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 AOC3-version1.1

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.

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.

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

Biofilm Damage in Drinking Water Distribution Progressive loss of flow capacity Increased pumping pressures Greater potential for scale & corrosion

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 AOC3-version1.1

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. AOC3-version1.1

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

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.

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. AOC3-version1.1

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 AOC3-version1.1

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.

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 AOC3-version1.1

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 AOC3-version1.1

Sensitivity of Luminous Bacteria to Various Nutrients in Water AOC3-version1.1

Correlation with the Standard (Van der Kooij) Test AOC3-version1.1

Applications Examples

AOC and related water quality data for various Norwegian raw and treated water samples AOC3-version1.1

Monitoring biofouling potential in industrial 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 AOC3-version1.1 Data presented in ppb carbon.

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

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) AOC3-version1.1

Early-AOC Test Procedure Step By Step

Early-AOC Test Protocol

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.

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.

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.

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.

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.

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).

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.

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.

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. AOC3-version1.1

Frequently Asked Questions

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

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.

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.

Thank You CheckLight Ltd. P. O Thank You CheckLight Ltd. P.O. Box 72 Qiryat-Tiv’on 36000 Israel Tel: 972 4 9930530 Fax: 972 4 9533176 info@checklight.biz http://www.checklight.biz AOC3-version1.1