A SIMPLIFIED ATP ASSAY FOR PLANKTONIC ORGANISMS WITH IMPROVED
EXTRACTION EFFICIENCY
A Thesis
Presented to
The Faculty of the Department of Marine Science
San José State University
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
by
Julie C. Kuo
August 2015
© 2015
Julie C. Kuo
ALL RIGHTS RESERVED
The Designated Thesis Committee Approves the Thesis Titled
A SIMPLIFIED ATP ASSAY FOR PLANKTONIC ORGANISMS WITH IMPROVED
EXTRACTION EFFICIENCY
by
Julie C. Kuo
APPROVED FOR THE DEPARTMENT OF MARINE SCIENCE
SAN JOSÈ STATE UNIVERSITY
August 2015
Dr. Nicholas W. Welschmeyer
Department of Marine Science
Dr. Kenneth H. Coale
Department of Marine Science
Dr. G. Jason Smith
Department of Marine Science
ABSTRACT
A SIMPLIFIED ATP ASSAY FOR PLANKTONIC ORGANISMS WITH IMPROVED
EXTRACTION EFFICIENCY
by Julie C. Kuo
The adenosine triphosphate (ATP) assay, a proxy of living biomass (used by
oceanographers for more than 50 years), was investigated for its potential as a ballast
water compliance monitoring tool. Though some ATP assays are reliable for estimating
living biomass in specific microbial communities, those assays may be less than optimal
for others. For this project, an ATP assay was developed to quantify ATP concentration
in most (if not all) aquatic microbial communities; it was also optimized for convenient
and rapid assessment of ballast water compliance for persons without a scientific
background (e.g., ship engineers, mates, ballast water inspectors). The newly developed
ATP assay, called “the P-BAC ATP assay,” was tested against the traditional boiling Tris
ATP assay and a 2nd generation ATP assay.
From this project, I concluded that the traditional Tris ATP assay underestimated
ATP concentrations by 2 to 4-fold in various natural aquatic microbial communities
compared to the P-BAC ATP assay. When compared to the 2nd generation ATP kit, PBAC extracted comparably at times and up to 3.4-fold more ATP. Furthermore, the PBAC ATP assay was successfully executed during full-scale shipboard treatment tests at
the Golden Bear Test Facility (California Maritime Academy, CSU), by measuring the
change in living biomass before and after administering the ballast water treatment.
ACKNOWLEDGEMENTS
I dedicate this thesis to my Mom and Dad. 我很感謝你們的愛, 奉獻, 和擁護
你擺在我生上. I am forever grateful to you! I would also like to thank my sister
(Annie) and brother (Jalongkabong) for being the best siblings I could ever ask for.
I would like to thank my advisor, Nick Welschmeyer, for funding my entire thesis
project, creating incredible opportunities for me to grow as a scientist, and his continual
push for me to “think outside the box”.
I would like to thank my committee members Kenneth Coale and Jason Smith for
their essential moral support, laboratory space and reagents, as well as their dedication
towards helping me meet deadlines.
I would like to thank my fellow Bio Ocea Labmates: Brian Maurer, Karen Parker,
Kelene Keating, Jeff Johnsen, Liz Lam, Nicole Bobco, April Woods, Jennifer Broughton,
Kurt Buck, and Sara Tanner who, in many ways, have contributed to the completion of
my thesis and good times.
Lastly, I would like to thank Ching-Chong Efan for your guidance, love, and
perspective on life.
v TABLE OF CONTENTS
ACKNOWLEDGEMENTS ................................................................................................ v
LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ............................................................................................................ x
Introduction ......................................................................................................................... 1
Invasive Species: an International Concern .................................................................... 1
Biomass Response Parameters Suitable for Monitoring Ballast Water .......................... 2
Oceanographic living biomass indicators. .................................................................. 3
Cell-specific vs. bulk fluorescein diacetate (bulk FDA) technique. ........................... 4
Adenosine triphosphate (ATP) and luminescence. ..................................................... 5
The ATP assay. ........................................................................................................... 5
Materials and Methods ...................................................................................................... 11
Sample Collection ......................................................................................................... 11
Phytoplankton Cultures and Maintenance .................................................................... 14
Reagents and ATP Extraction Procedures .................................................................... 15
Tris(hydroxymethyl)aminomethane hydrochloride (Tris). ....................................... 15
Ultrapure Milli-Q water (MQ). ................................................................................. 15
Sulfuric acid (SA). .................................................................................................... 16
Phosphoric acid (PA). ............................................................................................... 16
Benzalkonium chloride (BAC). ................................................................................ 17
Benzelthonium chloride (BTC)................................................................................. 18
vi Chlorhexidine digluconate (CHD). ........................................................................... 18
Triton X-100. ............................................................................................................ 18
Phosphoric acid and benzalkonium chloride (P-BAC). ............................................ 18
DMSO and benzalkonium chloride (D+P-BAC). ..................................................... 18
LuminUltra second generation ATP assay kit. ......................................................... 19
Sediment Extractions .................................................................................................... 20
Luciferase Light Reaction and Matrix-matched Response Factor ................................ 21
Results ............................................................................................................................... 23
Adenosine Triphosphate (ATP) Extractant Optimization............................................. 23
LuminUltra’s lysing agent “UltraLyse.” ................................................................... 23
Aqueous buffer.......................................................................................................... 25
Surfactants................................................................................................................. 25
Inorganic acid............................................................................................................ 27
Acid and surfactant combination. ............................................................................. 30
Extraction volume. .................................................................................................... 31
Extraction time. ......................................................................................................... 32
Luciferase Enzyme Compatability ................................................................................ 33
P-BAC light-quenching effects offset by dilution. ................................................... 34
ATP Preservation in P-BAC and UltraLyse Stored in -20°C ....................................... 37
P-BAC Validation on Algal Cultures and Environmental Samples ............................. 38
Algal cultures. ........................................................................................................... 38
Nearshore Monterey Bay. ........................................................................................ 39
vii Offshore Monterey Bay. ........................................................................................... 40
Trans-Pacific crossing: San Francisco, CA to Busan, South Korea, Manila,
Philippines, and Saipan, Northern Mariana Islands. ................................................. 41
Moss Landing Harbor sediment. ............................................................................... 43
Ballast Water Treated by Ultra-violet (UV) Irradiation ............................................... 46
Discussion ......................................................................................................................... 49
Algal Cultures and Environmental Samples Concentrated onto 0.7 µm GF/Fs ........... 49
Samples Concentrated onto 10 µm Nylon Filters and Sediment Samples ................... 50
Conclusion ........................................................................................................................ 52
References ......................................................................................................................... 53
Appendix A: ATP Extractants and Tested Conditions ..................................................... 56
viii LIST OF TABLES
Table 1. IMO regulatory standards for various organisms…………………………..……2
ix LIST OF FIGURES
Figure 1. Preliminary Data: Tris Compared to LuminUltra’s QGO-M ATP Extraction
Efficiency…………...……………………………………….…………….………..……10
Figure 2. Preliminary Data: Potential ATP Discarded by Cell-softening Agent………...10
Figure 3. Sampling Stations Between San Francisco & Busan and Busan & Manila…...12
Figure 4. Sampling Stations Between Manila and Saipan…………….……………...….13
Figure 5. LuminUltra Extraction Comparisons……………..……..……..…..……….…24
Figure 6. UltraLyse Extraction Time Optimum Compared to LumiClean+UltraLyse
extraction……………..………………………………..………………………..………..24
Figure 7. ATP Hydrolysis During MQ and Tris Extractions………..…..………………26
Figure 8. MQ vs. LumiClean+UltraLyse ATP Extraction……………...………….…….26
Figure 9. Extraction Comparisons Among Surfactants: BAC, CHD, BTC, and TritonX100……………..……………..……………..……………..…………………………..…27
Figure 10. 0.3% BAC Extraction Efficiency at 20C and 100C Over Time…….…..…....28
Figure 11. Effects of Vortexing and Bead-beating During a 40-minute Sample Extraction
by 0.5% ………..……………..……………..…………….……………………………..28
Figure 12. 1% BAC Compared to UltraLyse ATP Extraction Efficiency……..…….…..29
Figure 13. ATP Extraction Comparison Between LumiClean+UltraLyse, 5% Sulfuric
Acid, and 5% Phosphoric Acid…………...……..………….………………………...….29
x Figure 14. ATP Extraction Efficiency of Various BAC and P-BAC Concentrations…...30
Figure 15. Extraction Volume Effects on ATP Extraction Efficiency…...….……..……31
Figure 16. P-BAC Extraction Time Optimum……………..…….…..………………..…33
Figure 17. Luciferase Enzyme Signal Optimum……..……..……………..………….….34
Figure 18. Luciferase Sensitivity as a Function of P-BAC Dilution; Signal Normalized to
ATP Loaded…………….……...……………..………………………………….………36
Figure 19. Luciferase Sensitivity as a Function of P-BAC Dilution; Signal NOT
Normalized to ATP Loaded...………..……..…………………………...……………….36
Figure 20. Storage Effects of ATP Extracted in P-BAC and Ulyse…..…………………37
Figure 21. Comparison of ATP Extraction Efficiencies on Algal Cultures…………......39
Figure 22. Comparison of P-BAC_60min, UltraLyse_60min, and Boiling Tris ATP
Extraction Efficiency…..……………...……….…..…………….…..……………..……40
Figure 23. P-BAC vs. Tris Extracted ATP in Offshore Monterey Bay…..……..……….41
Figure 24. P-BAC vs. Tris Extracted ATP: Sampling Stations Plotted Against ATP
Concentration (SF Bay to Busan).…..……………...….…..……………………….……42
Figure 25. Comparison P-BAC vs. Tris Extracted ATP (SF Bay to Busan).…….….…..43
Figure 26. Comparison P-BAC vs. Tris Extracted ATP (Busan to Manila)……………..43
Figure 27. Comparison Between P-BAC and UltraLyse ATP Extraction Efficiency on
Sediment…..…………....………….…..…………….…..………………………………45
xi Figure 28. Comparison Between P-BAC and UltraLyse ATP Extraction Efficiency on
Sediment (with and without Bead-beating)…………………..………………………… 45
Figure 29. ATP Extraction Efficiency Comparisons (UV-treated and Untreated Ballast
Water)..…..……………..……….…..…………….………………………...…….…..…47
Figure 30. UltraLyse_60min vs. P-BAC_60min ATP Extraction Efficiency (All Samples
from UV BWMS…..………...……………….…….…..…………….…………….…….47
Figure 31. Tris vs. P-BAC_60min ATP Extraction Efficiency (All Samples from UV
BWMS)..…..…………….…………...……………….…..……………………..……….48
xii Introduction
Invasive Species: an International Concern
Invasive species in marine ecosystems have detrimental effects on native habitats.
They can trigger socioeconomic devastation and compromise human health through
pathogenic diseases (Ruiz et al., 1997). Documented cases include the zebra mussel
(Dreissena polymorpha) takeover in the Great Lakes of North America (Ludyanskiy et
al., 1993), Asian carp invasions in the U.S. Midwest river systems, and the Vibrio
cholerae epidemic in South and Central America in 1992 (McCarthy et al., 1994). These
alien invaders are inadvertently distributed through various vectors including 1) hull
fouling of ships, 2) introduction as commercial aquaculture, and 3) through ship
ballasting operations. Shipping vessels stabilize their transit by taking up and
discharging ballast water to counteract imbalances caused by the cargo loads. As
researchers and policy makers around the world continue to investigate impacts of marine
invaders brought on by deballasting operations, a consensus has been reached for
implementing ballast water discharge standards as a way to mitigate further destruction in
coastal habitats and the spread of disease. Although the International Maritime
Organization (IMO) had designated a set of ballast water management guidelines (Table
1), these standards are rarely enforced due to the inconvenience, inefficiency, and
complexity of current techniques for measuring organism viability. A practical solution
to this matter is to develop a user-friendly bioassay capable of producing a rapid and
reliable ballast water treatment response.
1 Table 1. IMO regulatory standards for each size class during foreign ballast water discharge. Maximum concentration allowable for ≥50 µm zooplankton is reported in “live organisms per meter cubed”, 10 – 50 µm cells are in “live organisms per mL”, and <10µm pathogenic bacteria is in colony-­‐forming-­‐units (CFU) (Regulation D-­‐2 of the BWM Convention). ≥ 50 µm
organisms
(Zooplankton)
10 - 50 µm
organisms
(Protists)
Indicator microbes
V. cholera
(01 & 0139
serotype)
E. coli
Enterococcus
Maximum
concentrat<10 live
<1
<250
<100
ion
<10 live
organisms/
CFU/100
CFU/100 CFU/100
allowable
organisms/mL
m3
mL
mL
mL
for
discharge
Biomass Response Parameters Suitable for Monitoring Ballast Water
Existing regulatory standards generated by the IMO are in numeric concentrations
(Table 1). Enumeration of ≥50 µm organisms is determined under a stereomicroscope,
implementing the poke-and-probe method; this technique is relatively straightforward as
movement is the identifying feature for a live organism. The indicator microbe category
utilizes pre-packaged kits, certified to accurately estimate colony-forming-units for
specific bacterium such as Escherichia coli, Enterococcus, and Vibrio cholera. The 10 –
50 µm size class, however, is more challenging to enumerate because the organisms are
much smaller and movement is less apparent. Therefore, molecular viability markers are
coupled with expensive and technical instrumentation (i.e. epifluorescence microscope,
flow cytometer) to quantify viable cells in this size class. Not only do these techniques
require hours of specialized training, but the instrumentation itself are inconvenient to
carry on and off ships. As a result, bioassays used for monitoring microbial productivity
2 in aquatic environments have been investigated as tool for routine assessment of ballast
water sterility.
Oceanographic living biomass indicators.
Measurements of viable biomass in oceanography aid scientists in describing
trophic web dynamics, biogeochemical carbon cycling, and other physiological
phenomena in the complex aquatic biosphere. Bulk viability assays for monitoring
cellular activity in marine environments include in situ and extracted chlorophyll
fluorescence, algal and bacterial grow-out techniques, particulate organic carbon (POC),
oxygen demand, nutrient depletion, adenosine triphosphate, and esterase activity via the
Bulk FDA technique just to name a few. Two disadvantages are associated with using
chlorophyll a (chl a) fluorescence as a viability indicator: 1) chl a is present only in
phytoplankton and 2) the pigment can stay intact in senescent cells, thereby
overestimating living biomass. Grow-out techniques can be tedious and take days or
weeks to acquire results. POC is generally an overestimate of living biomass, as it does
not distinguish between living carbon and detritus; it is estimated up to >70% of POC in
the mixed layer can be associated with non-living particles. Other techniques such as
oxygen, nitrogen, phosphate, and biosilica bioassays require technical training and
instrumentation for reliable results thereby making these techniques inconvenient. ATP
and esterase activity, however, have been shown to correlate significantly with the
metabolic activity of all living microbial activity in marine habitats and these techniques
have been optimized for rapid, simple, and reproducible detection of bulk viability.
3 Cell-specific vs. bulk fluorescein diacetate (bulk FDA) technique.
Esterase enzymes are a good proxy for metabolic activity in microorganisms
because they occur in all living organisms and are rapidly hydrolyzed upon cell death--an
important characteristic for the quantitative assessment of living biomass. Another
important characteristic associated with this proxy is its allometric behavior with
biovolume. For decades, FDA (a non-fluorescent compound) has been used in
conjunction with esterases to investigate viability in microorganisms and tissue. When
FDA is added to a sample containing live and dead cells, FDA freely permeates all cells.
In living cells, esterase enzymes cleave the acetate molecules from FDA, leaving behind
fluorescein, a fluorescent compound, which optimally excites at 490 nm (blue) and emits
at 513 nm (green). In dead cells, FDA will stay intact, since dead cells lack esterase; as a
result, these cells will not have fluorescent properties
Two FDA techniques exist for quantifying viability in the 10 – 50 µm size
category: cell-specific and Bulk FDA. The cell-specific FDA assay is utilized for
numeric counts of live organisms. When FDA is cleaved, fluorescein accumulates in
living cells, causing them to fluoresce green and become easily detected by
epifluorescence microscopy or flow cytometry. Over time, however, fluorescein leaks out
of the cell and into the medium, causing this assay to be difficult. The Bulk FDA
technique capitalizes on this caveat since the rate of leakage is constant and associated
with esterase activity. Though the Bulk FDA assay seems relatively advantageous as a
ballast water indicator, disadvantages exist in the abiotic production of fluorescein and
sensitivity towards pH and temperature.
4 Adenosine triphosphate (ATP) and luminescence.
ATP, much like esterase enzymes, is found in all metabolically active organisms
from bacteria to multi-cellular animals; it is maintained at fairly uniform levels in various
cells and it also has an allometric relationship to biovolume (Holm-Hansen, 1970; Karl
1980). The majority of ATP biosynthesized at any given time is in the cytoplasm and
mitochondria of both plants and animals. Following production, ATP is expended in
various biological functions including bioluminescence; this phenomena can be found in
prokaryotes and eukaryotes as a way to communicate for the purpose of attracting a mate,
hunting for prey as well as deterring predators. A template of the bioluminescent reaction
is given in Eq.1:
ATP+O2+luciferase→Oxyluciferin+AMP+PPi+CO2+ Light
(Eq. 1)
Extracted ATP is hydrolyzed by the luciferase enzyme and oxygen (O2) to produce
oxyluciferin, adenosine monophosphate (AMP), pyrophosphate (PPi), carbon dioxide
(CO2), and observable light. The light generated from the reaction can be measured by a
luminometer and computed for ATP concentration if ATP is the limiting component
within the reaction mixture.
The ATP assay.
For more than half a century, the chemiluminescent reaction has been studied and
optimized for a diverse range of applications including the evaluation of living biomass
in aquatic environments, biological wastewater treatment validation, surface sanitation
determination, microbial activity in microbrews, and the investigation of cell death in
prokaryotic and eukaryotic organisms (Berney et al., 2008; Hoefel et al., 2003; Holm-
5 Hansen and Booth, 1966; McElroy, 1947; Karl, 1980). Four assumptions are associated
with the application of the ATP assay in consideration of proxies for living biomass: 1)
all living organisms contain ATP, 2) ATP is rapidly hydrolyzed following cell mortality,
3) cellular ATP is positively related to cell volume (biomass) and 4) ATP is extracted
optimally and with uniform efficiency in all living cells. Assumption 1 is universally
accepted as any living cell requires ATP to function. Assumption 2 has been well
substantiated (Eguchi et al., 1997; Pedersen et al., 1999; Tiwari et al., 2002). Assumption
3 has been verified, notably in the seminal work by Holm-Hansen (1971). Assumption 4
has been the subject of numerous studies that parallel the development of ATP
methodologies over the last 50 years (Holm-Hansen and Booth, 1966; Holm-Hansen,
1970; Karl, 1980; Sinclair et al., 1979).
The three main steps for performing the ATP assay include 1) sampling
(capturing of living organisms), 2) ATP extraction, and 3) ATP measurement via the
luciferin-luciferase light reaction. The light emitted from the reaction is proportional to
the dissolved ATP concentration and can be accurately detected on a luminometer when
calibrated to an ATP standard. Though this assay has many applications in
environmental and clinical sciences, and is widely utilized to detect bulk viability,
researchers have encountered problems associated with inefficient cell lysis and
consequently, incomplete ATP extraction. Many ATP extraction procedures have been
developed over the years to overcome this issue; however, the subject of incomplete ATP
extraction persists as a point of contention among scientists measuring living biomass in
many environments (Karl, 1980).
6 One of the most established methods of ATP extraction is the boiling
tris(hydroxymethyl)aminomethane-buffer (Tris) method (Holm-Hansen and Booth, 1966;
Holm-Hansen, 1973). Though the Tris method has a strong historical presence in
environmental monitoring (Karl 1980), including routine application in the ongoing 25year Hawaii Ocean Time Series (Karl and Lukas n.d., HOTS Program, University of
Hawaii), the execution of the assay is somewhat inconvenient for routine measurements
of ballast treatment performance on board ship; especially for non-technical staff such as
the ship’s crew and port state control inspectors.
Biotech companies such as Kikkoman and Hygenia realized the inconvenience of
traditional ATP extraction methods and sought to develop convenient alternatives for
extracting and measuring ATP for surface sanitation in the food preparation industry and
water quality compliance and purity in pharmaceuticals.
The resultant ATP protocols condensed the procedures required to perform the
three essential steps of the ATP assay (see above) and termed the packaged results as “1st
generation ATP kits.” For example, sampling might include using a cotton swab to swipe
a surface or dip into a liquid to acquire the sample. The extraction procedure is reduced to
a single-step operation, which requires immersing the cotton swab in a proprietary
extractant and the luminescence assay might be made in a hand-held luminometer
utilizing the same ‘cuvette’ in which the extraction was run (Kikkoman Biochemifa
Company, Lumitester PD-30 Instruction Manual 2014). While the assays are simple to
perform, the accuracy of the results are lower than the conventional ATP assay systems;
reduced sample volume and light-quenching effects of the extractant on the luciferase
7 light reaction also contribute to significantly lower detection limits for routine plankton
work. Consequently, new companies such as LuminUltra, Ltd. (Canada), have worked
towards overcoming issues associated with 1st generation kits by developing stronger
lysing reagents and improving compatibility of the luciferase reagent with lysing reagent.
These new kits are referred to as “2nd generation ATP kits;” they provide a means to
analyze samples on conventional filters appropriate for routine particle capture in
planktonic work.
2nd generation ATP kits are purported to overcome issues associated with 1st
generation kits, while also preserving simplicity. These kits utilize cationic-surfactants
such as benzalkonium chloride (BAC) to extract ATP. BAC is a membrane solubilizing
surfactant (strong detergent) that can be found as a biocide in ship hull paints,
preservative in contact-lense solution, and disinfectant in common household products,
such as Lysol. LuminUltra‘s 2nd generation ATP kit, allows larger volumes of samples to
be analyzed (e.g., collected on filters) and provides a strong lysing agent for optimal ATP
release. The recommended ATP extraction procedure is a two-step process: for a
planktonic sample with mixed communities of natural microorganisms: 1) pass
proprietary cell-softening agent (LumiClean) through the filter, then pass the cell-lysing
agent (UltraLyse) into a tube containing dilutant (UltraLute) which nominally dilutes the
UltraLyse 10-fold. The dilution step reduces the unfortunate inhibitory effect of the BAC
extractant on the efficiency of luminescence in the enzymatic reaction scheme.
The results of a preliminary experiment comparing the conventional Tris ATP
extraction (Karl 1993) to the 2nd generation commercial reagents from LuminUltra, show
8 that Tris is 2.4-fold less effective at extracting ATP from a natural water sample (Figure
1). Furthermore, when LumiClean (the initial cell-softener) was included as part of the
extraction procedure, the difference increased to 3-fold; that is, the proprietary cellsoftner (LumiClean) also acted as an ATP extractant. This observation suggests that
boiling Tris extractions, frequently used in reporting aquatic living biomass and cellular
carbon estimates (Karl 1993, Sinclair et. al 1978, Hamilton and Holm-Hansen 1967,
Brezonik et al. 1975, Hunter and Laws 1981), may be yielding significant underestimates
of ATP. As a result, ratios of ATP to other biomass parameters commonly cited in
marine science ecological work (i.e.: chl a:ATP, C:ATP, ATP per cell, ATP:cell volume;
references) may require new calibrations exploiting the stronger (more desired) extraction
procedure such as the LuminUltra’s 2nd generation ATP assay kit. Though LuminUltra’s
2nd generation ATP kit could be used for these re-evaluations, it would require double the
effort and cost for a single sample, due to the necessity of having to measure two
reagents. Preliminary results show that if LumiClean is unread, up to 40% of the ATP
could be lost during the initial pass (Figure 2). This compromises the efficiency of the
protocol and makes it less appealing for ballast inspectors and scientists looking for a
rapid, inexpensive, and convenient viability technique. If the LuminUltra’s ATP
extractants could be combined into a single “super” extractant, the kit would be more
appealing. The purpose of this thesis is to develop an ATP assay for universal
applications including routine viability measurements in ballast water and oceanographic
research. The following set of objectives were used to guide this thesis project:
1) Simplify cellular ATP extraction while maintaining or exceeding maximum
9 extraction efficiency of a conventional and 2nd generation ATP assay
2) Evaluate commercial luciferin-luciferase enzyme reagents for compatibility
with the improved extractant
3) Validate improved ATP assay system with lab cultures, environmental
samples, and treated ballast water
Figure 1. The traditional boiling Tris extraction method (Holm-Hansen and Booth 1966) extracted
2.4-times less ATP than LuminUltra’s ATP kit which required only the ATP measurement of
UltraLyse. When LumiClean was measured for ATP and summed to UltraLyse ATP, the difference
was 3-fold more than Tris.
Figure 2. Potential ATP discarded by cell softening agent. ATP can be understimated by
10% and up to 40% in a sample by following LuminUltra’s instructions for discarding the
LumiClean filtrate and measuring only the UltraLyse filtrate.
10 Materials and Methods
Sample Collection
Environmental water samples were collected in clean 2 L polycarbonate bottles
and 8 L polypropylene carboys. Water samples from inshore and offshore locations,
ballast water, and surface sediment were used for the development and optimization of a
new ATP extractant. The samples were also used for comparing the traditional boiling
Tris extraction method to the new extractant and UltraLyse (LuminUltra’s patented celllysing agent).
Inshore samples were used to test various ATP extraction methods and identify
an optimal extractant. These samples included seawater from Moss Landing Harbor and
Lover’s Point on the Monterey Bay. Brackish water was collected from the Carquinez
Strait off the California Maritime Academy (CMA) and Kirby Park in Monterey County.
Freshwater environmental samples were acquired at Hudson’s Landing.
Offshore samples were opportunistically collected to assess the difference between
ATP extracted by the traditional boiling Tris method (Karl, 1993) and the extractant I
developed in this thesis. In March 2013, offshore samples in Monterey Bay were
collected using a CTD rosette deployed off the research vessel (R/V), Point Sur. Samples
were taken at various depths between the surface and 1000m with remotely operated
Niskin bottles. Other offshore samples include surface casts off the training ship (T/S),
Golden Bear, in May 2014 and June 2014, from a transect spanning SF Bay, California to
Busan, South Korea and Busan to Manila, Philippines to Saipan (Figure 3 and 4); this
effort was used to capture the transition of highly productive coastal waters to low
11 biomass oligotrophic waters as well as investigate the discrepancy between the ATP
extracted by boiling TRIS vs. my extractant formulation.
A B Figure 3. Sampling stations between San Francisco and Manila. A) Transect sampling stations from San Francisco Bay, California to Busan South Korea. Located in the upper right corner of this map is a high-­‐
resolution map of the sample stations near the departure port at CMA. Located in the lower left corner is a h igh-­‐resolution map of the sampling stations near to the destination port in Busan. B) Transect from Busan, Korea to Manila Philippines. 12 Figure 4. Sampling stations between M anila and Saipan. Transect sampling stations from M anila, Philippines to Saipan. Ballast water samples were acquired on the T/S Golden Bear during Type
Approval tests for a UV-irradiation ballast water management system (BWMS); trial
nomenclature has been changed for proprietary reasons. Ambient water was uptaken by
the ship’s pump, UV-irradiated prior to its introduction into the ship’s ballast tank, held
in the dark tank for 5-days, and UV-treated during its discharge back into the sea.
Control seawater was delivered directly into a ballast tank with UV treatment; it was also
held in the dark for 5-days and discharged similarly to UV-treated water. During the
uptake and discharge events, water was subsampled into carboys and processed for ATP
using boiling Tris, UltraLyse (from the LuminUltra ATP assay kit), and the new
extractant formulation.
All aquatic samples were concentrated onto 25 mm diameter filters either on 0.7
µm (pore size) glass-fiber filters (GF/F) or 10 µm nylon Millipore filters and were
performed in triplicates per sample. Cells were harvested onto GF/Fs by either a syringe
13 and plunger connected to a resusable syringe filter holder or by vacuum pump on a filter
rack; GF/Fs nominally captured cells ≥0.7 µm, which include bacteria. For 10 µm
filters, cells were harvested using gravity filtration. 10 µm filters captured organisms ≥10
µm and were used to assess the IMO 10 – 50 µm size category. Though samples were
not pre-filtered to remove >50 µm organisms, the particle size distribution in the ocean
suggests that >50 µm cells are negligable when filtering small volumes (i.e.: 100 mL)
because they are logarithimically less common than smaller organisms (Chisolm, 1992).
Lastly, sediment samples collected from Moss Landing Harbor during low tide
were extracted in P-BAC and UltraLyse. These samples were also tested for potential
bead-beating effects on extraction efficiency. Twenty and 100 µm Thermo Fisher
Scientific beads were used in conjunction with the BioSpec Bead-Beater.
Phytoplankton Cultures and Maintenance
Original phytoplankton culture stocks were purchased from the Carolina
Biological Supply and National Center for Marine Algae and Microbiota (NCMA). These
algal cultures were maintained in the Moss Landing Marine Laboratories’ (MLML)
Biological Oceanography Lab and used for ATP extractant validation tests. Marine algal
growth media was made by passing ambient seawater (local Monterey Bay) through a
0.22 µm continuous-flow filter, autoclaving and diluting commercial (Sigma, Inc.) F/2
Guillard’s marine enrichment solution 50-fold into the autoclaved filtrate. Freshwater
algal media was made using AlgaGro nutrient enrichment and laboratory RO water (0.22
µm filtered; 18.2 MΩ•cm) then autoclaved. Both marine and freshwater media were
autoclaved for 20 minutes and cooled to room temperature prior to decanting a small
14 volume of an older culture into the clean media for the initiation of new batch cultures.
The cultures were grown in natural light and ambient room temperature (18-22°C) on a
north-facing windowsill of the Biological Oceanography Lab at MLML; in some cases
batch cultures were maintained in temperature-controlled (15°C) continuous light
incubators (ca. 70 µmole photon m-2s-1 PAR photon flux intensity).
Reagents and ATP Extraction Procedures
All ATP extractants and extractant conditions can be found in Appendix A.
Tris(hydroxymethyl)aminomethane hydrochloride (Tris).
ATP extractions using boiling tris were based on the extraction procedure
developed by Holm-Hansen and Booth (1966). Tris was reconstituted and diluted to 20
mM using ultrapure MQ. The solution was titrated to pH 7.8 using 20 mM Tris base.
Following the pH adjustment, the Tris pH 7.8 was dispensed into glass vials in 4 mL
aliquots and autoclaved for 30 minutes. When the vials were cooled to room temperature,
they were capped and frozen at -20°C until sample processing. Prior to sample
processing, the Tris vials were thawed, loaded onto a heating block and brought to
100°C. Cells were harvested onto a filter (either a 0.7 µm GF/F or 10 µm nylon
Millipore filter) and immediately submerged in a Tris vial for 5 minutes at 100°C to
extract intracellular ATP. After the boiling extraction, the vial was cooled in a bucket of
crushed ice and later brought to room temperature for analysis. If the sample was not
analyzed immediately, it was stored at -20°C for later analysis.
Ultrapure Milli-Q water (MQ).
MQ ATP extractions were based on studies conducted by Yang et al. (1993). The
15 method of extraction was similar to the Tris extraction with regards to the extraction and
cooling process.
Sulfuric acid (SA).
ATP extractions using SA were based on the method developed by Karl and
Larock (1975) used for extracting ATP from organisms residing in soil and marine
sediments. SA was tested at concentrations including 250 mN (2.5% weight to weight),
500 mN (5% w/w), and 1 N (10% w/w) made from 10 N H2SO4 commercial stock
(Sigma-Aldrich reference #10002938). The buffer used to mix SA was autoclaved 25
mM tricine (pre-adjusted to pH 7.8 using 25 mM tris base). Ethylenedinitrilo-tetracetic
acid (EDTA) was not included in the final product because it significantly interfered with
the luciferase light reaction. Samples filters were submerged in 5 mL of extractant predispensed into 15 mL polystyrene Falcon tubes sitting on ice at 4°C. After the sample
filters were introduced to the extractant, they were vortexed at 10-second intervals for a
total of 60 seconds and allowed to extract on ice for 10 minutes. Afterwards, each
sample tube was vortexed for 10 seconds and centrifuged for 5 minutes at 4,000 rpm.
One mL of the supernatant from each tube was then subsampled into a 10 mL beaker,
diluted 10-fold by 25 mM Tris-tricine (pre-adjusted to pH 7.8 using 25 mM tris base),
and the mixture was titrated to pH 7.8 using NaOH pellets. After the pH adjustment, the
sample was immediately analyzed for ATP content.
Phosphoric acid (PA).
Phosphoric acid (PA) extractions were performed similar to the method developed
by Karl and Craven (1980) for naturally occurring microbial assemblages in aquatic
16 environments; this method is identical to Karl and LaRock’s (1975) sulfuric acid
extraction method except that phosphoric acid was used in place of sulfuric acid.
Phosphoric acid ATP extractions were tested at a concentration of 500 mM (5% w/w),
and 1 M (10% w/w), buffered by 25 mM tricine (pre-adjusted to pH 7.8 using 25 mM tris
base). Samples were immersed in 5 mL of each acid mixture, vortexed at 10-second
intervals for a total of 60 seconds and allowed to extract on ice for 10 minutes. Each
sample was then centrifuged for 5 minutes at 4,000 rpm, 1 mL of supernatant was
transferred into a 10mL beaker, and diluted 10-fold by 25 mM tricine (pre-adjusted to pH
7.8 using 25 mM tris base). Samples were then titrated to pH 7.8 using NaOH pellets and
analyzed immediately for ATP.
Benzalkonium chloride (BAC).
ATP extractions using the basic surfactant, BAC, were based on the methods
developed by Hattori et al.(2003) and Siro et al. (1982) for the intracellular ATP
extraction of bacteria and yeast cells. BAC concentrations tested include 8.4 mM (0.3%
w/w), 14 mM (0.5% w/w), 19.6 mM (0.7% w/w), and 28 mM (1% w/w) BAC. Buffers
tested for compatibility with BAC include autoclaved MQ, 25 mM tris (pre-adjusted to
pH 7.8 using 25 mM tris base), and 25 mM tricine (pre-adjusted to pH 7.8 using 25 mM
tris base). Sample filters were submerged for various time periods between 0 and 60
minutes at room temperature (19-22°C). BAC concentrations at 0.3% and 0.5% were
investigated for extraction efficiency at 100°C. At the end of the extractions, the samples
were diluted 10-fold and up to 100-fold, using 25 mM tricine (pre-adjusted to pH 7.8
17 using 25 mM tris base), to counteract light-quenching effects on the chemiluminescent
light reaction.
Benzelthonium chloride (BTC).
BZC extractions were performed similar to BAC extractions without the boiling
experiment.
Chlorhexidine digluconate (CHD).
CHD extractions were performed similar to BAC extractions without the boiling
experiment.
Triton X-100.
Extractions were performed similar to BAC extractions without the boiling
experiment.
Phosphoric acid and benzalkonium chloride (P-BAC).
Acid-surfactant extractions were inspired by Christen and Devol (1980). PA was
combined with BAC to test extraction efficiency, as they were the optimal extractants of
all extractants tested above. 1% (w/w) BAC was tested with PA at 2.5% (w/w) and 5%
(w/w) concentrations. Extraction times varied between 5 and 90 minutes to obtain the
extraction incubation optimum. At the end of the extraction period, samples were handshaken for 3 seconds, subsampled, and diluted 50-fold and 100-fold by 25mM tricine
(pre-adjusted to pH 7.8 using 25 mM tris base) prior to ATP measurements.
DMSO and benzalkonium chloride (D+P-BAC).
An attempt was made to mimic the benefits of LumiClean using DMSO. After
concentrating cells onto a syringe filter, 5 mL of 100% DMSO was passed through the
18 sample and collected in a 50 mL falcon tube and diluted 10-fold using 25 mM tricine
(pre-adjusted to pH 7.8 using 25 mM tris base). Following the DMSO pass, 1 mL of 1%
(w/w) BAC was passed through the same sample and captured in a microcentrifuge tube
where it was subsampled and diluted 100-fold by tricine for ATP analysis.
LuminUltra second generation ATP assay kit.
The LuminUltra 2nd generation ATP assay kit was conducted similar to the
procedure outlined in the manufacturer’s protocol (LuminUltra’s Quick Reference Guide,
Product #: QGOM-25/QGOM-100). Samples were concentrated onto a filter using a
syringe and plunger. 5 mL of LumiClean was passed through the concentrated sample,
collected in a 15 mL Falcon tube and diluted 10-fold prior to analysis. Following the
cell-softening step, the cell-lysing agent ‘UltraLyse’, was passed through the filter and
captured in a 15 mL polystyrene tube, prefilled with proprietary buffer ‘UltraLute’,
creating a 10-fold dilution to the filtrate. Though saving LumiClean was not in
LuminUltra’s standard protocol, it was shown to remove a significant amount of ATP and
therefore must be measured. Due to the inconvenience of having to measure LumiClean
and UltraLyse for a single sample, UltraLyse was investigated separately for its ATP
extraction potential. Given below is the nomenclature and corresponding method of
extraction using LuminUltra’s commercial reagents:
“LuminUltra Protocol” designates LuminUltra’s standard protocol, which
instructs discarding LumiClean and measuring ATP only in the UltraLyse filtrate
(LuminUltra’s Quick Reference Guide, Product #: QGOM-25/QGOM-100).
19 “LumiClean+UltraLyse” designates the sum of LumiClean and UltraLyse
extracted ATP.
“UltraLyse_60min” designates the immmersion of a sample in UltraLyse for 60
minutes.
“LumiClean+UltraLyse_60min” designates the sum of ATP extracted by a
LumiClean pass and subsequent immersion in UltraLyse for 60 minutes.
“UltraLyse_1x pass” designates a sample extracted for ATP by a single 1 mL pass
of UltraLyse without LumiClean.
“UltraLyse_3x pass” designates a sample extracted for ATP by passing the same
mL of UltraLyse through the filter three subsequent times. Again, LumiClean was not
involved with this protocol. This procedure was taken from First and Drake (2013).
Sediment Extractions
Sediment samples collected from Moss Landing Harbor during low tide events
were used to compare ATP extraction efficiencies between UltraLyse and the new
extractant. These samples were extracted by injecting 170 µL of sample directly into 1
mL of extractant, vortexing the mixture, and in one experiment, extracting for 60
minutes. In another experiment, the samples were extracted for 30 minutes and analyzed
for ATP. Then, 200 µg of 20 µm beads and 200 µg of 100µm beads were added to the
original extracting tube and mechanically extracted in the BioSpec Beadbeater for 3
minutes. Samples extracted in UltraLyse were centrifuged at 10,000 rpm for 5 minutes.
The supernatant was filtered using a 0.45 µm filter to remove suspended particulates that
obstructed luminometer readings, then diluted 10-fold using UltraLute, following the
20 manufacturer’s protocol for analyzing an UltraLyse extracted sample. The newly
developed extractant from this thesis, however, did not require centrifugation, nor prefiltration through 0.45 µm filter—it was simply diluted 100-fold by a 25 mM tricine (preadjusted to pH 7.8 using 25 mM tris base).
Luciferase Light Reaction and Matrix-matched Response Factor
Extracted ATP was measured on a Turner Designs 20/20 luminometer using
commercial luciferin-luciferase enzyme reagents including Promega Enliten, LuminUltra
Luminase, and LuciferaseX (a proprietary formulation). All enzymes, which are
temperature and light sensitive, were stored on ice and wrapped in foil to slow enzyme
deterioration during analysis. ATP measurements were made by reacting 50 µL of
enzyme to 100 µL of sample. Within 4 seconds of enzyme addition, the sample vial was
gently shaken and measured on the luminometer, for an integration period of 10 seconds.
The reaction between luciferin-luciferase and ATP generates a light signal, which
is directly proportional to the mass of ATP per volume contained in the sample matrix.
However, it is important to understand that the response is also 1) volume-dependent, 2)
reagent-dependent and 3) optically-dependent on the physical dimensions of the final
cuvette solution. Thus, the volumetric addition of enzyme and sample must be constant
as well as the optical qualities of the cuvette for all assays, including the ATP standard
for generating the response factor (RF). To be specific, the reagent chemistry and
volume of the standard must be in identical proportions to the sample matrix and volume
analyzed in the luminometer. The calibration coefficient, in luminometer light response
units (RLU) per ng of ATP per L (RLU/(ng/L)), is influenced by optical and chemical
21 properties of the matrix. For example, the same ATP mass loaded into two identical
analysis cuvettes, with one containing Tris and the other containing a surfactant, will not
yield equivalent luminescence responses due to the luciferase reaction with the chemical
properties of each extractant. Therefore, a RF must be generated for the sample extracted
in Tris and as well as for the sample extracted in the surfactant. Linearity in ATP
luminescent measurements can only be maintained with standardized 1) volumetric
dilutions, 2) cuvette shape and, 3) reagent chemistry within the combined “sample” plus
“enzyme” mixture.
22 Results
Adenosine Triphosphate (ATP) Extractant Optimization
Several types of ATP extraction reagents and methodologies were assessed to
identify the optimal ATP extractant formulation. Our optimal criteria were defined as a
formulation yielding highest and consistent ATP yields with fewest processing
manipulations. Reagent groups tested included aqueous buffers, inorganic acids, basic
surfactants, acidic surfactants and reagents from LuminUltra’s 2nd generation kit.
Extractant optimizations were tested on Moss Landing Harbor water and concentrated
onto GF/F (0.7 µm) filters, unless otherwise specified.
LuminUltra’s lysing agent “UltraLyse.”
One of the many UltraLyse trials involved a single UltraLyse pass (without
LumiClean), another involved three passess of UltraLyse through the sample (First and
Drake 2013), and lastly, a sample was immersed in UltraLyse for 30 minutes. The 30minute sample immersion was found to be most convenient, though it was still not as
effective as LumiClean+UltraLyse (Figure 5). Therefore, a time-series experiment was
conducted where independent samples from the same seawater sample were submerged
in UltraLyse. The results showed a 60-minute sample immersion in UltraLyse was
comparable to the sum of ATP extracted by LumiClean and UltraLyse (Figure 6).
Among the different ways the reagents were tested, a sample immersed in UltraLyse for
60-minutes was most efficient and simple to perform. The results were also comparable
to the sum of LumiClean and UltraLyse extracted. As a result, UltraLyse 60-minute
submersion extraction was used as a point of reference throughout this study.
23 Figure 5. LuminUltra extraction comparisons. LuminUltra reagents were assessed
for maximum extraction efficiency. ‘LClean+ULyse’ was optimal among the
variations tested. LuminUltra protocol. Extractant nomenclature is provided in the
Method’s section under LuminUltra 2nd generation ATP assay kit.
Figure 6. UltraLyse extraction time optimum compared to LumiClean+UltraLyse.
Moss Landing Harbor water concentrated onto a GF/F filter immersed in UltraLyse
for 60-minutes extracted similarly to the sum of ATP acquired from a single
LumiClean and UltraLyse pass. Extractant nomenclature is provided in the
Method’s section under LuminUltra 2nd generation ATP assay kit.
24 Aqueous buffer.
The standard historical method used as a point of reference was the boiling Tris
method (Holm-Hansen and Booth 1966). A comparison between ultrapure Milli-Q (MQ)
water- vs. Tris-extracted ATP resulted in MQ being 1.53-times more effective than Tris
in extracting intracellular ATP. The most optimal extraction time for both methods was 5
minutes at 100°C (Figure 7). Although MQ was more effective as an ATP extractant for a
natural seawater sample, ATP seemed to be hydrolyzed more rapidly in MQ extraction
solution—a 2-fold loss in ten minutes. Tris exhibited little to no loss over 20 minutes.
When ATP hydrolysis was tested using an ATP standard, both MQ and Tris seemed to
preserve the ATP similarly. This suggests there are components in the natural sample
that hydrolyze ATP, such as ionic metals and enzymes that Tris may be inhibiting.
Furthermore, when MQ is compared to LumiClean+UltraLyse ATP extractability, it falls
60% short of its competitor (Figure 8).
Surfactants.
Surfactants tested in this study include benzalkonium chloride (BAC),
chlorhexidine digluconate (CHD), benzethonium chloride (BTC), and Triton X-100.
BAC, BTC, and Triton X-100 were tested at four different concentrations between 0.1%
(w/w) and 1%. For these three surfactants, each concentration was tested two times: once
using 25 mM tricine (pre-adjusted to pH 7.8 using 25 mM Tris Base), and once using
MQ. CHD was tested at 0.1% and 0.3%, buffered only by MQ. Optimal extraction
incubation time was also determined for each concentration during these experiments.
The results from these experiments showed that a 20-minute sample filter immersion in
25 1% BAC, buffered by tricine, was optimal—about 40% more than 0.3% CHD, 50% more
than 0.7% BTC, and 75% more compared to 1% Triton (Figure 9).
Figure 7. Boiling MQ extracted 1.53-times more ATP than boiling Tris. Both
extractants optimally extracted at 5 minutes. ATP hydrolysis was apparent in MQ.
Figure 8. Boiling MQ extracts 60% less intracellular ATP compared to the sum of
ATP extracted by a single LumiClean and UltraLyse pass.
26 Figure 9. A sample filter extracted in 1% benzalkonium chloride (BAC) for 20
minnutes was optimal in extracting ATP compared to 0.3% chlorohexidine
digluconate for 20 minnutes (CHD), 0.7% benzethonium chloride (BTC) for 20
minutes, and 1% Triton X-100 for 5 minutes.
BAC extractions were coupled with physical agitation including vortexing,
boiling, and bead-beating in an attempt to increase ATP extraction efficiency.
Experimental results indicated no significant benefit was associated with boiling a sample
in 0.3% BAC, over a period of 30 minutes (Figure 10). No benefits were observed for
samples bead-beated for 4 minutes or vortexed for 10 minutes during a 40 minute
immersion in 0.5% BAC (Figure 11). A comparison of 1% BAC extractions and
UltraLyse_60min indicated that 1% BAC was 2-fold less effective than UltraLyse in
extracting ATP (Figure 12).
Inorganic acid.
Sulfuric acid (SA) extracted 5% more ATP compared to phosphoric acid (PA),
not including any surfactant mixtures (Figure 13). When compared to
27 LumiClean+UltraLyse, both SA and PA resulted in a 2.8-fold lower ATP concentration.
Increasing the concentration of SA to 10% showed no additional benefit for ATP
Figure 10. Extraction efficiency of 0.3% BAC was not improved when boiled at
100C.
Figure 11. Extraction efficiency of a sample filter immersed in 0.5% BAC was not
improved by bead-beating or vortexing.
extraction.
28 Figure 12. A sample extracted in 1%BAC for 60 minutes is 2-fold less efficient at
extracting ATP compared to UltraLyse.
Figure 13. Sample filters extracted in 5% (w/w) sulfuric acid tricine mixture and
%5 (w/w) phosphoric acid and tricine mixture were nearly 2.8-fold less successful
at extracting ATP compared to the sum of a single LumiClean (red)+UltraLyse
(blue) extraction.
29 Acid and surfactant combination.
PA and BAC were combined and tested at a 2% PA, 1% BAC target weight-toweight (w/w) mixture and 5% PA, 1% BAC. 25 mM tricine (pre-adjusted to pH 7.8 with
25 mM tris base) composed 97% and 94% of each mixture, respectively. Furthermore,
the pH of the extractant ranged from an acidic 1.2 to 1.5. In an experiment comparing
the extraction efficiency of 5% PA_1% BAC, 2% PA_1% BAC, 1% BAC, and 0.7%
BAC, the performance of 5% PA_1% BAC (P-BAC) at extracting ATP was optimal; it
exceeded that of 2% PA_1% BAC and 1% BAC by 20%, and 40% for 0.7% BAC (Figure
14). Vortexing did not enhance P-BAC TP extractions (P-BAC).
Figure 14. Benzalkonium chloride (BAC) at concentrations of 0.7% and 1% (w/w)
were tested against mixtures of phosphoric acid (PA) and BAC at concentrations of
2% PA with 1% BAC and 5% PA with 1% BAC. All extractant concentrations
were created using 25 mM tricine pre-adjusted to pH 7.8 using 25 mM tris base.
The results show 5% PA with 1% BAC mix as the optimal extractant among the
extractants tested
30 Extraction volume.
Moss Landing Harbor water was extracted in 1 mL and 2 mL of P-BAC,
independently, to identify the optimal extraction volume for the largest light response.
The same volume of seawater was concentrated onto GF/Fs, which were then submerged
into 1 mL of P-BAC (using 1.5 mL microcentrifuge tubes) or 2 mL of P-BAC (using a 5
mL polystyrene cuvettes). The samples were extracted for 60 minutes, prepped for ATP
analysis, and measured on the luminometer. The results revealed no significant
difference between ATP concentrations of samples extracted 1 mL and 2 mL of P-BAC
(Figure 16). However, the light output from the luminometer was 2-fold higher in the 1
mL extraction compared to the 2 mL extraction, thus indicating the 1 mL extraction
provides more sensitivity. As a result, all P-BAC extractions were performed using 1 mL
of the P-BAC extractant.
Extractant volume Figure 15. No significant difference was observed in the ATP extracted from the
same natural water sample extracted in 1 mL vs. 2 mL of extractant.
31 Extraction time.
Extraction incubation optimum for P-BAC was validated by five time-series
experiments where samples were measured at 5, 10, 20, 30, 40, 60, and 90 minutes using
independent extractions (Figure 16). The results showed that samples concentrated onto
GF/F filters and extracted in P-BAC for 20 minutes yielded between 80% to 100% of the
ATP optimum from the sample. Extraction times beyond 20 minutes also resulted in
relative ATP yields within between 80% and 100%. Extractions terminated at 10 minutes
resulted in 70-100% of the optimum. At 5 minutes, ATP yield ranged from 70 to 90% of
the optimum.
When P-BAC_60min ATP extractions were compared to LumiClean+UltraLyse
extractions using Moss Landing Harbor water, the results indicated the P-BAC extraction
recovered 1.38-times more ATP. In summary, P-BAC_60min extractions were optimal
for extracting ATP in Moss Landing Harbor water (concentrated onto 0.7 µm GF/Fs).
The optimal extraction time for a sample immersed in P-BAC is anywhere in between 20
and 90 minutes; the ATP extracted within this range is within 20% of the optimum.
32 Figure 16. A 20 minute or greater extraction yields an ATP concentration within
20% of the optimum. Extractions between 5 and 10 minutes yield ATP
concentrations within 30% of the optimum.
Luciferase Enzyme Compatability
Luciferase enzymes tested for compatibility with P-BAC included “flash” and
“glow” enzymes. Flash enzymes are known for their sensitivity and rapid results in
seconds. Its drawbacks, however, include a rapid loss in luminescence optimum within
seconds and inhibition in high ionic strength solutions. Most of the results produced in
this thesis are achieved by using the flash enzyme “Enliten”—its reactivity is instant and
extremely sensitive. Glow enzymes are generally lower in sensitivity and take 1 to 10
minutes to reach signal optimum. The benefit of using these types of enzymes is for its
resistance to high ionic strength reagents and long-lasting signal optimum; thus, they are
popular in ATP-related assays that involve measurements in 96-well plates where the
assay time could last several minutes. Glow enzymes used during this experiment
33 include Luminase and LuciferaseX. These enzymes were tested against Enliten for
sensitivity and compatibility with P-BAC. When Enliten was compared to Luminase and
LuciferaseX, it outcompeted both by 2-fold and 33-fold, respectively. With regards to
the luminescence half-life during analysis, Enliten lost half of its intensity at 5 minutes,
Luminase’ was at 12 minutes and LuciferaseX’s was at 35 minutes (Figure 17). Enliten,
Luminase, and LuciferaseX were further examined for compatibility with P-BAC by
identifying the lowest dilution factor necessary to obtain the largest signal.
Figure 17. Commercial luciferase enzyme reagents, Enliten, Luminase, and
LuciferaseX were tested for luminescence. Enliten provided the most signal per
mass loaded, though its half-life was the shortest among the enzymes tested in this
experiment.
P-BAC light-quenching effects offset by dilution.
Quantitative P-BAC dilutions were created by adjusting P-BAC and tricine
volumes, while holding ATP standard constant. This allowed the ATP concentration at
34 each dilution to be comparable with regards to signal output. The results indicated a 100fold dilution factor to P-BAC removed most, if not all, inhibitory effects of P-BAC on
Enliten and Luminase enzymes and a 50-fold dilution was required for LuciferaseX
(Figure 18). Enliten provided 1.8-times more signal than Luminase and 28-times more
than LuciferaseX. Though this experiment was able to identify the dilution necessary to
eliminate light-quenching effects of P-BAC, it does not incorporate the actual ATP
loaded during a sample extraction. The optimal dilution factor for a sample extracted in
P-BAC was a 50-fold dilution when using the Enliten and Luminase enzyme; for
LuciferaseX, it was a 20-fold dilution (Figure 19). Though LuciferaseX’s optimal
dilution factor was more reasonable than the others, the optimum luminescence is an
order-of-magnitude lower than Enliten and 6.8-fold less than Luminase. Thus, the
enzyme most compatible with the current P-BAC extractant is Enliten since it provides
the most sensitivity compared to the other enzymes tested.
35 Figure 18. P-BAC has light-quenching effects during the luciferase light reaction.
Using ATP standard mixed into P-BAC, light-quenching effects can be eliminated
by diluting the extractant 100-fold using tricine prior to reacting with Enliten or
Luminase and 50-fold with LuciferaseX.
Figure 19. For seawater samples extracted in P-BAC, the optimal dilution
factor after the extraction was 50-fold, prior to reacting with Enliten or
Luminase enzyme. For LuciferaseX, it was a 20-fold dilution factor.
36 ATP Preservation in P-BAC and UltraLyse Stored in -20°C
Ballast water samples from the UV-treatment tests were processed for ATP by
concentrating water samples onto GF/F 0.7 µm filters and extracting them in P-BAC and
UltraLyse for 60 minutes. The samples were measured for initial ATP concentration and
then stored at -20°C for seven days. At the end of the storage period, the samples were
thawed in room temperature water for 15-minutes and analyzed again for ATP. The
results indicated that P-BAC preserved ATP better than UltraLyse; P-BAC showed an
overall ATP preservation of 98% and UltraLyse showed 76% (Figure 20). This aspect of
P-BAC is useful when multiple samples require analysis, but at a later time. P-BAC has
Figure 20. P-BAC samples and UltraLyse (ULyse) extracted seawater samples were
extracted for ATP, analyzed, stored in -20C for 7 days, and re-analyzed. Samples extracted
in P-BAC preserved better than UltraLyse extracted samples.
37 the ability to preserve ATP directly in the extractant in -20°C for atleast 7 days.
P-BAC Validation on Algal Cultures and Environmental Samples
Algal cultures.
Algal cultures including a saltwater dinoflagellate (Prorocentrum sp.),
chlorophyte (Tetraselmis sp.), diatom (Thalassiosira sp.), and freshwater red alga
(Porphyridium sp.) were extracted by various ATP extraction methods including P-BAC,
LumiClean+UltraLyse and boiling Tris (Figure 21). Among the extractants tested, PBAC was most efficient at extracting ATP. For Prorocentrum, P-BAC extracted nearly
37% more ATP than LumiClean+UltraLyse and 61% more than Tris. For Tetraselmis, PBAC extracted 23% more ATP than LumiClean+UltraLyse and 58% more than Tris. For
Thalassiosira, P-BAC extracted 16% more ATP than LumiClean+UltraLyse and 26%
more than Tris. For Porphyridium, P-BAC extracted similarly to LumiClean+UltraLyse
and 60% more than Tris. This lack of proportionality between extractants indicates that
some algal species are more challenging to extract due to the cells’ physiological status
and perhaps the positioning of mitochondria in the cytoplasm. The overall conclusion is
that the P-BAC ATP assay extracts ATP optimally compared to LumiClean+UltraLyse
and Tris, in four different phyla of alga.
38 Figure 21. A comparison of ATP extractions on the Procorocentrum
sp.,Thalassiosira sp., Tetraselmis sp., and Porphyridium sp. by P-BAC,
LumiClean+UltraLyse, and boiling Tris.
Nearshore Monterey Bay.
Samples collected from the Monterey County area were extracted using the
boiling Tris, LumiClean+UltraLyse, and P-BAC method. A freshwater sample was
collected from Hudson’s Landing, brackish water from Kirby Park, and saltwater from
Lover’s Point, all within the same day. The samples were concentrated onto GF/F filters
and extracted for ATP. In all three water samples, the P-BAC_60min extraction was
optimal at extracting ATP compared to the other extractants. UltraLyse_60min
extractions were optimal for the freshwater sample from Hudson’s Landing, but less
39 optimal for the brackish water sample from Kirby Park and Lover’s point, extracting 23%
and 47% less ATP, respectively. Tris performed even worse at 10% of the relative ATP
extracted for Hudson’s Landing, 28% for brackish, and 29% for Lover’s Point (Figure
22). Though more samples with similar salinities need to be evaluated for ATP by these
extractants, these initial results indicate that the traditional Tris boiling method and
UltraLyse have various extraction difficulties in different water types. P-BAC, in
contrast, seems to be optimal at extracting ATP from a range of environmental samples.
Figure 22. A comparison of ATP extractions on a freshwater, brackish
water, and seawater sample using P-BAC, LumiClean+UltraLyse, and
boiling Tris resulted in P-BAC optimally extracting ATP from all three
samples.
Offshore Monterey Bay.
During the March 2014 Biological Oceanography class cruise on the R/V Point
Sur, multiple water samples were collected in the water column, from the sea surface to
40 1000 m using a CTD rosette. These samples were obtained using a CTD rosette equipped
with Niskin Bottles. Surface to 1000 m-depth seawater was collected and processed for
ATP using boiling Tris and P-BAC 60-minute extractions. The slope from the linear
regression of Tris vs. P-BAC was 2.7 (R2=0.99) (Figure 23). Results from the
comparison between traditional boiling Tris vs. UltraLyse_30min and P-BAC_60min
extractions suggest Tris is ineffectively extracting ATP from offshore environmental
samples, thus necessitating a revision of current estimates of cellular ATP content.
Figure 23. Offshore Monterey Bay surface to 1000 m depth samples, collected in
March 2014, were processed for ATP using Tris and P-BAC (60 minute)
extractions. The two datasets plotted against each other reveals UltraLyse as
extracting overall 2.7-fold more ATP compared to Tris .
Trans-Pacific crossing: San Francisco, CA to Busan, South Korea, Manila,
Philippines, and Saipan, Northern Mariana Islands.
ATP was also underestimated using the boiling Tris extraction method, when
compared to P-BAC, for samples opportunistically collected on the California Maritime
41 Academy T/S Golden Bear, during its transit from in San Francisco Bay, CA (late April
2014), across the Pacific, to Busan, South Korea (mid May 2014), Manila, Philippines
(late May 2014), and Saipan, Northern Mariana Islands (early June 2014). San Francisco
Bay to Busan samples were taken by pumping seawater from the hull intake (at 5 meters
below sea surface) up to the main deck at the same level of the analytical lab. Busan to
Figure 24. Surface samples were collected during the California Maritime Academy
2014 summer cruise. High biomass peaks can be observed at the beginning and end of
the transect. Furthermore, P-BAC was the optimal ATP compared to Tris.
Manila and Saipan surface waters were sampled by deploying a weighted surface bucket
several times while the ship was underway, thus filling an 8 L carboy which was mixed
and used for all subsequent assays. The samples were processed onto GF/Fs and extracted
using P-BAC and Tris (Figure 24, 25, 26). From Vallejo to Busan, the P-BAC to Tris
ATP ratio was 4.0 (R2=0.94), Busan to Manila was 2.2 (R2=0.85) and Manila to Saipan
was 3.4 (R2=0.94). Again, P-BAC was optimal at extracting ATP when compared to the
conventional boiling Tris method in various marine environments.
42 Figure 25. When the Tris and P-BAC ATP data from the SF Bay to Busan transect
were plotted against each other, the graph revealed a slope of 4.0, indicating P-BAC
was extracting on average 4-fold more ATP than Tris.
Figure 26. When the Tris and P-BAC ATP data from the Busan to Manila transect were
plotted against each other, the graph revealed a slope of 2.2, indicating P-BAC was
extracting on average 2.2-fold more ATP than Tris in these samples.
Moss Landing Harbor sediment.
Moss Landing Harbor sediment samples were added directly to extraction
reagents UltraLyse and P-BAC for 60 minutes. At the end of extraction, UltraLyse
43 samples were vortexed and centrifuged for 5 minutes at 10,000 rpm. All UltraLyse
extracted samples had to be passed through a 0.45 µm filter because many small particles
were still suspended in the supernatant after being centrifuged. P-BAC did not require
centrifugation or pre-filtration prior to analysis since the sample was diluted 100-fold by
tricine—a promising positive effect of the required large dilution factor. The difference
between P-BAC and UltraLyse ATP was a substantial 3.4-fold (Figure 27).
In an independent trial, P-BAC and UltraLyse extractions were accompanied by
bead-beating in an attempt to increase extraction efficiency. Moss Landing Harbor
sediment was mixed directly into P-BAC and UltraLyse by vortexing for 10 seconds.
The samples were then set aside for a 30-minute extraction. At the end of 30 minutes, the
UltraLyse samples were vortexed. UltraLyse samples were then centrifuged for 5
minutes, subsampled (100 µL), diluted 10-fold by UltraLute, and passed through a 0.45
µm filter prior to ATP analysis. The P-BAC samples were vortexed, subsampled (10
µL), and diluted 100-fold using tricine. 200 µg of 20 µm beads and 200 µg of 100µm
beads were then added to each of the original extraction tubes. The tubes were then
mechanically extracted for 3 minutes using a bead-beater, centrifuged, subsampled and
diluted prior to the second round of ATP analysis. The results indicated that beadbeating, using 20 µm and 100 µm beads were not successful in increasing extraction
efficiency for P-BAC nor UltraLyse. The signal readings between P-BAC and UltraLyse
showed P-BAC was 2.9-times higher in ATP concentration, slightly lower than the first
trial (Figure 28). Overall, P-BAC is more preferable for marine sediment ATP
extractions compared to UltraLyse because it optimally extracts ATP. Furthermore, P-
44 BAC’s dilution procedure aids in the removal of optical interference by suspended
sediment particulates, which makes it a much simpler protocol relative to UltraLyse.
Figure 27. Moss Landing Harbor surface sediment was processed for ATP by P-BAC
and UltraLyse (60 minute extractions). P-BAC resulted in 3.4-fold more ATP
compared to UltraLyse.
Figure 28. Moss Landing Harbor surface sediment was processed for ATP by P-BAC
and UltraLyse with and without beads. Again, P-BAC extracted significantly more
ATP (a 3-fold difference) compared to UltraLyse. No significant difference was
observed in sediment extracted with beads in both extractants.
45 Ballast Water Treated by Ultra-violet (UV) Irradiation
A commercial UV ballast water management system (BWMS) was tested on San
Francisco (SF) Bay water, aboard the T/S Golden Bear. Ten µm nylon filters from
Millipore were used for concentrating organisms so a comparison can be made to
numeric counts of the viable 10 – 50 µm organisms by flow cytometry. Figure 29 depicts
ATP concentrations of a control and UV-treated ballast water sample processed by three
ATP extraction methods: 1) P-BAC_60min, 2) UltraLyse_60min, and 3) the boiling Tris
method. All extraction methods measured a 100-fold reduction in ATP between the
control and treatment samples. P-BAC extracted 1.2-fold more ATP than UltraLyse and
2-fold more than Tris in the control for this particular sample. In treatment samples, PBAC and UltraLyse extracted similarly and 2-fold more than Tris. Seven experiments,
similar to the experiment depicted in Figure 29, were performed using the same UVtreatment system. The results are plotted in Figure 30. The slope of the linear regression
between UltraLyse to P-BAC ATP was 1.04 suggesting the ATP extraction efficiency for
organisms >10 µm were very similar between the two methods. The slope between PBAC and Tris ATP was 1.9, indicating a 2-fold greater extraction efficiency in P-BAC
compared to Tris (Figure 31). Overall, all three extraction methods were able to detect a
significant reduction in >10µm organismal biomass after the water was treated with UV.
The difference, however, was that P-BAC was optimal at extracting ATP compared to the
traditional boiling Tris method and comparable UltraLyse, a 2nd generation commercial
extractant.
46 Figure 29. Control and UV-treated water was processed for ATP using P-BAC_60min,
UltraLyse_60min, and the boiling Tris method. All extraction methods measured a
reduction in ATP of 100-fold from the control to treated discharge. In this experiment,
P-BAC extracted 1.2-fold more ATP than UltraLyse and 2-fold more than Tris in the
control. In treatment samples, P-BAC and UltraLyse extracted similarly and 2-fold
more than Tris.
( Figure 30. When the ATP data from P-BAC and UltraLyse extractions were plotted
against each other, a slope of near to one suggests a similar ATP extraction efficiency
between the two extractants.
47 ( Figure 31. When the ATP data from P-BAC and Tris extractions were plotted against
each other, a slope of two suggests ATP was underestimated by 2-fold using Tris
extractions.
48 Discussion
Ballast operators and inspectors require a rapid, reliable, and simple viability
indicator to monitor treated ballast water for regulatory compliance; the ATP assay,
utilizing P-BAC as the ATP extractant, is the ideal method for this purpose as other
extractants do not consistently extract ATP, nor are they efficient for persons without a
scientific background. The advantages of using P-BAC include 1) minimal ATP
degradation during storage, 2) optimal removal of cellular ATP from organisms in
aquatic and sedimentary environments, 3) a single-step extraction operation and 4) rapid
results. Furthermore, the P-BAC ATP assay may be used to improve estimates of living
biomass in oceanographic research and its applications are limitless.
Algal Cultures and Environmental Samples Concentrated onto 0.7 µm GF/Fs
P-BAC has been shown to optimally extract ATP in four morphologically
different algal species (Prorocentrum sp., Thalassiosira sp., Porphyridinium sp., and
Tetraselmis sp.), reared in single-species cultures (Figures 23). And though only a few
cultures were assessed in this thesis project, the lack of extraction ratio-consistency
indicates that some organisms are more challenging for some extractants, whereas PBAC seems to be consistently optimal.
Another set of samples P-BAC optimally extracted include environmental
samples such as freshwater, brackish, and multiple seawater samples concentrated onto
GF/Fs (0.7 µm). In freshwater, the P-BAC to UltraLyse ATP ratio was 1. In the brackish
sample, the ratio was 1.3 (n=1), and in the saltwater sample from Lover’s Point, the ratio
was 1.9 (n=1). The mean ratio of three independent Moss Landing Harbor collections
49 was 2.2 (n=3); this suggests the organisms in various environments pose a range of ATP
extracting difficulties for UltraLyse .
Similarly, P-BAC to Tris ratios were also inconsistent among different samples
concentrated onto GF/Fs. The ATP ratio between the two extractants observed in
Hudson’s Landing (freshwater) was nearly an order of magnitude at 9.1 (n=1), for Kirby
Park (brackish water) it was 3.6 (n=1), and for Lover’s Point (seawater) it was 3.5 (n=1).
P-BAC to Tris ATP ratio for offshore Monterey Bay samples (which included surface
seawater to 1000 m in depth) was 2.7 (n=14). The mean ratio observed from the
opportunistic sample collection off the T/S Golden Bear, during the California Maritime
Academy summer 2014 cruise, was 3.2 (n=73). These results indicate Tris may be
consistently underestimating ATP content by nearly 3-fold in aquatic samples
concentrated onto GF/Fs. Therefore, a reassessment of the standard oceanographic
methods for the measurement of ATP in oceanographic research should be pursued in
particular of microbial size fraction using highly efficient extraction reagents such as PBAC.
Samples Concentrated onto 10 µm Nylon Filters and Sediment Samples
For samples concentrated onto 10 µm filters, the P-BAC and UltraLyse ATP
extraction efficiency was comparable. On average, the ratio of ATP extracted between PBAC and UltraLyse for untreated ballast water from Carquinez Strait was 1.04 (n=7).
This suggests P-BAC and UltraLyse to be similar in their ATP extracting capabilities for
>10µm organisms in the Carquinez Strait. Though UltraLyse may be capable of
optimally extracting ATP from the larger microorganisms, it is important to note that
50 UltraLyse is limited to the >10µm size category as it does not optimally extract samples
concentrated onto GF/Fs, nor is its performance on sediment comparable to P-BAC, a
3.4-fold (n=2) difference.
The results from this thesis project suggest that the P-BAC ATP assay is an
optimal ATP extraction method for various aquatic environmental samples including
fresh, brackish, and seawater communities. Furthermore, it is optimal for microbial ATP
extractions in marine surface sediment where a highly diverse community of
microorganisms co-exist (i.e.: bacteria, autotrophic and heterotrophic protists). However,
the tradeoff for efficient ATP extraction in multiple different environments exists in the
reduced sensitivity of the assay due to the light-quenching effects of the extractant;
nonetheless, the P-BAC ATP assay provides reliable ATP measurements even for
samples from low productivity zones (i.e.: seawater from 1000 m in depth, oligotrophic
areas, UV-treated ballast water). Additionally, excessive sample volumes were not
necessary to acquire reliable luminescence measurements two or more orders of
magnitude above the blank; sample volumes that were used for P-BAC ATP extractions
were comparable to acetone-based chlorophyll a extractions. The increased sensitivity
and lower limits of detection of the P-BAC ATP assay will also enable studies associated
with ATP dynamics (production, decay) in a range of environments.
51 Conclusion
ATP is a reliable proxy of living biomass because it is ubiquitous in all living
cells, its strong correlation with cell carbon, and rapid hydrolysis upon cell death. For the
ATP assay to be reliable at measuring intracellular activity, it must be optimally extracted
from cells and be stabilized following extraction. Following these assumptions, the ATP
assay has been exploited by oceanographers to understand trophic level interactions,
physiology and bioenergetics of planktonic and sedimentary communities. It has also
been routinely applied in quality control of drinking water, compliance assessment of
waste-water sterilization, the evaluation of acute and chronic toxic doses of antibiotics in
microorganisms, the rate of increase of microbial biofouling, and much more. Currently,
this bioassay is being groomed for monitoring ballast water discharge compliance.
Changes in ATP levels following a treatment can represent the effectiveness of the
BWMS at meeting discharge standards. However, conventional ATP extraction
procedures can underestimate ATP and be inconvenient for routine assessment of ballast
water by a ship’s crew or ballast water inspectors. As a result, the P-BAC ATP assay
was developed to abate the short-comings of conventional, 1st generation, and even 2nd
generation ATP extraction methods. This assay was created for more reliable, rapid, and
convenient ATP measurements, geared towards persons with or without scientific
backgrounds. Though P-BAC’s initial purpose was to investigate living biomass in
aquatic environments, it would be interesting to apply this assay to the study of
extremophiles in deep-sea sediment or explore potential life on Mars or Europa.
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55 Appendix A: ATP Extractants and Tested Conditions
56