Final MSc thesis presentation

Development and Comparison of an Intracellular ATP Method for High Saline

Waters

Supervisor(s):Prof. Maria Kennedy, PhD, Prof.em. Jan C. Schippers, PhD, MSc

Mentor(s):Loreen O. Villacorte, PhD, MScSergio Salinas, PhD, MSc

Co-mentor Almotasem Abushaban , MSc

External examiner Zhong Yu, PhDUrban Water and Sanitation master programmeWater Supply Engineering specialization MSc final presentation Delft, 02April 2015

Almohanad Abusultan

2

Adenosine Triphosphate Adenosine triphosphate (ATP) is present in every living cell including bacteria. It is called a molecule of currency and it is an energy carrier ATP + H2O → ADP + Pi ΔG = -30.5 kJ/mol

Bioluminescence is the most widely used for ATP determination

3

ATP methods in aquatic environments • Many protocols have been developed to measure ATP in freshwater

1. Promega BacTiter GloTM Microbial Cell Viability Optimized Assay (Hammes et. al, 2010){LOD = 0.0001 nM ATP}

2. Het Waterlaboratorium (HWL) ATP protocol (Celsis products){ LOD = 1ngATP/L}

• These methods cant be applied in seawater because: Interference of salts which hamper the Luciferase/Luciferin reaction.

Source: (Van der kooij and Veenendaal, 2010).

4

ATP methods in saline water

ATP quantification in ballast water. (Van

Slooten, et al., 2015)

• Quantifies larger living organisms (10–50 μm),

• Uses a 10 μm membrane pore size for filtration .

• LOD: 2.5±0.5 cells/mL

ATPSaline

• Both fresh and seawater,

• Uses a 0.1 μm membrane pore size for filtration

• LOD: 0.2 ngATP/L

Filtration Rinsing Extraction

5

Problem statement

ATPSaline method was recently developed at UNESCO-IHE, However it is not yet neither verified nor compared in fresh or seawater.

6

Main goal

Further development and comparison an ATPSaline method for high saline waters.

7

Objectives

1. To compare ATPSaline method with the Promega optimized protocol in fresh water.

2. To investigate the correlation of ATPSaline with intact cells concentration (measured by FCM) in fresh and saline water.

3. To test and compare ATPSaline method with available intracellular ATP commercial kit (ATP Water Glo) in saline water.

4. To apply the ATPSaline as a monitoring tool in seawater desalination pilot plant.

8

Research methodology

Promega optimized protocol

Correlation with FCM


Fresh water

Seawater

Promega ATP Water

Glo

Zeeland seawater treatment pilot plant

Bacterial growth

calibration curve

Testing in seawaterApplication in seawaterComparison

9

ATPSaline vs. Promega optimized protocol

Method Promega optimized protocol ATPSaline

Reagents usedPromega reagent which comprise both lysing agent and luciferin-luciferase enzyme

LuminEX for ATP extraction Promega reagent for luciferin-

luciferase reaction

Separation of free and intra-cellular ATP

Total = PromegaFree = 0.1μm pre-filtration + Promega

Intracellular = 0.1μm retention + LuminEX extraction + Promega

Intracellular ATP Intracellular ATP = total ATP - free ATP Direct determination of Intracellular ATP

Application Applicable in freshwater only Applicable in both sea- and freshwater samples

Exposure time to light generating reagent 20 s 0 s

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1. Comparison of ATPSaline method in fresh water.

Fresh water samples were collected from Kralingen water plant (Evides, Rotterdam).

Both intact cell concentration using FCM and ATP concentration were measured.

ATPSaline is well correlated to Promega Optimized protocol and intact cell concentration.

ATPSaline is 22% higher than Promega Optimized protocol.

0.0 30.0 60.0 90.0 120.00.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

f(x) = 0.776059289343293 x − 3.172855400612R² = 0.947709512833734

Microbial ATP - ATPSaline (ng/L)

Mic

robi

al A

TP -

Prom

ega

optim

ized

pro

toco

l (n

g/L)

0.0 30.0 60.0 90.0 120.0 150.0 180.00.00

200,000.00

400,000.00

600,000.00

800,000.00

1,000,000.00

1,200,000.00

1,400,000.00

1,600,000.00

f(x) = 7784.88365953642 x + 27074.2446920142R² = 0.82230852840713

Microbial ATP - ATPSaline (ng/L)

Inta

ct c

ell(x

106

cel

l/m

L)

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A. Higher sampling volume (5000 µL) in ATPSaline compared to Promega Optimized protocol (500 µL) might increase the probability to capture larger organisms in ATPSaline.

B. Using different ATP standard products results in dissimilar calibration curves and/or ATP concentration.

C. The difference of sample exposure time to the light generating reagent (Promega BacTiter-GloTM reagent) between ATPSaline (0 s) and Promega optimized protocol (20 s).

Why ATPSaline gives higher results?

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A. Verification of capturing larger microorganisms.

• ATPSaline protocol.

• Canal water.

• Different sampling volumes.

• A linear relationship was observed between RLU values and filtered sample volume.

• ATP concentration is independent of sample volume.

0 2 4 6 8 10 12 14 160.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

f(x) = 63366.9641089109 x + 744.5R² = 0.99474284008567

f(x) = 75174.2537128713 x + 744.5R² = 0.998168749079214

0.1 um filter

Filtered canal water volume (ml)Re

lativ

e lig

ht u

nit (

RLU)

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B. Effect of ATP standard product on calibration curve • BioThema and Promega ATP

standards.

• Both have 100 nmol/L ATP concentration.

• ATPSaline calibration curve procedure were followed

0 50 100 150 200 250 3000.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

6.0E+05

7.0E+05

f(x) = 2417.78521723416 x + 815R² = 0.999067984896625

f(x) = NaN x + 815R² = 0

Promega ATP St.

ATP concentraiton (ng/L)Re

lativ

e lig

ht u

nit (

RLU)

• Linear relationship between ATP concentrations and RLU values.• An excellent linearity and low variation coefficient of not more than

(<8%).

• ATP standard material does not affect ATP calibration curve and hence does not affect the ATP values obtained from the calibration curves.

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C. Effect of sample exposure time to the light generating reagent

Source: Hammes et al., 2010

• In ATPSaline protocol, maximum ATP value is at 0s and luminescence signal lost over time.• In promega optimized protocol, two processes happens at the same time : ATP extraction

and light generating.• In promega optimized protocol, the promega reagent needs at least 20 s to extract all the

ATP in water sample and hence obtain maximum RLU. However, some of the luminescence signal will be lost during the extraction period.

0 100 200 300 400 5000

50000

100000

150000

200000

250000

300000

f(x) = 361879.735870835 x^-0.163186339546597R² = 0.984741251297551

Time (seconds)

RLU

X 10

00

20

100 ngATP/L ATP standard solution Tap water

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Conclusion

The last hypothesis may explain the 22% difference between ATPSaline and Promega Optimized protocol.

However it is not needed to apply 20 s wait in ATPSaline protocol because ATP is already extracted.

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2. Comparison of ATPSaline in seawater

0 25 50 75 100 125 1500.00

100,000.00

200,000.00

300,000.00

400,000.00

500,000.00

600,000.00

700,000.00

800,000.00

900,000.00

f(x) = 6347.99796601163 xR² = 0.968772660624579

ATP concentration (ng/L)

Inta

ct c

ell c

once

ntra

tion

(x 1

06 N

o/m

L)

0 1 2 3 4 5 6 7 8 90

0.5

1

1.5

2

2.5

3

0

2000

4000

6000

8000

10000

12000

Samples

ATP

Conc

entr

ation

(ng/

L)

Inta

ct c

ell (

cell/

mL

X100

0)

• ATPSaline shows a well correlation with intact cell concentration (R2 =0.93).

• ATPSaline is well correlated even at very low ATP concentrations.

• ATPSaline

• Intact cell concentration (FCM)

• Lab solution (ASW inoculated with 10000 bacterial cell /ml)

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Fresh water

Seawater

Promega ATP Water

Glo Testing in seawaterApplication in seawaterComparison

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3. Testing Promega ATP Water Glo and compare it to ATPSaline

• Promega Company is developing ATP Water Glo assay kit to measure ATP in aqueous samples like seawater, waste water, water in reservoirs etc.

Method ATP Water Glo ATPSaline

Reagents used Bacterial lysis for ATP extraction BacTiterGlo 2.0 for luciferin-luciferase

reaction

LuminEX for ATP extraction BacTiterGlo 1.0 for luciferin-

luciferase reaction

Separation of free and intra-cellular ATP

Intracellular = 0.2μm retention Intracellular = 0.1μm retention

Filtration rate Manual 300 L/m2/h

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3. Testing Promega ATP Water Glo & compare it to ATPSaline

Promega ATP Water Glo calibration curve was prepared at IHE lab and compared to the calibration curve provided by Promega

0 500 1000 1500 2000 25000

20000400006000080000

100000120000140000

f(x) = 58.1507646176912 x + 3541.04947526237R² = 0.999096823900292

ATP (F moles)

Rela

tive

light

uni

t (RL

U)

Maximum RLU value measured in IHE is almost half the value measured by Promega.

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• ATP Water Glo

• Seawater

• 0.1 and 0.2 μm filters

0.1 um 0.2 um 0.00

20.00

40.00

60.00

80.00

100.00

120.00

104.24

53.55AT

P co

ncen

trati

on (n

g/L)

• Higher ATP concentration can be measured using 0.1 µm filters than 0.2 µm.

• Effect of filter pore size on ATP Water Glo

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• 0.1 μm filters,

• Different calibration curves,

• Scheveningen seawater.

ATP Water Glo gives relatively similar ATP concentration to ATP Saline , however it has a very high standard deviation.

ATPSaline ATP Water Glo22.0

23.0

24.0

25.0

26.0

27.0

28.0

29.0

30.0

31.0

STD = 1.06

STD = 19.22

ATP

conc

entr

ation

(ng/

L)

• Comparison between ATPSaline and ATP Water Glo methods

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Parameter ATPSaline ATPSaline Modified

ATP extraction LuminEX Bacterial lysisluciferase/luciferin enzymatic reaction

BacTiter-GloTM 1.0

BacTiterGlo 2.0

ATPsaline ATPsaline Modified 0

10203040506070 STD = 6.10

STD = 44.70

ATP

conc

entr

ation

(n

g/L)

0 50 100 150 200 250 3000.0E+00

1.0E+04

2.0E+04

3.0E+04

4.0E+04

5.0E+04

6.0E+04

7.0E+04

8.0E+04

f(x) = 283.971425832869 x + 695R² = 0.99674479013986

f(x) = NaN x + 695R² = 0 ATPsaline

ATP concentraiton (ng/L)

Rela

tive

light

uni

t (RL

U)

Seawater sample

Using Promega Bacterial lysis and Promega BacTiter-Glo 2.0 chemicals in ATPSaline method is promising, however it needs more adaption and optimization

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Fresh water

Seawater

Promega ATP Water

Glo

Zeeland seawater treatment pilot plant

Bacterial growth

calibration curve

Testing in seawaterApplication in seawaterComparison

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4. Monitoring of Zeeland seawater treatment pilot plan

1st time: Zeeland seawater treatment pilot plant scheme (17th of Feb. 2015)

2nd time: Zeeland seawater treatment pilot plant scheme (11th of Mar. 2015)

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1st time Zeeland pilot plant monitoring using ATPSaline method and intact cell concentration (FCM) (17/02/2015).

Before

MS

After MS

Feed 10kd

Feed 150kd

Permeate 10kd

Permeate 150kd

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

0100000200000300000400000500000600000700000800000

ATPSaline measurementsFCM measurements

ATP

conc

entr

ation

ng/

L

Inta

ct ce

ll(x

106

cell/

mL)

ATP concentration decreases after each treatment step using

ATPSaline method.However, it was not the

case in intact cell concentration using FCM in all treatment

steps.

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2nd time Zeeland pilot plant monitoring using ATPSaline method (11/03/2015).

Typical trend is observed between

ATPSaline and intact cell

concentration (FCM)

Before strainer

After strainer

RSW heated 15

C

Feed both UF

10KDA permeate

150 KDA permeate

0.0

100.0

200.0

300.0

400.0

500.0

600.0

0

500000

1000000

1500000

2000000

2500000ATPSaline measurementFCM measurement

ATP

conc

entr

ation

ng/

L

Inta

ct c

ell (

x 10

6 ce

ll/m

L)

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5. Monitoring bacterial growth potential based on ATP and FCM.

Preliminary bacterial growth test based on ATPSaline method.

0 24 48 72 96 120 144 1680

20

40

60

80

100

120

140Blank 1 ug/L 5 ug/L 10 ug/L

Incubation time (Hrs)

ATP

conc

entr

ation

(ng/

L)

• Inoculated ASW• Different Glucose

concentrations• Daily measurement

(5 days)

There is no clear difference between the blank sample and the other concentrations.

0 24 48 72 96 120 144 1680

100,000200,000300,000400,000500,000600,000700,000800,000900,000

Blank1 ug C5 ug C10 ug C

Incubation Periods (hrs)

FCM

Inta

ct C

ells

(x 1

03 c

ells/

mL)

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Conclusion• Comparison of ATPSaline with Promega optimized protocol:

ATPSaline is well correlated to promega optimized Protocol (R2=0.95, n=13, p <<0.0001). However, microbial ATPSaline values are 22 % higher than Promega optimized protocol ATP concentration.

The highest Luminescence signal was found immediately after mixing the Luciferase/luciferin reagent (light generating reagent) with the sample and 22 % of Luminescence signals were lost within 20 seconds.

The dissimilarity of sample exposure time to the light generating reagent (BacTiter-GloTM reagent) in ATPSaline (0 s) and Promega optimized protocol (20 s) may be the main reason behind the difference between the two methods.

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ConclusionATPSaline shows an excellent correlation with intact cell concentration measured by

FCM in both freshwater (R2 = 0.82, n= 13 samples, p <<0.0001) and seawater (R2 = 0.93, n= 32 samples, p <<0.0001).

Relatively close ATP values were obtained by measuring seawater sample using ATP Water Glo assay and ATPSaline. However, an extremely high standard deviation was noticed in the ATP values using Promega ATP Water Glo assay.

A promising impression was made from using Promega Bacterial lysis and Promega BacTiter-Glo 2.0 reagents in ATPSaline method. However, this needs an additional adaption and optimization for the chemicals to ATPSaline method.

ATPSaline was successfully applied in a pilot scale seawater treatment plant (Zeeland seawater pilot plant), whereby a clear decrease in ATP concentrations after each treatment steps was observed.

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Recommendation• Further development and optimization of ATP Water Glo assay in order to

minimize the standard deviation.

• More testing and Comparison of the ATPSaline assay is required for the adaption of Promega Bacterial lysis and BacTiter Glo 2.0 chemicals

• Investigate the relationship between biofouling in RO membranes and ATP based on ATPSaline method.

• Moreover, ATPSaline can be used for many applications in the water sector. ATPSaline could be used as a basis for AOC determination test and to assess biofilm formation.

Acknowledgment

Documents

Final MSc thesis presentation