final msc thesis presentation
TRANSCRIPT
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
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 fresh- water samples
Exposure time to light generating reagent 20 s 0 s
10
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)
11
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?
12
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)
13
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.
14
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
15
1. Comparison of ATPSaline method in fresh water.
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.
16
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)
17
Promega optimized protocol
Correlation with FCM
Correlation with FCM
Fresh water
Seawater
Promega ATP Water
Glo Testing in seawaterApplication in seawaterComparison
18
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
19
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.
20
3. Testing Promega ATP Water Glo and compare it to ATPSaline
• 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
21
3. Testing Promega ATP Water Glo and compare it to ATPSaline
• 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
22
3. Testing Promega ATP Water Glo and compare it to ATPSaline
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
23
Promega optimized protocol
Correlation with FCM
Correlation with FCM
Fresh water
Seawater
Promega ATP Water
Glo
Zeeland seawater treatment pilot plant
Bacterial growth
calibration curve
Testing in seawaterApplication in seawaterComparison
24
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)
25
4. Monitoring of Zeeland seawater treatment pilot plan
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.
26
4. Monitoring of Zeeland seawater treatment pilot plan
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)
27
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)
28
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.
29
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.
30
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