbial degradation for MIB removal. Many of the studies mentioned

reported MIB removal to below analytical detection limits; however, these studies did not

clearly report the final concentration of MIB at the ng/1 levels commonly found in natural

waters or to a concentration that did not confer odor. To improve upon these methods,

additional studies are needed to further assess the MIB transformation potential of

microbial communities, particularly, the ability of cultures to deplete MIB at different

initial concentrations, including the ng/1 levels commonly found in natural waters. Also,

improved methods of identification of MIB-degraders that involve genetics-based

techniques are desirable because of their increased accuracy. By better understanding

MIB concentration effects on the activity of MIB-degrading bacteria and the phylogeny

of these microorganisms, drinking water facilities will be better able to design more

effective biologically based systems that cater to these specific requirements.














CHAPTER 3
CHARACTERIZATION OF A MICROBIAL CULTURE CAPABLE OF REMOVING
TASTE- AND ODOR-CAUSING 2-METHYLISOBORNEOL FROM WATER: A
MANUSCRIPT TO BE SUBMITTED TO WATER RESEARCH

Introduction

Tastes and odors are considered by most consumers to be significant factors when

determining potable water quality. The occurrence of taste and odor problems is

widespread and has been reported in Asia, Austrailia, North America and Europe

(Ashitani et al., 1988; Persson, 1983; Suffet et al., 1996; Zimmerman et al., 1995). Some

of the more prominent taste- and odor-causing substances are naturally occurring organic

compounds that produce an earthy-musty odor in drinking water (Rashash et al., 1997).

Many of these organic have been identified and include 2-isopropyl-3-methyoxy

pyrazine (IPMP), 2,3,6-trichloranisole (TCA), 2-isobutyl-3-methyoxy pyrazine (IBMP),

trans-1, 10-dimethyl-trans-9-decahol (geosmin) and 2-methylisobomeol (MIB). These

compounds are released by blue-green algae, typically from late spring through early fall,

in concentrations reported to be as high as 100 ng/1 (Tenauchi et al., 1995); however

concentrations ranging from 10 ng/1 can cause malodorous drinking water (Persson,

1980). While not a concern in terms of health impacts, the offensive odor and taste of

MIB may lead to psychosomatic effects, such as headaches, stress, or stomach upsets

(Young et al., 1996).

Water utilities typically rely on conventional treatment methods to remove taste-

and odor-causing compounds. These methods can effectively decrease IPMP, TCA,

IBMP and geosmin concentrations to below their odor thresholds; however, they are









typically unsuccessful in removing MIB to below its commonly accepted odor threshold

concentration (OTC) of 10 ng/1 (Ashitani et al., 1988). Recent studies have begun to

examine biological treatment as an alternative method for MIB removal. Ishida et al.

(1992) and Egashira et al. (1992) isolated several strains of bacteria from natural waters

that are capable of using MIB as a growth substrate, including Pseudomonas aeruginosa,

Pseudomonas pseudoalcaligenes, Bacillus sp., and Flavobacterium multivorum. Ishida et

al. (1992) conducted batch and continuous feed experiments studying the removal of

MIB by a bacterium isolated from Lake Kasumigaura in Japan. The results for the batch

experiments showed the ability of the bacterium to deplete MIB at the mg/1 and [tg/l

levels, while the continuous feed study showed the isolated strain was capable of

reducing influent MIB concentrations of 600 ng/1 to approximately 60 ng/1. The

researchers identified the isolated strain as a Bacillus sp. using phenotypic identification

tests. Egashira et al. (1992) examined the removal of [tg/l concentrations of MIB in

drinking water by a biological filter inoculated with surface water from Lake Biwa,

Japan. This study also reported the effects of temperature and pH on MIB depletion in a

packed column and identified isolated cultures from the filter media using a biochemical

test kit. No error was reported in the studies performed by either Egashira et al. (1992) or

Ishida et al. (1992); however, the trends of their results support the potential of MIB

depletion by bacteria.

Although many reports show positive findings on microbial depletion of MIB, the

potential for microbial communities to deplete MIB to below the OTC has not been

shown. Also, the effect of MIB concentration on microbial growth and metabolic activity

has not been fully explored. The purpose of this study was to further assess the MIB









transformation potential of microbial communities, particularly, the ability of cultures to

deplete MIB at different initial concentrations including the ng/1 levels commonly found

in natural waters. The effect of varying MIB concentration on microbial growth and

oxidation potential was also examined. The characterization and identification of the

isolated culture was conducted by using transmission electron and light microscopy to

examine fine cell structures and 16S rRNA gene sequencing to construct alignment

profiles and a neighbor joining phylogenetic tree.

Materials and Methods

Enrichment and Isolation of MIB Transforming Bacteria

Source water used as initial inoculum

The source of inoculum used in this study was Lake Manatee in Manatee County,

FL. Lake Manatee currently feeds the Manatee County Water Treatment Facility to the

supply of potable water for Manatee County. This reservoir experiences periods of

extensive algal blooms from late spring through early fall, resulting in average MIB

concentrations in the raw water of approximately 25 ng/1. Water samples were collected

in July 2000 in sterile glass bottles from the raw water source, which were then stored at

4 C until their use in the batch enrichment experiments.

Enrichment procedures

The enrichment for potential MIB-degrading bacteria was conducted by pumping 1

1 of feed solution of buffered mineral salts medium (MSM) (Table 3-1) (Izaguirre et al.,

1988) inoculated with 200 ml lake water and spiked with 6 mg/1 MIB (Wako Pure

Chemicals, LTD., Osaka, Japan) through anthracite-packed glass columns (Figure 3-1).

The feed solution was circulated through the columns at a flow rate of 0.5 ml/min by a

peristaltic pump for 5 days, at which time biological growth was visible on the anthracite.












Table 3-1: Components of mineral salts media (Izaguirre et al., 1988)
Concentration
Species (mg/
(mg/1)
NH4Cl 50
K2HPO4 100
MgSO4 50
CaCl2 20
FeC13 1

A control column was also run without added MIB. Further enrichment was then

achieved in batch enrichments with flasks containing 10 ml MSM, 6 mg/1 MIB, and 0.5

grams of anthracite that was removed from the biologically active columns. These flasks

were then incubated in an incubator shaker at 30 oC and 300 rpm until turbidity was

observed after approximately 3 days. Repeated transfers to fresh MIB-enriched medium

were performed before characterization was conducted.

Inoculum with MIB medium

Control (MSM Only)





Anthracite- Column Parameters
Length 9.7 cm
Bed Volume 76.3 ml
Flow Rate 0.5 ml/min






Effluent circulated
back to pump

Figure 3-1: A schematic of the anthracite column apparatus used to isolate a culture
capable of depleting MIB.









Solid culturing of the MIB-degrading culture was performed by streaking from the

liquid cultures onto agar plates composed of MSM medium and 1.5% (w:v) agar. Plates

were subsequently placed in a sealed 4-liter dessicator equipped with an uncovered

beaker containing 10 ml of 100 mg MIB/1 of deionized water and incubated at 30 OC.

Characterization of the Isolated Mixed Culture

Cellular morphology

The cellular morphologies of the strains present in the isolated mixed culture were

identified using light microscopy and transmission electron microscopy (TEM). The

liquid cultures were sampled during the stationary phase in order to check for the

presence of spores. For light microscopy observations, a pipet tip was touched to a

colony and then inserted into a drop of deionized water on a glass slide. The glass slide

was then examined and photographed using a Nikon Optiphot-2 microscope (Nikon,

Tokyo, Japan) with either differential interference contrast or phase contrast optics.

Liquid and solid cultures examined using TEM methods were prepared by first

placing samples on a formvar-coated 300 mesh copper grid and treating with a negative

stain. The liquid culture was prepared by placing 1 drop of the culture on the grid with

an equal amount of 1% aqueous uranyl acetate. The liquid was wicked off the grid with

filter paper after 2 minutes and rinsed once with deionized water. For the solid culture, a

pipet tip was touched to a colony, inserted into a drop of deionized water mixed with an

equal amount of 1% uranyl acetate on the grid, and wicked off the grid after 2 minutes as

previously described. The negatively stained sample grids were then observed and

photographed at 100 kv on a Zeiss EM10 electron microscope (Zeiss, Oberkochen, Germany).









Genotypic characterization

16S rRNA genetic analysis of the dominant species in the isolated culture was

performed by MIDI Labs Inc., (Newark, DE, USA) by using MicroSeq 500 16S

ribosomal RNA (rRNA)-based bacterial identification system (Applied Biosystems,

Foster City, CA). The 16S rRNA gene was PCR amplified from genomic DNA isolated

from the isolated culture. The PCR primers used for the amplification corresponded to E.

coli positions 005 and 1540 (full length packages) and 005 and 531 (500 base pair

packages). Amplification products were purified from excess primers and deoxy nucleotide

triphosphates (dNTPs) using Microcon 100 (Millipore, Billerica, MA, USA) molecular weight

cut-off membranes and checked for quality and quantity by running a portion of the

products on agarose gel.

PCR cycling parameters used were 95 C and 10 min for initial incubation, 95 C

and 30 seconds for melting, 72 C and 45 seconds for chain extension and 60 C and 30

seconds for annealing. Cycle sequencing of the 16S rRNA amplification products was

carried out using AmpliTaq FS DNA polymerase and dRhodamine dye terminators (Roche

Molecular Systems, Inc., Pleasanton, CA, USA). Excess dye-labeled terminators were

removed from the sequencing reactions using a Sephadex G-50 spin column (Amersham

Pharmacia Biotech, Piscataway, NJ, USA). The products were collected by centrifuge,

dried under vacuum and frozen at -200 C until they were ready to load. Samples were

then resuspended in a solution of formamide/blue dextran/EDTA and denatured prior to

loading. The samples were electrophoresed on an ABI Prism 377 DNA sequencer

(Applied Biosystems, Foster City, CA, USA). The results were analyzed using Applied

Biosystems DNA editing and assembly software (Applied Biosystems, Foster City, CA,

USA).









Sequences were compared with previously identified sequences in the Microseq

database using Microseq software (Applied Biosystems, Foster City, CA, USA) and in

the National Center for Biotechnology Information (NCBI) GenBank database using

BLAST (Altschul et al. 1990). Isolated culture sequences were aligned with closely

related sequences from the Microseq database based on the percent genetic difference of

the unknown culture strains with those of known strains. This alignment match was then

used to construct the neighbor joining phylogenetic tree.

Growth characterization of the isolated culture

Specific growth rates and generation times of the isolated culture were determined

from turbidity measurements using a UV-VIS spectrophotometer (Milton Roy Company,

Ivyland, PA, USA) at a 600 nm wavelength. To assess the effect of MIB concentration

on growth, initial MIB concentrations of 5, 10, and 20 mg/1 were prepared in triplicate in

MSM medium with 20% inoculum (v:v). These concentrations were chosen based on

previous publications reporting observed microbial growth on MIB in the given range

(Ishida and Miyaji, 1992; Izaguirre et. al, 1988). Chemical (MIB only) and biological

(cells only) controls were also included with each set of concentrations. The specific

growth rates were calculated using linear fits to the slope of the exponential growth phase

of each growth curve obtained.

MIB Depletion Potential of the Isolated Culture

Oxygen uptake studies

Oxidation has been reported as one significant microbial pathway for MIB

transformation (Tanaka et. al, 1996; Oikawa et. al, 1995). In order to assess the MIB

oxidation potential of the isolated culture, oxygen uptake analysis was first performed.

Cell suspensions of the isolated culture were prepared for the oxygen uptake experiments









by harvesting cultures at 34-log phase by centrifugation at 1.94 g for 25 minutes in a J2-

HS Beckman centrifuge (Beckman Coulter, Fullerton, CA, USA). Cells were washed

once in MSM to remove residual MIB and resuspended to a wet cell weight concentration

of 0.2 g/ml. The final cell suspension was stored in an ice bath during the oxygen uptake

experiments.

A 1.9-ml glass, water-jacketed reactor (Figure 3-2) was used at a constant

temperature of 30 C to measure the rates of oxygen consumption at various initial

substrate concentrations as described by Lindner et al. (2000). An electrolyte and

membrane-covered Clarke-type electrode (Instech Laboratories, Plymouth, MA, USA)

was inserted into the reactor using a ground-glass port with two rubber o-rings and was

connected to a biological oxygen monitor (Yellow Springs, Yellow Springs, OH, USA).

Monitor output was sent to an A/D converter board (DAS08-PGL, Computer Boards,

Mansfield, MA, USA) for data collection using Labtech Notebook software (Wilmington,

MA, USA). In all assays, the reaction chamber was filled with MSM before the addition

of cells or substrate. The electrode was calibrated daily (following manufacturer's

instructions) after application of fresh electrolyte and membrane. Runs for each

concentration were conducted in triplicate. Oxygen uptake rates were calculated

following the methods presented by Hitchman (1978). To verify oxygen uptake by the

isolated culture, controls included runs with MIB only and cells only, and all rates were

corrected for endogenous metabolism.









SCells + MSM + MIB
To DO Meter
and DAQ system H20 In



H20 Out
Figure 3-2: A schematic of the reactor used for the oxygen uptake experiments

MIB depletion studies

The protocol for the analysis of MIB depletion was developed using Solid Phase

Micro Extraction (SPME) procedures described in Standard Method 6040D (APHA,

2001). The MIB depletion potential of the isolated culture was examined at two MIB

concentrations, 5 mg/1 and 25 ng/1. These concentrations were chosen because the

highest rate of oxygen uptake was observed at 5 mg/l, and 25 ng/1 represents average

environmental concentrations observed in Lake Manatee during periods of high algal

bloom. Microcosms were prepared in triplicate by addition of a stock solution of 100

mg/1 MIB to flasks containing a mixture of 50 ml MSM and inoculum added to yield an

optical density of 0.5 (k = 600 nm). Controls included with each set of experiments were

a substrate control (MIB only) and a killed control with autoclaved cells. All microcosms

were incubated in an incubator shaker at 30 oC and 300 rpm and sampled at regular time

intervals for analysis. The 5 mg/1 and 25 ng/1 cultures were incubated until no change in

MIB concentrations were observed. Samples were collected from the 25 ng/1 cultures by

transferring 10 ml of the culture to a 20 ml scintillation vial containing 3 g of NaC1,

which was then immediately capped. Samples were taken from the 5 mg/1 MIB cultures

and then serially diluted in deionized water to ng/1 MIB concentrations, and these diluted

solutions were transferred to 20 ml scintillation vials as previously described. Sample









vials were stored at 4 C prior to analysis. All samples were analyzed by a Varian 3900

gas chromatograph (Varian, Inc., Palo Alto, CA, USA) with an ion trap ion detector, an

Equity 5 fused silica capillary column (Supelco, Bellefonte, PA, USA), and a Saturn

2100 mass spectrophotometer (Varian, Inc., Palo Alto, CA, USA) with an auto-sampling

system (CTC Analytics, Zweigen, Switzerland) fitted with a SPME fiber (Supelco,

Bellefonte, PA, USA). The fiber, coated with divinyl-carboxen-polydimethylsiloxane

cross-link, was injected into the headspace of each sample and allowed to equilibrate with

the aqueous solution for 30 minutes. The fiber was then removed and inserted into the

injection port of the GC/MS system where it was allowed to desorb for 5 minutes. Saturn

Workstation version 5.52 data acquisition software (Varian, Inc., Palo Alto, CA, USA)

was used for data analysis. Duplicates of each culture sample, a set of MIB standards,

and a blank were run during the depletion studies to regularly calibrate the system.

Results and Discussion

Characterization of the Isolated Mixed Culture

Cellular and colony morphology

Homogenous, cream-colored, opaque colonies were observed on the agar 36

hours after inoculation, and full growth development was reached after an additional 60

hours of incubation. As summarized in Table 3-2, the colonies, approximately 2 mm in

diameter, were round with a glassy surface, minimal elevation, and smooth edges.









Table 3-2. Colony and cellular characteristics of the dominant strain in the isolated
culture
Characteristics Isolated Culture
Colony Morphology
Size 2 mm
Shape Round
Color Cream
Topography Glassy
Full Development 96 hours
Elevation Minimal
Edge Smooth

Phase contrast light microscopy and transmission electron microscopy were used to

elucidate cellular morphologies and fine structure of the strains) present in the isolated

culture. Both techniques revealed that the isolated culture was composed of

predominantly bacillus-shaped bacteria (Figure 3-3). The average cell size was

approximately 5 am in length and 1 [tm in width. The presence of opaque structures,

denoted by arrows in Figures 3-3A and 3-3B, indicates morphological evidence of

sporulation. When observed under light microscopy the cells were highly motile, and

flagella were observed in the TEM micrographs (Figure 3-3B). Flagellation and

sporulation are cellular responses that are indicative of nutrient deprivation and allow the

cells to move towards nutrient-rich regions or to remain dormant in periods of nutrient

deprivation, respectively (Harwood, 1989). Both characteristics of these cells-

flagellation and sporulation- provide insight into the modes of survival that these cells

possess in their natural reservoir environment that undergoes seasonal changes in MIB

concentrations.


























Figure 3-3: Phase contrast photograph (A) and transmission electron photograph (B) of
the MIB-degrading isolated culture when grown on solid agar. Flagella and
sporulation denoted with arrows.

Genotypic characterization

The 16S rRNA phylogeny results (Figure 3-4) from the MicroSeq alignment

confirms placement of the isolated culture the Bacillaceae family of bacteria within a

branch dominated by Bacillus strains. The isolated culture showed a 1.68% genetic

difference (%GD) with its nearest relative Bacillusfusiformis (accession # AF2132169).

Although this is not sufficient enough to place the unknown to the species level, it does

match the isolated strain to the Bacillus genus level.

It is important to note; however, that results from the BLAST search of the

GenBank database showed that the closet match of the MIB unknown was with Bacillus

Sphaericus, at a 1% GD. Bacillus sphaericus and its sub-species Bacillusfusiformis are

both strictly aerobic, mesophilic bacilli that are capable of forming flagella and spherical

endospores in order to adapt to nutrient deprivation. Bacillus sphaericus is currently

applied in the remediation of other contaminants, including: urea herbicides (Doi and

McGloughlin, 1992).











Bacillus fusiformis
Bacillus sphaericus
6.34%
Bacillus insolitus
7.96%
Sporosarcina urae
8.4%
Bacilus pasteurif
8.65%
Bacillus lentus
8.99%
Bacillus circulans
%Bacillus flexus
Bacillus megarerium
9.89%
9% Staphylococcus sciuri sciuri


Figure 3-4: The neighbor joining phylogenetic tree of the isolated culture constructed
from the MicroSeq alignment report. The top 10 closest genetic matches of
the isolated culture are presented with their percent genetic difference. A
lower % difference indicates a closer match.

Growth characterization

The effects of varying initial MIB concentration on specific growth rates were

determined using the spectrophotometric methods as previously described. Figure 3-5

shows the growth curves of the cultures grown with initial MIB concentrations of 5, 10,

and 20 mg/1. The highest specific growth rate calculated from each curve corresponded

to an initial MIB concentration of 10 mg/l (0.067 + 0.015 hr1). A summary of the

specific growth rates and generation times calculated for each concentration is provided

in Table 3-3.












0.8

0.75

e 0.7

0.65 -
0

0.6


0.55

0.5
0 5 10 15 20
Time (hr)


Figure 3-5: Growth curves of isolated culture at initial MIB concentrations of 5 mg/1 (*),
10 mg/1 (0), and 20 mg/1 (A). All absorbance measurements were taken at a
wavelength of 600 nm.

Table 3-3: A summary of the growth kinetics for the isolated culture grown at 5, 10, and
20 mg/1 MIB.
MIB Concentration Specific Growth Rate (p) Generation Time
5 mg/1 0.045 + 0.005 hr- 15.64 + 1.73 hr
10 mg/I 0.067 + 0.0015 hr- 10.74 + 2.67 hr
20 mg/I 0.034 + 0.004 hr- 20.56 + 2.42 hr

MIB Depletion Potential of the Isolated Culture

Oxygen uptake studies

Figure 3-6 presents the oxygen uptake rates observed over a range of MIB

concentrations from 2.5 mg/1 to 20 mg/1. A maximum rate of 0.04 + 0.004 moles

02/s/ml was observed at 5 mg/1 followed by a dramatic decrease in rates at higher MIB

concentrations tested. As no effects on the probe were observed in controls with only

MIB, this behavior, reported in other oxygen uptake studies (Lindner et al., 2000, 2003),










suggests a toxicity effect on the cells. Whether this is caused by excessively high MIB

concentrations or formation of toxic intermediates is not known.



0.05
0.045
0.04 -
8 0.035
._ E
S 0.03
S 0.025
0Q
SE 0.02
0.015-
x E
O 0.01

0.005 -
0 k
0 5 10 15 20 25
MIB Concentration (mg/I)

Figure 3-6: Oxygen uptake rates for the isolated culture over a range of MIB
concentrations.

MIB depletion studies

The ability of the isolated culture to remove MIB at mg/l and ng/1 concentrations

was examined (Figure 3-7 and Figure 3-8). Five mg/l MIB, representing the high range

of concentrations, was chosen because this was the concentration where the maximum

rate of oxygen uptake was observed, as discussed previously. To represent the low and

perhaps more environmentally relevant range of MIB concentrations, 25 ng/1 was

selected.











80

70

60
"a
S50

S40

S30

20

10

04
0 20 40 60 80 100
Time (hr)

Figure 3-7: MIB depletion percent removal curves for isolated culture microcosms grown
with initial MIB concentrations of 25 ng/1 (*) and 5 mg/1 (*).

As shown in Figure 3-7, each culture was capable of decreasing the concentration

of MIB by over 50% in less than 60 hours. There was also no noticeable lag time

experienced by either culture before depletion occurred. This is most likely due to each

culture's acclimation to the initial concentrations through routine transfers conducted

prior to the depletion studies. The culture grown with an initial MIB concentration of 5

mg/1 (4.2 mg/1 measured) depleted MIB at an average rate of 0.35 + 0.004 mg/l/hr,

removing nearly 66% of the MIB to yield a final concentration of 1.8 + 0.02 mg/1 MIB

after 72 hours of incubation. The culture grown with an initial concentration of 25 ng/1

(28.5 ng/1 measured) removed MIB at a rate of 0.20 + 0.05 ng/l/hr and, after 96 hours of

incubation, had reduced the concentration of MIB by approximately 58% to 9.6 + 0.3

ng/1. While the isolated culture was capable of depleting MIB at both concentration

levels, it is interesting to note that it was able to remove MIB to below the OTC of 10

ng/1 in the 25 ng/1 microcosms and no odor was observed in the culture at the end of the









study. The controls used in this study did not show any significant cell or substrate

effects on MIB depletion and/or analytical recovery.





35 5

30
4
25

20 3
E
m 15 -

10
10
5

0 0
0 20 40 60 80 100 120
Time (hr)

Figure 3-8: MIB depletion curves for isolated culture microcosms grown with initial MIB
concentrations of 25 ng/1 (*) and 5 mg/1 (*).

Conclusions

Microorganisms indigenous to aqueous surface-water environments that

experience seasonal blue-green algae outbreaks may play an important role in cycling

taste- and odor-causing compounds released by these microorganisms. Degradation of

these compounds, such as MIB, may be a result of pure-culture or mixed-culture activity.

Furthermore, opportunity may exist in using these bacteria for enhanced biodegradation

for either in situ or ex situ applications. We obtained samples from a drinking water

reservoir servicing Manatee County, FL, as it was our hypothesis that, because of the

MIB outbreaks experienced in this reservoir on a seasonal basis, bacterial populations

capable of using MIB as a growth substrate could be isolated. We report in this study

successful isolation of a culture that is capable of degrading MIB to below odor threshold









levels. The predominant strain in this culture was matched to the Bacillus genus level

and most closely related by 16S rRNA analysis to Bacillus sphaericus. While previous

studies that focused on isolation of MIB-degrading bacteria have also identified Bacillus

strains (Ishida and Miyaji, 1992), these results showed the influence of MIB

concentration on microbial activity. The maximum growth rates were observed at 10

mg/1 MIB, whereas observed oxygen uptake rates were the highest at 5 mg/1 MIB.

Depletion of MIB was shown at 5 mg/1 and 25 ng/1 initial concentrations, and final

concentrations of MIB below the OTC of 10 ng/1 were observed in the latter case.

The implications of these results are that microbial populations can be derived

from natural water sources for removal of MIB and possibly other taste- and odor-

causing compounds to concentrations that render drinking water as "wholesome," defined

by Young et al. (1996) as both toxicologically and aesthetically acceptable. The culture

isolated in this study was capable of using MIB as a growth substrate at relatively high

and low concentrations of substrate, thus suggesting the ability of this culture to remove

MIB throughout the phases of a seasonal outbreak of blue-green algae. Future work on

this study should include further examination of the effectiveness of biological removal

of MIB from natural surface waters under water treatment operational conditions that

include continuous flow and variation in water characteristics.















CHAPTER 4
CONCLUSIONS

The focus of this project was to examine the potential for microbial transformation

of MB by a culture isolated from water collected from Lake Manatee in Manatee

County, Florida. This study was part of a larger project that included an investigation on

tailoring granular activated carbon specifically for MIB removal. This work included a

literature review and laboratory studies focusing on the isolation and characterization of

microbial systems capable of removing MIB. The hypothesis that drove the laboratory

phase of this project was that, because of the MIB outbreaks experienced in this reservoir

on a seasonal basis, bacterial populations capable of using MIB as a growth substrate

could be isolated.

In this project, a MIB-degrading bacterial culture was isolated from a Lake

Manatee water sample. The sample was used as an inoculum in a feed solution,

composed of MSM and 6 mg/1 MIB, which was passed through an anthracite column

until growth was observed. Subsequently, the isolated culture was characterized by using

growth kinetics to determine specific growth rates, light and TEM microscopy to

examine fine cell structures, and 16S rRNA phylogenetic analysis to assess the closest

match of the unknown isolate(s) to known bacterial strains. Finally, the MIB

transformation potential of the microbial community was assessed by using oxygen

uptake methods and batch microcosm experiments combined with SPME coupled with









GC/MS to determine the ability of the culture to oxidize and degrade MIB at different

initial concentrations and to deplete MIB to below the OTC.



The results of this study supported the hypothesis by showing that a bacterial

population was isolated from Lake Manatee that is capable of using MIB as a growth

substrate. The predominant strain in this culture was matched to the Bacillus genus level

and most closely related by 16S rRNA analysis to Bacillus sphaericus. While previous

studies that focused on isolation of MIB-degrading bacteria have also identified Bacillus

strains (Ishida et al., 1992), these results showed the influence of MIB concentration on

microbial activity. Maximum growth rates were observed at 10 mg/1 MIB, whereas

observed oxygen uptake rates were the highest at 5 mg/1 MIB. Depletion of MIB was

shown at 5 mg/1 and 25 ng/1 initial concentrations, and final concentrations of MIB below

the OTC of 10 ng/1 were observed after 96 hr in the latter case.

These results imply that microbial populations can be derived from natural water

sources for removal of MIB and possibly other taste- and odor-causing compounds to

concentrations that render aesthetically acceptable drinking water. The culture isolated in

this study was capable of using MIB as a growth substrate at over a relatively large range

of concentrations of substrate, thus suggesting the ability of this culture to remove MIB

throughout the phases of a seasonal outbreak of blue-green algae.

Currently, most drinking water facilities are equipped with powdered activated

carbon and oxidation processes, such as chlorination, to remove taste- and odor-causing

compounds. However, these technologies are insufficient in removing MIB.

Alternatively, biological treatment systems should be considered for the removal of this









compound. One possible biological system application is a biologically active granular

activated carbon filter to be used as a polishing step of the treatment process. This

system would have several advantages over conventional treatment, including the

removal of MIB through carbon adsorption and biodegradation, the production of

biologically stable water, the removal of trihalomethane precursors, and the extension of

activated carbon bedlife (Bitton, 1999).

Future work on this study should address questions concerning the effectiveness

of biological removal of MIB from natural surface waters. A closer study of the

biodegradation pathway(s) followed and toxicity mechanisms) should be pursued to

elucidate whether intermediates formed render odor and/or toxicity effects. Also, the

effect of water treatment operational conditions, including continuous flow and variation

in water characteristics, should be determined before a pilot-scale system is designed.

Finally, filtration media of different physical and chemical characteristics should be

assessed for optimal microbial growth. Answers to these questions will ensure that the

ultimate design of a large-scale biological system is the most effective in terms of

economics and performance.















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BIOGRAPHICAL SKETCH

I began studying at the University of Florida in August 1997. The

multidisciplinary approach to solving problems for the health and well being of society

drew me to the field of environmental engineering. As I progressed through my B.S.

degree in environmental engineering, I found particular interest in classes covering the

biological treatment of contaminants. When I completed my B.S. degree in 2001, I knew

I wanted to further explore these subjects; therefore, I decided to attend graduate school

for a master's degree in environmental engineering with a focus on biological

remediation. The challenge of pursuing this degree has been unparalleled; however, the

skills and experience I have obtained along the way, I know, will benefit me for the rest

of my life. I look forward to continuing my education in environmental engineering

when I begin work as a potable water engineer this June.