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

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.

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.

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

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 Under Microaerobic Conditions………………………………………………………………….108

6.1.2 Rhamnolipid Production: Rhamnolipid Production and Cell Growth Under the Regulation of rhl Quorum-sensing System…………………………….109

6.1.3 Degradation and Production Kinetics of Autoinducer in Stationary Phase and Effects of C4-HSL on Rhamnolipid Production………………………109

6.2 Recommendation for Future Study…………………………………………....110

6.2.1 Reasons for Observation of Decreasing Denitrification Rate During the Course of Fermentation Process…………………………………………...110

6.2.2 rpoS Gene Effects on Cell Metabolism……………………………….…...112

REFERENCES………………………………………...……………………………….113

LIST OF TABLES

Table Page

1.1: Comparison of Biosurfactant with Synthetic Surfactant………………………...... 3

2.1: Major Types of Biosurfactants Produced by Microorganisms…...... 15

2.2: Possibilities of Biosurfactant Produced by Microorganisms...... 18

2.3: Genes Controlled by las System ...... 41

LIST OF FIGURES

Figure Page

2.1: Four Types of Rhamnolipids Found in Pseudomonas species. MW Represents the Molecular Weight...... 24

2.2: The Oxidation of the Alkane to the Corresponding Fatty Acid...... 26

2.3: Gluconeogenesis: Intermediary Metabolism Relating to Biosurfactant Precursor Synthesis from Hydrocarbon Substrates. Key Enzymes Are A, Isocitrate lyase; B, Malate Synthase; C, Phosphoenolpyruvate Carboxykinase; D, Fructose-1,6- bisphosphatase...... 28

2.4: Formation of dTDP-Rhamnose...... 29

2.5: Biosynthesis of Rhamnolipids...... 32

3.1: Experimental Setup...... 54

3.2: Comparison of Cell Concentration Profiles Under Non-denitrifying as Well as Anaerobic and Microaerobic Denitrifying Conditions ...... 55

3.3: Nitrate Profiles Under Anaerobic and Micro-aerobic Conditions...... 56

3.4: Cell Concentration in Growth Phase...... 59

3.5: Profiles of Steady State Cell , Phosphate and Rhamnolipid Concentrations...... 62

3.6: Profiles of Specific Rhamnolipid Productivity and Cell Yield from Phosphorus. .... 64

3.7: Profiles of Cell and Rhamnolipid from Glucose...... 65

4.1: Nutrient Effect on Cell Growth...... 73

4.2: Nitrate Concentration Profiles in Different Systems ...... 76

4.3: Proposed Mechansim of the Regulation of Cell Growth by Quorum Sensing System...... 77

4.4: Cell Growth Profiles with and without the Addition of Autoinducer...... 79

4.5: Effect of rhlI Gene on Rhamnolipid Production...... 81 xii

4.6: Effect of rhlR Gene on Rhamnolipid Production ...... 82

4.7: Effect of Autoinducer Concentration on Rhamnolipid Production...... 84

4.8: Rhamnolipid Profiles with Various Amount of Spent Medium addition...... 85

5.1: Experimental Setup...... 95

5.2: Cell Concentration and Rhamnose Concentration Profiles in Batch Fermentation...... 96

5.3: Autoinducer Profiles of Various Systems...... 99

5.4: Degradation Kinetics of Autoinducer in Stationary Phase...... 100

5.5: Autoinducer Profile in Stationary Phase...... 102

5.6: Autoinducer Concentration Profile.in Batch Fermentation of Wild-Type of PAO1 ...... 104

5.7: Autoinducer Concentration Profile in rhlR Mutant ...... 106

6.1: Profiles of Specific Cell Growth rate and Specific Nitrate Consumption Rate with Wild Type PAO1 and rhlI mutant……………………………………...111

xiii CHAPTER I

INTRODUCTION

1.1 Background

Surfactants possess both hydrophilic and hydrophobic structure moieties, which in turn impart many unusual surface/interfacial properties. The ability of a surfactant to reduce the surface tension of water depends on its molecular structure. Synthetic surfactants are commonly produced using a variety of organic chemistry methods, depending on the type and structure of the molecule desired. The commercial importance of surfactant is evident from the increasing trend in their production and a variety of industrial applications. The surfactant industry has grown about 300% within the U.S. chemical industry during the last decade [1].

When a surfactant is present at low concentration in a system, it has the property of adsorbing onto the surfaces or the interfaces of the system. It also has the property of altering to a marked degree of surface or interfacial free energy of interfaces. The term interface indicates a boundary between any two immiscible phases; the term surface denotes an interface where one phase is a gas, usually air.

The interfacial free energy is the minimum amount of work required to create that interface [2]. The interfacial free energy per unit area is what was measured when the interfacial tension between two phases was determined. It is the minimum amount of

1 work required to create unit area of the interface or expand it by unit area. When

thesurface tension of a liquid was measured, the interfacial free energy per unit area of

the boundary between the liquid and the air above it was measured. Surfactants can

significantly change the amount of the work required to expand the interfaces. Therefore,

surfactants usually act to reduce interfacial free energy rather than increase it.

Picture a low concentration of surfactant molecules in dilute solution. If

exogenous surfactant is added to the solution, the surfactant will be adsorbed at the

surface until saturation is reached and the surface tension becomes constant with

increasing concentration. If further surfactant is added to the solution, the surfactant

molecules remain in the bulk of the solution but these hydrophobic heads will still be

repelled from the water. They can form spherical assemblies known as micelles where

the interior of the micelle resembles a separate hydrocarbon phase. The concentration at

the first form of micelle is known as the critical micelle concentration (CMC).

The total use of synthetic surfactants in the United States in 1982 was 2.5 million

metric tons [2]. It has been reported that the U.S. surfactant industry has grown about

300% within the last decade [1]. A comparison of biosurfactants and synthetic surfactants is shown in Table 1.1 [3]. Not only are biosurfactants potentially very effective but also offer some distinct advantages over the highly used synthetic surfactants. Biosurfactants exhibit highly specificity which are consequently suited to new applications, temperature stability, nontoxicity, and a broad range of structures. In addition, the chemical and physical properties of biosurfactants could be modified genetically, biologically or chemically for specific application [3].

Table 1.1

Comparison of biosurfactants with synthetic surfactants

Surfactant Surface Tension Critical Micelle 1990 Cost Cost at (mN/m) (mg/L) ($/Kg)a CMC ($/L) Producing Organism R. erythropolis 37 15 12.2 0.183

P. aeruginosa 29 15 5.9 0.089

T. bombicola 37 82 2.8 0.229

B. subtilis 27 11 (0.01mM) 20.32 0.224

Anionic Synthetics Detergent alkylate dodecylbenzene (LABS) 47 590 1.03 0.608

Sodium Lauryl Sulfate 37 2023-2890 0.95 1.92-2.74 (SLS or SDS)

a Biosurfactant costs are calculated on the basis of raw material accounting for 35% of the total cost, assuming Kerosene, molasses and soybean meal as substrate.

1.1.1 Applications of Biosurfactants

Interest in biosurfactants has increased considerably in recent years, as they are

potential candidates for many commercial applications [4]. Biosurfactants are substances

widely used in various industrial processes such as pharmaceutical, cosmetic, petroleum,

water treatment and food production. For example, they are normally employed in

various petroleum industrial processes, including emulsification and de-emulsification,

separation, formation of low viscosity emulsion products to transport heavy crude,

emulsion washing, formation of slurries, corrosion inhibition, oil recovery, and

hydrocarbon bio-degradation promotion [5]. The well-known case of using rhamnolipid biosurfactants to enhance the removal of spilled oil was occurring at Alaskan gravel [6] and the results demonstrated that rhamnolipids can release oil to a significantly greater extent (2 to 3 times) than the pressurized water alone.

In the personal care market, biosurfactants are attractive because of their low

toxicity, excellent moisturizing properties, and skin compatibility. For example, the

addition-polymerized product of sophorolipid biosurfactants with propylene glycol have

specific compatibility to the skin and found commercial utility as skin moisturizers [7].

Some of the biosurfactants show anti-microbial, anti-fungal, and/or antiviral activities at

very low concentrations [8]. Because of their low toxicity, they can be used as antibiotics

and bio-pesticides. For instance, the use of rhamnolipid surfactants for the treatment of

leaves of Nicotiana glutinosa infected with tobacco mosaic virus led to a 90 % reduction in the number of lesions [9]. It has also been found that 1% of rhamnolipids decreased the potato virus X content of systematically infected N. tabacum L. Sansun by 46 % and

secondarily infected leaves by 43 % [9].

In the food-related industries, rhamnolipid biosurfactants can be used to clean and sanitize food processing equipment, to wash fruits and vegetables, to assist removal of pesticides and wax coating, and to solubilize flavor oil in bakery and ice cream [193]. In the pharmaceutical industry, rhamnolipids can be used as emulsifiers for drug formulation and carrier of liposomes for controlled release [192]. Rhamnolipids are also used as an ingredient in ointment and cream for dermatological diseases. In addition, rhamnolipids are employed in oral and parenterally applied medication to treat auto- immune diseases. Rhamnolipids are proposed to have the potentials for the wound healing, anti-depression, and anti-rejection of organ transplantation as well [10]. In the water treatment, rhamnolipids are used to separate and recover oil. Rhamnolipids can also form either complexation or chelation with metals to reduce water contamination [7].

The sugar L-rhamnose, which is a part of rhamnolipids, is used as a fine chemical in scientific and industrial settings. Rhamnose is widely occurring in bacterial polysaccharides [11], lipopolysaccharides [12], and in leaves and flowers of poison ivy

Taxicodendron radicans [13]. The most common L-rhamnose-containing natural products are quercitrin and niringin [10]. The present methods for the commercial preparation of rhamnose require extraction of quercitrin from oak bark. Quercitrin is composed of aromatic aglycone quercetine and rhamnose, recovered by the hydrolysis of these molecules. Several disadvantages accompany the labor-intensive processes for the production of rhamnose from quercitrin, including the production of large quantities of toxic, aromatic waste products and the needs for the toxic or corrosive chemicals in the

extraction process [7, 14]. Biological production of biosurfactants is, therefore, of importance.

1.1.2 Production of Biosurfactants

The future of biosurfactants will be governed by the net economic gain between its production cost and application benefits. In biosurfactant production, fermentation step is the major operation in determining its cost of production. Generally, biosurfactants are produced by growth on hydrocarbon substrates; however, hydrocarbon substrates are poor choice from the aspects of feedstock and process costs. Recent advances in genetic engineering have offered the possibility of altering the substrate requirement and increasing the productivity of the organism. One of such examples is the transfect of P. aeruginosa strain by inserting plasmid containing lac promoter from E. coli. Thus it is possible to produce rhamnolipid from whey, a waste product from dairy industry [15]. Other significant achievements in the field during the last few years include biosurfactant production by immobilized cells [16] and by continuous system and cheaper continuous biosurfactant recovery [17]. Of immediate importance is the synthesis of sugar ester by lipase. This class of biosurfactant is receiving renewed interest because of its mildness to skin and eyes [18].

In spite of the above developments, the key factors towards the success of a biosurfactant will be the development of cheaper and large–scale production process, the use of low-cost raw materials, a high product yield, and superactive, highly specific, and selective biosurfactants for specific applications. However, many biosurfactants are

commercially attractive. Only one biosurfactant-Emulsan, a lipopolysaccharide emulsifier produced by Acinetobacter sp., has been commercilized [19].

Rhamnolipids is one group of the biosurfactants attracting more and more attention because of its potential applications. Rhamnolipid production has been limited by a severe foaming problem caused by the strong aeration and rhamnolipid produced.

Therefore it becomes a very important issue to handle the aeration rate to alleviate the foaming problem. However, reducing the aeration rate was unfavorable for the cell growth. The low yield of rhamnolipid production attributed from the unfavorable cell growth represents the major barrier to bring this product to market.

1.1.3 Relation to Bioremediation

In addition to the industrial and ecological significance of biosurfactants, knowledge of microbial hydrocarbon metabolism is of critical importance to understanding and design of in-situ biodegradation/bioremediation of non-aqueous phase liquid contaminants (NAPLs) in the environment. Among the natural bacterial genus,

Pseudomonas are capable of degrading various groups of hydrocarbons, i.e., n-alkanes

[20, 21, 22, 23], cycloalkanes [24], aromatics [25, 26], and polyaromatics [27]. They have also been found to be among the most commonly occurring microorganisms in petroleum-contaminated soils and groundwater. For example, Ridgway et al. [28] isolated approximately 300 gasoline-degrading bacteria from the well water and core material from a shallow coastal aquifer contaminated with unleaded gasoline.

Pseudomonas were found to make up 86.9% of the bacteria identified, with P.

aeruginosa being the most prevalent.

Besides direct hydrocarbon degradation by P. aeruginosa, remediation of other

groups of NAPLs may benefit from its production of extracellular rhamnolipids. One key difficulty in remediation of NAPLs-contaminated sites results from their being either

adsorbed or trapped by capillary action within the pores of soil matrices [29]. This not

only retards the remediation rates but often limits the extent of removal attainable. Being

very effective surfactants, rhamnolipids have the potential to mobilize, by emulsification and/or solubilization, the otherwise unavailable contaminants for remediation [30]. In fact, the phenomenon of oil mobilization by biosurfactants has been commonly observed in the field and reported as one of the peculiar aspects of in situ bioremediation [29].

During acclimation of the native aquifer population and soon after startup of remedial system, it is often the case that the operator will see increased levels of dissolved hydrocarbons in the monitoring samples taken at the extraction wall. This will occur as a spike in the analysis for target compounds. Sometimes the effect is dramatic and can amount to several orders of magnitude above observed baseline levels.

The metabolism of hydrocarbon by Pseudomonas species is, however, very

complicated. The aspects of hydrocarbon degradation and biosurfactant production of

Pseudomonas species clearly need careful investigation in order to identify the key

factors that govern the reduction of costs for all aspects of biosurfactant production, and

detail the possibility for design of in situ biodegradation/bioremediation of organic

contaminants in the environment. As a continuous study of previous work carried out to

produce rhamnolipid with various long-chain hydrocarbons and proof of concept of

rhamnolipid production with denitrifying P. aeruginosa in this lab, the proposed study was described in the following section.

1.2 Scope of Research

This research examines the rhamnolipid production under denitrifying microaerobic conditions by Pseudomonas aeruginosa (ATCC 9027). In this study, the key parameters such as the nitrate delivery in cell growth phase and stationary phase and the phosphorous addition to maintain the culture activity are also explored. In addition, rhamnolipid production under the regulation of quorum-sensing system is investigated. The effects of autoinducer on cell growth and rhamnolipid production are evaluated with P. aeruginosa

PAO1 and its rhlI and rhlR mutants as well. Autoinducer degradation and production kinetics in stationary phase are described in this study. The possibility of using high cell concentration to improve rhamnolipid production is also evaluated.

1.3 Objectives of Research

The ultimate goal of the research project is to develop a relatively cheap process for rhamnolipid production by microaerobic denitrifying Pseudomonas aeruginosa and to understand the role of rhl quorum-sensing system played in regulating the rhamnolipid production. The specific research objectives are to:

1. Identify the key parameters of manufacturing process for rhamnolipid production by

microaerobic denitrifying P. aeruginosa.

2. Explore the relationship between the rhl quorum-sensing system and rhamnolipid

production, and possibility of enhancing rhamnolipid production by high biomass

culture.

3. Develop mathematical models to describe the autoinducer degradation and production in stationary phase to further enhance rhamnolipid production.

1.4 Structure of the Dissertation An extensive survey of existing literature on topics closely related to this research work along with a description of related processes is given in Chapter II. The characterization of rhamnolipid production process is described in Chapter III. In

Chapter III, cell growth under aerobic, microaerobic, and anaerobic conditions are discussed. In addition, the nitrate delivery rates in cell growth phase and stationary phase are covered and the addition of phosphorous to maintain the culture activity for rhamnolipid production is investigated as well. The rhamnolipid production under the regulation of rhl quorum-sensing system is described in Chapter IV. In this chapter, how the rhlI gene, part of the rhl quorum-sensing system, affects the cell growth is discussed and how the rhl quorum-sensing genes affect the rhamnolipid production is investigated.

Furthermore, high biomass culture for rhamnolipid production is also presented in this chapter. In Chapter V, mathematical models for the autoinducer synthesis and degradation in the stationary phase are conferred. The rhamnolipid production affected by the addition of autoinducer is given in this chapter as well. In Chapter VI, conclusions summarized based on the experimental results are drawn. Recommendations for further studies of this research project are also given in this chapter.

CHAPTER II

LITERATURE SURVEY

Biosurfactants such as rhamnolipids have attracted more and more attention because of their potential uses as drug for organ transplantation, wound healing and depression, bio-pesticides and bio-remediation as well. This research aims to understand the biosynthesis of rhamnolipids with Pseudomonas aeruginosa and to explore how the rhl quorum-sensing system regulates the rhamnolipid production and cell density by P. aeruginosa PAO1. A mathematics model to determine the degradation and production of the signaling molecule, autoinducer (butynol-homoserine lactone) in stationary phase is also described in this work. The fundamental details for the common topics related to this study are given in this chapter. A brief overview of the structure and properties of biosurfactants is described in the beginning and followed by sections describing the general biosynthesis of biosurfactants and the specific biosynthetic pathway of rhamnolipids. At the end, the concept of quorum-sensing system is introduced.

2.1 Structures and Properties of Biosurfactants

Some bacteria, yeast and fungi are able to produce biosurfactants as a membrane-

binding metabolite or a secreted product in the culture media. The molecular structures

of these biosurfactants comprise a hydrophilic portion, which may consist of mono-, oligo-, or polysaccharides, amino acids or peptides or carboxylate or phosphate groups, and a hydrophobic portion, which is composed of saturated or unsaturated (hydroxy) fatty acids such as α-alkyl-β-hydroxy fatty acid [31]. Examples include the mycolic acid, glycolipid, polysaccharide-lipid complex, lipoprotein or lipopeptide, phospholipid, or the microbial cell surface itself [32]. The emulsifying properties of these substances are due to the existence of hydrophobic and hydrophilic moiety within the same molecular, which allows them to interact between two phases with different physicochemical characteristics. In addition to lowering the interfacial tension, the formation of an ordered molecular film at the interface can also dominate the interfacial rheological behavior and mass transfer [33].

The efficiency and effectiveness of these compounds are determined by their ability to reduce the surface tension of an aqueous solution and by the measurement of the critical micelle concentration (CMC) or concentration of biosurfactants necessary to achieve the lowest possible surface tention [34]. The properties of these compounds may vary depending on their sources. For example, the lipopeptides biosurfactant, a glycolipid, from B. licheniformis JF-2 is stable at a temperature up to 75ºC for at least

140 hours [35]. It is stable at pH values ranging from 5.5 to 12 but slowly loses activity under more acidic conditions. This JF-2 producing biosurfactant also has strong capability to reduce the surface tension of aqueous solutions and the interfacial tension against decane or octane to 0.01 mN⋅m-1, which is favorable comparing with the values

obtained from commercial synthetic surfactants [36].

The physicochemical properties of biosurfactants, reduction of surface tension, and the stability of emulsions formed by biosurfactants are the important factors in the search of a potential biosurfactant for a specific application. If economically competive, biosurfactants have the potential to replace synthetic surfactants, as they possess similar structural and physical properties, and are produced by renewable substrates, with the advantage of being degradable [37].

The main classes of biosurfactants are glycolipids such as lipoamino acids and lipopeptides and polymer like lipoproteins and lipopolysaccharides etc. The most commonly identified and characterized biosurfactants are glycolipids and lipopolysaccharides [38]. Glycolipids contain carbohydrates and long-chain aliphatic acids or hydroxy aliphatic acids. Among them, sophorolipids and rhamnolipids are wildly studied [39]. Certain species of Pseudomonad such as P. aeruginosa UI 29791 are known to produce large amounts of the glycolipids, rhamnolipids (46 g/l), which contain one or two molecules of rhamnose linked to one or two molecules of β-hydroxydecanoic acid [40]. The presence of biosurfactants is commonly detected by the reduction of the interfacial/surface tension or by the thin-layer chromatography techniques.

Surfactants can be classified as anionic, cationic, zwitterionic, and nonionic types depending on the nature of their hydrophilic groups [41]. Anionic surfactants such as rhamnolipids possess negative charges at their surface-active state as cationic surfactants carry positive charges. Zwitterionic surfactants contain both positive and negative charges in their molecules, and the non-ionic surfactants have no ionic charges with it.

The ionic properties have significant effects to the biosurfactants’ behaviors, for example, the adsorption phenomena on charged surfaces.

The efficiency of a surfactant in reducing the surface/interfacial tension is based on its critical micelle concentration (CMC). Many physical properties of the surfactant solution such as surface tension, osmotic pressure, electrical conductivity, light scattering, and refractive index, change abruptly in the critical point of CMC [41]. All additional surfactants added above the CMC associate readily to form colloidal clusters in the solution such as micelles, bilayers and vesicles [33], leading to the insignificant changes to those physical properties above the CMC.

2.2 Synthesis of Biosurfactants

Many microorganisms including bacteria, yeast, and fungi are able to produce a

complex mixture of biosurfactants when they used different substrates as their carbon

sources. The major types of biosurfactants produced by microorganisms are summarized

in Table 2.1. The biosurfactants such as rhamnolipids were mainly produced from

bacterial fermentation [42]. Microbial metabolism is an integrated process that required

the anticipation of numerous enzymes of the microorganisms. The metabolic pathways

of microorganism involved in the synthesis of these two groups of precursors for biosurfactant production are diverse. In many cases, the first enzymes that are unique to the biosynthetic pathways for the synthesis of these precursors are regulatory enzymes.

Therefore, in spite of the wide spectrum of interfacially active compounds, there are some common features for biosynthesis of biosurfactants and their regulations [31].

A biosurfactant molecule contains hydrophobic and hydrophilic moieties. The

Table 2.1: Major Types of Biosurfactants Produced by Microorganisms

Biosurfactant type Producing microbial species A. Glycolipids Trehalose mycolates Rhodococcus erythropolis Arthrobacter paraffineus Mycobacterium phlei Trehalose esters Mycobacterium fortitum Micromonospora spp. Mycobacteriun smegmatis Mycobacterium paraffinicum Rhodococcus erythropolis Mycolates of mono-, di- Corynebacterium diphtheriae and trisaccharide Mycobacterium smegmatis Arthrobacter spp. Rhamnolipids Pseudomonas spp. Sophorolipids Torulopsis bombicola Torulopsis petrophilum Torulopsis apicola Candida spp.. B. Phospholipids and Fatty Acids Phospholipids and fatty acids Candida spp. Corynebacterium spp. Micrococcus spp. Acinetobacter spp. Phospholipids Thiobacillus thiooxidans Aspergillus spp. C. Lipopeptides and Lipoproteins Gramicidens Bacillus brevis Polymyxins Bacillus polymyxa Omithine-lipid Pseudomonas rubescens Thiobacillus thooxidans Cerilipin Gluconobacter cerinus Lysin-lipid Agrobacterium tumefaciens Streptomyces sioyaensis Surfactin, subtilysin Bacillus subtilis Peptide-lipid Bacillus licheniformis D. Polymeric Surfactants Lipoheteropolysaccharide Arthrobacter calcoaceticus RAG-1 Heteropolysaccharide A. calcoaceticus A2 Polysaccharide-protein A. calcoaceticus strains Candida lipolytica Manno-protein S. cerevisiae Carbohydrate-protein Candida petrophilum Mannan-lipid complex Candida tropicalis Mannose/erythrose-lipid Shizonela melanogramma Ustilago maydis Carbohydrate-protein-lipid complex Pseudomonas spp. Pseudomonas fluorescens Debaryomyces polymorphus E. Particulate Biosurfactants Membrane vesicles Acinetobacter sp. H01-N Fimbriae A. calcoaceticus Whole cells Variety of microbes

hydrophilic moieties in biosurfactants show a greater degree of complexity and involve a number of biosynthetic pathways. Three major activities required to regulate the production of biosurfactants are (1) the induction of surfactant synthesis by hydrocarbons, (2) the catabolic repression by water-soluble primary substrates, and (3) the regulation by the nitrogen source or a multivalent cation.

Glycolipid biosurfactants are commonly considered as sugar-containing lipids in which both moieties may be linked either glycosidically (i.e. as an ether or more correctly as a hemiacetal link) as in the sophorose, rhamnose, and cellobiose lipids, or via acylation

(i.e. as an ester link) as in the acylpolyols, trehalose lipids, and sugar mycolates [36].

The biosynthetic pathways to these lipids must include the generation of both the lipophilic (fatty acid) and the hydrophilic (carbohydrate) moieties. In general, the biosynthesis of biosurfactants, especially for glycolipids such as rhamnolipids biosynthesis, may fall into one of the following pathways according to Haferburg et al.

[43] and Syldatk and Wagner [31]:

1. Both the carbohydrate and the lipid moiety are synthesized independently of the

growth substrate.

2. The synthesis of the lipid moiety depends on the hydrophobic carbon source, that is, it

is a derivative of the hydrophobic carbon source and the carbohydrate is synthesized

de novo.

3. The carbohydrate moiety reflects the carbon source being used for growth or

maintenance and the lipid moiety is synthesized de novo.

4. The synthesis of both residues depends on the carbon substrates being used.

The specific details of the biosynthesis pathway of rhamnolipids are discussed in the next section (Section 2.3). In general, the production of biosurfactants can be growth correlated, as in the case of many cell wall-bound compounds. On the other hand, as in the case of many extracellular ionic surfactants, a major production phase can rarely be observed before the stationary phase of cell growth. The details are summarized in Table

2.2.

2.2.1 Microorganisms for Rhamnolipid Production

Rhamnolipids were found for the first time in 1946 in Pseudomonas pyocyanea culture after growth on glucose. The structure of rhamnolipid was not elucidated until

1965. The microbial species that is known of its capability to produce rhamnolipids is

Pseudomonas aeruginosa. Various P. aeruginosa strains, specifically, DSM 2874,

ATCC 9027, ATCC 10145, UI 29791, etc., have been reported to be excellent producers of rhamnolipids [44, 45, 46, 47]. Rhamnolipids can also be produced by P. fluorescens or P. putida by genetically cloning the essential genes from P. aeruginosa for the synthesis of rhamnolipid-producing enzymes, but not by Escherichia coli due to incapable of producing precursors for rhamnolipid synthesis [48]. P. aeruginosa isolated from the soil contaminated with polycyclic aromatic hydrocarbons (PAHs) [49], from the sludges contaminated by hydrocarbons adjacent to petrochemical industries or crude oil spills [5] were found to furnish rhamnolipid production. The production of rhamnolipids can be performed in batch, continuous, or semi-continuous processes. To optimize rhamnolipids production various approaches have been employed including the growing,

Table 2.2: Possibilities of biosurfactant production by microorganisms.

1. Cell growth-associated production of biosurfactants 1.1) Induction of production by lipophilic substrates 1.2) Increase of production by optimization of medium composition 1.3) Increase of production by optimization of environmental influences as pH, temperature, aeration, agitation speed, etc. 1.4) Increase of production by addition of reagents, which cause a change of cell wall permeability as penicillin, ethambutol, EDTA, etc. 1.5) Increase of production by addition of reagents which cause a detachment of cell wall-bound biosurfactants into the medium as alkanes, kerosene, EDTA, etc.

2. Biosurfactant production by growing cells under growth-limiting conditions 2.1) Production under N-limitation 2.2) Production under limitation of multivalent cations 2.3) Increase of production under growth-limiting conditions by a change of environmental conditions such as pH or temperature

3. Biosurfactant production by resting cells 3.1) Production by resting free cells 3.2) Production by resting immobilized cells 3.3) Production by resting immobilized cells with simultaneous product removal

4. Biosurfactant production by growing, resting free, and resting immobilized cells with addition of precursors

free resting and immobilized cells [38]. The highest production was obtained with the free resting cells. However, the final titer was in the range of 4-5 g/L.

2.2.2 Nutrients and Limiting nutrients for Rhamnolipid Production

Various species utilize different primary nutrients such as carbon sources to

produce rhamnolipids. The commonly used carbon sources for rhamnolipids production

are n-paraffin [50], glycerol [51], proteose peptone-glucose salts [52], ethanol [53],

stearic acid [54], corn oil [55], etc. Pseudomonas species was report to utilize hydrocarbons for growth and to produce rhamnolipids [56, 57]. However, the microorganism could not digest saturated hydrocarbons in the absence of oxygen. This was observed when using hexadecane as the carbon source for P. aeruginosa ATCC

10145 [60]. This characteristic of Pseudomonas aeruginosa could limit the selection of

carbon sources. Different cultivation conditions and medium compositions influence the

productivity and crude product composition of rhamnolipids [54, 58]. Arino et al.

reported the influence of various nitrogen and carbon sources on the rhamnolipid

production from glycerol [49]. It was found that the nitrogen concentrations around 3 – 5

g/L with the mix substrate of glycerol and ethyl dodecanoate yielded the highest

rhamnolipid production. The best rhamnolipid production was reported mostly under

aerobic nitrogen-limiting conditions, in the presence of excess carbon and phosphorus

concentrations [59, 55, 38]. Magnesium limitation also causes rhamnolipid production

with the highest yield obtained when the carbon-to-magnesium ratio in the fresh feed to

continuous culture is 364 or higher [61]. Guerra-Santos et al. [61] reported

that the change of potassium, sodium, or calcium concentrations has no effect on the rhamnolipid production. The optimal sodium chloride concentration was 100 mM when

P. aeruginosa DSM 2874 was grown on glucose medium [54]. Linhardt et al.[55] suggested the optimal iron concentrations of 50-100 μg/L for the production of rhamnolipids by P. aeruginosa UI29791 in the corn oil medium. However, rhamnolipid production from glycerol was reported to be inhibited by organic acid salts such as sodium succinate, sodium citrate, and sodium acetate [51].

2.2.3 Physical Condition Effects

The effect of agitation on the cell growth on hydrocarbon was investigated by

Velankar et al. [62]. It was found that the growth in exponential phase increased with the

increase of agitation. This could be partially due to the interfacial area between the

nutrient pool and gas phase and thus enhances the mass transfer coefficient. The system

without agitation does not provide good oxygen mass transfer to the cells throughout the

medium, and might result in cell death due to oxygen depletion. Different types of

agitation also affect the rhamnolipid production. As an example, a higher rhamnolipid

production was achieved by the rotary- agitation type when compared to the lateral type

at 150 rpm and the control without agitation [5].

P. aeruginosa can produce rhamnolipids in the temperature ranging from 23 to

43°C, with the best production reported in the range of 27 – 37°C [54, 61]. The

cultivation pH influences the degree of foam formation, which was typically found in

rhamnolipid biosynthesis. Above a pH of 6.5, foam formation was much more severe

than that at lower pH values. The optimum pH for rhamnolipid production is also strain- dependent, for example, the best of pH between 6.2 – 6.4 was suggested by Guerra-

Santos et al. [61], while minimal pH effect between 6 and 7 was suggested by Arino et al.

[49].

2.2.4 Difficulty of Biosurfactant Synthesis

A serious problem in biosurfactant production under aerobic condition is foams.

With the conventional submerged aeration, very stable foams are generated in the

presence of rhamnolipids at concentrations greater than 0.1 g/L [60]. This causes serious

problems, including high expenditures for foam control, complexity of down stream

processing and the limitation of productivity as well.

Due to rapid foaming in large volume and high foam stability, a mechanical foam

breaker fails to control the foam problem. On the other hand, chemical antifoam agents

may affect downstream processing, especially the performance of filtration units [63].

The possible influence of the addition of antifoam agents on cell metabolism and the

pollution of the reactor effluent have to be considered as well. This serious foaming

problem could be eliminated if there is no or less need of aeration for the culture’s

respiration. P. aeruginosa is a facultative aerobe that can use either oxygen or nitrate as

the terminal electron acceptor. Nitrate addition was therefore used to replace the aeration

in the process for meeting the cells’ respiration demand. Dr. Ju et. al. have shown the

possibility of anaerobic rhamnolipid production in their work [80]. The details of using

nitrate as electron acceptor instead of oxygen are presented in section 2.4.

2.3 Metabolic Pathway of Rhamnolipid Synthesis

The understanding of rhamnolipid biosynthetic pathway is essential to the

improvement of its overall production in the fermentation. Rhamnolipids were first

reported to be produced from the fermentation of glycerol by P. aeruginosa in 1949 [64].

Hauser and Karnovsky further investigated the biosynthesis of rhamnolipids and its

production in 1954 [65, 51]. In 1985, the physical and chemical properties of

rhamnolipids were further characterized by Syldatk et al. [44].

It is known that nitrogen deficiency can induce many microbes to shift

metabolism leading to overproduction of certain metabolites [38]. Rhamnolipids, the brownish solid material, are also parts of them. They consist of one or two molecules of

rhamnose and one or two molecules of β-hydroxydecanoic acid as shown in Figure 2.1.

Besides the abundant 3-hydroxydecanoic acid moiety shown in Figure 2.1, 3-

hydroxyoctanoic, 3-hydroxydodecanoic and 3-hydroxydodecenoic acid moieties have

also been observed in rhamnolipid structures most recently [49].

2.3.1 Substrate Metabolic Pathways

In general, the formation pathway of glycolipid biosurfactants depends on the

microorganism. The sugar moiety and the lipid moiety of rhamnolipids by P. aeruginosa

are synthesized de novo [31]. Three possible mechanisms by which the bacteria can

consume the lipophilic substrates are by the uptake of dissolved hydrocarbons, by direct

contact with the surface of the substrates, or by the uptake of emulsified and/or

solubilized hydrocarbon. The mechanism of uptake of dissolved hydrocarbons is only

applicable to the short-chain hydrocarbons that can dissolve in a sufficient amount for the microbial consumption. The direct contact mechanism applies when the cells bind to the droplets of the substrate. The uptake of emulsified/solubilized hydrocarbons normally occurs in the presence of extracellular surfactants [66].

The oxidation of alkanes starts with the initial oxidative step that is catalyzed by a complex hydroxylase (monooxygenase) linked to an electron carrier system. The reaction results in the formation of corresponding fatty alcohol, as shown in Equation 2.1.

+ R – CH3 + O2 + 2NADH → R – CH2OH + 2NAD + H2O……….....(2.1)

This long-chain fatty alcohol product is further oxidized to the corresponding fatty

aldehyde and fatty acid by the alcohol dehydrogenase and the aldehyde dehydrogenase

enzymes, respectively. The conversion of alkanes to the corresponding fatty acids is

depicted in Figure 2.2. These enzymes, which have been characterized by Clarke and

Ornston [67], are localized in the cytoplasmic membrane. It is postulated that the

emulsified alkane passes through the cell’s outer membrane by diffusion and is oxidized

to the corresponding fatty acid before it enters the cytoplasm [68].

When the substrates for microbial growth are non-carbohydrate substances, such

as alkanes, alkenes, fatty acids, acetate, or ethanol, many important sugars must be

synthesized from the pool of intermediates in the central pathways of metabolism. These

sugars, for example, hexoses, pentoses, tetroses and trioses, are crucial for the synthesis

of structural entities of the cell or for the biosynthesis of amino acids, proteins, purines,

O CH CH O 2 C CH CH2 COOH HO O CH (CH ) 3 2 6 (CH2)6

OH OH CH3 CH3 Rhamnolipid 1

O CH CH2 COOH HO O CH 3 (CH2)6 Rhamnolipid 2 OH OH CH3

O CH CH O 2 C CH CH2 COOH HO O CH (CH ) 3 2 6 (CH2)6

CH CH OH O 3 3 HO O CH3 Rhamnolipid 3 OH OH

O CH CH2 COOH HO O CH 3 (CH2)6

CH OH O 3 HO O CH 3 Rhamnolipid 4

OH OH Figure 2.1: Four types of rhamnolipids found in Pseudomonas species. MW represents the molecular weight.

pyrimidines, and nucleic acids [66].

Fatty acids obtained from the oxidation of alkanes can be further metabolized by a variety of metabolic pathways. The oxidation via β-oxidation is common in bacteria.

The β-carbon, i.e., the second carbon from the carboxyl group of the fatty acid, is oxidized and the carbon chain is broken. Coenzyme A is attached to each piece to form acetyl-CoA, which is metabolized through the tricarboxylic acid (TCA) cycle to obtain additional energy [69]. Part of the acetyl-CoA obtained is used for the synthesis of sugars by gluconeogenesis, which is the reversing process of glycolytic sequence of sugar breakdown (Figure 2.3). Rhamnose, the sugar portion of rhamnolipids, is formed by this gluconeogenesis pathway. It is 6-deoxymannose and is found in several bacteria [70]. It is synthesized by an NADPH-linked deoxythymidine phosphate (dTDP)-D-glucose oxidoreductase (Figure 2.4) with the other enzymes that involve not only the reduction at

C6 of glucose but also inversion of the configuration at C3, C4, and C5. The dTDP- rhamnose is used directly as the substrate for the condensation with the lipid moiety to form rhamnolipids. Since the carbon chain of fatty acids is broken by β-oxidation, the formation of shorter-chain fatty acids is highly possible. Two types of fatty acids required for rhamnolipid synthesis, β-hydroxydecanoic acid and β-hydroxydecanoyl-β- hydroxydecanoic acid, are formed through this pathway [31].

Alkane CH3(CH2)nCH3 O2 Monooxygenase H2O

Fatty Alcohol CH3(CH2)nCH2OH

Alcohol Dehydrogenase

Fatty Aldehyde CH3(CH2)nCHO

Aldehyde Dehydrogenase

Fatty Acid CH3(CH2)nCOOH

β -Oxidation

Acetyl- Fatty Acid Synthesis Gluconeogenesis CoA

Figure 2.2: The oxidation of the alkane to the corresponding fatty acid.

2.3.2 Rhamnolipid Formation

The synthesis of rhamnolipids in vivo using glycerol and sodium acetate as the substrates was investigated by Hauser and Kanovsky [65, 71, 72]. It was found that, in the system using glycerol and sodium acetate as the mixed substrate, the carbons from glycerol can furnish all carbons in both sugar and acid parts of rhamnolipids, while the carbon from acetate can supply only the carbon of the β-hydroxydecanoic acid part. The rhamnose moiety seemed to be derived by the condensation of two 3-carbon units formed from glycerol and the carbon of fatty acid seemed to be synthesized by fatty acid biosynthesis pathway from 2-carbon units.

The de novo rhamnolipid synthesis by the extracted enzymes from P.

aeruginosa has been investigated by Burger et al. [73, 74]. These enzymes, called

rhamnosyl transferases, catalyze the reactions between the precursors of the sugar and the

acid parts in order to form rhamnolipids. The sugar part, which is the rhamnosyl donor,

is thymidine diphosphate L-rhamnose (TDP-L-rhamnose) and the acid part is β-

hydroxydecanoyl-β-hydroxydecanoate. The synthesis reactions are:

2-β-Hydroxydecanoyl-CoA + H2O →

β-hydroxydecanoyl-β-hydroxydecanoate + 2 CoA (2.2)

TDP-L-rhamnose + β-hydroxydecanoyl-β-hydroxydecanoate →

TDP + L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (2.3)

TDP-L-rhamnose + L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate

• Polysaccharides Glucose • Cellobiose • Trehalose • Sophorose Glucose 6-P • Rhamnose • Mannose

Fructose 6-P D

Fructose 1,6 bis P Pentose phosphate

Glyceraldehyde 3-P Dihydroxyacetone-P

Phosphoenolpyruvate Sn-Glycerol 3-P C

Oxaloacetate Lipids

Malate

Acetyl-CoA β-oxidation succinate

Fatty acids B 2-oxoglutarate Citrate

A Glyoxylate Isocitrate

Figure 2.3: Intermediary metabolism relating to biosurfactant precursor synthesis from hydrocarbon substrates. Key enzymes are A, isocitrate lyase; B, malate synthase; C, phosphoenolpyruvate carboxykinase; D, fructose-1,6-bisphosphatase.

CH2OH O O O OH HO O P O P O Thymidine OH - O- O

NADPH

+ NADP

HO O O O CH 3 O P O P O Thymidine - HO HO O- O

Thymidine-diphospho-rhamnose (TDP-rhamnose)

Figure 2.4: Formation of dTDP-rhamnose.

→ TDP + L-rhamnosyl- L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (2.4)

Burger et al. [73] found that the synthesis of rhamnolipids proceeds by sequential glycosyl transfer reactions as shown in Equations 2.2 – 2.4, and each is catalyzed by a specific rhamnosyl transferase. The enzyme activity of the TDP-L-rhamnosyl-L- rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate rhamnosyl transferase works best at pH 6.7 [73]. Ochsner et al. [75, 76] and Pearson et al. [77] have further studied the genes involved in rhamnolipid biosynthesis and confirmed the mechanism demonstrated by

Burger et al. [76]. The rhlR and rhlI genes were identified as the regulatory genes which are necessary for the transcriptional activation of the rhlA and rhlB genes that encode the rhamnosyltransferase enzyme. The rhlA gene was found to be expressed slowly in the exponential growth phase, but it increases 20-fold during the stationary phase. The rhlB protein is the catalytic protein of the rhamnosyltransferase. The binding of activated rhlR protein to target sites upstream of the rhlA enhances transcription of the rhlA and rhlB operan and subsequently form the rhamnosyltransferase (rhlAB), which is the key enzyme capable of rhamnolipid biosynthesis. The biosynthesis of rhamnolipids is shown in Figure 2.5.

The major rhamnolipids are L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β- hydroxydecanoate and L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate, which are referred as rhamnolipids 3 and 1 (Figure 2.1), respectively [78].

2.4 Denitrification The anaerobic reduction of nitrate and nitrite to nitrous oxide or elemental nitrogen is

termed denitrification, and the physical process is called anaerobic respiration.

Denitrification is important because of its role in the regeneration of fixed nitrogen. It has become of particular interest in recent years due to increased cost of nitrogen fertilizer and potential reduction of crop yields because of this microbiological phenomenon [79]. In addition, the generation of gaseous nitrogen oxides has become a concern because of the potential effects on the ozone layer of the upper atmosphere [79].

With these factors as impetus, basic knowledge in the area of denitrification has expanded rapidly in a relatively short time.

The most predominant denitrifying bacteria in our environment have been reported to belong to the genus Pseudomonas [80]. Species of Pseudomonas are nonfermenting organisms capable of generating energy only by respiration. The mechanism and regulation of electron flow and ATP synthesis under denitrifying conditions are considered to be process similar to aerobic respiration. It is clear, however, that different spectra of cyto-chromes and enzymes are required for anaerobic respiration (79). The process of denitrification is thought to occur in a stepwise manner

- - as follows: NO3 → NO2 → NO→ N2O→ N2. As would be expected, many of the

enzymes of pathway are closely associated with cytochromes in Pseudomonas

aeruginosa.

Several themes run constant. The presence of oxygen suppresses the enzymes

necessary for denitrification [81, 82]. Once depressed, the quantity of these enzymes is

O HO O O O HO CH C O CH COOH CH3 CH 2 CH 2 P O P O Thymidine O + (CH 26) (CH 26) HO HO - - CH CH O O 3 3 Thymidine-diphospho-rhamnose (TDP-rhamnose) beta-Hydroxydecanoyl-beta-hydorxydecanoate

Rhamnosyltransferase 1

TDP

rhamnosyl- O transferease

O CH CH2 C O CH CH2 COOH rhl rhl rhl rhl HO O A B R I CH (CH ) (CH ) Genes 3 26 26 CH CH autoinducer 3 3 synthetase HO HO

Rhamnolipid 1 L-rhamnoosyl- beta-hydroxydecanoyl- beta-hydroxydecanoate TDP-rhamnose

Rhamnosyltransferase 2

TDP

O CH CH2 C O CH CH2 COOH HO O CH 3 (CH 26) (CH 26) CH CH 3 3 HO O

HO O Rhamnolipid 3 CH3 L-rhamnosyl-L-rhamnosyl beta-hydroxydecanoyl- beta-hydroxydecanoate HO HO 32

Figure 2.5: Biosynthesis of Rhamnolipids. directly affected by the initial nitrate concentration in the culture [81]. Anaerobiosis diminishes the α-type cyto-chromes drastically and stimulates production of c-type cyto- 32

chromones in most denitrifying bacteria [79]. Finally, nitrite accumulates in the culture media, usually before the onset of visible gas production. Nitrite may or may not inhibit further reduction of nitrogen oxides, depending on the species of bacteria and the culture conditions [83, 84].

One important denitrification aspect, which has been largely ignored, is the transport of nutrients by denitrifiers during anaerobic respiration and its relationship to the enzymes and the cyto-chrome system. Evidence has been presented to demonstrate coupling between anaerobic and transport of lactose and amino acids and anaerobic electron transfer in isolated membrane vesicles of E. coli [85]. More recently, it has been shown that amino acid transport can be coupled to electron transfer by using nitrate as an electron acceptor in the obligate anaerobe Veillonella alcalescens [86]. Several energy donors and components of the membrane-bound anaerobic electron transport chain of

Veillonella alcalescens were also identified. Virtually, no information is available, however, on the active transport of solutions in bacteria growing under denitrifying conditions.

Pseudomonas aeruginosa possesses an inducible active transport system for glucose as well as inducible enzymes for the Enter-Doundroff pathway and the oxidative portion of hexose mono-phosphate-pentose cycle [87, 88]. Guymon and Eagon [87], however, demonstrated that in Pseudomonas aeruginosa glucose and gluconate were actively transported as the free sugars. Evidence has been found, moreover, which implicates the phosphoenopyruvate-phosphotransferase system as the mechanism by which fructose is transported by Pseudomonas species [89]. Thus, with the sole

exception of fructose, sugars are considered to be accumulated by a carrier-mediated active transport system in P. aeruginosa.

2.4.1 Nitrite Accumulation

Many investigators have observed that nitrite can inhibit the denitrification,

especially at high concentration. Accumulation of extracellular nitrite has been reported

during denitrification in pure cultures, implying that, under some conditions, denitrifying

bacteria transport the nitrite intermediate out of the cell and later take the extra-cellular

nitrite back into the cell for complete denitrification. Beltach and Tiedje [90] described a model that determined the accumulation of extracellular nitrite based on experiments with cell suspensions of Alcaligene spp., Pseudomonas fluorescens, and a

Flavobacterium isolated. Recently, Kornaros et al [91] have shown that nitrite accumulated in cultures of Pseudomonas denitrificans, ATCC 13867, when nitrate was

present. Spikes of 30 mg/L NO3-N added after denitrification of nitrite had begun

caaused a 61 % decrease in the nitrite reduction rate.

Rijn et al. [92] confirmed that nitrate inhibit nitrite reduction in Pseudomonas

stutzeri isolated from a denitrifying fluidized bed reactor. They observed that for

Pseudomonas stutzeri the level of nitrite accumulation was determined by the number of

electrons supplied per mole of carbon/energy substrate compound, with greater

accumulation of extracellular nitrite with relatively oxidized substrates like acetate,

compared with butyrate. The authors suggested that, when substrate electrons were limited, nitrate seemed to be the preferred electron acceptor, compared with nitrite. Competition for substrate electrons between nitrate and nitrite reductases also has been reported in Paracoccus denitrificans and Pseudomonas flourescens [93, 94]. Research

using pure cultures of denitrifying bacteria suggests that one mechanism for the transient accumulation of extracellular nitrite during denitrification is a competitive advantage for electrons of nitrate reductase over nitrite reductase. Then, by a mechanism, which is not understood, intracellular nitrite is transported out of the cells temporarily. Finally, the denitrifying bacteria take in the nitrite by-product for further reduction.

2.4.2 Inhibition of Denitrification Inhibition of denitrification by high concentrations of nitrate has been reported

[95]. Some researchers have suggested that nitrate substrate inhibition of denitrification is actually due to the toxicity of accumulated nitrite, specifically the unionized nitrous acid species [96, 97, 98]. Abelin and Seyfried [99] found that inhibition of denitrification occurred when the concentration of HNO2 reached 0.13 mg/L (0.04 mg/L N), and

suggested that the concentration of nitrous acid, not nitrite, controls the inhibition of

denitrification. More recently, Glass et al. [100] found that, at even near-neutral pH

values of 6 and 7, nitrite concentrations of 30 and 250 mg/L NO2-N, respectively, could

inhibit denitrification. Because inhibition occurred at neutral pH, and decreased only

with increasing pH to at least 8, it was thought that HNO2 species was the likely cause

[100]. Other researchers have reported that, at pH values below 7, denitrification rates

decrease significantly [101, 100].

2.4.3 Oxygen Effects on Denitrification

The enzymes of nitrate respiration are only formed when specific conditions for

denitrification are present [102, 103]. Anaerobic conditions alone are sufficient to

partially derepress all of the enzymes involved in the nitrate respiration, but the presence

of nitrate or nitrite intensifies the response [104]. The synthesis of denitrifying enzymes

is repressed in the presence of oxygen [105, 106, 82]. Oxygen blocks the synthesis of respiratory nitrate reductase in E. coli. at the level of transcription and at some later step of enzyme formation [106]. Although the derepression of denitrifying enzymes is thought to require strict anaerobiosis, the activity of some of the performed enzymes in the pathway may persist under aerobic conditions [102, 104].

It has been known that oxygen inhibits the reduction of nitrate and the formation of nitrogen gas by denitrifying bacteria [107, 108]. Addition of oxygen to cultured cell suspensions or reaction mixtures containing extracts of denitrifiers results in inhibition of the pathway at some level and thus a rapid loss of the capacity for reduction of nitrate and other nitrogen oxides [90, 109, 106, 110]. Until recently there has been very limited work of quantitative nature showing the magnitude of oxygen inhibition in relation to the partial pressure of oxygen during nitrate respiration. Furthermore, the significance of this inhibitory effect remains unclear because the site at which oxygen inhibits nitrate respiration has not been well defined. The effects of the oxygen on nitrate respiration have been studied mainly by growing bacteria under anaerobic conditions, measuring nitrate reduction rates, introducing oxygen, and observing any change in the rates of nitrate respiration. As a result of this methodology, the effect of oxygen on nitrate entry into the cell has been overlooked as a major potential regulatory mechanism in nitrate respiration.

2.5 Quorum Sensing System Microorganisms are consistently subjected to a myriad of environmental stimuli.

Such cues include change of temperature, osmolarity, pH, and nutrient availability. In response, bacteria have developed multiple systems that allow adaptation to these environmental fluctuations. For instance, two-component signal transduction schemes 36

allow microorganisms to sense and respond to multiple environmental factors by the activation or repression of specific target genes [111]. Similarly, the expression of assorted sigma factors in response to various signals enables transcriptional specificity in bacteria [112]. Alteration to DNA topology, protein-mediated or otherwise, can also result in changes to the transcriptional profile of a microorganism [113, 114].

A further layer of microbial sensing and response mechanisms has been recently uncovered in the form of cell-to-cell communication via the use of small signal molecules. Once thought of as a rare phenomenon, restricted to a few obscure examples, it is now increasingly apparent that an extensive range of microorganisms has the ability to perceive and respond to the presence neighbouring microbial populations. Numerous signaling molecule-mediated sensing and response pathways have now been defined and many fall within the scope of a form of regulation that is commonly known as quorum- sensing. The term quorum-sensing was first used in a review by Fuqua et al. [115]. The process relies on the production of a low-molecular-mass signaling molecule, the extracellular concentration of which is related to the population density of the producing organism. The signaling molecule can be sensed by cells and this allows the whole population to initiate a concerted action once a critical concentration has been achieved.

Several chemical classes of microbially derived signaling molecule have now been identified. Broadly, these can be split into two main categories: (1) amino acids and short peptides, commonly utilized by gram-positive bacteria [116, 117, 118] and (2) fatty acid derivatives, frequently utilized by gram-negative bacteria. In this section, the wide range of quorum sensing systems that employ N-acyl homoserine lactones as the

signaling molecule with which to control the expression of diverse physiological functions will be described in chapter 4.

2.5.1 Quorum Sensing in Pseudomonas aeruginosa

In recent years, the microorganism on which most quorum-sensing-related studies

have been initiated is Pseudomonas aeruginosa, reflecting its importance as a common

opportunistic human pathogen. The basis of the pathogenicity of P. aeruginosa is its

ability to produce and secrete multiple extracellular virulence factors such as proteases,

haemolysins, exotoxin A, exoenzyme S and pyocyanin. These exofactors are collectively

capable of causing extensive tissue damage in humans and other mammals [119, 120].

2.5.1.1 las Quorum Sensing System in Pseudomonas aeruginosa

Regulation of the genes encoding the exoproducts is primarily dependent upon a signaling system that encompasses at least two sets of LuxRI homologues. The first hint

that P. aeruginosa was controlling gene expression in a cell density-dependent manner

came with the report by Whooley et al [121] that exoprotease production occurred in a

growth-associated manner. The mechanism responsible for this control was uncovered

when Gambello and Iglewski [122] complemented an elastase-deficient P. aeruginosa

strain. They showed that this elastase-negative strain contained the lasB gene, which

encodes elastase, but didn’t produce a lasB mRNA. The subsequent search for a positive

regulator that would complement elastase phenotype of this strain led to the discovery of

the lasR gene [122]

The lasR gene encodes a 293-amino-acid homolog of the prototypical quorum- sensing transcriptional activator LuxR of Vibro fisheri [123]. The LasR protein, like other LuxR homologs, has two highly conserved regions. One is the putative autoinducer-binding region, which constitutes the amino two-thirds of the protein, and the other is the putative DNA-binding region which consists of a helix-turn-helix motif located in the carboxyl third of the protein [122]. The lasR gene is transcribed as a monocistronic operon [122], which is directly followed on the P. aeruginosa chromosome by the lasI gene [124].

The lasI gene encodes LasI, a 201-amino-acid homolog of the prototypical V. fischeri LuxI autoinducer synthase protein [124]. LasI was shown to direct the synthesis of N-(3-oxododecanoyl)-L-homoserine lactone, which is the autoinducer molecule of the las quorum-sensing system [125].

2.5.1.1.1 Properties of LasR and 3-(oxododecanoyl)-L-Homoserine Lactone After the components of las quorum-sensing system were defined, some of their properties were determined. Deletion analysis of lasR suggested that the 3-

(oxodocanoyl)-L-homoserine lactone interaction site was within the amino acids 3 to 155,

and that transcriptional activation by the LasR required only the carboxyl-terminal

portion of LasR from amino acids 160-239 [126]. The ability of LasR binding to the lasB

promoter region was determined [127], and it has been shown that 3-(oxodocandoyl)-L-

homoserine lactone will specifically bind to cells expressing LasR (128). Passador et al

also showed that the length of the acyl side chain was the most important factor that

affected the ability of 3-(oxodocandoyl)-L-homoserine lactone analogs to activate or bind

to LasR. As acyl side-chain length decreased from the normal 12-carbon length of 3-

(oxodocandoyl)-L-homoserine lactone, the ability to activate or bind to LasR decreased, indicating that the LasR/3-(oxodocandoyl)-L-homoserine lactone. Interaction was one of high specificity. To study specificity between quorum sensing systems from different species, Gray et al. [129] performed interchangeability studies in which different combinations of the las and lux quorum sensing systems were reconstituted in E. coli.

They found that these two systems were specific concerning the autoinducer required to activate LasR or LuxR (3-oxo-C12-HSL or 3-oxo-C6-HSL, respectively), and that some cross binding of promoters could occur (LasR-3-oxo-C12-HSL could effectively activate the lux promoter and Lux-3-oxo-C6-HSL could partially activate the las promoter).

2.5.1.1.2 Genes Controlled by the las System

Together, the LasR and 3-(oxodocandoyl)-L-homoserine lactone has proved to

control a number of P. aeruginosa virulence genes as given in Table 2.3 [130, 131, 132].

After LasR’s role for elastase production was discovered, it was shown that this protein was also required for the production of two other P. aeruginosa proteases, LasA protease

and alkaline protease [130]. Gambello et al [131] also found that LasR was required for

full expression of exotoxin A. Seed et al [132] showed that LasR and 3-(oxodocandoyl)-

L-homoserine lactone controlled transcription of monocistronic lasI operon indicating

that this autoinducer synthase gene was controlled by a positive feedback loop. This suggested that when a threshold concentration of 3-oxo-C12-HSL was reached, LasR-3- oxo-C12-HSL complex could induce the production of more 3-oxo-C12-HSL. Seed et al. also discovered that 10-fold more 3-oxo-C12-HSLwas required for LasR to activate lasB than the amount needed to activate lasI, indicating that a hierarchy existed with respect to

the order of gene activation by 3-oxo-C12-HSL. In this hierarchy, lasI could be activated before lasB, causing the 3-oxo-C12-HSL concentration to increase so the lasB and other

LasR-3-oxo-C12-HSL-controlled genes could be activated. Finally, the truly global role of the las quorum-sensing system was strengthened with the report of Chapon-Herve et al. [133] that demonstrated LasR-3-oxo-C12-HSL activated the xcpP and xcpR genes, which encode proteins of the Pseudomonas aeruginosa general secretory pathway.

2.5.1.2) Characterization of the P. aeruginosa rhl Quorum-Sensing System

It became apparent that P. aeruginosa contained another quorum-sensing system

Table 2.3: Genes Controlled by las System

Gene Controlled by Enzymes regulated Function of Enzyme las System by las System

lasA LasA Elastolytic activity las Quorum- sensing System lasB LasB Elastolytic activity lasR LasR Binding to 3-oxo- C12HSL to regulate the expression of lasI and rsaL lasI LasI Binding to LasR enzyme to regulate the expression of lasI and rsaL

when Ochsner et al [134] discovered a second P. aeruginosa LuxR homolog. The gene

that encoded this homolog was named rhlR for its ability to regulate the production of

rhamnolipids, which acts as a biosurfactant/hemolysin. Pearson et al [135] reported that

P. aeruginosa produced a second autoinducer molecule, N-butyryl-L-homoserine lactone 41

(C4-HSL), that affected lasB expression. The components of rhl quorum-sensing system fell into place when Ochsner and Reiser [136] reported that the rhlI gene, which encodes a LuxI homolog, was located directly downstream from rhlR. The rhlI gene was subsequently shown to direct the synthesis of C4-HSL [137].

The rhl system has not been studied to the extent that the las system has, but it appears to be a typical quorum-sensing system. Pearson et al [138] showed that C4-HSL binds specifically to cells expressing rhlR, but unlike the binding of 3-(oxodocandoyl)-L- homoserine lactone to LasR-expression cells [128], this binding is significantly enhanced by overexpression of the chaperone GroESL. The binding of C4-HSL to RhlR- expression cells suggests that the RhlR-C4-HSL complex is responsible for activating genes controlled by the rhl system.

The genetic organization of the rhl system is relatively similar to that of the las system in which rhlI is located directly downstream from rhlR. However, unlike the las system, one of the primary targets of the rhl system, rhlAB, is located directly upstream from rhlR. The rhlA and rhlB genes, which encode for a rhamnosyltransferase required for rhamnolipid production [139], were shown to be cotranscribed on a single mRNA

[138] while rhlR and rhlI are transcribed from their own promoters [140].

2.5.1.2.1 Genes controlled by the rhl Quorum-Sensing system

Ochsner et al [134] found that rhlR was not only involved in rhamnolipid

production, but also responsible for full elastase activity. Winson et al [137] reported that

expression of rhlI was positively controlled by RhlR and C4-HSL to create a typical

positive autoregulation loop in which RhlR-C4-HSL activated the production of more

C4-HSL. Pearson et al [138] constituted the rhl system in E. coli to develop a C4-HSL

bioassay that proved RhlR and C4-HSL were required and sufficient to activate rhlA transcription. Latifi et al [141] reported that RhlR-C4-HSL could activate rpoS, which encodes a stationary phase sigma factor involved in the regulation of numerous stress- response genes. However, a reversed result was reported by Whiteley et al [142].

2.5.1.3 Intracellular Communication Between Quorum-sensing System

With the realization of quorum-sensing systems in P. aeruginosa, Pearson et al

(138) found that LasR-3-(oxodocanoyl)-L-homoserine-lactone preferentially activated

lasB over rhlA and that RhlR-C4-HSL preferentially activated rhlA over lasB. These

results indicated that the R proteins and autoinducers of the las and rhl system were not

interchangeable and that there was relatively high specificity with regard to the quorum-

sensing-controlled promoters that each system could activate. Moreover, it was apparent

that these two systems were not completely independent of each other. The first

indication that these systems actually were communicating came when Pearson et al.

[135] presented data that suggested LasR may control the production of C4-HSL.

The link between the las and rhl system was finally elucidated when Latifi et al.

[141] showed that LasR-3-oxo-C12-HSL activated the transcription of rhlR. This

implied that there existed a hierarchy of Pseudomonas aeruginosa quorum sensing in

which the las system lies upstream of the rhl system in a signaling cascade [141]. The

transcriptional control of rhlR expression by the las system was confirmed by Pesci et al.

[143], who also found that the las system controlled RhlR at yet another level. They

showed that 3-oxo-C12-HSL could block C4-HSL from binding to cells expressing rhlR.

This was undesirable because it has been shown that C4-HSL will not block 3-oxo-C12-

HSL from binding to cells expressing LasR, and that 3-oxo-C12-HSL will not activate

RhlR [128, 138]. This blocking was shown to have a biological effect as 3-oxo-C12-HSL significantly inhibited the ability of RhlR and C4-HSL to activate rhlA [143].

These data suggested that the las system controlled RhlR at post-translational level. This meant that the post-translational control of RhlR by 3-oxo-C12-HSL could occur before rhlI was induced, when the concentration of 3-oxo-C12-HSL would be higher than the concentration of C4-HSL. The excess 3-oxo-C12-HSL could block the association of RhlR and C4-HSL until enough RhlR and/or C4-HSL were present to overcome the blocking effect. This would provide P. aeruginosa with two mechanisms to ensure that the rhl system is activated after the las system, implying that only the las system directly senses a quorum. It has been speculated that perhaps multiple levels of

RhlR control are required because this protein affects a critical gene(s) [143]. The demonstration that rpoS is positively regulated by RHlR-C4-HSL adds greater importance to RhlR regulation and could explain the multiple levels that exist [141].

2.5.2) Regulation of LasR

The addition of rhlR to the growing list of LasR-controlled genes means that lasR

is at the top of the P. aeruginosa quorum-sensing hierarchy. When this is considered, the

regulation of lasR is obviously of great importance to P. aeruginosa quorum sensing.

Pesci et al. [143] found that in P. aeruginosa, lasR was expressed at a basal level of

transcription until becoming activated in the last half of log phase growth. This result

was in contrast to that of Latifi et al. [141] who reported that lasR is expressed

constitutively in P. aeruginosa. Both studies appear to be carefully documented so the

reason for the conflict of data is not apparent. Pesci et al. [150] demonstrated that in P. aeruginosa, lasR is partially expressed in the absence of 3-oxo-C12-HSL, and mildly up- regulated by 3-oxo-C12-HSL. In similar studies, Latifi et al. [141] reported that LasR negatively autoregulates lasR transcription in E. coli. Some of the confusion about the lasR regulation may have been answered with a report by Albus et al. [144] on the control of lasR. They showed that lasR transcription could begin in two different locations that are 30 bp apart. The control of lasR expression by two different promoters that potentially could activate transcription under different conditions might be the reason for the puzzling data obtained during the study of lasR regulation. Albus et al. [144] also showed that lasR transcription is positively regulated through a cyclic AMP receptor protein concensus sequence centered 77 and 47 bp upstream from the two respective lasR transcriptional start sites. Vfr, which is the P. aeruginosa homolog of cyclic AMP receptor protein, was required for lasR expression and was able to bind to the lasR promoter region, indicating that it could directly affect LasR regulation [144]. Finally, the discovery of a potential lux box sequence in the lasR promoter region adds another possible mode of regulation for this gene [144]. When all data are considered, the regulation of lasR is obviously multi-factorial and will require much future research before being completely understood.

2.5.3 Quorum Sensing and Pseudomonas aeruginosa Virulence

P. aeruginosa is an human pathogen that primarily infects immuno-compromised

individuals, but it is also a serious problem for cystic fibrosis patients who are generally

not considered to be immuno-compromised [145]. These properties combined with the

ubiquitous nature of this pathogen cause it to be a major source of nosocomial infections

[145]. The ability of Pseudomonas aeruginosa to also infect plants and insects [146,

147] underscores the capacity of this organism to adapt to changes in its environment and to produce a variety of virulence factors. As discussed previously, a number of

Pseudomonas aeruginosa virulence factors, including elastase, LasA protease, alkaline protease, exotoxin A, and rhamnolipid, as well as critical genes such as xcpP, xcpR, and rpoS are controlled by the las and/or rhl quorum-sensing systems. The reason that P. aeruginosa controls many of its virulence factors through quorum sensing is an often discussed topic, with one of the more common speculative answers being that perhaps quorum sensing allows the coordinated timing of virulence factor expression so their production occurs when sufficient cell numbers are present to overwhelm host defenses.

With so many importance genes under the quorum-sensing umbrella, it was not surprising to learn that a P. aeruginosa lasR mutant was significantly less virulent than the wild-type strain in a neonatal mouse model of infection [148]. This indicated that

LasR was required for full virulence of P. aeruginosa. Another interesting observation that links quorum sensing to virulence in a different way was that 3-oxo-C12-HSL stimulated respiratory epithelial cells to produce interleukin-8, a potent activator of the inflammatory response [149]. This suggests 3-oxo-C12-HSL acts directly as a virulence factor in addition to its role as an indirect virulence factor as part of the LasR-3-oxo-C12-

HSL activation team.

46 CHAPTER III

CHARACTERIZATION OF FERMENTATION PROCESS FOR RHAMNOLIPID

PRODUCTION UNDER DIFFERENT RESPIRATION CONDITIONS

Rhamnolipids, a group of glycolipids produced from gram-negative bacteria such

as Pseudomonas aeruginosa, are the most effective surfactant known today [151].

Rhamnolipids are known for their potential applications in enhanced oil recovery [152] and in mobilizing non-aqueous phase liquid contaminants in soils and aquifers [153, 154] due to their capability to effectively emulsify and solubilize hydrocarbons. Rhamnolipids have also been proposed as pesticides because of their antiviral, antibacterial, and antifungal activities [155, 156, and 157]. In addition, rhamnolipids can serve as the source of rhamnose [158], which is currently produced by extraction of quercitrin and rutin from oak bark or other plants. The extraction process employs toxic, corrosive chemicals and generates large amounts of hazardous aromatic wastes. The rhamnolipids, which can be easily hydrolyzed to form rhamnose, represent an enviroment-friendly alternative to the current production process.

A considerable amount of effort has been devoted toward process development for large-

scale rhamnolipid production with aerobic microbial fermentation. The goal, however,

remains illusive due to severe foaming problem during the course of fermentation, which

led to early termination of process and poorer productivity [159, 160]. A silicon antifoam

has been reported to control the foaming problem successfully

47 [160]. Nonetheless, the addition of antifoam increased the production cost and

complicated the downstream recovery and purification, especially in the filtration unit

[161].

To effectively alleviate foaming problem without complicating the downstream

processes and increasing the cost of the final product, a novel approach has based on

anaerobic respiration or denitrification, has been introduced by Ju et al. [162] for the

rhamnolipid production. In their studies, nitrate was used as the electron acceptor in place of oxygen and phosphorus was selected as the limiting nutrient because nitrate

could be assimilated as N source [60, 163], rendering the commonly used N-limitation

impossible under denitrifying condition. A higher yield of rhamnolipids could be

achieved [164].

It has been mentioned that nitrate and nitrite could have adverse effects on cell

metabolism when they reached certain critical levels [165]. Therefore, maintaining

suitable nitrate and nitrite concentrations by compensating the nitrate for respiration

needs becomes a challenging work for successful process development. In addition,

nutrient limitation frequently leads to an early deterioration of cell activity. The minimal

concentration of limiting nutrient required to maintain the cell activity is thus also an

important factor to the production process. In the previous study of Ju et al. [162], they

focused on the fundamental studies such as the effects of different limiting nutrients and

aerobic versus anaerobic conditions on rhamnolipid productivity.

In this chapter, studies were made to compare the cell growth under surface aeration

without nitrate respiration, anaerobic denitrification and micro-aerobic

48 denitrification and to develop a suitable way of delivering nitrate to make up the nitrate consumed by cell respiration. At the end of this chapter, a continuous culture was used to study the effect of phosphorus concentration in maintaining the cell activity without attenuating the rhamnolipid productivity.

3.1 Materials and Methods

3.1.1 Microorganism

The microorganism used throughout this study was Pseudomonas aeruginosa

from American Type Culture Collection (Manassas, VA). The culture was grown in 3%

TSB (tryptic soy broth) medium prior to being transferred to other media for different

study purposes. The stock culture was lyophilized in 10 % skim milk and maintained at

40 C for cell banking

3.1.2 Media

The composition of the pre-culture medium as summarized in the following:

NH4H2PO4, 3 g/L; K2HPO4, 2 g/L; MgSO4•7H2O, 1 g/L; FeSO4, 0.00045 g/L; and

glucose, 20 g/L. The typical basal medium for rhamnolipid production had the following

composition: NH4Cl, 4 g/L; KH2PO4, 2 g/L; NaCl, 0.5 g/L; MgSO4•7H2O, 0.3 g/L;

CaCl2, 0.01 g/L; MnCl2•4H2O, 0.01 g/L; FeSO4, 0.1 g/L; KCl, 10 g/L; NaNO3, 3.1 g/L;

and glucose, 20-65 g/L.

49 3.1.3 Methods

3.1.3.1 Dry Cell Weight Analysis

After the cells were washed by distilled water to remove the coated materials on

the cell surface, the cells collected by centrifugation were resuspended in deionized

water. They were then dried to constant weight at 110°C and weighted to determine the

cell dry weight (CDW) concentration.

3.1.3.2 Ammonium and Nitrate Analyses

The ammonium and nitrate analyses were made using an ammonia electrode (M-

44325, Markson Science Inc.). The sample was diluted to the proper range, i.e. 1 – 1000

+ - ppm for NH4 -N and 1-20 ppm for NO3 - N, and poured into a U-tube with a small

magnetic stirring bar for mixing. The ammonia electrode was inserted through one end

of the U-tube and the reagents were added from the other end. 0.3 mL of an ionic-

strength adjusting solution (ISA) and 1.5 mL of 10 N NaOH were added. The steady mV

reading was recorded and converted to the ammonium concentration using a pre-

established calibration curve. For nitrate analysis, 0.3 mL of titanous chloride solution

(20%, Fisher Scientific Co.) was added to reduce nitrate (and nitrite) to ammonia. The

next steady reading (mV) was taken. It corresponded to the combined ammonium, nitrate, and nitrite concentrations present in the medium.

50 3.1.3.3 Rhamnolipid Analysis

To quantify the rhamnolipids produced, the supernatant of sample was adjusted to

pH 2.0 with 1 N HCl and extracted with ethyl acetate at room temperature. The organic

phase was then dried at 40°C and the rhamnolipids hydrolyzed into rhamnose and lipids

(i.e. hydroxylalkanoic acids) in 5 mL of 2 N HCl for 6 h. Ethyl acetate was used to

remove the lipids from the aqueous phase. The remaining rhamnose-containing aqueous

phase was analyzed for the rhamnose concentration, using the standard anthrone method:

3.3 mL of the anthrone solution (2 g/L of anthrone in concentrate sulfuric acid) was

prepared and mixed with 1.7 mL of the aqueous solution at 5°C. The mixture was heated at 95°C for 16 minutes and measured for the absorbance at 625 nm.

3.1.3.4 Glucose Analysis

The glucose concentrations were determined by using the enzymatic glucose assay kit from Sigma Diagnostics (Procedure No. 510). The analysis involved the simultaneous use of two enzymes, glucose oxidase and peroxidase. A chromogenic oxygen acceptor, o-Dianisidine, was included to form an intense brown color when oxidized by peroxidase. The glucose concentration was proportional to the intensity of the color formed. The intensity was measured with a UV-Vis spectrophotometer at 425-

475 nm.

3.1.3.5 Phosphorus Analysis

The phosphate concentration was determined by using the Stannous Chloride

51 Method (4500-P D) described in Standard Methods for the Examination of Water and

Wastewater (17th Edition). The cell-and particle-free samples reacted with ammonium

molybdate under acid conditions to form molybdatephosphoric acid. The

molybdatephosphoric acid was then reduced by stannous chloride to intensely colored molybdenum blue. The color intensity was measured with UV-Vis spectrophotometer at

690 nm.

3.1.4 Experimental Setup

The experiment was conducted in a 2-L flask containing 1.5 L of medium. The

flask was equipped with pH probe and controller for automatic addition of either NaOH

or nitric acid-sodium nitrate solution. Only surface aeration was employed, by passing

humidified air through the headspace of the flask using an air pump. For continuous

culture, a fresh medium was fed to the flask at different dilution rates. The effluent was

collected in a large vessel. The experimental setup was shown in Figure 3.1.

3.2 Results and Discussion

3.2.1 Cell Cultivation Under Aerobic, Anaerobic, and Microaerobic Conditions

Conventionally, rhamnolipid production has been carried out under aerobic

conditions. The fermentation had to use low cell concentrations because of the severe

foaming problem caused by submerged aeration of the rhamnolipids-containing broth

[60, 166]. With the alternative approach of anaerobic respiration to avoid the severe

foaming problem, higher cell concentration might be employed to achieve higher

52 productivity.

To compare the cell concentrations attainable, experiments were simultaneously

conducted under three different conditions. In the first system, the nitrate-free medium was surface-aerated and magnetically stirred with higher speed. No anaerobic respiration

was expected. In the second system, nitrogen gas was passed through the headspace to

ensure the anaerobic condition and sodium nitrate was added periodically to maintain cells’ anaerobic respiration. In the third system, surface aeration was combined with the nitrate addition for mixed microaerobic respiration and denitrification. The pH was all

controlled at 6.5 ± 0.1 with either 1 N NaOH or 1 N HCl. Samples were taken daily for

the measurement of cell concentration and nitrate concentration. The experimental

results are shown in Figures 3.2 and 3.3.

The results shown in Figure 3.2 indicated that the cultures under anaerobic and

microaerobic denitrification conditions reached higher cell concentrations than the

pH Cll

Pump

Fresh Feed

Pump

Bioreactor

Acid Tank Harvest Tank

Figure 3.1 Experimental Setup

54 10

Microaerobic Denitrification Cell Concentration (g/L) Non-Denitrification Anaerobic Denitrification

0.1 0 100 200 300 400

Batch Culture Time (h)

Figure 3.2 Comparison of Cell Concentration Profiles Under Non-denitrifying as well as

Anaerobic and Micro-aerobic Denitrifying Conditions

55 4000

3000 -N mg/L) 3

2000

1000

Nitrate Concentration (NO Anaerobic Denitrification Microaerobic Denitrification 0 0 100 200 300 400

Figure 3.3 Nitrate Profiles under Anaerobic and Micro-aerobic Conditions

56 culture in nitrate-free medium. Apparently, the oxygen arrested the cell growth in the

latter system. Note also that the maximum cell concentrations reached were about the

same level in the anaerobic and microaerobic denitrification systems, but the cells grew

faster in the system of combined microaerobic respiration and denitrification.

The results given in Figure 3.3 revealed that the nitrate concentration gradually

accumulated in the cultures, which might cause negative effects on cell growth. In a

systematic study on the effects of nitrate concentration on the cell growth, the optimal

cell yield of batch culture of Pseudomonas aeruginosa grown under anaerobic conditions

- was reported to be obtained with 100 mM nitrate (i.e., 1,400 mg/L of NO3 -N) in the

initial medium [167]. Controlling the instantaneous nitrate concentration in the broth is

likely a critical factor to achieving high cell concentration for rhamnolipid production.

3.2.2 Rhamnolipid Production with Phosphate Limitation under Aerobic and Anaerobic

Conditions

Microbial production of rhamnolipids was widely evaluated by investigators

under aerobic conditions. As mentioned earlier, the most serious limitation in such

system was the serious foaming problem, which led to earlier termination of production

process and, thus, poor productivity. The rhamnolipid production under anaerobic conditions presents an attractive alternative because it could eliminate the foaming

problem and avoid complicating the downstream process due to the addition of antifoam

agent. However, anaerobic denitrification is less energy favorable compared to aerobic

57 respiration. The denitrifying fermentation might behave differently from the

conventional process in cell growth and rhamnolipid synthesis. Batch experiments were

therefore carried out to explore the difference between the aerobic and anaerobic rhamnolipid production.

Experiments were conducted in 2L flasks with either surface aeration for aerobic

rhamnolipid production or addition of nitrate for the anaerobic denitrifying process.

Cells were harvested in late exponential growth phase and then split into two 2L flasks

containing phosphorus-free medium under aerobic and anaerobic conditions,

respectively. The sample was taken daily to trace the cell mass and rhamnolipid

concentrations. The experimental results are summarized in Figure 3.4. The cell

concentration profile indicated that the cell activity did not last very long and decreased

dramatically after 24 h of cultivation under the surface-aerated condition. The finding

could result from the combined effects of oxygen limitation for cell respiration and

phosphorus starvation. On the other hand, the cell concentration maintained for 4 to 5

days under anaerobic denitrification before starting to decrease. The results indicated

that the cells needed phosphorus to prolong the culture longevity beyond 4 to 5 days of

stationary-phase cultivation. The supplementation of phosphorus to the culture

for maintenance of cells was found to be an important factor for long-term rhamnolipid

production.

The rhamnolipid productivity was found much lower under the anaerobic

condition compared with that under the aerobic condition. The average specific

rhamnolipid production rate was 12 mg/g-cell•hr under the anaerobic condition and 36

58 10

Time(hr) vs 1:2

Cell Concentration (g/L) Time(hr) vs 1:3 2 Time(hr) vs 1:4

0 0 50 100 150 200 250

Culture Age (hour)

Figure 3.4 Cell Concentration in Growth Phase

59 mg/g-cell•hr under the aerobic condition. The specific rhamnolipid production rate

under the anaerobic condition is therefore about 1/3 of that under the aerobic condition.

This finding is consistent with the previous report by Ju et al. [60]. The conversion

yield of rhamnolipid from glucose was about 15 % under the anaerobic condition and

30% under the aerobic condition, respectively. The efficacy of synthesizing

rhamnolipids from glucose under the aerobic condition was 100% higher compared with

that under the anaerobic condition. The observation could at least be partially attributed

to the lower energy efficiency associated with anaerobic denitrification when cells used

nitrate as the electron acceptor for respiratory purpose. Nevertheless, the foaming

problem was effectively alleviated under anaerobic respiration without addition of any

antifoam, which could be costly and could complicate the downstream purification and

recovery processes.

3.2.3 Continuous Culture Study on Effects of Phosphorus Concentration

Rhamnolipids are mainly produced when cells are in the stationary phase. Cell

entered the stationary phase typically because of the depletion of the limiting nutrient.

The stationary-phase cell activity would thus affect the productivity and cost of rhamnolipids. How to prolong the longevity of stationary-phase culture by maintaining a

critical concentration of nutrient could be very important factor to achieving effective

rhamnolipid production. A continuous culture was conducted to study the effects of the

phosphorus concentration present in the system.

60 The experiment was conducted in a 2L flask with a fixed working volume of

1.45L. Cells were first allowed to grow to stationary phase in the phosphorus-limiting medium. A feed medium was then continuously added into the reactor to start the

continuous culture. The composition of the feed medium was the same as that of the

growth medium, except containing 20 g/L of glucose and 70 mg/L of phosphorus. The

dilution rates studied were 0.025, 0.083, 0.133, and 0.2 h-1, respectively. Samples were

taken from the flask daily for following the concentrations of cell mass, phosphorus,

glucose, and rhamnolipids. The biomass profile of cell concentration was used to assure the reach of steady state. The experimental results are summarized in Figures 3.5, 3.6

and 3.7.

The steady-state cell concentration was found to decrease with increasing dilution

rate as shown in Figure 3.5. The decrease of steady-state cell concentration was faster

than that in a typical continuous culture. The Monod constant, K, for phosphorus was

found to be around 15 mg/L, when the experimental results were fitted with Monod

Equation, i. e.,

μ • S μ = max (3.1) K + S

where μ is the specific cell growth rate, μmax is the maximal specific cell growth rate, S is

the substrate concentration, and K is the Monod constant. The steady-state cell

61 2 100 50

80 40

60 30

1 50

40 20 Cell Concentration (g/L)

Rhamnolipid Concentration (g/L) 30 Phosporus Concentration (mg/L) 20 10

0 0 0 0.00 0.05 0.10 0.15 0.20

-1 Dilution Rate (h )

Figure 3.5 Profiles of Steady State Biomass, Phosphate and Rhamnolipid

Concentrations

62 concentration (X) of a continuous culture in steady state can be described as Equation

(3.2)

• DK sx (SYX 0/ −= ) (3.2) μm − D where Yx/s is the coefficient of cell yield from limiting nutrient, S0 is the phosphorous concentration in the fresh feed medium, and D is the dilution rate. Yx/s can be calculated and plotted against D as shown in Figure 3.6. Based on Equation 3.2, the value of Yx/s was found to decrease with increasing dilution rate. As phosphorus concentration was higher at higher dilution rate, the finding indicated that the cells contained lower phosphorus contents when grown in low phosphorus environment. Malmacrona-Friberg et al. [173] and Wanner et al. [174] have reported the same phenomenon of varying specific P contents with the concentration of P (as limiting nutrient) in K. pneumoniae, E. coli, and Arthrobacter sp..

On the other hand, the specific rhamnolipid productivity was quite stable (13 mg/g-cell•h) at the dilution (growth) rates ≤ 0.083 h-1 but decreased to zero when the dilution (growth) rate increased to 0.2 h-1 as shown in Figure 3.6. The information revealed that the threshold growth (dilution) rate for rhamnolipid production was somewhere between 0.133 and 0.2 h-1.

The cell yield from glucose was found relatively constant, about 35%, with various dilution (growth) rates, ranging from 0.025- 0.2 h-1 as shown in Figure 3.7.

However, the production yield of rhamnolipids from glucose decreased with the increase

16 55 )

14 3 50

45 10

8 40

6 35

4 Cell Yield from Phosphorus (x10 Phosphorus from Yield Cell

30 2 Specific Rhamnolipid Productivity (mg/g-cell/h) (mg/g-cell/h) Productivity Rhamnolipid Specific

0 25 0.00 0.05 0.10 0.15 0.20 0.25

-1 Dilution Rate (h )

Figure 3.6 Profiles of Specific Rhamnolipid Productivity and Cell Yield from

Phosphorus

0.50 0.16

0.14 0.40 0.12

0.10 0.30

0.08

0.20 0.06

Cell Yield from Glucose (g/g) 0.04 0.10 Rhamnolipid Yield from Glucose (g/g) 0.02

0.00 0.00 0.00 0.05 0.10 0.15 0.20 Dilution Rate (h-1)

Figure 3.7 Profiles of Cell and Rhamnolipid Yields from Glucose

65 of dilution (growth) rate. Additional glucose was consumed for cell growth and for production of other metabolites, leading to a poorer yield at higher dilution (growth) rate.

3.3 Conclusions

Based on the experimental results, the following conclusions are drawn:

(1) A much higher cell concentration was achieved (10-13 g/L) under the condition of

combined microaerobic respiration and denitrification when compared to the

traditional non-denitrifying culture, where the cells grew to only about 1-2 g/L,

(2) The minimum pecific cell growth rate required to maintain the maximum specific

rhamnolipid productivity was around 0.08 h-1 and calculated maximal phosphorus

consumption rate for maximum specific rhamnolipid productivity was around 2

mg/g-cell•h.

66 CHAPTER IV

ROLE OF RHL QUORUM-SENSING SYSTEM IN CELL GROWTH,

RHAMNOLIPID PRODUCTION AND DENITRIFICATION

The opportunistic human pathogen Pseudomonas aeruginosa produced a variety of virulence factors, including exotoxin A, alkaline protease, alginate, phospholipases, and extracellular rhamnolipids through the regulations of las and rhl quorum sensing systems [136]. Among these virulence factors, rhamnolipids have attracted more and more attentions because of their potential applications in the areas of medicine, agriculture and food additives [175, 8, and 9]. Rhamnolipids are synthesized by biocatalysts, rhamnosyltransferases, whose productions are regulated through the rhl quorum sensing system [134]. rhl quorum sensing system is composed of rhlR and rhlI genes. The rhlR gene encodes a transcriptional protein [134], RhlR, which binds with autoinducer (C4-HSL), which is synthesized by the RhlI protein [137]. The complex formed by the binding of RhlR and C4-HSL regulates the expression of rhlAB gene, which encodes the rhamnosyltransferases for the rhamnolipid production [176].

Traditionally, rhamnolipid fermentation was conducted under aerobic conditions with various cultures, including those employing continuous culture [171, 172, 152], immobilized cells [177, 178], and resting cells [177]. All of these studies suffered from heavy foaming even at rhamnolipid concentration as low as 0.1 g/L [60, 166]. The serious foaming problem caused an earlier cutoff of the production process, thus leading

67 to a significantly lower productivity. A number of efforts have been made to overcome the foaming problem by either adding an effective antifoam reagent or using a mechanical foam breaker [166, 179]. However, these methods have been proved to be insufficient in handling the foam problem. The addition of chemical antifoam reagent was reported to complicate the downstream recovery and purification processes, especially in the filtration unit [161].

As it was mentioned in previous chapter, the foaming problem can be improved by using denitrification as an alternative respiratory route. In that approach,

Pseudomonas aeruginosa ATCC 10145 was introduced as the rhamnolipid producer

[162]. In Chawala et al. study [162], the rhamnolipid productivity under anaerobic denitrifying condition was reported to be about 1/3 of that under the aerobic condition.

Foaming problem, however, was eliminated by this novel alternative, but the setback in specific rhamnolipid productivity and the culture longevity needed to be further improved.

rhl quorum sensing system, however, has been known to regulate rhamnolipid production for years, but employing rhl quorum sensing system in fermentation process for rhamnolipid production without exogenous addition of autoinducer has never been achieved due to the limited cell concentration. This chapter discusses the role of rich medium in supporting cell growth and also examines the rhamnolipid production under aerobic and micro-aerobic conditions. The effects of autoinducer on cell growth and effects of quorum sensing system on rhamnolipid production were investigated by using wild type Pseudomonas aeruginosa PAO1, rhlI and rhlR mutants. Rhamnolipid

68 production under the regulations of rhl quorum-sensing system was also executed by using Pseudomonas aeruginosa ATCC 9027. Subsequently, a high biomass culture coupled with the use of rhl quorum sensing system to regulate the rhamnolipid production was employed for long-term cultivation and rhamnolipid production.

4.1 Materials

4.1.1 Organisms

The microorganism used to study rhamnolipid production was Pseudomonas

aeruginosa ATCC 9027 from American Type Culture Collection (Manassas, VA). The

culture was grown at 3% TSB media prior to being transferred to different media for

specific experiments. The bacterial cells were transferred to 10 % skim milk and

moisture was removed at low temperature and pressure by using a lypholyzer. The

culture was then maintained at 4o C. Pseudomonas aeruginosa PAO1, rhlI and

rhlR mutants were used to study the effects of rhl quorum-sensing system on cell growth

and rhamnolipid production. The cultures were maintained on the agar slants of LB

medium and sub-cultured regularly.

4.1.2 Media

The compositions of the media for pre-culture can be summarized as: NH4H2PO4

(3 g/L); K2HPO4 (2 g/L); MgSO4•7H2O (1 g/L); FeSO4 (0.00045 g/L); and glucose (20

g/L).

The compositions of typical basal medium used for rhamnolipid production are:

NH4Cl (2 g/L); KH2PO4 (2 g/L); NaCl (0.5 g/L); MgSO4•7H2O (0.3 g/L); CaCl2 (0.01 g/L); MnCl2•4H2O (0.01 g/L); FeSO4 (0.1 g/L); KCl (10 g/L); NaNO3 (3.1 g/L); and

glucose concentration was varied from 20 g/L to 70 g/L depending on the studying

purpose.

4.2 Methods

4.2.1 Dry Cell Weight Analyses

After the cells were washed by distilled water to remove the coated materials on the cell surface, the collected cells were suspended again in de-ionized water. They were then dried on the aluminum-weighing dish (Fisher Scientific) at 110°C for 3 hours before measuring the cell dry weight (CDW).

4.2.2 Ammonium and Nitrate Analyses

The ammonium and nitrate analyses were made using an ammonia electrode. The

ammonium and nitrate concentrations can be measured accurately in a wide range of 1 –

+ - 1000 ppm for NH4 -N and a narrow range less than 20 ppm for NO3 - N. The sample

was diluted to the proper range and poured into a U-tube with a small magnetic stirring

bar for mixing. The ammonia electrode (M-44325, Markson Science Inc.) was inserted through one end of the U-tube and the reagents were added from the other end. 0.3 mL of an ionic-strength adjusting solution (ISA) and 1.5 mL of 10 N NaOH were added. The steady reading (mV) was recorded and converted to the ammonium concentration using

a pre-established calibration curve. For nitrate analysis, 0.3 mL of titanous chloride

70 solution (20%, Fisher Scientific Co.) was added to reduce nitrate (and nitrite) to ammonia. The next steady reading (mV) was taken. It corresponded to the combined ammonium, nitrate, and nitrite concentrations present in the medium.

4.2.3 Rhamnolipid Analysis

To quantify the rhamnolipids, the supernatant of sample was adjusted to pH 2.0 with 1 N

HCl and extracted with ethyl acetate of double volume at room temperature. The organic

phase was then dried at 40°C. The residue was then hydrolyzed in 5 mL of 2 N HCl for 6

hours. The acid hydrolysis reaction breaks the rhamnolipids into rhamnose and lipids

(i.e. hydroxylalkanoic acids). Ethyl acetate was used to remove the lipids from the

aqueous phase. This rhamnose containing aqueous phase is quantified for the

concentrations of rhamnolipids as rhamnose concentration, following the standard

anthrone method. 3.3 mL of the anthrone solution (2 g/L of anthrone in concentrate

sulfuric acid) was prepared and mixed with 1.7 mL sample solution at 5°C. The mixture

was heated at 95°C for 16 minutes, before measuring the absorbance at 625 nm.

4.2.4 Glucose Analysis

The glucose concentrations were determined by using the enzymatic glucose assay kit obtained from Sigma Diagnostics (Procedure No. 510). The glucose oxidase method is based on the simultaneous use of the enzymes, glucose oxidase and peroxidase, in two concurrent reactions. A chromogenic oxygen acceptor, o-Dianisidine, is able to

71 form a more intense brown color when oxidized by peroxidase. The original glucose concentration is proportional to the intensity of the color formed. This intensity can be measured with UV-Vis spectrophotometer at wavelength between 425-475 nm.

4.2.5 Phosphorous Analysis

Phosphorous concentrations were analyzed with the method of Fiske and

SubbaRow, introduced first in 1925. The assay kit was purchased from Sigma

Diagnostics (Procedure No. 670). Cell and particle free sample was treated with 20% of

trichloroacetic acid to remove protein and lipid phosphorous. Ammonium molybdate

was used to react with the sample in an acid condition to form phosphomolybdate. A

mixture of sodium bisulfite, sodium sulfite and 1-amino-2-naphthol-4-sulfonic acid

reduces the phosphomolybdate to form a phosphomolybdenum blue complex. The

intensity of the color is proportional to the phosphate concentration and is measured with

UV-Vis spectrophotometer at wavelength 690 nm.

4.3 Results and Discussion

4.3.1 Effect of Rich Medium on Cell growth

Rich medium has been widely used to enhance the cell growth without further

dilution of the cell mass with the addition of the essential nutrients. To avoid the

undesirable side effects caused from the rich medium, experiment was conducted in 15-

ml test tubes containing different concentrations of rich medium for 24 hours without any

pH control and nutrient addition. In this study, the concentrated basal medium ranging

1.1

1.0

0.9

0.8 Biomass (g/L) 0.7

0.6

0.5 1x 2x 3x 4x 5x 6x 8x 10x Concentrated nineral Nutrient

Figure 4.1 Nutrient Effect on Cell growth

73 from 1X to 10X was used to examine the effects of the mineral nutrient on the cell growth with the measurement of cell concentrations. The experimental results are given in Figure 4.1. The biomass reading from different concentration of rich medium showed a bell shape variation. The results suggested that the medium with 3X to 5X concentration would be the most suitable medium for cell growth. The individual nutrient effects on cell growth could be very complex, however, this experiment was conducted to find a simple and easy way to compose the suitable medium for cell growth.

After comparing the biomass of 1X basal medium with that of the rest of the other rich medium after 24 hours of cultivation, no apparent adverse effects on cell growth were found in this experiment. To achieve high biomass without any dilution effects from the nutrient replenishment, 5X-concentrated rich medium was used as the initial growth medium for all the following studies.

4.3.2 Effects of Anaerobic Denitrification by Spent Media

Microbial production of rhamnolipids was widely evaluated by investigators

under aerobic conditions. As discussed earlier, the most notorious limitation of aerobic

production of rhamnolipids was the serious foaming problem, which led to earlier termination of production process and thus resulted in a poorer productivity. The efficacy of the selected antifoam reagent to alleviate the foaming problem traps the researchers in a dilemma situation because the antifoam reagent could complicate the downstream purification processes and increase the cost of the products. However, the rhamnolipid production under anaerobic conditions presents an attractive alternative

because it could effectively eliminate the foaming problem causing by the aeration and also avoid complicating the downstream process due to the addition of antifoam reagent.

Although anaerobic rhamnolipid production represents an attractive alternative of aerobic rhamnolipid synthesis, the typical results found in a regular anaerobic denitrifying fermentation with Pseudomonas aeruginosa suggest that the specific denitrification rate decreases in parallel with the specific cell growth rate. Since rhamnolipids are mainly produced when cells are in stationary phase, cellular denitrification in stationary phase needs to be investigated. To explore the effects of spent media on denitrification, experiments were conducted in 50-mL test tubes. Pseudomonas aeruginosa culture was grown to stationary phase and then split into 4 50-mL test tubes containing 45 mL of culture. One of the test tubes was heated at 80 degree C for 15 minutes before experiment. Another one of the test tubes was centrifuged to replace the spent media with fresh media. The other one of the test tubes was centrigued to remove the supernatant and the supernatant was mixed with ethyl acetate at ratio of 1 to 1 for 15 minutes and followed by phase separation. The separated supernatant was mixed with the cells again before the study. The rest of the test tubes was remained the same

(without any treatment). Samples were taken daily to measure the denitrification activity.

The results shown in Figure 4.2 indicating that the denitrification activity was completed inhibited in the test tube without ant treatment. The denitrification activity was resumed in the test tube with fresh media while the denitrification activity in the test tubes with heat pre-treatment and ethyl acetate extraction were partially resorted. These results suggested that inhibitory factor(s) presented in the spent media stop denitrification.

CHAPTER V

DEGRADATION AND SYNTHESIS KINETICS OF QUORUM-SENSING AUTOINDUCER IN PSEUDOMONAS AERUGINOSA FERMENTATION

Pseudomonas aeruginosa is a human pathogen that is often responsible for

hospital-acquired infections in urinary tracts, blood streams, and surgical wounds [190].

In addition, P. aeruginosa is responsible for high mortality rate with ventilator-associated

pneumonia [120]. The pathogenicity of this bacterium has traditionally been associated

with the numerous virulence factors produced via the cell-density dependent gene

expression mechanisms, quorum sensing, that monitor the cell density, respond to

environmental changes, and allow cell-to-cell communication. Accordingly, the P. aeruginosa virulence factors are not normally produced until a high cell density is achieved although the expression of the virulence genes also depends on the environmental stimuli such as nitrogen availability or osmolarity [138].

The paradigm of the gram-negative quorum-sensing system is the lux system from

Vibrio fischeri . The general model of quorum sensing involves production of N-acyl

homoserine lactone (autoinducer, AI), a diffusible molecule synthesized by LuxI type of

AI synthase or acylhomoserine synthase. The AI is produced at a basal level at low cell

density; consequently, its concentration in the medium increases with cell growth. Upon

reaching a threshold concentration, AI binds to its specific target protein, a LuxR-type

transcriptional activator. The AI-regulator complex then typically induces the production

87 of more AI (thus the name, autoinducer) and expression of other genes that are under

its control.

P. aeruginosa contains two quorum-sensing systems, the las and rhl systems, as

shown in Figure 1 [122, 124, 130]. In the las system, the AI synthase (LasI) directs the

synthesis of a P. aeruginosa AI (PAI1), N-(3-oxododecanoyl) homoserine lactone or

ODDHL [125], which binds with the lasR-encoded transcriptional activator, LasR [122].

Similarly, the rhl system consists of the transcriptional activator protein RhlR [134] and

the AI synthase RhlI that directs the synthesis of a second AI (PAI2), N-butyryl

homoserine lactone or BHL [135]. The regulation of two quorum-sensing systems is

complex and involves Vfr, a CRP homologue, as well as a two-component regulator

GacA [144]. In addition, the two systems are not independent: the production of PAI2 is

promoted not only by the PAI2-RhlR complex but also by the PAI1-LasR complex from

the other quorum-sensing system [126]. The PAI1 production is also regulated through

both regulatory cascades [190].

The overlapping and interactive nature of the two quorum sensing systems is also seen in the genes that are regulated. The activator LasR:OddHL complex does not only induce the production of several virulence factors, such as the LasA and LasB proteases, alkaline protease, exotoxin A but also has a positive effect in regulating the expression of secretion proteins XcpP and XcpR [130, 131]. The RhlR:BHL complex also induces expression of the LasA and LasB proteases as well as the Xcp system. In addition, the

RhlR:BHL complex controls the expression of rhlAB and rhlG. The genes rhlAB encode

for rhamnosyltransferase, which catalyzes the transfer of (one or two) rhamnose from

TDP-L-rhamnose to β-hydroxyalkanoate for rhamnolipid synthesis [73]. The more

88 recently identified gene rhlG, homologous to the fabG gene in the general fatty acid

synthesis, codes for the enzyme RhlG that controls the synthesis of the hydroxyalkanoate

used specifically in rhamnolipid production [191].

Being biodegradable and biocompatible [32], rhamnolipids are environment-

friendly surfactants. They are known to have antibacterial, antifungal and antiviral activities [9, 156, 157], and find potential medical applications in wound healing, depression, and organ transplants [175]. Many studies have been conducted on rhamnolipid production in P. aeruginosa fermentation [157, 5]. Active rhamnolipid production typically occurs with resting cells or during the stationary phase of batch culture [38, 162]. Regulated by the rhl system, rhamnolipid synthesis should depend

strongly on the kinetics of production and degradation of the autoinducer PAI2. Current

literature reports on PAI2 synthesis are scarce and contradictory: while some reported

that the autoinducer is optimally produced in rich medium during the stationary phase

[186, 187], another showed a rapid degradation once the batch culture entered the

stationary phase (AI concentration dropped by 80% in 2 h) [188]. In this work, P.

aeruginosa PAO1 and its rhlR(-) and rhlI(-) mutants were used to investigate the kinetics

of PAI2 degradation and production. The study focused mainly on the kinetics during the

stationary phase, where active production of rhamnolipids took place. The effect of PAI2

on specific rhamnolipid productivity was also evaluated using the rhlI(-) mutant culture

added with various autoinducer concentrations.

89 5.1 Materials and Methods

5.1.1 Microorganisms and Media

P. aeruginosa PAO1 and its isogenic rhlI and rhlR null mutants,SS165 and

SS227, were used in this study. The cultures were maintained on the agar slants of LB

medium (Becton Dickinson, MD) and subcultured regularly. Chromobacterium

violaceum (CV026) was used to detect autoinducer production (Renee and Kendall,

2000). CV026 was cultivated as previously described (Renee and Kendall, 2000). The

growth medium for P. aeruginosa cultures had the following composition (in g/L):

glucose, 40; NH4Cl, 10; K2HPO4, 5.5; NaCl, 1.5; MgSO4, 1.5; CaCl2, 0.05; MnCl2, 0.1;

FeSO4, 0.5; and NaNO3, 3.1.

5.1.2 Methods

5.1.2.1 Molecular Biological Methods

Plasmids were purified with QIAprep spin miniprep columns made by Qiagen

(Santa Clarita, CA). DNA fragments were excised from agarose gels and purified using

the Qiaex II DNA gel extraction system (Qiagen) according to the manufacturer’s

instructions. Restriction enzymes and DNA modification enzymes were purchased from

New England Biolabs (Beverly, MA). For DNA amplifications using Perkin Elmer

GeneAmp 2400, Pfu from Strategene (La Jolla, CA) was used. Oligonucleotides were purchased from Operon, Inc. (Alameda, CA). DNA was introduced into E. coli or P. aeruginosa either by electroporation or by conjugation. A standard electroporation procedure was used for E. coli using the E. coli Gene Pulser by Bio-Rad (Hercules, CA).

P. aeruginosa electroporation was performed as previously described [183].

90 5.1.2.2 Construction of P. aeruginosa rhlI Mutant

To construct a P. aeruginosa rhlI null mutant, the suicide plasmid pSS28, which

carries the rhlI302::aacCI null allele was generated. Briefly, the complete rhlI gene and

flanking sequences was PCR amplified from P. aeruginosa PAO1 chromosome as a 890 bp XbaI-HindIII DNA fragment using the thermostable DNA polymerase Pfu

(Strategene, La Jolla, CA) to avoid errors, and the resulting fragment was cloned into

pUC19 to generate pSS13. The PCR amplified rhlI gene carried on pSS13 was verified

via DNA sequencing. To construct a null allele, pSS13 was partially digested with

EcoRI, which cuts once within the vector sequence and once within the rhlI ORF, and the

protruding ends were converted to blunt ends with T4 DNA polymerase in the presence

of dNTP. The aacCI gene which encodes gentamicin resistance (Gmr) was isolated from

pUCGM1 (Schweitzer) as a SmaI fragment, was inserted into the blunted site in rhlI to

generate the rhlI302::aacCI allele. pSS28 was introduced into P. aeruginosa PAO1 by electroporation, and the rhlI mutant, SS227, was isolated as having Gmr (encoded by the

rhl302::aacCI allele) but lacking carbenicillin resistance (Cbr) encoded by the plasmid

vector, following a double cross-over event. The presence of the rhlI302::aacCI allele in

SS227 was verified by PCR analysis.

5.1.3 Autoinducer Analysis

5.1.3.1 Preparation of Autoinducer Extract

Samples (2 ml) taken throughout the P. aeruginosa growth cycle were extracted

thrice with equal volume of ethyl acetate containing 0.01% glacial acetic acid for 5

minutes. The extract was then dried under sterile air and stored at –20 °C.

91 5.1.3.2 Bioassay

bioassay was carried out according to the procedure of Renee and Gray (Renee

and Gray, 2000). CV026 was cultivated in a 30 0C incubator with 200-rpm agitation

overnight. The culture was then diluted with sterile medium to an optical density at 660

nm (OD660) of 0.002. One milliliter of this culture suspension was added to each sterile culture tube containing either a synthetic standard or a dried sample extract to be tested

for autoinducer activity. The standards were prepared from a sample of synthetic

autoinducer (N-butanoyl-homoserine lactone) obtained from Dr. Eberhard (Ithaca

College, Ithaca, NY). The assay cultures were grown overnight at 30 °C in a shaker (200

rpm). A 200-μl sample was taken from each culture and placed in a 2-ml Eppendorf

tube. For cell lysis, the tube was added with 200 μl of 10% sodium dodecylsulfate, mixed for 5 second, and incubated at room temperature for 5 minutes. Violacein was quantitatively extracted from this cell lysate into 900 μl of butanol, with 5-s vortex mixing and centrifugation at 13,000 rpm (i.e. approximately 14,400 g) for 5 minutes in a microcentrifuge. The supernatant was collected and measured for its optical density at

585 nm (OD585). Normalized to the optical density of the original CV026 suspension

used in the assay, the violacein unit was calculated as the value of (OD585/OD660) × 500.

5.1.3.3 Cell Dry Weight

The cells were washed once with deionized water to remove the slimy materials

sometimes attached to the cell surface. The cell pellet was then resuspended in deionized

water and dried to constant weight in an aluminum weighing-dish at 110 °C for at least 3

92 h. The cell dry weight (CDW) concentration of the sample was calculated accordingly.

5.1.3.4 Cell Protein Analysis

For protein analysis, the cells were collected by centrifugation and the pellet was washed once with deionized water to remove the organics that may interfere with the analysis. The washed cell pellet was resuspended in 3 ml of 0.2 N NaOH and boiled at 100 °C for 20 minutes. The lysate was then analyzed by the standard Lowry’s method (Rosen, 1978) using the diagnostic kit from Sigma (690-A, St. Louis, MO). A spectrophotometer (UV/VIS spectrophotometer, Perkin-Elmer Lambda 3B) was used to determine the absorbance at 650 nm.

5.1.3.5 Rhamnolipid Analysis The broth sample taken from P. aeruginosa fermentation was centrifuged to collect the supernatant. The supernatant was adjusted to pH 2.0 with 1 N HCl and extracted with ethyl acetate of double volume at room temperature. The organic phase was dried at 40 °C and the residue hydrolyzed in 5 ml of 2 N HCl for 6 h. The acid

hydrolyzed rhamnolipids into rhamnose and lipids (i.e. hydroxylalkanoic acids). Ethyl

acetate (5 ml) was added to extract the lipids from the aqueous phase, which was then

analyzed for the rhamnose concentration by the standard anthrone method (162).

5.2 Experimental Setup Experiments were conducted in cell-containing and cell-free systems. The wild

type strain of P. aeruginosa PAO1 was grown to stationary phase (around 8-9 g/L) and

its rhlI mutant was also grown to high cell concentration simultaneously in 2-L conical

93 flasks. The rhlI mutant culture was then switched to continuous process with a

0.025 h-1 dilution rate to maintain the cell activity and supply active cells for kinetic

study. The operating scheme of experiment is given in Figure 5.1.

5.3 Results and Discussion

5.3.1 Cell Growth Study

Figure 5.2 summarizes the results of cell growth and rhamnolipid production

obtained in batch fermentations of wild-type PAO1 and its rhlI and rhlR null mutants.

The growth profiles of the two mutants were similar to that of the wild-type. On the other hand, no appreciable rhamnolipid synthesis was observed with the two mutants, consistent with the known role of RhlR:PAI2 complex in controlling the synthesis of enzymes responsible for rhamnolipid production [134, 191]. Lacking of either RhlR or

PAI2 in the mutants was shown to prevent active rhamnolipid synthesis.

5.3.2 Autoinducer Degradation in Stationary Phase

Synthesis and degradation of autoinducers may occur simultaneously throughout

the culture span by Amy et al. [127). Our initial focus was on the kinetics in stationary

phase where most rhamnolipids were produced and the cell concentration remained

relatively constant. It has been reported that the homoserine lactone autoinducers are

optimally produced in rich media during the stationary phase while only minute amount

Cell-Free System

Centrifuge and Collect Supernatant

Harvested Culture

Mixed with PAO1 Batch Culture Supernatant

Harvested Culture Feed Medium Centrifuge and Collect Cells

Effluent rhlI Mutant of PAO1 Cell-Containing Continuous Culture System

Figure 5.1 Experimental Setup

95 10

0.8

0.6

0.4 4 Cell Concentration(g/L) Cell Conc.( rhlR)

Cell concen. (rhlI) 0.2 Rhamnose Concentration (g/L) 2 Wild-Type PAO1 Rhamnose(rhlR) rhamnose (rhlI) rhamnose (PAO1)

0 0.0 024681012

Time (day)

Figure 5.2 Cell Concentration and Rhamnose Concentration Profiles in Batch Fermentation

96 are produced in standard TSB (tryptic soy broth) medium [186, 187]. On the other hand,

a contradictory observation of rapid decrease of autoinducer concentration in stationary

phase has also been reported [188]. In the latter, P. aureofaciens PGS12 was cultivated

in nutrient broth medium at 28 °C. The hexanoyl-homoserine lactone (HHL) autoinducer

was produced during the growth phase but about 80% of the accumulated HHL

disappeared in 2 h after the culture reached the stationary phase. To better understand the

phenomenon of autoinducer production and degradation in the current work, experiments

were designed to examine the autoinducer degradation and synthesis separately.

The following experiments were first conducted to study the autoinducer

degradation. Both the wild-type P. aeruginosa PAO1 and its rhlI null mutant were cultivated as described. The parent strain PAO1 was grown in a 2-L batch culture

to provide the autoinducer-bearing broth; the latter in a continuous culture (dilution rate =

0.025 h-1) to provide active mutant cells at a constant metabolic state. The wild-type culture reached the stationary phase at Day 6 with a cell concentration of approximately 9 g/L. Two broth samples (50 mL) were taken at Days 6 and 7, respectively, and centrifuged to collect the cell-free medium. Two aliquots (50ml each) cell-free spent medium were used in the subsequent experiment for following the change of autoinducer concentration. One aliquot was mixed with the rhlI(-) cells harvested from the

continuous culture, to make a cell concentration of 8.81 g/L; the other was kept as a cell-

free control. The use of rhlI(-) cells was to ensure no autoinducer synthesis occurred

during the experiment so that the degradation could be examined apart from biosynthesis.

Samples were withdrawn from the systems at 0, 30, 60, 90, 120, 210, and 300 min and

measured for the autoinducer concentrations. The experimental results are shown in

97 Figures 5.3 and 5.4.

The results indicate that autoinducer degradation was cell-associated. For both

Days 6 and 7 samples, there were no apparent trends of change in the autoinducer concentrations in the cell-free systems, except the random fluctuations presumably

caused by the sensitive bioassay. On the other hand, clear degradation profiles were

observed in the systems containing rhlI(-) cells. As shown in Figure 5.4, the degradation can be well described empirically by the following kinetics, with a first-order dependency on the autoinducer concentration ([AI]):

AId ][ −= AIk ][ (1) dt d where kd is the degradation constant. Upon integration,

− d tk [= ][] o eAIAI (2)

where [AI]o is the autoinducer concentration at the beginning of the degradation

experiment. The best-fit value of kd, averaged from both sets of experiments, was 0.195

± 0.009 h-1. Being cell-associated, the degradation would also depend on the cell

concentration: faster at higher cell concentrations. Although the dependency is yet to be

determined because only one cell concentration was evaluated in this work, it is plausibly

180

Violacein Unit (PAO1) 160 Violacein Unit (rhlI)

140

120

100

Violacein Unit Violacein 60

0 0 50 100 150 200 250 300 350 Time (Minute)

Figure 5.3 Autoinducer Profiles of Various Systems

0.24

0.22

0.20

0.18

0.16

0.14

0.12

Autoinducer Concentration (mg/L) Concentration Autoinducer 0.10

0.08 0 50 100 150 200 250 300 350

Time (Minute)

Figure 5.4 Degradation Kinetics of Autoinducer in Stationary Phase

100 first order also, i.e.,

AId ][ −=−= AIxkAIk ][][ dt d ,spd where x is cell concentration and kd,sp is the specific degradation constant, per unit cell concentration. Under the current study condition, the value of kd,sp for P. aeruginosa

PAO1 was calculated to be 0.024 ± 0.001 L/(g cells-h).

5.3.3 Autoinducer Synthesis in Stationary Phase

Without knowing the exact mechanism of autoinducer degradation (albeit cell- associated), the degradation could not be eliminated experimentally to allow for independent evaluation of the kinetics for autoinducer synthesis. The synthesis kinetics had to be derived mathematically,

AId ][ r += r synthesis dt ndegradatio where d[AI]/dt is the net change rate of autoinducer concentration in the fermentation with simultaneous autoinducer synthesis and degradation.

A batch fermentation of wild-type P. aeruginosa PAO1 was carried out with pH maintained at 6.5 ± 0.1. Daily samples were taken for measurements of the cell concentration and, during the stationary phase, the autoinducer concentration. The results are shown in Figure 5.5. The autoinducer concentration decreased in the stationary phase but much slower than that reported by Seveno et al. [188]. For easy comparison, the net

AI concentrations were also empirically fit with an exponential decay equation (i.e. first- order kinetics),

101

10 150 0.25

8 120 0.20

6 90 0.15

Biomass (g/L) 4 60 0.10 Violacein Unit Violacein

2 30 0.05

0 0 0.00 0 2 4 6 8 10 12 14 Time (day)

Figure 5.5 Autoinducer Profile in Stationary Phase

102 − tk [= ][] o eAIAI (3)

The net decay constant k was 0.0066 ± 0.0004 h-1. Note that this value is much smaller

-1 than the kd value obtained earlier, i.e., 0.195 h . Autoinducer synthesis was thus found to continue in the stationary phase but the rate was slightly slower than the rate of degradation, leading to the net decrease in autoinducer concentration.

The same experiment was repeated and the results obtained are shown in Figure 5.6. The autoinducer profile in stationary phase was very similar and the best-fit net decay constant k was 0.0058 ± 0.0004 h-1. The phenomenon was thus reproducible, with an average k value of 0.0062 h-1.

5.3.5 Autoinducer concentration Profile in Batch Fermentation of Wild-Type PAO1

The autoinducer concentration was followed throughout the batch fermentation as shown in Figure 5.6. Unlike in the stationary phase, the autoinducer concentration increased during the growth phase. The increase was slower during the exponential growth phase (till day4 (89 h)) and became faster in the transition stage of slower growth, where the rpoS gene was activated [189]. The RpoS protein has also been reported to have a role of positive regulation on autoinducer synthesis by enhancing the expression of rhlI gene [189]. Seveno et al. [188] also reported that the maximum autoinducer concentration was achieved in the transition phase.

On the other hand, the autoinducer concentration changed from increase to decrease once the culture reached the stationary phase. Some unidentified mechanism was involved to slow down the autoinducer synthesis or promote its degradation, or both.

103

200 10

180

160 8

140

120 6

100

Violacein Unit 80 4 Cell Density (g/L)Cell Density

40 2

0 0 0 2 4 6 8 1012141618

Time (day)

Figure 5.6 Autoinducer Concentration Profile in Batch Fermentation of Wild-Type PAO1

104 More research work is needed to explore more details.

5.3.6 Autoinducer Concentration Profile in Batch Fermentation of rhlR(-) Mutant

The mechanism of gene regulation reported in the literature for autoinducer PAI2 synthesis through the las and rhl quorum-sensing systems is given in the article published by Dockery and Keener [190]. Two features are most notable: (1) the complex of LasR protein and PAI1 autoinducer from the las system promotes the expression of rhlR gene;

(2) the complex of RhlR protein and PAI2 autoinducer promotes (auto-induces) the rhlI gene for PAI2 synthesis. Apparently, the rhlR gene and its encoded RhlR protein play an essential role in regulating the PAI2 synthesis. To more quantitatively evaluate this regulation, batch fermentations were carried out using the rhlR(-) mutant. Without the synthesis of RhlR protein, the interaction between las and rhl systems are disrupted and the effect of auto-induction by RhlR-PAI2 complex should be eliminated. The PAI2 synthesis in rhlR(-) fermentation would thus result solely from the constitutive expression of the rhlI gene, and the different behaviors between the wild-type culture and the rhlR(-) mutant would indicate the effect of the auto-induction.

Experimental results of the batch fermentation are shown in Figure 5.7.

Autoinducer production was slower in the growth phase compared with the wild type strain. Only one third of the autoinducer produced in wild type strain was observed in the rhlR mutant. This could be attributed to the interior induction of autoinducer production regulated by the complex of RhlR and C4-HSL was inactivated. The results also indicate that most of the autoinducer (C4-HSL) produced contributed from the interior induction

105

60 10

50 8

40 6

4 Cell Density (g/L) Cell Density 20

Autoinducer Concentration (mg/L) 10

0 0 024681012

Time (day)

Figure 5.7 Autoinducer Concentration Profile in rhlR Mutant

106 loop whereas two third of the autoinducer was produced through this regulatory loop. The overall autoinducer concentration in stationary phase was also fitted with first order degradation kinetics and the overall degradation constant kor was found to be

0.0144 (1/h). Because the overall accumulation constant kor is larger compared with that of the wild type strain, faster degradation kinetics was found in the stationary phase with rhlR mutant. This was due to a lower autoinducer production rate resulting from the inactivated interior induction loop by silencing rhlR gene and accompanied with a stable degradation rate when compared with that of the wild type strain.

5.4 Conclusions

Based on the experimental results shown in this chapter, the following conclusions can be summary:

(1) The autoinducer (C4-HSL) degradation was found to be the cell related and is an

intracellular activity.

(2) The autoinducer production and degradation are proportional to the autoinducer

-1 concentration. The degradation constant kd was found to be 0.0062 (h) .

(3) The rhlR mutant was found to produce less autoinducer when compared with wild

type strain (1/3).

(4) Autoinducer was found to play an essential role in regulating rhamnolipid

production.

107

1000

800

600

400 Spent (mg/L) E.A (mg/L) Heat(mg/L) Nitrate Concentration (mg/L) 200 Fresh (mg/L)

0 0 5 10 15 20 25 Cultivation Time (hour)

Figure 4.2 Nitrate Concentration Profiles in different System

rhlA rhlB rhlR

(+)

Rhamnosyltransferase RhlR AI

RhlR

(+) rhlI rpoS

Figure 4.3 Proposed Mechanism of the Regulation of Cell Growth by Quorum-Sensing System

4.3.3 Autoinducer Effects on Cell Growth

Population density-dependent gene activation by the LuxR-LuxI family of

transcriptional regulators is commonly observed in a diverse group of gram-negative

bacteria (115, 180). However, autoinducer (C4-HSL) was proposed as a key regulator for

cell growth. Controversial results are still floating around the literature. An experiment

was conducted to verify the proposed hypothesis, as seen in Figure 4.3. The experiment

was carried out in a 2L flask containing 1.5L of medium with rhlI mutants. The pH was

controlled at 6.5 ±0.1 with automatic addition of either 1N NaOH or mixed solution of

HNO3 and NaNO3. The surface aeration was employed to create a micro-aerobic

condition for the cells. A 50 mL aliquot of sample was harvested from the culture after

40 hours cultivation. The harvested culture was then split into two parts. Each part

contained 25 mL aliquot of the harvested culture in a 250 mL flask. The concentrated

autoinducer was added into one of the system to a final concentration of 0.07 mg/L. The

cultures in these two systems were then aerobically grown with strong agitation. The cell

densities of these two systems were monitored for another 36 hours. The experimental

results are summarized in Figure 4. 4. The results shown in Figure 4.4 indicate that the

cell growth was affected by the addition of autoinducer. No cell growth was observed in

the system containing 0.07 mg/L of C4-HSL, however, the cells showed a strong growth

tendency in the other system. These results suggested that the rhlI gene played an

essential role in regulating the cell growth. These results were also consistent with the

results reported with Pseudomonas aeruginosa and Rhizobium leguminosarum [181,

182].

8 Time (hour) vs rhlI(g/L) Time (hour) vs rhlI+AI (g/L) 7

3 Cell Concentration (g/L) 2

0 0 20406080100

Culture Age (hour)

Figure 4.4 Cell Growth Profiles with and without the Addition of Autoinducer

4.3.4 Effects of rhl Quorum Sensing System on rhamnolipid production

To explore the effects of rhl quorum sensing system on rhamnolipid production,

Pseudomonas aeruginosa PAO1 wild type and its rhlI and rhlR mutants were used for

this study. Experiments were carried out in 2 2L conical flasks containing 1.5L medium

with wild type and rhlI mutant of Pseudomonas aeruginosa PAO1, respectively. The pH

was maintained at 6.5±0.1 with automatic addition of either 2N of NaOH or 2N of HNO3 and NaNO3 mixture. The samples were taken daily for nitrite concentration, specific

nitrate consumption rate, specific cell growth rate, cell concentration and rhamnolipid

analysis. The experimental results are shown in Figure 4.5. It was found that the cell

growth rate of rhlI mutant was slightly higher than that of wild type PAO1 species. The

rhamnose profile also revealed that the rhamnolipid production was almost inhiited in the

culture of rhlI mutant. In contrast, the rhamnolipid concentration was accumulated to 1.3

g/L in the culture of PAO1 wild type species. A similar experiment was also conducted

for rhlR mutant. The experimental results are summarized in Figure 4.6. As it’s seen in

the Figure 4.6, the rhamnolipid production was also inhibited during the course of

experiment. The rhamnose profile indicated that the rhlR gene also play an important

role in regulating rhamnolipid production. These results strongly supported the

observation [136] that rhl quorum sensing system plays an essential role in regulating

rhamnolipid production.

4.3.5 Autoinducer Effects on Rhamnolipid Productivity

Autoinducer (C4-HSL) was recognized as an important component to enhance the

100 10 2

Biomass (PAO1) Biomass (rhlI) 80 8 Rhamnose (PAO1) Rhamnose (rhlI) Nitrite (rhlI)

60 6

40 4 Biomass (g/L) Rhamnose Concentration (g/L) Nitrite Concentration (mg/L) Nitrite Concentration 20 2

0 0 0 0 20406080100120140160180

Time (hr)

Figure 4.5 Effect of rhlI Gene on Rhamnolipid Production

100 10 2

Biomass (PAO1) Biomass (rhlR) 80 8 Rhamnose (PAO1) Rhamnose (rhlR) Nitrite (rhlR)

60 6

40 4 Biomass (g/L) Biomass Rhamnose Concentration (g/L) Nitrite Concentration (mg/L) 20 2

0 0 0 0 20406080100120140160180

Time (hr)

Figure 4.6 Effect of rhlR Gene on Rhamnolipid Production

82 expression of rhlAB gene, which encodes a rhamnosyltransferase for rhamnolipid production. Therefore, autoinducer concentration could play an essential role in regulating rhamnolipid productivity by affecting the enzyme production in the culture.

To explore the effects of autoinducer on rhamnolipid productivity, the following experiments were conducted for this goal. Experiments were conducted in 125-ml shake flasks containing 8-9 g/L of rhlI mutant cell concentration. The rhlI mutant cells were harvested from the culture in the early exponential phase to assure the cell activity. The conditioned medium containing C4-HSL autoinducer was harvested in the early stationary phase from the wild type culture and was used in this study post autoinducer concentration was determined. 3 ml, 5 ml, 7 ml, 13 ml and 18 ml of conditioned medium were separately added to the systems containing 100 ml of conditioned medium and fresh medium at the beginning of the experiment. Samples were taken from the flasks for the measur ement of rhamnolipid concentration every 10-12 hours.

The experimental results are given in Figure 4.7 and 4.8. The results shown in

Figure 4.7 unveiled that the more autoinducer was used in the system, the more rhamnolipid was produced in the system. This observation is consistent with the proposed mechanism that rhlI gene or C4-HSL plays an essential role in regulating rhamnolipid production. The maximal rhamnolipid productivity was calculated from all systems and used to create a plot by against autoinducer concentration shown in Figure

4.8. A saturation concentration of autoinducer was found whereas 13 to 18 % of the conditioned medium or a corresponding 0.075 to 0.09 mg/L of C4-BHL autoinducer was employed to the system. This result also uncovered that the minimal required

2 Rhamnolipid Productivity (mg/L/h) Rhamnolipid Productivity

0 02468101214161820

Initial AI Concentration (%)

Figure 4.7 Effect of Autoinducer Concentration on Rhamnolipid Production

0.5

18% of AI0 13% of AI 0.4 0 7% of AI0

5% of AI0

3% of AI0 0.3

0.2

0.1 Rhamnose Concentration (g/L) Rhamnose Concentration

0.0 0 1020304050

Time (hour)

Figure 4.8 Rhamnolipid Profiles with Various Amount of Spent Medium Addition

85 autoinducer to maintain the maximal rhamnolipid production is about 13 % of the

4.4 Conclusions

Based on the experimental results, the following conclusions can be dawn:

(1) Rich medium can be employed in the system to avoid frequent nutrient addition

responsible for causing a lower cell concentration and titers. No apparent side

effects were observed in the system

(2) The rhamnolipid productivity under microaerobic conditions is around 1/3 of that

under aerobic condition

(3) Autoinducer was found to play an essential role in regulating the cell density and

rhamnolipid production

(4) Effects of rhl quorum-sensing system on denitrification is very profound and

needs to be explored in more details.

CHAPTER VI

CONCLUSIONS

In this study, the possibility of using Pseudomonas aeruginosa ATCC 9027 to produce rhamnolipids under micro-aerobic (combined aerobic and anaerobic) conditions was evaluated. The understanding of how rhl quorum-sensing system of Pseudomonas aeruginosa PAO1 regulates rhamnolipid production and cell growth was also included in

this research work. The research started with the investigation of key parameter

identification for rhamnolipid production under micro-aerobic conditions (Chapter 3)

followed by the rhamnolipid production and cell growth under the regulation of rhl

quorum-sensing system (Chapter 4). The kinetic study of autoinducer (C4-HSL) was

discussed at the end of this research work (Chapter 5). The experimental findings and

conclusions obtained from each part of this study are summarized in the followings.

6.1) Conclusions

6.1.1) Characterization of Rhamnolipid Fermentation Process under Micro-aerobic

Conditions

High cell concentrations (10-13 g/L of dry cell weight) can be achieved with the micro-aerobic culture while 1-2 g/L of cell concentrations was achieved in the aerobic

culture. The optimal composition of the mixture of sodium nitrate and nitric acid

solution used to compensate the nitrate consumed for the cell respiration and

108 organic acid produced in the system is 1:2 in cell growth phase. The suitable composition of the mixture of sodium nitrate and nitric acid solution used to compensate the nitrate consumed for the cell respiration and organic acid produced in the system during the course of cultivation is 1:1 in stationary phase. To maintain the maximal rhamnolipid production, the cell growth rate needs to be maintained in between 0 and 0.08 h-1. The maximal phosphorous consumption rate for cell activity is around 2 mg/g•cell•h.

6.1.2) Rhamnolipid Production: Rhamnolipid Production and Cell Growth under the Regulation of rhl Quorum-sensing System

Nutrient limitation is not a necessary way to force the cells into the stationary phase for rhamnolipid production. In contrast, rhl quorum-sensing system was found to regulate cell growth in a cell-density-dependent manner. High rhamnolipid concentrations can be achieved through the regulation of rhl quorum-sensing system.

The specific rhamnolipid productivity with the cells under the regulation of rhl quorum- sensing system was found to be 100% higher than that regulated by limiting nutrient. No rhamnolipid production was observed in the culture either with rhlI or rhlR mutant. High cell density culture can be employed for rhamnolipid production.

6.1.3) Degradation and Production kinetics of Autoinducer (C4-HSL) in Stationary

Phase and Effects of C4-HSL on Rhamnolipid Production

The degradation of autoinducer (C4-HSL) was found to be cell related and to take place in intracellular of Pseudomonas aeruginosa PAO1. The autoinducer degradation rate was found to be first order kinetics of its concentration and autoinducer was produced not only in the growth phase but also in the stationary phase. The autoinducer 109 production rate was first order kinetics of autoinducer concentration, too in the stationary

phase. Only one third of autoinducer was produced with culture of rhlR mutant of

Pseudomonas aeruginosa PAO1when compared with that of wild type culture. RhlR

gene of rhl quorum-sensing system plays an essential role in regulating autoinducer

production.

6.2) Recommendation for Future Study

6.2.1) Reasons for Observation of Decreasing Denitrification Rate During the Course

of Fermentation Process

For some unknown reasons cells’ respiration rate and specific cell growth rate

decreased with the increasing of culture span as it is given in Figure 6.1. The specific

denitrification rate decreases in parallel with the specific cell growth rate. In most of the denitrification study, nitrite, the toxic intermediate of denitrification process, accumulation was considered as the main reason to slow down the denitrification that observed in this research work. But the nitrite profile indicated that the maximal nitrite concentration found in this study was 16 mg/L, which was low enough to cause any negative effects on cell activity. Some preliminary study was conducted to clarify if any repressor was produced during the course of fermentation process. The results shown in

Figure 6.2 and 6.3 indicated that denitrification was resumed after the conditioned (spent) medium was replaced with fresh medium, but no further cell growth was observed in the systems with various physical and chemical treatments to remove the repressor produced in the conditioned medium. These results suggested that one or more repressors existing in the spent medium to cause the decrease of denitrification rate.

110

0.12 35 -N) 0.10 30 3

) SCGR Wild-Type

-1 SNCR Wild-Type SCGR rhlI 0.08 25 SNCR rhlI

0.06 20

0.04 15

0.02 10 Specific Cell Growth Rate (D 0.00 5 Specific Nitrate Consumption Rate(mg/L NO Consumption Rate(mg/L Specific Nitrate

0 0246810

Time (day)

Figure 6.1 Profiles of Specific Cell Growth rate and Specific Nitrate Consumption Rate with Wild Type PAO1 and rhlI Mutant

111 A proposed mechanism indicated RpoS protein positively regulates the expression of nitrate reductase, but negatively regulates the expression of nitrite reductase. If this postulation is truth, nitrate consumption rate should be increased in parallel with the nitrite accumulation. In contrast, a slower nitrate consumption rate and low nitrite profile was observed in this study. Therefore, the reason to cause the decrease of specific denitrification rate need to be clarified to further maintain cell activity and optimize rhamnolipid production.

6.2.2) rpoS Gene Affects Cell Metabolisms

Since rpoS gene is attributed as one of the reasons to program cells entering into stationary phase, the metabolism affected by rpoS gene may need to be clarified if rpoS gene is knocked out for cell growth purpose. The understanding of rpoS gene effects on cell growth and cell metabolism could be further used to improve rhamnolipid production and simplify the rhamnolipid fermentation process.

112

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