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Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

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Page 1: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of

Biofouling/ Bacterial Re-Growth Potential

Version 1.3.1

Page 2: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Agenda

Definitions - biofilm, biofouling, biostability

Damage due to biofilm and factors affecting its build up

The role of carbon-based nutrients

AOC measurement

Bioluminescence-based AOC testing

Application examples (drinking water, dead sea, seawater, industrial water

purification)

Summary - advantages & benefits

Logistics

Q & A

Page 3: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Definitions:

Biofouling - the undesirable accumulation of micro-organisms, algae

and diatoms, plants, and animals on surfaces, such as piping,

reservoirs with untreated water, storage tanks with finished water.

Biofilm - a complex structure adhering to surfaces that are regularly

in contact with water, consisting of colonies of bacteria and usually

other microorganisms such as yeasts, fungi, and protozoa that

secrete a mucilaginous protective coating in which they are encased.

Bio-stability - the ability to limit regrowth in drinking water. It depends

on the concentration of disinfectant residual and on the concentration

of substrate required for the growth of microorganisms.

Page 4: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Biofilms in Water Systems Affect:

Water quality (contamination by released microorganisms)

Health (release of bacteria in water and bacteria-containing aerosols)

Hydrodynamic parameters (clogging, friction & hydraulic resistance)

Material (covering of surfaces, change of surface properties, microbially

influenced corrosion [MIC])

Biofilms are responsible for billions of dollars in lost industrial productivity, as

well as product and capital equipment damage each year.

Page 5: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Biofilm Damage in Desalination Systems

Once formed, biofilms can be very difficult to remove either through disinfection or chemical cleaning leading to:

Energy waste

Degraded salt rejection

Shortened membrane life

Page 6: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Biofilm Damage in Drinking Water Distribution

Progressive loss of flow capacity

Increased pumping pressures

Greater potential for scale & corrosion

Page 7: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Factors Causing Biofilm Growth

Presence of microbial nutrients in the water

Characteristics of pipe wall, such as roughness

Microbial and chemical quality of the water entering the system

Water temperature and pH

Low disinfectant residual level in the water

Velocity of the water

Page 8: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Nutrient Availability

To grow, organisms must derive from the environment all the substances that

they require to synthesize cell material and generate energy.

For coliform and heterotrophic bacteria, the principle nutrient sources are

phosphorus, nitrogen and organic carbon in a ratio of about 1:10:100

(P:N:C).

Hence, organic carbon is often a growth-limiting nutrient.

Most organic carbon compounds in water supplies are natural in origin,

derived from living and decaying vegetation.

These may include humic and fulvic acids, polymeric carbohydrates, proteins,

and carboxylic acids.

Page 9: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Carbon in Drinking Water is Measured in Various Ways:

TOC - total organic carbon, which is the total

amount of soluble and insoluble organic

carbon compounds present in the water.

DOC - dissolved organic carbon, which is the

soluble fraction of TOC.

BDOC - biodegradable organic carbon

measured by reduction in DOC after 3 weeks.

AOC - assimilable organic carbon, which is

the fraction of DOC that can be readily

digested and used for growth by aquatic

micro-organisms.

TOC

DOC

BDOC

AOC

Relative concentration illustration

Page 10: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

The Importance of AOC Measurements

Not all organic compounds present in water support microbial growth. Hence, it is important to be able to quantitatively measure the fraction that is assimilable.

Total or dissolved organic carbon (TOC, DOC) proved inadequate for that purpose; it has been shown (Van der Kooij et al, 1982; Werner and Hambsch, 1986,1988) that the fraction of the total organic carbon pool which is available for biodegradation can be very small and is generally highly variable.

Page 11: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

The Standard AOC Method

A bioassay developed by Van der Kooij (1982):

Inoculation of a water sample with two microorganisms (P17, NOX)

whose growth is monitored and maximum yield determined after 5-7

days.

Based on known yield coefficients, the equivalent amount of carbon

(usually expressed in µg of acetate-carbon/L) is calculated.

The method is labor and materials-intensive and care is needed to

properly handle water samples to avoid contamination with

extraneous organic material.

Page 12: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Application of the AOC Test in Drinking Water Systems

Researchers in the Netherlands have found that growth of HPC bacteria is:

Limited at AOC levels of <10µg/L

Occurs occasionally at AOC levels of 20-50 µg/L

Always occurs at AOC levels of >50 µg/L

Page 13: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Advantages of Using a Bioassay Based on Luminous Bacteria

The freeze-dried preparations of luminous bacteria are stable for long periods.

The hydrated bacteria regain their full level of in-vivo luminescence.

Luminescence may be easily measured by readily available luminometers.

Page 14: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Applications & Benefits

The Early-AOC test could serve to :

Monitor water at various pre- & post-filtration steps

Optimize timing of filter backwash

Provide early warning of membrane bio-fouling

Predictive indication of bacterial re-growth

Early warning of changes in bio-stability

Reduce operation & maintenance costs

Page 15: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

AOC Test – Features & Advantages:

Principle – starved luminous bacteria are not emitting light; once exposed to the water sample, the development of luminescence reflects the concentration of utilizable organic carbon compounds

Bioassay – freeze-dried luminous bacteria (Vibrio harveyi -drinking water applications; Vibrio fischeri - seawater applications)

Equipment –luminometer, pipettor, tips, water bath/incubator

Fast – results obtained within 2 hours

Sensitive – a variety of assimilable organic compounds can be detected at a wide concentration range (10-1000 ppb)

Reliable – very good correlation was obtained against the standard AOC test

Cost effective – low price encourages more frequent testing, for close monitoring of water treatment processes and optimization of costly disinfection treatment

Page 16: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Sensitivity of Luminous Bacteria to Various Nutrients in

Water

AOC TEST

1

10

100

1000

1 10 100 1000

AOC CONC. (ppb)

Luminescence

Glucose

Aminoacide

Yeast.ex

K-acetate

tap water 40-1%

Page 17: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Correlation with the Standard (Van der Kooij) Test

Page 18: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Applications

Examples

Page 19: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

AOC and related water quality data for various Norwegian raw

and treated water samples

Page 20: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Monitoring biofouling potential inindustrial water purification systems

Plant A, a problem was spotted in the GAC step (#3). The elevated level of AOC was probably due to microbial accumulation. Early detection allowed protection of expensive RO membranes.

Using the AOC Test May Reduce Operation & Maintenance Costs

Data presented in ppb carbon.

Page 21: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Kit Components & Accessories

Each kit holds the following items:

Biosensor vials

Concentrated & dilute Assay Buffers

Hydration Buffer

Carbon cocktail standard

Empty test vials

Additional equipment needed:

Water bath & heating plate

Repeat pipettor

Pipettor & tips

Vortex

Page 22: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Logistics

Once received - open box immediately and transfer:

Buffers box to 4°C -

[ DO NOT FREEZE!]

Bacteria box to freezer (-10°C) -

Note- expiration date is taped on each box (e.g., EXP 01/09)

Page 23: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Early-AOC Test Procedure

Step By Step

Page 24: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Early-AOC Test Protocol

Page 25: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Important Tips for Successful Operation

Follow test protocol precisely - do not try to change volumes of reagents and buffers.

As reference clean water, use water that is <5ppb TOC, such as Milli-Q Element/Gradient/Synthesis Grade.

Keep Assay Buffer in either a disposable sterile plastic tube, or rinsed and heat-treated (8hrs , 250°C) glassware.

Do not touch tips with bare hands to reduce the risk of contamination. It is advisable to run samples in duplicates, at least in the first runs, to make sure

test is performed accurately. When running many samples, make sure to take a reading of the controls at the

beginning and again at the end of the reading session, as light keeps increasing with time.

During the dilution procedure, be careful not to dip the tip too much into the liquid and make sure you do not drag air bubbles.

If one out of the 3 negative control tubes exhibits a significantly different result than the other two, disregard it and calculate the average of 2 out of the 3 tubes.

Page 26: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Step 1 - Getting Ready:Serial dilution of sample

Place clean marked tubes in line on a suitable rack. Dispense 1.75ml of the tested sample into the first tube. Add 0.25ml of concentrated Assay Buffer. Mix well by vortex.

Dispense 1ml of the diluted Assay Buffer in tubes #2-14. Transfer 1ml aliquot from the first tube to the 2nd tube (I.e., first double dilution). Mix well.

Repeat this step 5 times to reach a final dilution of about 75 fold in the 7th tube. Discard 1ml from the final vial in the sample dilution set.

Leave 3 tubes as negative control (holding only diluted Assay Buffer). In order to maintain similar aeration conditions, make sure to mix the buffer in these tubes by mock pipetting up and down.

Page 27: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Step 1 (cont.) - Getting Ready:Preparing the Standard

The last 4 tubes will serve as positive control.

Prepare the 5ppm daily-fresh stock of carbon cocktail solution in the following manner: add 0.1ml of the 5mg/ml (5000ppm) stock into 0.9ml diluted assay buffer (Solution A; 500ppm). Mix well by carefully pipetting up and down.

Prepare another tube with 0.99ml of diluted assay buffer and dispense into it 0.01ml from solution A (Solution B; 5ppm). Mix well by carefully pipetting up and down.

From Solution (B), dispense 10, 20, 40, 80 µl to 1st, 2nd, 3rd, 4th tube, respectively.

Page 28: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Step 2 - Preparing the Bacteria:Hydration & Pre-incubation

Hydrate freeze dried bacteria by rapid addition of 0.5ml cold Hydration Buffer.

Mix well by vortex.

Incubate in water bath for 30 minutes at 28°C.

Page 29: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Step 2 (cont.) - Preparing the Bacteria:Dispensing

Use a repeat pipettor to fill the syringe with the bacteria suspension.

Make sure no air bubbles are trapped in the syringe.

Rapidly and carefully dispense 10µl of the hydrated bacteria into each tube.

Mix well by vortex.

Page 30: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Step 3 - Incubation

Place tube rack at 28°C (preferably in a water bath).

Incubate for 60-150 minutes (or when two lowest concentrations of the standard solution emit significantly higher luminescence (>2xSD) than the calculated average of the negative control).

Page 31: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Step 4 - Data Recording

Place luminometer next to water bath.

Measure obtained luminescence by sequentially removing each tube from bath to luminometer and back.

Calculate the AOC value of the tested sample (in carbon equivalent units) using the provided Excel module.

Choose the time point that exhibited the highest sensitivity of the Standard. Do not calculate the average obtained at different reading time points.

Page 32: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Excel Module - Data Analysis

Standard curve is plotted to obtain the linear relation between given carbon equivalent concentration and light level.

Light level in the sample dilution set is plotted against sample concentration to determine linear range and lowest detectable value.

Page 33: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Re-shipment

Just before re-shipping to end user insert into the shipment box-

- Buffers box

- Bacteria box

- Tubes sack

- User guide & Excel CD

Use the fastest route (preferably on ice) to end user.

Make sure the receiving end handles the shipment properly.

Page 34: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Frequently Asked Questions

Page 35: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Q & A

Q: Are luminous bacteria dangerous? Do I need to be a trained microbiologist in order to be able to conduct CheckLight's assays?

A: Luminous bacteria are not pathogenic and are harmless. No special skill is required to carry out the different tests other than very basic laboratory techniques (pipetting, dilutions etc) and equipment (pipettor, tips, luminometer).

Q: What is the danger in having high level of nutrients in drinking water?

A: High nutrient levels in drinking water may lead to the following drinking water problems:

• Increased levels of microbes, including opportunistic pathogens, in the bulk water, as well as in the pipe biofilm and sediments.

• Loss of disinfectant residual through reactions between disinfectant and nutrients.

• Production of toxic and/or carcinogenic DBPs through reactions between disinfectant and nutrients.

• Unreliability of total coliform sampling due to increased growth of heterotrophic bacteria, resulting in false-positives or false-negative coliform tests. Coliform sampling may also become unreliable due to stimulated growth on pipe biofilms and sediments. These increased numbers may not be represented in coliform samples of bulk drinking water.

• Development of aesthetic problems

Page 36: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Q & A

Q: What are the benefits of obtaining rapid information on AOC levels?

A: Acquiring this vital information would enable the water utility provider to take timely preventative action to avoid bacterial re-growth, optimally control its disinfection program and reduce the presence of excessive Disinfection By Products (DBPs).

Q: How might chlorinated water affect luminescence?

A: Chlorine is usually introduced into drinking water systems in order to avoid microbial contamination. Since luminous bacteria used in the assay are also sensitive to this treatment, sodium thiosulfate is included in the assay buffer to de-chlorinate the sample before adding the bacteria.

Q: Why is there a control in each assay?

A: Readings of the negative control are needed in order to obtain the background reading of the cells without the sample. In addition, a set of positive controls is run in order to calibrate the system and provide the proper “translation” of light units to carbon equivalent units.

Page 37: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Q & A

Q: Can I play around with the volumes of bacteria, buffers and other assay conditions?A: No. It is extremely important to follow the test protocol instructions to the word. Since the test is very

sensitive, any seemingly minor variations result in poor reliability.

Q: Can I reuse the provided test vials?A: Due to the high sensitivity of the assay, care should be taken to keep all vials, plastic tips, and

pipettes extremely clean. Do not reuse test vials and do not wash glassware pipettors or pipette tips with detergent, acid, or solvents.

Q: What is the shelf life of the reagents?A: The shelf life of the freeze dried bacteria is one year when stored in a deep-freezer

(-10°C to 20°C). Reagent should not be stored in a self-defrosting freezer, which defrosts by warming up periodically. The assay buffers should be stored in a regular refrigerator (4°C) and under no circumstances should they be frozen.

Page 38: Rapid Determination of AOC (Assimilable Organic Carbon) - Early Warning of Biofouling/ Bacterial Re-Growth Potential Version 1.3.1

Thank You

CheckLight Ltd.P.O. Box 72

Qiryat-Tiv’on 36000Israel

Tel: 972 4 9930530Fax: 972 4 [email protected]

http://www.checklight.biz