ONLINE MONITORING OF AEROBIC DENITRIFICATION OF PSEUDOMONAS AERUGINOSA BY NAD(P)H FLUORESCENCE A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Qing Xia December, 2005 ONLINE MONITORING OF AEROBIC DENITRIFICATION OF PSEUDOMONAS AERUGINOSA BY NAD(P)H FLUORESCENCE Qing Xia Thesis Approved: Accepted: _______________________________ _______________________________ Advisor Department Chair Lu-Kwang Ju Lu-Kwang Ju _______________________________ _______________________________ Committee Member Dean of the College Bi-min Zhang Newby George K. Haritos _______________________________ _______________________________ Committee Member Dean of the Graduate School Ping Wang George R. Newkome _______________________________ Date ii ABSTRACT In cystic fibrosis airway infection, Pseudomonas aeruginosa forms microaerobic biofilm and undergoes significant physiological changes. It is important to understand the bacterium’s metabolism at microaerobic conditions. Continuous cultures of P. aeruginosa (ATCC 9027) maintained at different dissolved oxygen concentrations (DO) and three different dilution rates (D) were studied for the effects of DO and D on various culture properties, especially on aerobic respiration and denitrification. The DO was varied from 0 mg/L (completely anoxic condition) to 2.2 mg/L, and measured with optical sensors that could accurately determine very low DO based on oxygen-quenched luminescence. The studied dilution rates were 0.026 h-1, 0.06 h-1 and 0.13 h-1. The strain was found to perform aerobic denitrification; while the specific nitrate and nitrite reduction rates decreased with increasing DO, denitrification persisted even at relatively high DO levels (1-2.2 mg/L) at different D. In the presence of nitrate, the Monod constant for DO (i.e., the critical DO at which the specific oxygen uptake rate (OUR) is half of the maximum rate) was practically zero (< 0.001 mg/L) for this P. aeruginosa strain. Aerobic denitrification appeared to function as an electron-accepting mechanism supplementary or competitive to aerobic respiration. The shift of culture’s respiratory mechanism was also clearly detected with a fluorometer targeting at intracellular NAD(P)H, i.e., the reduced coenzymes nicotinamide adenine dinucleotides (phosphate). Comparatively, the NAD(P)H fluorescence was highest at the anoxic, iii denitrifying condition (NFUDN), lowest at fully aerobic conditions (NFUOX), and intermediate fluorescence (NFU) at conditions where both denitrification and aerobic respiration occurred. Representing a quantitative measure of the culture’s “fractional approach” to the fully denitrifying state, the normalized fractions (NFU - NFUOX)/(NFUDN - NFUOX) were correlated with the calculated fractions of electrons accepted by denitrification. The denitrification-accepted fractions of electrons increased with the NFU fractions: the increases were gradual at larger DO levels (DO ≥ 0.1 mg/L), but much sharper at lower DO at three different dilution rates. The fluorescence fraction changed more rapidly than the electron fraction at very low DO levels (< 0.001 mg/L). The results demonstrated that online NAD(P)H fluorescence was a feasible technique for effective monitoring and quantitative description of the microaerobic state of microorganisms. iv ACKNOWLEDGEMENTS I would like to express my deep appreciation to my academic advisor, Dr. Lu- Kwang Ju, for his encouragement, precious suggestions, sufficient patience to guide, and financial support during my master’s study. I am also thankful to Dr. Bi-min Zhang Newby and Dr. Ping Wang for serving as my committee members as well as for their suggestions on this work. I also appreciated Mr. Fan Chen’s help for the experiment setup and development. I am also grateful for Mr. Nathan, K. Klettlinger and Mr. Nicholas, J. Hammilton’s helps in all analytical experiments. I would like to thank my group members, Ms. Lin Huang and Ms. Shuyan Qiu, for their friendship and assistance. I am eternally grateful to my beloved husband Fan Chen for his love, understanding, and whole-hearted support during entire graduate study at the University of Akron. I also thank my parents and sisters for their continuous support from China during my study. v TABLE OF CONTENTS Page LIST OF FIGURES ........................................................................................................... ix CHAPTER I INTRODUCTION...................................................................................................... 1 1.1 Scope of Research................................................................................................ 4 1.2 Research Objectives............................................................................................. 5 1.3 Structure of Thesis ............................................................................................... 5 II LITERATURE SURVEY........................................................................................... 7 2.1 Biofilm Formation ............................................................................................... 7 2.2 Two Luminescent Techniques ........................................................................... 10 2.2.1 Fluorescence ............................................................................................. 10 2.2.2 NAD(P)H Fluorescence............................................................................ 12 2.2.3 Influence of Various Factors on Fluorescence in Solutions ..................... 15 2.2.4 Luminescence Technique for DO Measurement ...................................... 18 2.2.4.1 The Principles of the Fiber-optic Oxygen Meter .................................. 18 2.3 Biosurfactant Production................................................................................... 20 2.3.1 Structures and Properties of Biosurfactants.............................................. 20 2.3.2 Synthesis of Biosurfactants....................................................................... 21 vi 2.3.2.1 Microorganisms for Rhamnolipid Production ...................................... 21 2.3.2.2 Choice of Carbon Substrates and Limiting Nutrients........................... 22 2.3.2.3 Temperature and pH Effects ................................................................. 23 2.3.2.4 Foaming in Biosurfactant Fermentation ............................................... 23 2.3.3 Rhamnolipid Types and Rhamnolipid Production.................................... 24 2.3.3.1 Rhamnolipid Formation........................................................................ 25 2.4 Respiration ......................................................................................................... 27 2.4.1 Aerobic Respiration .................................................................................. 27 2.4.2 Anaerobic Respiration .............................................................................. 29 2.4.2.1 Denitrification....................................................................................... 29 2.4.3 Microaerobic Denitrification .................................................................... 32 III MATERIALS AND METHODS ............................................................................. 34 3.1 Microorganism and Medium.............................................................................. 34 3.2 Experimental Setup and Continuous Culture..................................................... 35 3.3 Analytical Methods............................................................................................ 37 3.3.1 Cell Concentrations and Cell Dry Weight Analysis ................................. 39 3.3.2 Ammonium and Nitrate Analysis ............................................................. 39 3.3.3 Nitrite Analysis......................................................................................... 40 3.3.4 Glucose Analysis ...................................................................................... 40 3.3.5 Rhamnolipids Analysis ............................................................................. 41 3.3.6 DO and OUR Measurement...................................................................... 41 3.3.7 Culture Fluorescence Measurement.......................................................... 42 3.3.8 Calculations............................................................................................... 43 vii IV AEROBIC, MICROAEROBIC, ANAEROBIC DENITRIFICATION OF PSEUDOMONAS AERUGINOSA............................................................................ 46 4.1 Introduction........................................................................................................ 46 4.2 Cell Metabolism and Respiration Mechanism at D = 0.026h-1, D = 0.06 h-1 and D = 0.13 h-1 ................................................................................................. 48 4.2.1 Materials and Methods.............................................................................. 48 4.2.2 Cell Properties in Continuous Cultures Maintained at Different DO and D……………………………………………………………………..49 4.2.3 Respiration Mechanism and Energy Generation at Different DO and D . 66 4.2.4 Culture Fluorescence ................................................................................ 70 4.2.5 Examine Transition of Electron-Accepting Mechanism Using NAD(P)H Fluorescence...........................................................................