An Introduction to an Unconventional Microbiological Monitoring Tool.
A. Barnett, E.I.T – LuminUltra Technologies Ltd.
D. Tracey, P.Eng – LuminUltra Technologies Ltd.
The Water Sector
2
Drinking Water
Run a process in which pollutants
are removed from water making it
safe to drink.
Water leaving the facility is of high
quality however issues can arise
within the distribution system:
• Loss of residual
• Biofilm Formation
• Nitrification
Water Resource Recovery
Removing pollutants/nutrients from
water making it safe to return to the
environment.
Microorganisms are used to remove
different nutrients. However, can be
affected by things such as:
• Low Amount of Living Organisms
• Toxicity
• Bulking
The Water Sector – The Problem
3
Drinking Water
• Loss of Residual
• What is the disinfection going
after?
• Biofilm Formation
• What can occur if the
disinfection residual drops?
• Mitigation Effectiveness
• How did the mitigation action
effect the system?
Water Resource Recovery
• Low Amount of Living Organisms
• How does the quantity of
living organisms compare to
the total solid?
• Toxicity
• Has something entered the
system causing the organisms
to react badly?
• Bulking
• Is something promoting
filamentous organism growth?
The Water Quality Toolbox
4
Physical
(E.G Color, Turbidity,
Temperature)
Chemical
(E.G pH, Disinfection,
BOD)
Microbiological
(E.G Plate Counts, DNA, Microscope)
The water quality toolbox within the
drinking water industry is made up of
3 parts:
• Physical Analysis
• Chemical Analysis
• Biological Analysis
Each section brings unique and
valuable insight to how the system is
performing
The Water Sector – The Tools
5
Drinking Water
• Physical
• Turbidity
• Color
• Alkalinity
• Chemical
• Disinfection Residual
• pH
• Hardness
• Microbiological
• Plate Counts
Water Resource Recovery
• Physical
• Settleability
• Total Suspended Solids
• Temperature
• Chemical
• BOD
• Nitrogen
• Phosphorous
• Microbiological
• Microscope
• DNA
• Plate Counts
Inside the Prokaryote Cell
6
DNAHighly Specific and Readily Targetable
RNASpecific but Difficult to Handle
Protein/EnzymeCan be Specific But Need Antibodies or
Other Recognition Molecules
Membrane (Lipid)Typically Not Specific
ATP MoleculeEnergy Carrier Non-
Specific
What is ATP?
7
ATP = Adenosine Triphosphate
• Primary energy carrier of all
living cells.
• Energy is stored in the third
Phosphate bond.
• If the cells needs energy it
will cleave that bond forming
ADP.
How is ATP Measured?
8
• As seen in the tails of fireflies, the
reaction of ATP with Luciferase
emits light.
• This light is measured using a
luminometer and reported in
RLU’s.
• The extracting and analysis of
ATP takes ~ 5 minutes.
ATP Luciferase Enzyme Light
(Luminase™)
𝐴𝑇𝑃 + 𝑂2 + 𝑙𝑢𝑐𝑖𝑓𝑒𝑟𝑖𝑛𝑀𝑔𝐿𝑢𝑐𝑖𝑓𝑒𝑟𝑎𝑠𝑒
++
𝐴𝑀𝑃 + 𝑃𝑃𝑖 + 𝑜𝑥𝑦𝑙𝑢𝑐𝑖𝑓𝑒𝑟𝑖𝑛 + 𝑙𝑖𝑔ℎ𝑡
ATP in the Water Sector
9
Drinking Water
Why would I use a non-specific
microbial test?
• Identify areas of total biological
risk.
• Assess if turbidity is physical or
biological.
• Troubleshoot and see how
mitigation action affects the
system.
Water Resource Recovery
Why would I use a non-specific
microbial test?
• Identify the % of TSS that is
living.
• See if the total amount of
organisms suddenly decreases.
• Determine the percentage of
organisms that are filamentous.
10
Industry Standard – HPC
• HPCs take at least 48 hours to obtain results.
• HPCs are known to detect <1% of the total
population1.
• Information is only provided on organisms that can
grow…
• …in the media used
• …at the temperature provided
• …within the incubation time allowed
• Injured or unhealthy cells grow more slowly or not
at all.
1 Maki et al., 1986; Siebel et al., 2008
Drinking Water – Finding the Source
A Customer Called:
• Water quality leaving the
facility was excellent!
• Disinfection residuals quickly
disappeared without a clear
explanation.
• ATP was used to troubleshoot.
Resolution:
• An un-listed valve was leaking
allowing biofilm to build up and
enter the system.
11
Drinking Water – Turbidity
• Visual inspection and turbidity
can indicate high risk in some
case.
• However in other cases,
immediate assessment is not
so easy.
• For samples such as this, one
cannot quickly assess risk and
must therefore wait until
microbiological tests are done.
12
Drinking Water – Turbidity
Scenario 1:
• The turbidity + biological
activity is high pointing to signs
of biological issue.
• After flushing for 30 minutes,
the turbidity dropped while the
ATP remained high.
13
0
0.5
1
1.5
2
2.5
0 30 60 120
Tu
rbid
ity (
NT
U),
Ce
llu
lar
AT
P (
pg
/m
L)
Flush Duration (minutes)
Turb = 8.2 NTU
Scenario 2:
• Turbidity was at 8.2 while the
ATP level was quite low
indicating this is not a
biological issue.
• After flushing 30 minutes, both
measurements are low.
Drinking Water – Disinfection
Total Chlorine:
• As expected, there is a strong
relationship between free
chlorine and biological activity.
• However, Free Chlorine does
not always indicate low
biological activity.
• Microorganism can grow and
thrive within biofilms that form
an EPS layer protecting it from
disinfection.
• EPS = Extracellular Polymeric
Substances
14
0
5
10
15
20
25
30
35
40
45
0 0.5 1 1.5 2 2.5 3
Cellu
lar A
TP, cA
TP
(pg
/mL)
Free Chlorine, FAC (mg/L)
Drinking Water – Disinfection
Distribution System Audit:
• Both chlorine and ATP results
were taken at points moving
away from the plant.
• Shown in West 3, while
Chlorine is still high the
biological activity has already
raised to a high-risk level.
• It was determined that actions
needed to be taken in the
west end.
15
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0.01
0.1
1
10
100
1000
10000
To
tal C
l 2(m
g/L
)
log1
0 c
AT
P(p
g/m
L)
cATP (ng/mL) Total Cl2 (mg/L)
Drinking Water – Disinfection
Chlorine:
• As the system is flushed, the
chlorine increases across the
west end.
• With a goal of 1.0 mg/L
achieved, the flushing could
stop.
16
ATP:
• Biological activity decreased
during the flush as expected.
• However, while chlorine
indicated the flushing could
stop, ATP told a different story.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Initial Cross Cxn Flush 1d Flush 3d Flush 17d
To
tal C
l 2(m
g/L
)
Plant West 4 West 5 West 6 West 7 West End
0.01
0.1
1
10
100
1000
10000
Initial Cross Cxn Flush 1d Flush 3d Flush 17d
log
10
cA
TP
(pg/m
L)
Plant West 4 West 5 West 6 West 7 West End
How to Take a Test – Drinking Water
17
• Filter the water to catch the
organisms.
• Push an extraction fluid
through the filter and into a
dilution tube.
• Mix the dilution fluid with the
firefly enzyme and place into a
luminometer to take a reading.
• Mix the firefly enzyme with the
calibration fluid to measure the
enzyme strength and convert
the units to a concentration.
ATP in the Water Sector
18
Drinking Water
Why would I use a non-specific
microbial test?
• Identify areas of total biological
risk.
• Assess if turbidity is physical or
biological.
• Troubleshoot and see how
mitigation actions affect the
system.
Water Resource Recovery
Why would I use a non-specific
microbial test?
• Identify the % of TSS that is
living.
• See if the total amount of
organisms suddenly decreases.
• Determine the percentage of
organisms that are filamentous.
Water Resource Recovery Sample
19
Along with organics and inorganics, there are a few different types of
ATP molecules that need to be identified:
• Cellular ATP (cATP): ATP found within living organisms.
• Dissolved ATP (dATP): ATP found in the environment.
• Total ATP (tATP): The combination of cellular and dissolved ATP.
Water Resource Recovery – TSS/VSS
20
While these parameters are useful for identifying the flow of solids, they
have some inherent interferences when looking to measure just the
biological population.
Due to these interferences, they will react slower to change.
Total Solids Inventory
• What are some of the benefits
of having a higher percentage
of active biomass?
• Better Setting
• Reduced Bulking
• Improved O2 Transfer
Efficiency
21
To
tal S
olid
s In
ve
nto
ryExample Wastewater Sites
Dead Biomass & Non-Biological Solids
Living Biomass
Improving the Active Biomass Ratio
• A plant was experiencing
issues with bulking.
• The typical solids inventory
was ~ 7000 mg/L
• Through some scheduled and
closely monitored wasting
events the:
• Total solids reduced by
33%
• Active biomass ratio
increased from 11% to
25%
22
0
1000
2000
3000
4000
5000
6000
7000
8000
12-May-10 18-May-10 21-May-10 26-May-10
To
tal S
olid
s I
nve
nto
ry, T
SS
(m
g/)
Living Biomass Dead Biomass & Non-Biological Solids
0
5
10
15
20
25
30
Active
Bio
ma
ss R
atio (
%)
12-May-10 18-May-10 21-May-10 26-May-10
Toxicity Monitoring
• Microorganisms are
susceptible to inhibition from
toxic materials.
• When something like this
enters the facility, the
organisms can become
stressed and die.
• By mixing some influent and
activated sludge, we can
determine the affect each
stream has on the organisms.
23
Bioreactor
Stream A
Stream B
Stream C
Stream ABC Effluent
0
500
1000
1500
2000
2500
3000
0 1 4 8Cellu
lar A
TP, cA
TP
(ng/m
L)
Time (h)
Stream A Control Stream B Stream C
Not So Nice Surprises
• A plant received waste from
~10 industrial clients and was
surprised with toxic upsets
without warning.
• Following January 5th the
plant saw:
• 57% decrease in Living
Biomass
• 21% decrease in TSS
• >200% increase of BOD
24
More Solids ≠ More Organisms
• Once the toxic event was
identified, the bioreactor was
reseeded to make up for lost
organisms.
• Although the reseeding
increased the TSS, the living
biomass quickly fell.
• After attempting to reseed
twice, it was determined the
toxicity was entrenched in the
sludge.
25
Filamentous Organisms
• Filamentous organisms are an important part of the settleability of the
sludge blanket.
• However if too many filamentous organisms grow, it can lead to bulking
and eventually carry-over.
• Excessive Filamentous growth can be cause by:
• Influent fats/lipids
• Poor O2 Transfer Efficiency
• Low F/M
26
Floc Bulking Event
• A facility treating waste from a
nearby pulp and paper mill
experienced frequent floc
bulking events.
27
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
50
100
150
200
250
300
350
400
450
fbA
TP
(%
)
SS
VI (m
L/g
)
SSVI Alarm fbATP Alarm
• A floc bulking event was
identified when:
• SSVI > 250ml/g (February 12th)
• % fbATP > 50% (January 29th)
Floc Bulking Event
• A chemical treatment plan was
conducted using Ferric
Sulphate to assist in settling
and Hypochlorite to destroy the
filamentous organisms.
28
• When the Hypochlorite was
removed, the filamentous
organisms increased
dramatically.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0
50
100
150
200
250
300
350
400
450
fbA
TP
(%
)SS
VI (m
L/g
)
Hypochlorite Added Ferric Sulphate Added Hypochlorite and Ferric Sulphate Added
Floc Bulking Event
• The issue was determined to
be an increased in volatile fatty
acids entering the facility from
the pulp and paper mill.
29
• Once the root cause of the
issue was resolved, the levels
returned to normal.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0
50
100
150
200
250
300
350
400
450
fbA
TP
(%
)SS
VI (m
L/g
)
Hypochlorite Added Ferric Sulphate Added Hypochlorite and Ferric Sulphate Added
How to Take a Test – Clean Water
30
• As mentioned earlier, the test
is broken into tATP and dATP.
• For both measurements only
have to complete 1 calibration.
• Mix the calibration fluid with
the firefly enzyme and place
into a luminometer to take a
reading.
• For the tATP test:
• Invert the sample to ensure the
organisms are well dispersed.
• Placed 1mL of sample into an
extraction tube.
How to Take a Test – Clean Water
31
• Once the beads settle, the
solution can then be mixed with
the firefly enzyme.
• Place the reaction into the
luminometer to take a reading.
• Incubate for 1 minute.
• Then pour the extraction tube
contents into a dilution tube
that contains resign beads.
How to Take a Test – Clean Water
32
• Incubate for one minute.
• Mix some of the stabilized fluid
with the firefly enzyme.
• Place the reaction into the
luminometer to take a reading.
• For the dATP test:
• Invert the sample to ensure the
organisms are well dispersed.
• Placed 1mL of sample into the
stabilizer tube.
Summary – What is ATP?
33
ATP = Adenosine Triphosphate
• Primary energy carrier of all
living cells.
• Energy is stored the third
Phosphate bond.
• If the cells needs energy it
will cleave that bond forming
ADP.
Why Was It Chosen?
34
DNAHighly Specific and Readily Targetable
RNASpecific but Difficult to Handle
Protein/EnzymeCan be Specific But Need Antibodies or
Other Recognition Molecules
Membrane (Lipid)Typically Not Specific
MetaboliteHard to Find Specific Targets
ATP MoleculeEnergy Carrier Non-
Specific
Adding a New Tool
35
Drinking Water
• Physical
• Turbidity
• Color
• Alkalinity
• Chemical
• Disinfection Residual
• pH
• Hardness
• Microbiological
• Plate Counts
• ATP
Water Resource Recovery
• Physical
• Settleability
• Total Suspended Solids
• Temperature
• Chemical
• BOD
• Nitrogen
• Phosphorous
• Microbiological
• Microscope
• DNA
• Plate Counts
• ATP
ATP in the Water Sector – Summary
36
Drinking Water
• Found the source of high
biological risk.
• Determined if a turbidity event
was biological.
• Compared disinfection to
biological activity.
• Determined the effectiveness of
a flushing activity.
Water Resource Recovery
• Looked at measuring the
biological activity of activated
sludge.
• Saw a facility keep the living
population while decreasing the
overall solids.
• Determined the effectiveness of
a reseeding following a toxic
event.
• Identified a floc bulking event
and assessed the mitigation
tactics.
Adam Barnett, E.I.T
Dave Tracey, P.Eng
Questions?