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- 1 - Contract n°EVK1-CT-2002-00108 Deliverable 02 version 2 Handbook for analytical methods and operational criteria
- 1 - Contract n°EVK1-CT-2002-00108 Deliverable 02 version 2 Handbook for analytical methods and operational criteria
- 1 - Contract n°EVK1-CT-2002-00108 Deliverable 02 version 2 Handbook for analytical methods and operational criteria
- 1 - Contract n°EVK1-CT-2002-00108 Deliverable 02 version 2 Handbook for analytical methods and operational criteria
- 1 - Contract n°EVK1-CT-2002-00108 Deliverable 02 version 2 Handbook for analytical methods and operational criteria

Contract n°EVK1-CT-2002-00108

- 1 - Contract n°EVK1-CT-2002-00108 Deliverable 02 version 2 Handbook for analytical methods and operational criteria
- 1 - Contract n°EVK1-CT-2002-00108 Deliverable 02 version 2 Handbook for analytical methods and operational criteria

Deliverable 02 version 2

Handbook for analytical methods and operational criteria for biofilm reactors

Updated November, 2005

Non confidential

Contact persons :

http://www.safer-eu.com

sylvain.fass@nancie.asso.fr

Jean-Claude.Block@pharma.uhp-nancy.fr

EVK1-CT-2002-00108 Surveillance and control of microbiological stability in drinking water distribution networks Handbook for analytical methods and operational criteria for biofilm reactors

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SAFER

EVK1-CT-2002-00108

Surveillance and control of microbiological stability in drinking water distribution networks

Revised

Handbook for analytical methods and operational criteria for biofilm reactors (Version

2.0)

WP2. Tools for biofilm monitoring

Prepared by:

Ilkka Miettinen National Public Health Institute Department of Environmental Health Kuopio, Finland

Gabriela Schaule IWW Rhenish-Westfalian Institute for Water Department of Applied Microbiology Mulheim, Germany

DATE: 13h November, 2005

EVK1-CT-2002-00108 Surveillance and control of microbiological stability in drinking water distribution networks Handbook for analytical methods and operational criteria for biofilm reactors

 

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Content

1 INTRODUCTION

5

2 STRUCTURE

5

3 MICROBIOLOGICAL METHODS

6

3.1 Enumeration of culturable mic roorganisms (water/biofilm)

6

3.2 Total cell number

6

3.3 Coliform bacteria and E. coli (water and biofilm)

7

3.4 Dehydrogenase activity with redox stain 5-cyano-2,3-ditolyl tetrazolium chloride (CTC)

(water/biofilm)

8

4

CHEMICAL METHODS

9

4.1 Measurement of adenosine triphosphate (ATP)

9

4.2 Total organic carbon /dissolved organic carbon

11

4.3 Modification of non-purgeable organic carbon (NPOC) analyses (water)

12

4.4 Assimilable organic carbon (AOC) (water)

13

4.5 Total phosphorus

16

4.6 Microbially available phosphorus (MAP) (water)

17

4.7 Total nitrogen

20

4.8 Assessment of nucleic acid damages by chlorination using fluorochrome staining (SYBR-

II or PI) and flow cytometry

21

4.9 Amino acid

26

4.10 Protocol use for biofilm extraction and dispatch samples to:

26

5 QUALITY CONTROL AND QUALITY ASSURANCE

27

5.1 Quality control and quality assurance in CR4

27

5.2 Quality control and quality assurance in CR6

27

5.3 Quality control and quality assurance in CR9

27

6 BIOFILM MONITORING DEVICES

29

6.1 Propella (The common biofilm monitoring device for all partners)

29

6.2 Rotating Annular Reactor (modified RotoTorqueTM) [CR4]

34

6.3 Biofilm generator

39

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6.4 Flow cell reactor

40

6.5 Pipeline biofilm collector

41

6.6 Differential turgidity measurement

43

6.7 Fibre optic devices (FOS, and FluS sensors)

44

6.8 Electrochemical, nanovibration, and capacitive monitors

47

6.8.1 Mechatronic Surface Sensor - MSS

47

6.8.2 Capacitive sensors

47

6.9

Biofilm formation monitoring using ATR-FTIR sensor

48

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1 Introduction

The objective of workpakage 2 is to provide monitoring systems for assessing biofilm

development in situ, on-line and non-destructively. Research will be organised on two

levels: one will aim to intercalibrate reactors for biofilm build-up, the second will aim

to develop biofilm monitoring devices. The main challenge is to establish devices

having both high sensitivity, rapid response potential, and an early warning capacity.

The second deliverable of workpackage 2 is to make a handbook for basic

methodologies. This handbook will describe the common protocols of all basic

analytical methods, which are used by partners participating to SAFER programme.

This information enables the exchange of information and comparison of different

methods.

The handbook contains information about methods used for characterisation of the

water , biofilm sampling and biofilm characterisation.

2 Structure

Within three chapters the different methods are listed starting with the microbiological

methods (1), followed by the chemical (2) and the physical methods (3) which are

used within the working groups of SAFER.

If for any of the methods an EN ISO Standard is available, this method will be the

basis and will be eventually modified by the partners. The modifications are listed.

The general structure of the method description:

1. Scope /principles

2. References

3. Definitions

4. Materials

5. Procedure

6. Expression of results

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3

Microbiological methods

3.1

Enumeration of culturable microorganisms (water/biofilm)

A) European Standard: EN ISO 6222 (Water quality – Enumeration of culturable

micro-organisms – Colony count by inoculation in a nutrient agar culture medium).

See original instructions from the pdf-file “HPC ISO6222.pdf” from SAFER ftp-site.

B) Number of colony forming units on R2A nutrient agar

Scope

The bacterial colony forming units (CFU) are enumerated by spread plate method for heterotrophic plate counts.

Reference

Reasoner DJ and Geldreich EE (1985). A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microb. 49: 1-7.

Procedure

Heterotrophic plate counts (HPC) are estimated by spread plating method using 0.1 mL or 1 mL sample. The medium is R2A-agar (Difco, USA) (Reasoner and Geldreich 1985). R2A-agar plates are incubated for 7 days at 22 ± 2 ° C before the colony

forming units (CFU) are counted by eye.

Membrane filter method will be used if the cell density of the sample is too low for direct spread plate. The membrane filters have a diameter of 47 mm, a pore size of 0.2 µ m. The sample up from 10 mL will be filtered and the membrane filter placed on one R2A agar plate. Be careful: air bubbles under the filter should be avoided.

Expression of results

Results are expressed as the mean number of bacterial CFU per mL of water sample

3.2 Total cell number

Scope

This method describes a procedure for counting all bacteria in water and homogenised and/or diluted biofilm samples using the dye 4´, 6-diamidino-2- phenylindole (DAPI). There are other fluorochromes which can be used for the same purpose e.g. Acidine Orange and Syto 9. Acridine Orange is the dye which is often used by the semiconductor industry to estimate the total cell number in ultra pure water. The procedure follows in this case the ASTM Standard Test Method Designation F 1095-8. The advantage of Acridine Orange is that it will show bright cells with the disadvantage that the stain is much more unspecific to noncellulare material.

The total cell number of biofilms can be evaluated directly without removing the biofilm by using the Confocal Laser Scanning Microscope. Staining will be performed the same way like water samples (filter method). Counting is performed with epiflourescence microscope.

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References

Hobbie, J.E., R.J. Daley, Jaspers, S. (1977): Use of Nucleopore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33: 12225-1228. ASTM Standard Test Method: Designation: F 1095 – 8 (Reapproved 1994). Rapid enumeration of bacteria in Electronic-Grade Purified Water Systems by Direct Count Epifluorescence Microscopy.

Materials

4´, 6-Diamidino-2-phenylindole (DAPI), black Nucleopore filter (pore size 0.2 µ m), non-fluorescent immersion oil.

All reagents should be filtered 0.2 µm. Procedure

Place one black polycarbonate membrane filter (0.2 µ m pore size, laser beamed) on

top of the sampling port, assuring that the shiny side of the filter is facing upwards.

Filter an aliquot of the sample and stop the filtration process immediately when the

through a cellulose nitrate filter with the pore size of

sample is filtered through. Supplement with 1 mL DAPI (10 µ g/mL) and add 1 mL

Triton X-100 (0.1 %). The final concentration should be 5 µ g/mL. The incubation

time should be 15 to 20 minutes. Then the supernatant is filtered through and the

filter with the stained bacteria placed in a petri dish or any other box to let it air dry. If

the filter will be stored more than 2 days, add formaldehyde (1 % v/v) to the DAPI

solution.

The air dried filter is prepared for the microscope by embedding it in immersion oil on

the surface of a clean microscope slide; like a sandwich the filter is between the

immersion oil and a clean glass coverslip.

The enumeration of bacteria and other microorganismns are performed with a

magnification of at least 1000 fold in a epifluorescence microscope. All blue stained

cells are counted in randomly chosen microscopic viewing fields delineated by the

eyepiece micrometer. There should be 10-50 cells per viewing field. In minimum 300

bacteria should be counted or so many viewing fields that the coefficient of variation

of < 30% is obtained.

3.3 Coliform bacteria and E. coli (water and biofilm)

See the original instructions from the PDF-file “ E.coli colforms ISO9308.pdf” from the

ftp-site of SAFER.

Alternative methods

Two alternative membrane filtration media (chromogenic Harlequin, and mEndo LES)

can be used for E. coli and coliform counting.

See instructions of chromogenic agars from ftp-site of SAFER: “Harlequin coli

agar.pdf / Oxoid coli agar.pdf / Cromocult agar.pdf / Tergitol agar.pdf”.

Also Colilert Quantitray (IDEXX) method can be used if considered necessary for

E.coli/coliform counting. The colilert analyses follows the manufacturer´s instructions.

For Colilert, the sample size used is 100 ml. For further instructions see PDF-file:

“Colilert.pdf” from the ftp-site of SAFER.

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with redox stain 5-cyano-2,3-ditolyl

tetrazolium chloride (CTC) (water/biofilm)

Scope

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) can be applied for direct

epifluorescent microscopic counting of dehydrogenase active (metabolically active)

bacteria. CTC is noncolorless and nonfluorescent. It is water soluble and can pass

solubilised in water the cell wall and will be intracellular reduced via electron

transport system. Inside the cell CTC will be reduced via electron transfer. The

reduced form of tetrazolium salts are called formazan. "CTC-formazan" molecules

are not water soluble. They accumulate intracellularly and build up o type of crystall

which is fluorescent (maximum at 520 nm) after excitation with fluorescent light (465

nm). It is possible to counterstain bacterial cells with DAPI after the incubation

procedure with CTC.

Bacteria with a low level of dehydrogenase might not be able to form a large crystal.

To get a information about the number of potentially active cells, nutrients might be

added during the incubation time with CTC to activate the cells.

References

Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF (1992), Use of a fluorescent redox probe for direct visualisation of actively respiring bacteria, Appl. Environ. Microbiol. 58, 6, 1801-8 Schaule G., Flemming H-C, Ridgway HF (1993). Use of 5-Cyano-2,3-ditolyl tetrazolium chloride for quantifying planctonic and sessile respiring bacteria in drinking water. Appl. Envir. Microbiology 59: 3850-3857.

3.4 Dehydrogenase

activity

Material

Black polycarbonate membrane filter (pore size 0,2 µ m) and a filtration set

Epifluorescent microscope and nonfluorescent immersion oil

5-cyano-2,3-ditolyl tetrazolium chloride (CTC, Polysciences). Reactant solution of CTC in tubes can be kept frozen at 4ºC or have to be freshly prepared.

4´, 6-diamidino-2-phenylindole (DAPI). Procedure for water samples

Add CTC solution to the water sample to a final concentration of 4 mM CTC and

incubate the mixture for 4 hours in the dark between 22 and 28ºC.

Addition of nutrients (R2A is useful for drinking water samples).

If there are many bacteria present the sample should be agitated to avoid anoxic conditions. Filter the sample after the incubation time through a black polycarbonate membrane

filter (pore size 0.2 µ m) and wash slightly with water. Either the filter is now placed in

a box to be air dried or supllememnted with 1 mL of DAPI (5 µ g/mL) to stain all

bacteria. DAPI will be filter through after 25 minutes. Then the filter is air dried.

The dried filter will be placed with nonfluorescent immersion oil on glass microscope

slides. The examination is performed at 1000 magnification using an epifluorescent

microscope. Count the red fluorescent cells as dehydrogenase active cells and the

blue ones (DAPI) as total cell number.

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4

Chemical methods

4.1

Measurement of adenosine triphosphate (ATP)

Scope

The survival of a cell necessitates a continual energy input to allow the

accomplishment of all the metabolic activities. One of the energising molecules

commonly determined is a nucleotide which is a marker of the bacterial biomass :

adenosine triphosphate (ATP). By hydrolysis of the 3 phosphate bonds, ATP can

release some energy, which is directly usable by the cell. The measurement of ATP

is carried out in 4 successive stages described below :

1. pre-concentration of the sample

2. extraction of the cellular ATP

3. enzymatic determination by bioluminescence

4. ATP quantification and expression of results.

The measurement of ATP is carried out with an apparatus and products from the

LUMAC company. For all the stages, followed protocols are those prescribed by this

company.

Procedure

Pre-concentration of samples to be analysed

The method used is a membrane filtration with a moderate pressure in a view to limit

the bacterial stress. The sample is first filtered on a cellulosic membrane and then

rinsed with an demineralised, sterile and apyrogenic water. Bacteria retained by the

membrane then suffer a reactivation phase according to the LUMAC protocol: input

on the membrane of 500 µ L of peptoned bubble without ATP (LUMACULT, ref. 9233-

1) during a contact time equal to 15 minutes.

In our experiments, samples (between 8 and 20 mLin volume) were filtered on a

sterile membrane in cellulose acetate with a porosity of 0.45 µ m. The pre-

concentration was applied both on the immersion waters in contact with tested

materials and on the sonication product of the biofilm present on materials.

ATP extraction

Among many extraction products used for the determination of the ATP, we choose a

detergent commercialised by LUMAC : the NRB for which the chemical

composition is always a manufacturing secret.

The membrane is first soaked in 500 µ L of LUMACULT. Then 500 µ L of extraction

product (NRB) is added and mixed by soft agitation during 30 seconds. The

determination of the ATP is then carried out with 200 µ L of this mixture. A negative

check sample is included, replacing the sample by 8 to 10 mLof demineralised,

sterile and apyrogenic water.

Enzymatic measurement of ATP

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The most common quantification method of the bacterial ATP is based on an

enzymatic reaction and on bioluminescence detection. The ATP determination

method is based on the use of the Luciferase (an enzyme extracted and purified from

the glow-worm (Photinus pyralis) and its substrate named Luciferine.

In presence of Mg++ ions, oxygen and ATP and with the luciferase as a catatyst, the

luciferine is oxidized. The reaction is endergonic: a consumption of ATP molecules

occurs with production of photons. Their emission is proportional to the quantity of

the consumed ATP: they are quantified by a bioluminometer (LUMAC, ref. M2500). In

our experiments, the ATP determination was implemented with a commercial kit from

LUMAC.

The photometer produces some Relative Light Unit (RLU) during the enzymatic

reaction. The conversion of these RLU into ATP concentration in the sample is

obtained by proportioned additions of standard ATP.

Proportioned additions consist of an initial measurement of ATP on the sample to be

analyse. Then a known quantity of ATP is introduced in the same measurement cell,

and a second measurement of ATP is carried out. The bioluminometer contains a

data colection program: all the reaction times are first stored and proportioned

additions are allowed.

Stages of bacterial ATP quantification:

1. concentrated sample on membrane + NRB + LUMACULT,

2. 100 µ L of the enzymatic complex Luciferine – Luciferase (LUMIT PM) are automatically added by an integrated pump system,

3. integration of photons produced for 10 seconds,

4. results of the quantity of ATP in the sample in RLU (RLU 1 ),

5. addition of 20 µ L of standard ATP (LUMAC) with a know concentration,

6. integration for 10 seconds,

7. results of the quantity of total ATP in RLU (sample ATP and standard ATP) in the analysed volume (RLU 2 ).

Expression of results

As ng ATP per unit.

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4.2 Total organic carbon /dissolved organic carbon

European Standard CEN 1484 : “Water analysis - Guidelines for the determination of total organic carbon (TOC) and dissolved organic carbon (DOC). See PDF-file “TOC DOC CEN1484.pdf” from the SAFER tfp-site.

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4.3 Modification of non-purgeable organic carbon (NPOC) analyses

(water)

Non-purgeable organic carbon (NPOC) is analysed by a method which is a modification of a CEN 1484 “ Water determination. Guidelines for analyses of total organic carbon (TOC) and dissolved organic carbon (DOC)” standard.

Non-purgeable organic carbon (NPOC) is determined from water samples which are acidified (pH 3) with hydrochloric acid and purged with nitrogen gas (10 minutes) before the analyses. The content of organic carbon is analysed by Shimadzu TOC- 5000/5050 -analyzer. The combustion temperature is +680 °C.

Dissolved organic carbon (DOC) is analysed in a similar way as NPOC, except that the water samples are prefiltered with 0,22 µ m syringe filters and no nitrogen purging is used.

Detection limit is 0,3 mg/L. Method is accredited.

Uncertainty of quantitative determination for different concentrations:

< 10 mg/l 15 % and > 10 mg/l 10 %

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4.4 Assimilable organic carbon (AOC) (water)

a. The Dutch standard method NEN 6271. See the original description « AOC NEN

6271.pdf-file » from the ftp-site of SAFER.

b. AOC method modification

Scope

The AOC bioassay using Pseudomonas fluorescens P-17 and Aquaspirillum sp.

NOX involves growth to a maximum density of a small inoculum in a batch culture of

pasteurized test water. Pasteurization inactivates native microflora. The test

organisms are enumerated by spread plate method for heterotrophic plate counts

and the density of viable cells is converted to AOC concentrations by an empirically

derived yield factor for the growth of P. fluorescens P-17 on acetate-carbon and

Aquaspirillum sp. NOX on oxalate-carbon as standards. The number of organisms at

stationary phase is assumed to be the maximum number of organisms that can be

supported by the nutrients in the sample and the yield on acetate carbon is assumed

to equal the yield on naturally occurring AOC (van der Kooij 1982, Kaplan and Bott

1989). The underlying assumption of the AOC bioassay is that the bioassay

organism(s) represent the physiological capabilities of the distribution system

microflora. In some waters (e.g humic waters) inorganic nutrients regulate bacterial

growth (Miettinen et al., 1999). Thus, to ensure that carbon is limiting bacterial

growth, enough of inorganic nutrients are added in sample of test water.

In theory, concentrations of less than 1 µ g C/L can be detected. In practice, organic

carbon contamination during glassware preparation and sample handling imposes a

limit of detection of approximately 5 to 10 µ g AOC/L. High concentration of metals

(esp. Al, Cu) is toxic for strain P. fluorescens, which makes this procedure unsuitable

for waters containing these metals.

References

Kaplan L.A., Bott T.L. 1989. Measurement of assimilable organic carbon in water distribution systems by a simplified bioassay technique. In Advances in Water Analysis and Treatment, Proc. 16 th Annu. AWWA Water Quality Technology Conf., Nov. 13-17, 1988, St. Louis, Mo., p. 475. American Water Works Assoc., Denver, Colo. Miettinen I.T., Vartiainen T. and Martikainen P.J. 1999. Determination of assimilable organic carbon in humus-rich drinking waters. Water Res. 33 (10): 2277-2282. Reasoner D.J., Geldreich E.E. 1985. A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microbiol. 49:1-7. Swanson K.M.J., Busta F.F., Peterson E.H., Johnson M.G. 1992. Count methods. In Compendium of methods for the microbiological examination of foods. Vanderzant C., Splittstoesser D.F., eds. APHA, Washington, 75-95. Van der Kooij D., Visser A., Oranje J.P. 1982. Multiplication of fluorescent pseudomonads at low substrate concentrations in tap water. Antonie van Leeuwenhoek 48:229-243.

Materials

Incubation vessels - 100 ml volume borosilicate glass vials (larger volumes are advisable) with caps.

Hot water bath (65-70 °C)

Continuously adjustable pipettes capable of delivering between 10 and 100 µ L, between 200 and 1000 µ L and between 1000 and 5000 µ L.

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Eppendorf vials or glass tubes for dilution series.

Glass test tubes and Vortex mixer.

Petri dishes (disposable, plastic). Reagents

Sodium acetate stock solution, 400 mg acetate-C/L. Dissolve 2.267 g CH 3 COONa . 3H 2 O (or 1.71 g CH 3 COONa) in 1 L organic-carbon free, deionized water. Transfer to 100-mL vials, cap and autoclave. Store at 5 ° C. Solution may be held for up to 12 months.

Sodium thiosulfate solution. Dissolve 30 g Na 2 S 2 O 3 in 1 L deionized water. Transfer to 100 mL vials, cap and autoclave.

Buffered water.

R2A agar (Reasoner and Geldreich 1985)

Organic-free water.

Mineral salts solution. Dissolve 4.55 g (NH 4 ) 2 SO 4 , 0.2 g KH 2 PO 4 , 0.1 g MgSO 4 . 7H 2 O, 0.1 g CaCl 2 . 2H 2 O, 0.2 g NaCl in 1 L organic-free water. Transfer to 100-mL vials, cap and autoclave.

Cultures of test strains Pseudomonas fluorescens P-17 (ATCC 49642) and Aquaspirillum sp. NOX (ATCC 49643). Preparation of incubation vessels

Wash 100-mL vials with phosphate-free detergent, rinse with hot water, immerse in

0.1 N HCl for 2 h, and rinse with deionized water three times, dry, cap with foil, and

heat to 550 ° C for 6 h or 250 ° C for 8 h. Use same cleaning procedure for all

glassware.

Procedure

Preparation of stock inoculum

Prepare turbid suspension of P. fluorescens P-17 and Aquaspirillum sp. NOX by

transferring colonies from R2A agar plates into 2 to 3 mL of filtered (pore size

0.2 µ m) and autoclaved sample to obtain a final concentration of approximately 10 8

cfu/ml (e.g. it corresponds to 0.10 absorbance of microbe mixture at 420 nm). The

microbe mixture is diluted with autoclaved water 10 -3 - 10 -4 and inoculated into

pasteurised (30 min, 65° C) water. Any fresh water that supports growth of P.

fluorescens P-17 can be used. Before inoculation, water is added 1/1000 final dilution

of mineral salts solution and final concentration of 1000 µ g acetate C/L.

Incubate at room temperature (25 ° C) until the viable cell count reaches the

stationary phase. The stationary phase is reached when the viable cell count,

measured by colony forming units (spread plate method), reaches its maximum

value. Store stock cultures not more than 12 months at 5 ° C. After the stationary

phase is reached, make a viable count of the culture (spread plate) to determine the

appropriate volume of inoculum to be added to each bioassay vessel.

Preparation of incubation water

Collect 500 mL sample in an organic-free vessel and pour into two parallel 100 ml

vials. Neutralize samples containing disinfectant residuals with 100 µ L sodium

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thiosulfate solution added to each vial. Add 100 µ L of mineral salts solution to the each vial. Cap vials and pasteurize in 65-70 °C water bath for 30 min.

Inoculation and incubation

Cool, inoculate with stock inoculum of P. fluorescens P-17 and Aquaspirillum sp. NOX (final concentration of bacteria 500-1000/ml) by removing cap and using a

carbon-free pipet. Plastic, sterile tips for continuously adjustable pipets are suitable.

° C in the dark for 9 days. If higher

temperature is used, maximum growth is reached earlier, which shortens the incubation time (see below).

Enumeration of test bacteria

Bacterial concentrations are analysed on incubation days 7, 8 and 9. If higher temperature is used, maximum is reached earlier (should be tested beforehand). Shake vigorously the vials and make dilution series. Mechanical shaker (Vortex) may be used to shake the dilutions. Plate at least 3 dilutions (10 -2 , 10 -3 , 10 -4 and 10 -5 ) (dilutions depends on assumed AOC concentrations) on R2A agar. Incubate plates at room temperature for 3 days and score the number of colonies of each strain.

P. fluorescens P-17 colonies appear on the plates first; they are 2 to 4 mm in diameter with diffuse yellow pigmentation. Aquaspirillum sp. NOX colonies are small (1 to 2 mm diameter) white dots. It may be necessary to count P. fluorescens and Aquaspirillum sp. colonies at different dilutions. Sample vials on three separate days. Count all colonies on selected plates containing 25 to 250 colonies of each bacterium and compute colony counts (Swanson et al. 1992).

Determination of the yield of P. fluorescens P-17 and Aquaspirillum sp. NOX. The growth yield of the test bacteria is determined using sodium acetate as a substrate individually for P. fluorescens P-17 and Aquaspirillum sp. NOX. It is acceptable to use the previously derived empirical yield values of 6.9 x 10 6 CFU P. fluorescens P- 17/ µ g acetate-C and 2.1 x 10 7 CFU Aquaspirillum sp. NOX/ µ g acetate-C at 15 °C.

Expression of results

Average the viable count results for the average density over 3-day period (or take a maximum value) and calculate concentration of AOC as the product of the of the viable counts and the inverse of the yield:

Result = µ g AOC/L = [(P. fluorescens CFU/mL)(1/yield) + (Aquaspirillum sp. NOX

CFU/mL)(1/yield)] (1000mL/L)

Inoculated samples are incubated at 15

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4.5 Total phosphorus

ISO DIS 6878 (Water quality – Determination of phosphorus – Ammonium molybdate spectrometric method) - See the original instructions from the PDF-file “ ISO_DIS 6879 determination of phosphorus.pdf” from the ftp-site of SAFER.

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4.6 Microbially available phosphorus (MAP) (water)

Scope

The MAP bioassay using Pseudomonas fluorescens P-17 involves growth to a

maximum density of a small inoculum in a batch culture of pasteurized test water.

Pasteurization inactivates native microflora. The test organism is enumerated by

spread plate method for heterotrophic plate counts and the density of viable cells is

converted to MAP concentrations by an empirically derived yield factor for the growth

of Ps. fluorescens P-17 on phosphate-phosphorus as standard. The number of

organisms at stationary phase is assumed to be the maximum number of organisms

that can be supported by the nutrients in the sample. The yield on phosphate-

phosphorus (PO 4 -P) is assumed to be equal the yield on naturally occurring MAP.

Ps. fluorescens P-17 has phosphatase activity. The underlying assumptions of the

MAP bioassay are that the carbon and inorganic nutrients, with the exception of

phosphorus, are present in excess, i.e., that phosphorus is limiting (Lehtola et al.,

1999). Concentrations 0.08-10 µ g MAP/L can be detected (Lehtola et al., 1999).

High concentration of metals (esp. Al, Cu) are toxic for strain Ps. fluorescens, which

makes this procedure unsuitable for waters containig these metals.

References

Lehtola M.J., Miettinen I.T., Vartiainen T., Martikainen P.J. 1999. A new sensitive bioassay for determination of microbially available phosphorus in water. Appl. Environ. Microbiol. 65:2032. Reasoner D.J., Geldreich E.E. 1985. A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microbiol. 49:1. Swanson K.M.J., Busta F.F., Peterson E.H., Johnson M.G. 1992. Count methods. In Compendium of methods for the microbiological examination of foods. Vanderzant C., Splittstoesser D.F., eds. APHA, Washington, 75-95.

Materials

-

Incubation vessels - borosilicate glass vials (volume at least 100 ml, 250-500 ml is recommend because of more effective shaking) with caps.

-

Hot water bath (65-70 °C)

-

Continuously adjustable pipettes capable of delivering between 10 and 100 µ L, between 200 and 1000 µ L ,and between 1000 and 5000 µ L.

-

Eppendorf vials or glass tubes for dilution series

-

glass test tubes

-

Vortex mixer

-

Petri dishes (plastic)

-

Sodium

acetate stock solution, 400 mg acetate-C/L. Dissolve 2.267 g

CH 3 COONa . 3H 2 O (or 1.71 g CH 3 COONa) in 1 L organic-carbon free, deionized water. Transfer to 100-mL vials, cap and autoclave or pasteurize at 60 ° C for 35 min. Store at 5 °C. Solution may be held for up to 6 months.

-

Sodium thiosulfate solution. Dissolve 30 g Na 2 S 2 O 3 in 1 L deionized water. Transfer to 100 mL vials, cap and autoclave.

-

Buffered water.

-

R2A agar (Reasoner and Geldreich 1985)

phosphorus-free water. Alternatively, use HPLC-grade bottled water. - Mineral salts solution. Dissolve 0.48 g NH 4 NO 3 , 0.1 g MgSO 4 . 7H 2 O, 0.1 CaCl 2 . 2H 2 O, 0.1 g NaCl, 0,1 g KCl in 1 L phosphorus-free water. Transfer to 100-mL vials, cap and autoclave.

-

g

- Culture of test strain Pseudomonas fluorescens P-17 (ATCC 49642).

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Preparation of incubation vessels:

Wash vials with phosphate-free detergent, rinse with hot water, immerse in 0.1 N HCl for 2 h, and rinse with deionized water three times, dry, cap with foil, and sterilize e.g. by heat treatment. Use same cleaning procedure for all glassware.

Procedure

Preparation of stock inoculum Prepare turbid suspension of Ps. fluorescens P-17 by transferring colonies from R2A agar into 2 to 3 mL filtered (0.2 µ m), autoclaved sample. Use colonies not older than one week. The autoclaved media water can be any water that supports growth of Ps. fluorescens P-17. Before inoculation water is added of 150 µ L/lmineral salts solution,

1000 µ g acetate-C/L and pasteurised (30 min, 65 °C).

Incubate at room temperature (25 ° C) until the viable cell count reaches the stationary phase. The stationary phase is reached when the viable cell count, as measured by spread plates, reaches maximum value. Store stock cultures for not more than 12 months at 5 °C.

Preparation of sample water Collect water samples in an glass vessels and pour 100 mL into Erlenmeyer vessels. Use two parallel vials/vessels for MAP measurement. Neutralize samples containing disinfectant residuals with 50 µ l sodium thiosulfate solution for 100 ml sample. 100

µ L of mineral salts solution and 400 µ L of sodium acetate solution are added to the

each vial. Cap vials and pasteurize in 65-70 °C water bath for 30 min.

Inoculation and incubation Cooled waters are inoculate with approximately 1000 colony forming units (CFU)/mL (usually 2 drops of stock inoculum by pasteur pipette, concentration should be tested beforehand) of Ps. fluorescens P-17. Use the following equation to calculate volume of the inoculum:

(1000 CFU/mL) x (100 mL/vial) volume of inoculum = ---------------------------------------------- CFU/mL stock inoculum

Inoculated samples are incubated at 15 ° C in the dark for 8 days. If higher temperature is used, maximum growth is reached earlier, which shortens the incubation time (see below).

Enumeration of test bacteria Bacterial concentrations are analysed on incubation days 4-8 (starting from 4 th day and continuing until 8 th day). If higher temperature is used, maximum is reached earlier (3-7 days, should be tested beforehand). Shake vigorously the vials and make

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dilution series. Mechanical shaker (Vortex) may be used to shake the dilutions. Plate at least 3 dilutions (dilutions depend on assumed MAP concentrations) on R2A agar. Incubate plates at room temperature for 3 days and score the number of colonies. Count all colonies on selected plates containing 25 to 250 colonies of each bacterium and compute colony counts.(Swanson et al., 1992).

Expression of results

The maximum microbial growth number (CFU/ml) is converted to phosphorus concentration by using the yield factor. In determining of the yield factor, maximum growth (cfu) of Ps. fluorescens is related to different concentrations of Na 2 HPO 4 . The yield factor is derived from the slope of the line when cell growth is plotted against PO 4 -P concentration (Lehtola et al., 1999). Also, previously derived empirical yield value of 3.73 x 10 8 CFU Ps. fluorescens P-17/ µ g PO 4 -P can be used (Lehtola et al.,

1999)

The maximum plate counts are transformed with a conversion factor into the amount of phosphorus:

µ g MAP/L =

(CFU/mL) x (1000 mL/L)

(Measured yield factor or 3.73 x 10 8 CFU/µ g PO 4 -P*/L)

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4.7 Total nitrogen

European Standard: ENV 12260 (Water quality – Determination of bound nitrogen (TN b ) – following oxidization of nitrogen oxides).

See the original instructions from the PDF-file “ Nitrogen EN 12260.pdf” from the ftp-

site of SAFER.

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4.8 Assessment of nucleic acid damages by chlorination using

fluorochrome staining (SYBR-II or PI) and flow cytometry

Objectives

We aim to develop a sensitive and rapid method to assess the intracellular injuries

caused by disinfectants such as chlorine. The method combines cell staining with a

fluorochrome which efficiency depends on the nucleic acid integrity, and flow

cytometry for an objective quantification of the fluorescence changes. This

deliverable describes the procedure. The method gives a relative results which allow

to assess the intracellular injuries before and after bacterial chlorination.

Background

The bacteriological quality control of drinking water based on culture methods has

two important drawbacks: firstly, it is time-consuming method because bacteria

colonies on nutritive agar medium require from 1 to almost 15 days of incubation to

be conveniently observed. This prevents any use of such methods for an on-line

follow-up of the water bacteriological quality. Secondly, culture methods lead to an

important underestimation of the viable bacteria number: only a fraction of the

bacteria colony is cultivable (Roszak and Colwell, 1987). This fraction does not

represent the whole viable bacteria number, and more importantly underestimation is

worsened by the oxidant stress induced by disinfectants (hypochlorite, chloramine).

An alternative process to culture methods for controlling the disinfection efficiency

could use fluorescent dyes, which nucleic acid staining efficiency can be affected by

several processes, including chlorine disinfection. Many fluorescent probes are

commercially available and allow a good staining of all bacteria and rapid counting by

flow cytometry.

The main difficulty to apply fluorescence assay to the water quality disinfection after

chlorination is then to find a nucleic acid fluorescent probe that can (i) be a good

marker of damaged cells (or uninjured ones) as previously reported by Saby et al.

(1997) and Phe et al. (2004), (ii) be excited at 488 nm, the mostly used argon-laser

blue line in flow cytometers and (iii) lead to quantitative results. Besides, the water

quality control requires a quantitative result, and a high sensitivity, which means that

the probe should stain bacteria with very high complex rate constant and high

fluorescent quantum yield after excitation at 488 nm.

Here, we have selected SYBR Green II RNA Gel Stain (SYBR-II) and as propidium

iodide (PI) for quantitative staining of nucleic acids damages by chlorine. We have

defined the concentration of fluorochrome to be used, and the best contact time for

staining bacteria. At last, we have documented the way to count fluorescent events

and to measure fluorescence with flow cytometer.

Materials and reagents

SYBR Green II RNA gel stain (Molecular Probes, S-7586) Propidium Iodide (Sigma, P-4170) 12 ×75 mm plastic Falcon tube (Becton Dickinson- 352054)

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Aluminium paper Tube racks (Fisher, A 73 764 481) Protective gloves Vortexer Procedure The protocol is organized in 4 steps:

* Sampling water in aseptic conditions and chlorination neutralization.

* Bacterial staining with SYBR Green II or with propidium iodide (PI).

* Samples treatment analysis with flow cytometer.

* Treatment of data recorded by flow cytometry thanks to a software which allows to translate files in flow cytometry language into files in ASCII. Sampling

Sampling of water must be carefully done (i.e. aseptically) in order to prevent

contamination with dead or alive cells. Residual oxidant must be neutralized with

sodium thiosulfate.

Bacterial staining with SYBR-II or with PI

SYBR-II is a fluorochrome which stains efficiently nucleic acids. Its chemical formula

is covered by a patent and its mode of staining on nucleic acids is unknown. This

staining is done with a quantum yield higher than the other fluorochromes (Lebaron,

1998). SYBR-II is a membrane-permeant (Herrera et al., 2002) and has a low

intrinsic fluorescence, there is no need to destain to remove free dye (Haugland,

2002). This fluorochrome emits a green fluorescence (513 nm) when it is excited by a

flow cytometer argon laser at 488 nm, so the fluorescence is detected by the FL1

channel of flow cytometer FACSCalibur.

PI is a membrane-impermeant fluorochrome: it crosses only structurally damaged

membrane, so it is usually used as an indicator of membrane permeability. The

chemical structure of PI is a phenanthridine structure which allows binding to DNA by

intercalating between the bases with little or no sequence preference and with a

stoichiometry of one dye per 4-5 base pairs of DNA (Haugland, 2002). PI also binds

to RNA. PI emits a red fluorescence (617 nm) when it is excited by a flow cytometer

argon laser at 488 nm, so the fluorescence of PI is detected by the FL3 channel of

flow cytometer FACSCalibur.

One milliliter of sample in Falcon sterile tube is mixed with 0.5 µ L of the SYBR-II

solution or with PI solution (final concentration 10 µ g/mL). Samples are incubated

during 30 minutes in the dark, at room temperature (22±1°C). After staining step,

sample is immediately analysed by flow cytometer.

Counting of the total cell number and determination of the relative

fluorescence by flow cytometry

The counting of the fluorescent total cells is determined by combining staining with

SYBR Green II (or with PI) and flow cytometer (FACSCalibur, Becton Dickinson, San

Jose, California). Bacterial samples are analyzed by flow cytometer (FACSCalibur,

Becton Dickinson) with a theoretical flow rate of 40 µ L/min and the signals are

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treated with a BDCellQuest software (Becton Dickinson). The real flow rate is

checked by the difference in weight of the analysed tube before and after analysis. In

order to not saturate detection systems, dilutions are necessary in order to have a

counting under 2000 events/s.

All analysis is done in two steps. First, analyse an unstained sample in order to

determine the background and the self-fluorescence of the sample. Second, repeat

the analysis with stained sample by fluorochrome.

Analysis of sample without fluorochrome (SYBR-II or PI)

One millilitre of the sample is introduced in a sterile tube adapted for flow cytometer

(Becton Dickinson). This sample is analyzed by flow cytometer and the different

settings of sensitivity and amplification of FSC and SSC parameters are adjusted in

order to placed background and self-fluorescence of the sample in the bottom left

square with as coordinates (x,y) (2; 2) of the cytogram FL1 = f (SSC) for SYBR-II

(Figure 1) (or FL3 = f (SSC) for PI) thanks to settings.

FSC

FSC

BBaackgroundckground Background
BBaackgroundckground Background

SSC

SSC

FL1 FL1 Background Background
FL1
FL1
Background
Background

SSC

SSC

Figure 1: Example of cytogram of a sample without SYBR-II.

Analysis of stained sample with SYBR-II or with PI

The first aim is to localize and to discriminate the bacterial population on background

using different parameters which are on the one hand, the forward scatter (FSC) and

the other hand the side scatter (SSC). It is this second parameter which is measured

because flow cytometer has a better sensitivity for this parameter than for FSC. The

bacterial sample is isolated using the graphical palette of the BDCellQuest software

(Becton Dickinson).

A new acquisition is initiated and the display mode carries out under histogram

reporting on the X axis either FL1 (for SYBR-II staining) or FL3 (for PI staining) or

SSC, and in the ordinate axis the number of counted events.

The acquisition time depends of the number of cells counted and the discrimination

of

the bacterial populations. A zonation is realized around the population of interest

on

the cytogram FL1 = f (SSC) for SYBR-II staining or FL3 = f (SSC) for PI staining.

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The difference between before and after analysis weigh allows us to determine the analysed volume per minute. Thanks to a determination of a real flow rate and the number of fluorescent cells, the total number of bacteria per millilitre can be determined.

Single cell analysis

Flow cytometers generally store data in specialized flow cytometry standard (FCS) format, in which the intensities of each scatter or fluorescence parameter are encoded in a specialized, compact binary format that cannot be displayed in a word processor, or imported easily into other software.

Conversion to plain text ASCII puts the intensities values in plain text that can be viewed and edited in a word processor or used in a spreadsheet or generic data plotting software. "ASCII" designates a standard method of coding plain text or numbers on computers. Most textual information on computers is stored in ASCII. "Plaint text" files, conventionally named ending with .txt, contain only the text in ASCII.

The conversion of FCS listmode data files to plain text ASCII is done by Median Fluorescence Intensity (MFI) software. MFI is a software for analysis of flow cytometry data, it is a DOS program, so it works only on PC and not on Macintosh. MFI can be downloaded freely by Internet at this adress :

http ://www.umass.edu/microbio/mfi. The procedures of installing and operating MFI program can be found at this adress :

http://www.umass.edu/microbio/mfi/install.htm#running.

Table 1 represents a concrete example of a MFI-generated ASCII list mode data from FCS file.

Table1 : Example of ASCII list mode data from FCS file

Channel

FSC

SSC

FL1

0

0

0

929

1

0

0

1

2

0

2

14

3

0

438

1

4

0

77

2

56

99

229

0

57

109

264

0

58

95

233

0

69

210

49

0

70

230

59

0

71

183

39

0

Each line represents a single event. The numbers in the columns labelled FSC, SSC and FL1 represent the scatter and fluorescence intensities for each event. If imported into generic scientific plotting or spreadsheet software, the above plain text file can be used as input to generic plotting software, mathematics or statistics software, or spreedsheets.

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MFI software allows to improve the signal treatment of data recorded by flow cytometry (fluorescence intensity mean of population of interest) in order to get single cell analysis.

References

Haugland, R.P. (2002). Handbook of fluorescent probes and research products, 9th edition. Eugene : Molecular Probes Inc., 679 pages. Herrera G., A. Martinez, M. Blanco and J.E. O'Connor (2002). Assessment of Escherichia coli B with enhanced permeability to fluorochromes for flow cytometric assays of bacterial cell function. Cytometry 49 : 62-69. Lebaron P., N. Parthuisot and P. Catala (1998). Comparison of blue nucleic acid dyes for flow

cytometric enumeration of bacteria in aquatic systems. Appl. Environ. Microbiol. 64 (5) : 1725-

1730.

Phe M.H., M. Dossot and J.C. Block (2004). Chlorination effect on the fluorescence of nucleic acid staining dyes. Water Res. 38 : 3729-3737. Phe M.H., M. Dossot, H. Guilloteau and J.C. Block. Use of nucleic acid staining dyes to assess chlorinated drinking water bacteria injuries by flow cytometry. Water Res. (submitted). Saby S., I. Sibille, L. Mathieu, J.L. Paquin and J.C. Block (1997). Influence of water chlorination on the counting of bacteria with DAPI (4',6- diamidino-2-phenylindole). Appl. Environ. Microbiol. 63 : 1564-1569.

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4.9 Amino acid

4.10 Protocol use for biofilm extraction and dispatch samples to:

Extraction of biofilm

Fill a glass flask exempt from organic matter with ultra-pure water (about 7 mL of water per cm 2 of coupon) acidified by HNO 3 (pH < 2).

Place the coupon on the surface of the water (see figure below)

Ultrasonic probe Ultrasonic probe 15 15 W –10 min W –10 min Glass vial Glass
Ultrasonic probe
Ultrasonic probe
15
15
W –10 min
W –10 min
Glass vial
Glass vial
(20 mL)
(20 mL)
Material (coupon)
Material (coupon)
(biofilm support)
(biofilm support)
Acidified (HNO
Acidified (HNO 3 , pH<2)
, pH<2)
3
biofilm
biofilm
Ultrapure water
Ultrapure water

(7 mL per cm 2 of coupon)

of coupon)

(7 mL per cm

2

Extract biofilm with a ultrasound probe ( 3mm, 15 W during 10 minutes with 50% of rate of activation; apparatus : Sonifier II BRANSON model or similar)

Extract the biofilm of two coupons by sample if it is possible

Dispatch of sample

Use glass flasks exempt of organic matter for expedition of sample. The volume of flask must be small to decrease risk of contaminations. Flasks must be closed with Teflon caps or caps covered with Teflon.

Acidify the samples of water ( ultra pure HNO 3 , pH < 2).

Store samples at 4°C and send in icebox via express transport.

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5

Quality control and quality assurance

5.1

Quality control and quality assurance in CR4

The Laboratory of Microbiology and Chemistry has status of accredited testing

laboratory permit by DAR (the German Accreditation Service). The Laboratory of

Microbiology and Chemistry complies with the standard EN ISO/IEC 17025 and thus

will also operate in accordance with ISO 9001 and ISO 9002.

The main analytical methods and the test equipment used in the laboratory of Microbiology and Chemistry are described in specific Standard Operating Procedures (SOP). The competence of the personnel is provided by internal audits and continuous appropriate education and training.

5.2 Quality control and quality assurance in CR6

The quality control system of KTL/ Department of Environmental Health is designed

to satisfy the internal managerial needs. Two official international quality standards

are used in the organisation. The Laboratory of Chemistry has status of accredited

testing laboratory permit by FINAS (the Finnish Accreditation Service). Laboratory of

Chemistry complies with the standard EN ISO/IEC 17025 and thus will also operate

in accordance with ISO 9001 and ISO 9002. The laboratory of Toxicology complies

with the OECD Principles of Good Laboratory Practice. The Toxicity Testing Unit is

approved in the national GLP compliance Program and inspected on a regular basis.

The quality system of Department of Environmental Health is created based on the Principles of GLP and the standard EN ISO/IEC 17025. The Department has common Standard Operating Procedures, which are establish by the Management and Quality Assurance Unit. Internal audits concern the common procedures of the Department and the main processes of the laboratories.

The main analytical methods and the test equipment used in the laboratory of Environmental Microbiology are described in specific SOPs (so called SOP MB). The competence of the personnel is provided by continuous appropriate education and training.

5.3 Quality control and quality assurance in CR9

International Depository Authority Microbial Strain Collection of Latvia (MSCL) in practice follows the principles listed in "Guidelines for the Establishment and

Operation of Collections of Cultures of Microorganisms" /2nd Edition , June 1999; Revised by the World Federation for Culture Collections (WFCC)/ with the purpose of promoting high standards of scientific service in microbiological laboratories. CABRI QUALITY GUIDELINES (Demonstration Project ERBBIO4-CT96-0231, co-funded by

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grant from DGXII of the Commission of the EU) Part I has been adopted in MSCL and it covers procedures that as far as possible quarantee:

adherence of CABRI to international European or national regulations as

well as to ethical and safety standards in the field of biotechnology;

authenticity of biological materials;

purity of cultures or absence of contaminants;

quality-controlled processing of cultures;

accuracy of data collected and supplied;

punctuality and adherence to delivery standards.

The competence of the personnel is provided by annual Training courses and workshops organized by ECCO( European Culture collection organization) as well by appropriate education.

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6

Biofilm monitoring devices

6.1

Propella (The common biofilm monitoring device for all partners)

PROPELLA ® THE DYNAMIC CONCEPT TO STUDY INTERACTIONS BETWEEN WATER AND MATERIALS

Possible uses:

Measurement of surface biological colonisation

Corrosion

Entartrement and deposits

Salting out contaminants by materials

Biological and chemical stability of water

Surface and water disinfection testing

Fields of application

Research studies

Testing of materials

In situ biofilm, corrosion measurements on water plants and distribution networks

Introduction

In drinking water distribution networks, as in numerous industrial processes, the degradation of water quality (biological, chemical contamination) and/or exposed surfaces (corrosion, scaling) is explained by the interaction between the liquid and material phases. It is difficult to predict the intensity of these water-material interactions and in many cases it is necessary to expose the material to water in order to evaluate the compatibility of the two products. Contrary to static tests, Propella® was built to simulate in the laboratory a piece of pipe transporting liquid such as potable water, thermal waters, etc.

Principles of PROPELLA®

The Propella® reactor provides an original and efficient solution to undertake tests on a laboratory scale, and in particular:

to control independently the hydraulic residence time and the speed of water circulation

to control hydraulic water flow which is indispensable to reproduce transfers between solid and liquid phases

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to study water characteristics and exposed surfaces

Propella® is a perfectly mixed reactor, in which the liquid is pushed by a propulsive propeller through an internal tube (see the illustration below). The liquid flows along the canalisation section studied, as in a real pipe. It is easy to impose a defined Reynolds number by fixing the circulation rate in the pipe. The hydraulic residence time is inversely proportional to the alimentation flow of the reactor.

How the PROPELLA functions

The reactor can test real canalisation sections or only coupons of materials, with or without continuous flow. The flow rate near the pipe is controlled by the rotation speed of the propeller. According to needs, sampling devices can be put on the studied pipe to measure surface properties. These devices can be sampled without emptying or stopping the reactor. Except for the studied canalisation section, all materials in contact with water are of inox 316l and Teflon to insure the chemical inertia of the system. These materials can be adapted however to other needs.

The reactor allows, among others :

to model disinfectant use in drinking water distribution networks, and the influence of pH, temperature, laminar or turbulent flow, bacterial deposits (biofilm), pipe material, etc.

to study the salting out of mineral and/or organics by the pipe material, and also to model bacterial growth with or without disinfectant.

The reactor permits :

to modify and/or maintain the fluid characteristics by introducing reactive into the reactor ;

to modify and/or maintain a defined agitation characterised by a Reynolds number, and also a direction flow ;

to quantify microbial deposits on pipewalls and sample a part of the colonised surface of the reactor in contact with water ;

to test different materials used in real drinking water distribution networks ;

to work at different temperatures by the presence of an internal thermoregulation system.

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Characteristics

water supply motor exit coupons biofilm sampling section of devices material under study vane double
water supply
motor
exit
coupons
biofilm
sampling
section of
devices
material under
study
vane
double
internal
cylinder
water for
thermoregulation

Figure 1: Scheme of Propella

Surface/volume relation identical to a real pipe

Volume : approx. 2.23 L (generally 100 x 500 mm)

Flow variable speed from 0.05 to 0.5 m/s (typically 0.2 m/s)

Inox or glass pipes available to study specifically water

Material : inox 316L and Teflon (except studied pipe)

Up to 20 sampling devices per pipe

Possibility to connect reactors in series

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Setup examples

32 Setup examples Figure 2: Pictures of Propella Propella© reactor, dimensions Internal cylinder : height 460

Figure 2: Pictures of Propella

Propella© reactor, dimensions

Internal cylinder : height 460 mm, internal diameter 44,0 mm, external diameter 72,5

mm (thickness of material = 14,25 mm)

External cylinder : height 500 mm, external diameter 110,0 mm, internal diameter

93,4 mm, thickness of material = 8,3 mm

Normal procedure to clean PVC/PEHD coupons

3 hours soaking in a detergent solution (Aquet, Polylabo, reference 64528; a non- ionic, neutral pH, no-phosphates and biodegradable detergent or equal detergent, concentration 1%) and then careful brushing by hand

rinsing thoroughly with tap water

steeping in a chlorine solution (20 mg/L) during 15 minutes

rinsing two times with distilled sterile water without bacterial cells

drying in an oven (60°C), then storing in a sterile place

then ready to use

Normal procedure to clean Propella reactor

De-assemble completely the Propella reactor

Only for the external envelope (PVC or PEHD one): 1 hour in a chlorine solution (100 mg Cl2/L) and then careful brushing

Rinse thoroughly with tap water

Rinse two times with distilled sterile water without bacterial cells.

Wait until the propella reactor is completely dry

Re-assemble Propella reactor, then ready to use

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Sampling of biofilm from a Propella Procedure is described in a separate file on the ftp-site of SAFER “Propella_sampling.doc (3.6 MB)

Biofilm removal protocol PVC coupons that are colonized with biofilms are taken from the sampling devices without discontinuing the water flow within the distribution system. They are then placed in sterile flasks containing 25 ml of bacterial cell-free distilled water. Less than 30 minutes later, the biofilm is dispersed by a gentle sonication (2 min. ultrasounds at 2 W, 20 KHz; Bioblock Scientific Vibra cell, model 72401; probe model 72403,

Bioblock, USA, diameter 3mm). The probe is placed 1 cm above coupon, inside bacterial cell-free distilled water. The bacterial content of the resulting suspensions must be analysed within one hour.

DON'T FORGET TO PLACE

THE

STERILE

FLASK

CONTAINING

THE

COUPON IN

ICE DURING

SONICATION

TO

AVOID

INCREASE

O F

TEMPERATURE

 
TO AVOID INCREASE O F TEMPERATURE   Figure 3: Dispersing the biofilm from Propella coupons To

Figure 3: Dispersing the biofilm from Propella coupons

To determine sonication efficiency:

Repeat the sonication as above by placing the coupon in a new sterile flask containing 25 ml of bacterial cell-free distilled water. If the count is less than 1% of bacteria compared with the first sonication, you can estimate one sonication is sufficient. Otherwise do two/three successive sonications.

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6.2 Rotating Annular Reactor (modified RotoTorqueTM) [CR4]

Scope The RotoTorque TM (Rotating Annular Reactor) (Characklis, 1990) is a useful device for biofilm growth under defined conditions. It was modified by Griebe and Flemming (1996) and later again by Schulte and Wingender (2000). The Rotating Annular Reactor can be applied in research studies, in the testing of materials or cleaning procedures and in in situ measurements in water plants and distribution networks.

References Griebe, T., Flemming, H.-C. 2000. Rotating annular reactors for controlled growth of biofilms. In: Flemming, H.-C., Szewzyk, U., Griebe, T. (Eds.), Biofilms, Lancaster, Pennsylvania, Techonomic Publishing Company, pp. 23-40.

Schulte, S. 2003. Efficacy of hydrogen peroxide against biofilm bacteria. PhD theses of the University of Duisburg-Essen.

Materials

The following list summarises experimental variables that are important in the selection, design and construction of all biofilm devices when different research questions will be addressed.

Physical parameters:

Flow velocity + Shear stress

Temperature

Surface properties, composition and characteristics of the internal materials

Hydraulic residence time Chemical parameters:

Substrate composition and concentration

Bioproducts in the biofilm matrix and bulk liquid phase

Redox potential

Inorganic ions

Organic and inorganic particles

Biological parameters:

Microorganism type (algae, protozoa, bacteria, viruses, etc.)

Defined or undefined culture

Mixed or pure culture

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Engine Influent extractable Screw for the extraction of of coupons test-surfaces (Coupons) rotating inner
Engine
Influent
extractable
Screw for the extraction of
of coupons
test-surfaces
(Coupons)
rotating
inner cylinder
with
recirculation
tubes
outer cylinder
with uptake
for
the
outlet

Figure 4: Scheme of the Rotating Annular Reactor after Griebe and Flemming (1996). In the figure there are shown two focus levels. The water containing inner space is marked light blue

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36 Table 1: Dimensions of the Rotating Annular Reactor Inner cylinder: unit     Height

36

Table 1: Dimensions of the Rotating Annular Reactor

Inner cylinder:

unit

   

Height

cm

 

18,0

Bore

cm

 

10,2

Exposed area (vertical)

 

cm_

 

576,8

 

Exposed area (horizontal)

 

cm_

 

157,1

Total area

cm_

 

733,9

Outer cylinder:

     

Height

cm

 

20,6

Bore

cm

 

11,30

 

Exposed area (vertical)

 

cm_

 

731,1

Exposed area (horizontal)

 

cm_

 

100,3

Total area

cm_

 

831,4

Coupons:

   

Number

   

12

Width

   

1,5

Length

cm

 

22,0

Exposed length

cm

 

20,1

Exposed area

cm_

 

30,9

Total area (x 12)

 

cm_

 

370,8

 

Percentage on the total area of the outer cylinder

 

%

 

44,6

Recirculation tubes:

       

Number

   

4

Angle

°

 

80

Length

cm

 

18,5

Bore

cm

 

1,0

Exposed area

cm_

 

231,47

 

Total Rotating Annular Reactor:

     

Volume

mL

 

ca. 650 dependent on the rotation speed

Exposed total area (without recirculation tubes)

 

cm_

 

1565,3

Percentage of the coupons on the total area

 

%

 

23,68

Specific area

cm_/cm_

 

2,76

Table 2: Measurements of the middle rotating velocities and Reynolds- numbers in the Rotating Annular Reactor with different revolutions per minute

Revolutions

ri calculated

Horizontal velocity

Vertical velocity

Total velocity.

Re

per min

[U min -1 ]

[m s -1 ]

[m s -1 ]

 

[m s -1 ]

[m s -1 ]

-

50

0,13

0,10

 

0,01

0,10

514

200

0,53

0,44

 

0,03

0,45

2226

600

1,60

1,29

 

0,08

1,29

6439

ri: rotating velocity of the inner cylinder

Re Reynolds number

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Re = 11,846 x revolutions/min010002000300040005000600070000200400

x revolutions/min 010002000300040005000600070000200400 Figure 5: Reynolds-number of the annular reactor as a

Figure 5: Reynolds-number of the annular reactor as a function of rotating velocity.

Procedure

Sterilisation and operation of the Rotating Annular Reactor

Prior to the experiments the reactor system including all tubes is either sterilised by

autoclaving for 20 minutes at 121°C or disinfected by biocides with 1000 mg/L

hydrogen peroxide for 2 hours at a rotation speed of the inner cylinder of 400 rpm.

The sterile Rotating Annular Reactor is then fed with drinking water flowing with

50 mL/h corresponding to a residence time of 12 hours.

Sampling the biofilm from the test-surfaces

)

When they are colonised with biofilms they are taken from the sampling devices

without discontinuing the water flow within the distribution system. They are analysed

for bacterial density directly by using epifluorescence microscopy. The biofilm is

stained with 4´, 6-diamidino-2-phenylindole (DAPI) having a final concentration of 5

µ g/mL. On each slide, at least 300 bacterial cells must be counted at 1.000 x

magnification on the coupon.

For indirect enumeration the biofilm bacteria are scraped mechanically with a razor

blade and disaggregated on a vortex for 3 minutes. With this bacterial suspension the

total cell number (see chapter total cell number) and the heterotrophic plate count

(see chapter enumeration of culturable microorganisms) are performed.

Coupons can be made out of different materials (PVC, stainless steel, copper,

Setup examples

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38 Figure 6 : Images of Rotating Annular Reactors (modification of Schulte and Wingender, 2000) under

Figure 6 : Images of Rotating Annular Reactors (modification of Schulte and Wingender, 2000) under operation conditions

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6.3 Biofilm generator

The schematic representation of the biofilm generator is given on Figure 7.

of the biofilm generator is given on Figure 7. Figure 7: Chemostat lab biofilm generator Diagram

Figure 7: Chemostat lab biofilm generator

Diagram of the second stage model biofilm system with multiple assemblages of coupons suspended from rigid titanium wire inserted through silicone rubber bungs in the top ports. The weir system is used to maintain the volume at the required level. Temperature, oxygen and pH probes are not shown.

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6.4 Flow cell reactor

The biofilms are formed on several adhesion slides placed within flow cell reactors (which have a semi-circular cross section), where drinking water can flow under different hydrodynamic conditions. The adhesion slides, which can be made in different sizes and of different materials, are glued to rectangular pieces of PMMA properly fitted in the apertures of the flow cell. The equipment is connected to a side stream in a drinking water system or a simulated drinking water system, as schematically represented in the Figure 8.

water system, as schematically represented in the Figure 8. Figure 8: Linear flow through reactor for
water system, as schematically represented in the Figure 8. Figure 8: Linear flow through reactor for

Figure 8: Linear flow through reactor for biofilm

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6.5 Pipeline biofilm collector

Biofilm monitoring system used in CR9 The biofilm monitoring systems consist for Pipeline biofilm collector and Propella and

a plug flow reservoir. The system is connected directly to the water supply system of

Riga. Water from the water system water is collected in the reservoir that ensures

constant pressure and flow in the biofilm reactors (Figures 9 and 10).

The same device will be used as the reference biofilm samplers in CR6 (Finland) and

CR9 (Latvia). The differences in the devices/test systems between CR6 and CR9 are

described in the text.

Maintenance

Biofilm collectors are 10 cm long PVC pipes, which are connected together one after

another with ball valves and stainless steel loops. Cleaning of the pipeline system

parts follows the Propella cleaning procedure. The pipeline system is installed on

stainless steel frame. The amount of the PVC pipes in one biofilm collector will vary

in different tests: 7 samplings with 3 replicates equals to 21 subsamples. Water flow

through the pipes will be adjusted to 500 ml/min (= 0.1 m/s) using flow meters/valves.

Before sampling, ball valves at both sides of a PVC pipe are closed and the pipe

samples full of water are removed and replaced with new pipes. Valves are opened

and water flow is connected back after the sampling.

Biofilm sampling

Part of water (1.5 ml) is removed from the detached pipe and a spoonful of sterile

glass beads are inserted into the pipe. To detach biofilm from the inner side of the

pipe, the pipe-valve system containing water and glass is vortexed for 20 minutes.

This water-biofilm sample is combined with the water removed before vortexing.

Finally the pipe is rinsed with sterile deionized water (5 ml) (CR9: pipe is rinsed twice

= total 10 mL) which is combined with the vortexed water-biofilm sample.

CR6: the test system CR6 uses tap water of Kuopio city as feed water. Pipeline device is a system connected directly to the tap water system In Propella tap water flows first into a small glass vessel, where it is pumped with peristaltic pump into the device.

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42 Figure 9. Schematic drawing of biofilm monitoring system in CR9 C A B Figure 10.

Figure 9. Schematic drawing of biofilm monitoring system in CR9

C A B
C
A
B

Figure 10. Photos of (a) biofilm monitoring system with (b) reservoir and (c) inlet and sewerage.

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6.6 Differential turgidity measurement

DTM is an optical measurement system consisting of two measurement cells – pairs

of optical windows (Figure 11). One pair is continuously cleaned in order to prevent

biofilm building. The fouling effect on the non-cleaned cell can be directly measured if

the signal of the cell with clean surface is subtracted.

Flow irection
Flow irection

detector

with clean surface is subtracted. Flow irection detector Light source detector Light source Figure 11: Schematic

Light

source

detector

Light

source

Figure 11: Schematic view of a DTM device

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6.7 Fibre optic devices (FOS, and FluS sensors)

FOS and FluS are both fiber optic based devices. Optical fiber heads are implemented in the water system.

Substratum Optical Fiber
Substratum
Optical Fiber

Figure 12: Schematic view of a FOS

The Fibre Optical Device (FOS)

The principle of the fibre optical device is based on the determination of the local

concentration of light scattering particles depositing on a tip of an optical fibre. The

backscattered light quantified and the signal is related to the amount of material. This

signal is essential a sum parameter composed of the contributions of all light scattering

materials. However, it can be specified by calibrating with microorganisms and by

changing of the system by addition of nutrients or increase of shear forces; this will

influence the signal in a predictive way. The concentration is determined by the

scattering signal.

The measuring head Light from a monochromatic or quasichromatic source is transmitted through optical

fibres to the system in question. The backscattered light is transmitted by a parallel

fibre to a detector. In a water system, the sensor is integrated evenly into the inner

surface and experiencing the same flow conditions as the rest of the internal wall.

Therefore, it can be considered as representative for the situation. Material

depositing on the surface will contribute the strongest signal of backscattered light

while particles in the water phase give a much lower signal. This has been

demonstrated in the calibration experiments carried out in WP 1. The diameter of the

measuring fibre is about 0,2 mm. Although it is made of glass, it has been observed

that the deposit forming on it is not distinguishable from that forming on other

surfaces such as stainless steel.

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Figure 13 shows the basic configuration of the sensor head. The distal end of a

single fibre, cut and polished perpendicularly to its optical axis, is used as test

surface. WATER Integrated in SURFACE BIOFILM
surface.
WATER
Integrated in SURFACE
BIOFILM

Figure 13 Schematical depiction of the configuration of the sensor head. The device is integrated into the surface which is monitored for deposition of material

The FOS has been calibrated and the results were presented at one of the cluster

meetings which are documented in the protocols. The algorithm for the software has

been changed in order to allow distinguishing between biotic and abiotic material on

the basis of selection of specific wavelengths of the reflected light. A chip and a

sender will be implemented and the data can be transferred via satellite to remote

operators.

During the work performed in WP 1, the sensor was implemented into an Annular

Reactor (arrows in figure 14):

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46 Figure 14: 3 FOS implemented in an Annular reactor EVK1-CT-2002-00108 Surveillance and control of microbiological

Figure 14: 3 FOS implemented in an Annular reactor

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6.8

Electrochemical, nanovibration, and capacitive monitors

6.8.1

Mechatronic Surface Sensor - MSS

The basic idea behind this monitor is to utilise the nanovibrations to evaluate the amount of the biofilm through mathematic analysis of the signal. This technique utilizes the direct and converse electromechanical properties of smart materials, allowing simultaneous actuation and sensing.

Until now, this technique has been successfully used in the diagnosis of plate metal defects, namely in the aerospace industry, and in soil consistence analysis in civil engineering. The monitor is a half-tube flow cell (see figure 15) and the sensors are on the outer flat surface of this flow cell.

Water
Water

Figure 15: Mechatronic Surface Sensor

Sensors

6.8.2 Capacitive sensors

Capacitive sensors are analogous, non-contact devices. A capacitive bridge is built from internal capacitance and the capacitances created by the proximity of the object (the biofilm) to be measured. The dielectric is directly related to the distance/medium in front of the sensor plate; ultra-precise electronics convert the capacitance information into an analog signal. Very precise sensors can be made, up to de resolution of 0.01 nanometer.

The monitor is identical to the one containing the mechatronics surface sensors (a half-tube flow cell) and the capacitive sensor will be inserted on the flat surface of this flow cell.

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6.9 Biofilm formation monitoring using ATR-FTIR sensor

Scope The development of the biofilms is monitored on a germanium crystal, which is compatible with vibrational spectroscopic measurements, in a continuously drinking water fed flow chamber included in an open circuit (Figure 16). To improve the sensitivity and the delay of response, the crystal is firstly covered by a Luria Bertani (5g/L) conditioning film, and then colonised by two hours exposure to Pseudomonas fluorescens (Pf) suspension, as bacterium model frequently isolated in drinking waters. Absorption bands for proteins (Amide II band) in the vibrational spectra are chosen as probes, because these macromolecules are major constituents of bacteria and biofilms.

Peristaltic Trash flask pump Sample flask ATR flow cell
Peristaltic
Trash flask
pump
Sample flask
ATR flow cell

Figure 16: Experimental device for the infrared biosensor

ATR/FT-IR analysis ATR/FT-IR spectra are measured between 4000 and 800 cm -1 on a Bruker Vector 22 spectrometer equipped with a KBr beam splitter and a DTGS (deuterated triglycine sulphate) thermal detector. The resolution of the single beam spectra is 4 cm -1 . The number of bi-directional double-sided interferogram scans is 100, which corresponds to a 1 min accumulation. The interferograms are apodised with the Blackman-Harris 3-Term function. No smoothing but a baseline correction is subsequently applied.

The ATR flow cell is a SPECAC cell (Eurolabo, ref. 11160) designed to enclose a horizontal trapezoid crystal. The incidence angle of the ATR crystal is 45°, which allows six internal reflections on the upper face in contact with the sample (figure 17). The compartment of the spectrometer containing the flow cell is continuously purged with dry and decarbonated air provided by a Balstom compressor for removing water

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vapour and carbon dioxide. FT-IR measurements are made at room air-conditioned

temperature (21°C +/- 2 °C). Irradiance throughout the empty cell is about 11 % of

the full signal (without the ATR accessory). ATR spectra are shown with an

absorbance scale corresponding to log(R reference /R sample ), where R is the internal

reflectance of the device. The ratio of the single beam ATR spectrum of the

conditioned film (A) + bulk species in the studied water (B) to the spectrum of (A) +

(B) + bacteria gave the absorbance scale spectrum of the bacteria attached on the

Ge crystal. When necessary, the contribution of water vapour due to variation in

relative humidity in the room was eliminated. The corresponding spectrum was

obtained separately by calculating the difference between spectra of "wet" and dry

air. Despite the absorbance scale used the ATR spectra is not strictly proportional to

the absorption coefficients as no further correction is applied.

IR sourcedetectorentranceexitGe crystalBiofilm
IR sourcedetectorentranceexitGe crystalBiofilm

Figure 17: Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) flow through cell

Biofilm study in flow cell

The flow cell (figure 2) is successively and continuously supplied with (i) ethanol (75

% for 4 hours) to clean the system, (ii) non pyrogenic sterile water (1 hour at 40

ml/min and 10 hours at 0.7 ml/min) to remove the ethanol (control by recording

spectra) (iii) Luria Bertani (LB) nutritive medium to deposit a conditioned organic film

(during 5 to 10 hours), (iv) the bacterial suspension (2 hours) which allowed bacteria

to adhere to the surface of the crystal, (v) the tested 0.2 µ m filtered water for 24

hours. Ethanol, bacterial suspension and water samples are passed through the ATR

flow cell by using an up-stream Gilson Minipuls 3 pump with a flow of 44 mL h -1

(hydraulic residence time approximately 4 min). The infrared spectra of the hydrated

dynamic biofilm are automatically collected every 15 minutes for the first hour and

then every hour.

Pseudomonas fluorescens bacteria suspension

Pseudomonas fluorescens (Pf) strain was purchased at Institut Pasteur (CIP6913,

Paris). Stock cultures were prepared with cryogenic balls in glycerol: a 24h-culture of

Pf realised on R 2 A (Difco, 218263) agar plate, identified by API 20NE strip (number

0147555). Then, the bacteria were scraped, and suspended in the cryogenic tube. It

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was homogenised with vortex, and staid at rest 3 hours before removing the glycerol with a sterile Pasteur pipette. They kept frozen at -80°C.

Standardized cultures were obtained by growing the organisms at 28°C, under stirring (350rpm) in Luria Bertani broth (LB Broth Miller, Difco, 244620) for two consecutive periods (the first 24h and the second 10 hours). The cells were collected at the end of their exponential phase of growth. The suspension was centrifuged and washed two times with sterile saline solution (NaCl 9 0 / 00 ). Then, the pellet was resuspended in NaCl 9 0 / 00 and a LB solution at 5g/L was inoculated to obtain a bacterial suspension whose absorbance was about 0.31 at 620nm. After 1h, under stirring (350rpm), at room temperature (21°C), this suspension was flowing into the open circuit.

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