Change of Peptides and Free -Amino Acids Contents during Nanjing Dry-Cured Du...
Final MSc thesis presentation
1. 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
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
2
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.
3
Source: (Van der kooij and Veenendaal, 2010).
4. ATP methods in saline water
4
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.
5
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.
7
9. ATPSaline vs. Promega optimized protocol
9
Method Promega optimized protocol ATPSaline
Reagents used
Promega 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 = Promega
Free = 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.
10
y = 0.78x - 3.17
R² = 0.95
n = 13
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
0.0 30.0 60.0 90.0 120.0
MicrobialATP-Promegaoptimizedprotocol
(ng/L)
Microbial ATP - ATPSaline (ng/L)
y = 7784.9x + 27074
R² = 0.82
n = 22
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
0.0 30.0 60.0 90.0 120.0 150.0 180.0
Intactcell(x106cell/mL)
Microbial ATP - ATPSaline (ng/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).
11
Why ATPSaline gives higher results?
12. A. Verification of capturing larger microorganisms.
• ATPSaline protocol.
• Canal water.
• Different sampling volumes.
12
• A linear relationship was observed between RLU values and filtered
sample volume.
• ATP concentration is independent of sample volume.
y = 75174x + 744.5
R² = 0.9956
y = 63367x + 744.5
R² = 0.9883
0.00E+00
2.00E+05
4.00E+05
6.00E+05
8.00E+05
1.00E+06
1.20E+06
0 5 10 15 20Relativelightunit(RLU)
Filtered canal water volume (ml)
0.1 um filter 0.22 um filter
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
13
y = 2380x + 815
R² = 1
y = 2417.8x + 815
R² = 0.9985
0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
6.0E+05
7.0E+05
0 50 100 150 200 250 300
Relativelightunit(RLU) ATP concentraiton (ng/L)
Promega ATP St.
Biothema ATP St.
• 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
14
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
50
100
150
200
250
300
0 100 200 300 400 500
RLUX1000
Time (seconds)
20
100 ngATP/L ATP standard solution Tap water
15. 1. Comparison of ATPSaline method in fresh water.
15
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
y = 6348x
R² = 0.9293
n = 32
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
0 25 50 75 100 125 150
Intactcellconcentration(x106No/mL)
ATP concentration (ng/L)
16
0
2
4
6
8
10
12
0
0.5
1
1.5
2
2.5
3
0 5 10
Intactcell(cell/mLX1000)
ATPConcentration(ng/L)
Samples
• 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)
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.
18
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
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y = 58.151x + 3541
R² = 0.9991
0
20000
40000
60000
80000
100000
120000
140000
0 500 1000 1500 2000 2500
Relativelightunit(RLU)
ATP (F moles)
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
20
104.24
53.55
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
0.1 um 0.2 um
ATPconcentration(ng/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.
21
ATP Water Glo gives relatively similar ATP concentration to ATP Saline ,
however it has a very high standard deviation.
STD = 1.06
STD = 19.22
0.0
10.0
20.0
30.0
40.0
50.0
60.0
ATPSaline ATP Water Glo
ATPconcentration(ng/L)
• Comparison between ATPSaline and ATP Water Glo methods
22. 3. Testing Promega ATP Water Glo and compare it to ATPSaline
22
Parameter ATPSaline ATPSaline Modified
ATP extraction LuminEX Bacterial lysis
luciferase/luciferi
n enzymatic
reaction
BacTiter-
GloTM 1.0
BacTiterGlo
2.0
STD = 6.10
STD = 44.70
0
20
40
60
80
100
ATPsaline ATPsaline Modified
ATPconcentration(ng/L)
y = 2294.1x + 695
R² = 0.9951
y = 283.97x + 695
R² = 0.9947
0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
6.0E+05
7.0E+05
0 50 100 150 200 250 300
Relativelightunit(RLU)
ATP concentraiton (ng/L)
ATPsaline
ATPsaline Modified
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
24. 4. Monitoring of Zeeland seawater treatment pilot plan
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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).
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0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
Before MS After MS Feed 10kd Feed
150kd
Permeate
10kd
Permeate
150kd
Intactcell(x106cell/mL)
ATPconcentrationng/L
ATPSaline measurements
FCM measurements
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).
26
Typical trend is
observed
between
ATPSaline and
intact cell
concentration
(FCM)
0
0.5
1
1.5
2
2.5
0.0
100.0
200.0
300.0
400.0
500.0
600.0
Before
strainer
After
strainer
RSW
heated 15 C
Feed both
UF
10KDA
permeate
150 KDA
permeate
Intactcell(x106cell/mL)
ATPconcentrationng/L
ATPSaline measurement
FCM measurement
27. 5. Monitoring bacterial growth potential based on ATP and FCM.
Preliminary bacterial growth test based on ATPSaline method.
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0
50
100
150
200
0 24 48 72 96 120 144 168
ATPconcentration(ng/L)
Incubation time (Hrs)
Blank 1 ug/L 5 ug/L
• Inoculated ASW
• Different Glucose
concentrations
• Daily measurement
(5 days)
There is no clear difference between the blank sample and the other concentrations.
0
100
200
300
400
500
600
700
800
900
1,000
0 24 48 72 96 120 144 168
FCMIntactCells(x103cells/mL)
Incubation Periods (hrs)
Blank
1 ug C
5 ug C
10 ug C
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.
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29. Conclusion
ATPSaline 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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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.
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