an introduction to an unconventional microbiological ... · be an increased in volatile fatty acids...

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?


• 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


• 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.



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)


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)


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


microbial test?

• Identify areas of total biological

risk.

• Assess if turbidity is physical or

biological.

• Troubleshoot and see how

mitigation actions affect the

system.



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.


31

• Once the beads settle, the

solution can then be mixed with

the firefly enzyme.

• Place the reaction into the


• Incubate for 1 minute.

• Then pour the extraction tube

contents into a dilution tube

that contains resign beads.


32

• Incubate for one minute.

• Mix some of the stabilized fluid

with the firefly enzyme.

• Place the reaction into the


• 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


• 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.


• 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

[email protected]

Dave Tracey, P.Eng

[email protected]

Questions?

mailto:[email protected]

mailto:[email protected]

an introduction to an unconventional microbiological ... · be an increased in volatile fatty acids...

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