got ballast? got organisms?
Detecting Low Numbers of Viable Organisms
by Measuring ATP
Russ Herwig
School of Aquatic and Fishery Sciences
College of the Environment
University of Washington
herwig@uw.edu
Pacific Ballast Water Group - Seattle, WA
February 16, 2012
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It’s nice to be back…
Thank you. Thank you.
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Washington Ballast Water Work Group
Pacific Ballast Water Group
MAKING A DIFFERENCE:
ONE DAY AT A TIME
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University of Washington: 125 years old
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UNIVERSITY OF WASHINGTON
12 YEARS OF BALLAST WATER RESEARCH
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First Funding Agencies
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U.S. Fish and Wildlife Service, Department of Interior
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National Sea Grant, Department of Commerce
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October 1, 2001 to September 30, 2003
$38,505.00
S/T Tonsina Science Team
U.S. Fish and Wildlife Service, Department of Interior
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September 29, 2000 to December 31, 2005
$199,905.14
Ballast Water Monitoring and Treatment
November 1, 2001 to December 31, 2004
$29,894.56
U.S.G.S. Cooperative Agreement
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June 1, 2002 to December 31, 2004
$243,412
U of W Ballast Water Treatment Research
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ATP Project Personnel
• Sara Kelly, Research Scientist.
Now working in a UW virology
research lab.
• Rachel Thompson,
Undergraduate Student. Now
working at USGS Northwest
Fisheries Science Center with
Dr. Maureen Purcell.
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Russell Herwig
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Geometric mean densities of coastal
and oceanic zooplankton in West
Coast and Trans Pacific trip types
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Discovery and History of ATP
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1929 - Discovered by German chemist Karl Lohmann
1939 to 1941 - Fritz Lipmann (USA) shows that ATP is main bearer of
chemical energy in the cell. He coins phrase "energy-rich phosphate
bonds". Nobel Prize in Medicine or Physiology 1953
1937 - Herman Kalckar (Denmark) establishes that ATP synthase
linked with cell respiration.
1948 - Alexander Todd (UK) synthesizes ATP chemically. Nobel Prize
in Chemistry 1957
1958 - Mildred Cohn (USA) uses nuclear magnetic resonance (NMR)
that showed 3 peaks for ATP. One for each phosphorus group
– Battled sexual discrimination for much of her career. Forced to work
outside jobs to pay for her equipment.
– Still attending departmental seminars up to age of 95
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1961 - Peter Mitchell (UK) shows that cell respiration leads to differing
concentrations of hydrogen ions (pH) inside and outside the
mitochondrial membrane (“chemiosmotic hypothesis”). Nobel Prize in
Chemistry 1978
1981 - John E. Walker (UK) determines DNA sequence of genes
encoding the proteins in ATP synthase. Nobel Prize in Chemistry 1997
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High Energy Biochemicals
Anhydride bonds
Ester bond
Anhydride bond
Adenosine triphosphate (ATP)
Phosphoenolpyruvate
Anhydride
bond
Thioester
bond
Acetyl
Coenzyme A
Acetyl-CoA
Acetyl phosphate
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Pathways Leading to ATP Formation
Intermediates in the
biochemical pathway
Energy-rich
intermediates
Substrate-level phosphorylation
Energized
membrane
Less energized
membrane
Oxidative phosphorylation
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ATPase
F1
In
b2
Membrane
C12
Fo
Out
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Smooth endoplasmic reticulum
Microtubules
Mitochondrion
Rough endoplasmic
reticulum
Flagellum
Cytoplasmic
membrane
Mitochondrion
Ribosomes
Microfilaments
Peroxisome
Golgi complex
Lysosome
Chloroplast
Nucleus
Nuclear envelope
Nuclear pores
Nucleolus
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Conundrum of Determining Living
Organisms in (Treated) Ballast Water
• Somewhat easier to do in
other environments and with
other animals
• Microscopic organisms at
relatively high starting
densities
• Microscopic organisms at
very low ending
concentrations following
ballast water treatment
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Zooplankton at High Concentrations
Not too much a problem if motile, > 50
Control
Jeff Cordell
Following ozone treatment
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Motility, Even in Protists, 10 to 50 µm
Peridimium (dinoflagellate)
Paramecium
Protists: unicellular eukarya (have mitochondria and other
organelles)
Brook Biology of Microoganisms
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Motility in Bacteria, < 10 µm
Salmonella
Brook Biology of Microorganisms
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Laboratory Experiments
Extraction and Measurement of ATP in Zooplankton
Sample Collection  Separation by microscopy 
Organisms added to
solvent (DMSO) and
crushed with sterile pestle
to release ATP
Samples can be frozen and
assayed at a later date
Samples are then combined
with Promega ATP reagent
and light output is read on
luminometer
Transferred to
individual microfuge
tubes
Determine ATP
concentrations
from standard
curve
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Methods Development
• Work with individual species of zooplankton and
phytoplankton
• Pick ATP extraction solvent
• First set of tests performed with individual species
prepared as 1, 3, and 5 individuals per tube
– 3 or 4 replicate preparations
• Second set of tests performed with individual
organisms that were separately extracted
– Large amount of ATP per individual zooplankton
– 10 replicates of 1 individual that was extracted
• Method for killing organisms: 60°C for 10 minutes
– Organisms examined for motion
– Motion = alive = viable
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Collection of Freshwater Zooplankton
• Zooplankton collected using a
plankton net
– Vertical tow
– Horizontal tow
• Lake Washington Ship Canal
on UW campus
• 73 µm mesh net
• Placed collected zooplankton in
jar and returned to lab
• Picked and sorted freshwater
candidate species (Hyaella,
Daphnia) for ATP assays
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Promega GloMax® 20/20 Luminometer
• Single tube luminometer
• Photomultiplier tube to measure light
output
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Protocol Summary for Multiple
Individuals - Artemia and Tigriopus
• Artemia and Tigriopus californicus
– Artemia = brine shrimp
– Tigriopus = local marine copepod
• Artemia hatched overnight, held for 24 hours
• T. californicus was collected in Puget Sound
• Standard ATP curve prepared before each test using DMSO as
solvent
• Extracted with 1 ml DMSO. Organisms crushed with micro-pestle.
Held for 5 minutes.
• Frozen immediately or performed ATP assay
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Artemia
48 hour nauplii for ATP analyses
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ATP DMSO standard for Artemia
R2 = 0.9976
1.0E+07
1.0E+06
1.0E+05
RLU
1.0E+04
1.0E+03
1.0E+02
1.0E+01
1.0E+00
1.0E-17
1.0E-16
1.0E-15
1.0E-14
1.0E-13
1.0E-12
ATP (moles)
• ATP quantities linear from 10-16 to 10-12 moles
• 5 x 10-14 to 10-10 grams of ATP (10 to 10,000 ng [nanograms])
• In later standard curves preparations, did not prepare 10-17 mole standard.
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Levels of ATP in Artemia
6.0E-11
ATP (moles)
5.0E-11
4.0E-11
3.0E-11
2.0E-11
1.0E-11
0.0E+00
0
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5
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Number of Organisms
• Different quantities (1, 3, 5) of Artemia were mixed together and extracted.
• Large variation between replicate preparations with same number
• Tighter replication in later experiments
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Tigriopus californicus
(copepod)
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Tigriopus californicus (Tiger Pods)
Tiger Pods Video
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ATP Levels in Tigriopus californicus
5.0E-10
4.5E-10
ATP (moles)
4.0E-10
3.5E-10
3.0E-10
2.5E-10
2.0E-10
1.5E-10
1.0E-10
5.0E-11
0.0E+00
0
1
2
3
4
5
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Number of Organisms
• Preparation of 1, 3, 5 T. californicus together and separately extracted.
• Note variability between averages for the different preparations
• 10-10 moles = 500 ng ATP
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Protocol Summary for Preparing
Single Individuals - Daphnia and
Hyaella
• Daphnia pulacara and Hyaella
• Daphnia were collected using plankton net that we used to
sample Lake Washington Ship Canal (freshwater) by UW
campus
• Hyaella collected from Eurasian milfoil (invasive aquatic plant)
growing in Lake Washington Ship Canal by UW campus
• Zooplankton were sorted and picked from samples with aid of
dissecting microscope
• Single organisms were placed in microfuge tubes
– 10 single individuals separated and extracted
• ATP extraction procedure similar as previously described
– Promega Enlighten kit
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Daphnia pulacara
(cladoceran)
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ATP per Individual Daphnia
7.0E-10
ATP (moles)
6.0E-10
5.0E-10
4.0E-10
3.0E-10
2.0E-10
1.0E-10
0.0E+00
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3
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5
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8
9
10
Individual
• Approximate 4-fold difference in ATP content for Daphnia collected from same site at
same time. (10-10 moles = 5 x 10-8 grams or 500 µg)
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Hyaella
(amphipod)
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ATP in Individual Hyaella
1.4E-09
ATP (moles)
1.2E-09
1.0E-09
8.0E-10
6.0E-10
4.0E-10
2.0E-10
0.0E+00
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5
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10
Individual
• Approximate 4-fold difference in ATP concentration per organism collected
from same site at same time (10-9 moles or 5 x 10-7 grams [500 ng])
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ATP (moles)
Average Amount of ATP in Different
Zooplankton Species
9.0E-10
8.0E-10
7.0E-10
6.0E-10
5.0E-10
4.0E-10
3.0E-10
2.0E-10
1.0E-10
0.0E+00
HK
Daphnia
Artemia
Tigriopus
Daphnia
Hyaella
Species
• HK = heat killed. 5 minutes at 60°C.
• Data presented on linear scale.
• Larger zooplankton have more ATP per individual.
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Future Work
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Current Work and Future Plans
• Altering conditions of ATP extraction protocol
– Increasing extraction time
– Extracting at higher temperature
– Use/not use pestle to mash organisms
• Using “glow” ATP reaction vs. “flash” reaction test assay
• Validating and testing enumeration method with fractionated
communities of organisms
– > 50 µm
– > 10 to ≤ 50 µm
– 0.2 to ≤ 10 µm
• Testing with different ballast water (freshwater and marine)
treatments
– Heat treatment
– Oxidizing biocides
• What happens to ATP during these treatments?
– UV light
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Fractioning Samples for Analysis
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Enumeration Method 1
ATP Threshold Test
Collection of Sample
Fractionation of Sample
<10 µm
10 to 50 µm
>50 µm
Determine Quantity of ATP
Determine Quantity of ATP
Determine Quantity of ATP
Above Threshold
Below Threshold
Above Threshold
Below Threshold
Above Threshold
Below Threshold
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Limitations?
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Viruses
Head
Collar
Tail
Tail pins
Endplate
Tail fibers
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Bacterial Spores and Resting Stages
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Acknowledgements
• National Sea Grant Program - Ballast Water
Management Demonstration Program
• Promega
– Technical representatives
• Jeff Cordell
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