Real-time Monitoring of Microbial Activity in

Drinking Water
14th of May 2020

Sjoerd van der Knoop MSc BaSc

Vladimir Pavlita
Webinar will start in 15 minutes (please ensure your PC sound is active)
Real-time Monitoring of Microbial Activity in
Drinking Water
14th of May 2020

Sjoerd van der Knoop MSc BaSc

Vladimir Pavlita
Webinar will start in 10 minutes (please ensure your PC sound is active)
Real-time Monitoring of Microbial Activity in
Drinking Water
14th of May 2020

Sjoerd van der Knoop MSc BaSc

Vladimir Pavlita
Webinar will start in 5 minutes (please ensure your PC sound is active)
Disclaimer

Hach webinars are educational in nature and not an endorsement of any treatment 
technologies or processes.

This document contains information owned by Hach. 

Personal use, copying or distribution is allowed for webinar participants for their own
internal purposes, when mentioning Hach as the source.

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EZ Series – Drinking Water
On‐line Monitoring of Bacterial and 
Pathogen Load in Drinking Water 
Applications  
Agenda

• Hach and the EZ Series

• What industries monitor biological activity and in which process steps? 

• How is microbial activity currently being monitored?

• Traditional vs online measurements

• The practical use of ATP in the field

When the EZ Series were added to the Hach portfolio

TITRATION COLORIMETRY

VOLTAMMETRY ION‐SELECTIVE E. CHEMILUMINESCENCE

Technology portfolio

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 EZ Series: 75 new analyzers, a platform overview

• Iron / Manganese • Alkalinity
• Low Range Hardness • Hardness
COLORIMETRY TITRATION
• Nitrate / Nitrite • Ammonium 
• Orthophosphate • Sulfate

ION SELECTIVE SPECIALIZED

ELECTRODE METHODS VOLTAMMETRY

• Fluoride • ATP Chemiluminescence • Arsenic

• Chloride • Total Phosphorus / Nitrogen • Mercury
• Sulfide • WWTP Influent Toxicity • Lead
• Ammonium • Volatile Fatty Acids • Cadmium

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 Parameters covered by EZ Series in Drinking water production
Finished water
Alkalinity
6 key parameters: Hardness
Iron
Manganese
• Iron
• Manganese
• Alkalinity
Disinfection
• Hardness
• Aluminum
Settling
• ATP Aluminum
Post‐treatment
Alkalinity

Filtration
Iron
Oxidation Manganese

Coagulation / Flocculation
Alkalinity
Raw Water Intake Hardness 
Alkalinity
ATP
Hardness
Iron
Manganese
Where and why is microbial activity determined in
Drinking Water?
Where measure microbial activity in DW?
Filter
Beds

Chlorine
Flash Flocculation Settling Addition
Ozone Contactors
Plant Mixers Basin Basin (Ammonia
(Pre-Chlorination)
Intake Addition) Clear
Well

Filter Wastewater Backwash

Water
Tower
To
Booster Distribution
Station System

House
Why measure microbial activity?
Raw water, 
Pre‐
Oxidation
• Monitoring of groundwater contamination during 
rainfall events
• Microbial load in the surface water
• Indicator for early detection and treatment of 
cyanobacterial blooms
• Monitoring of pre‐oxidation treatment step
 Why measure microbial activity?
Post‐
Filtration

• Biofiltration steps monitoring
• Monitoring during dynamic events 
(e.g. backwashing)
• Bacteria growth on the sand filters
• Membranes fouling control
Why measure microbial activity?
Disinfection &
Clear Well

• Monitoring and optimisation of disinfection 
treatments steps
• Efficiency of the disinfection process check
• Contact time in the Clear Well
Why measure microbial activity?
Distribution 
Network

• Monitoring of the bacteria regrowth in the distribution network
How is microbial activity currently monitored?
Traditional approach to determine microbial activity

Bacterial contamination is traditionally detected by heterotrophic plate counting (HPC) 
or dip‐slides (traditional lab methods).

These cultivation‐based methods are limited or influenced by various factors:
• Sampling frequency (contaminations between samplings is not detected)
• Species selective culture media (higher costs)
• Cultivability of different bacteria (0.1 ‐ 1% of all bacterial species)
• Availability and accuracy of laboratory personnel

1. Covers regulatory requirements.
2. Sampling time + incubation time + handling time = slow response time

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How is microbial activity used in the process?

• Multi‐day measurement used to correct first steps of the process

Treatment /  Storage and 
Intake
Storage transport 

1. Prevent high bacterial loads to come in with intake water.
2. Optimize treatment when it happens.
3. Prevent regrowth in the storage tanks – clear well.

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ATP Chemiluminescence Reaction

ATP assays using luciferin/luciferase reactions allow to assess microbial activity in water. 
• The ASTM D4012 (Standard Test Method for Adenosine Triphosphate Content of 
Microorganisms in Water) was developed as a quick and sensitive alternative to 
plate counting.

Higher ATP content Higher light output Higher bacterial load

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EZ7300 Series – differentiate between living and dead bacteria

Question: What is one of the main challenges in measuring the microbial load (contamination) in 
water?
Answer: In order to have a clear picture of the microbial load it is important to differentiate ATP 
portions within living cells from non‐living cells.

ATP in the water source can be located…
Either inside bacteria or other cells
EXTRACELLULAR
= intracellular
Or freely in the water surrounding the cells 
= extracellular or free ATP INTRACELLULAR

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EZ7300 Series – Proprietary sample pretreatment
Sonicator for cell membrane disruption Dark cell + refrigerator (4°C )

DETECTOR UNIT

SONICATOR ‐ LYSIS

GRAB SAMPLE PUMP

SAMPLE INLET
SAMPLE IN

SAMPLE PUMP
CLEANING COOLING BLOCK
CALIBRATION 

DRAIN PUMP
ACCLIMATIZED CHAMBER
COOLING WATER LOOP

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EZ7300 Series – General

What? 
The first microbiology analyzer using the ATP firefly assay 
and complying with international standard method ASTM 
D4012‐81

The on‐line microbiology analyzer delivers operators:
• Fast results (7‐10 minutes ≈ real‐time data.)
• Automation through panel PC
• Analytical performance
• Reliability for up to 8 streams to lower investment cost
– Stream selection with modbus or digital input

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EZ7300 Series – High analytical performance

• Complete ATP recovery: detection of intracellular, extracellular and total ATP
– 3 output values for SCADA system + alarm relais
• Rapid measurement: 7 ‐ 10 minutes (including lysis)
– Can be changed by user – own decision.
• Smart features: automatic calibration and 3‐step cleaning protocol
– 20 pg/mL or 200 pg/mL
• Low limit of detection (LOD): 0.5 pg/mL 
• Extended reagent stability by refrigeration
• Factory configured, tested and calibrated

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EZ7300 Series – Proprietary sample pretreatment

What is the relevance of measuring the different ATP portions?
• Extracellular ATP or free ATP is the portion of ATP released by dead cells.
• Total ATP is obtained after lysis of the biomass by sonication of the sample.
• Intracellular ATP is the portion of ATP from the metabolically active (living) organisms.

Cleaning
1. Rinsing after each sample
2. Cleaning with HCl to break down any biofilm
3. Cleaning with NaOH to hydrolyze remaining ATP released
4. Rinsing with sample

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EZ7300 – Analytical Specifications

ANALYTICAL SPECS

Standard range 0.5 – 200 pg/mL

Method Standard method ASTM D4012‐81 
Determination of adenosine triphosphate (ATP) by means of 
chemiluminescent reaction using luciferin and luciferase
Precision < 4% full scale range on standard test solutions
DW around 2 pg/mL
Surface water around 500 pg/mL
Analysis time 10 minutes incl. lysis

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The practical use of ATP in the field
Clean water applications

Stream Standard solution pg/mL ATP RLU

total 10 pg/mL ATP in tris buffer 10,15 42355
free 10 pg/mL ATP in tris buffer 9,93 41462
total 5 pg/mL ATP in tris buffer 4,86 20318
free 5 pg/mL ATP in tris buffer 5,11 21365
total 1 pg/mL ATP in tris buffer 1,02 4328
free 1 pg/mL ATP in tris buffer 0,96 4056
total 0.5 pg/mL ATP in tris buffer 0,53 2286
free 0.5 pg/mL ATP in tris buffer 0,50 2178
total 0.1 pg/mL in tris buffer 0,12 570
free 0.1 pg/mL in tris buffer 0,10 479
total 0.05 pg/mL ATP in tris 0,06 313
free 0.05 pg/mL ATP in tris 0,05 272
total nanopure water 0,00 90
free nanopure water 0,00 80

• Factory calibration of the analyzer with ATP standard solution 
• Excellent correlation is obtained between actual and expected results in the 
measuring range of 0 ‐ 10 pg/mL; Extension to 1000 pg/mL

27
Optimization of a biological filter and plant risk mitigation

The objectives:

• Monitor in real time the microbial load of the 
influent and effluent of their biological filter

• Determine the efficiency of the biological filter in 
removing biomass (microbial load)

• Trouble shoot/mitigate risk (corrosion) in other parts 
of the plant using the grab sample line of the 
analyzer

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Reverse Osmosis performance monitoring and optimisation

The objectives:

• Identify better, realistic surrogates for use in 
monitoring RO performance for reuse

• Achieve higher log removal credits than 
conductivity and TOC

• Increasing confidence in RO’s ability to remove 
high levels of pathogens

• Measuring online ATP in feed and permeate 
helps to calculate log removal values

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Field Data: Water Disinfection

Example: influence of disinfection program on ATP portions 
1400

1200

1000

800
ATP total
RLU

600
ATP free

400 ATP intra‐cellular

200

0
0 2 4 6 8 10 12 14 16 18
‐200
Time
Post treatment: ATP intracellular ≈ 0    : all bacteria have been killed by the lysing biocide.
ATP total =  ATP free : all ATP from the bacteria has been released.

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Field Data: Biocide Application

Results of the EZ7300 are used to generate a trendline for each process line being monitored. 
Deviation from the set range will alert the user to take appropriate steps for corrective action.

31
Field Data: River Water Monitoring

The microbial load of the 
raw water was monitored 
by means of ATP analysis.

The graph shows that the 
ATP analyzer can be used 
to monitor and optimize 
the pre‐oxidant dosing. 

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Field Data: Drinking Water Disinfection Control

The residual 
microorganism were 
monitored by means of 
ATP analysis.

The graph shows that the 
ATP analyzer can be used 
to monitor and optimize 
the disinfection dosing. 

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Research Paper: Using ATP to evaluate bacteria regrowth in storage

Conclusions
An online ATP approach confirmed results from discrete ATP measurements. The continuous 
measurement of intracellular ATP led to more accurate interpretation of microbial regrowth and 
enhanced identification of ATP peak and growth phases of indigenous bacteria.

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Summary: ATP in drinking water production

• The EZ7300 series ATP analyzer can be used as a direct warning system to indicate bacterial activity 
on drinking water plants.
– Prevent delayed plate test results and take action when needed. 

• Sample water at fixed intervals, directly from your system without manual sampling.

• Create digital track record in your SCADA system rather than offline data storage.

• Optimize your plant operation.
• Improve your treatment strategy.
• Prevent unwanted exposure to consumers.

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 Pavlita, Vladimir van der Knoop, Sjoerd
Hach Lange s.r.o. Dr. Lange Nederland B.V.
Zastrcena 8 Laan Van Westroijen 2a
CZ‐14100 Prague NL‐4003 AZ  Tiel
vladimir.pavlita@hach.com sjoerd.vanderknoop@hach.com

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