IIHUIW)HUM/IN!HUI!“”WWW!“WIJIHIIHHI 133 279 THS THESlB llllllllll llllllllllllllll Ulllll'llll 3 1293 01701 9203 This is to certify that the thesis entitled The Role of Trace Copper in the Transformation of Carbon Tetrachloride by Pseudomonas Stutzeri KC presented by Hae Kyung Kim has been accepted towards fulfillment of the requirements for M. S. degree inEnvironmental Engineering 521954; é/c/Li Major professor Date May 1998 0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN REFURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 1M animus-m4 THE ROLE OF TRACE COPPER IN THE TRANSFORMATION OF CARBON TETRACHLORIDE BY PSEUDOMONAS STUTZERI KC By Hae Kyung Kim A THESIS Submitted to Michigan State University In partial fulfillment of the requirements For the degree of . MASTER OF SCIENCE Department of Civil and Environmental Engineering 1998 ABSTRACT THE ROLE OF TRACE COPPER IN THE TRANSFORMATION OF CARBON TETRACHLORIDE BY PSEUDOMONAS STUTZERI KC By Hae Kyung Kim Pseudomonas stutzeri KC secretes a low-molecular-weight factor when grown under iron limiting conditions [4,7]. This factor fortuitously transforms carbon tetrachloride to carbon dioxide, formate and other non-volatile products under denitrifying conditions without the production of chloroform. The factor can be activated for CT transformation by diverse cell types. Maximized and reliable transformation of CT is only achieved when trace exogenous copper is combined with the secreted factor and active cells. This work demonstrates that exogenous copper participates at the reaction level in CT transformation. The reaction requires only catalytic amounts of copper - more than ~ 6.4 M of copper did not increase the rate of transformation. In fact, increasing level of copper inhibited transformation and cell growth. Cell types sensitive to copper mediated CT transformation at faster rates that did cell types resistant to copper. ACKNOWLEDGMENTS I would like to express my appreciation, first of all, to Dr. Craig S. Criddle for giving me opportunity and encouragement to pursue my experiments. His persistent support, guidance, and patience led me where I am today. Special thanks goes to Dr. Mike Dybas for his help on various experiment procedures and advice and to Dr. Robert Hickey for his valuable critical review of this thesis. This work was supported by the NSF Center for Microbial Ecology at Michigan State University under NSF grant BIR-9120006. Last but not least, I thank my parents for their love and supports. TABLE OF CONTENTS Page LIST OF TABLES ............................................................................. vi LIST OF FIGURES ............................................................................ vii CHAPTER 1 — INTRODUCTION ..................................................... 1 CHAPTER 2 — MATERIALS AND METHODS ..................................... 4 Materials and Methods ................................................ 4 Organisms ............................................................... 4 Chemicals ............................................................... 4 Media ................................................................... 4 Groundwater ........................................................... 5 Analytical Methods .................................................... 5 CHAPTER 3 —— EFFECT OF INITIAL PH AND COPPER CONCENTRATION ON GROWTH ......................................................... 7 Materials and Methods ................................................ 7 Effects of Copper on Growth ......................................... 7 Effect of pH on Growth ................................................ 9 Results and Discussion ................................................ 10 CHAPTER 4 — DETERMINATION OF THE EFFECTS OF TRACE COPPER ON THE BIODEGRADATION OF THE CARBON TETRACI-ILORIDE. .................................................... 22 Preparation of partially purified culture supernatant ................ 22 Cell factors ............................................................... 22 Modeling ................................................................... 23 iv Results and Discussion ................................................... 25 Kinetics of CG. transformation ......................................... 25 Effects of Copper ........................................................ 27 Copper toxicity ........................................................... 29 Stimulated indigenous flora ............................................. 33 CHAPTER 5 — ISOLATION OF COPPER EFFECT ON BIOMOLECULE ...... 37 Materials and Methods ................................................. 37 Modeling ................................................................... 38 Results and Discussion ................................................. 38 CHAPTER 6 — ENGINEERING APPLICATION ................................... 42 CHAPTER 7 — CONCLUSIONS ....................................................... 45 FUTURE WORK RECOMMENDATIONS ................................................ 47 LIST OF REFERENCES ...................................................................... 48 APPENDIX A ................................................................................... 50 APPENDD( B ................................................................................... 54 APPENDIX C ................................................................................... 57 APPENDIX D ................................................................................... 60 APPENDIX E .................................................................................... 63 APPENDD( F .................................................................................... 64 APPENDIX G .................................................................................... 66 LIST OF TABLES Table 4.1 Comparions of kinetic coeflicients for transformation of CT by P .stutzeri KC using biomolecule secreted from SGM with 0.32 M of copper and P. fluorescens grown without copper. Table A-1 Effect of initial medium pH on optical density during aerobic growth of P. fluorescens in synthetic groundwater. Table A-2 Effect of copper concentration on optical density during aerobic growth of P. fluorescens in synthetic groundwater. Table A-3 Effect of initial medium pH on optical density for denitrifying growth of P. fluorescens in synthetic groundwater. Table A-4 Effect of copper concentration of optical density on denitrifying growth of P. fluorescens in synthetic groundwater. Table A-5 Efl‘ect of copper concentration on protein production for aerobic and denitrying growth of Pseudomonas KC in synthetic groundwater. Table A-6 Efi‘ect of copper concentration on protein production for aerobic and denitrifying growth of Pseudomonas KC in Schoolcraft groundwater Table A-7 Effect of copper concentration on protein production for copper-sensitive and copper-tolerant Pseudamonas sam'ngae in synthetic groundwater. Table A-8. Efl‘ect of initial medium pH on protein production for copper-sensitive and copper-tolerant Pseudomonas sym’ngae in synthetic groundwater. Table 3-] Original data used for determination of biomass for P. fluorescens in SGM with and without copper for modeling. Table B-2 Original data used for determination of biomass for Pseudomonas syringae in SGM with and without copper. Table B-3 Data and calculations used for determining the observed yield of Pseudomonas fluorescens, Pseudomonas stutzeri KC, and Pseudomonas Syringae grown in SGM without copper using largest dry weight value. Table 01 Second order rate coeflicient and transformation capacity by secreted factor produced in synthetic groundwater without copper. Table C-2 Second order rate coefficient and transformation capacity by secreted factor produced in synthetic groundwater with copper. Table C-3 Second order rate coefficient and transformation capacity by secreted factor produced in synthetic groundwater with copper and stimulated indigenous flora grown with and without copper in Schoolcrafi groundwater. Table D-l First order rate coefficient and decay coeflicient by secreted factor produced in synthetic groundwater without copper. Table D-2 First order rate coefficient and decay rate by secreted factor produced in synthetic groundwater with copper. Table D-3 First order rate coeficient and decay coeficient by secreted factor produced in synthetic groundwater with copper and stimulated indigenous flora grown with and without copper in Schoolcrafi groundwater. Table E] Second order rate coefficient and transformation capacity by secreted factor produced in synthetic groundwater in absence of copper. Table E-2 Second order rate coefficient and transformation capacity by secreted factor produced in synthetic groundwater in presence of copper. Table F -1 First order rate coeficient and decay coeficient by secreted factor produced in synthetic groundwater in absence of copper. Table F-2 Second order rate coeficient and transformation capacity by secreted factor produced in synthetic groundwater in presence of copper. LIST OF FIGURES Figure 3.1 Effects of pH on aerobic growth of Pseudomonasfluorescens in synthetic groundwater. Figure 3.2 Effects of trace copper on aerobic growth of Pseudomonas fluorescens in synthetic groundwater. Figure 3.3 Efiects of pH on denitrifying growth of Pseudomonas fluorescens in synthetic groundwater. Figure 3.4 Efl‘ects of trace copper on denitrifying growth of Pseudomonas fluorescens in synthetic groundwater. Figure 3.5 Efl°ects of trace copper on aerobic growth of Pseudomonas stutzerz’ KC in synthetic groundwater. Figure 3.6 Efl‘ects of trace copper on denitrifying growth of Pseudomonas stutzeri KC in synthetic groundwater. Figure 3.7 Effects of trace copper on aerobic growth of Pseudomonas stutzeri KC in Schoolcrait groundwater. Figure 3.8 Efi‘ects of trace copper on denitrifying growth of Pseudomonas stutzeri KC in Schoolcrafi groundwater. Figure 3.9 Effects of pH on aerobic growth of copper-sensitive Pseudomonas syringae in synthetic groundwater. Figure 3.10 Effects of pH on aerobic growth of copper-tolerant Pseudomonas syringae in synthetic groundwater. Figure 3.11 Effects of copper on aerobic growth of copper-sensitive Pseudomonas .sjm'ngae in synthetic groundwater. Figure 3.12 Efl‘ects of copper on aerobic grth of copper-tolerant Pseudomonas syringae in synthetic groundwater. Figure 4.1 Transformation of CT by biomolecule secreted from SGM with 0.32 M of copper and Pseudomonasfluorescens grown from SGM without copper. Figure 4.2 Second order coefficients for CT degradation for P. fluorescens gown with and without copper using biomolecule from a KC culture gown without copper. Figure 4.3 CT transformation capacity for P. fluorescens gown without copper and using secreted biomolecule from a KC culture gown without copper. Figure 4.4 CT transformation capacity for P. fluorescens gown with copper and using secreted biomolecule from a KC culture gown without copper Figure 4.5 Second order coeflicients for CT degadation for P. fluorescens gown with and without copper using biomolecule from a KC culture gown with copper Figure 4.6 CT transformation capacity for P. fluorescens gown without copper and using secreted biomolecule from a KC culture gown with copper Figure 4.7 CT transformation capacity for P. fluorescens gown with copper and using secreted biomolecule from a KC culture gown with copper Figure 4.8 Second order rate coeflicients for CT degadation for indigenous flora gown with and without copper using biomolecule fi'om a KC culture gown without copper. Figure 4.9 CT transformation capacity of indigenous flora gown with and without copper using biomolecule fi'om a KC culture gown without copper. Figure 4.10 Second order rate coefficients of CT degadation for indigenous flora gown with and without copper and biomolecule from a KC culture gown with copper. Figure 4.11 CT transformation capacity of indigenous flora gown with and without copper and using biomolecule from a KC culture gown with copper. Figure 5.1 Pseudo-second order rate coefficients for copper-sensitive and copper-tolerant P. syringae strain gown without copper using secreted biomolecule fi'om a KC culture gown in the absence of copper. Figure 5.2 Pseudo-second order rate coefficients for copper-sensitive and copper-tolerant P. syringae strains gown without copper using secreted biomolecule flour a KC culture gown in the presence of copper. Hc LIST OF SYMBOLS first order decay coefficient, hr '1 total mass of substrate in the system active organism concentration (protein), mg/L initial active-biomass concentration, mg/L initial concentration of substrate in the aqueous phase, mg/L theoretical or true biomass transformation capacity, mg substrate / mg protein second order rate coefi'icient, L/mg protein-hr first order rate coeficient, hr'l time, hours VL/ (V L+HcVG) Henry’s constant (-) CHAPTER 1 INTRODUCTION Previous research on Pseudomonas stutzeri KC has established that the degradation of carbon tetrachloride (CT) is a cometabolic process including two key elements. One of these elements is a small (S 500 Da) extracellular molecule secreted under iron-limiting conditions. The second is a cell factor(s) that can be supplied by diverse cell types, including Pseudomonasfluorescens and Escherichia coli [23: Tatara unpublished data]. Production of the cell associated factor is a robust trait of many microorganisms and is not dependent on iron or oxygen levels. By contrast, production of the extracellular molecule is achieved by strain KC only under iron limiting ( S 1.0 uM) conditions [4,7]. Copper also plays a role in the transformation of CT. Copper is required for the nutrition of nearly all organisms. It mainly serves as an inorganic component within enzymes [22]. The role of copper for CT degadation by strain KC presents an interesting paradox. At pH 8.2, 6.4 uM copper is toxic for aerobic gowth, but this level of copper supports degadation rates that are higher than rates obtained at lower concentrations. Under most circumstances, the free cupric ion appears to be responsible for toxic efi‘ects on cell gowth [8,19]. In order to maximize the rates of CT degadation for field applications, the level and role of copper should be clearly I understood. Previous research provides some evidence for the direct participation of copper at the reaction leveL It includes the data obtained from experiments with Pseudomonasfluorescens and inhibited CC14 transformation by cyanide, a metalo-center inhibitor [23,24]. One possible role of copper is as a component in the active site of the secreted lactor. When cells are gown in the absence of copper or at very low copper concentrations, they tend to lack the potential to degade CT unless extra copper is added (24: Tatara et al.). Another possible scenario is direct coupling of copper with CT; with reduced copper acting as an election donor for CT reduction. The range of pH values over which CT degadation occur is broad, but the optimum for CT transformation is approximately 8.5 as measured by Tatara [23]. Copper speciation likely influences the effect of pH. Biotransformation experiments performd with fixed copper concentrations and varied pH levels can conceivably isolate the effects of copper speciation revealing toxic species and changes in biomolecule fimction at the reaction level. The primary objective of this research was to investigate the role of copper in the kinetics of CT degadation and to ascertain at what level it participates in the transformation. It may be possible to inject the secreted biomolecule directly into a contaminated site along with added copper rather than injecting KC cells. Because the cells required to regenerate the secreted factor can be native to the contaminated site, ecological and transport issues raised by the introduction of non-native organisms are avoided. CHAPTER 2 MATERIALS AND METHODS Organisms. Pseudomonas sp. strain KC (DSM deposit no. 7136, ATCC deposit number 55595), derived originally from aquifer solids from Seal Beach, CA (Criddle et aL, 1990), was routinely maintained on nutrient agar plates. The copper sensitive and tolerant strains of Pseudomonas syringae Al487 and Al 513R were obtained fi'om University of California, Berkeley (Courtesy, S. Lindow). They were routinely cultured on King’s medium B (KB) at 30° C before inoculated to nutrient broth (3). Pseudomonas fluorescens (ATCC deposit no. 13525) was obtained from the culture collection of the Microbiology Department at Michigan State University. Chemicals. All chemicals used were ACS reagent gade (Aldrich or Sigm Chemical Co.). All water used in reagent preparation was deionized 18 Mohm resistance or geater. Carbon tetrachloride (99 % purity) was obtained fi'om Aldrich Chemical Co., Milwaukee, WI. Media. Synthetic goundwater medium (SGM) contained (per liter deionized water): 0.455 g of NaSiO; 9H20, 0.16 g of Na2C03, 0.006 g of NaSOi, 0.02 g of KOH, 0.118 g of MgClz o6H20, 0.0081 g of CaClz 02 H20, 6.81 g of KHzPO4, 0.8 g NaOH, 1.6 g 4 NaNO3, 1.6 g acetate, and 1 ml of trace element solution. The trace element solution contained (per liter deionized water): 0.021 g LiClz, 0.08 g CuSO. 05H20, 0.106 g ZnSO4o7H20, 0.06 g TiCh, 0.03 g KBr, 0.03 g KI. 0.629 g MDCIzO4H20, 0.036 g SnC12-2H20, and 0.3 g FeSO4o7H20. In certain experiments, copper was omitted fi'om the trace element solution. The pH of SGM was adjusted to 8.2 by addition of NaOH pellets followed by titration with 1M NaOH, then autoclaved at 121 °C for 25 min. Nutrient broth and nutrient agar (Difco) plates were prepared according to manufacturer’s instructions. Nutrient broth cultures were gown aerobically at 20 °C 150 rpm to stationary phase (12 hours). 1 % (v/v) ofthis culture was used as the inoculum for experiments to evaluate the effects of trace copper. Groundwater. Groundwater fi'om a CT-contaminated aquifer in Schoolcrafi, MI, was used to prepare an enrichment of indigenous flora. Groundwater samples were obtained manually by withdrawing goundwater with a Teflon bailer from a 2 inch steel well screened at 30 feet below the water table. Groundwater samples were stored in pre- sterilized, sealed Nalgene carboys or in Wheaton bottles equipped with Teflon lined caps at 4 °C. Analytical methods. Carbon tetrachloride was assayed by gas chromatography (GC) by removing a headspace sample for analysis. A five-point external calibration curve was prepared by diluting a primary standard (8.2 ug of CT per ul‘of methanol) in SGM medium to achieve concentrations bracketing that of assay samples. Assay and calibration samples were carried out in 28 ml serum tubes (Bellco Glass no. 2048-00150) sealed with S Teflon lined butyl rubber septa (West Co. no. 935326) and aluminum crimp seals. Nitrate, nitrite, and acetate ions were assayed by ion chromatogaphy (Dionex model 20001SP ion chromatogaphy with suppressed conductivity detection equipped with a Dionex Ionpak AS4-A anion exchange column and utilizing a 1.8 mM bicarbonate/ 1.7 mM carbonate mobile phase at a ml/min). Chromatogams were recorded and data integated using Turbochrom 3 software (Perkin Elmer Corp). External standard cah'bration curves, which bracketed the concentrations of the test samples, were prepared by diluting primary ion standards into deionized water having at least 18 Mohm resistance. Measurements of pH were made with an Orion model 720A pH meter. Optical density measurements were measured at 660 nm using a Shimadzu UV -160 spectrophotometer. CHAPTER 3 EFFECT OF INITIAL pH AND COPPER CONCENTRATION ON GROWTH Materials and Methods. Effects of Copper on Growth. To insure that toxicity did not limit the rate of CT degadation at high copper concentration, studies were performed evaluating the efl‘ects of copper on the growth rate of Pseudomonasfluorescens, Pseudomonas stutzeri KC, and Pseudomonas syringae. Starter cultures were prepared by transferring cells fi'om nutrient agar plates to previously autoclaved nutrient broth using aseptic technique. After 12 hours ofgowthinnutrient broth, a1 %(v/v) inoculumwastransferredto SGMor Schoolcraft goundwater. Copper was added just prior to inoculation as CuSO. 05H20. A gowth curve for Pseudomonasfluorescens in SGM was obtained using 28 ml balch tubes and 250 ml flakes for denitrifying and aerobic gowth conditions, respectively. Balch tubes containing 5 ml of SGM were passed through an anaerobic hood interlock three times to remove oxygen fi'om the headspace then sealed with rubber stoppers. Balch tubes and flasks containing SGM were autoclaved for 20 minutes and allowed to equilibrate for at least for 1 day before nutrient broth inoculation. This procedure prevented excess phosphate precipitation alter autoclaving. Optical density values at 660 nm (ODm) were obtained fi'om aerobic samples containing 0, 0.32, 3.2, 6.4, 9.6, 12.8 M copper for approximately every 6 hours for 40 hours. OD“ values were also obtained fiom denitrifying samples containing 0, 0.32, 3.2, 6.4, 9.6 uM copper approximately every 5 days for 27 days. Initial and final pH was measured for each sample. Growth of strain KC in SGM and Schoolcraft goundwater was evaluated using SOO-ml Wheaton bottles and 250-ml flasks for denitrifying and aerobic gowth conditions, respectively. Bottles with loosened caps were passed through an anaerobic hood interlock three times to remove oxygen fi'om the headspace. Aerobic and denitrifying cultures gown in Synthetic Groundwater containing 0 - 12.8 M copper were sampled approximately every 4 hours for 36 hours. Total protein accumulation was monitored using the modified Lowry protein analysis at 660 nm [14]. Schoolcrafi goundwater was amended with acetate (750 mg/L) and nitrate (450 mg/L) for denitrifying growth and with acetate alone for aerobic gowth. After pasteurization at 65 °C for 8 hours, phosphate was also added to give a final concentration of 10 mg/L. Samples of culture gown in amended Schoolcrafi goundwater were taken approximately every 4 hours for 36 hours, and total protein accumulation was monitored using the modified Lowry protein analysis. Initial and final pH for each sample was measured. Pseudomonas Mugae was gown aerobically in a shake flask with SGM containing copper concentrations that depended upon the strain : a copper sensitive strain A1487 provided by S. Lindow from University of California, Berkeley was routinely cultured on King’s medium B (KB) at 30° C before inoculated to nutrient broth [3]. After 12 hours gowth in nutrient broth, a 1 % (v/v) inoculum was transferred to SGM and gown at 0, 0.32, 1.28, 3.2, 12.8, 32 [1M copper ; the copper tolerant strain A1513R (provided by S. Lindow) was gown at O, 0.32, 1.28, 12.8, 32, 64M. Samples were taken approximately every 12 hours for 114 hours. Protein accumulation was monitored by Lowry at 660 nm after all samples reached stationary phase [14]. Initial and final pH were measured. Effect of pH on Growth. Growth of Pseudomonas fluorescens and Pseudomonas syringae as a function of initial medium pH was determined by preparing synthetic goundwater at various pH levels. NaOH pellets and 1M NaOH solution were used to adjust solution pH to the desired level. Growth curves were obtained for Pseudomonas fluorescens in SGM 28-ml balch tubes and a 250-ml shake flask at pH values of 6.5, 7, 7.5, 8, 8.5. Each sample was prepared in triplicate. Anaerobic samples were rendered anoxic by passage through an anaerobic glove box, where the samples were sealed under an atmosphere of 98% N2 and 2% H2. For aerobic cultures, optical density was measured every 6 hours for 42 hours; for denitrifying culture, optical density was measured approximately every 5 days. Initial and final pH were measured for each sample. Copper sensitive and copper tolerant Pseudomonas syringae strains were gown aerobically at initial pH values of 6.5, 7, 7.5, 8, 8.2, 8.5 and monitored for gowth by the Lowry protein assay. Samples of culture gown in 250-ml flakes were removed approximately every 12 hours for 114 hours. After all samples reached stationary phase, protein accumulation was monitored by the Lowry assay at 660 nm. Results and Discussion Rapid transformation of CCl4 requires cell factors, notably membranes and NADH, which may be obtained fi'om many different cell types plus a secreted biomolecule produced by Pseudomonas stutzeri' KC [23]. In the presence of secreted factor, aerobically-gown Pseudomonasfluorescens transformed CT at a pH of 8.2 in synthetic goundwater (T atara, 1996). The maximum gowth rate under aerobic conditions (Figures 3.1 and 3.2) was about 10 times geater than the gowth rate under denitrifying conditions at pH 7 (Figure 3.3 and 3.4). Increasing levels of added copper had a profound efi‘ect on the gowth of cells gown under aerobic but not under denitrifying conditions. 0,6- f/ A 04‘ ' "$‘s g / "i i--.."‘ 8 :1 85 U ’ o E ’ "'x 6.5 § 02- 75 V .§ I ' "x" a .1" 0 =7; . ' ' 15 30 45 Time(bours) -0.2 Figure 3.1 Efi‘ect of pH on aerobic growth of Pseudomonas fluorescens in synthetic goundwater. Labels on plot indicates initial pH. 10 0.6 - A 0 § 0.4 - s \ z. . .5 0.32 8 g I 3.2 8. , 0.2 - 6.4 “A ., 9 6 0 15 30 45 Time (hours) Figure 3.2 Efl‘ect of trace copper on aerobic gowth of Pseudomonas fluorescens in synthetic goundwater. Labels indicate added copper concentration in micromolar units. 11 A possible explanation for copper toxicity in the aerobic cultures is copper-catalyzed production of toxic forms of oxygen during respiration, such as superoxide or hydroxyl radicals. Superoxide can cause oxidative destruction of lipids and other biochemical components [6,15]. The hydroxyl radical OH- also attacks many of the organic substances present in cells and can cause cell lysis. 0.1- '8 Optical Density (A660) 0.02 30 Time (days) -0.02 Figure 3.3 Efl‘ect of pH on anoxic growth of Pseudomonas fluorescens in synthetic goundwater. Labels indicate initial pH 12 0.1- Optical Density (A660) 44. i 0 10 Time (days) 20 Figure 3.4 Effect of trace copper on anoxic gowth of Pseudomonas fluorescens in synthetic groundwater. Labels indicate added copper concentration in micromolar units. Unlike P. fluorescens, the gowth rate of Pseudomonas stutzeri KC under denitrifying conditions was as fast as its gowth rate under aerobic conditions (Figure 3.5 and 3.6). Like P. fluorescens, however, copper tolerance of strain KC was higher under denitrifying conditions than under aerobic conditions. Low concentrations of copper inhibited gowth, but to a lesser extent than observed for Pseudomonasfluorescens. 250-1 § u—s LII O l 0.32 Protein (jig/ml) § 6.4 O . .—-‘ V g T ' T 9.6 ‘ 12 . 24 6 0 Time (hours) 3 Figure 3.5 Efl‘ect of copper on aerobic growth of Pseudomonas stutzeri KC in synthetic goundwater. Labels on plots indicate added copper concentration in micromolar units. ‘ 14 250 a 200 - "E 150 - °' .32 .5 O 2 32 ° .1 5.4 36 Time (hours) Figure 3.6 Efl'ect of copper on anoxic gowth of Pseudomonas stutzen' KC in synthetic goundwater. Labels on plots indicate added copper concentration in micromolar units. 15 In Schoolcrafi goundwater, gowth of strain KC was more inhibited than in synthetic goundwater, probably because of higher backgound levels of trace metals in the goundwater (Figure 3.7 and 3.8). 250 - 200 ‘ Protein (pg/ml) C.‘ (3 § 50- 0 12 Time (hOIII'S) 24 36 Figure 3.7 Efl‘ect of copper on aerobic gowth of Pseudomonas stutzeri KC in Schoolcrafl groundwater. Labels on plot indicate added copper concentration in micromolar units. 16 250 - 200 « "33150 - 0.32 °:‘t’ E 9:: /—‘ 5:100 - , I, ' / 3.2 50 « 0 I A I 12 Time (ham) 24 36 Figure 3.8 Effect of copper on anoxic gowth of Pseudomonas stutzeri KC in Schoolcraft groundwater. Labels on plot indicate added copper concentration in micromolar units. 17 Maximum gowth rate was obtained at pH 7.5 for both the copper sensitive Pseudomonas syringae strain Al487and the copper tolerant strain A1513R. Low levels of copper inhibited gowth of strain Al487. For Gram-negative bacteria, copper -resistance is associated with a unique set of proteins : CopA and CopC in the periplasmic space, CopB in the outer membrane. These proteins are induced by copper. Their likely function in copper resistance is sequestration of copper ions in the periplasm, preventing entry of copper ions into the cytoplasm [18]. 200 - Proteig (pg/m1) 8 I l 0 40 . 80 120 Time(hours) Figure 3 .9 Efi‘ect of pH on aerobic gowth of copper-sensitive Pseudomonas syringae in synthetic goundwater. Labels on plot indicate initial pH. 200- Protein (pg/ml) 8 80 120 Time(hours) Figure 3.10 Efl'ect of pH on aerobic gowth of copper-tolerant Pseudomonas Mngae in synthetic goundwater. Labels on plot indicate initial pH. 19 200- 0 4“ ”2 :é; /‘ 1.28 am / 5 ‘ 32 a . 8 f/ e 1:? / \ . . é??- -_ “(\k 128 32 0 ' . - 0 40 Time (hours) so 120 Figure 3.11 Efl‘ect of copper on aerobic gowth of copper-sensitive Pseudomonas syringae in synthetic goundwater. Labels on plot indicate added copper concentrations in micromolar units. 200- 12.8 /‘. "E 3‘: 00 2:1 ‘ f 3 1. l e 32 a. .» ,4, 4.7. ‘I/ I” I ‘5’ 4' 64 O I I — 0 40 Time (hours) 80 120 Figure 3.12 Efl‘ect of copper on aerobic gowth of copper-tolerant Pseudomonas syringae in synthetic goundwater. Labels on plot indicate added copper concentrations in micromolar units. 21 CHAPTER 4 DETERMINATION OF THE EFFECTS OF TRACE COPPER ON THE BIODEGRADATION OF CARBON 'I'ETRACHLORIDE Preparation of partially purified culture supernatant. In order to determine whether the presence of trace copper afl‘ects the transformation of carbon tetrachloride at the reaction level, cultures of strain KC gown with and without added trace concentrations of copper were gown for approximately 24 hours in Synthetic Groundwater Medium (SGM), then fi'actionated by centrifirgation and ultrafiltration. Following an initial screening for CCli transformation activity, cells were harvested by centrifugation (15 minutes, 5,000 rpm), and the supernatant filtered through a 0.2 uM filter 3 times. Filtered supernatant was further fractionated by filtration through Amicon 10, 000 molecular cut- ~ ofl’ filters in a Coy anaerobic glove box (95% N2 5% H2 atmosphere). The resulting filtrate was then lyOphilized. CT transformation assays were performed by dissolving the Iyophilized fraction in 4.5 ml SGM, combining the resulting solution with 0.5 ml samples of cell washed factor, under a N2 atmosphere, spiking with CCL (10 ug/ liter), and assaying CCL levels by gas chromatogaphy, as described in Chapter 2. Cell factors. Pseudomonas fluorescens cells gown in the presence and absence of trace copper SGM were harvested at OD 660 values of approximately 0.192 and 0.187 22 respectively. Cell and suspended fiactions were separated by centrifugation (10 min, 3000 rpm). Cell pellets were resuspended to one tenth their original volume in fresh medium of the identical composition used for gowth. To evaluate the efi‘ect of copper, cell and supernatant combinations of Pseudomonas stutzeri strain KC and Pseudomonas fluorescens were mixed. Copper was added to selected samples as CuSO. -5H2O fi'om 80mg/L or 800mg/L stock solutions to give the desired final concentration. Each sample was prepared in triplicate, rendered anoxic by passage through an anaerobic glove box, sealed under an atmosphere of 98% N2 and 2% H2, spiked with 10 ul of a 20 mg/L aqueous stock solution of CT, placed on a shaker, and assayed for CT degadation by GC analysis of the headspace. To prepare enrichments of indigenous microflora from Schoolcraft goundwater, two 500- ml samples of Schoolcrafi goundwater were placed into sterile IOOO-ml flask and amended with 500 mg/L acetate and 430 mg/L nitrate. One flask received 0.32 M copper. After 5 days of shaking, both groundwater samples received an additional spike of 500 mg/L acetate. Stimulated indigenous flora were harvested at optical densities of 0.234 and 0.279 for cultures gown in the absence and presence of trace copper, respectively, and centiifiiged to separate cells fiom supernatant. Cell supernatant combinations of Schoolcraft indigenous flora and Pseudomonas slutzeri KC were prepared in the same way as described previously for Pseudomonasfluorescens. 23 Modeling. Tatara (1995) demonstrated that disappreance of CG, and appearance of products could be quantified with a simple model in which transformation activity is assumed to decay with time: -Wa=k'XCaq-e—kdr= Vaq Meme-kw k (Vaq+Hch) (1) where M can motal mass of CCL in the system = Caq(Vaq+Hch), k= first order rate coeflicient (min ’1), k4 = first order decay coefficient air"). Equation (1) can be integated to obtain M as a function of time: = 11 V09 .. - (2) M Moexp[kd(V04+H0V0¢1)(exp( k4) 1)] The endogenous decay term kd includes loss of activity caused by cell death and by depletion of reducing power required for monooxygenase activity. Another simplified case is obtained when product toxicity is the dominant factor causing loss of transformation activity. F exp(—k'AFt) (3) X0 - 91170 exp(—k'AF t) M=Mo Where Clo is initial concentration of substrate in the aqueous phase (mg/L), F = Xo — Clo IT. Disappearance of the target compounds was modeled using both equations (2) and (3). Kinetic parameters were estimated by nonlinear regession using Statistics (MathSofl, Gaithersburg, MD). 24 Results and Discussion Kinetics of CC14 transformation. The dependence of CCh transformation rates on concentration of copper and disappearance of CCI. activity was evaluated by plotting the logarithm of mass of C0. versus time. A simple model invoking first-order degadation with respect to CC14 concentration failed to describe the transformation (Figure 4.1). However, the endogenous decay model of equation 2 did describe the data, suggesting gadual decay on depletion of a key reactant. In the endogenous decay model, loss of transformation activity is controlled by time, and is not afi‘ected by the amount of nongowth substrate transformed. This contrasts with the transformation capacity model of equation 3 that also successfully described the data. In this case, the decay of transformation activity is directly proportional to the amount of nongowth substrate transformed. Table 4.1 Comparison of kinetic coeflicients for transformation of CT by P. stutzeri KC using biomolecule secreted from SGM with 0.32 M of copper and P. fluorescens gown with copper. Best fit for the parameters of equation 3 (Xo =187.08 mg/L and Henry’s constant = 1). Equation “1de k'ang-hr) mu) 1:. Tog/mg) “mm" (M coeficient r2 First-order 12.8 0.016 2.99 NA’ NA 0.881 2 12.8 0.046 8.18 1.255 NA 0.989 3 12.8 0.039 6.9 NA 0.19 0.991 NA“ indicates that data is not available. 25 -o.4 - a E —0.8 . -l.2 . . 0 l time(hours) Figure 4.1 Transformation of CT by biomolecule secreted from SGM with 0.32 M of copper and Pseudomonasfluorescens gown fi'om SGM without copper. Fitting parameters are summarized in Table 4.1. 26 Effects of Copper. Examination of CT degrading activity with selected combinations of cell and fi'eeze-dried fractions of P. stutzeri KC revealed a distinct efl'ect due to the presence of copper. Tatara (1995) reported previously that rapid transformation of CC14 was only obtained with samples containing added copper and omission of only 1 uM copper was suficient to prevent CC14 transformation. The present work confirmed this finding. As shown in Figure 4.2, the efl‘ect of added copper on CT degadation was most striking in samples of freeze-dried fractions gown without copper. Samples fiom fi'eeze- dried fiactions gown without copper did not degade CT to any appreciable extent, but when exogenous copper was added, rapid and appreciable degadation of CT was observed. Exogenous copper added to samples gown in the presence of copper also increased the rate of CT degadation, but not to the same extent as samples gown without copper. 0.15 - 0.1 - P .fluorsecens gown w/Cu k' 0.05 - . / ‘ / P .fluorsecens gown 0 . w/o Cu I ‘ 1 0 1 10 100 Copper Concentration (11M) Figure 4.2 Second order coeficients for CT degadation for P. fluorescens gown with and without copper using biomolecule from a KC culture gown without copper (Biomolecule was harvested from 2 difl‘erent cultures: biomolecule from the first culture for experiments with 0, 0.32, 3.2 uM Cu”; biomolecule from the second culture was used for experiments with 0.64, 6.4 and 25.6 m cu”). 27 (10006'- 0 , . j 0 l 10 100 Copper concentration (uM) Figure 4.3 CT transformation capacity for P. fluorescens gown without copper and using secreted biomolecule from a KC culture gown without copper (Biomolecule was harvested from 2 difi‘erent cultures: the first culture for experiments with O, 0.32, 3.2 uM Cu”; biomolecule fi'om the second culture was used for experiments with 0.64, 6.4 and 25.6 M Cu”). (1008‘1 Tc 0.004 a o, , m I I 0 l 10 100 Copper Concentration (uM) Figure 4.4 CT transformation capacity for P. fluorescens gown with copper and using secreted biomolecule fi'om a KC culture gown without copper (Biomolecule was harvested from 2 different cultures: the first culture for experiments with 0, 0.32, 3.2 uM Cu”; biomolecule from the second culture was used for experiments with 0.64, 6.4 and 25.6 M Cu”). 28 Copper toxicity. Copper addition just prior to initiation of the CT degadation assay markedly increased the extent of CT degadation, and this was true for cells gown in the presence or absence of copper. At low copper concentrations, CT transformation activity increased linearly as exogenous copper concentration was increased. Beyond a certain level, however, additional copper provided no benefit. Copper concentrations over the range of 6.4 uM did not increase degadation activity significantly. A reduced rate of activity was observed at 25.6 M. The rate of CT degadation and the efl‘ects of copper concentration depended upon the method of biomolecule production. Freeze-dried fractions gown in SGM without copper exhibited geater sensitivity to exogenous copper concentrations and higher tolerance to high copper concentrations. On the other hand, freeze-dried fi'actions gown in SGM with copper did not show the same sensitivity to copper concentrations. Transformation activity remained stable at copper concentrations of 0.64 M to 12.8 1.1M, but rapidly declined at higher levels. When microorganisms encounter excessive copper concentrations, detoXification is mediated by various mechanisms include energy dependent emux, intracellular sequestration, and extracellular complexation [10]. The presence of copper in the medium can induce proteins that bind the copper and thereby detoxify it. Competition between such proteins and the secreted factor may explain the higher activity levels of biomolecule in synthetic goundwater in the absence of copper. 29 0.08 - 0.06 - P fluorsecens gown kt 0.04 .. 0.02 ‘ P .fluorsecens gown w/o Cu 0 I I TI 0 l 10 100 Copper Concentration (uM) Figure 4.5 Second order coeficients for CT degadation for P. fluorescens gown with and without copper using biomolecule fiom a KC culture gown with copper (Biomolecule was harvested fi'om 2 difl‘erent cultures: biomolecule fi'om the first culture for experiments with 0, 0.32, 3.2 uM 012+; biomolecule fi'om the second culture was used for experiments with 0.64, 6.4 and 25.6 M Cu”). 30 0.12 - 0.08 - l Tc 0.04 - 0 fl 0 l 10 100 Copper Concentration (uM) Figure 4.6 CT transformation capacity for P. fluorescens gown without copper and using secreted biomolecule from a KC culture gown with copper (Biomolecule was harvested fiom 2 difl‘erent cultures: the first culture for experiments with 0, 0.32, 3.2 uM Cu”; biomolecule from the second culture was used for experiments with 0.64, 6.4 and 25.6 M Cu”). 0.0006 - 0.0004 ‘ Tc ‘ 0.0002 '1 .h. 0 r I I 0 l 10 100 Copper Concentration (uM) Figure 4.7 CT transformation capacity for P. fluorescens gown with copper and using secreted biomolecule fi'om a KC culture gown with copper (Biomolecule was harvested from 2 difl‘erent cultures: the first culture for experiments with 0, 0.32, 3.2 uM Cu”; biomolecule fiom the second culture was used for experiments with 0.64, 6.4 and 25.6 M Cu”). 31 Previous research has established that rapid transformation of CC14 requires both cell factors and a secreted factor present in supematant of Pseudomonas stutzerr' KC. This study establishes that the role of copper is at the reaction level. Rapid and reliable CC]. transformation was obtained when cells fi'actions were reconstituted with a fi'eeze-dried supernatant fiaction from strain KC, confirming the need for both extracellular and intracellular factors. Micromolar levels of copper dramatically enhanced CCL. transformation, suggesting that copper participates directly in the reaction, perhaps in a catalytic role. The results of this work support the conclusion that transformation of ca. by strain KC proceeds by a complex mechanism. The transformation appears to involve three factors: a cell fiaction, a supematant fiaction, and copper. Evidence for this hypothesis include the following observations: (1) the fi'eeze-dried supernatant did not transform CT unless cell factors were provided; (2) the freeze-dried fiaction harvested fi'om culture gown in SGM without copper did not exhibit high transformation rates, even though cell factors were provided; (3) and the rate of transformation and persistence of activity increased as the added copper increased to an upper limit, depending on the method used to prepare fi'eeze-dried fiactions. Tatara (1996) demonstrated that a cytoplasmic component is not required for transformation, and that NADH does not reduce the secreted factor directly. A possible role for copper is in the reduction of the secreted factor possibly in conjunction with some component of the cell membrane. Alternatively, copper may be an essential component of the secreted factor. The secreted factor may fimction as a mediator of electron transfer between the cell and the CClt molecule. Of significance is the fact the 32 rate of transformation of secreted factor does not increase linearly as the concentration of copper is increased. Above a certain concentration, additional copper does not increase the transformation rate. At high copper concentrations, decreased transformation rates were observed. At low levels, copper may provide electrons for CCh reduction, but at high levels, reduction or regeneration of the secreted biomolecule may be shut down by copper toxicity. Stimulated indigenous flora. Regeneration of activity by organisms indigenous to the Schoolcrafi aquifer was examined at various copper concentrations. As shown in Figure 4.8, indigenous flora successfully regenerated the activity of a fi'eeze-dried fraction containing the secreted biomolecule. This finding supports previous reports (Tatara; 1995) and indicates that the ecological and transport issues raised by the introduction of KC non-native organism might be avoided by stimulating indigenous flora and adding the secreted factor (plus trace copper). The activity observed with the stimulated flora was geater than that observed with Pseudomonasfluorescens. For samples containing biomolecule harvested from culture gown with copper, the rate of CT degadation activity suddenly increased at an exogenous copper concentration of 3.2 M and remained high thereafter. On the other hand, freeze-dried fiactions from cultures gown in the absence of copper SGM had lower rates of transformation for added copper level exceeding 6.4 uM. 33 0.06 - 0.04 ~ indigenous flora grown w/o Cu .. \ 0.02 - «r- ? indigenous flora grown w/Cu O r I I 0 l 10 Copper concentration (pM) Figure 4.8 Second order rate coeficients for CT degradation for indigenous flora grown with and without copper using biomolecule from a KC culture grown without copper. 0.15 - 0.1 Tc indigenous flora gown 0.05 _ w/Cu . Copper Concentration (uM) . . Figure 4.9 CT transformation capacity of indigenous flora gown With and Without copper using biomolecule from a KC culture gown without copper. 34 0.06 - 0.04 d k' 0.02 - indigenous flora gown w/o Cu O , fl 0 l 10 Copper concentration (uM) Figure 4.10 Second order rate coeflicients of CT degadation for indigenous flora gown with and without copper and biomolecule from a KC culture gown with copper. 0.15 - 0.1 4 Tc indigenous flora gown 0.05 I H—fi 0 l 10 Copper Concentration (uM) Figure 4.11 CT transformation capacity of indigenous flora gown with and without copper and using biomolecule fi'om a KC culture gown with copper. 35 Compared to pure cultures like Pseudomonas fluorescens, the mixed culture of Schoolcrafi aquifer flora had a relatively high standard deviation, perhaps reflecting variability in the populations present. 36 CHAPTER 5 ISOLATION OF COPPER EFFECT ON BIOMOLECULE Materials and Methods. To better isolate the effects of copper on the secreted biomolecule produced by P. stutzeri KC, copper tolerant and sensitive Pseudomonas syringae strains were obtained fi'om the University of California, Berkeley (Courtesy, S. Lindow). The copper sensitive strain Al487 and the copper tolerant strains A1513R were originally isolated from almond trees and bean plants respectively. They were routinely cultured on King’s medium B (KB) at room temperature before inoculation to nutrient broth (3). Pseudomoms syn‘ngae strains were gown in the absence of trace copper in synthetic goundwater for 32 hours and harvested at OD 660 values of approximately 0.2. Cell and suspended fractions were separated by centrifugation (10min, 3000rpm). Cell pellets were resuspended to one tenth their original volume in fresh medium of the identical composition used for gowth. CT transformation assays were performed by adding 0.5 ml of Pseudomonas syringae culture to 4.5 ml of lyophilized supernatant containing secreted biomolecule produced by Pseudomoms Mutzeri strain KC, as described in Chapter 4. Copper was added to select samples as CuSO4 05H20 using a 80mg/L solution or 800mg/L to achieve final concentrations of O, 0.32, 3.2, 32, 64 M. Each sample was prepared in triplicate, rendered anoxic by passage through an anaerobic glove box, sealed under an atmosphere of 98% N2 and 2% H2, Spiked with 10 ul of a 20 37 mg/L aqueous stock solution of CT, placed on a shaker, then monitored for CT degadation by injection of headspace samples on a gas chromatogaph. Modeling. Assuming product toxicity is the dominant factor causing loss of transformation activity, the disappreance of the target compounds was modeled with equation (3) in Chapter 4, where X0 is the initial Pseudomonas syringae (mg/L). The observed yield on acetate for both cultures was calculated using dry weight measurements obtained by filtering a known volume through a pre-weighted 0.2 pm pore filter membrane (Gelman Sciences). Membranes were dried 6 hours at 104 °C and weighed. Ion chromatogaphy was used to measure the acetate consumption. The observed yield was calculated by dividing the increased in dry weight biomass concentration by the total acetate consumed over the identical time period. Actual substrate data at specific time points were used, but dry weight measurements were interpolated from a plot of dry weight versus time. This was necessary because substrate and dry weight data time points were taken at different times. Raw data and supporting calculations are provided in Appendix B. Results and Discussion As shown in Figure 5.1, copper sensitive and copper tolerant strains exhibited difi‘erent patterns of transformation at varied copper concentrations. The rate was low when no copper was added to sensitive cells and suddenly increased as concentration of copper increased. The rate did not increase significantly beyond 3 M and finally decreased. 38 Tolerant cell did not show as large an increase in rates, with little decrease in the rate of transformation at higher copper levels. In fact, the rate of CT transformation was highest at the highest copper concentration (Figure 5.1). This difference was even more apparent for copper sensitive cells combined with secreted factor fiom strain KC cells gown with copper (Figure 5.2). In this case, the rate of transformation decreased drastically for 32 M copper. The CT degadation rate of copper tolerant cells decreased as the added copper concentration increased. Examination of CT degading activity with selected combinations of cell and freeze-dried fractions of P. stutzeri KC indicated that the presence of copper in the gowth medium of strain KC influenced the transformation pattern obtained. When copper was not present, the rate of degadation and sensitivity toward trace amount of copper addition was higher than when the secreted factor was produced in the presence of copper. This suggests that copper has additional indirect efl‘ects on the transformation, beyond its direct efl‘ects at the reaction level. When microorganisms encounter excessive copper, detoxification is mediated by various biochemical mechanisms including energy dependent efllux, intracellular sequestration, and extracellular complexation [9]. The presence of copper can induce the production of proteins that detoxify copper by chelating it. These induced proteins may compete with secreted factor. This may explain higher activity for biomolecule produced in synthetic goundwater in the absence of copper. The rationale for use of copper sensitive and copper tolerant cells was that copper tolerant cells would transform CT more rapidly at high copper concentrations. This was not 39 observed. Instead, rapid transformation was associated with limited transformation capacity. This may indicate that toxic intermediates are produced causing cell damage or death during CT degadation. Alternatively, copper sensitive cells may have higher rate of degadation because copper tolerant cells produce chelating proteins that compete with the secreted factor for copper. 0.09 7 0.06 4 o I I I I 0 l 10 100 Copper concentration (uM) Figure 5.1 Pseudo-second order rate coefficients for copper-sensitive and copper-tolerant P. syringae strain gown without copper using secreted biomolecule flour a KC culture gown in the absence of copper. 0.03 1 sensitive 0.02 :_ k0 tolerant 0.01 ‘- 0 l 1 1 0 l 10 100 Copper concentration (pM) Figure 5.2 Pseudo-second order rate coeficients for copper-sensitive and copper-tolerant P. syringae strains gown without copper using secreted biomolecule fiom a KC culture gown in the presence of copper. 41 CHAPTER 6 ENGINEERING APPLICATION The introduction of novel organisms into a new environment is termed bioaugnentation. Many important scientific issues must be considered in evaluating the potential ecological consequences of the planned introduction of organisms into the environment. These include survival and reproduction of the introduced organisms, interactions with other organisms, and the effects of the introduced organisms on ecosystem firnction. When considering the environmental application of microorganisms, ecological issues are critical. This study presents a generally favorable outlook for use of the secreted factor produced by P. stutzeri KC in field applications. Use of the secreted factor with biostimulated indigenous flora avoids problems associated with the ecology and transport of non-native organisms. Although mixed cultures of indigenous flora fiom Schoolcraft aquifer had a high capacity for CT transformation, the pattern of transformation depended on the biomolecule production environment. The role of mixed cultures of aquifer flora on CT degadation should be investigated more thoroughly. Additional work is needed to identify the specific factor(s) that initiate decay of regeneration of the secreted factor and reduction of copper (II) to copper (I) in order to 42 provide electrons for CT transformation. Instead of adding cupric copper, CT transformation may be evaluated with direct cuprous addition. This thesis has addressed the catalytic role of copper on biochemical components and processes responsible for cometabolism of CCl4 degadation. It has raised several questions: What is the secreted factor made of? Why is it produced? What are the factors preventing it fiom being produced while gowing in synthetic goundwater, and is there any way to minimize inhibitory factors preventing its activity? How can we prevent decay of activity during filtration? Once we obtain these fundamental answers about generation and optimization of purification and storage conditions, many engineering applications of these secreted factors are likely to become apparent. Another question of interest is: What are the differences between the efl‘ects of copper obtained in the lab and its efl‘ects in field application? Addition of trace copper to goundwater to stimulate CT degadation leads to a paradox: higher copper addition may be needed to achieve high rates of CT degadation, but high copper concentrations may also kill KC or indigenous flora. Therefore, close monitoring and research before application is recommended in order to enhance CT degadation. A factor that severely limited this research was consistent production of the secreted factor. A small quantity of biomoelcule was produced for each study. Since activity of the biomolecule is not consistent from culture-to-culture, it is difiicult to conclude that difl'erences in CT transformation were due to various cell components. Also the increase 43 in pH caused by addition of freeze dried supernatant may lead to some unknown effect on CT transformation. CHAPTER 7 CONCLUSIONS . Trace amounts of copper are needed to enhance the rates of transformation of CT. . Copper affects the CT transformation at the reaction level. . Above a certain concentration, copper exerts toxic or inhibitory effects that reduce transformation, even in copper-tolerant cells. . Growth of cells in media without copper resulted in the production of secreted factor with higher transformation activity. 45 When the secreted factor was produced by cells gown in synthetic goundwater with 0.32 M of copper: Copper-tolerant P. syringae had lower CT transformation rates compared to the copper-sensitive P. syringae; except at high copper addition (32 uM). CT transformation activity was observed at low copper levels (range of 0 uM to 6.4 uM ). Copper-tolerant P. syringae had a high CT transformation capacity, suggesting reduced toxicity. . When the secreted factor was produced by cells gown in synthetic goundwater without copper: Higher rates of CT transformation were observed for both copper-tolerant and copper- sensitive cells. The highest transformation capacity observed during these experiments was obtained at 0.32 M copper for the copper-tolerant cells and when no copper was present for the copper-sensitive cells. The rates of CT transformation for copper-tolerant P. syringae did not increase in proportion to increment of copper concentrations; a decrease in rates was observed at the highest copper concentration tested (32 1.1M). Rates of CT transformation for copper-sensitive P. syringae decreased as the amount of exogenous copper increased. FUTURE WORK RECOMMENDATIONS . Investigate possible role and applications of copper for other cometabolic processes. . Increase the degadation time for copper-tolerant and copper-sensitive P. syringae. . Determine how medium components affect secreted factor production and identify methods for maximizing its activity. . Enumeration of gowth curve according to time interval to improve accuracy. . Evaluate CT degadation of indigenous flora gown under denitrifying conditions. 47 LIST OF REFERENCES . Cha, 1.8., and DA Cooksey. 1991. Copper resistance in Pseudanranas syringae mediated by periplasmic and outer membrane proteins. Microbiology 88:8915-8919. . Chang, W., and CS. Criddle.1995. Biotransformation of HCFC-22, HCFC-l42b, HCFC-123, and HFC-134a by methanotrophic mixed culture MMl. Biadegradatian 6: 1-9. . Criddle, CS. 1993. Kinetics of Cometabolism. Biotechnology and Bioengineering, 41:1048-1056. . Criddle, C.S., J.T.DeWitt, D.Grbric Galic, and PL. McCarty. 1990. Transformation of carbon tetrachloride by Pseudamanas sp. strain KC under denitrification conditions. App]. Environ, Microbial. 56(11):3240-6. . Criddle, CS, 1989. Reductive Halogenation in Microbial and Electrolytic Model Systems, Stanford University. . Diguiseppi, J and I.Fridovich. 1983. Oxy Radicals and their scavenger systems. Vol.1: Molecular Aspects. Elsevier Science Publishing Co., Inc. . Dybas, M.J., G.M.Tatara, and CS. Criddle. 1995. Localization and characterization of the carbon tetrachloride transformation activity of Pseudamanas sp. strain KC Appl. Environ. Microbial. 61(2):758-762 . Gadd, GM. and AlGrifiths 1978. Microorganisms and Heavy Metal Toxicity Microbial Ecology, 42303-317 . Gordon, AS. et al 1992. Growth, Copper-Tolerant Cells, and Extracellular Protein Production in Copper-Stressed Chemostat Cultures of Vibria alginabzricus. App]. Environ. Microbiol, 59(1):60-66 10. Harwood-Sears, V. and AS. Gordon. 1990. Copper-Induced Production of Copper- Binding Supernatant Proteins by Marine Bacterium Vibria alginoblticus App]. Environ. Microbial. 56(5): 1327-1332. 11. Huges, M.N.,and RK. Poole. 1991. Metal speciation and microbial gowth-the hard (and sofi) facts. J. of Genera] Microbial. 137, 725-734 48 12. Knoll, W.H. 1996. Factors Influencing the Competitive Advantage of Pseudamams sp. strain KC for Subsequent Remediation of A Carbon Tetrachloride Impacted Aquifer, Michigan State University. 13. Lewis, TA and R.L.Crawford. 1993. Physiological factors afi‘ecting carbon tetrachloride dehalogenation by the denitrifying bacterium Pseudarnams sp. strain KC. App]. Environ. Microbial. 59: 1635-1641 14. Markwell, M.A, S.M. Haas, NE. Talbert, and LL. Bieber. 1981. Protein Determination in Membrane Lipoprotein Samples: Manual and Automated Procedures. Methods Enzymol., 72: 296-301. 15. McCord, 1M. 1979. Superoxide dismutase and oxygen toxicity, 109-123. Elsevier Holland, Inc. 16. Menkissoglu, O. and SE Lindow,. 1991. Chemical Forms of Copper on Leaves in Relation to the Bactericidal Activity of Cupric hydroxide Deposits on Plants. Phytopathology, 81: 1263-1270 17. Menkissoglu, O. and SE. Lindow, 1991. Relationship of Free Ionic Copper and Toxicity to Bacteria in Solutions of Organic Compounds. Phytopathology, 81 : 1258- 1263 18. Mills, S.D.et al 1993. A Two-Component Regulatory System Required for Copper- Inducible Expression of the Copper Resistance Operon of Pseudamams syringae. J of Bacteriology. 175 (6) 1656-1664. 19. Petersen, R 1982. Influence of Copper and Zinc on the Growth of a Freshwater Alga, Scenedesmus quadricaudrr. The sigrificance of Chemical speciation. Environ. Sci. Technol. 16(8): 443-447. 20. Schecher, W.D. 1994. Mineql+: A Chemical Equilibrium Progam for Personal Computers P&G Co. Hallowell, Maine. 21. Sneathen, mark 1994. Theoretical and Experimental Competitiveness of Pseudamanas stutzeri strain KC, Michigan State University 22. Stanier, RY. et al 1986. 5th ed. The Microbial World by Prentice Hall, NJ 07632 23. Tartara, gegory michael 1996. Physiology of Carbon Tetrachloride Transformation by Pseudamanas stutzeri strain KC, Michigan State University 24. Tartara, G.M, M.J., Dybas, and CS. Criddle, 1993. Efl‘ects of medium and trace metals on kinetics of carbon tetrachloride transformation by Pseudamanas sp. strain KC. Appl. Environ. Microbiol. 59:2126-2131. 49 Appendix A EFFECT OF INITIAL PH AND COPPER CONCENTRATION ON GROWTH FOR PSEUDOMONAS FL U ORESCENS, PSEUDOMONAS KC, AND PSEUDOMONAS S YRHVGAE Table A-1 Efi‘ect of initial medium pH on optical density during aerobic gowth of Pseudamanasfluarescens in synthetic goundwater.l 1mm“ Final Culture pH‘ Final Optical Density @ Culture pH 40hrs.(A660) 6.483 6.470 :1: 0.0012 0.255 :1: 0.036 6.971 8.448 i 0.0495 0.497 :t 0.058 7.468 9.035 :1 0.0828 0.494 1: 0.027 ‘ 8.054 9.07 :1- 0.0265 0357 i 0.012 8.392 8.90721: 0.0181 0314 i 0.016 Table A-2 Efl‘ect of copper concentration on optical density during aerobic gowth of Pseudamanasfluarescens in synthetic goundwater.2 (initial pH 8.172) Copper concentration (11M) Final Culture 9H2 Fm Optrcal Densrty @ 42 hrs. (A660) 0 9.0701 0.002 0.357 i 0.034 0.32 8.948 i 0.0195 0.337 :1: 0.001 3.2 8.743 :1: 0.0322 0.185 :1: 0.032 6.4 8.712 i 0.0635 0.071 i 0.017 9.6 8.363 1' 0.0513 0.013 :1: 0.002 12.8 8.227 1: 0.0981 0.008 :1: 0.004 1. Approximately after 40 hrs. 2. Approximately after 42 hrs. 50 Table A-3 Efl‘ect of initial medium pH on optical density for denitrifying gowth of Pseudamanasfluarescens in synthetic goundwater.3 Initial Final can... p113 Final Optical Density Culture pH @27days.(A660) 6.483 6.33 1 0.0013 0.004 i 0.002 6.971 7.37 i 0.0032 0.031 i 0.012 7.468 8.07 i 0.0635 0.036 t 0.008 8.054 8.35 :1: 0.0298 0.046 i 0.004 8.392 8.46 i 0.0281 0.047 :t 0.012 Table A-4 Efl'ect of copper concentration of optical density on denitrifying gowth of Pseudamanasfluarescens in synthetic goundwater.3 (initial pH 8.15) - Final Culture pH3 Final Optical Density @27 Copper concentratron (uM) days. (A660) 0 8.57 :1: 0.0024 0.013 :t 0.005 0.32 8.42 i 0.0325 0.009 i 0.002 3.2 8.43 1- 0.0315 0.008 t 0.001 6.4 8.21 :1: 0.0365 0.007 :1: 0.002 9.6 8.17 i 0.0213 0.003 :1: 0.002 3. Approximately afier 27 days. Table A-5 Efl‘ect of copper concentration on protein production for aerobic and denitrifying gowth of Pseudamanas KC in synthetic goundwater.4 (initial pH 8.185) Copper Final Culture pH4 Protein Concentration @32 hrs. concentration (pg lml) (uM) aerobic denitrifying aerobic denitrifyinj 0 8.89 i 0.0127 8.83 i 0.0151 163.55 :1: 11.55 114.36 i 6.42 0.32 9.09 :1: 0.023 8.97 d: 0.035 121.46 :1: 1.31 153.03 :1: 1.31 3.2 8.86 :1: 0.021 9.01 :t 0.009 124.6 :1: 4.03 99.03 :t 4.03 6.4 8.32 :t 0.013 9.18 i 0.015 105.46 1: 3.43 83.89 i 4.31 9.6 8.42 :1: 0.018 8.459 :1: 0.0013 4.79 :1: 0.16 7.79 :l: 0.16 12.8 8.27 :1: 0.012 8.28 i 0.008 5.36 :l: 0.72 8.36 :l: 0.72 51 Table A-6 Effect of copper concentration on protein production for aerobic and denitrifying gowth of Pseudamanas KC in Schoolcrafi goundwater.4 Copper Final Culture pH“ Protein Concentration@32 hrs. concentration (11g lml) (11M) aerobic denitrifying aerobic denitrifying 0 8.93 i 0.013 8.83 i 0.012 134.27 :1: 25.21 101.97 :1: 15.21 0.32 8.91 i 0.03 9.07 i 0.031 119.79 i 14.09 128.69 :1: 14.09 3.2 8.52 i 0.003 8.68 i 0.008 97.03 i 4.03 83.69 :1: 4.03 6.4 8.32 i 0.01 8.43 :1: 0.017 6.36 i 0.72 8.46 :1: 0.72 9.6 8.36 i 0.003 8.35 i 0.007 5.79 i 0.16 3.4 i 0.16 4. Approximately after 32 hrs. Table A-7 Effect of copper concentration on protein production for copper-sensitive and copper-tolerant Pseudamanas syringae in synthetic goundwater.’ (initial pH 8.109) Copper Final Culture pH Protein Concentration@l 14 hrs. concentration (11g lml) (liM) Sensitive Tolerant Sensitive Tolerant 0 9.71 :1: 0.014 9.5 :1: 0.028 120.14 :1: 9.96 109.26 :1: 3.09 0.32 9.67 i 0.001 8.35 :1: 0.007 141.79 :1: 1.64 128.04 i 10 1.28 9.66 i 0.001 9.21 :1: 0.014 125.3 d: 6.59 112.33 1: 3.47 3.2 9.56 i 0.028 *NA 91.79 :I: 9.6 NA 12.8 8.13 i 0.003 9.645 t 0.035 12.14 i: 4.12 158.82 1 16.07 32 8.11 i: 0.013 9.115 :1: 0.078 1.07 i 1.05 93.39 :1: 3.96 64 NA 8.13 i 0.028 NA 3.03 :1: 0.79 *NA denotes data that was not available. 52 Table A-8 Efi‘ect of initial medium pH on protein production for copper-sensitive and copper-tolerant Pseudamanas syringae in synthetic goundwater.s Protein Concentration @114 hrs. Initial Final Culture pH (pg / ml) Culture pH Sensitive Tolerant Sensitive Tolerant 6.426 6.415 :1: 0.007 6.435 i 0.021 14.1 i 0.52 6.07 i 0.06 6.932 9.01 i 0.085 8.88 i 0.24 106.92 :1: 6.09 95.47 :1: 11.07 7.493 9.46 i: 0.03 9.44 i 0.028 131.31 i 11.96 125.98 :1: 10.17 7.958 9.575 t 0.035 9.52 i 0.028 107.8 1 18.31 119.68 i 1.34 8.109 9.51 i 0.014 9.545 :1: 0.035 97.1 1 22.72 111.25 :1: 0.21 8.350 9.61 :1: 0.014 9.555 :1: 0.007 101.92 i 18.54 104.42 :1: 16.76 5. Approximately afier 114 hrs. 53 Appendix B ORIGINAL DATA AND CALCULATIONS FOR BIOMASS USED ON MODELING Table B-1 Original data used for determination of biomass for Pseudamanasfluarescens in SGM with and without copper for modeling. (Samples in triplicate) Pseudamanasfluarescens Time ‘ Avera We' ' Stdev Weight Avera OD2 Stdev OD W/o Cu W/Cu W/o Cu W/Cu W/o Cu W/Cu “”0 Cu W/ Cu 0 0.0001 0.0001 0 0 0 0.001 0 1.7E-5 8 0.0007 0.0007 5.77E-5 1.03E-11 0.0196 0.022 0.0052 0.003 16 0.0009 0.001 5.77E-5 5.77E-5 0.1032 0.092 0.01 0.01 24 0.0032 0.0025 0.00015 0.00015 0.33 0.242 0.01 0.019 36 0.0042 0.0034 0.00025 0.0001 0.387 0.381 0.012 0.012 OD versus biomass concentration for P. fluorescens in SGM w/o copper: Biomass = 0.0099* OD + 0.0002, 12 = 0.9797 OD versus biomass concentration for P. fluorescens in SGM w/copper: Biomass = 0.0084* OD + 0.0003, 12 = 0.9805 Table B-2 Original data used for determination of biomass for Pseudamanas syringae in SGM with and without copper. (Samples in triplicate) Payringae Copper Sensitive Time , Average Weighti Stdev Weight Avera or)2 Stdev on W/o Cu W/Cu W/o Cu W/Cu W/o Cu W/Cu W/o Cu W/ Cu 0 0.0001 0.0001 0 0 0 0.001 0 1.7E-5 24 0.00075 0.00105 7.07E-5 7.07E-5 0.0177 0.02 0.0038 0.0023 36 0.0019 0.0023 0.00014 0.00014 0.101 0.155 0.0053 0.034 58 0.0041 0.0032 0 0.00014 0.424 0.358 0.024 0.042 P. syringae Copper Tolerant Time Ave e We' Stdev Weiglt Ave OD Stdev OD W/o Cu W/Cu W/o Cu W/Cu W/o Cu W/Cu W/o Cu W/ Cu 0 0.0001 0.0001 0 0 0 0.001 0 1715-5 24 0.00065 0.00085 0.00035 7.07E-5 0.0177 0.02 0.0037 0.0023 36 0.0022 0.0017 0 0.00014 0.103 0.124 0.013 0.023 58 0.0039 0.004 0.00014 0.00028 0.403 0.471 0.0025 0.0393 1. Units in g/10ml. 2. Optical density measured in A660. OD versus biomass concentration for copper-sensitive P. syringae in SGM w/o copper: Biomass = 0.0079* OD + 0.0008, 13 = 0.9746 OD versus biomass concentration for copper-sensitive P. syringae in SGM w/copper: Biomass = 00058" or) + 0.0012, r2 = 0.9695 OD versus biomass concentration for copper-tolerant P. syringae in SGM w/o copper“. Biomass = 0.0072* OD + 0.0011, .1 = 0.9462 OD versus biomass concentration for copper-tolerant P. syringae in SGM w/copper: Biomass = 0007* or) + 0.0007, 13 = 0.9971 55 Table B-3 Data and calculations used for determining the observed yield of Pseudamanas fluorescens, Pseudamanas stutzeri KC, and Pseudamanas S yringae gown in SGM without copper using largest dry weight value. (Samples in triplicate) Pseudamanasfluarescens Initial pH Acetate consumed Dry Weight Observed Yield Final pH@ (mM) (mg/L) (M acetate) 40 hrs 7 6.78 480 0.407 8.96 8.2 6.73 460 0.404 9.084 Pseudamanas KC Initial pH Acetate consumed Dry Weight Observed Yield Final pH@ (HIM) (mm) (mills/258m) 40h“ 7 6.89 530 0.413 8.86 8.2 6.65 500 0.399 9.278 Copper sensitive Pseudamanas Syringae Initial pH Acetate consumed Dry Weight Observed Yield Final pH@ (mM) (mam (Mam) 5 days 7 6.58 490 0.395 8.756 8.2 6.47 470 0.388 9.125 Copper tolerant Pseudamanas Syfingae Initial pH Acetate consumed Dry Weight Observed Yield Final pH@ (HIM) Lilia/L) 018311328 acetate) 5 days 7 5.68 440 0.341 8.723 8.2 5.99 450 0.360 9.108 56 Appendix C EFFECT OF COPPER ON SECOND ORDER RATE COEFFICIENT AND TRANSFORMATION CAPACITY BY SECRETED FACTOR PRODUCED FROM PSEUDOMONAS KC Table C-l Second order rate coeficient and transformation capacity by secreted factor produced in synthetic goundwater without copper. Biomoleculel 381%; (32:33“ K'( L / mg-h) Tc (pg CT/mg) 1'2 -Cu2 + Cu3 0 0.03 i 0.002 0.32 i 8E-02 0.983 0.32 0.042 a 0.0075 2.8 a 4.1 0.983 0.64 0.077 a 0.0181 0.2 a 215-02 0.987 3.2 0.071 i 0.0195 0.47 :1: 0.2 0.95 6.4 0.083 :1: 0.0524 0.3 i 0.2 0.983 12.8 0.068 a 0.0087 0.2 :1: 113-02 0.99 25.6 0.04 a 0.0109 0.1 :1: 713.03 0.87 - Cu‘ 0 0.02 :t 0.003 0.24 : 5E-02 0.99 0.32 0.039 i0.0075 0.38 5:02 0.98 0.64 0.071 a 0.0424 0.32 a 0.2 0.99 3.2 0.059 a 0.0186 0.26 513.02 0.99 6.4 0.044 :1: 0.018 2.8 a 0.1 0.977 12.8 0.032 2: 0.0072 0.47 :1: 215-02 0.977 25.6 0.018 a 0.0027 0.24 i 513.02 0.90 57 Table C-2 Second order rate coefficient and transformation capacity by secreted factor produced in synthetic goundwater with copper. Biomoleculel egg/it; C2313“ K’( L / mg‘h) Tc (pg CT/mg) r2 +Cus + Cu3 0 0.025 a 0.0028 0.23 :1: 75.02 0.977 0.32 0.04 a 0.0044 2.1 a 2502 0.987 0.64 0.053 a 0.006 0.23 a 4502 0.987 3.2 0.058 t 0.0063 0.21 : 8E-03 0.983 6.4 0.045 i: 0.005 0.27 :t 45-02 0.99 12.8 - 0.034 a 0.007 0.34 :t 0.2 0.99 25.6 0.012 a 0.003 0.21 :1: 8E-02 0.907 - Cu‘ 0 0.017 a 0.0006 0.29 i 35-02 0.99 0.32 0.03 50.0047 0.19 1515-02 0.98 0.64 0.045 a 0.001 2.89 :1: 4.5 0.99 3.2 0.041 t 0.0153 0.23 57502 0.99 6.4 0.028 a 0.008 0.63: 0.4 0.99 12.8 0.03 a 0.001 0.16 :1: 75-03 0.977 25.6 0.008 3: 0.002 33.2 i 57.3 0.95 l. Biomolecule harvested in 2 difl‘erent cultures : a first set for experiments with 0, 0.32, 3.2 11M Cu” ; a second set for experiments with 0.64, 6.4 and 25.6 M Cu” 9'93“.“ 58 Secreted factor produced in synthetic goundwater without copper. P. fluorescens gown in 0.32 M synthetic goundwater. P. fluorescens gown in synthetic goundwater in absence of copper. Secreted factor produced in 0.32 [AM synthetic goundwater. Table C-3 Second order rate coeficient and transformation capacity by secreted factor produced in synthetic goundwater with copper and stimulated indigenous flora gown with and without copper in Schoolcrafi goundwater. Growth Cu added Biomolecule condition (11M) K'( L / mg*h) Tc (ug CT/mg) r2 -Cu‘ + Cu7 0 0.01 a 0.005 101 :1: 2.8 0.79 0.32 0.01 i 0.001 3.2501 n 0.2 0.81 3.2 0.03 i 0.007 1.35.01 5 6E-03 0.97 . ' 6.4 0.02 :1: 0.002 35.01 a 0.2 0.88 - cu8 0 35-03 :1: 15-04 67 a 57.4 0.72 0.32 0.02 50.005 1.1501 :1: 25-03 0.96 3.2 0.04 :1: 0.007 1350157503 0.98 6.4 0.01 :1: 45-04 60 :1: 57.2 0.93 +0? + Cu’ 0 0.01 a 0.001 33 :1: 57.4 0.85 0.32 0.02 :1: 0.002 0.2: 75-02 0.93 3.2 0.04 a 0.015 0.3 i 0.1 0.97 6.4 0.04 a 0.009 0.2 i 0.1 0.96 - Cu“ 0 0.01 a 0.001 67 a 56.6 0.90 0.32 0.02 50.005 0.2 :1: 0.1 0.98 3.2 0.03 a 0.012 0.3 :1: 0.1 0.98 6.4 0.04:1: 0.007 0.1 i 2502 0.98 6. Secreted factor produced in synthetic goundwater without copper. 7. Indigenous flora gown in 0.32 M synthetic goundwater. 8. Indigenous flora gown in synthetic goundwater in absence of copper. 9. Secreted factor produced in 0.32 M synthetic goundwater. 59 Appendix D EFFECT OF COPPER ON FIRST ORDER RATE COEFFICIENT AND DECAY COEFFICIENT BY SECRETED FACT OR PRODUCED FROM PSEUDOMONAS KC Table D-l First order rate coeflicient and decay coeficient by secreted factor produced in synthetic goundwater without copper. . Biomoleculel cgrrreritfn Cm“ K(min‘l) Kd (min'l) 1"2 -Cu’ + on3 0 0.098 a 0.014 0.0098 :1: 0.0032 0.987 0.32 0.2 :1: 0.0359 0.022 :1: 0.00078 0.98 0.64 0.2 :1: 0.035 0.019 a 0.0046 0.983 3.2 0.246 a 0.0466 0.017 a 0.008 0.983 6.4 0.31 :1: 0.068 0.037 a 0.011 0.99 12.8 0.26 i 0.029 0.034 t 0.0063 0.983 25.6 0.23 a 0.01 0.055 a 0.0079 0.99 - Cu‘ 0 0.08 a 0.0097 0.011 1 0.0022 0.983 0.32 0.124 a 0.014 0.0066 :1: 0.00115 0.99 0.64 0.241 1 0.0641 0.022 a 0.0073 0.99 3.2 0.186 a 0.0135 0.0135 a 8.7504 0.99 6.4 0.16 a 0.038 0.0199 :1: 0.0073 0.96 12.8 0.173 a 0.013 0.035 :1: 0.00396 0.99 25.6 0.16 a 0.029 0.04 :1: 0.011 0.987 Table D-2 First order rate coeflicient and decay rate by secreted factor produced in synthetic goundwater with copper. . . Growth Cu added . .1 . .. Biomolecule condition (11M) K(mln ) Kd (min ) 1’2 +00‘ + Cu3 0 0.085 :1: 0.012 0.012 :1: 0.0044 0.97 0.32 0.121 :1: 0.014 0.014 :1: 0.005 0.98 0.64 0.157 :1: 0.0069 0.015 1 0.00032 0.987 3.2 0.17 i 0.042 0.017 i 0.0095 0.987 6.4 0.152 :1: 0.015 0.015 :1: 0.0027 0.99 12.8 0.158 :1: 0.0124 0.023 :1: 0.0022 0.99 25.6 0.044 :1: 0.0045 0.0098 :1: 0.0008 0.947 - Cu‘ 0 0.072 a 0.015 0.0096 i 0.0054 0.987 0.32 0.092 i 0.017 0.0096 1 0.0055 0.957 0.64 0.137 i 0.0006 0.011 :1: 0.0006 0.99 3.2 0.17 :1: 0.025 0.022 t 0.007 0.977 6.4 0.123 t 0.011 0.01121: 0.004 0.99 12.8 0.13 :1: 0.005 0.023 :t 0.002 0.99 25.6 0.03 :1: 0.0047 0.008 :1: 0.00073 0.97 Biomolecule harvested in 2 difl‘erent cultures : a first set for experiments with 0, 0.32, 3.2 nM Cu” ; a second set for experiments with 0.64, 6.4 and 25.6 M Cu2+ Secreted factor produced in synthetic goundwater without copper. P. fluorescens gown in 0.32 M synthetic goundwater. P. fluorescens gown in synthetic goundwater in absence of copper. Secreted factor produced in 0.32 1.1M synthetic goundwater. fl 9'95”!" 61 Table D3 First order rate coemcient and decay coefficient by secreted factor produced in synthetic goundwater with copper and stimulated indigenous flora gown with and without copper in Schoolcraft goundwater. Biomolecule 631%?“ (timed K(min") Kd (min'l) 1'2 -Cn‘ + Cu1 0 0.061 a 0.03 0.0246 :1: 0.018 0.887 0.32 0.135 :t 0.055 0.0375 5. 0.0194 0.91 3.2 0.31 :1: 0.119 0.037 a 0.0159 0.93 6.4 0.162 a 0.035 0.031 i 0.007 0.87 - Cn‘I 0 0.025 :1: 0.01 0.024 a 0.0058 0.87 0.32 0.154 a 0.052 0.037 :1: 0.016 0.98 3.2 0.31 i 0.063 0.036 a 0.0067 0.97 6.4 0.089 a 0.045 0.021 a 0.0169 0.96 +Cn9 + C1? 0 0.21 a 0.078 0.039 a 0.011 0.92 0.32 0.099 5: 0.038 0.008: 0.0087 0.94 3.2 0.35 i 0.009 0.03 :t 0.00052 0.983 6.4 0.35 a 0.153 0.03 a 0.0146 0.987 - Cu“ 0 0.148 a 0.085 0.0144 1 0.002 0.96 0.32 0.153 i 0.0176 0.029 5 0.00052 0.977 3.2 0.295 :1: 0.144 0.022 a 0.0115 0.987 6.4 0.3 a 0.061 0.023 :1: 0.0026 0.99 6. Secreted factor produced in synthetic goundwater without copper. 7. Indigenous flora gown in 0.32 M synthetic goundwater. 8. Indigenous flora gown in synthetic goundwater in absence of copper. 9. Secreted factor produced in 0.32 11M synthetic goundwater. 62 Appendix E EFFECT OF COPPER ON SECOND ORDER RATE COEFFICIENT AND TRANSFORMATION CAPACITY BY SECRETED FACTOR PRODUCED FROM PSEUDOMONAS KC Table E-l Second order rate coefiicient and transformation capacity by secreted factor produced in synthetic goundwater in absence of copper. Cell Cu added Biomolecule factor (11M) K'( L / mg‘h) Tc (pg (717mg) 1’2 -cnI Sensitive2 0 0.007 t 0.0005 6.5 i 56 0.96 0.32 0.036 :t 0.0167 0.3 a: 0.2 0.97 3.2 0.052 i 0.0173 0.18 a 15-02 0.99 32 0.049 a 0.033 0.17 :1: 25-02 0.99 64 0.041 a 0.011 0.11 5; 8E-02 0.98 Tolerant3 0 0.02 a 0.0096 0.12 :1: 35-02 0.79 0.32 0.028 :1: 0.0153 0.35 d: 0.1 0.98 3.2 0.032 a 0.006 0.29 :1: 0.2 0.96 32 0.055 :1: 0.016 0.2 a 25.02 0.99 64 0.056 a 0.01 0.16 :1: 15-02 0.97 Table E-2 Second order rate coeficient and transformation capacity by secreted factor produced in synthetic goundwater in presence of copper. Biomolecule ffcigr Cmed K'( L / mg‘h) Tc (pg CT/mg) r2 +cn‘ Sensitive2 0 0.024 :1: 0.0048 0.29 i 0.2 0.98 0.32 0.022 a 0.0033 3.3 :1: 57.5 0.99 3.2 0.026 :1: 0.0011 0.4 :1: 65-02 0.98 32 0.006 :1: 0.001 99.4 i 0.5 0.94 Tolerant} 0 0.008 a 0.0025 2435000 :1: 421800 0.95 0.32 0.012 :1: 0.0007 1859000 :t 322000 0.99 3.2 0.014 :1: 0.0014 63 a 55 0.97 32 0.009 a 0.0003 2497000 :1: 217900 0.97 1. Secreted factor produced in synthetic goundwater without copper. 2. Copper-sensitive P. syringae gown in synthetic goundwater without copper. 3. Copper-tolerant P. syringae gown in synthetic goundwater without capper. 4. Secreted factor produced in synthetic goundwater containing 0.32 M copper. 63 Appendix F EFFECT OF COPPER ON FIRST ORDER RATE AND DECAY COEFFICIENT BY SECRETED FACTOR PRODUCED FROM PSEUDOMONAS KC Table F-l First order rate coemcient and decay coeficient by secreted factor produced in synthetic goundwater in absence of copper. Biomolecule ffcilcllr Chas/30d K(min") Kd (min") 12 -Cu‘ Sensitive2 0 0.035 :1: 0.0017 0.0051 :1: 0.00176 0.86 0.32 0.21 i 0.073 0.031 a 0.0089 0.983 3.2 0.258 :1: 0.1 0.032 5: 0.0145 0.99 32 0.165 i 0.056 0.023 a 0.0086 0.99 64 0.165 :1: 0.0206 0.04 a 0.0041 0.88 Tolerant} o 0.28 :1: 0.138 0.096 :1: 0.023 0.917 0.32 0.154 a 0.114 0.0195 x 0.021 0.99 3.2 0.33 :t 0.131 0.043 a 0.014 0.99 32 0.244 a 0.067 0.035 :1: 0.0075 0.983 64 0.329 a 0.12 0.049 a 0.0169 0.877 Table F-2 Second order rate coeficient and transformation capacity by secreted factor produced in synthetic goundwater in presence of copper. Cell Cu added Biomolecule factor (11M) K(min") Kd (mind) ‘2 +Cn‘ Sensitive2 0 0.133 a 0.0165 0.017 a 0.0042 0.987 0.32 0.1 :1; 0.012 0.0072 :1: 0.0024 0.99 3.2 0.137 r 0.026 0.012 i 0.0046 0.98 32 0.034 :1: 0.01 0.0084 :t 0.0056 0.96 Tolerant3 o 0.031 a 0.01 0.0005 i 0.00057 0.957 0.32 0.055 t 0.0019 0.0035 :1: 0.0032 0.987 3.2 0.066 i 0.012 0.0061 :1: 0.0046 0.967 32 0.042 a. 0.0097 0.0049 :1: 0.00845 0.97 l. Secreted factor produced in synthetic goundwater without copper. 2. Copper-sensitive P. syringae gown in synthetic goundwater without copper. 3. Copper-tolerant P. syringae gown in synthetic goundwater without copper. 4. Secreted factor produced in synthetic goundwater containing 0.32 1.1M copper. 65 Appendix G PRELIMINARY STUDY 9 Effect of Initial medium pH on CT transformation The range of pH values over which CT degadation occurs is broad, but the optimum for CT transformation is approximately 8.5 by Tatara [23]. An assay was conducted using a fixed copper concentration and varied pH to examine the effect of speciation of copper and change of biomolecule fiinction at the reaction level. This pH range was first tested using the gowth of Pseudamanas fluorescens to make sure there was no negative effect. Copper concentrations of 0.64, 6.4, 12.8, 25.6 1.1g in form of CuSOa oSHzO were added to SGM prior to increasing the pH to 6.3 and 7.3 in order to achieve speciation of copper. A better understanding of copper speciation in the CT transformation and toxicity could be obtained by experiments like those performed in this work, but the care must be taken to account for the increase in pH caused by addition of fi'eeze-dried supernatant. 0 Competition theory One of the possible scenario for secreted factor produced by P.3tutzeri KC could be to compete with other cells for nutrients. In order to verify this, biomolecule was added to starting exponential period of P. fluorescens gowing in SGM. Plates count data showed that there was no significant difl‘erence on cell number on P. fluorescens in the absence or presence of biomolecule. However, addition of freeze-dried supernatant delayed production of the geenish-yellow pignents (siderophores) normally produced by P. fluorescens in the stationary phase. 67 "‘1111111111111111“