interaction between micro-algae and quorum sensing...

Faculty of Bioscience Engineering Academic year 2009 – 2011

INTERACTION BETWEEN MICRO-ALGAE AND

QUORUM SENSING MOLECULE DEGRADING

BACTERIA

Ace Vincent Bravo Flandez I

Promoter Prof.dr.ir. Peter Bossier

Supervisor Natrah Fatin Mohd Ikhsan

Thesis submitted in partial fulfillment of the requirements for the academic

degree of Master of Science of Aquaculture

i

COPYRIGHT

The author and promoter give permission to put this thesis to disposal for

consultation and to copy parts of it for personal use. Any other use falls under

the limitations of copyright, in particular the obligation to explicitly mention the

source when citing parts out of this thesis.

Gent, Belgium, 26th August 2011

Promoter

Prof. dr. ir. Peter Bossier

Supervisor

Natrah Fatin Mohd Ikhsan

Author

Ace Vincent Bravo Flandez I

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ABSTRACT

Bacterial diseases are the bottleneck for the development of aquaculture

sector. Many pathogenic bacteria have been recently identified to resist any

antibiotic treatment. Studies shows that many aquaculture gram-negative

pathogenic bacteria regulate their virulence factor through quorum sensing

molecules, a small organic and freely diffusible called N-acyl homoserine

lactones (AHLs). The aim of the present study was to isolate AHL-degrading

bacteria from microalgae. In this study, 3 strains have been isolated (T2, I3

and C2) from selected microalgae (Tetraselmis suecica, Isochrysis affinis

galbana (T-Iso), Chaetoceros muelleri) and proven to degrade exogenous

AHLs. The three bacterial strains which showed high AHL degradation

activities were selected for further studied: (i) interaction of microalgae and

QS molecule degrading bacteria and (ii) In vivo challenged test of selected

aquatic organism. In vivo result showed that combination of both AHL-

degrading bacteria to specific microalgae significantly increases the survival

of the tested aquatic organism. In conclusion, AHL-degrading bacteria might

be use together with green water techniques and acts as biocontrol to fight

against bacterial diseases.

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ACKNOWLEDGEMENTS

I would like to express my sincere appreciation and gratefulness;

To the Flemish Interuniversity Council (VLIR) for the scholarship grant that

enable me to pursue Master of Science in Aquaculture at Ghent University.

To Prof.Dr Patrick Sorgeloos for accommodating me and accepting me as a

student at the Laboratory of Aquaculture and Artemia Reference Centre

(ARC), Ghent Belgium

To my promoter Prof .Dr.ir Peter Bossier, for allotting his valuable time to

read, correct and discuss ideas with me to improve my thesis manuscript

To, Natrah my supervisor thank you very much for your guidance, reminders,

corrections and shared ideas/scientific knowledge/experiences, despite your

busy schedule and deadlines you have always been there to answer all my

queries and guided me to make my research work better. You have been a

good mentor.

To Dr.ir.Tom Defroit, for unselfishly sharing his scientific expertise during and

after the study.

To the entire ARC staff, assisting me while conducting my Laboratory

experiment, to our MSc. coordinators Bart Van Delsen and Sebastian

Vanopstal for making us comfortable with our 2 year stay in Belgium

To my colleagues thank you for the camaraderie, fun, laughter, motivation and

shared knowledge throughout our study period. I wish everybody’s success.

To my fellow Filipino Students thanks you for the companionship and making

my stay in Belgium more enjoyable see you all in the Philippines

To my Papa, Mama, Brother, Sister and beloved Hera thanks for all the

prayers, love, support and inspiration as I achieve my goals in life.

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NOTATION INDEX

AI-2 Autoinducer 2

AIP Autoinducer peptide

AHL N-acyl homoserine lactone

F/2 F/2 medium, Guillard and Ryther 1962, Guillard 1975

IO Instant Ocean

HAI-1 Harveyi Autoinducer 1

HHL N-hexanoyl-L-homoserine lactone (HHL)

MA Marine Broth

MB Marine Agar

OHHL N-(3-oxo-hexanoyl)-homoserine lactone

QS Quorum Sensing

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TABLE OF CONTENTS

COPYRIGHT..................................................................................................... i

ACKNOWLEDGEMENTS............................................................................... iii

NOTATION INDEX ......................................................................................... iii

TABLE OF CONTENTS .................................................................................. v

LIST OF FIGURE.......................................................................................... viii

LIST OF TABLES ............................................................................................ x

CHAPTER 1: INTRODUCTION ....................................................................... 1

CHAPTER 2: LITERATURE REVIEW............................................................. 3

2.1 Importance of Algae ............................................................................. 3

2.1.1 Characterization of Microalgae ........................................................ 3

2.1.2 Nutritional importance of microalgae ............................................... 4

2.1.2.1 Pigments ................................................................................... 5

2.1.2.2 Fatty Acids ................................................................................ 5

2.1.2.3 Tocopherols, sterols and protein............................................... 5

2.1.2.4 Polysaccharides........................................................................ 6

2.1.2.5 Vitamins, mineral and antioxidants ........................................... 6

2.1.3.6 Pharmaceuticals and biologically active compounds................ 7

2.1.3 Uses in aquaculture ......................................................................... 7

2.2 Interactions between bacteria and algae ......................................... 11

2.2.1 Photosynthetic extracellular exudate of algae ............................... 11

2.2.2 Stimulation of bacterial growth by algae ........................................ 13

2.2.3 Stimulation of algal growth by bacteria .......................................... 13

2.2.4 Algicidal and pathogenic interaction of bacteria towards algae ..... 14

2.3 Quorum sensing in the marine environment ................................... 15

2.3.1 Production of QS signals of gram – and gram + by marine bacteria

................................................................................................................ 16

2.3.2 Disruption of bacterial cell-to-cell communication as a novel

strategy to fight bacterial infection .......................................................... 24

CHAPTER 3: MATERIALS AND METHODS................................................ 29

vi

3.1 In vitro: Isolation of AHL-degrading bacteria from algal culture .. 29

3.2 Quorum sensing (QS) bacterial strains growth condition.............. 30

3.3 Quorum sensing molecules .............................................................. 30

3.4 Bacterial density determination ........................................................ 31

3.5 Detection of hexanoyl homoserine lactones (HHL) ........................ 31

3.6 AHL degradation assay...................................................................... 31

3.7 Co-culture of algae and QS signal degrading bacteria (Algae and Bacteria interaction)................................................................................. 32

3.7.1 Source of Microalgal strains........................................................... 32

3.7.2 Axenic culture of microalgae in seawater with antibiotic................ 32

3.7.3 QS signal degrading strain growth condition ................................. 33

3.7.4 Algae and QS bacteria interaction ................................................. 33

3.7.5 Statistical analysis.......................................................................... 34

3.8 In vivo test: Artemia experiment ....................................................... 34

3.8.1 Axenic culture of microalgae in Instant Ocean............................... 34

3.8.2 Gnotobiotic culture of Artemia........................................................ 34

3.8.3 Challenge tests .............................................................................. 35

3.8.4 Survival and growth of Artemia ...................................................... 36


3.9 In vivo test: Mussel experiment: ....................................................... 36

3.9.1 Mussels D-veliger larvae................................................................ 36

3.9.2 Bacterial pathogenic strain............................................................. 36

3.9.3 QS signal degrading bacterial strain .............................................. 37

3.9.4 Challenged tests ............................................................................ 37


CHAPTER 4: RESULTS................................................................................ 38

4.1 In vitro experiment: ............................................................................ 38

4.1.1 Isolation of AHL-degrading bacteria from algal culture .................. 38

4.1.2 Detection of hexanoyl homoserine lactones (HHL)........................ 38

4.1.2 AHL degradation assay.................................................................. 39

4.1.3 Bacterial density during 72 hour AHL degradation assay .............. 42

4.2 Algae and QS bacteria interaction .................................................... 44

4.2.1 Algal growth dynamics ................................................................... 44

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4.2.2 Dynamics of bacterial growth......................................................... 45

4.3 In vivo experiment:............................................................................. 47

4.3.1 Artemia challenge test ................................................................... 47

4.3.2 Mussel challenge test .................................................................... 48

CHAPTER 5: DISCUSSION .......................................................................... 51

5.1 Enrichment of AHL degrading bacteria from microaglae............... 51

5.2 AHL degradation activity of isolated AHL-degrading bacteria ...... 52

5.3 Links between microaglae and bacterial community, growth,

function and activity................................................................................ 54

5.3.1 Relative fluorescence differences ................................................. 54

5.3.2 Relationship between Quantum yield (Φ), accessory pigment and

nutrient availability .................................................................................. 56

5.4 Beneficial effects of algae and bacteria interaction towards aquatic organism ................................................................................................... 58

CHAPTER 6: CONCLUSION ........................................................................ 61

REFERENCES: ............................................................................................. 62

ANNEX 1: ...................................................................................................... 72

ANNEX 2: ...................................................................................................... 75

ANNEX 3: ...................................................................................................... 77

viii

LIST OF FIGURES Figure 1. Simplified schematic diagram of interaction relationship between

algae and bacteria in aquatic environment. Mineralization of dissolve organic compund (DOM) in a form of bacterial action are thought to stimulate algal growth. Likewise, production of extracellular exudate and algal detris by algae able to sustained the growth of bacterial community. While, antagonistic factors can inhibit the growth vice versa.................. 12

Figure 2. Representative of chemical structure of different autoinducer

molecules identified in Gram-positive and Gram-negative bacteria........ 20 Figure 3. Production of AHL signal by Gram-negative bacteria. The LuxI

protein produced the AHL signal. AHL signaling molecules diffuse freely through the plasma membrane. AHL signal increases as bacterial population increases. If certain threshold in reached, the AHL signal binds to the LuxR protein, a cognate response regulator. The LuxR-AHL transcriptional co-activator complex also will binds at the LuxICDABE promoter as a result this activates transcription of this operon (redrawn after Asad and Opal 2008)...................................................................... 21

Figure 4. A general model for peptide-mediated quorum sensing in many

Gram-positive Bacteria. A peptide signal precursor locus is translated into a precursor protein, subsequently, cleaved producing the processed autoinducer peptide (AIP). The AIP signal is get transported out of the cell via ATP binding cassette an ABC transporter. When extracellular concentration of AIP reached a certain threshold, a sensor kinase detects the increasing AIP concentration. The sensor kinase protein is activated to phosphorylate the cognate response regulator. The phosphorylated response regular activates the transcription of target genes. (redrawn after Miller and Bassler, 2001). ............................................................... 22

Figure 5. The hybrid two-component quorum sensing system of Vibrio

harveyi. The QS signal HAI-1 (an AHL) and AI-2 is get biosynthesis by LuxM and LuxS, respectively. HAI-1 and AI-2 are both detected at the cell surface. LuxN receptor protein is responsible for detection of HAI-1 and LuxP-LuxQ receptor protein is responsible for AI-2 detection. In the absence of QS signal, both receptor proteins autophosphorylate and transfer phosphate from LuxU to LuxO. The phosphorylated LuxO is an active repressor for the target genes. At high cell density, LuxN and LuxPQ interact with their respective QS signal and change from kinases to phosphatases that drain phosphate away from LuxO via LuxU. The dephosphorylated LuxO is inactive. Subsequently, LuxR binds the LuxCDABE promoter and activates transcription (redrawn after Miller and Bassler 2001).......................................................................................... 23

Figure 6. Inactivation of AHL via pH-mediated lactonolysis ........................... 25 Figure 7. HHL standard curve of CV026 in AHL quenching assay ................ 39

ix

Figure 8. Detection of HHL degradation: samples after 72 h contact between axenic QS signal degrading strains grown in MB supplemented with HHL. 10µl of the supernatant of each QS strains was spotted in three parts over buffered LB that already spread with reporter strain CV026........... 40

Figure 9. N-hexaoyl-L-homoserine (HHL) degradation activity by the axenic

QS signal degrading bacteria inoculated at 108 CFU ml-1 (high cell density). Monitoring of HHL concentration at different time points. The data points are the mean values of the 3-spotted replicates of each QS degrader bacterial supernatant. T2: QS degrader isolated from Tetraselmis suecica; I3-3: QS degrader isolated from Isochrysis affinis galbana (I3); C2-1: QS degrader isolated from Chaetoceros muelleri (C2); P6000/Control: P3/pME6000/Marine Broth medium only: as negative control and P6863: P3/pME6863 as positive control .............................. 41

Figure 10. N-hexaoyl-L-homoserine (HHL) degradation activity by mixed

culture of QS signal degrading bacteria inoculated at 108 CFU ml-1 (high cell density). Monitoring of HHL concentration at different time points. The data points are the mean values of the 3-spotted replicates of each mixed sample QS degrader bacterial supernatant. T2: QS degrader isolated from Tetraselmis suecica; I3: QS degrader isolated from Isochrysis affinis galbana; C2: QS degrader isolated from Chaetoceros muelleri; P6000/Control: P3/pME6000/Marine broth medium only: as negative control and P6863: P3/pME6863 as positive control .............................. 42

Figure 11. 48 h bacterial density determination of axenic QS signal degrading

bacteria inoculated at 108 CFU ml-1 (inoculated at high density) T2: QS degrader isolated from Tetraselmis suecica; I3-3: QS degrader isolated from Isochrysis affinis galbana (I3); C2-1: QS degrader isolated from Chaetoceros muelleri (C2); Control: marine broth medium only............. 43

Figure 12. 48 h bacterial density determination of axenic QS signal degrading

bacteria inoculated at 106 CFU ml-1 (inoculated at high density). T2: QS degrader isolated from Tetraselmis suecica; I3-3: QS degrader isolated from Isochrysis affinis galbana (I3); C2-1: QS degrader isolated from Chaetoceros muelleri (C2). Control: marine broth medium only............. 43

Figure 13. Relative fluorescence of selected microalgae incubated in F/2

medium (+ silica for Diatom culture) with and without a QS degrading isolate...................................................................................................... 46

Figure 14. Quantum yield reading on the 15th and 18th day of algae cultivation.

Tetra, Iso and Chaeto only: represent as the control treatment of microalgae Tetraselmis suecica, Isochrysis galbana and Chaetoceros muelleri; Tetra and T2: Tetraselmis suecica and QS T2; Iso and I3: Isochrysis galbana and QS I3; Cheato and C2: Chaetoceros muelleri and QS C2; All QS (represent the 3 QS degrader cultivated together with the each selected microalgae). *: Significant difference in Quantum yield between the control and the treatment of interest (PT-test< 0.05)............. 47

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LIST OF TABLES

Table 1. Major classes and genera of micro-algae cultured in aquacultur (modified from De Pauw and Persoone, 1988)......................................... 7

Table 2. Identified quorum sensing signals and virulence factors controlled by

QS system in marine and pathogenic bacteria (Dobretsov et al. 2009).. 18 Table 3: Composition of F/2 medium ............................................................. 33 Table 4. Percentage survival (mean ± standard error), individual length (IL

mean ± standard deviation) and Vibrio harveyi BB120 concentration (cfu ml-1) of Artemia after 48 hours of post-exposure with Vibrio harveyi BB120 (105 cfu ml-1). Axenic algae and QS signal degrading bacteria were added at 106 cells ml-1 and 107 cfu ml-1, respectively. All Artemia were fed with dead (autoclave) LVS3 (107 cfu ml-1) at the start of the experiment. ............................................................................................. 48

Table 5. Percentage survival of mussel larvae (means ± standard error of the

three replicates) after 72 hours of post exposure with Vibrio harveyi BB120 and Vibrio anguillarum LMG4437, pathogen, axenic algae and QS degrader strains were added 106 CFU/ml, 106 cells/ml and 107 CFU/ml, respectively. Each treatment was supplemented with F2 medium. ........ 49

Table 6. Percentage survival of mussel larvae (means ± standard error of the

three replicates) after 72 hours of post infection with Vibrio harveyi BB120 and Vibrio anguillarum LMG4437, pathogens, axenic algae and QS degrader strains were added 106 CFU/ml, 106 cells/ml and 107 CFU/ml, respectively. Each treatment was supplemented with F2 medium plus Silica. ...................................................................................................... 50

1

CHAPTER 1: INTRODUCTION

Bacteria and micro-algae are numerically dominant organism and ubiquitous

around aquatic ecosystem, their metabolism largely controls pelagic energy

flow and nutrient cycling (Cole, 1982). Bacteria or micro-algae, produces

novel secondary metabolites having potential application in disease treatment

specifically in aquaculture and pharmaceutical industry. For example, bacteria

living on the surfaces of marine micro-algae or “phycosphere” produced

secondary metabolites, which can inhibit the growth of other competitive

bacteria. In this case, highly competitive environment where space and

availability of nutrients are limited might be a selective force, which may lead

to the evolution of a variety of effective adaptation in several algal-associated

bacteria (Boyd et al., 1999). Additionally, Kanagasabhapathy et al., (2006)

conducted an experiment on the antibacterial activities of isolated bacteria

from brown algae. They concluded that 20% of isolates exhibited antibacterial

activity and belong the members of Bacillus. They suggest that the members

of Bacillus are successful competitors with other microorganisms for surface,

space and nutrient in the marine environment. Additionally, many researchers

have suggested that positive effects of bacteria on culture microalgae have

been documented (Munro et al., 1995; RicoMora and Voltolina, 1998;

Hirayama, 1996). Bacterial community played a crucial part in stable mass

culture of diatoms. Fukami et al., (1997) mentioned that bacterial biofilm on

surfaces are one of the important factors affecting the benthic diatoms

attachment.

Meanwhile, in aquaculture micro-algae are extensively used as a nutritional

food source (EPA and DHA) of many commercially important aquatic

organisms. It also produces bioactive compound, which can deter the growth

of pathogenic bacteria. Lio-Po et al., (2005) demonstrated the effects of

micro-algae (Chaetoceros calcitrans and Nitzchia sp.) on the growth of

luminous Vibrio sp; they suggested that complete inhibition was obtained 24 h

after exposure. Indeed, it is known that some micro-algae have bactericidal

effects against pathogenic Vibrio sp.

Chapter 1 Introduction

2

In addition, bacteria-algae interactions also have important role in the oxygen

and carbon dioxide balance in the culture (Pruder, 1983). Epifanio, (1979),

investigated the synergistic interaction with mixed algal diets in bivalve and

molluscs. It was also documented that significant growth of bivalve was

observed when good algae were added to unfiltered seawater containing silt,

instead of the same algae in filtered seawater. They speculated that bacteria,

other microorganism even extracellular compounds are responsible in

increasing the nutritional content of the algae food in nature (Walne, 1970).

Aquaculture is still the fastest growing food production sector in the world

(FAO, 2009); with the increasing world population there will be a proportional

increase in food demand. Due to this increase, aquaculture is the key to

solve this shortage of food supply in the future. However, aquaculture has

been facing bacterial disease outbreaks as a main problem over the last

decade causing threat to the development (FAO, 2007). That is why additional

research is needed to elucidate the determinant of disease outbreaks and

create preventive measure to decrease bacterial diseases. To date, limited

studies have been conducted on bacteria-algae interactions, specifically those

bacteria that able to degrade quorum sensing (QS) signal molecules, without

affecting the growth of other pathogenic bacteria. Natrah et al., (2011a) have

shown that freshwater algae Chlorella saccharophila have the ability to inhibit

the production of QS regulated virulence factors. Finally, the challenge now is

to find ways on how we can decrease bacterial diseases using algae and

bacterial interaction. However, little is known about the factors controlling QS

in algae bacteria interaction. Therefore, the objectives of this study were to

isolate AHL degrading bacteria from selected microalgae and to establish

interactions and relationship between microalgae and quorum sensing (QS)

signals degrading bacteria.

3

CHAPTER 2: LITERATURE REVIEW

2.1 Importance of Algae

2.1.1 Characterization of Microalgae Microalgae refer to photosynthetic prokaryotic or eukaryotic microorganism

that possesses a unicellular or simple multicellular structure, which allows it to

grow rapidly and survive in harsh conditions. Microalgae size ranges from a

few micrometers to more than 100 µm depending on the species. (FAO,

1996). Microalgae are ubiquitous in nature, mainly found distributed in the

aquatic environment as well as in the terrestrial environment. Furthermore,

microalgae are free living, but a number of microalgae live in symbiosis with

other organisms. There are 30,000 of estimated microalgae species that have

been explored and analyzed for its enormous benefits. (Richmond, 2004 and

Mata, 2010). These microalgae are kept in collection, investigated for

chemical contents and cultivated in industrial quantities.

Commonly studied microalgae belongs to the group of Cyanophyta (blue-

green), Chlorophyta (green) and Bacillariophyta (diatoms). These major

groups of micro algae are known for its biotechnological relevance has and

have been used as nutritional supplements for humans and feed additives for

animals (Mata, 2010 and Gouveia et al., 2008). Microalgae gained

tremendous interest in the scientific community due to its massive biological

resource that satisfies animal and human needs. Various products and new

applications emerge from microalgae utilization.

Microalgae possess a well-balanced chemical composition thus it is used to

enhance the nutritional content of food and animal feed. Moreover, they are a

good source of highly valuable bioactive compounds such as polyunsaturated

fatty acids, pigments, antioxidants and pharmaceuticals. In the aquaculture

industry, microalgae are vital as feed and as life support system in the early

life stages of cultured aquatic organisms. Microalgae are known to effectively

remove/utilize excess nutrients (pollutants) in the aquatic environment. The

Chapter 2 Importance of microalgae

4

biofixation capacity of microalgae is promising in reducing/recycling excess

atmospheric CO2; this is a natural mechanism to combat global

environmental heating and climate change Furthermore, microalgae are

foreseen as a sustainable feedstock for biodiesel production thus it possess a

potential in replacing vegetable crops as oil source (Gouveia et al., 2008)

2.1.2 Nutritional importance of microalgae The widespread utilization of microalgae for human and animals needs

depicts its high nutritional content. Though different microalgae species vary

considerably in their nutritional value but a proper selection of mixture of

microalgae remains to be a good source of nutrition for larval animals. The

nutritional value of a microalgae species can be influenced by several factors;

such as its size, shape, digestibility (cell wall), biochemical composition and

the consumers need.

Furthermore, the intrinsic chemical composition of algae can be modified by a

wide range of environmental factors such as temperature, illumination, pH

value, mineral contents, CO2 supply, or population density, growth phase and

algae physiology (Gouveia et al., 2008). As cited by Brown (2002) microalgae

proximate composition as influenced by its growth phase shows that in late

logarithmic growth phase contain 30-40% protein, 10-20% lipid and 5-15%

carbohydrates, meanwhile in stationary phase it can change significantly and

when nitrate is limited carbohydrates increases at the expense of protein. But

there is no strong correlation between the proximate composition of

microalgae and nutritional value as long as a discreet selection of mixed algal

diet will provide adequate concentration of vitamins, fatty acids for

aquaculture animals (Brown, 2002)

The capacity of microalgae to biosynthesize metabolize, accumulate and

secrete a variety of primary and secondary metabolites (pigments, fatty acids,

tocopherols and sterols, proteins, polysaccharides, vitamins and minerals,

antioxidants and pharmaceutical and other biologically active compounds)

makes it more interesting to be thoroughly investigated. These valuable


5

substances possess a diverse potential in food, pharmaceutical and

cosmetics industries.

Adapted from Gouveia et al., (2008) review in novel products from microalgae

2.1.2.1 Pigments Algae are characterized by their conspicuous color pigment, each algae

possess its own or combined pigments. Widely known pigments are

chlorophyll, phycobiliproteins and carotenoids. These natural pigments can

have potential role as antioxidants, food and pharmaceutical colorant, anti-

inflammatory, neuroprotective, and hepatoprotective.

2.1.2.2 Fatty Acids Several microalgae can synthesize essential polyunsaturated fatty acids

particularly Linolenic acid, arachidonic acid, Eicosapentaenoic acid and

docosahexaenoic acid. Animals are unable to synthesize these long chain

fatty acids and only higher plants and microalgae, which supply the whole

food chain. Polyunsaturated fatty acids are widely known for its various

neutraceutical and pharmaceutical applications. In our dietary needs, source

of PUFA is the marine fish, however fish stocks are at present a declining

resource due to continued anthropogenic fishing malpractices. Microalgae are

a promising source of PUFA due to its superior lipid stability and naturally rich

in antioxidants carotenoids and vitamins. The high nutritional value and its

ability to synthesize and accumulate PUFA only proves that microalgae play

an important role in aquaculture (Patil et al., 2006, Gouveia et al., 2008)

2.1.2.3 Tocopherols, sterols and protein

Tocopherols are present in photosynthetic and non-photosynthetic tissues of

higher plants including algae. Studies suggest that bivalve’s growth rates are

related to the kind and amount of sterols contained in the algae diet.

Moreover, polyhydroxysterols contained in marine organisms are said to

possess an anticancer, cytotoxic and other biological activity.


6

Microalgae are considered an unconventional source of protein due to the

high protein content of numerous microalgae species. The cells can

synthesize all amino acids thus it’s capable of providing essential ones to

human and animals. Free amino acid synthesized by microalgae varies

between species and influenced by its growth condition and phase. The

synthesize protein or amino acids maybe a byproduct of algal process to

produce other useful chemicals and future genetic enhancement could

produce sufficiently high concentration of amino acids (Spolaore et al., 2006

and Gouveia et al., 2008).

2.1.2.4 Polysaccharides The forms of Carbohydrates found in microalgae are starch, glucose, sugars

and other polysaccharides. The high digestibility of these microalgae

carbohydrates sets no limit in using dried whole microalgae as feeds

(Spolaore et al., 2006). Sulphated galactan exopolysaccharide produced by

unicellular red algae Poryphyridium cruentum and Chlamydomonas mexicana

are used to replace carageenan and soil conditioner respectively. Moreover,

these highly sulphated algal polysaccharides have pharmacological properties

on the stimulation of human immune system (Gouveia et al., 2008).

2.1.2.5 Vitamins, mineral and antioxidants The microalgae biomass is characterized as source of all the essential

vitamins and balanced minerals. The widely known Spirulina contains high

levels of vitamin B12 and iron making it appropriate for nutritional supplements.

On the other hand, vitamin content can be influenced by the microalgae’s

genotype, growth phase, nutritional status of alga, light intensity and other

environmental factors, therefore it can be subjected to manipulation in it

culture condition, strain selection or genetic engineering (Gouveia et al.,

2008). The efficient development of protective system of microalgae against

reactive oxygen and free radicals created a high interest in using microalgae

as natural antioxidants. Several algae exhibits strong antioxidant activity

through its methanolic microalgae crude extracts as compared from α-

tocopherol (Gouveia et al., 2008).

Chapter 2 Uses in aquaculture

7

2.1.3.6 Pharmaceuticals and biologically active compounds Microalgae is a relatively large reservoir of novel compounds, numerous of

these compounds manifests biological activity which are characterize with

unique, interesting structure and functions. For decades, the marine

microorganisms’ Cyanobacteria have been investigated for new

pharmaceutical and antibiotics. As of 2001 there are 424 screened

compounds that includes lipoproteins (40%), alkaloids, amides and other

compounds. The biological activity involves cytotoxic, antitumor, antibiotic,

antimicrobial (antibacterial, antifungal, antiprotozoa) antiviral (anti-HIV) as well

a biomodulatory effects such as immunosuppressive and anti-inflamamatory.

The reported cytotoxic effect of these compounds maybe related to defense

mechanisms manifested by an organism in the highly competitive marine

environment where they lack immune system thus it produces secondary

metabolites such as toxins (Gouveia et al., 2008).

2.1.3 Uses in aquaculture

Microalgae play a significant role for most aquatic animals in the aquaculture

industry. It is the primary food source for most invertebrates for their whole life

cycle. In commercial and experimental invertebrate hatcheries, installation of

microalgae production system is necessary for larvae production and

domestication. Commonly known microalgae consumers in aquaculture are

filter feeders (mollusc larvae, juveniles and broodstock), fish larvae,

crustacean larvae and live prey (rotifers, Artemia) fed to late larvae and

juveniles of fish and crustacean species (Brown et al., 1997; and Bastien,

2006).

Table 1. Major classes and genera of micro-algae cultured in aquacultur (modified from De Pauw and Persoone, 1988)

CLASS GENUS AREA OF APPLICATION

Bacillariphyceae Skeletonema Penaeid shrimp,bivalve mollusc,bivalve postlarvae

Thallasiosira Penaeid shrimp, bivalve larvae, bivalve postlarvae

Phaeodactylum Penaeid shrimp, bivalve larvae, bivalve

postlarvae, brine shrimp, freshwater prawn larvae


8

Chaetoceros Penaeid shrimp,bivalve larvae, bivalve postlarvae,, brine shrimp

Cylindrotheca Penaeid shrimp Bellerochea bivalve postlarvae Actinocyclus bivalve postlarvae Nitzchia brine shrimp Cyclotella brine shrimp

Isochrysis Penaeid shrimp,bivalve larvae, bivalve postlarvae,, brine shrimp

Haptophyceae Pseudoiisochrysis dicrateria

bivalve larvae, bivalve postlarvae ,freshwater prawn larvae

Chrysophyceae Pavlova bivalve larvae, bivalve postlarvae, brine shrimp,marine rotifers

Tetraselmis Penaeid shrimp, bivalve larvae, bivalve postlarvae, abalone larvae,brine shrimp,

marine rotifers Prasinopyceae Pyramimonas bivalve larvae, bivalve postlarvae Micromonas bivalve postlarvae Chromoonas bivalve postlarvae Cryptopyceae Cryptomonas bivalve postlarvae Rhodomonas bivalve larvae, bivalve postlarvae

Xanthophyceae Chlamydomonas bivalve larvae, bivalve postlarvae,freshwater zooplankton,marine rotifers,brine shrimp

Chloprophyceae Chlorococcum bivalve postlarvae Olisthodiscus bivalve postlarvae Cyanophyceae Carteria bivalve postlarvae

Dunaliela bivalve larvae, bivalve postlarvae,marine rotifers

Spirulina Penaeid shrimp, bivalve postlarvae,marine rotifers,brine shrimp

The high productivity of a hatchery strongly depends on the quality and

quantity of the food source. Microalgae can be useful as feed for aquaculture

species if it possess several important key attributes such as appropriate size

for ingestion and digestion (range from 1-15 µm for filter feeders,10-100 µm

for grazers), rapid growth rate, suitable for mass culture, stable in culture to

any physico-chemical (temperature, light, nutrients) fluctuations in the

hatchery system and with high nutrient content and nontoxic (Brown, 2002).

Microalgae are fed in several forms; traditionally live microalgae were

intensively cultured used to feed in commercial mollusc hatcheries but due to

its high operational cost alternative production procedures emerge. The

proposed alternatives simplified and decreased cost of production. Microalgae

pastes, spray dried microalgae, frozen biomass are just a few of non-living


9

substitutes that were tested in hatcheries. Nutrition wise, live microalgae

remains the best food source for larvae as it has higher nutritive value, good

digestibility and possess natural bacterial flora that has a positive health

effects to larvae (Bastien, 2006).

Meanwhile, suboptimal effects by live microalgae substitutes include lower

growth, higher mortalities and low level/absence of highly unsaturated fatty

acids. Meanwhile, low ingestion/digestion by larvae and poor physical

behavior thus can be used as substrate for pathogenic bacteria. Nevertheless,

this array of substitutes can be used as supplement and backup food source

when live algae portion are in short supply (Bastien, 2006). Additionally,

Borowitzka (1997) cited that in Canada and USA wet algal concentrates could

be a good alternative feed for it maintains original composition in a long

storage period as compared to dried algae.

Microalgae are not only used as feed but also as a quality-enhancing agent of

the quality of cultured species. The carotenoids and astaxanthin found in

microalgae enhances pigmentation on various fish and shellfish. Consumers

prefer natural pigmentes over synthetically manufactured. In salmonoids

pigments from microalgae enhances flesh color which result in high quality

product. Spirulina and Dunaliella sp. are commonly used to pigment

crustaceans and shrimp respectively. Furthermore, algal carotenoids may

function as growth hormone but needs for further investigation (Borowitzka,

1997). In oyster production, microalgae is used for oyster refining process

where it comes in contact with naturally or artificially grown algae to improve

final product quality (Mueller-Fuega, 2000).

Green water technique has long been used during culture of both shrimp and

fish larvae in hatcheries together with the zooplankton prey. Most commonly

used algae species are the Nannochloropsis oculata and Tetraselmis suecica.

The presence of dense microalgae population contributes in stabilizing and

improving the quality of the culture environment such as light attenuation

(shading effect), oxygen and ammonia balance, pH stabilization, excretion of


10

vitamins or growth promoting substances, probiotic effect and stimulating

immunity. Additionally in fish hatcheries it is also used for live prey production

for the growing fish larvae. The presence of microalgae allows quick recovery

of live prey population in times of collapse, improve nutritional quality of live

prey, regulate bacterial population from Vibrio , thus leads to better results on

survival, growth and transformation index (Mueller-Fuega, 2000; Borowitzka,

1997; and Neori, 2011). Indeed microalgae is undeniably important in the

aquaculture industry.

Chapter 2 Algae and Bacteria Interactions

11

2.2 Interactions between bacteria and algae In aquaculture, micro-algae are use as essential food source and feed

additive in commercial rearing of aquatic animal (Borowitzka, 1997; Muller-

Feuga, 2000), especially in all stages of marine bivalve molluscs, shrimp and

prawn larvae, marine finfish and crustaceans. Although Jones et al., (1987)

and Heras et al., (1994) substitute of live food microalgae such as

microcapsules and yeast-based diet exist in the market. However, live algae

are still the best nutritional source and preferred food in hatchery (Borowitzka,

1997).

The application of micro-algae not only increases the quality of live food, it

also serves to establish a phycosphere (Cole, 1982; Sapp et al., 2007); a

unique symbiosis that develop from the combination of a specific micro-algae

and bacteria. Nevertheless, successful establishment of symbiont bacteria

remains enigmatic. To date, little is known about the factors controlling algae

and bacteria interactions in aquatic ecosystem. Recently, it has been reported

that association of bacteria and algae might be due to spatial, temporal

(Grossart et al., 1999) and organic matter produced by different types of

algae, which cause shifts in bacterial species composition.

Meanwhile, it is known that bacteria that live attached to algal surfaces and

that consume extracellular products consequently participate in

biogeochemical cycling and play an important part in the microbial loop (Sapp

et al., 2007) (Figure 1). A correlation between algae and bacterial biomass

was demonstrated by Rooney-Varga et al., (2005) suggesting that the

dynamics of these two communities are linked together, and changes in the

phytoplankton community. They concluded that specific interactions between

algae and attached bacteria may occur and that such interaction could be

important in controlling the composition of both communities.

2.2.1 Photosynthetic extracellular exudate of algae It is known that phytoplankton and bacterial communities are thoughts to be

loose or tight association. A report from (Aota & Nakajima, 2001) stated that

when competition for phosphorus is severe, the amount of phytoplankton


12

decreases, and in turn, extracellular organic carbon (EOC) released from

phytoplakton decrease. Thus bacterial growth may be simultaneously limited

by carbon and phosphorus. If carbon limitation becomes more severe,

bacterial growth is mainly limited by EOC. Therefore, competition for

phosphorus will be reduced. Thus, mutualistic relation could be expected due

to carbon flow from phytoplankton to bacteria. Futhermore, (Bell et al., 1974)

investigated the assimilation of extracellular products of Skeletonema

costatum by bacterial isolates which indicate the role of exudates as a carbon

source. Moreover, Whittaker and Feeny (1971) mentioned that marine

bacteria are capable of utilizing algal extracellular products. Sapp et al.,

(2007) and Descy et al., (2002) pointed out that phytoplankton exudates is

the important source of carbon and may contribute to the base of the

microbial food web in aquatic ecosystem.

Figure 1. Simplified schematic diagram of interaction relationship between algae and bacteria in aquatic environment. Mineralization of dissolve organic compund (DOM) in a form of bacterial action are thought to stimulate algal growth. Likewise, production of extracellular exudate and algal detris by algae able to sustained the growth of bacterial community. While, antagonistic factors can inhibit the growth vice versa.


13

2.2.2 Stimulation of bacterial growth by algae Bell et al., (1974) demonstrated that Pseudomonad grows well to a high

steady state with co-culture of Skeletonema costatum both batch and

continuous culture. While in the absence of alga viable bacterial count was

significantly lower. Thus, suggest that Pseudomonad growth is most like

supported by the extracellular organic compound and dead algal cell. It has

been stated that the role of extracellular organic compound (EOC) secreted

by algae sustained the creation and maintenance of a phycosphere effect,

showing that microbial ultilization of these compound can result in stimulation

of physiologically specific bacterial types, likewise bacterial mineralization

developed phytoplankton population (Bell et al. 1974).

Cole (1982) enumerated that several processes are involved in the transfer of

organic material from algae to bacteria.

1. Bacteria may parasitize an algal cell

2. Bacteria may obtain nutrition during the decomposition of a dead algal

cell. Releasable nutrients in such a form of bacterial action.

3. Or organic material released from alga during cell growth may also be

available to bacteria.

Additionally, recent research suggest that 14% of the estimated bacterial

production was accounted for by algal excretion. They reported that major

proportion of algal exudates are used by bacteria (Brock and Clyne, 1984).

Suggesting that extracellular organic carbon (EOC) play a major role in

stimulating the growth of bacterial population.

2.2.3 Stimulation of algal growth by bacteria To our knowledge, bacteria plays an important role in remineralization of

organic compound to simplier molecules that can be easily assimilated by

phytoplankton. Cole, (1982) mentioned that phytoplankton growth could be

supported by allochthonously supplied nutrients in the absence of in situ

remineralization by heterotrophs. Meanwhile, Oswald, (1986) reported that


14

carbon often limits that growth of algae in sewage treatment plant, but

sources of carbon may be available to algae in waste ponds with the renewal

of carbon that are fix and released by bacteria at the bottom of the pond.

2.2.4 Algicidal and pathogenic interaction of bacteria towards algae A yellow pigmented Pseudoalteromonas peptidysin strain (class

Proteobacteria, gamma subdivision), an interesting bacterium known to have

a potent algicidal effects on several harmful algal bloom. It was documented

by Lovejoy et al. 1998 that this strain Y bacterium produces water-borne

algicidal compounds, that it acted a rapid cell lysis and death of Gymnodinium

catenatum, Chattonella marina and Heterosigma akashiwo. The same result

was obtained by Skerratt et al. (2002) were 5 bacterial isolates secret algicidal

extracellular exudates which can inhibit algal gorwth. They demonstrated that

algicidal and inhibitory activity of 5 bacterial isolates was not regulated via

AHL quorum sensing circuit, but rather it was controlled by means of AI-2

(autoinducer-2) quorum sensing system.

Chapter 2 Quorum sensing in the marine environment

15

2.3 Quorum sensing in the marine environment Quorum sensing is a system of cell-to-cell communication among bacterial

kingdom. Where, a singled bacterium releases their communicable chemicals

that attach to regulator proteins and activate them so that the regulator protein

can switch particular gene expression on or off. These communicable

chemicals are based on self-generated signal molecules called autoinducers

(Table 1). Different bacteria use different autoinducers such as N-acyl

homoserine lactones (AHLs), which are synthesized by Gram-negative

bacteria, while Gram-positive bacteria synthesize g-butyrolactones

(autoinducer peptide).

In the marine environment, it appears that planktonic organism; vertebrates

and invertebrates associated with marine bacteria are capable in producing

quorum-sensing signals. In recent study by Huang et al., (2007), it was

investigated that production of AHL signals found near marine subtidal biofilm.

The detection of AHL signals was base on 2 reporter strains,

Chromobacterium violaceum CV026 (produces purple pigment in response to

AHLs with short alkanoyl C4 to C8 acyl side chain) and Agrobacterium

tumefaciens A136 (produces β-galactosidase in response to AHLs with acyl

side chain of C6 to C12). Results indicate that 2 day-old biofilm induced

coloration of C. violaceum CV026 while A. Tumefaciens A136 did not show

blue coloration. On the contrary, the 4 and 6 day old biofilm induced blue

coloration on A. Tumefaciens A136, but did not induced purple coloration on

C. violaceum CV026. The study suggested that during the early stage of

biofilm development short alkanoyl or 3-oxo-alkanoyl side chain (C4 to C8)

AHLs were produced. Meanwhile, 4 and 6 day old biofilm produced a long

chain (>C6) AHLs signals were it was strongly detected by the reporter strain

A. Tumefaciens A136. They concluded it was probable that different types of

AHLs were produced in subtidal biofilm of different ages (Huang et al., 2007).

In addition, Gram et al., (2002) investigated the production of quorum sensing

signal in marine-snow, free living bacteria and associated bacteria from

planktonic diatoms (Thalassiosira rotula and Skeletonema costatum). Based

on the result, four of 43 bacterial isolates were positive to produce acylated


16

homoserine lactone (AHLs) compound were it activated the AHL reporter

strains (Chromobacterium violaceum-CV026, Agrobacterium tumefaciens and

Escherichia coli-pSB403). Four of the AHL-producing bacteria were identified

by 16S ribosomal DNA gene sequence analysis as α-Proteobacteria

(Roseobacter spp.) and γ-Proteobacteria (Marinobacter spp). Further

investigation was made on Roseobacter strains, Based on bioassay–coupled

thin layer chromatography showed that 7-day old Roseobacter strains culture

produces N-hexaoyl-1-homoserine lactone (C6-HSL) and N-octanoyl-

homoserine lactone (C8-HSL). Furthermore, Taylor et al., (2004) conducted

an experiment on bacteria associated with marine sponges for the production

of AHL molecule. They found activation of the two reporter strains

(Chromobacterium violaceum-CV026, Agrobacterium tumefaciens) was

observed which would suggest that AHL producing bacteria are present on

marine sponges. The result was further confirmed by gas chromatography

and mass spectrometry (GS-MS), and demonstrated the production of C6-

HSL ans N-(3-oxo)-hexanoyl-homoserine lactone (3-oxo-C6-HSL) by Vibrio

sp. (tentative identified as Vibrio campbellii by 16S ribosomal DNA).

Decho et al., (2009) reported the production of AHLs by marine microbial

mats (stromatolites). The mats are composed mostly of cyanobacteria and

sulfate-reducing bacteria. Characterization of AHL was done using

dichloromethane to extract the AHL compound and analyzed by liquid

chromatography/mass spectrometry (LC/MS). The study revealed that a wide

range of AHL (from C4- to C14-HSL), were extracted from the microbial mats.

2.3.1 Production of QS signals of gram – and gram + by marine bacteria

It is believed that quorum sensing is involved in many important bacterial

phenotypes such as biofilm formation, motility, swarming, antibiotic

resistance, sporulation, pathogenicity and virulence, which are essential for

the successful establishment of symbiotic or pathogenetic relationship with

their respective host (Gonzalez and Keshavan, 2006). Cell to cell

communication begins with the production of QS signal molecules, were QS

bacteria can detect and respond to the accumulation of autoinducer molecule.


17

As a population density of producing autoinducer grows, the extracellular

concentration of autoinducer signal molecule increases with increasing

bacterial density. When autoinducers reached a crucial threshold level,

bacterial population responds specifically in gene expression resulting in the

synchronous activation of certain bacterial phenotype.

Generally, for a molecule to be classed as a quorum sensing signal whether

Gram-negative or Gram-positive bacteria (see figure 2 and table 2), there are

four important criteria that need to be met (Diggle et at. 2007).

1. The production of the quorum-sensing signal should take place

either during specific stages of growth or response to particular

environmental changes

2. The quorum-sensing signal should accumulate in the extracellular

environment and be recognized by a specific bacterial receptor.

3. The accumulation of a critical threshold concentration of the

quorum-sensing signal should stimulate a concerted response.

4. The cellular response should extend beyond the required to

metabolize or detoxify the molecule.


18

Table 2. Identified quorum sensing signals and virulence factors controlled by QS system in marine and pathogenic bacteria (Dobretsov et al. 2009)

Autoinducers

Bacteria

Signal synthase

Phenotypes and virulence factors controlled by QS

Reference

N-acyl homoserine lactones 3-oxo-C6-HSL 3-oxo-C10-HSL 3OH-C4-HSL C4-HSL

Vibrio fischeri Vibrio anguillarum Vibrio harveyi Pseudomonas aeruginosa

LuxI VanI LuxM RhII

Light production Virulence Bio-luminescence and biofilm production Biofilm maturation and adhesion

Nealson et al. 1970 and Eberhard et al. 2004 Defoirdt et al. 2004 Waters and Bassler 2005 Waters and Bassler 2005

3-oxo-C12-HSL Pseudomonas aeruginosa LasI Virulence production Pearson et al. 1994 3-oxo-C8-HSL Agrobacterium tumefaciens TraI Conjugation transfer of the

virulence plasmid Cha et al. 1998 and Fuqua et al. 2001

C4-HSL and C6-HSL Aeromonas hydrophyla, Aeromonas salmonicida

AhyI AsaI

Biofilm formation Enzyme production

Swift et al. 1999

C6-HSL Chromobacterium violaceum CvI Violacein, antibiotics and enzyme Cha et al. 1998 C4-HSL Seratia marcescens SwrI Swarming Miller and Bassler 2001 C6-HSL, Oxo-C6-HSL and C8-HSL

Yersinia enterocolytica Y. pseudotuberculosis

YenI YpsI

Motility aggregation Miller and Bassler 2001

C6-HSL, C8-HSL Roseobacter spp Marinobacter sp.

? ?

? ?

Gram et al. 2002

C6-HSL, 3-oxo-C6-HSL Sponge associated Vibrio sp. (Vibrio campbellii)

? ? Taylor et al. 2004

AHLs Silicibacter-Ruegeria subgroup

? ? Mohamed et al. 2008


19

Table 2. (Continued) Autoinducers

Bacteria

Signal synthase

Phenotypes and virulence factors controlled by QS

Reference

C12-HSL, C6-HSL 6-day old biofilm Vibrio alginolyticus

? ? Huang et al. 2007

Autoinducer peptide (AIP) Group 1 thiolactone

Staphylococcus aureus

AIP-I

Virulence

Lyon et al. 2000

Cyclic thiolactone Staphylococcus aureus AIP-II Virulence Zhang and Dong 2004 Group III thiolactone Staphylococcus aureus AIP-III Virulence Lyon et al. 2000 Group IV thiolactone Staphylococcus aureus AIP-IV Virulence Lyon et al. 2000 ADPITRQWGD Bacillus subtilis ComX Sporulation Waters and Bassler 2005 ERGMT Bacillus subtilis CSF Competence

Sporulation Waters and Bassler 2005

EMRLSKFFRDFILQRKK S. pneumonie CSP Competence Waters and Bassler 2005 γ-butyrolactones γ-butyrolactone

Streptomyces griseus

A-factor

Induce biosynthesis of antibiotics Waters and Bassler 2005

Diketopiperezines (DKP) Cyclo(Ala-l-Val) and cyclo(I-Pro-I-Tyr)

Pseudomonas aeruginosa

?

Cross species communication

Holden et al. 1999

Autoinducer-2 Furanosyl borate diester

Vibrio harveyii

AI-2

Luminescence

Chen et al. 2002

Unidentified signal CAI-1 V. cholerae Virulence Miller et al. 2002 V. parahaemolyticus CqsA Virulence Miller et al. 2002 Vibrio harveyii Henke and Bassler 2004 (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran

Salmonella enterica sv Typhimurum

AI2 Virulence gene expression Miller et al. 2004


20

Acyl homoserine lactone (AHL)

Autoinducer peptide (AIP)

Autoinducer-2 (AI-2)

Figure 2. Representative of chemical structure of different autoinducer molecules identified in Gram-positive and Gram-negative bacteria.


21

2.3.1.1 Acyl Homoserine Lactones (AHLs)

Figure 3. Production of AHL signal by Gram-negative bacteria. The LuxI protein produced the AHL signal. AHL signaling molecules diffuse freely through the plasma membrane. AHL signal increases as bacterial population increases. If certain threshold in reached, the AHL signal binds to the LuxR protein, a cognate response regulator. The LuxR-AHL transcriptional co-activator complex also will binds at the LuxICDABE promoter as a result this activates transcription of this operon (redrawn after Asad and Opal 2008).

Acyl homoserine lactone system is widely used among gram-negative

bacteria as the sole signal molecules (Natrah et al., 2011b and Asad and

Opal, 2008) and was first described in Vibrio fisheri (Nealson, 1977). AHL

molecules are organic compound that are highly soluble and freely diffusible

through the plasma membrane (Figure 3). Gram-negative bacteria produces

different type AHL molecules, which vary in the N-acyl side chain length (from

4 to 18 carbons), with the degree of saturation, and the number of oxygen

substitution. The L-isomeric structure of the homoserine lactone ring is

common to all AHLs molecules (Asad and Opal, 2008). In addition, the

number of acyl group connected to the homoserine lactone moiety confers

species specificity to each AHL molecule. The LuxI protein is responsible in

production of a specific acylated homoserine lactone (AHL) signaling

molecules. As the bacterial population density increases, extracellular

concentration of AHL increases. When a critical threshold is reached, the AHL


22

signals are detected and bind to the cognate LuxR protein, a response

regulator. The LuxR-AHL transcriptional co-activator complex binds at the

LuxICDABE promoter and activates or inactivates transcription of this operon.

2.3.1.2 Peptide Auto-inducers

Figure 4. A general model for peptide-mediated quorum sensing in many Gram-positive Bacteria. A peptide signal precursor locus is translated into a precursor protein, subsequently, cleaved producing the processed autoinducer peptide (AIP). The AIP signal is get transported out of the cell via ATP binding cassette an ABC transporter. When extracellular concentration of AIP reached a certain threshold, a sensor kinase detects the increasing AIP concentration. The sensor kinase protein is activated to phosphorylate the cognate response regulator. The phosphorylated response regular activates the transcription of target genes. (redrawn after Miller and Bassler, 2001).

Peptide-mediated quorum sensing has been found exclusively in Gram-

positive bacterial species. This type of QS system is somewhat related to AHL

system in gram-negative bacteria. Instead, of using AHL as signal molecules

Gram positive bacteria use oligopeptide autoinducers as the primary signaling

molecules. It starts with the peptide precursor locus getting translated as

precursor peptide signals, which are subsequently cleaved to produce the

processed autoindcer peptide (AIP) shown in figure 4. Frequently, AIP contain

side-chain modification such as isoprenyl group (Bacillus subtilis) or thio-

lactone rings (Stahylococcus spp.) for intra-species communication (Henke


23

and Bassler, 2004). The processed AIP is then secreted out of the cell via

ATP-binding cassette (ABC) transporter. When extracellular concentration of

AIP reached a certain threshold, the sensor kinase detects the extracellular

concentration of AIP and allows to autophosphorylates on a conserved

histidine residue (H) and subsequently, transmits sensory information via

phosphorylation of a cognate two-component response regulator protein. The

response regulator is phosphorylated on a conserved aspartate residue (D).

Lastly, phosphorylation of the response regulator activates the control

transcription of quorum-sensing target genes which eventually drives the

expression of bacterial phenotype (Henke and Bassler, 2004; Miller and

Bassler, 2001).

2.3.1.3 QS system hybrid for Vibrio harveyi extra Autoinducer-2

Figure 5. The hybrid two-component quorum sensing system of Vibrio harveyi. The QS signal HAI-1 (an AHL) and AI-2 is get biosynthesis by LuxM and LuxS, respectively. HAI-1 and AI-2 are both detected at the cell surface. LuxN receptor protein is responsible for detection of HAI-1 and LuxP-LuxQ receptor protein is responsible for AI-2 detection. In the absence of QS signal, both receptor proteins autophosphorylate and transfer phosphate from LuxU to LuxO. The phosphorylated LuxO is an active repressor for the target genes. At high cell density, LuxN and LuxPQ interact with their respective QS signal and change from kinases to phosphatases that drain phosphate away from LuxO via LuxU. The dephosphorylated LuxO is inactive. Subsequently, LuxR binds the LuxCDABE promoter and activates transcription (redrawn after Miller and Bassler, 2001)

Chapter 2 Quorum sensing disruption

24

An HAI-1 (AHL 3-hydroxybutanoyl-homoserine lactone) QS signaling

molecule in Vibrio harveyi is known as AHL, a signal that are use in many

Gram-negative bacteria. A HAI-1 molecule for Vibrio harveyi is use for intra-

species communication. However, it is known that Vibrio harveyi have an

extra cell-to-cell communication for inter-species communication called

LuxS/PQ system or autoinducer 2 (AI-2) denoted as furanosyl borate diester,

3a-methyl-5,6-dihydrofuro-[2,3-d][1,3,2]dioxaborole-2,2,6,6a-tetraol (Surette

et al. 1999; Taga and Bassler 2003). Unlike other gram-negative quorum

sensing systems in which AHL molecules are detected by cytoplasmic

response regulator (LuxR), detection of HAI-1 and AI-2 in V. harveyi occurs in

the periplasm via cognate response regulator LuxN and LuxPQ respectively,

shown in figure 5 (Taga and Bassler, 2003). The AI-2 synthesis, called LuxS

and LuxS homologues produce the molecule 4,5-dihydroxy-2,3-pentanedione

(DPD), which undergoes a variety of spontaneous chemical rearrangements

to form the final AI-2 (Camilli and Bassler, 2006: Irie and Parsek, 2008; Henke

and Bassler, 2004). This catalysis of DPD is widespread across bacterial

kingdom (both Gram-positive and Gram-negative), pathways for AI-2

biosynthesis (Gonzales and Keshavan, 2006). In addition, this reflects that AI-

2 quorum sensing circuit is used for interspecies bacterial communication

(Miller and Bassler 2001). Different species of bacteria recognize distinctly

rearranged DPD moieties, which allows bacteria to respond to AI-2 derived

from their own DPD and also to that produced by other bacterial species

(Camilli and Bassler, 2006).

2.3.2 Disruption of bacterial cell-to-cell communication as a novel strategy to fight bacterial infection

2.3.2.1 pH-mediated AHL lactonolysis From a review of the literature, Decho et al., (2009) found various fluctuation

of pH determined the AHLs having different acyl-chain length and were

equally susceptible to pH-mediated hydrolysis. The result showed differential

degradation of AHLs molecule ranged from 30min to > 100 hrs as a function

of acyl-chain length (the value is expressed in half life, t1/2). This result


25

concurs with the previous work (Voelkert and Grant, 1970; Michels et al.,

2000 and Yates et at., 2002). Moreover, It appears that AHLs molecule having

longer acyl-chains (C12-C16) were significantly less susceptible to hydrolysis

of lactone compare to shorter acyl-chain (<C10).

A parallel study conducted by Yates et al., (2002) reported the fate of C3-HSL

and C4-HSL, and was pH influenced the reaction of both AHLs molecules.

Results indicate that C3-HSL ring remained intact at pH 2 and was completely

opened at pH 7. Meanwhile, C4-HSL lactone ring remained intact until pH 5 to

6 and was completely hydrolyzed at pH 8 to form N-butanoylhomoserine (the

open-ring of C4-HSL) as shown in figure 6. Adjusting the pH to 2 can reverse

lactonolysis. They also suggest that the ring of C4-HSL was more stable than

that of C3-HSL molecule. Lastly, they postulated that neither HSL nor C3-HSL

will be useful as quorum sensing signal molecules since they rapidly

hydrolyze at pH below physiological level, thus, suggesting that C4-HSL is

likely to be the shortest-chain of AHLs QS signal that are useful to bacterial

cell-to-cell communication (Yetes et al., 2002).

Figure 6. Inactivation of AHL via pH-mediated lactonolysis

2.3.2.2 Chemical inactivation of QS signals On the other hand, Borchardt et al., (2001) demonstrated the effects of

oxidized halogen antimicrobials hypochlorous and hypobromous acids on

AHL molecules. Result showed that these antimicrobials rapidly react and

destroyed 3-oxo-acyl HSLs activity, while acyl HSLs not possessing the β-

keto group was unaffected. Moreover, the author also manifested despite high

concentration of polysaccharide biofilm component present, rapid inactivation

3-oxo HSLs could occur. In a collaborative study, Michels et al., (2000)


26

verified the inactivation of 3-oxo acyl HSL mechanism by liquid

chromatography and mass spectrometry. It showed that the reaction kinetics

is largely influenced by the pH of the reaction mixture. At pH 6, it was found

out that acyl HSL molecule containing 3-oxo moiety reacts quickly with both

hypochlorous and stabilized hypobromous acids, yielding a 2,2-dihalo-3-oxo

HSL molecule. Subsequently, the acyl chain is hydrolyzed, yielding a products

of the reaction are 2,2-dihalo-N-ethanoyl-L-homoserine lactone and carboxylic

acid. At pH 3, both dihalogenated and monohalogenated are detected.

Whereas at pH 8, the lactone ring 2,2-dihalo-N-ethanoyl-L-homoserine

lactone is hydrolyzed, yielding 2,2-dihalo-N-ehtanoyl-L-homoserine.

2.3.2.3 Interference of AI-2 mediated QS signaling

Xavier and Bassler (2005) reported a significantly greater bioluminescence

disappearance of V. harveyi when E. coli-LsrR- strain (AI-2 importer) was

added in the medium. In addition, mixing V. harveyi with E. coli LsrR- strain

demonstrate that production of light by V. harveyi was reduce due to the

constitutive removal or chemical interconversions of AI-2. These findings

imply that induction of Lsr genes in E. coli results in assembly of the V.

harveyi AI-2 transporter and subsequent consumption of AI-2 molecule. They

concluded that the interference with AI-2 QS-signaling affects the entire QS

gene expression. Furthermore, Defoirdt et al., (2006) investigated AI-2

disruption using natural and synthetic brominated furanones. In vivo result

shows that pathogenicity of Vibrio campbelli towards Artermia was disrupted

upon introduction of 20 mg/Liter of synthetic furanone. This suggests that AI-2

signaling was disrupted when funanones compound was added in the

medium.

2.3.2.4 QS signals biodegradation by bacterial AHL lactonase and

acylase Several AHL-degrading enzymes identified across bacterial kingdom have the

potential to be used as quorum quenchers (QQ). These enzymes are capable

to degrade of AHL molecules, which resulted in reduced activity of QS signals

(Dong et al., 2000; Dong and Zhang, 2005; Bauer and Robinson, 2002). The


27

mechanism of enzymatic biodegradation of AHL lactonase, hydrolyzed the

lactone ring, yielding in acyl-homoserine. Meanwhile, AHL acylase cleaves

the acyl-group resulting in homoserine lactone and fatty acid. Apparently, AHL

lactonase and acylase enzymes are used by bacterial in order to interrupt QS

signaling of other species, probably used as a defense mechanism against

antibiotic-producing bacteria in the ecological niche (Gonzalez and Keshavan,

2006) sources of carbon, nitrogen and energy (Uroz et al. 2007; Leadbtter

and Greenberg 2000) and biocontrol (Rasmussen and Givskov, 2006; Dong

and Zhang, 2005).

2.3.2.5 Interference with QS antagonists From a review of the literature, Dobretsov et al., (2009) found various bacteria

produce QS signals that, in turn, can interfere with QS signaling in other

bacteria. More specifically, Swift et al. (1999) reported that 3-oxo-C12-HSL at

a concentration 10 µM inhibited virulence factor production by the aquatic

pathogens Aeromonas hydrophila and A. salmonicida. In addition, AHL

molecules (C6-HSL) could stimulate the production of the pigment violacein,

exoprotease and chitinase in QS regulated Chromobacterium violaceum,

while this phenotype could also be inhibited by the QS signal from

Pseudomonas aeruginosa (3-oxo-C12-HSL). The stimulatory or inhibitory

effects was linked to the structure of the acyl side chain of the molecules.

Meanwhile, some bacteria are able to produce dipeptides substance

(diketopiperazines, DKPs) that can act as AHL structural mimicry and affect

QS system of bacteria by binding to AHL receptor proteins (Dobretsov et al.,

2009). At the same time, Chlamydomonas reinhardtii, a unicellular fresh water

chlorophyte, was also found to secrets a number of substances that mimic the

activity of bacterial quorum sensing (Teplitski et al., 2004). It was documented

that compound secreted by the alga stimulated both independently and

synergistically by an AHL (3-oxo-C4-HSL= AHL) and by a furanosyl borate

diester (AI-2).

To date, many studies have been conducted on halogenated furanones that

are naturally produced by the red alga Delisea pulchra furanones have been


28

shown to possess inhibitory activity against AHL-mediated signaling. Previous

work has shown that furanones from D. pulchra and their synthetic analogues

are capable of disrupting QS-regulated behaviors in gram-negative bacteria

(Defoirdt et al., 2004). A study conducted by, Manefield et al., (1999) showed

that halogenated furanones, at the concentration produced by algae, are

capable of displacing OHHL molecules from the cognate LuxR receptor

protein.

Chapter 3 Materials and methods

29

CHAPTER 3: MATERIALS AND METHODS

3.1 In vitro: Isolation of AHL-degrading bacteria from algal culture

Five test specimens of marine micro-algae cultured in xenic condition were

tested for isolation of bacterial AHL degraders. All the xenic micro-algal

strains Tetraselmis suecica, Isochrysis affinis galbana (T-Iso), Chaetoceros

muelleri and Pavlova lutheri were obtained from the algal culture collection of

Laboratory of Aquaculture & Artemia Reference Center. Meanwhile, water

samples for an outdoor pond algal culture dominant with Skeletonema sp

were also included. Fifty µl from each of the non-axenic cultured algae were

transferred to a sterile 50 ml erlenmeyer flask containing 5 ml of minimal

culture medium (30g NaCl l-1), following the addition of 50 mg l-1 N-hexanoyl-

L-homoserine lactone (HHL) and 50 mg l-1 N-(3-oxo-hexanoyl)-homoserine

lactone (OHHL) in each Erlenmeyer flask. Isolation of QS signal degrading

bacteria was done using AHL molecules (HHL and OHHL) as the sole

sources of nitrogen and carbon. Erlenmeyer flask was covered with aluminum

foil to prevent the growth of algae. For the control, each flask would be without

the addition of HHL and OHHL. The cultures were placed on a shaker (120

r.p.m.) at 28 °C. The isolation was performed in four consecutive cycles; 72

hrs incubation for the 1st cycle, and 48 hrs for the 2nd to 4th cycle. At the end

of each cycle, 50µl of each sample was transferred to a new flask containing

5ml of fresh medium, and 100µl of each sample were plated in a non selective

medium marine agar (MA, Difco Laboratories, Detroit, USA) were number of

colonies were observed and counted.

Bacterial colony growing at the end of the fourth cycle with addition of AHL

molecules would be bacterial QS signals degrading strain, were then

inoculated to marine agar (MA) plates and incubated at 28°C for 48 h. Pure

bacterial colonies with different morphologies were selected and re-grown in

marine broth (MB, Difco Laboratories, Detroit, USA) and stored at -80°C with

40% glycerol for pure stock.


30

The QS signals degrading strain were made resistant to 50mg l-1 rifampicin.

Pure QS strain were grown till high density, then 10-8 cfu ml-1 of QS strain

were inoculated to the Erlenmeyer flask containing Marine broth with 50mg l-1

rifampicin and was placed on a shaker (120 r.p.m.) at 28°C. After, one percent

volume of the inoculated QS strain with 50mg l-1 rifampicin was then

transferred to a fresh Marine broth containing with 50mg l-1 rifampicin. The QS

strains rifampicin resistant were then preserved in 40% glycerol at -80°C.

3.2 Quorum sensing (QS) bacterial strains growth condition Chromobacterium violaceum CV026 was used as a reporter strain, which

contains a plasmid (protein regulator CViR) that produces purple pigmentation

(violacein) in response to exogenous HHL. This strain cannot produce AHL,

but able to detects AHL’s molecule with acyl-side chains of 4 to 8 carbon

atoms. This strain was grown in Luria-Bertani (LB) medium supplemented

with 20 mg L-1 of kanamycin and was placed at 28°C with constant agitation

(120r.p.m).

Two strains were used P3/pME6000 (negative control), Pseudomonas strains

carrying a plasmid without aiiA gene and P3/pME6863 (positive control),

Pseudomonas strain harboring a plasmid carrying aiiA gene from the soil

bacterium Bacillus sp. A24, responsible for AHL degradation. These strains

were grown in marine broth (MB) supplemented with 20 mg L-1 of tetracycline

and were incubated in the shaker (120r.p.m) for 24 h at 28°C.

For the pathogenic strain, Vibrio harveyi BB120 and Vibrio anguillarum

LMG4437 strains were grown in marine broth (MB, Difco). Bacterial growth

was measured using spectrophotometer (Thermo Spectronic) at OD550.

3.3 Quorum sensing molecules N-hexanoyl-L-homoserine lactone (HHL) molecule (Fluka) and N-(3-oxo-

hexanoyl)-homoserine lactone (OHHL) molecules (Fluka) were used in this

study.


31

3.4 Bacterial density determination Bacterial density in the suspension was checked using a spectrophotometer

(Thermo Spectronic). The optical density (OD) was measured at a wavelength

of 550 nm. The bacterial density was determined according to McFahrland

standard (Bio Merieux, France) by using the following formula:

Bacterial density (cfu ml-1) = 1.2 x109 x OD550 x df

3.5 Detection of hexanoyl homoserine lactones (HHL) Standard curve of different HHL concentration were developed using different

concentration of HHL. Different dilution series were prepared with the

following HHL concentration; 1, 2.5, 5, 7.5 and 10 mg l-1. Reporter strain

Chromobacterium violaceum (CV026) was cultured for two days in buffered

LB (pH 6.5) broth containing 20mg l-1 kanamycin. 100µl of the reporter strain

were then spread evenly on Luria Bertani (LB) agar plates and subsequently

10 µL of each of HHL concentration was dropped to the center of the buffered

LB agar plates (pH 6.5). The plates were then incubated for 24 hours at 28°C.

The diameter of the purple violacein zone was measured and correlated to the

concentration of HHL. This will be the basis in determining the concentration

of the HHL in the supernatant of the QS signal degrading strain.

3.6 AHL degradation assay Each of this axenic AHL degrading strain was performed in 50ml Erlenmeyer’s

flasks containing 5 ml of buffered Marine broth medium (pH 6.5)

supplemented with 10mg l-1 HHL. The AHL degrading strains were inoculated

into MB medium at 106 CFU ml-1 (Low density AHL assay) and 108 CFU ml-1

(high density AHL assay; in pure and mixed culture isolate). Pseudomonas

P3/pME6000 and P3/pME6863 strains were inoculated as a negative and

positive control, respectively, and grown under the same culture condition.

Addition of one negative control was added with no bacteria in the medium.

The flasks were placed on a shaker (120r.p.m.) at 28°C.


32

Degradation of HHL was assessed at 0, 3, 6, 9, 12, 24, 48, and 72 h. At

regular time intervals, 200µl samples from each culture were taken and

centrifuged for 10 min at 5000r.p.m. and stored at -20°C. The HHL

concentration in the cell-free supernatant was determined. Ten µl of the cell-

free supernatant were spotted in 3 parts of the buffered Luria Bertani agar (pH

6.5), on which 100µl of CV026 culture (Optical density = 2) were already

spread over the buffered LB agar. The plate was incubated for 24-48hrs at

28°C. The diameter of the purple violacein zone was measured and the

residual concentration of HHL in the supernatant was determined based on

the standard curve.

3.7 Co-culture of algae and QS signal degrading bacteria (Algae and

Bacteria interaction)

3.7.1 Source of Microalgal strains Three axenic microalgae species were used in this study, Tetraselmis

suecica, Isochrysis affinis galbana (T-Iso) and Chaetoceros muelleri, which

were kindly provided by the Culture Collection of Algae and Protozoa (CCAP,

Dunstaffnage Marine Laboratory, Scotland), Collection of Algae University

Gottingen (SAG) and Provasoli-Guillard National Center of Marine

Phytoplankton (CCMP). All samples were deposited at the Laboratory of

Aquaculture & Artemia Reference Center, University of Ghent, Belgium.

3.7.2 Axenic culture of microalgae in seawater with antibiotic

Each axenic micro-algal strain was cultivated in F/2 (Table 3) medium (+Silica

for diatom) with addition of 5 types antibiotic (kanamycin 100µl ml-1, ampicillin

250µl ml-1, gentamicin 50µl ml-1, neomycin 500µl ml-1, and streptomycin 50µl

ml-1) in 200ml sterilize Schott bottle. Autoclave seawater was previously

added in the F/2 medium with a salinity of 30 ppt for optimal growth of algae.

The Cultures were kept under constant illumination and filtered sterile aeration

at 20-22°C. Sample were taken after 10 days after inoculation, algal densities

were determined by using Burker haemocytometer. The axenic algae were

centrifuged and re-suspended in a 50ml Falcon tube containing 10ml of F/2


33

medium and this procedure was done twice to remove the residual antibiotic.

All handlings were performed in a laminar-flow hood to maintain axenity.

Table 3: Composition of F/2 medium Ingredients Stock solution

concentration (g/l of distilled water

Volume in 1L of filtered sea water (salinity of 30 g/L)

NaNO3 75 g /L 1 ml NaH2PO4.H20 5 g/L 1 ml F2 Trace Metal Solution - 1 ml F2 Vitamin solution - 0.5 ml Filtered seawater Salinity 30 g/L 1 L NaSiO3.9H20 30 g/L 1 ml for diatom culture

3.7.3 QS signal degrading strain growth condition Three bacterial strains (all strains were isolated from xenic algae) were

selected for their positive or negative effect toward algae (algae and bacteria

interaction). Pure QS signal degrading strain were already stored at -80°C

with 40% glycerol and were cultured on marine broth (MB). The cultured QS

were plated on MA and placed on incubator 48h at 28°C. Later, one colony

was picked and re-grown on MB. This method is to verify that only single

strain (colony) will be used for algae and bacteria interaction. Bacterial

density was determined by spectrophotometer (Thermo Spectronic), the QS

signal degrading bacteria were washed twice with autoclaved F/2 medium

(+Silica for diatom) before start of the experiment.

3.7.4 Algae and QS bacteria interaction Each of the axenic micro-algae and their respective axenic QS degrading

isolate were both inoculated in 250ml Erlenmeyer flask containing 50ml F/2

medium (+Silica for diatom) at 104 cells ml-1 and 102 cfu ml-1, respectively. As

control treatment, axenic algae and QS degrading isolate were only

inoculated in F/2 medium with the final concentration of 104 cells ml-1 and 102

cfu ml-1, respectively. The Erlenmeyer flask was placed on the shaker

(120r.p.m.) with constant illumination (4000lux) at 20-22°C for 15 days. All

parameter such as chlorophyll measurement, Optical density, algae counting

and bacterial plating were monitored every 3 days intervals.


34

3.7.5 Statistical analysis To test for differences in algal and bacterial growth between various

treatments, collected data were analyzed using independent sample T-test

and was performed using the Statistical Package for the Social Sciences

(SPSS) software, version 17.0. Mean relative fluorescence of algae was

tested at 0.05 level of significance.

3.8 In vivo test: Artemia experiment

3.8.1 Axenic culture of microalgae in Instant Ocean Three axenic micro-algal strain (Tetraselmis suecica, Isochrysis affinis

galbana (T-Iso) and Chaetoceros muelleri) were cultivated in F/2 medium

(+Silica for diatom) added in autoclave artificial seawater containing 35g l-1 of

Instant Ocean systhetic sea salt (Aquarium Systems Inc., Sarrebourg, France)

in 200ml sterilize Schott bottle. The cultures were kept under constant

illumination and filtered sterile aeration at 20-22°C. Sample were taken after

10 days after inoculation, algal densities were determined by using Burker

haemocytometer.

3.8.2 Gnotobiotic culture of Artemia

Experiments were performed with high quality cysts of Artermia franciscana,

originating from Great Salt Lake, Utah, USA (EG Type, INVE Aquaculture

NV, Belgium). 200 mg of cysts were hydrated on falcon tube containing 18ml

of tap water for 1 h, in the course of hydration of cysts aeration was also

provided. Sterile cysts and nauplii were obtained via decapsulation, according

to the procedure from Marques et al. (2004a,b). During decapsulation, 660 µl

NaOH (32%) and 10 ml of NaOCl (50%) were added to the hydrated cyst

suspension. The reaction was stopped after 2 min by adding 14 ml of

Na2S2O3 (10g l-1). Installation 0.22 µm filtered sterile aeration was provided.

And all manipulation ware carried out under laminar flow hood.

The decapsulated cysts were washed carefully with autoclave artificial

seawater containing 35g l-1 of Instant Ocean synthetic sea salt (Aquarium


35

Systems Inc., Sarrebourg, France) over a 100 µm sieve sterile net and

transferred to a sterile 50ml screw cap falcon tube (TRP, γ-irradiated)

containing 20 ml autoclave artificial seawater and hatched for 24 h on a rotor

(4 min-1) at 28°C with constant illumination. After 24 h, Artemia nauplii

hatched, 30 nauplii were picked and transferred to sterile 50ml glass tubes

containing 20ml filtered and autoclaved synthetic seawater. The Artemia

nauplii suspension was fed with 200µl dead (autoclave) LVS3 at a density of

approximately 107 cfu ml-1, axenic micro-algae and their respective QS signal

degrading bacterial isolate was added. Three replicates were performed per

treatment. The glass tube were placed on a rotor at 4 min-1, and exposed to

constant illumination at 28°C for 48 h.

The QS signal degrading bacteria were grown in marine broth for 48 h.

Subsequently bacterial density were determined by spectrophotometer (OD

=1), bacterial cells of interest were washed twice by autoclave synthetic

seawater (35g l-1 of Instant Ocean synthetic sea salt). 200µl of the bacterial

cultures (109 cfu ml-1) was added in each treatment, so each treatment

containing 107cells ml-1 of the QS signal degrading bacteria.

3.8.3 Challenge tests The pathogen Vibrio harveyi BB120 was inoculated in marine broth for 24 h

and placed on the shaker (120r.p.m) at 28°C. The luminescence of the Vibrio

harveyi BB120 was verified after 24 h of incubation. The presence of

luminescence represents high bacterial density. The pathogen Vibrio harveyi

BB120 cells were washed with synthetic sea water (35g l-1 of Instant Ocean

synthetic sea salt), additionally the OD550 was adjusted to 0.1 (108 cfu ml-1)

and stored for 1 day at 4°C prior to addition to Artemia culture. The axenic

micro-algae, QS signal degrading bacteria and Vibrio harveyi BB120 density

used in all challenge tests were 106 cells ml-1, 107 cells ml-1 and 105 cfu ml-1,

respectively.


36

3.8.4 Survival and growth of Artemia

The percentage survival of Artemia was scored 48 h after the addition of the

strains of interest (micro-algae, QS strain and BB120). Subsequently, live

Artemia were fixed with Lugol’s solution to measure their individual length (IL),

using a dissecting microscope equipped with a drawing mirror, a digital plan

measure and the software Artemia 1.0.

3.8.5 Statistical analysis For this experiment, the mean survival and IL of larval Artemia was compared

to the mean survival and IL in the pathogen control treatment. The differences

in survival and IL of Artemia culture in different conditions were investigated

with independent sample T-test, and analysis of variances (ANOVA), Tukeys

multiple comparison range, respectively. Statistical Package for the Social

Sciences (SPSS) software, version 17.0 was used. Mean survival and

Individual length (IL) were tested at 0.05 and 0.01 level of significance.

3.9 In vivo test: Mussel experiment: 3.9.1 Mussels D-veliger larvae

Mussel D-veliger larvae (Mytilus edulis) were used in this experiment, mussel

larvae were obtained from Mieke Eggermont, Nancy Nevejan and Aaron

Plovie. The larvae were immediately washed two times with artificial seawater

containing 35g l-1 of Instant Ocean synthetic sea salt (Aquarium Systems

Inc., Sarrebourg, France) over a 30µm sterile sieve and allowed to

acclimatize in artificial seawater (Instant Ocean).

3.9.2 Bacterial pathogenic strain The pathogenic strains Vibrio harveyi BB120 and Vibrio anguillarum

LMG4437 were inoculated in MB and placed on shaker (120r.p.m.) at 28°C for

24h. The overnight cultures were diluted to adjust the OD550 0.1 (108 cfu ml-1).


37

3.9.3 QS signal degrading bacterial strain From -80°C stock QS signal degrading bacteria were re-grown in marine broth

for 48 h. After, bacterial density was determined by spectrophotometer

(OD550=1). 50µl of QS signal degrading bacterial culture was added in each

treatment, so each treatment containing 107cfu ml-1 of the QS signal

degrading bacteria

3.9.4 Challenged tests Six aliquots were taken randomly from the base population for counting.

Subsequently, total number of mussel larvae from a base population was

determined, and it was adjusted to the desired base population (30 D-veliger

larvae ml-1). During the challenge test, 1ml from the base population

approximately containing 30 larvae was transferred to a 6 well transparent

microplate containing 4ml of artificial seawater (35g IO l-1). Each treatment

was done in triplicate. Afterward, bacterial pathogenic strain, QS signal

degrading bacteria and axenic micro-algae were added in each treatment with

the density of 106 cfu ml-1, 107 cfu ml-1 and 106 cfu ml-1, respectively. Survival

of mussel larvae was monitored after 72 h of incubation.

3.9.5 Statistical analysis For this experiment, the mean survival of mussel larvae was compared to the

mean survival in the pathogen control treatment. The differences in survival

were analyzed by an independent sample t-test, using Statistical Package for

the Social Sciences (SPSS) software, version 17.0. Mean survival and

Individual length (IL) were tested at 0.05 and 0.01 level of significance

38

CHAPTER 4: RESULTS

4.1 In vitro experiment:

4.1.1 Isolation of AHL-degrading bacteria from algal culture

In the present investigation, 11 different strains of potential bacterial QS

degraders were collected from 5 species of microalgae. These

microorganisms were selected following the AHL enrichment procedure. For

the preliminary screening, all of the bacterial strain were collected and tested

for their AHL degradation activity for 24 h. The results showed that 3 strains

corresponding to 18% of the total strains isolated inhibited the

Chromobacterium violaceum CV026 to produce purple pigmentation

(violacein). These strains degrade HHL within 24 h, were selected for more

detailed study. The bacterial isolates from each algal sample were given a

corresponding bacterial code; T2, I3 and C2 isolated from Tetraselmis

suecica, Isochrysis affinis galbana (T-Iso) and Chaetoceros muelleri,

respectively.

4.1.2 Detection of hexanoyl homoserine lactones (HHL) Diameter of violacein (purple pigment) produced by Chromobacterium

violaceum CV026 strain correlated proportionally to the amount of HHL

concentration tested at five different concentrations (1, 2.5, 5, 7.5 and 10ppm)

(Figure 7). The concentration of HHL was determined according to the

standard curve equation with regression coefficient R2 value higher than 0.9.

Chapter 4 In vitro: AHL degradation assay

39

Figure 7. HHL standard curve of CV026 in AHL quenching assay

4.1.2 AHL degradation assay The QS degrading bacteria (T2, I3 and C2) were inoculated at 106 CFU ml-1

and 108 CFU ml-1 (for pure and mixed culture AHL assay) in buffered marine

broth (MB) supplemented with 10 mg l-1 HHL. HHL concentration was

checked every 3 h interval at 0, 3, 6, 9, 12, 24, 48 and 72h.This experiment

will enable us to draw conclusion on whether the isolates are able to degrade

AHL’s molecules in nutrient rich environment.

4.1.2.1 Single pure QS degraders Axenic QS degrader strains (T2, I3 and C2) were tested for their AHL

degradation activity for 72 h. The test strains were inoculated at 108 CFU ml-1,

grown in a buffered MB medium (pH= 6.5) supplemented with 10mg l-1 HHL

and was placed on the shaker (120r.p.m) for monitoring. Each test strains

were monitored and subjected for 3 h interval at 0, 3, 6, 9, 12, 24, 48 and 72h

for HHL concentration. This will show us how fast the degradation activity is

each of the QS degrading isolates in MB medium. Based on HHL degradation

experiment, positive control P3/pME6863 showed highest degradation activity

where HHL concentration was below detection limit within 6h (Figure 9).

Meanwhile, QS strain I3 and C2 degraded HHL below detection limit after 12

h, while T2 degraded HHL after 24 h of incubation. In addition, it was evidently

demonstrated that C2 was the strongest HHL degrader among the three QS

y = 3.393x -‐ 2.469 R² = 0.957

0

2

4

6

8

10

12

0 1 2 3 4

HHL concen

tra�

on m

g l-‐1

Average diameter (cm)

AHL conc. Linear(AHL conc.)


40

degrader isolates tested. Inoculated at 106 CFU ml-1, a fast HHL degradation

activity could still be demonstrated resulting in a concentration of HHL below

detection limit within 12h (data not shown).

Figure 8. Detection of HHL degradation: samples after 72 h contact between axenic QS signal degrading strains grown in MB supplemented with HHL. 10µl of the supernatant of each QS strains was spotted in three parts over buffered LB that was already spread with reporter strain CV026.


41

4.1.2.2 Mixtures of different QS degraders In a parallel experiment, HHL concentration was below detection limit within 3

h of incubation when the three QS degrading bacteria were mixed together

(Figure 10). Whereas, mixing only two QS degraders showed 50% reduction

of HHL concentration for the first 3h and was completely degraded after 6h,

comparable to positive control P3/pME6863. For the negative control

P3/pME6000, there was an apparent 50% decrease of HHL concentration

after at least 72 hours throughout the experiment, which was probably due to

chemical inactivation of HHL.

Figure 9. N-hexaoyl-L-homoserine (HHL) degradation activity by the axenic QS signal degrading bacteria inoculated at 108 CFU ml-1 (high cell density). Monitoring of HHL concentration at different time points. The data points represent the mean values of the 3-spotted replicates of each QS degrader bacterial supernatant. T2: QS degrader isolated from Tetraselmis suecica; I3: QS degrader isolated from Isochrysis affinis galbana (I3); C2: QS degrader isolated from Chaetoceros muelleri (C2); P6000/Control: P3/pME6000/Marine Broth medium only: as negative control and P6863: P3/pME6863 as positive control

0

2

4

6

8

10

12

0hr 3hrs 6hrs 9hrs 12hrs 24hrs 48hrs 72hrs

HHL concentration mg l-1

Time (h)

T2 I3 C2

P6000 P6863 Control


42

Figure 10. N-hexaoyl-L-homoserine (HHL) degradation activity by mixed culture of QS signal degrading bacteria inoculated at 108 CFU ml-1 (high cell density). Monitoring of HHL concentration at different time points. The data points represent the mean values of the 3-spotted replicates of each mixed sample QS degrader bacterial supernatant. T2: QS degrader isolated from Tetraselmis suecica; I3: QS degrader isolated from Isochrysis affinis galbana; C2: QS degrader isolated from Chaetoceros muelleri; P6000/Control: P3/pME6000/Marine broth medium only: as negative control and P6863: P3/pME6863 as positive control

4.1.3 Bacterial density during 72 hour AHL degradation assay Figure 11 and 12 show the optical density of QS degrading strain during 72 h

HHL degradation assay. Bacteria were inoculated at 108 CFU ml-1 and 106

CFU ml-1 for high and low density, respectively. Based on the result, it seems

that I3 reflected the highest growth among the QS strain tested (both high and

low density). However, there are no significant differences in growth among

the QS degraders.

0

2

4

6

8

10

12

0hr 3hrs 6hrs 9hrs 12hrs 24hrs 48hrs 72hrs

HHL concentration mg l-1

Time (h)

T2 I3 C2 I3 T2 I3 C2 T2 C2 P6000 P6863 Control


43

Figure 11. 48 h bacterial density determination of axenic QS signal degrading bacteria inoculated at 108 CFU ml-1 (inoculated at high density) T2: QS degrader isolated from Tetraselmis suecica; I3: QS degrader isolated from Isochrysis affinis galbana (I3); C2: QS degrader isolated from Chaetoceros muelleri (C2); Control: marine broth medium only.

Figure 12. 48 h bacterial density determination of axenic QS signal degrading bacteria inoculated at 106 CFU ml-1 (inoculated at low density). T2: QS degrader isolated from Tetraselmis suecica; I3: QS degrader isolated from Isochrysis affinis galbana (I3); C2: QS degrader isolated from Chaetoceros muelleri (C2). Control: marine broth medium only.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 hrs 3 hrs 6 hrs 9 hrs 12 hrs 24 hrs 48 hrs

Cell density (OD 550)

Time (h)

T2

I3

C2

Cont

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 hrs 3 hrs 6 hrs 9 hrs 12 hrs 24 hrs 48 hrs

Cell density (OD 550)

Time (h)

T2

I3

C2

Cont

Chapter 4 In vitro: Algae and bacteria interactions

44

4.2 Algae and QS bacteria interaction 4.2.1 Algal growth dynamics To study algae and bacteria interaction, we used series of co-culture

experiment of Tetraselmis suecica, Isochrysis affinis galbana (T-Iso) and

Chaetoceros muelleri and 3 QS degrader isolate T2, I3 and C2. Determination

of algal fluorescence and their respective bacterial density load at different

incubation time points was measured. In relation to algal growth

(measurement of relative fluorescence), Tetraselmis suecica and

Chaetoceros muelleri exhibited significant differences between axenic and

xenic culture during all stages of incubation. Meanwhile, Isochrysis affinis

galbana (T-Iso) resulted in no significant difference between axenic and non-

axenic culture throughout the experiment (Figure 13C).

Furthermore, addition of 3 QS degrader (T1, I3 and C2) in Tetraselmis

suecica culture resulted in significantly higher growth in the presence of the 3

bacteria after 9 day (Figure 13E). The same result was obtained when T2 was

added alone to Tetraselmis suecica (Tetra and T2) culture. In contrast,

axenic Tetraselmis suecica (Tetra) culture grows slower and remained

significantly lower throughout 15th day of incubation. Relative fluorescence of

xenic Tetraselmis suecica (All QS in Tetra) culture decreased towards the end

of the experiment. Indicating, that 18th day cell went into the senescence

phase.

Meanwhile, relative fluorescence of xenic Chaetoceros muelleri (Chaeto and

C2) culture was continuously higher when incubated together with QS

degrader C2 from 9th to 15th day (Figure 13A). However, the differences were

insignificant. Interestingly, QS degrader C2 grown and added on axenic

Chaetoceros muelleri culture had more significant effect on algal growth on

the 18th day of incubation. A significant difference was also observed in

quantum yield reading during the 18th day of incubation (Figure 14). This

confirmed the result on relative fluorescence that indeed there was

significantly difference. In contrast, a minimum effect on growth was obtained


45

when Chaetoceros muelleri microalga was incubated with mixed population of

3 AHL-degrading bacteria (All QS in Chaeto). Interestingly, the quantum yield

reading was increased. Hence the latter two observations seem to contradict

each other.

4.2.2 Dynamics of bacterial growth In parallel, bacterial numbers (CFU ml-1) of axenic QS degrader and mixed

QS degrader isolate showed pronounced differences with respect to algal

species and incubation time point (figure 13B,D and F). Bacterial density in

the Isochrysis affinis galbana (T-Iso) and Chaetoceros muelleri with their

respective QS degrader I3 and C2, slightly increase during the 3rd and 9th day

of incubation and rapidly decreased thereafter. Meanwhile, pronounced

increases were observed on bacterial growth when all QS degraders isolated

were joined together with each algae (Isochrysis affinis galbana (T-Iso) and

Chaetoceros muelleri).

Further, numbers of bacteria in Tetraselmis suecica culture were similar to

those in axenic (T2) and xenic (All Qs in Tetra) culture. Meanwhile, highest

bacterial numbers were detected in the axenic bacteria (T2) and mixed culture

of 3 different QS (All QS in F/2).


46

A Chaetoceros muelleri B

C Isochrysis affinis galbana (T-Iso) D

E Tetraselmis suecica F

Figure 13. Relative fluorescence of selected microalgae incubated in F/2 medium (+ silica for Diatom culture) with and without a QS degrading isolate. Three-day interval were set in order to monitor the growth of algae and bacteria, using chlorophyll measurement (Excitation 410 nm/Emission 670 nm) and bacterial plating (in marine agar), respectively.

00

2,000

4,000

6,000

8,000

10,000

Fluorescence

Chaeto Chaeto and C2 All QS in Chaeto

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

Bacterial denstiy CFU

ml-1

C2

Chaeto and C2

All QS in Chaeto

All QS in F/2 +Si

00

200

400

600

800

1,000

1,200

Fluorescence

Iso Iso and I3 All QS in Iso

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07


ml-1

I3

Iso and I3

All QS in Iso

All QS in F/2

00

5,000

10,000

15,000

20,000

25,000

30,000

35,000

0 3 6 9 12 15 18

Fluorescence

Time (day)

Tetra Tetra and T2 All QS in Tetra

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

0 3 6 9 12 15 18


ml-1

Time (day)

T2

Tetra and T2

All QS in Tetra

All QS in F/2

Chapter 4 In vivo: Challenged test

47

Figure 14. Quantum yield reading on the 15th and 18th day of algae and bacteria cultivation. Tetra, Iso and Chaeto only: represent as the control treatment of microalgae Tetraselmis suecica, Isochrysis galbana and Chaetoceros muelleri, respectively, and Tetra and T2: Tetraselmis suecica and QS T2; Iso and I3: Isochrysis galbana and QS I3; Cheato and C2: Chaetoceros muelleri and QS C2; All QS (represent the 3 QS degrader cultivated together with the each selected microalgae). *: Significant difference in Quantum yield between the control and the treatment of interest (PT-test< 0.05).

4.3 In vivo experiment:

4.3.1 Artemia challenge test

From table 4, addition of Tetraselmis suecica (Tetra), Chaetoceros muelleri

(Chaeto) and with their respective QS degrader isolate T2 and C2 to the

culture water resulted a significantly high survival of challenged Artemia and

equally performed as good as to unchallenged Artemia. Meanwhile, significant

differences were observed for challenged Artemia that was supplied with

either axenic algae or QS degrading strain only. Interestingly, T2 QS degrader

showed unexpected result where it demonstrated a high percent survival, was

comparable to that in the unchallenged Artemia. The results suggest, better

performance can be obtained in challenged Artemia when both algae and QS

degrader are present in the culture water.

* *

*

*

* *

0

0.1

0.2

0.3

0.4

0.5

0.6

15th day 18th day

Quantum

yield

Time (day)

Tetra only

Tetra and T2

Iso only

Iso and I3

Chaeto only

Chaeto and C2

All QS in Tetra

All QS in Iso

All QS in Chaeto


48

Table 4. Percentage survival (mean ± standard error), individual length (IL mean ± standard deviation) and Vibrio harveyi BB120 concentration (cfu ml-1) of Artemia after 48 hours of post-exposure with Vibrio harveyi BB120 (105 cfu ml-1). Axenic algae and QS signal degrading bacteria were added at 106 cells ml-1 and 107 cfu ml-1, respectively. All Artemia were fed with dead (autoclave) LVS3 (equivalent to 107 cfu ml-1) at the start of the experiment.

Treatment Survival (%) IL (mm) cfu ml-1 BB120 only a

61 ± 4 0.82 ± 0.16 b 7.2 x 106

BB120 + Isochrysis galbana 85 ± 2 * 1.01 ± 0.17 a 6.6 x 106

BB120 + QS I3 58 ± 1 ns 0.77 ± 0.15 b 6.3 x 106 BB120 + Isochrysis galbana + QS I3

79 ± 3 * 1.06 ± 0.19 a 6.9 x 106

BB120 + Chaetoceros muelleri 87 ± 2 * 1.00 ± 0.15 a 5.1 x 106

BB120 + QS C2 88 ± 3 * 0.85 ± 0.13 ab 4.1 x 106 BB120 + Chaetoceros muelleri + QS C2

90 ± 2 ** 0.92 ± 0.22 ab 4.2 x 106

BB120 + Tetraselmis suecica 94 ± 6 * 0.96 ± 0.14 ab 1.4 x 106 BB120 + QS T2 90 ± 0 ** 0.83 ± 0.16 b 6.2 x 106 BB120 + Tetraselmis suecica + QS T2 99 ± 1 ** 0.98 ± 0.18 a 2.0 x 106 * Significantly different in survival between the pathogen control and the treatment of interest (PT-test< 0.05). ** Significantly different in survival between the pathogen control and the treatment of interest (PT-test< 0.01). ns Not significantly different at 0.01% level of significance a Survival and individual length of unchallenged Artemia nuplii was 99 ± 1 and 0.96 ± 0.15, respectively Means of individual length (IL) showing the same superscript letter are not significantly different (PTukey> 0.01) 4.3.2 Mussel challenge test

Table 5 shows the mean percentage survival of mussel larvae challenged with

Vibrio harveyi BB120 and Vibrio anguillarum LMG443 supplemented with F/2

medium and scored after 72 h. Result showed, that the performance of

challenged mussel larvae supplemented with QS degrading isolate (I3) and

axenic algae (Isochrysis affinis galbana = Iso) shows a high significant

difference (Pvalue< 0.01) when compared to the pathogen control (mussel

larvae challenged with Vibrio harveyi BB120 and Vibrio anguillarum LMG443)

This result confirmed that algae and QS degrading strain in combination show

significant effect in protecting mussel larvae to fight infection. Meanwhile,

addition of either axenic Isochrysis affinis galbana (Iso) or QS degrading

strain I3 challenge mussel with Vibrio anguillarum LMG4437 resulted a

significant difference (Pvalue< 0.05) when compared to pathogen control. In


49

contrast, mussel larvae that were supplemented with either axenic algae

(Isochrysis affinis galbana) or QS degrading strain I3 showed no significant

difference (Pvalue >0.01) for mussel challenge with Vibrio harveyi BB120.

Table 5. Percentage survival of mussel larvae (means ± standard error of the three replicates) after 72 hours of post exposure with Vibrio harveyi BB120 and Vibrio anguillarum LMG4437, pathogen, axenic algae and QS degrader strains were added 106 CFU/ml, 106 cells/ml and 107 CFU/ml, respectively. Each treatment was supplemented with F2 medium.

Treatment Survival (%) BB120 only a 33 ± 6 BB120 + Isochrysis galbana 36 ± 5 ns BB120 + QS I3 41 ± 8 ns BB120 + Isochrysis galbana + QS I3 62 ± 3 ** LMG4437 only LMG4437 + Isochrysis galbana

12 ± 1 42 ± 7 *

LMG4437 + QS I3 51 ± 12 * LMG4437 + Isochrysis galbana + QS I3 69 ± 3 ** * Significantly different in survival between the pathogen control and the treatment of interest (PT-test< 0.05). ** Significantly different in survival between the pathogen control and the treatment of interest (PT-test< 0.01). ns Not significant different between the pathogen control. a Survival of unchallenged mussel D-veliger larvae was 86 ± 1

Table 6, showed similar experimental set-up to table 5 where the only

difference is that silica was supplemented in each treatment to optimize the

growth of Chaetoceros muelleri and QS degrading strain (C2). Among the

mussel challenge, addition of axenic Chaetoceros muelleri and QS degrader

C2 showed a significantly higher percent survival compared to pathogen

control (mussel larvae post exposure with Vibrio harveyi BB120 and Vibrio

anguillarum LMG443). Whereas, addition of QS degrader C2 in the culture

water somewhat decreased the pathogenicity of Vibrio anguillarum LMG4437.

However, mussel larvae challenged with Vibrio harveyi BB120 and

supplemented with QS degrader C2 shows no significant difference when

compared to pathogen control. It is probably that QS degrader C2 did not

protect mussel from Vibrio harveyi BB120.


50

Table 6. Percentage survival of mussel larvae (means ± standard error of the three replicates) after 72 hours of post infection with Vibrio harveyi BB120 and Vibrio anguillarum LMG4437, pathogens, axenic algae and QS degrader strains were added 106 CFU/ml, 106 cells/ml and 107 CFU/ml, respectively. Each treatment was supplemented with F2 medium plus Silica.

Treatment Survival (%) BB120 only a 30 ± 3 BB120 + Chaetoceros muelleri 71 + 2 ** BB120 + QS C2 22 ± 7 ns BB120 + Chaetoceros muelleri + QS C2 64 ± 2 ** LMG4437 only LMG4437 + Chaetoceros muelleri

0 62 ± 1 **

LMG4437 + QS C2 58 ± 2 ** LMG4437 + Chaetoceros muelleri + QSC2 69 ± 3 ** * Significantly different in survival between the pathogen control and the treatment of interest (PT-test< 0.05). ** Significantly different in survival between the pathogen control and the treatment of interest (PT-test< 0.01). ns Not significant different between the pathogen control. a Survival of unchallenged mussel D-veliger larvae was 79 ± 4.

Furthermore, we tried to statistically proved the interaction by using 2-way

ANOVA between 3 algae and 3 AHL-degrading bacterial relationship, but we

failed to obtain normal distribution among the sample treatments also

transformation of the data failed to produce a normal distribution of data.

51

CHAPTER 5: DISCUSSION

5.1 Enrichment of AHL degrading bacteria from microaglae In the present study, we have successfully isolated three (T2, I3 and C2) AHL-

degrading bacteria that are closely associated from three species of

microalgae (Tetraselmis suecica, Isochrysis affinis galbana -T-Iso and

Chaetoceros muelleri). It was documented that bacteria can loosely or tightly

associated to phytoplankton within the “phycosphere”. Cole (1982) stated that

a phycosphere is a zone sorrounding the algal cell, where microoganisms are

attached and are influenced by algal extracellular products. Isolates of pure

AHL-degrading bacteria from algal cultures were obtained using AHL

enrichment procedure that was pioneered by Tinh et al., (2007b), based on

the utilization of AHL molecules served as the carbon and nitrogen sources

for the growing AHL-degrading bacteria, by subsequently plating on the

marine agar. Based on the enrichment procedure, we have confirmed that the

three isolated bacterial strain support the view that these bacteria are capable

to degrade AHL molecules. Exogenously added HHL and OHHL molecules

was utilized as a carbon and nitrogen sources. This study confirms previous

findings of a soil bacterium, Variovorax paradoxus, able to grow in limited

nutrient condition and using AHL signal molecules as the energy sources

(Leadbetter and Greenberg, 2000).

Furthermore, we have proven that AHL-degrading bacteria coexist within the

phycospere of microaglae in the aquatic environement. Similar result have

been found in the case of terrestrial plant, Uroz et al., (2003) conducted

experiment showing that AHL-degrading bacteria are present in the

rhizosphere (analogous to phycoshpere in aquatic ecosystem). The same

result was reached by Tinh et al., (2007). They obtained bacterial enrichment

cultures (ECs) isolated from the gut of Pacific white shrimp Panaeus

vannamae. They reported that the enrichment culture (EC’s) able to interferes

with the V. harveyi HAI-1 autoinducers quorum sensing mechanism in vitro.

While in vivo experiment demonstrated a significant improvement of growth

rate of rotifers (Brachionus plicatilis) challenged to pathogenic V. harveyi. This

Chapter 5 AHL degrading bacteria

52

would suggest that a wide variety of AHL-degrading bacteria could exist in

diverse environment resulting in complex host-microbes interaction.

5.2 AHL degradation activity of isolated AHL-degrading bacteria

We would like to acknowledge from this research project that this is the first

attempt to isolate AHL-degrading bacteria from microalgae.

In this study, Chromobacterium violaceum CV026, a mini-Tn5 mutant beta-

proteobacterium was used as the reporter strain. CV026 it is known to

detects and responds to a range of AHLs molecules (usually having C4 to C8

acyl side chain), by inducing the synthesis of violacein, a purple pigment

antibiotic. Mc Clean et al., (1997) mentioned that HHL signal molecule is the

most active molecule in inducing the synthesis of violacein, the natural C.

violaceum AHL. That is why C. violaceum CV026 is the most convenient tool

for biological assay in conducting this research.

In the HHL degradation assay, we found out that every bacteria we have

isolated have differences in the degree of HHL degradation activity. The HHL-

degrading abitilities of the three isolated strains (T2, I3 and C2) as well as the

mixed culture of strains were assessed by using HHL-degradation assay

(inoculation of AHL-degrading bacteria and addition of 10ppm HHL). The,

detection of the remaining HHL in the supernatant was determined every 3h

interval. The rate of inactivation (degradation) of HHL in the supernatant can

be correlated to the production of violacein in the CV026 plate by measuring

the diameter of the purple pigment. Based on the fate of HHL molecule during

the HHL assay experiment, we report that indeed there was degradation

activity of HHL molecule by AHL-degrading bacteria, hence, bacterial cell-free

supernatant that are spotted on CV026 plates exhibited no induction of purple

pigment (violacein).

The degradation properties of pure strain and mixed culture differed with

respect to their HHL degradation kinetics. In fact, results showed that the

Chapter 5 AHL degrading bacteria

53

mixed culture of 3 strains (T2, I3 and C2) degraded the exogenously added

HHL after 3h of incubation. While 2 strains mixed culture degraded HHL after

6h of incubation. This result agree with the previous findings of Dang et al.,

(2009) and Tinh et al., (2007). This implied that mixed culturing the AHL-

degrading bacteria would result in strong degradation activity of AHL

molecules. This results is supported by Flagan et al. (2003), that cocultivation

of V. paradoxus VAI-C and Arthrobacter strains VAI-A in AHL containing

medium, resulted to higher growth yield compared to monoculture strain

under the same culture condition. The authors found out that Arthrobacter

strains VAI-A efficiently utilized AHL as nitrogen source, compared to V.

paradoxus VAI-C which can assimilate lactone nitrogen very slowly. They

postulated that microbial consortia may have a synergistic effects towards the

AHL signal molecule turnover and mineralization. We therefore speculated

that the 3 AHL-degrading strains, may contain different mechanism to

inactivate the HHL signal molecule. It is possible that the 3 AHL-degrading

bacterial strains have specific enzymes to inactivate the HHL molecule. For

instance, strains carrying genes coding for lactonase and acylase (both

degrade AHL molecules) have been described. Dong et al., (2000) found a

Bacillus sp. that has a aiiA gene that is responsible in opening the lactone

ring. Similar result was obtained by Park et al., (2003) they reported that the

AhID gene from Arthrobacter sp. degrade various AHLs. On the other hand,

acylase enzyme inactive the HHL via cleaving the molecule and

subsequently, obtaining carboxyl group and homoserine lactone moiety.

Romero et al., (2005) demonstrated that AiiC gene from Anabaena sp.

inactivate the AHL molecules via acylase.

Furthermore, the best known acylases enzyme are acc gene from Shewanella

sp. (Morohoshi et al., 2005); AhIM from Streptomysec sp. (Partk et al., 2005)

and AiiD from R. Eutropa (Lin et al., 2003). Thus, it is possible that these 3

AHL-degrading bacteria that we isolated on microalgae possess more than

one type of AHL-degrading enzymes.

Chapter 5 Links between algae and bacteria

54

5.3 Links between microaglae and bacterial community, growth, function and activity

In our present study, we aim to determine the impact of AHL-degrading

bacteria on the growth of microalgae. Stimulative and inhibitory effects of

microbial communities on the growth of microaglae is a complex phenomenon

involving quantitative and qualitative aspects. Bacterial density benefited by

the secondary metabolites (extracellular exudates) that are produced by

microalgae, or bacterial cells increase when the growth rate of the microalgae

decrease, where excretion of organic products from senescent microalgae

certainly will be incorporated and used as substrate for the growing bacteria.

Meanwhile, microalgae could also be stimulate via mineralization of bacteria,

best known example is the transformation of particulate (POC) to dissolve

organic carbon (DOC)(Grossart et al., 2006).

5.3.1 Relative fluorescence differences

Our results shows that specific interactions between microalgae and AHL-

degrading bacteria strongly depend on the species of microalgae. This was

supported with the previous findings by Grossart and Simon (2007),

suggesting that the presence of the bacterial community and of specific

population have distinct effects on the growth and organic matter release of

algae. This might explain our result, that the differences of relative

fluorescence on the three microalgae (Tetraselmis suecica, Isochrysis affinis

galbana-T-Iso and Chaetoceros muelleri) were affected with the

presence/absence of bacterial community during cultivation. Bacterial

community significantly influences the development of microalgal growth. To

our knowlegde, bacterium with antagonistic effects play important role in

controling the dominance of some algal species. Skerratt et al., (2002)

reported that species specifc bacteria (Bacillus cereus, Pseudoalteromonas,

Cellulophaga lytica and Firmicutes) exhibited predatory and algicidal abilities

to antagonized harmful algae (Gymnodinium catenatum). For example,

production of proteases that is produced by Pseudoalteromonas that used to

lysed a algal cell, or by directly attacking the algal cell that is used by


55

Cellulophaga species as mode of action to control the growth of algae. In

contrast, bacteria also supply growth factors (nitrogen, phosphorus and

vitamins) that promotes phytoplankton growth, suggesting that bacteria and

algae interaction could be species specific.

In our study, we postulated that the increased of relative fluorescence of

Tetraselmis suecica with the addition of bacteria (co-cultivation and all QS

mixed culture) on the 9th and 15th day was due to stimulatory substances that

was produced by bacterial community (T2)(Figure13E). We assumed that

bacterial mineralization of T. suecica debris and lysed cellular components

are the important process for supplying T. suecica with nutrients. Grossart

(1999) reported that high growth and increased ectoenzyme activities

(aminopeptidase) of bacteria in the presence of alga indicate that enhanced

bacterial mineralization of organic particle which are rich in carbon and

nitrogen, led to increased of phytoplankton growth. However, we cannot

exclude the idea that bacteria also compete with microalgae for nutrients and

space. Probably, towards the end of the experiment no significant difference

of relative fluorescence between monoculture (axenic T. suecica) and co-

culture (T. suecica with the addition of bacterium QS T2) of T. suecica were

observed because of competition for nutrient. The decrease of relative

fluorescence on the 18th day, however, resulted in accelerated degradation of

senescent T. suecica cells and increased colonization of AHL-degrading

strain (T2) as shown in figure 13E.

Additionally, the relative fluorescence of Chaetoceros muelleri added with

AHL-degrading bacteria (QS C2), where we observe slight increase of relative

fluorescence on the 9th and 15th day. However, there was no measurable

difference on the relative fluorescence between monoculture (axenic C.

muelleri) and co-culture (C. muelleri with the addition of bacterium QS C2) of

C. muelleri culture. However, towards the end (18th day) of the experiment

rapid degradation of C. muelleri in both treatments were observed.

Interestingly, in the treated culture of C. muelleri (C. muelleri + QS C2) the

relative fluorescence on 18th day was significantly higher (P < 0.05) compared


56

to the control (axenic culture C. muelleri). On the other hand, C. muelleri

growth was retarded with the presence of the 3 AHL-degrading strains. This is

possible that competition of nutrients and space influenced a significant

reduction of relative fluorescence in all stages of C. muelleri growth (added

with 3 QS degrading bacteria). We firmly suggest the stimulation/inhibition of

algal growth by bacterial community is largely influences by environmental

conditions. Thus, nutrient limitation can alter algae and bacteria interaction.

5.3.2 Relationship between Quantum yield (ΦΦ), accessory pigment and

nutrient availability

Photosynthesis is a quantum process. In oxygenic photosynthesis, it is

defined as the maximum rate of O2 evolved or CO2 fixed per mole of photons

absorbed inside the reaction center (photosynthetic apparatus) at irradiances

which are subsaturating to photosynthesis (Cleveland et al., 1989) Therefore,

a quantum yield of 0.1 O2 (mol quanta) corresponds to a quantum

requirement of 8 quanta for every molecule of oxygen evolved. Since for each

molecules of oxygen in the reaction center, two molecules of water are

photochemically oxidized, leading to the production of four electrons and four

protons (Babin et al. 1995). It is been postulated by many biological

oceanographers that the quantum yield is always constant. However, it must

be stressed out those environmental factors, such as nitrogen availability and

nitrogen redox state (Cleveland et al. 1989), temperature (Sosik and Mictchell,

1994) and photoprotectant pigment (zeaxanthin) (Bidigare et al., 1989 and

Babin, 1995) can affects the photosynthetic activity or reduction in the

quantum yield of microalgae.

Babin et al., (1995) enumerated the potential reasons for a reduction in the

maximum quantum yield.

1. Photosynthetic organism contains pigments that absorb

photosynthetically available radiation (PAR) but do not transfer the

absorbed excitation energy to a photochemical reaction center.

2. Numbers of photochemically compotent reaction centers can vary as a

function of irradiance (light) or nutrient status


57

3. Cyclic electron flow around either photosystem 1 or photosystem 2

permits photochemical utilization of absorbed excitation energy without

concomitant production of O2 or reduction of CO2.

Our results show support the above statement, firstly, the difference of

quantum yield obtain among cultivated microalgae can be manifested by the

difference pigments contain in each microalgae. For example, we observe

that the quantum yield reading of Chaetoceros muelleri is always lower when

we compare the quantum yield reading of both Tetraselmis suecica,

Isochrysis affinis galbana-T-Iso cultures. To our knowledge, Chaetoceros sp

contain extra pigment called xanthophyll, Kashino and Kudoh (2003) reported

that this pigment act as a photoprotection mechanisms for algal

photosynthesis systems under excess irradiance. Meanwhile, Bidigare et al.,

(1989) demonstrated that zeaxanthin (xanthophyll caroteniods) serves an

important function as a photoprotectant pigment in Synechococcus clone

WH7803 (coccoid marine cyanobacteria), and as such can significantly

decreases (20 to 40%) on the quantum yield for photosynthesis. They

concluded that carotenoid serves as photoprotectant, and are capable to

quenching the triplet state of photosensitizing molecules; single oxygen; and

free radicals, all of which are potentially destructive in the photosynthetic

system.

Babin et al., (1995) mentioned that nutrient always play an important role in

determining the photosynthetic yield in nature. Thus, based on our finding the

difference on quantum yield between axenic and xenic (microalgae and QS

bacteria) culture of microalgae, were best explain by the nutrient (nitrogen)

availability and nitrogen redox state on the culture medium. It is possible that

bacterial mineralization production by AHL-degrading bacteria contribute

significant difference of quantum yield among cultivated xenic microalgae. In

xenic condition, we assume that ammonium regeneration rates are higher and

microalgae growing rapidly, which resulted in higher quantum yield reading.

On the onther hand, Cleveland et al., (1989)

Chapter 5 Beneficial effects of algae and bacteria interaction,

58

reported that higher nitrate concentration may result on lower quantum yield,

hence, reduction of nitrate to ammonium competes with CO2 fixation for

photochemically produced reductant. As a consequence, qauntum yield will

be approximately 25% lower for growth in nitrate compared to ammonium.

5.4 Beneficial effects of algae and bacteria interaction towards aquatic organism In this study, the objective of these experiment was to determine the

beneficial effects of microalgae and AHL-degrading bacteria toward Artemia

and mussel larvae, by close monitoring their survival during the exposure to

selected pathogenic bacteria. It is known the most Gram-negative bacteria

used QS systems for expression of certain phenotype (e.g. virulence factor

production). The QS regulated production of serine protease (a virulence

factor) on Aeromonas salmonicida is best documented by Swift et al., (1997).

They reported that the secretion of serine protease makes the bacteria

species virulent. Authors try to elucidate the mechanism behind QS systems.

Rasch et al., (2004) figure out on how to inhibit bacterial cell-to-cell

communication in order to stop the pathogenicity without having effect on the

growth and survival of the pathogenic bacterium. However, the downside of

using compound such as furanone as quorum sensing inhibitor (QSI) is that a

small amount of furanone has a detrimental effect on target species such as

rainbow trout (Rasch et al., 2004) and Artemia franciscana (Defoirdt et al.,

2006).

Our present study, we investigated the use of QSI by using bacteria that are

naturally occurring on the algae “phycosphere” and capable in degrading QS

signaling molecule (AHL). We used this approach to control opportunistic

bacteria, whose virulence factor might be QS regulated (AHL). The

combination of AHL-degrading bacteria and microalgae marked good

responses on the performance (percent suvival) of challenged Artemia

franciscana and mussel larvae. Enhanced survival of Artemia and mussel

treated with AHL-degrading bacteria and microalgae was comparable with the

control treatment (unchallenged aquatic organism). Thus, it is not possible to


59

say that mircroalgae served as good nutritional input because of the short

term experimental on the growing aquatic organism; and audacious to put that

some microalgae produced antibacterial substances that deter the growth of

pathogenic bacteria Hence this experiment does not not allow us to establish

that QSI by algae was the mode of action. In accordance with the previous

statement, this support that green aglae Tetraselmis suecica control the

proliferation of Vibrio spp. in shrimp hatchery (Regunathan and Wesley,

2004), luminous bacteria (Vibrio harveyi) are inhibited by green water

technique (dominated by Chlorella spp.)(Huervana et al., 2006) and

Skeletonema costatum inhibited the growth Vibrio anguillarum (Naviner et al.,

1999).

However, we also found the effects of bacteria on the performance of Artemia

and mussel larvae, survival of aquatic organism was more pronouced if AHL-

degrading bacteria were added in the culture medium. Several possibility that

might explain for such protection by AHL-degrading bacteria towards aquatic

organism. It is possible that an increased survival of Artermia sp. and mussel

larvae against pathogenic bacteria, was due to the presence of the AHL-

degrading bacteria (T2, I3 and C3) on the culture medium, and their abilities

to interfer/degrade the QS molecules. These authors support with our first

speculation, they found out that expression of biofim formation (Morohoshi et

al 2008) and exoprotease activity (Morohoshi et al 2005) of V. anguillarum

and Aeromonas sp. were disrupted, respectively. And they found out that the

activity was due AHL-acylase (aac) by Shewanella sp. strain MIB010.

However, it is hard to validate our first argument since our pathogen strain (V.

harveyi) virulence factor are control with multi-channel QS system. Defoirdt et

al. (2005) demonstrated that AI-2 QS system control the virulence of V.

harveyi towards Artemia sp. That is why we come up with our second

assumption, thus, might possible that our AHL-degrading bacteria not only

degrade the AHL molecule, but it also interfere with other QS system. Xavier

and Bassler (2005) reported a significantly greater bioluminescence

disappearance of V. harveyi when E. coli-LsrR- strain (AI-2 importer) was

added in the medium. In addition, mixing V. harveyi with E. coli LsrR- strain


60

demonstrate that production of light by V. harveyi was reduce due to the

constitutive removal or chemical interconversions of AI-2. These findings

imply that induction of Lsr genes in E. coli results in assembly of the V.

harveyi AI-2 transporter and subsequent consumption of AI-2 molecule.

61

CHAPTER 6: CONCLUSION

Much work has been done to determine the effects of AHL degrading bacteria

on the biodegradation of organic signaling molecules produced exclusively by

gram-negative pathogenic bacteria.

In this study, we have Isolated 3 bacterial strains that are capable of

degrading the exogenous AHLs molecule. These strains were isolated

following the AHL-degrading enrichment procedure that was pioneered by

Tinh et al., (2007), where AHLs molecules serve as sole sources of carbon

and nitrogen for the growing of AHL degrading bacteria. Our study

demonstrated that administration of both AHL degrading bacteria to the

specific microalgae showed significant increase in percent survival of the

Artemia and mussel. This would suggest that green water techniques and

addition of microorganism capable of degrading AHL (and have probiotic

potential) protected the cultured aquatic organism against invasion of

pathogenic bacteria. However, it’s remained to be established that QSI and

more in particular AHL degradation is the mode of action of the isolated

bacteria.

Interestingly, the outcome of In vivo test of mussel on AHL degrading strain

T2 was striking, it showed significant increase of percent survival of

challenged Artemia with Vibrio harveyi BB120. Several explanations can be

formulated with such protection by T2 strains. Their effect was even more

pronounce when both AHL degrading strain T2 and Tetraselmis suecica are

inoculated together. That is why we proposed that further investigation should

be made for the AHL-degrading bacteria strain to obtained concrete evidence

of the mode of action of these strains toward the pathogen. An increased

understanding of the QS degrading bacteria and algae interactions within the

“phycospheres” will help us more precise manipulation of the microbial

ecology. With advanced molecular techniques (DGGE, Real time PCR) and

well-developed gnotobiotic animal system (Artemia) we could identify the

specific, host-microbe interaction in the future.

62

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Annex 1: Artemia challenge test protocol Tom Defoirdt

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ANNEX 1: ARTEMIA CHALLENGE TEST PROTOCOL I.Materials

Autoclaved Instant Ocean 35 g/L in distilled water LVS3 (feed source) pathogenic bacterium Marine Broth or Marine LB (yeast extract 5 g/L, tryptone 10 g/L, Instant

Ocean 35 g/L) Marine Agar or Marine LB Agar (Marine LB + 15 g/L agar) Falcon tubes or glass tubes1 NaOH 32% NaOCl 50% Na2S2O3 10 g/L sterile 100 µm sieve, recipient and spatulum sterile 0.22 µm Millipore filter pipettes (1 mL, 20 mL) and micropipettes 200 mg cysts

II. Preparation of feed and pathogen Preparation of feed

1. Grow LVS3 on Marine Agar or Marine LB Agar plates at 28°C 2. Scrape the grown cells off the plates with an inoculation loop and

bring into sterile sea water 3. Homogenize the suspension (vortex, pipetting up and down) 4. Measure OD550 and set the suspension at OD550 of approx. 1 5. Autoclave the suspension; autoclaved LVS3 can be stored for

months prior to use 6. Add 200 µL of feed suspension for 20 nauplii ~107 cells per mL

Artemia culture water Preparation of pathogen

1. Inoculate 5 mL fresh Marine Broth or Marine LB with 10 µL pathogen culture from -80°C

2. Incubate overnight or for 24h 3. Measure OD550 4. Set at OD550 of approx. 0.1 (dilute in fresh sea water) ~108 cells

per mL 5. (Optional: dilute another 10 times in fresh sea water) 6. Add 20 µL of pathogen suspension to 20 mL of Artemia culture

water ~105 cells per mL Artemia culture water (105 in case of extra dilution)

III. Decapsulation and hatching 1 It could be that nauplii stick to the sides of falcon tubes; in that case it is better to use glass tubes.


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Hydration (non-sterile)

1. Bring 200 mg cysts in a falcon tube (doesn’t need to be sterile) with 18 mL tap water

2. Aerate for 1h Decapsulation (from now on sterile laminar flow hood)

1. Stop aeration 2. Disinfect tubing for sterile aeration and install sterile aeration (see

figure); don’t aerate the cyst suspension yet 3. Add 660 µL NaOH + 10 mL NaOCl to cyst suspension 4. Bring cyst suspension in new, sterile falcon tube 5. Start aeration of the suspension 6. Sample cysts regularly with 1000 µl micropipette 7. Add 14 mL Na2S2O3 to stop reaction if cysts sink2 8. Wash over 100 µm sieve with sea water 9. Bring cysts in falcons with 30 mL sea water. Note: cysts will be

orange and sticky after decapsulation; clump formation is normal. 10. From now on, only open falcon tubes under laminar flow hood!

Figure 1 : sterile aeration of cyst suspension Hatching

1. Put the falcon with decapsulated cysts on a rotor with constant light; 28°C

2. Incubate like this for at least 24h IV. Challenge

2 Or until they don’t go to the surface so quickly anymore; normally this takes around 2- 2.5 minutes. It’s better to stop too early (then the shell will not be completely removed, but the cysts will be sterile) than too late (then the Artemia would be killed).

Sterile tubing (disinfected inside and outside)

Millipore filter

Sterile 1 mL pipette

Airpump

Tubing

Falcon tube


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Start of challenge 1. Bring groups of 20 nauplii in sterile falcon tubes with 20 mL sea

water 2. Add feed (200 µL LVS3 suspension for 20 nauplii) 3. Add pathogen (20 µL of pathogen suspension per 20 mL culture

water) 4. (Optinal: add treatment; e.g. add probiotic bacteria)

Sampling

1. Measure survival 2 days after addition of pathogen (optional: extra measurement after 1 day). To count, pour the culture water on the lid of a petri dish or a 96-well plate and remove nauplii with a micropipette as you count them.

2. Bring 1 mL culture water of control treatments into 9 mL fresh growth medium and incbate for at least 2 days at 28°C to check for contamination

3. (Optional: take samples for PCR analysis to check for contamination)

4. (Optional: homogenize nauplii and plate on Marine Agar or Marine LB Agar to count number of pathogens per nauplius)

5. (Optional: plate culture water on Marine Agar or Marine LB Agar to count number of pathogens per mL culture water)

Setup of experiment

Treatments: control (no pathogen added; only feed); challenge (both pathogen and feed added); extra treatments (pathogen + feed + extra treatment, e.g. probiotic bacteria)

Each treatment at least in triplicate Timing: see scheme

Day Handling -- - Prepare media

- Prepare LVS3 feed suspension -1 - Inoculate pathogen

- Decapsulation and start hatching 0 - Prepare pathogen suspension

- Start challenge (1) - (Optional: count survival)

- (Optional: plate homogenized nauplii and/or culture water)

2 - Count survival - Inoculate fresh medium with control culture water - (Optional: plate homogenized nauplii and/or culture water)

4-5 - Check medium for turbidity sterility of nauplii? Scheme: timing of a challenge test

Annex 2: QS degraders enrichment Natrah Ikhsan

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ANNEX 2:

ENRICHMENT OF BACTERIA FROM ALGAE CULTURE AS QS DEGRADERS GENERAL INFORMATION Tinh et al. ASSAY STANDARD OPERATION PROCEDURE 1. Required Equipments and Materials

Sterile Eppendorf tubes 15 mL sterile Falcon tube OHHL (Stock solution 1000 ppm) and HHL (Stock solution 2500 ppm) Special algae medium with the exclusion of nitrate, vitamin & trace

elements. Only phosphate is added. Adjust to pH 6 using HCL. Marine Luria Bertani agar plates Non-axenic algae culture Micro-centrifuge Aluminium foil 10 µl-200 µl pipettes; multi channel pipette and disposable tips

2. Protocol

1. Put 1 mL of non-axenic algal culture in a sterile Eppendorf tubes 2. Centrifuge in microcentrifuge for 15 minutes at 5000 rpm 3. Collect 50 µl of the supernatant and inoculate in the special algae

medium (5 mL) 4. Add HHL and OHHL at a final concentration of 50 ppm each in the

falcon tube 5. Cover the falcon tube with aluminium foil to avoid the growth of

algae. 6. Control would be falcon tube without addition of AHL 7. Incubate at 28°C 8. Leave for 3-4 days- FIRST CYCLE 9. Repeat the cycles for 2-3 cycles until the bacterial growth in the

control tube is totally gone. 10. After the cycle is finish, keep the enrichment culture in -80°C

*The shorter the cycle the better so there is no possibility of bacteria that can use the hydrolyzed AHL. Bacteria that use hydrolyzed AHL will later show a less degradation activity when tested with the biosensor.

Annex 2: QS degraders enrichment Natrah Ikhsan

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AHL degradation assay Materials

Marine agar (buffered with MOPS) HHL (Stock solution of 2500 ppm) Biosensor Chromobacterium violaceum CV026 Normal LB plates (without salts) buffered with MOPS

2. Protocol

1. Grow enrichment culture in mLB overnight 2. Dilute bacteria to OD = 0.1 3. Inoculate 100 µl each to 5 mL of buffered mLB 4. Incubate with 10 ppm HHL for 24 hours 5. The next day, centrifuge and filter sterilize to collect the supernatant 6. Put 10 µl in the middle of normal buffered LB spread with CV026 7. Compare the degradation activity with a standard curve of (0, 1, 2.5,

5, 7,5 and 10 ppm). Example of a standard curve is as below

y = 0.266x + 1.492 R² = 0.975

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 2 4 6 8 10 12

Annex 3: Co-culture of algae and QS bacteria Natrah Ikhsan

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ANNEX 3: CO-CULTURES OF ALGAE AND QS BACTERIAL DEGRADERS GENERAL INFORMATION ASSAY STANDARD OPERATION PROCEDURE 1. Required Equipments and Materials

Fluorimeter Spectrophotometer Haemocytometer (Algal density-cells/mL) Aqua Pen LB+ SW plates (Bacterial density-CFU/mL) Axenic algae – Isochrysis (T-Iso), Tetraselmis suecica, Pavlova lutheri,

Chaetoceros muelleri Bacterial QS degraders from Isochrysis (T-Iso), Tetraselmis suecica,

Pavlova lutheri, Chaetoceros muelleri, Skletonema costatum (outdoor pond)

Bacterial QS (Loan’s LT strain) F2 Algal medium (With silicate for diatom) Schott bottle vs Erlenmeyer flask

2. Protocol

1. Dilute axenic algal culture 100x in Schott bottle (200mL) or Erlenmeyer (50 mL total volume). Count the first density (0 day)

2. Final concentration of bacteria will be around 102 CFU/mL (absorbance & plating at 0 day)

3. Bacteria from non-axenic algal cultures will be collected through centrifugation at 3000 ppm for 5 minutes.

4. For each algae, treatments will include a. Axenic algae only b. Axenic algae + QS degraders c. QS degraders only

5. Research parameters will be* a. Chlorophyll measurement (Excitation 410 nm/Emission 670 nm) b. Optical density (550 nm) c. Quantum yield (Aqua pen) d. Bacterial plating e. Algal counting

6. Supernatant from axenic algae only and axenic algae + QS degraders will be collected at late exponential and late stationary, extracted with EtOAc and tested for QSI activities.

*All the parameters will be checked every three days for about 18 days

interaction between micro-algae and quorum sensing...

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