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Please Note: The author retains the copyright while the New Jersey Institute of Technology reserves the right to distribute this thesis or dissertation Printing note: If you do not wish to print this page, then select “Pages from: first page # to: last page #” on the print dialog screen The Van Houten library has removed some of the personal information and all signatures from the approval page and biographical sketches of theses and dissertations in order to protect the identity of NJIT graduates and faculty. ABSTRACT BIODEGRADATION OF NAPHTHALENE USING PSEUDOMONAS putida (ATCC 17484) IN BATCH AND CHEMOSTAT REACTORS by Jay Boyd Best Kinetic experiments were conducted using Pseudomonas putida (ATCC 17484) to determine the rate of growth when naphthalene was provided as a sole carbon source in suspended biomass systems. Batch and chemostat reactors were used under three sets of conditions: non-aerated, aerated at 29.5° C, and aerated at 25.5° C. A number of concentrations were tested under each set of conditions. Data regression was used to determine parameters for Monod (non-inhibitory), Andrews (inhibitory), and zero-order kinetics. Although the Andrews fit of the data provided a slightly lower sum-of-squares error (SSE), the amount of data scatter and the weak inhibition made the Andrews fit only marginally better. In addition, extremely small values of the saturation constant (Ks < 0.01mg/L) made the use of a zero-order kinetic constant nearly identical with a Monod model for naphthalene concentrations above 0.05 mg/L. Particularly troublesome experimental problems included inconsistent biomass measurements by optical density and wall growth. Suggested biokinetic parameters are: µ = 0.0074 min-1, Ks = 0.0022 mg/L, and K.1 = 31 mg/L (for Andrews model); µmax =0.0057 min-1, and Ks = 0.00088 mg/L (for Monod model); and k0= 0.0058 min-1 (for the zero-order model) BIODEGRADATION OF NAPHTHALENE USING PSEUDOMONAS purida (ATCC 17484) IN BATCH AND CHEMOSTAT REACTORS by Jay Boyd Best A Thesis Submitted to the Faculty of the New Jersey Institute of Technology in Partial Fulfillment of the Requirements of the Degree of Master of Science Department of Chemical Engineering, Chemistry, and Environmental Science October 1997 APPROVAL PAGE BIODEGRADATION OF NAPHTHALENE USING PSEUDOMONAS putida (ATCC 17484) IN BATCH AND CHEMOSTAT REACTORS Jay Boyd Best Dr Gordon Lewandowski, Thesis Advisor ( Dart Chairperson and Distinguished Professor, Department of Chemical Engineering, Chemistry, and Environmental Science, MIT Dr. Piero M. A rmenante, Committee Member Date Professor, Department of Chemical Engineering, Chemistry, and Environmental Science, MIT Dr. David Kafkewitz, Committee Member ate Professor, Department of Biological Sciences, Rutgers University - Newark BIOGRAPHICAL SKETCH Author: Jay Boyd Best Degree: Master of Science in Chemical Engineering Date: October 1997 Undergraduate and Graduate Education: • Master of Science in Chemical Engineering, New Jersey Institute of Technology, Newark, New Jersey, 1997 • Bachelor of Science in Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, 1989 Presentations and Publications: • Best, Jay B. "Economic Analysis of the New Toxicity Characteristic Leaching Procedure," Proceedings of Superfund 1990, Washington DC, November 1990. • Best, Jay B. and Simone, Deborah "Evaluations of High Concentrations of VOCs in Landfill Gas: A Case Study of the Rose Hill Regional Landfill Superfund Site," Proceedings of the New England Environmental Expo, Boston, Massachusetts, October 1994. iv To my wife and daughter. V ACKNOWLEDGMENT There are a number of people with out whose help this project would have been impossible. I would like to thank Professor Gordon Lewandowski for his guidance and support as my thesis advisor, Professors David Kafkawitz for serving on my thesis committee and providing troubleshooting when I encountered problems, and Piero Armenante for serving on my thesis committee and providing the inspiration to switch to the Chemical Engineering program. Many people played a role in my enrollment at NJIT in the chemical engineering program. I would like to thank former Professor Vital Aelion for encouraging me to attend NJIT, and Professor Richard Trattner for allowing me to attend and ensuring my initial departmental support. Additional thanks go to: Dilip Mandal, who has provided me extraordinary help throughout my experimental work; and Gwen San Agustin and Clint Brockway who on several occasions were able to troubleshoot various analytical systems for me. Finally, this would not have been possible without a grant from the Northeast Hazardous Substance Research Center at NJIT. TABLE OF CONTENTS Chapter Page 1 INTRODUCTION 1 2 LITERATURE REVIEW 3 3 OBJECTIVES 9 4 EXPERIMENTAL APPARATUS AND REAGENTS 10 4.1 Shake Flasks 10 4.2 Jacketed-Batch Reactors 10 4.3 Chemostats 11 4.4 Analytical Equipment 11 4.5 Reagent Preparation 13 4.5.1 Nutrient Broth 13 4.5.2 Inorganic Growth Medum 13 4.5.3 Naphthalene-Saturated Growth Medium 13 4.5.4 Calibration Standards 14 5 EXPERIMENTAL PROCEDURES 15 5.1 Selection of Culture 15 5.2 Naphthalene Sampling and Analysis 16 5.2.1 Collection of Samples 16 5.2.2 Naphthalene Analysis by HPLC 17 5.3 Biomass Concentration Measurements by Optical Density 5.4 Shake Flasks 5.4.1 Preliminary Measurements 22 TABLE OF CONTENTS (continued) Chapter Page 5.4.2 Multi-Concentration Shake Flask Experiments -)3 5.5 Jacketed Reactor with Aeration 5.5.1 Measurement of Naphthalene Loss by Abiotic Mechanisms )5 5.5.2 Measurement of Naphthalene Loss due to Biodegradation 26 5.6 Chemostats 28 6 RESULTS AND DISCUSSION 30 6.1 Shake Flasks 30 6.I.1 Preliminary Measurements 30 6.1.2 Multi-Concentration Experiments 32 6.2 Jacketed-Batch Reactor with .Aeration 35 6.2.1 Naphthalene Loss by Abiotic Mechanisms 35 6.2.1.1 Naphthalene Loss Due to Volatilization 36 6.2.1.2 Naphthalene Loss due to Adsorption to the Reactor Surfaces 37 6.2.2 Naphthalene Loss Due to Biodegradation 39 6.3 Chemostat 45 6.4 Determination of Kinetic Parameters 49 6.5 Summary 56 7 CONCLUSIONS AND RECOMMENDATIONS 61 viii TABLE OF CONTENTS (continued) Chapter Page APPENDIX A RE-ANALYSIS OF DATA FROM BUITRON AND CAPDEVILLE 1993; AND GOLDSMITH AND BALDERSON 1989 66 APPENDIX B OPTICAL DENSITY CALIBRATION FOR MEASUREMENT OF BIOMASS 69 APPENDIX C ESTIMATION OF MONOD PARAMETERS FROM PRELIMINARY SHAKE-FLASK EXPERIMENTS 72 APPENDIX D GROWTH/DEGRADATION CURVES FOR SHAKE- FLASK EXPERIMENTS 80 APPENDIX E SEMI-LOG PLOTS OF SHAKE-FLASK DATA 84 APPENDIX F THEORETICAL AND EXPERIMENTAL EVALUATION OF STRIPPING LOSSES. 88 APPENDIX G GROWTH/DEGRADATION CURVES FOR JACKETED- BATCH EXPERIMENTS 89 APPENDIX H PREDICTED NAPHTHALENE AND BIOMASS CONCENTRATION CURVES FOR BATCH EXPERIMENTS 107 REFERENCES 113 ix LIST OF FIGURES Figure Page 2-1 Naphthalene Degradation Pathway 5 4-1 Schematic Diagram of Jacketed-Batch and Chemostat Reactors 12 5-1 Comparison of Naphthalene Degradation in Each Culture 16 5-2 Typical Chromatogram of Sample Containing Naphthalene 19 5-3 Typical Calibration Curve 19 6-1 Comparison of Naphthalene Concentrations in Preliminary Shake-Flask Experiments. 31 6-2 Experimental Biomass and Naphthalene Concentrations for a Typical Shake Flask Experiment 33 6-3 Experimental Shake-Flask Results 34 6-4 Volatilization Loss Parameter (k) as Function of Aeration Rate (Qair) at 29.5°C in 2-Liter Reaction Vessel 38 6-5 Naphthalene Concentration Due to Desorption 39 6-6 Specific-Growth Rates from Jacketed-Batch Experiments at 29° C 42 6-7 10/96 45 Biomass and Naphthalene Measurements 43 6-8 Specific-Growth Rates Determined from Jacketed-Batch Experiments Conducted at 25° C 44 6-9 Biomass and Naphthalene Concentrations in "11/96 - Batch 11" 47 6-10 Specific-Growth Rate Determined by Chemostat at 29.5° C 48 6-11 Specific-Growth Rate Determined by Chemostat at 25.5° C 50 6-12 Parameter Fitting of Non-Aerated Experimental Data 53 6-13 Parameter Fitting of Aerated Experimental Data at 29.5° C 54 6-14 Parameter Fitting of Aerated Experimental Data at 25.5° C 55 6-15 Parameter Fining of All Experimental Data 57 6-16 Simulation vs. Experimental Biomass and Naphthalene Concentrations 61 LIST OF TABLES Table Page 2-1 Naphthalene Degradation Rates Provided in Literature 8 4-1 Growth Medium Composition 13 4-2 Working Standard Concentrations and Dilutions 14 5-1 HPLC Parameters for Naphthalene Analysis 18 5-2 Optical Density Calibration Measurements 22 5-3 Preliminary Shake Flask Experiments 22 5-4 Experimental Dilutions for Multi-Concentration Shake Flask Experiments )4 5-5 Summary of Jacketed Batch Reactor Experiments 5-6 Summary of Chemostat Experiments '79 6-1 Summary of Shake-Flask