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Rhamnolipids Production with Denitrying Pseudomonas RHAMNOLIPIDS PRODUCTION WITH DENITRYING PSEUDOMONAS AERUGINOSA A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Chun Chiang Chen May, 2006 RHAMNOLIPIDS PRODUCTION WITH DENITRYING PSEUDOMONAS AERUGINOSA Chun Chiang Chen Dissertation Approved: Accepted: Advisor Department Chair Dr. Lu-Kwang Ju Dr. Lu-Kwang Ju Committee Member Dean of the College Dr. Amy Milsted Dr. George K. Haritos Committee member Dean of the Graduate School Dr. Teresa Cutright Dr. George R. Newkome Committee Member Date Dr. Stephanie Lopina Committee Member Dr. Ping Wang ii ABSTRACT Rhamnolipids causes severe foaming during its production by conventional aerobic fermentation of Pseudomonas aeruginosa. This problem necessitates the reduction of aeration, which in turn limits the cell concentration employable and productivity achievable in the process. As a continual work to the previous study conducted by Chayabutra and Ju [162], a mixed-mode operation of aerobic and anaerobic fermentation was examined for its potential to minimize foaming and for its related problems when implemented rhamnolipid production. The key factors investigated in this study included: [1] the method for nitrate delivery that would minimize the inhibitory or toxic effects of high nitrate concentration on cell metabolism; [2] the phosphorous supplementation to maintain specific rhamnolipid productivity while cells were still growing; and [3] the effects of quorum sensing systems, a nature population control mechanism, on cell growth and rhamnolipid production. It was found that in the micro-aerobic process, amixed solution of sodium nitrate and nitric acid could be used to meet cell respiration needs and support cell growth to a relatively high concentration (> 10 g/L). A 5-fold increase in cell concentration was achieved in this study when compared to the typical aerobic fermentation. According to iii the results from the phosphorous-limiting continuous cultureconducted in the study, high specific rhamnolipid productivity could be maintained when the specific cell growth rate was lower than 0.08 (h-1). It was also observed that, an early onset of stationary phase took place in the P. aeruginosa culture when there was no apparent nutrient limitation. The phenomenon was attributed to the effect of quorum-sensing systems of the bacterium. The rhl quorum-sensing system (involving rhlR and rhlI genes) was known to regulate cell growth and rhamnolipid production. The degradation and synthesis kinetics of the rhlI gene-derived product, an autoinducer, were therefore evaluated in this study. The autoinducer was found to be unstable in the fermentation broth and its degradation could be empirically described with a first-order decay kinetics. To maintain the maximal rhamnolipid productivity, at least 13% of the peak autoinducer concentration (v/v) needs to be added in the fermentation broth in the beginning. The micro-aerobic rhamnolipid fermentation overcomes foaming problem, that retards the productivity achievable and market applicables. The autoinducer degradation and synthesis kinetics could have a medicinal application through the development of a stable autoinducer analogue to control the population of P. aeruginosa that could cause death rate in the hospitals. iv ACKNOWLEDGMENT I would like to take this opportunity to express my sincere gratitude to my dissertation advisor, Dr. Lu-Kwang Ju, for his invaluable guidance, inspiration, and tremendous patience during this study. I am also grateful Dr. Amy Milsted, Dr. Ping Wang, Dr. Teresa J. Cutright, and Dr. Stephanie Lopina for serving on my doctoral committee, precious suggestions and encouragement. I thank Dr. Iradie Lieke for her assistance during her visiting in University of Akron, Dr. Sang-Jin Suh for providing mutated strains to complete this work, Dr. A. Eberhard for providing pure autoinducer for sample analysis, and many others for dedicating their input during this study. I also want to express my genuine appreciation to my parents for their endless support and love during many years in graduate school. v TABLE OF CONTENTS Page LIST OF TABLES………………………………………………………………………..ix LIST OF FIGURES……………………………………………………………………….x CHAPTER I. INTRODUCTION…………………………………………………………………. …1 1.1 Background………………………………………………………………………..1 1.1.1 Application of Biosurfactants ……………………………………………….. 4 1.1.2 Production of Biosurfactants …………………………………………………6 1.1.3 Relation to Bioremediation……………………………………………………7 1.2 Scope of Research…………………………………………………………………9 1.3 Objectives of research….………………………………………………………….9 1.4 Structure of Dissertation…………………………………………………………10 II. LITERATURE SURVEY…………….. …………………………………………….11 2.1 Structures and Properties of Biosurfactants……………………………………...11 2.2 Synthesis of Biosurfactants………………………………………………………14 2.2.1 Microorganisms of Rhamnolipid Production………………………………...17 2.2.2 Nutrients and Limiting Nutrient for Rhamnolipid Production………….. ….19 2.2.3 Physical Conditions Effects………………………………………………….20 2.2.4 Difficulty of Biosurfactant Synthesis………………………………………...21 2.3 Metabolic Pathway of Rhamnolipid Synthesis…………………………………..22 2.3.1 Substrate Metabolic Pathways……………………………………………….22 2.3.2 Rhamnolipid Formation……………………………………………………...27 vi 2.4 Denitrification…………………………………………………………………….30 2.4.1 Nitrite Accumulation…………………………………………………………34 2.4.2 Inhibitation of Denitrification………………………………………………...35 2.4.3 Oxygen Effects on Denitrification…………………………………………….36 2.5 Quorum Sensing System…………………………………………………………37 2.5.1 Quorum Sensing System in Pseudomonas aeruginosa………………………..38 2.5.1.1 las Quorum Sensing in Pseudomonas aeruginosa………………………..38 2.5.1.1.1 Properties of LasR and 3-(oxododecanoyl)-L Homoserine Lactone…39 2.5.1.1.2 Genes Controlled by the las System………………………………….40 2.5.1.2 Characterization of the P. aeruginosa rhl Quorum Sensing System……...41 2.5.1.2.1 Genes Controlled by the rhl Quorum Sensing System……………….42 2.5.1.3 Intracellular Conmunication Between Quorum Sensing System………...43 2.5.2 Regulation of LasR…………………………………………………………...44 2.5.3 Quorum Sensing and Pseudomonas aeruginosa Virulence…………………..45 III. CHARACTERIZATION OF FERMENTATION PROCESS FOR RHAMNOLIPID PRODUCTION UNDER DIFFERENT RESPIRATION CONDITIONS………….47 3.1 Materials and Methods…………………………………………………………..49 3.1.1 Microorganism……………………………………………………………….49 3.1.2 Media………………………………………………………………………...49 3.1.3 Methods……………………………………………………………………...50 3.1.3.1 Dry Cell Weight Analysis……………………………………………….50 3.1.3.2 Ammonium and Nitrate Analysis………………………………………..50 3.1.3.3 Rhamnolipid Analysis……………………………………………………51 3.1.3.4 Glucose Analysis………………………………………………………...51 vii 3.1.3.5 Phosphorous Analysis……………………………………………………51 3.1.4 Experimental Setup…………………………………………………………52 3.2 Results and Discussion…………………………………………………………..52 3.2.1 Cell Cultivation Under Aerobic, Anaerobic and Microaerobic Conditions…52 3.2.2 Rhamnolipid Production with Phosphorous Limitation Under Aerobic and Anaerobic Conditions………………………………………………………..57 3.2.3 Continuous Culture Study on Effects of Phosphorous Concentration………60 3.3 Conclusions……………………………………………………………………...66 IV. ROLE OF RHL QUORUM SENSING SYSTEM IN CELL GROWTH, RHAMNOLIPID PRODUCTION AND DENITRIFICATION……………………67 4.1 Materials…………………………………………………………………………69 4.1.1 Organism…………………………………………………………………….69 4.1.2 Media………………………………………………………………………..69 4.2 Methods…………………………………………………………………………70 4.2.1 Dry Cell Weight Analysis…………………………………………………..70 4.2.2 Ammonium and Nitrate Analysis…………………………………………...70 4.2.3 Rhamnolipid Analysis………………………………………………………71 4.2.4 Glucose Analysis……………………………………………………………71 4.2.5 Phosphorous Analysis………………………………………………………72 4.3 Results and Discussion………………………………………………………….72 4.3.1 Effects of Rich Media on Cell Growth……………………………………...72 4.3.2 Effects of Anaerobic Denitrification by Conditioned Media……………….74 4.3.3 Autoinducer Effect on Cell Growth…………………………………………78 4.3.4 Effects of rhl Quorum Sensing System on Rhamnolipid Production……….80 viii 4.3.5 Autoinducer Effect on Rhamnolipid Productivity……………………………..80 4.4 Conclusions………………………………………………………………………...86 V. DEGRADATION AND SYNTHESIS KINETICS OF QUORUM SENSING AUTOINDUCER IN PSEUDOMONAS AERUGINOSA FERMENTATION…….87 5.1 MaterialS and Methods………………………………………………………….90 5.1.1 Microorganism and Media…………………………………………………...90 5.1.2 Methods……………………………………………………………………...90 5.1.2.1 Molecular Biological Methods…………………………………………..90 5.1.2.2 Construction of P. aeruginosa rhlI Mutant……………………………...91 5.1.3 Autoinducer Analysis………………………………………………………..91 5.1.3.1 Preparation of Autoinducer Extraction…………………………………..91 5.1.3.2 Bioassay…………………………………………………………………92 5.1.3.3 Cell Dry Weight Analysis……………………………………………….92 5.1.3.4 Cell Protein Analysis…………………………………………………….93 5.1.3.5 Rhamnolipid Analysis……………………………………………………93 5.2 Experimental Setup………………………………………………………………93 5.3 Results and Discussion…………………………………………………………..94 5.3.1 Cell Growth Study…………………………………………………………...94 5.3.2 Autoinducer Degradation in Stationary Phase……………………………….94 5.3.3 Autoinducer Synthesis in Stationary Phase………………………………...101 5.3.4 Autoinducer Concentration Profile in Batch Fermentation of Wild-Type PAO1………...……………………………………………………………..102 5.3.5 Autoinducer Concentration Profile in Batch Fermentation of rhlR(-) Mutant…………………………………………………………………..….103 5.4 Conclusions………………………………………………………….…………107 ix VI. CONCLUSIONS…………………………………………………………………..108 6.1 Conclusions…………………………………………………………………….108 6.1.1 Characterization of Rhamnolipid Fermentation Process
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