Final MSc thesis presentation

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Application and validation of methods to evaluate biological fouling in seawater RO membranes based on adenosine triphosphate (ATP) and flow cytometry (FCM)

Development and Comparison of an Intracellular ATP Method for High Saline WatersSupervisor(s):Prof. Maria Kennedy, PhD, Prof.em. Jan C. Schippers, PhD, MScMentor(s):Loreen O. Villacorte, PhD, MScSergio Salinas, PhD, MScCo-mentorAlmotasem Abushaban , MScExternal examinerZhong Yu, PhD

Urban Water and Sanitation master programmeWater Supply Engineering specialization MSc final presentation Delft, 02April 2015Almohanad Abusultan

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

Bioluminescence is the most widely used for ATP determination



ATP methods in aquatic environments Many protocols have been developed to measure ATP in freshwater Promega BacTiter GloTM Microbial Cell Viability Optimized Assay (Hammes et. al, 2010){LOD = 0.0001 nM ATP}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.3

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


ATP methods in saline water4


Problem statementATPSaline method was recently developed at UNESCO-IHE, However it is not yet neither verified nor compared in fresh or seawater.5


Main goal6


ObjectivesTo compare ATPSaline method with the Promega optimized protocol in fresh water.To investigate the correlation of ATPSaline with intact cells concentration (measured by FCM) in fresh and saline water.To test and compare ATPSaline method with available intracellular ATP commercial kit (ATP Water Glo) in saline water.To apply the ATPSaline as a monitoring tool in seawater desalination pilot plant.7


Research methodology8

Promega optimized protocol

Correlation with FCM

Correlation with FCMFresh waterSeawaterPromega ATP Water Glo Zeeland seawater treatment pilot plant

Bacterial growth calibration curve ATP Saline


ATPSaline vs. Promega optimized protocol9MethodPromega optimized protocolATPSalineReagents usedPromega reagent which comprise both lysing agent and luciferin-luciferase enzyme LuminEX for ATP extractionPromega reagent for luciferin-luciferase reaction Separation of free and intra-cellular ATPTotal = PromegaFree = 0.1m pre-filtration + PromegaIntracellular = 0.1m retention + LuminEX extraction + PromegaIntracellular ATPIntracellular ATP = total ATP - free ATPDirect determination of Intracellular ATP ApplicationApplicable in freshwater onlyApplicable in both sea- and fresh- water samplesExposure time to light generating reagent 20 s0 s


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.10


1. Comparison of ATPSaline method in fresh water.

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

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

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).

11Why ATPSaline gives higher results?


A. Verification of capturing larger microorganisms.ATPSaline protocol.Canal water.Different sampling volumes.12A linear relationship was observed between RLU values and filtered sample volume.

ATP concentration is independent of sample volume.


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 followed13Linear relationship between ATP concentrations and RLU values.An excellent linearity and low variation coefficient of not more than (