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, MSc Sergio Salinas, PhD, MSc Co-mentor Almotasem Abushaban , MSc External examiner Zhong Yu, PhD Urban Water and Sanitation master programme Water Supply Engineering specialization MSc final presentation Delft, 02April 2015 Almohanad Abusultan
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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
Intracellular ATP Intracellular ATP = total ATP - free ATP Direct determination of Intracellular ATP
Application Applicable in freshwater only Applicable in both sea- and fresh- water 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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1. Comparison of ATPSaline method in fresh water.
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.
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.