Handbook SAFER V02light

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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

Contract n°EVK1-CT-2002-00108

Deliverable 02 version 2Handbook for analytical methods and

operational criteria for biofilm reactors

Updated November, 2005

Non confidential

http://www.safer-eu.comContact persons : [email protected]

[email protected]



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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

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Content

1 INTRODUCTION 5

2 STRUCTURE 5

3 MICROBIOLOGICAL METHODS 6

3.1 Enumeration of culturable microorganisms (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



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 476.8.1 Mechatronic Surface Sensor - MSS 476.8.2 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 ofbacteria 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 µm. 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 byfluorescence microscopy. Appl. Environ. Microbiol. 33: 12225-1228.ASTM Standard Test Method: Designation: F 1095 – 8 (Reapproved 1994). Rapidenumeration of bacteria in Electronic-Grade Purified Water Systems by Direct CountEpifluorescence Microscopy.

Materials

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

All reagents should be filtered through a cellulose nitrate filter with the pore size of0.2 µm.

Procedure

Place one black polycarbonate membrane filter (0.2 µm 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

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

Triton X-100 (0.1 %). The final concentration should be 5 µg/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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3.4 Dehydrogenase activity 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 probefor direct visualisation of actively respiring bacteria, Appl. Environ. Microbiol. 58, 6, 1801-8Schaule G., Flemming H-C, Ridgway HF (1993). Use of 5-Cyano-2,3-ditolyl tetrazoliumchloride for quantifying planctonic and sessile respiring bacteria in drinking water. Appl. Envir.Microbiology 59: 3850-3857.

Material

• Black polycarbonate membrane filter (pore size 0,2 µm) and a filtration set

• Epifluorescent microscope and nonfluorescent immersion oil

• 5-cyano-2,3-ditolyl tetrazolium chloride (CTC, Polysciences). Reactant solution ofCTC 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 anoxicconditions.

Filter the sample after the incubation time through a black polycarbonate membrane

filter (pore size 0.2 µm) 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 µg/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 µL 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 µm. 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 µL of LUMACULT. Then 500 µL 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 µL 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 µL of the enzymatic complex Luciferine – Luciferase (LUMIT PM) areautomatically 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 (RLU1),

5. addition of 20 µL 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 theanalysed volume (RLU2).


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 µm 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 µg 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 µg 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 distributionsystems by a simplified bioassay technique. In Advances in Water Analysis and Treatment,Proc. 16th 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 organiccarbon 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 ofbacteria 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. InCompendium 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 atlow substrate concentrations in tap water. Antonie van Leeuwenhoek 48:229-243.

Materials

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

• Hot water bath (65-70 °C)

• Continuously adjustable pipettes capable of delivering between 10 and 100 µL,between 200 and 1000 µL and between 1000 and 5000 µL.



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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 gCH3COONa.3H2O (or 1.71 g CH3COONa) in 1 L organic-carbon free, deionizedwater. Transfer to 100-mL vials, cap and autoclave. Store at 5 °C. Solution maybe held for up to 12 months.

• Sodium thiosulfate solution. Dissolve 30 g Na2S2O3 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 (NH4)2SO4, 0.2 g KH2PO4, 0.1 gMgSO4

.7H2O, 0.1 g CaCl2.2H2O, 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) andAquaspirillum 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 µm) and autoclaved sample to obtain a final concentration of approximately 108

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 µg 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 µL sodium



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thiosulfate solution added to each vial. Add 100µL 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.

Inoculated samples are incubated at 15 °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 106 CFU P. fluorescens P-

17/µg acetate-C and 2.1 x 107 CFU Aquaspirillum sp. NOX/µg acetate-C at 15 °C.


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

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



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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 (PO4-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 µg 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 fordetermination 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 ofbacteria from potable water. Appl. Environ. Microbiol. 49:1.Swanson K.M.J., Busta F.F., Peterson E.H., Johnson M.G. 1992. Count methods. InCompendium 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 isrecommend because of more effective shaking) with caps.

- Hot water bath (65-70 °C)- Continuously adjustable pipettes capable of delivering between 10 and 100 µL,

between 200 and 1000 µL ,and between 1000 and 5000 µL.- 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

CH3COONa.3H2O (or 1.71 g CH3COONa) in 1 L organic-carbon free, deionizedwater. Transfer to 100-mL vials, cap and autoclave or pasteurize at 60°C for 35min. Store at 5 °C. Solution may be held for up to 6 months.

- Sodium thiosulfate solution. Dissolve 30 g Na2S2O3 in 1 L deionized water. Transferto 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 NH4NO3, 0.1 g MgSO4

.7H2O, 0.1 gCaCl2

.2H2O, 0.1 g NaCl, 0,1 g KCl in 1 L phosphorus-free water. Transfer to100-mL vials, cap and autoclave.

- 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 µm), 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 µL/lmineral salts solution,

1000 µg 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 µl sodium thiosulfate solution for 100 ml sample. 100

µL of mineral salts solution and 400 µL 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 4th day

and continuing until 8th day). If higher temperature is used, maximum is reached

earlier (3-7 days, should be tested beforehand). Shake vigorously the vials and make



19

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).


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 Na2HPO4. The

yield factor is derived from the slope of the line when cell growth is plotted against

PO4-P concentration (Lehtola et al., 1999). Also, previously derived empirical yield

value of 3.73 x 108 CFU Ps. fluorescens P-17/µg PO4-P can be used (Lehtola et al.,

1999)

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

of phosphorus:

µg MAP/L = (CFU/mL) x (1000 mL/L)

(Measured yield factor or 3.73 x 108 CFU/µg PO4-P*/L)



20

4.7 Total nitrogen

European Standard: ENV 12260 (Water quality – Determination of bound nitrogen

(TNb) – following oxidization of nitrogen oxides).

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

site of SAFER.



21

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)



22

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 totranslate 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 µL of the SYBR-II

solution or with PI solution (final concentration 10 µg/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 µL/min and the signals are



23

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.

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.

FSC

SSC

BackgroundFSC

SSC

BackgroundBackground

SSC

Background

FL1

SSC

Background

FL1



24

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 FL10 0 0 9291 0 0 12 0 2 143 0 438 14 0 77 2

... ... ... ...56 99 229 057 109 264 058 95 233 0

... ... ... ...69 210 49 070 230 59 071 183 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.



25

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 coliB with enhanced permeability to fluorochromes for flow cytometric assays of bacterial cellfunction. Cytometry 49 : 62-69.Lebaron P., N. Parthuisot and P. Catala (1998). Comparison of blue nucleic acid dyes for flowcytometric 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 nucleicacid staining dyes. Water Res. 38 : 3729-3737.Phe M.H., M. Dossot, H. Guilloteau and J.C. Block. Use of nucleic acid staining dyes toassess 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 waterchlorination on the counting of bacteria with DAPI (4',6- diamidino-2-phenylindole). Appl.Environ. Microbiol. 63 : 1564-1569.



26

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 cm2 of coupon) acidified by HNO3 (pH < 2).

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

• 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 HNO3, pH < 2).

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

Ultrasonic probe15 W –10 min

Material (coupon)(biofilm support)

Acidified (HNO3, pH<2)Ultrapure water

(7 mL per cm2 of coupon)biofilm

Glass vial(20 mL)

Ultrasonic probe15 W –10 min

Material (coupon)(biofilm support)

Acidified (HNO3, pH<2)Ultrapure water

(7 mL per cm2 of coupon)biofilm

Glass vial(20 mL)



27

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.


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.


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



28

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.



29

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



30

• 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.



31

Characteristics

motor

biofilmsamplingdevices

water forthermoregulation

vane

doubleinternalcylinder

section ofmaterial understudy

water supply exit

coupons

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



32

Setup examples

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 equaldetergent, 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...



33

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

C O N T A I N I N G T H E

COUPON IN ICE DURING

SONICATION TO AVOID

I N C R E A S E O F

TEMPERATURE

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.



34

6.2 Rotating Annular Reactor (modified RotoTorqueTM) [CR4]

Scope

The RotoTorqueTM (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



35

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

Influent

Engine

extractable

test-surfaces

(Coupons)

rotating

inner cylinder

outlet

with

recirculation

tubes

Screw for the extraction ofcouponsof coupons

outer cylinder

with uptakef o r t h e

coupons



36

Table 1: Dimensions of the Rotating Annular Reactor

Inner cylinder: unit

Height cm 18,0Bore cm 10,2Exposed area (vertical) cm_ 576,8Exposed area (horizontal) cm_ 157,1Total area cm_ 733,9

Outer cylinder:

Height cm 20,6Bore cm 11,30Exposed area (vertical) cm_ 731,1Exposed area (horizontal) cm_ 100,3Total area cm_ 831,4

Coupons:

Number 12Width 1,5Length cm 22,0Exposed length cm 20,1Exposed area cm_ 30,9Total area (x 12) cm_ 370,8Percentage on the total area of the outer cylinder % 44,6

Recirculation tubes:

Number 4Angle ° 80Length cm 18,5Bore cm 1,0Exposed area cm_ 231,47

Total Rotating Annular Reactor:

Volume mL ca. 650dependent on the

rotation speedExposed total area (without recirculation tubes) cm_ 1565,3Percentage of the coupons on the total area % 23,68Specific 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

Revolutionsper min

ri calculated Horizontal velocity Vertical velocity Total velocity. Re

[U min-1] [m s-1] [m s-1] [m s-1] [m s-1] -50 0,13 0,10 0,01 0,10 514200 0,53 0,44 0,03 0,45 2226600 1,60 1,29 0,08 1,29 6439

ri: rotating velocity of the inner cylinder

Re Reynolds number



37

Re = 11,846 x revolutions/min010002000300040005000600070000200400600revolutions/min

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

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

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

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

µg/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.

Setup examples



38

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



39

6.3 Biofilm generator

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

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.



40

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.

Figure 8: Linear flow through reactor for biofilm



41

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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Figure 9. Schematic drawing of biofilm monitoring system in CR9

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

inlet and sewerage.

BA

A

CA



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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.

Figure 11: Schematic view of a DTM device

detector

Lightsource

e

detector

Lightsource

ee

Flow irection



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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 areimplemented in the water system.

Biofilm

Bacteria

IIluminating light

Backscatteredlight

Optical Fiber

Illuminatedbiofilm area

EPS

Substratum

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 headLight 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.

Figure 13 Schematical depiction of the configuration of the sensor head. Thedevice is integrated into the surface which is monitored for deposition ofmaterial

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):

WATER

BIOFILMIntegrated in SURFACE



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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 Sensors

Figure 15: Mechatronic Surface Sensor

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.

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

Sample flask

Trash flask

ATR flow cell

Peristalticpump



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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(Rreference/Rsample), 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

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 µm 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 R2A (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 90/00 ). Then, the pellet was

resuspended in NaCl 90/00 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.

Handbook SAFER V02light

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