Northeast Fisheries Science Center Reference Document 05-17
Modification of chemically-defined medium ASP12
for picoplankter Aureococcus
anophagefferens,
with limited comparison of physiological requirements
of New York and New Jersey isolates
by John B. Mahoney
National Marine Fisheries Serv., 74
Magruder Rd., Highlands, NJ 07732
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publication date November 2005;
web version posted December 19, 2005
Citation: Mahoney JB. 2005. Modification of chemically-defined medium ASP12 for picoplankter
Aureococcus anophagefferens, with limited comparison of physiological requirements of New
York and New Jersey isolates. US Dep Commer, Northeast Fish Sci Cent Ref Doc. 05-17; 38
p.
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ABSTRACT: Two enriched sea water media used for initial culturing of the picoplankter Aureococcus
anophagefferens in this laboratory had varied and moderate at
best suitability. Utility of both apparently could be
affected greatly by difference in the quality of the sea water they
were prepared with. One was especially so affected because,
found subsequently, it lacked a supplement essential for the picoplankter. Occasional
precipitation made it additionally problematic. To enhance
vigor and long-term survival of A. anophagefferens in batch
culture and support experimental studies, a proven chemically-defined
medium, ASP12, was modified for the species. The
addition of selenium to original recipe ASP12 permitted
good growth of A. anophagefferens. All ASP12 constituents
except vitamins were then tested to determine levels most beneficial
for A. anophagefferens. The vitamins required by the
species, but not minimal concentrations necessary for optimal
growth, were determined. Additional nutrients found beneficial
for the species by other researchers, and some constituents of other
media found to have nutritional benefit for various phytoplankton
species, were evaluated and adopted if beneficial. Medium chelation,
medium pH, and effects of some plant hormones on growth of the species
were assessed. This process ultimately provided a very suitable
defined medium for A. anophagefferens.
Most testing of medium constituents and physical conditions affecting
growth was done with an isolate from Great
South Bay, New York. Equivalent testing of
some of the constituents and physical factors with an A. anophagefferens strain
isolated from Barnegat Bay, New
Jersey, permitted a limited comparison. The two strains appear physiologically
similar in many respects but have differences in tolerances to some of
the constituents. Salinity and temperature tolerances determined
for the New York strain in this study are in general
agreement with those previously reported for the strain by other researchers;
comparison of tolerances of New York and New
Jersey strains to these factors indicates they are basically similar.
KEY WORDS: Aureococcus anophagefferens culture, harmful algal blooms, New
York Bight.
INTRODUCTION
Beginning in 1985 and continuing for over a decade blooms of a toxic
picoplankter, Aureococcus anophagefferens, so-called “brown
tides,” recurred in New York Bight waters, specifically in eastern
Long Island, New York, coastal embayments (Nuzzi and Waters, 1989, 2004),
and in the New Jersey Barnegat Bay-Little Egg Harbor system and some
contiguous or adjacent bays (Olsen and Mahoney, 2001; Mahoney et al.,
2003b). The blooms caused actual or probable serious perturbation
to various components of the biota in both epicenters. Deleterious
effects of Long Island blooms were well substantiated; their detriment
to, e.g., bay scallop (Argopecten irradians) was especially severe
(e.g., Bricelj and Lonsdale, 1997). This paper describes modification
of a
classic chemically-defined phytoplankton culture medium for A. anophagefferens to
support experimental studies, maintain high culture vigor, and enhance
its long-term batch culture survival.
Enriched sea water (NW) phytoplankton media, especially when prepared
with water from the collection site, can be most suitable for isolation
and at least initial culturing (Guillard, 1995). The first isolation
of the picoplankter Aureococcus anophagefferens (Cosper, 1987),
a relatively difficult species to culture, employed ‘f/2’ NW
medium (Guillard and Ryther, 1962). Initial culturing of Cosper’s A.
anophagefferens BT-1 isolate in this laboratory likewise employed ‘f/2’ medium,
prepared with sea water from Middle Atlantic Bight locales, including
various eastern Long Island bays where the species bloomed, and various
shelf locales off the northeast U.S. Utility of ‘f/2’ in
this laboratory for BT-1 varied batch-to-batch and was moderate at best. This
appeared linked to where the sea water was collected, with the most successful
batches prepared with Middle Atlantic Bight offshore water; e.g., from
the shelf break in the vicinity of the Hudson Canyon, or from Georges
Bank in the Gulf of Maine. Sea water from the latter area was the
most consistently favorable; it is well known that a sea water batch
may be favorable or unfavorable for a phytoplankton species depending
on presence or absence of essential nutrients or inhibitory substances. Occasional
precipitation made ‘f/2’ medium additionally problematic. The
two problems experienced with this medium were recognized decades ago
to be common defects of NW media (Provasoli et al., 1957). A.
anophagefferens was cultured next in this laboratory in ‘K’ NW
medium (Keller et al., 1987). ‘K’ medium, developed
primarily for culture of oceanic ultraplankton, is a modification of
medium ‘f’ (Guillard, 1975). The recipe changes are:
a ten-fold increase in chelator concentration; an organic instead of
inorganic phosphorus source; lowering of copper concentration; and additions
of selenium, ammonium chloride, and a pH buffer. The authors reported
this medium to not be prone to precipitation, which they believed was
probably due to high chelator concentration. Growth in ‘K’ medium
generally was better than in ‘f/2’ medium but again medium
suitability varied batch-to-batch, from good to unacceptably low. Attempts
at this laboratory to make the culture axenic at this time failed, apparently
due to high antibiotic intolerance. Marginal health of the culture
contributing to the intolerance was suspected.
Provasoli et al. (1957) advocated artificial sea water (AW) media for
study of phytoplankton nutritional requirements, and avoidance of natural
sea water media limitations due to the complex and variable composition
of sea water. Provasoli and colleagues (e.g. Provasoli et al.,
1957; Provasoli and McLaughlin, 1963) developed reproducible, non-precipitating
AW media designed to be utilized by a wide variety of marine algae. They
expected none would be best for particular phytoplankton species. A.
anophagefferens growth in various of these media in this laboratory
was nil or varied, so clearly none tried was suitable, much less best,
in its original formulation. One of these, ASP12 (Provasoli,
1964), was selected for modification for A. anophagefferens because
of its known excellence for a variety of marine algae. For example,
besides phytoplankton, Provasoli employed ASP12 for multicellular
marine algae. ASP12 NTA (ASP12 with nitrilotriacetic
acid as additional chelator) was one of the three most suitable AW media
of 20 tested for red seaweed species (Provasoli, 1964). Iwasaki
(1967) employed slightly modified ASP12 NTA for culture of
the Conchocelis phase of the seaweed Porphyra tenera.
Optimization/enhancement of ASP12 for batch culture of A.
anophagefferens was the primary goal of the study. Comparison
of physiology of Long Island and New Jersey A. anophagefferens strains
became a secondary focus. Testing of various nutrients with New
York and New Jersey strains supported limited comparison of their relative
efficacy and tolerance. Comparison of tolerances of the New York
and New Jersey strains to salinity and temperature was of particular
interest because of similar or different bloom regulatory importance
of these factors in the respective bloom epicenters. The comparison
is not conclusive but serves at least to direct attention
to the question of applicability of findings from research on Long
Island A. anophagefferens blooms
to New Jersey outbreaks.
METHODS
Initial A. anophagefferens study at this laboratory, especially
trials of various media, employed the BT-1 culture, isolated in 1986
by E. Cosper from Great South
Bay, New York, as received from Cosper. A.
anophagefferens cultures obtained from the Provasoli-Guillard Center
for Culture of Marine Phytoplankton (CCMP) were employed subsequently. Most
ASP12 optimization/enhancement testing was done with strain
CCMP 1784 (= BT-1). CCMP 1794, isolated by R. Andersen from Barnegat
Bay, New Jersey, in 1997, and CCMP 1984, an axenic isolate derived from
CCMP 1784 in 2000 by M. Berg, were secondary test cultures. CCMP
1784 and CCMP 1794 are non-axenic. Strain designation is used guardedly
in this report. Stabile
et al. (2000) found CCMP 1784 and CCMP 1794 isolates to be genetically
different. However, Stabile (personal communication) considered it not
possible to extrapolate the level of genetic difference to physiological
difference. How representative test cultures are of dominant strains
in blooms in New York Bight coastal waters for over a decade is
uncertain. CCMP 1784 and CCMP 1794 are genetically different from
most isolates and bloom samples studied by Stabile et al. (2000) with
some exceptions: CCMP 1784 was not statistically genetically different
from a West Neck Bay, New York,
bloom sample, and CCMP 1794 was not statistically genetically different
from a Great South Bay, New York, bloom sample.
Batch culture methodology used in this study primarily followed that
developed by Provasoli, McLaughlin, and Hutner at Haskins Laboratories,
New York (e.g., Hutner et al., 1950; Hutner and Provasoli, 1951; Provasoli
et al., 1957; McLaughlin, 1958); Droop, Marine Station, Millport, Scotland
(e.g., Droop, 1958a; Provasoli et al., 1957); and Guillard, Bigelow Laboratory
for Ocean Sciences, West Boothbay Harbor, Maine (e.g., Guillard, 1975;
Guillard and Keller, 1984; Guillard and Morton, 2003). Glassware
was cleaned by a soak for 24 hours or longer in 1% Micro detergent; rinsing
followed by machine wash (final rinse, deionized water); a soak with
10% HCl for at least 12 hours; and a second machine wash. It was
then silanized (Schenck, 1984) with Prosil-28 or Glassclad-18, rinsed,
and again machine washed. Glassware silanization was repeated as
needed, usually after two or three uses; need for this was judged by
the amount of beading of water on glassware walls. Culture vessel
caps were washed similarly but were not silanized. Plastic pipette
tips were cleaned by separate ~24-hour soaks in Micro detergent and 10%
HCl, each followed by deionized water rinse.
For reagent solutions and media preparation, laboratory centrally-supplied
~17 megohm resistivity water which had passed through ion exchange and
activated carbon columns, was further purified by passage through a Barnstead
B-Pure system with activated carbon and ion exchange columns. It
was then filtered through a 0.2 µM polycarbonate filter, microwave-sterilized
in 2-liter Teflon® bottles (Keller et al., 1988), and refrigerated
until use. The filtration and microwaving were done primarily to
avoid microbial contamination of the preparation water and is not expected
to be necessary for medium prepared for routine culturing. The
recipe for ASP12 was
that of Provasoli (1964). Medium constituent chemicals were reagent-grade
unless ultra-pure. Ultra-pure chemicals, including sodium
chloride, magnesium chloride, vanadium, silver, and nickel, were used
in some tests. Reagent
solutions rarely were retained to depletion. They were used up
to 3-4 months for culture maintenance medium; solutions were remade for
test media unless batch preparations were close. Other than phosphorus
and silicon stock solutions which were in borosilicate bottles, reagent
stock solutions were in Teflon® or
polycarbonate bottles; all were refrigerator-stored. Provasoli
reported ASP12 constituents as percent concentrations. Percent
concentrations were retained for some of the tests, but molar amounts
were used for most. All constituent levels are reported as molar concentrations. Culture
vessels for A. anophagefferens maintenance and medium enhancement
tests were mostly 25 x 150 mm borosilicate screw cap test tubes with
polypropylene caps. Media were in 20 ml amounts in the test tubes. Four
replicates were used in most tests.
Medium sterilization regime varied. Medium for culture maintenance
and some tests (e.g., major salts) was autoclave-sterilized. Droop,
in Provasoli et al. (1957) recommended “light autoclaving” (i.e.,
15 PSI for 1 minute to reduce the possibility of medium precipitation
and avoid having to lower pH to prevent precipitation. He found
this satisfactory for media in test tubes. The mild autoclaving
regime for the present study was ~3 PSI, 105°C for 10 minutes in
an AMSCO (STERIS) Model 3031 autoclave “pure steam” stainless
steel system, with the steam generator supplied ~17 megohm resistivity
deionized water. For many tests (e.g., for metals and vitamins)
the basic growth medium was autoclave-sterilized, and test solutions
for post-autoclaving aseptic addition were microwave-sterilized (Keller
et al., 1988). Nutrient, etc., solutions to be microwave-sterilized
were prepared in 400 ml amounts in 500 ml Teflon® bottles; pH of
test solutions was adjusted to ~8 prior to microwaving. During
microwaving, solutions were cooled to ≥25°C between
the four timed sessions. For certain tests (e.g., metals screening),
basic medium and test solutions were microwave-sterilized separately. Sterilization
regimes for particular tests are identified in the text.
Following autoclave-sterilization, media were stored at room temperature
for ~12 hours and then refrigerator-stored. After microwave-sterilization
of media or test solutions for aseptic additions, they were also refrigerator-stored. Aseptic
dispensing of media and test solutions and culture inoculations were
done by Eppendorf or Oxford
pipettor in a Nuaire biological safety cabinet, using cotton-plugged
pipette tips that had been pressure cooker-sterilized in vials. Inoculations
were made at least 24 hours after test media preparation, as recommended
by Morel et al. (1979). To ensure vigor of the inoculum, stock
cultures were transferred regularly, using as inoculum the culture having
the greatest cell abundance in the current log phase transfer batch. Special
preparation of inocula for certain tests is outlined in the respective
sections. Experiment test tubes were randomly inoculated and distributed
randomly while spaced uniformly, 20 tubes in 40 place racks for incubation. The
term “serial transfer” in this report refers to transfer
of culture grown in a specific test medium into the same medium.
Culture incubation temperature was routinely 18°C. Light bank
illumination from below through a pane of glass (wire reinforced 1/4
inch, pebbled upper surface) provided -50 µE-2 m-2 sec-1 (measured
with a Li-Cor Model 185 quantum radiometer/photometer) from cool white
fluorescent lamps. Lighting was computer-programmed for a 12/12
hour light/dark cycle, with dimming control to adjust light intensity
and simulate 15-minute dawn and dusk periods. The light/dark cycle
was routine for this laboratory’s primary light banks and not optimized
for A. anophagefferens.
Cell abundance or yield of A. anophagefferens maintenance and
test batch cultures was assessed indirectly by measurement, primarily
at 2-3 day intervals, of in vivo fluorescence (Brand et al., 1981),
using a Turner Designs Model 10 fluorometer. Fluorescence measurements
are adequate to estimate cell populations of A. anophagefferens cultures
(Dzurica et al., 1989). Fluorometer readings were taken 4-5 hours after
start of light period. Each test culture was moderately vortex-mixed
just before being measured. A single mixing pulse of 3-4 seconds
sufficed for CCMP 1784 and CCMP 1794. Additional mixing of CCMP
1984 (e.g., three pulses of 5-6 seconds) was often necessary to disperse
cells aggregated in a floc at tube bottom; cell aggregation was most
common in older cultures with high cell abundance. Floc formation
was not seen in non-axenic cultures.
When growing vigorously, non-axenic CCMP 1784 and CCMP 1794 cultures
characteristically reached maximum cell abundance after ~2 weeks of incubation,
had a stationary phase of about a week’s duration, and then declined;
the decline generally was gradual. Axenic CCMP 1984 achieved comparable
cell abundance during similar incubation, and generally survived weeks
longer than the non-axenic cultures. Prior to disposal, all A.
anophagefferens cultures were microwave-sterilized.
To assess the CCMP 1784 culture microbial contamination, standard agar
plate streaking technique was used at this laboratory to isolate contaminants. Minitek
Bacterial Differentiation and supplementary tests performed on contaminant
isolates at the NEFSC Milford Laboratory (Kapareiko, internal report)
indicated a complex of seven bacterial and one yeast species. The
origin of this contaminant burden and whether it changed over time are
unknown. CCMP did not attempt to purify the culture after it was
deposited by E. Cosper (Andersen, personal communication). Microbial
contaminant diversity of CCMP 1794 was not assessed.
The alga/contaminant relationship in the non-axenic A. anophagefferens cultures
is only partly known. Provasoli (1964) suggested that associated
bacteria might benefit an alga in culture by supplying growth factors. Bacteria
growth enhancement of alga growth, as well as bacteria toxicity to alga,
have been reported (e.g., Berland et al., 1970; Berland et al., 1972). A
certainty is that microbial contaminants in the CCMP 1784 and 1794 cultures
eventually seriously stress A. anophagefferens, i.e., following
long-term incubation (e.g., ~30 days). Apparently, the picoplankter
can compete with the contaminants while it is growing vigorously, but
eventually it is overwhelmed. Similar process was described by
Soli (1963), who found that three diatom species in bacterized unialgal
cultures grew abundantly after transfer into fresh medium, but when diatom
growth ceased bacteria multiplied rapidly and overwhelmed the diatoms. Incubation
of non-axenic A. anophagefferens cultures at 10°C prolonged
survival. Comparison of culture growth at 10°C with that in
parallel culture at 18°C suggested this most likely was due primarily
to bacterial growth reduction rather than a slowing of A. anophagefferens growth. Because
of apparent microbial contaminant influence on culture survival at least,
some of the results obtained with non-axenic A. anophagefferens test
isolates may best be considered presumptive. Provasoli et al. (1957)
warned that assays of bacterized cultures cannot provide precise interpretation,
particularly when organic substances are in question, but also regarding
mineral and trace constituents. Only some of the tests could be
repeated when the axenic isolate CCMP 1984, of parent culture CCMP 1784,
became available.
Interpretation of test results was made on qualitative comparison of
growth response, prolongation of stationary phase, and culture long-term
survival. Except for temperature tests,
it is assumed that, in addition to or alternative to increased cell abundance,
prolongation of culture survival was related to medium composition. Inhibition
of microbial contaminants in some cultures by certain enrichments rather
than stimulation of A. anophagefferens growth is an alternative
possibility. Correlation of fluorescence measurements to culture
cell abundances with population microscope enumerations could only be
done occasionally. Therefore, extrapolation to cell numbers and
determination of growth rates are not possible. Fluorescence measurements
likely less accurately reflect cell populations in tests of varied physical
conditions, e.g., light and temperature (Brand et al., 1981), and when
cultures become nutrient-limited. This could be addressed only
by conservative interpretation of test results. Methodology details
for tests are mentioned in the particular sections for those who may
wish to repeat tests to obtain quantitative data. Format for tables
in this report follows that of Guillard and Keller (1984), with molar
concentrations in written-out exponential form.
Results with axenic CCMP 1984 were generally more reliable than those
obtained with the non-axenic isolates, e.g., through greater consistency
among test replicates. Although apparently not as serious a complication
as bacterial contamination, some results with the axenic isolate also
may be problematic due to cell aggregation as the culture aged. Assessed
by the unaided eye with the culture vessel held to a light source, CCMP
1984 could form cell floc even when the culture had daily vortex-mixing. Cell
aggregation can cause microscope enumeration of A. anophagefferens to
be highly inaccurate (Mahoney et al., 2003a), and important effect of
cell clumping on fluorometer measurements is a possibility. Mixing
prior to fluorometer readings was always at least largely effective in
dispersing the floc, but sometimes refractory small clumps visible to
the unaided eye remained, and considerable cell aggregation could be
present even when cell floc was not apparent to the unaided eye. For
example, as seen by the unaided eye, vortex mixing appeared to fully
disperse cells of a two-month-old CCMP 1984 culture that had aggregated
in a floc at the bottom of the culture tube. Microscope examination
of the culture at 400X magnification detected occasional clumps of ~30
to 300 cells, although most cells were solitary. Effect, if any,
of greater vortexing of CCMP 1984 on culture cell abundance was undetermined;
it did not reduce culture long-term survival. Suggesting cell fragility, A.
anophagefferens has
an exocellular polysaccharide-like layer but lacks a cell wall (Sieburth
et al., 1988). Also suggesting fragility, Anderson et al. (1989),
when developing an A. anophagefferens enumeration protocol, found
considerable disruption of cells unless they were suitably fixed. Apparent
normal increase of cell abundance of CCMP 1984, as determined by fluorometer
assessments through culture incubation, suggests relatively vigorous
vortex mixing at least did not cause major disruption of cells. Perhaps
the exocellular polysaccharide-like layer supports structural integrity
of live cells. It is assumed that fluorometer assessments provided
sufficient accuracy despite the complications. Electronic counting
was not attempted. Precluding this with the non-axenic cultures
was closeness in size of the picoplankter and culture microbial contaminants. Cell
aggregation precluded accurate electronic counting of the axenic culture.
Optimization of ASP12 medium for A. anophagefferens entailed
empirical assessment of requirement and optimum concentration of
27 original medium constituents including macro- and micro-nutrients,
vitamins, chelator, and pH buffer (except for buffer tests, the medium
never was prepared with more than half original recipe buffer concentration). Provasoli
et al. (1957) referred to this process as ‘tailoring’ a medium
for a species. Some alternative or additional nutrients and growth
factors, and non-nutritional medium constituents including metals, vitamins,
plant hormones, pH buffers, and chelators, also were evaluated. Medium
modification was generally cumulative, with indicated change from one
test most often incorporated into the basal medium for the next. Modifications
were adopted conservatively; e.g., the lowest concentration of a constituent
that would support a given growth improvement was selected even when
the species would tolerate higher concentrations. Growth inhibition
obtained with a constituent concentration, even if just slight or temporary,
caused a non-inhibitory concentration to be selected. Notwithstanding
incremental changes, intermediate versions of the medium are designated
ASP12. Except for chelator tests, simultaneous study
of multiple medium constituents, such as was done with major inorganic
ions by Droop (1958a), was not done. The final recipe is designated
ASP12A (Tables 1 and 2). Abbreviations NW and AW for
natural seawater media and artificial seawater media, respectively, were
adopted from Harrison et al. (1980).
RESULTS
AND DISCUSSION
ASP12 BASIC DESIGN CHARACTER
One of the concerns of Provasoli and coworkers in development of AW
media (e.g., Provasoli et al., 1954; Provasoli et al., 1957) was avoidance
of precipitation of the medium and associated sequestering of various
nutrients. For this practical consideration they often lowered
the general salinity and reduced concentrations of calcium, magnesium,
and phosphorus. ASP12, developed primarily for oceanic
dinoflagellates, is an exception to this. Many of the concentrations
of ASP12 major constituents are close to reported averages
in sea water (Goldberg, 1963; Bruland, 1983). These average concentrations
accompany test results; the values listed by Goldberg (1963), converted
to molar, are those available around the time ASP12 was developed. Because
ASP12 medium was intended for batch culture, some concentrations
naturally are greatly in excess of those in sea water.
SELENIUM: PRIMARY ASP12 MODIFICATION FOR A. anophagefferens
Early trials of various NW and AW media for A. anophagefferens in
this laboratory showed that if transferred from ‘K’ medium
to ASP12, it would grow reasonably well when the inoculum
was in culture maintenance amounts (0.5 ml or 0.75 ml of culture for
20 ml of medium), but not if the inoculum was reduced to test volume
(0.2 ml). Culture transfer from ‘f/2’ medium or ASP12 to
ASP12 had little success even with the larger inoculum. Nutrient
carry-over from ‘K’ medium to ASP12 seemed a likely
explanation. Comparison of constituents
revealed provision of selenium in ‘K’ medium but not in ‘f/2’ medium. This
was suspected to be the major nutritional difference between the media. Growth
of some phytoplankton is stimulated by selenium (e.g., Pintner and Provasoli,
1968), and it is essential for others (e.g., Price et al., 1987). Dzurica
et al. (1989) concluded that selenium is not a major requirement for A.
anophagefferens, although the element enhanced its growth. Addition
of selenium in ‘K’ medium level, 10-8 M, to ASP12 (unmodified
except for pH buffer reduction) resulted in its basic suitability for A.
anophagefferens. Suitability of a proven medium is to be expected
unless the species has a nutritional requirement not satisfied by the
medium. Selenium obviously is an indispensable enrichment for A.
anophagefferens growth in ASP12. Subsequently, Cosper
et al. (1993) reported that selenium is critical for A. anophagefferens growth.
Microwave-sterilized selenium, aseptically added to autoclave-sterilized
ASP12, was tested in concentrations of 1 to 5 x 10-8 M,
with 10-8 M increments. The full range was comparably
suitable for CCMP 1784 and its axenic isolate CCMP 1984. CCMP 1794
apparently has a slightly greater selenium requirement than the New
York strain, and is slightly less tolerant to the highest concentration. When
general cell abundance of CCMP 1794 was maximum in the test, yield supported
by the lowest and highest selenium levels was approximately 20% lower
than with intermediate levels. Selenium addition of 2 x 10-8 M
is adopted for ASP12A. It is expected that adding selenium
to ‘f/2’ medium would very likely improve its utility
for A. anophagefferens.
ASP12 MAJOR CONSTITUENTS, ORIGINAL, NEWLY ADOPTED OR CONSIDERED
Sodium chloride
Sum of major salts shows ASP12 has an open-ocean salinity
of 34.4 PSU. Results of salinity tolerance tests in ASP12 (discussed
below) indicated its salinity could be reduced 2 to 6 PSU. Avoidance
of possible medium precipitation through lowering NaCl concentration
was the primary concern. The long documented contamination of reagent
grade chemicals with impurities, e.g., certain metals (Pintner and Provasoli,
1963; Morel et al., 1979), was additional incentive. NaCl reduction
from 4.79 x l0-1 M to 4.28 x l0-1 M had little
if any effect on A. anophagefferens growth, and the lower concentration
is adopted for ASP12A. In one instance, however, when A.
anophagefferens CCMP 1784 was incubated in a batch of ASP12 having
either the original or reduced NaCl level, the original concentration
apparently was slightly more beneficial for long-term culture survival. Substitution
of AR grade NaCl and MgCl2 · 6H2O with
Baker Ultra Pure chemicals resulted in no discernible change in growth,
suggesting that any element stimulatory or essential to A. anophagefferens not
supplied as a medium constituent is introduced in sufficient amounts
in other salts, etc. The original NaCl concentration could be retained
if medium precipitation is not a problem.
Magnesium
In sea water, magnesium exhibits a nearly constant ratio to salinity,
and is present in an average concentration of 53.2 mM (Bruland, 1983);
Goldberg (1963) reported the average concentration to be 55.53 mM. The
ASP12 original recipe provides 48 mM magnesium, added as MgSO4 · 7H2O
and MgCl2 · 6H2O. Magnesium optimum
concentration for A. anophagefferens CCMP 1784 was tested, using
MgCl2 · 6H2O, in the range 25 to 70 mM,
with 5 mM concentration increments. Na2SO4 · 10H2O
was substituted as sulfur source. McLachlan (1964) used a similar
procedure for testing effects of magnesium on growth of a variety of
phytoplankton species. McLachlan (1973), because of extreme deliquescence
of MgCl2 · 6H2O, recommended titration
of a solution of the compound to determine concentration. Instead,
this compound and Na2SO4 · 10H2O,
which is also deliquescent, were dried at 70°C for ~1 hour before
weighing for medium preparation. Medium and test magnesium additions
were autoclave-sterilized together. Despite the test inoculum being
acclimated to 48 mM, magnesium concentrations higher than 45 mM were
slightly inhibitory through week 1 of incubation; the inhibition was
absent by day 11 and beyond. When general cell abundance was at
or close to maximum at day 11 of incubation, yield was highest with 40
mM magnesium; yield with 30, 35, and 45 mM was slightly lower and comparable,
and slightly further decreased with 25 mM. Cell abundances, moderately
below maximum, were comparable with all Mg test concentrations and had
little or no decline during week 3 of incubation. A. anophagefferens can
flourish with a wide range of magnesium concentrations, but based on
slightly greater day 11 cell abundance with 40 mM, and the early incubation
partial inhibition with concentrations 45 mM and higher, 40 mM is adopted
for ASP12A. In a preliminary test in which the same
concentrations of magnesium as above were obtained by varying concentrations
of both MgSO4 · 7H2O and MgCl2 · 6H2O
while maintaining their ratio, and with sulfur concentration allowed
to vary, the more favorable magnesium concentration range was 35 to 50
mM, with 40 mM slightly superior; week one growth inhibition was absent
below 60 mM.
Potassium
ASP12 potassium recipe concentration is 9.38 mM, which is
close to its reported average concentration in ocean water, 10.2 mM (Bruland,
1983) and 9.72 mM (Goldberg, 1963). Potassium concentration best
for A. anophagefferens CCMP 1784 was tested in the range 5 to
15 mM, with concentration increments of 1 mM. Medium and test potassium
additions were autoclave-sterilized together. Potassium antagonist
TRIS buffer (Provasoli et al., 1957), as usual, had half original recipe
concentration in the tests. No major yield differences throughout
incubation showed that a wide range of potassium concentrations are suitable. Slightly
greater yield at 11 to 13 days of incubation when the test general maximum
cell abundances were attained, greater uniformity of cell abundance among
test culture replicates, plus slightly decreased culture decline through
day 17 when the test was terminated, however, suggested 11 mM to be most
beneficial. This level is adopted for ASP12A. In a separate
test for minimum potassium required, levels in the 2.55 to 7.67 mM range
resulted in reduced long-term culture survival.
In culture, A. anophagefferens cells are in suspension during
growth phase, but eventually most sink to the bottom of the culture vessel. Hayward
(1970) suggested that loss of monovalent ions from the cells, particularly
potassium, may be a contributory factor to similar sedimentation of the
diatom Phaeodactylum tricornutum. Replicate CCMP
1784, CCMP 1794, and CCMP 1984 cultures were grown for 2 weeks, then
half of them were supplemented with 7.3 mM potassium, and all were further
incubated. Based on fluorometer measurements taken pre- and post-vortex
mixing, periodic through incubation, no difference in cell sedimentation
was apparent between potassium-supplemented and unsupplemented cultures.
Potassium concentration in the medium apparently is not a determinant
of A. anophagefferens sedimentation.
Sulfur
Bruland (1983) reported the average concentration of sulfur in sea water
to be 28.2 mM; the Goldberg (1963) average is just slightly lower, 27.6
mM. Added as MgSO4 · 7H2O, the
ASP12 recipe sulfur concentration is 28.397 mM. Droop
(1958a) advised that SO4 concentration can be reduced if the
nutritional need of the organism is satisfied. Sulfur requirement
of A. anophagefferens CCMP 1784 was tested in the range 0 to 40
mM, with 5 mM concentration increments. The source compound was
Na2SO4 · H2O, dried at 70°C
for ~1 hour before weighing for medium preparation. The medium
and Na2SO4 · H2O additions
were autoclaved together. The test inoculum was grown in full-strength
ASP12. With no sulfur supplementation growth was severely
retarded throughout test incubation. Maximum cell abundances maintained
in days 11 through 15 of incubation were comparable with sulfur in the
range 5 to 20 mM; 10 to 20 mM concentrations provided slightly superior
long term (through 28 days) culture survival. Levels of 20, 25,
and 30 mM resulted in slight inhibition during week 1; levels of 35
and 40 mM caused slight growth inhibition throughout incubation and shortened
long-term culture survival. Based on these results, sulfur concentration
is lowered to10 mM for ASP12A. Sulfur concentration
might more conveniently be adjusted (only one chemical would have to
be dried) by adding MgSO4 · 7H2O at
10 mM and lowering MgCl2 · 6H2O to 30
mM, but this was not tried.
Calcium
The reported average calcium concentration in ocean water is 10.3 mM
(Bruland, 1983) and 9.98 mM (Goldberg, 1963). The ASP12 recipe
concentration matches the latter value. Optimum calcium concentration
for A. anophagefferens CCMP 1784 was tested in ASP12,
prepared with original recipe chelator and metals concentrations, in
a 2.49 to 14.96 mM range with increments of 2.49 mM. Medium and
test calcium additions were autoclave-sterilized together. Levels
of 2.49 and 4.98 mM were inadequate, permitting only slight initial growth
which was followed shortly by culture collapse; 7.47 mM supported moderate
culture growth and longer culture survival. The original recipe
level, 9.98 mM, basically was suitable. However, slightly higher
cell abundance through most of the incubation term (day 4 to day 22)
and slightly improved long-term culture survival was obtained with 12.47
to 14.96 mM. Calcium concentration is increased to 12.47 mM for
ASP12A. The
concentration could be lowered to original level without major detriment
to A. anophagefferens growth if, e. g., medium precipitation is
encountered or less chelation is necessary.
Fluoride
Fluoride, a major ion in sea water, with reported average sea water
concentration of 68 µM (Bruland, 1983) or 68.5 µM (Goldberg,
1963), is not a constituent of ASP12 or a normal constituent
of other Provasoli media. It is a component of various media developed
by other researchers including: Aquil, 7.14 x 10-5 M, (Morel
et al., 1979); EASW, 6.57 x 10-5 M, (Harrison et al., 1980);
and ‘AK’, 10-5 M, (Keller et al., 1987). The
latter authors reported that fluoride was beneficial for culture of oceanic
ultraplankton species. Aquil AW medium, with varied iron and chelator
and with the addition of selenium, has been used to culture A. anophagefferens (Cosper
et al., 1993), indicating the species at least could tolerate fluoride
at Aquil recipe level. Effect on A. anophagefferens growth
was tested with fluoride at 1.052, 2.63, and 5.26 x 10-5 M,
and 1.05 x 10-4 M. Microwave-sterilized fluoride solutions
were added aseptically to autoclave-sterilized medium. Fluoride
at all test levels was beneficial throughout incubation; it increased
maximum yield and prolonged culture survival of A. anophagefferens isolates
CCMP 1784, 1794, and 1984. Growth with fluoride at 5.26 x 10-5 M
was slightly superior to that with the lower concentrations, and comparable
to that with the highest concentration; 5.26 x 10-5 M is adopted
for ASP12A. A possible secondary benefit of fluoride
supplementation is reduction of cell floc formation in older cultures
of axenic CCMP 1984. Unaided-eye observation suggested cell floc
to be less prevalent in fluoride-supplemented cultures, and when present
it appeared more amenable to disaggregation by vortex mixing.
Carbon
Inorganic carbon average concentration in sea water is 2.3 mM (Bruland,
1983). Other than the organic carbon portion of Na2 · glycero · PO4,
carbon is not a recipe nutrient constituent of ASP12 and reliance
for carbon in the medium is on diffusion of carbon dioxide into it. McLachlan
(1964) reported that addition of 2 mM of sodium bicarbonate to various
AW and NW media, including ASP (Provasoli et al., 1957), enhanced growth
of most phytoplankton species tested; concentrations greater than 2 mM
were not additionally beneficial and were inhibitory to one test species,
the diatom Skeletonema costatum. Bicarbonate effect on growth
of A. anophagefferens CCMP 1784 was tested in the range 1 to 6
mM, with 1 mM increments. Microwave-sterilized bicarbonate was
aseptically added to autoclave-sterilized medium. Results indicated
that under the culture conditions, bicarbonate supplementation was basically
unnecessary for maximum cell abundance and long-term culture survival,
but it did enhance growth moderately in ~week 1 of incubation. NaHCO3 concentrations
of 1 to 2 mM were approximately equally beneficial for A. anophagefferens. Similar
to the findings of McLachlan (1964) for S. costatum, the higher
concentrations were progressively inhibitory. NaHCO3 in
concentration of 1 or 2 mM is suggested as an optional supplement
for culture of A. anophagefferens in ASP12A.
ASP12 MINOR CONSTITUENTS, ORIGINAL OR CONSIDERED
Silicon
Silicon concentration in sea water ranges <1 to 180 µM, with
an average concentration of 100 µM (Bruland, 1983); Goldberg’s
(1963) reported average concentration is close to this (106.5 µM). ASP12 recipe
includes 527.8 µM silicon. Provasoli’s inclusion of
silicon in ASP12, which is required by diatoms, silicoflagellates,
and some chrysomonads (although the medium primarily was developed for
oceanic dinoflagellates) reflects its intended general utility. A.
anophagefferens CCMP 1784 requirement for silicon was tested first
in silanized borosilicate test tubes, in medium without silicon addition,
and with silicon additions in the range 25 to 200 µM, with 25 µM
increments. Microwave-sterilized
silicon solutions were added aseptically to autoclave-sterilized medium. Growth
over a 20-day incubation term was similar throughout the test. Following
this, serial transfers were made into the same test regime. A.
anophagefferens growth during a 33-day incubation again was similar
over the silicon supplementation range, but cell abundance of cultures
without silicon supplementation was slightly lower, particularly in early
incubation. Serial transfer from the second test was made into
the test regime with polycarbonate centrifuge tubes substituted for borosilicate
test tubes. Cell abundance of silicon-unsupplemented cultures was
greatly reduced; silicon in as low as 25 µM concentration
(the lowest tested)
adequately supported A. anophagefferens growth. These
results indicate that A. anophagefferens requires silicon. Other
researchers (LaRoche et al., 1997) concluded that it does not require
this element. Silicon
in 25 µM concentration is included as an ASP12A constituent,
despite the apparent likelihood that the borosilicate culture vessels
routinely used would contribute sufficient silicon for cell abundance
only slightly lower than that in silicon-supplemented medium.
Nitrogen
Nitrogen concentration in sea water ranges 1 to 45 µM, with an
average concentration of 30 µM (Bruland, 1983); Goldberg (1963)
reported a moderately higher average concentration, 38.5 µM. ASP12 recipe
NaNO3 level is 1.176 mM, approximately 39 times the Bruland
average. Test NaNO3 concentrations were 1.17, 2.35,
and 5.88 x 10-4 M, and concentrations in 2.94 x 10-4 M
increments from 8.82 to 23.52 x 10-4 M. Medium and test
nitrogen additions were autoclaved together. Test inoculum was
prepared by growing A. anophagefferens CCMP 1784 in ASP12 having
one tenth the recipe level of NaNO3; 200 ml of this depletion
medium in a 500 ml Erlenmeyer flask was inoculated with a 20 ml culture;
after five days 10 ml of this culture was used to inoculate a second
such flask which after incubation served as inoculum. Growth was
absent without nitrogen supplementation, indicating the depletion was
adequate and atmospheric ammonium diffusion into the medium was not sufficient
for growth. 1.17 x 10-4 M supported moderate cell abundance
through days 9 to 11 of incubation; the cultures then declined with apparent
bleaching of cells. Cultures having 2.35 to 23.52 x 10-4 M
supplements all reached their respective cell abundance maxima by day
11. Cell abundance supported then by 2.35 x 10-4 M was
below general maximum but considerable; within two days these cultures
evidenced pronounced decline with apparent bleaching of cells. NaNO3 concentrations
of 5.88 and 8.88 x 10-4 M supported cell abundances by day
11 nearly comparable or comparable, respectively, with those associated
with higher nitrogen levels; these cultures then began a gradual decline. Cell
abundance maxima supported by NaNO3 in 1.17 to 2.35 mM enrichments
supported comparable yield maxima which were sustained 2 to 5 days longer
than with lower concentrations. The ASP12 medium NaNO3 recipe
level is suitable for A. anophagefferens, but 1.47 mM prolonged
maximum yield ~3 days longer and slightly improved long term culture
survival. Higher concentrations were not additionally beneficial. NaNO3 concentration
is increased to 1.47 mM for ASP12A medium.
NH4Cl is not a constituent of ASP12 medium. It
is included in ‘K’ medium (Keller et al., 1987), which supports
growth of A. anophagefferens, in recommended maximum concentration
of 5 x 10-5 M. It was tested as sole nitrogen supplementation
for A. anophagefferens CCMP 1784 in levels of 2, 3, 4, 5, 6, 8,
10, 15, and 20 x 10-5 M. The inoculum was prepared as
before. CCMP 1784 did not grow without nitrogen supplementation. Maximum
yield, at days 12 to 14, was greater with increasing NH4Cl
concentrations to 10 x 10-5 M; the latter concentration also
provided the best long-term culture survival. As to be expected,
yield was much lower than that supported by NaNO3 in higher
concentrations. Partial inhibition in week 1 of incubation occurred
with 8 and 10 x 10-5 M NH4Cl. Concentrations
of 15 and 20 x 10-5 M resulted in initial growth lag, followed
by low-level growth, then severe culture decline in less than two weeks. NH4Cl
in the test concentration range, as supplement to NaNO3 in
1.47 mM concentration, had no apparent effect on cell abundance with
2 to 6 x10-5 M concentrations, but again partial early growth
inhibition occurred with 8 and 10 x 10-5 M concentrations,
and severe general inhibition with the higher concentrations. Results
show utilization by A. anophagefferens of ammonium at low levels
but do not support its addition to ASP12A. Comparison
of NH4Cl
utilization by CCMP 1784 and CCMP 1794 revealed greater NH4Cl
toxicity to the latter. Tested with 10-5 M, 3 x 10-5 M,
and 5 x 10-5 M NH4Cl, CCMP 1794 grew with the two
lower levels but 5 x 10-5 M resulted in severe growth lag
and delayed maximum cell abundance for approximately a week.
Phosphorus
Phosphorus concentration in sea water ranges 1 to 3.5 µM, with an average
concentration of 2.3 µM (Bruland, 1983), and 2.26 µM. (Goldberg, 1963). ASP12 recipe
provides a total of 79 µM of phosphorus from combined additions of K3PO4 and
Na2 · glycero · PO4; the
medium has a nitrogen : phosphorus ratio of ~15 : 1. The two phosphorus
compounds initially were tested singly in concentrations (µM) of 1, 3,
7, 10, 12, 15, and in 5 µM increments from 20 to 60 µM. Medium
and test phosphorus additions were autoclaved together. CCMP 1784
test inoculum was prepared in ASP12 medium having one-tenth
original recipe levels of K3PO4 and Na2 · glycero · PO4:
4.7 µM and 3.2 µM, respectively.
Cultures unsupplemented with phosphorus had very slight growth and collapsed
in approximately one week. K3PO4 in 1 µM
concentration supported low-level growth and apparent culture survival
for approximately two weeks. Three µM supported moderate
cell abundance and culture viability through approximately three weeks,
although with pronounced decline in the third week. The ample growth
response with at least the next-to-lowest level indicates that autoclaving
the complete medium did not make phosphorus unavailable, or at least
not the majority of it. Five µM improved general growth and
yield, and moderately extended culture viability. Close to maximum
yield was obtained with 7 µM in two weeks, and culture viability
persisted through the full incubation term of 31 days. Concentrations >7 µM
provided respective peak yields two days earlier than the lower ones. Levels
of 10 and 12 µM, respectively, provided cell abundances slightly
lower than maximum or at maximum, respectively. Levels ≥15 µM
better maintained the culture long-term. No inhibition was noted
to 60 µM,
which is well below inorganic phosphorus concentrations (3.22 to 6.44
mM) which Provasoli et al. (1957) reported to be toxic. K3PO4 in
40 µM concentration, although levels as low as 15 µM basically
were suitable, is selected for ASP12A because this level supported
slightly better long-term culture survival.
Initially, the A. anophagefferens strains tested did not optimally
utilize inorganic phosphorus when previously grown in medium having recipe
level of the organic source. Grown in the latter then transferred
to inorganic phosphorus-only medium, CCMP 1784 had a slight growth lag
that persisted through week one of incubation. CCMP 1794 and especially
CCMP 1984 had more severe growth lags, persisting approximately two weeks. No
growth lag occurred when the isolates were transferred from medium having
only inorganic phosphorus, in which they had acclimated, to the same
medium.
At the1 µM level, Na2 · glycero · PO4 supported
slight growth of CCMP 1784 for approximately a week; culture collapse
followed rapidly. Concentrations ≥35 µM caused partial
inhibition during ~week one. Other than this, response was similar
to that with the inorganic compound. For approximately the same
amount of phosphorus, the inorganic and organic phosphorus sources also
were used equally well by A. anophagefferens CCMP 1794 and CCMP
1984. Because
the species does not require organic phosphorus, this constituent could
be eliminated from ASP12A medium. However, the organic
phosphorus compound is highly soluble, so besides increasing medium versatility,
inclusion of both inorganic and organic phosphorus sources serves to
provide ample phosphorus concentration while helping avoid precipitation. No
major difference in long-term culture survival was seen when bacterized
CCMP 1784 and CCMP 1794 strains were grown with either inorganic or organic
phosphorus, which suggests microbial contaminants did not benefit from
the organic carbon, or they benefited in such a way that the picoplankter
was not caused extra detriment. There is sufficient justification, therefore,
for retention of both K3PO4 and Na2 · glycero · PO4. As
with K3PO4, Na2 · glycero · PO4 in
15 µM concentration basically could be adequate but based on slightly
better long-term culture survival with higher levels, and the reason
noted below, 20 µM is selected for ASP12A.
With the compounds in equal molar combination, cell abundance supported
by ≥3 ≤60 µM phosphorus was similar to that with either compound
alone for the same amount of phosphorus. Partial inhibition occurred
with >60 µM during the first week of incubation, although CCMP 1784
tolerated concentrations to 120 µM. To lower phosphorus concentration
from 79 µM to 60 µM, the concentration chosen for ASP12A medium,
Na2 · glycero · PO4 is reduced
most because it was inhibitory in concentrations ≥35 µM, whereas
no inhibition by K3PO4 was found with all levels
tested. The results suggest that ASP12A phosphorus
concentration might be ~halved if desired without major detriment to
culture growth and survival.
Vitamins
Potential complications in determining phytoplankton vitamin requirements
include inability to obtain vitamin-free test media so that the vitamin
may appear only stimulatory rather than essential (Droop, 1958b), and
the possibility that bacteria in algal cultures may supply vitamins (e.g.,
Berland et al., 1970). Provasoli et al. (1957) advised that tests
of vitamins in bacterized phytoplankton cultures can be indicative but
may not be completely reliable. Because of this, primary reliance
for vitamin requirements was on tests with the axenic isolate.
ASP12 recipe includes vitamin B12, thiamine, and
biotin. Because the three A. anophagefferens isolates flourished
in ASP12 over many transfers it is assumed that its vitamin
requirement is satisfied by the recipe group under the conditions of
culture. Also, enrichment with a mixture of the three recipe vitamins
plus 13 others (Vitamins 8; Provasoli et al., 1957) did not increase
axenic CCMP 1984 cell abundance. Water for medium preparation for
vitamin testing being filtered through activated charcoal cartridges
presumably removed vitamins, if any were present. Filtration, microwave
sterilization, and refrigerator storage of medium preparation water in
a closed Teflon® container until used, eliminated or minimized microbial
contaminant presence or growth in it. Microwave-sterilized vitamin
additions were added aseptically to autoclave-sterilized medium.
Axenic CCMP 1984, grown in ASP12 having full vitamin supplementation
then inoculated into medium having only single vitamin additions, grew
comparably with all vitamin regimes during two weeks of incubation. Apparently,
stored reserve and/or carry-over with the inoculum (the latter introducing
amounts ≤ than in a 1 : 100 dilution of recipe vitamin levels)
sufficed. Comparably high cell abundance was prolonged post 2 weeks
of incubation by vitamin B12 and thiamine. Two serial
transfers of these CCMP 1984 cultures into medium with vitamin supplementation
limited to vitamin B12 showed that the isolate would not grow
without thiamine. Transfer of CCMP 1984 inoculum grown with combined
vitamin B12 and thiamine supplementation to medium having
either thiamine or thiamine plus vitamin B12 supplementation
resulted in comparable cell abundance. Again, apparently stored
reserve and/or carry-over of vitamin B12 with the inoculum
sufficed. Transfer of thiamine-only supplemented culture from this
test to medium supplemented with thiamine or thiamine plus vitamin B12 resulted
in growth only with the combined vitamins. Therefore, CCMP 1984
requires vitamin B12 and thiamine. Similar results were
obtained for non-axenic CCMP 1784 and CCMP 1794. The similarity
of vitamin test results for the three isolates suggests that the bacterial
flora in the bacterized cultures is not supplying the required type
or level of vitamin. These vitamins are those most commonly required
(vitamin B12 more so than thiamine) by marine auxotrophic
algae (Provasoli et al., 1957; Provasoli, 1963). Minimum and optimum
vitamin B12 and thiamine concentrations required were not
determined. Doubling of thiamine concentration appears beneficial
but this was not confirmed. Utilization of vitamin B12 analogues
and thiamine moieties (Provasoli, 1964) was not determined.
Biotin elimination did not affect cell abundance. The three isolates
grew well through several incubations in medium having only vitamin B12 and
thiamine supplementation. Biotin replacement after the latter incubations
did not result in noticeable change in culture condition. Although
included in Table 1 as an ASP12A medium constituent, biotin
possibly could be eliminated for A. anophagefferens culture. It
is retained because it is not detrimental and its utility was not tested
with a range of culture conditions. The level of vitamin B12 used
by Iwasaki (1967) in his modification of ASP12, five times
the original recipe level, is adopted for ASP12A. This
is done as a precaution. This level of vitamin B12 and
higher are common in other Provasoli AW media (Provasoli et al., 1957). Concentrations
of the other two vitamins are original recipe levels. As done in
this laboratory, autoclaving of complete ASP12A for routine
culturing did not destroy or apparently diminish efficacy of the medium
vitamin complement.
Hormones
Bentley (1960) reported substances with biological activity similar
to auxins of higher plants, and Mowat, nee Bentley (1964) reported
gibberelin-like substances in marine phytoplankton. Plant hormones
including kinetin, indolacetic acid, and gibberellic acid increased growth
of the macroscopic alga, Ulva lactuca (Provasoli, 1958). These
hormones also increased growth of the Conchocelis phase of seaweed Porphyra
tenera (Iwasaki, 1965). Reviews of the question of whether
plant hormones control development of macroalgae concluded either that
they do (Bradley, 1991), or that available data are equivocal (Evans
and Trewavas, 1991). Effect of 10-10,
10-9, 10-8, and 10-7 M concentrations
of kinetin, indolacetic acid, and gibberellic acid on growth of A. anophagefferens CCMP
1784 was tested. Microwave-sterilized hormone solutions were added
aseptically to autoclave-sterilized medium. No A. anophagefferens growth
inhibition or enhancement resulting from these hormone supplements was
apparent.
ASP12 ORIGINAL
METALS CONSTITUENTS
ASP12 includes two metal mixes: PII, consisting
of a chelator, ethylenedinitrilotetraacetic acid (EDTA), and iron, boron,
manganese, zinc, and cobalt; and SII, consisting of bromine, strontium,
rubidium, lithium, molybdenum, and iodine (Table
2). Possibly because it
was developed for oceanic dinoflagellates, ASP12 has 1.0%
addition of PII metal mix, rather than 3.0% more commonly used in various
other Provasoli AW media. PI metal mix at 3.0% was best for most
species, but some species required additional (Provasoli et al., 1957). (The
difference between PI and PII mixes is elimination of copper in the latter). This
suggested that greater than recipe amount might be beneficial for A.
anophagefferens, although judging from long-term culturing
of A. anophagefferens in ASP12, PII and SII metals
mixes in recipe level were basically adequate.
The possible benefit for A. anophagefferens from increased metals
levels was approached through testing of individual PII and SII metals
rather than the whole mix. It was assumed that this would avoid
metals excess and a possible chelation problem. Potential complications
in tests of metals requirements include metal contaminants in reagent
grade chemicals used in media preparation, and nutrient carry-over with
test inocula (e.g., Morel et al., 1979). Pintner and Provasoli
(1963) advised that reliable results from tests of trace metal requirements
can only be had by virtual elimination of them. To at least partly
achieve this, potential growth limitation by individual PII and SII metals
was tested by a depletion assay in which A. anophagefferens CCMP
1784 was grown in ASP12 lacking in-turn supplementation with
one, followed by post-incubation serial transfers twice into the same
medium, each transfer being 1 : 100 dilutions of the culture volume. Water
for ASP12 medium preparation was purified by passage through
ion exchange columns, but metals removal from the complete medium in
this manner (such as used in Aquil medium preparation; Morel et al.,
1979), was not done. The ASP12 chelator : trace metals
ratio, normally 2.7 : 1, was not balanced in the assays. The most
extreme balance change was from manganese omission which resulted in
a ratio of ~10 : 1. Medium and test metals were microwave-sterilized
separately and added aseptically to autoclave-sterilized culture tubes. During
autoclaving, the tubes contained deionized water, which was emptied just
before medium dispensing. Test inocula were grown in ASP12 prepared
with normal enrichment levels. Reduction of concentrations of metals
by the depletion procedure, but likely not elimination of them, is not
considered a major complication because the goal was enhancement of culture
vigor and long-term survival rather than determination of absolute nutritional
requirements.
Assay results suggested all PII metals could be in greater concentrations. The
most important potential growth limiter, evident in the initial screening,
was cobalt. Slight growth reduction due to deficiency of iron and
manganese was apparent in the first serial transfer. Boron and
zinc limitation was not apparent until the second serial transfer. Strontium
was the only SII metals mix constituent identified as relatively deficient,
although not until the second serial transfer. ASP12 bromine
recipe concentration of 1.25 x 10-4 M is considerably less
than its sea water concentration, 8.4 x 10-4 M (Bruland, 1983)
or 8.13 x 10-4 M (Goldberg, 1963), but the depletion assay
indicated additional is not needed. Underestimation of trace metal
limitation by the assays is a possibility because simultaneous limitation
by more than one metal can be more severe than limitation by one metal
alone (Murphy and Guillard, unpublished data; Sunda, unpublished data;
both cited in Brand et al., 1983).
Tests of optimum concentrations of the individual PII and SII metals
identified as potentially growth limiting were made next. The original
recipe ASP12 chelator concentration was not adjusted when
these metals were tested in concentrations higher than original; change
of chelator : metals ratio to 1.7 : 1 was the most extreme, but this
is only slightly below the lower limit of the range 2 to 3 : 1, which
Provasoli et al. (1957) found most useful. Microwave-sterilized
metals solutions were added aseptically to autoclave-sterilized medium; A.
anophagefferens CCMP 1784 inoculations were made ~24 hours later. Some
tests were repeated for isolates CCMP 1794 and CCMP 1984. Concentrations
of sea water metals constituents in Goldberg (1963), converted to molar,
show values available at the time ASP12 was developed, although
the medium metal mixes were developed some years earlier (Provasoli et
al., 1957) and were partly based on even earlier studies by other researchers. Some
of the Goldberg (1963) values are considerably different than those provided
two decades later by Bruland (1983). Bruland stated that post-1975,
more accurate values of sea water constituents were developed, including
concentrations of many trace elements found to be lower by factors of
10 to 1000 than those previously accepted. He considered the primary
reasons for this to be major advances in instrumental analysis and analytical
chemistry, and elimination or control of contamination during sampling,
storage, and analysis.
Average concentration of cobalt in sea water is 0.02 nM (Bruland, 1983). Goldberg
(1963) reported a much higher level, 8.48 nM. Cobalt concentration
in ASP12 is 1.7 x 10-7 M. Cobalt was tested
in concentrations of 1.7, 1.95, 2.2, 2.7, and 3.2 x 10-7 M. Based
on increased cell abundance and uniformity of growth of CCMP 1784 among
eight test replicates, 2.2 x 10-7 M was the most beneficial
level and is selected for ASP12A. The two higher levels
resulted in partial growth inhibition in early incubation. Other
testing suggested that cobalt likely is the limiting metal constituent
for the species in ‘K’ medium also.
Average concentration of iron in sea water is 1.0 nM (Bruland, 1983);
Goldberg (1963) reported this as 1.79 nM. ASP12 recipe
iron level is 1.79 x 10-6 M. Tested in multiples of
recipe level, two- or threefold increases stimulated higher cell abundances
and greater long-term survival of CCMP 1784 and axenic CCMP 1794. Doubling
of iron concentration was beneficial for CCMP 1984; the three-fold level
was slightly inhibitory although it prolonged culture survival. Iron
in 3.58 x 10-6 M concentration is selected for ASP12A
medium. The recipe amount in PII metal mix is unchanged, and the
additional iron is supplied separately as FeEDTA, primarily to avoid
precipitation in the metals mix.
Boron is a major constituent of sea water, having an average concentration
of 0.416 mM (Bruland, 1983), comparable to 0.426 mM reported by Goldberg
(1963). ASP12 recipe boron level is 1.85 x 10-4 M. Tested
in concentrations of 1.85, 2.77, 3.7, and 4.62 x 10-4 M,
the three higher boron levels increased cell abundance and long-term
survival of CCMP 1784, CCMP 1794, and axenic CCMP 1984 to about the same
degree. Because there was no apparent additional benefit of the
two higher levels, boron concentration of 2.77 x 10-4 M is
selected for ASP12A.
Average manganese concentration in sea water is 0.5 nM (Bruland, 1983). Goldberg
(1963) reported a much greater level, 36.4 nM. ASP12 recipe
manganese level is 7.28 µM. Tested at recipe level, and with 9.1
and 12.75 µM, the higher levels promoted roughly comparable increase
of cell abundance and lengthened culture survival of CCMP 1784, CCMP
1794, and CCMP 1984. Manganese concentration is increased to 9.1
µM for ASP12A.
Average zinc concentration in sea water is 6 nM (Bruland, 1983); Goldberg
(1963) reported a much greater average concentration, 1.53 x 10-7 M. Zinc
concentration in ASP12 is 7.65 x 10-7 M. Tested
in concentrations of 0.76, 0.92, 1.07, and 1.22 µM, CCMP 1784 long-term
culture survival was enhanced greatly with Zn at the two higher levels. Zn
concentration of 1.07 µM is selected for ASP12A.
Strontium is a major constituent of sea water, having an average concentration
of 90 µM (Bruland, 1983), or 91 µM (Goldberg, 1963). Its ASP12 recipe
level is 22.83 µM, far less than average sea water concentration. Tested
in 22.8, 34.2, 45.6, 57, and 68.4 µM concentrations, the four higher
strontium concentrations comparably enhanced long-term survival of CCMP
1784. Strontium concentration of 34.2 µM is selected for ASP12A.
In routine culturing of A. anophagefferens in ASP12 medium
(original recipe except for selenium addition and reduced buffer), cobalt
apparently would be the only metal in PII metals mix likely to become
limiting. PII
metals mix modification might be restricted to this metal alone. Motivation
to increase levels of the other constituents primarily was to defer potential
limitation. However, their increased enrichment improved growth
and/or survival of isolate CCMP 1784. The SII metals that the
screening did not indicate to be required by A. anophagefferens in
the medium as prepared are not deleted. Their removal did not improve
growth in metals assays; the tests are limited in scope, and only medium
modifications that enhanced A. anophagefferens growth are considered
necessary.
ADDITIONAL METALS CONSTITUENTS
Arsenic and vanadium, found beneficial for A. anophagefferens (BT-1
= CCMP 1784) by Dzurica et al. (1989), and various metal constituents
of other phytoplankton media (Keating, 1985; Keating, personal communication;
Keller et al., 1987; Guillard, 1995), also were assessed for benefit
to A. anophagefferens in ASP12. Metals concentrations
used by Dzurica et al. (1989) or concentrations in various published
media were test starting levels. Microwave-sterilized metal test
solutions were added aseptically to autoclaved medium.
Average sea water concentration of arsenic is 23 nM (Bruland, 1983);
Goldberg (1963) reported this to be 40 nM. Tested in 14, 28, 42,
and 56 nM concentrations, 42 nM was most beneficial for A. anophagefferens CCMP
1784, especially with regard to yield uniformity and long-term culture
survival, and this concentration is adopted for ASP12A. Dzurica
et al. (1989) reported 10-8 M arsenic to be beneficial for
this strain.
Average sea water concentration of vanadium is 30 nM (Bruland, 1983);
Goldberg (1963) reported this to be 40 nM. Tested with A. anophagefferens CCMP
1784, vanadium at 10-8 M, the level used by Dzurica et al.
(1989), did not appear beneficial. Tested subsequently with axenic
CCMP 1984 in 1, 2, and 5 x 10-8 M concentrations, vanadium
enhanced long-term culture survival. The highest concentration
was slightly inhibitory; the intermediate concentration was more beneficial
than the lower level and is adopted for ASP12A.
Average sea water concentration of aluminum is 20 nM (Bruland, 1983);
Goldberg (1963) reported this to be 3.7 µM. Aluminum effect on
growth of A. anophagefferens CCMP 1784 was tested in concentrations
of 3.7, 7.4, 11.1, and 14.8 x 10-7 M. Aluminum
in 11.1 x 10-7 M concentration increased long-term culture
survival and is adopted for ASP12A. The lower concentrations
were not noticeably beneficial, and the highest concentration was slightly
inhibitory.
When preparing ASP12A, it was convenient to add solutions
of the additional metal constituents individually in 1.0 ml amounts for
a liter of medium. Combining them in a mix was not tried. Metals
also tested that had no apparent benefit for growth or survival of A.
anophagefferens CCMP 1784 in ASP12 as prepared, and under
the culture conditions, include copper, silver, nickel, cadmium, chromium,
lead, tin, tungsten, and barium. If A. anophagefferens requires
or uses any of this group or other metals that were not tested, this
need apparently was satisfied by contaminants in the reagents used in
medium preparation or metabolic substitution; introduction of metals
from the glassware or during autoclave sterilization are other possibilities
but considered less likely.
MEDIUM pH AND NON-NUTRITIONAL
CONSTITUENTS
pH
Provasoli (1964) recommended a pH range of 7.8 to 8.0 for ASP12. A.
anophagefferens CCMP 1784 was tested in pH-adjusted medium that
24 hours after autoclave sterilization had, respectively, pH values
of 8.0, 8.4, 8.5, 8.6, and 8.8. Levels 8.4 through 8.6 were more
favorable than the lowest; the highest pH was unfavorable. A
possible explanation for slightly better A. anophagefferens growth
with higher pH is greater CO2 absorption into the medium
(Provasoli et al., 1957) and somewhat greater EDTA chelation efficiency
(Chaberek et al., 1955). Medium precipitation with high pH is
a concern (Provasoli et al., 1957), so routinely ASP12A
pH was set at 8.4 before autoclaving, which provided ~8.2 after autoclaving. Microwave
sterilization resulted in less decrease. Overall, medium pH ranged
~8.2 to 8.3. The species flourishes in nature with pH in the
range recommended by Provasoli for ASP12. Water pH at various
sites in the New Jersey Barnegat Bay-Little Egg Harbor estuarine system
during a major A. anophagefferens bloom ranged 7.8 to 8.1 (unpublished
data).
pH Buffer
Provasoli et al. (1957) found TRIS (Tris (hydroxymethyl) aminomethane)
buffer was inhibitory in a concentration of 24.78 mM, but was not toxic
in 8.26 mM concentration for any of the phytoplankton species they tested. Subsequently,
Provasoli and McLaughlin (1963) reported TRIS concentration of 8.26
mM to be generally adequate, and that three Gyrodinium spp. tolerated
concentrations to 49.56 mM. The original recipe ASP12 TRIS
concentration is 8.26 mM. Decades ago tests at this laboratory
of Heterosigma
akashiwo (initially identified as Olisthodiscus luteus) TRIS
tolerance showed this concentration to be toxic to this species. Thereafter,
TRIS in 4.13 mM concentration was adopted. This
level has been satisfactory for maintaining medium pH; e.g., no medium
precipitation from autoclaving or large pH change during culture incubation,
and it apparently was not toxic to any species cultured in this laboratory. A.
anophagefferens CCMP 1784 tolerance to TRIS in ASP12 was
tested in multiples of the concentration in ‘K’ medium (10-3 M). In
the initial test series A. anophagefferens tolerated
TRIS well to 5 mM; 6 mM caused slight early growth inhibition, and
after cell abundance peaking, culture decline was greater than with lower
levels. The highest concentration, 10-2 M, resulted
in even greater early inhibition, a short period of low level of growth,
then pronounced culture decline. Serial transfers into the same
test range showed A. anophagefferens tolerance to 7 mM, suggesting
the species can acclimate to a higher level. Despite the species
tolerance of 5 mM without acclimation, a TRIS level of 4.13 mM is recommended
for ASP12A medium. Droop (1958b) and McLachlan (1964)
recommended glycylglycine and glycine buffering as a substitute for TRIS. McLachlan
(1973) cautioned that glycylglycine can be used only with axenic cultures.
This buffering was tried, nevertheless, with CCMP 1784 and CCMP 1794,
and the result was overgrowth by bacterial culture contaminants. It
was not assessed with the axenic culture.
Chelation
The primary chelator in ASP12 is EDTA, added as a component
of PII metals mix (Table 2). EDTA provision in ASP12 is
relatively moderate, being one third the level in various other Provasoli
media. Additional EDTA is not provided for trace metals in the
second metals mix in ASP12, SII, which except for lithium
are in ≤5 µM concentrations (Table 2). The ratio of chelator
to trace metals in PII mix is 2.7 : 1, within the range of 2 to 3 : 1
found most useful by Provasoli et al. (1957). The range of chelator
to metals ratios common in marine phytoplankton media is 0.8 to 2.7 :
1 (McLachlan, 1973).
Provasoli et al. (1957) did not favor EDTA : metals ratios greater than
3 : 1 because of the possibility of metal deficiencies and the active
binding of EDTA with calcium and magnesium at pH’s greater than
8.0. For increased chelation in ASP12 medium, Provasoli
employed nitrilotriacetic acid, NTA; the medium so modified is designated
ASP12NTA. Provasoli et al. (1957) described NTA
as a weaker metal chelator and McLachlan (1973) considered NTA less effective
than EDTA for most purposes. NTA is provided in 5.23 x 10-4 M
concentration in ASP12NTA, in contrast to EDTA in 2.7 x 10-5 M. In
ASP12NTA, therefore, the NTA : trace metals ratio, with the
molar total of trace metals in original PII mix used as basis for
calculation, is ~52 : 1. (The ratio of NTA to EDTA is
~19 : 1.) An advantage of NTA is that its calcium and magnesium
salts are highly soluble, which lessens the likelihood of precipitation
and concomitant loss of micronutrients from solution (Provasoli et al.,
1957). Also, McLachlan (1973) reported common use of NTA in marine
media to prevent precipitation of sodium glycerophosphate at elevated
temperatures.
Comparison of growth of A. anophagefferens CCMP 1784, CCMP 1794,
and CCMP 1984 in ASP12 and ASP12NTA, with original
recipe chelator and metals levels, showed that the latter was superior,
indicating ASP12 is underchelated for the species. A
10 : 1 EDTA : trace metals enrichments ratio in ‘K’ medium
favored culture of oceanic ultraplankton, but the medium is not recommended
for culture of coastal phytoplankton due to the possibility of metal
deficiencies (Keller et al., 1987). ‘K’ medium was
moderately suitable for A. anophagefferens in this laboratory.
Also,
a 10 : 1 chelator : trace metals ratio is used in chemically-defined
Aquil medium (Morel et al., 1979), which as modified by Cosper et al.
(1993) to include selenium, is likewise suitable for A. anophagefferens.
It is certain, therefore, that the species can grow in highly chelated
medium.
EDTA and NTA were tested singly and in combination, and in a range of
chelator : trace metal ratios: 1, 3, 7, and 10 : 1, with the trace metal
concentrations in unmodified PII metals mix used for the ratio calculations. Growth
of A. anophagefferens CCMP 1784 improved with increasing EDTA
: trace metals ratios from 1 : 1 to 7 : 1. The highest ratio tested,
10 : 1, resulted in growth retardation in week one of incubation but
subsequently was suitable. NTA in 3 : 1 chelator : trace metals
ratio was slightly superior to EDTA in the same ratio. In 7 : 1
ratio growth with EDTA and NTA was comparable through day 10 of incubation,
but subsequently culture growth and survival was superior with EDTA. Comparable
growth of CCMP 1784 was obtained in medium with EDTA : trace metals in
7 :1 ratio or with Provasoli’s
combination of EDTA : trace metals in 2.7 : 1 ratio plus 5.23 x 10-4 M
NTA. Similar results were obtained for CCMP 1794 and CCMP 1984.
EDTA as sole chelator in a ratio with PII trace metals of 7 : 1, or
EDTA in 2.7 : 1 ratio if coupled with NTA, is optional for ASP12A
medium (Table 2); the first regime is the one in current use in this
laboratory. In both regimes the concentration of EDTA is adjusted
for trace metals increases in PII metals mix, but not for trace metals
in SII metals mix (rubidium, lithium, iodine, and molybdenum), the additional
iron as Fe EDTA, or arsenic, aluminum, and vanadium enrichments (Table 2). With EDTA as sole chelator, compensation for the Fe EDTA addition
would require an EDTA increase from 8.53 x 10-5 M to 8.8 x
10-5 M to maintain a 7 : 1 chelator : metals ratio; utility
of this was not tested. Selenium and molybdenum are influenced
little if at all by chelation (Guillard, 1995).
The empirically determined chelation regimes for ASP12A are
suitable for A. anophagefferens but may not be optimal. Chelator
: metals ratios intermediate between 3 : 1 and 7 : 1, and between 7 :
1 and 10 : 1, were not tested. Suggesting an EDTA : trace metals
ratio higher than 7 : 1 might be favorable is that the 10 : 1 ratio (the
highest tested) was only temporarily growth-retarding. The EDTA
correction for trace metal increases may be superfluous for the alternative
combined EDTA and NTA chelation, given the large NTA supplementation. Provasoli’s
NTA level of 5.23 x10-4 M in ASP12NTA, unquestionably
arrived at through exhaustive testing, has proved beneficial in a variety
of media (Provasoli et al., 1957). The ratio tests suggest, however,
that the concentration likely could be lowered for A. anophagefferens. This
was not pursued. EDTA was superior to NTA in 7 : 1 chelator : metals
ratio, but this may not preclude NTA utility as sole chelator if its
level is appropriately adjusted. The effect of different pH’s
on chelation was not tested. The concentration of TRIS buffer in
ASP12A
medium, which also acts as a chelator although weaker than EDTA (e.g.,
Morel et al., 1979), has been halved, with undetermined effect on chelation. Greater
efficacy of citric acid than EDTA and NTA was found by Cosper et al.
(1993) when A. anophagefferens was transferred into medium with
selenium enrichment following two serial transfers in medium without
selenium. The selenium depletion likely introduced major stress
to the picoplankter, so the basis for chelator comparison is not ideal. Moreover,
use of citric acid is limited to axenic cultures.
To explore what effect chelation of specific ASP12 constituents
might have on A. anophagefferens growth, various chelators
suggested by S. Hutner (personal communication), including ethylene-bis(oxyethylene-nitrilo)tetraacetic
acid (EGTA) (which selectively complexes calcium); tripolyphosphate (complexes
iron, calcium, magnesium); sulfosalicylate and desferoxamine (complex
iron); and triethylenetetramine (complexes copper and nickel), were evaluated
as substitutes or supplements to EDTA and NTA. No chelator or combination
benefited growth of CCMP 1784 more than EDTA and NTA.
TEMPERATURE,
SALINITY AND LIGHT CONDITIONS AFFECTING GROWTH
Described in Chizmadia et al. (1984) and Hunchak-Kariouk et al. (1999),
the lagoonal Barnegat Bay-Little Egg Harbor estuarine system -- the New
Jersey epicenter for A. anophagefferens blooms -- extends ~70
km along the New Jersey coast. It ranges from ~2.0 to 6.5 km in width.
Depths are shallow, averaging 1.3 m in the northern half and ≥2.0
m in Little Egg Harbor. The eastern portion, averaging <1 m
in depth, is generally shallower than the middle and western portions,
which range to ~4.0 m deep. Tidal connections to the Atlantic Ocean are
through the Bay Head-Point Pleasant Canal and the Manasquan River at
the northern end; Barnegat Inlet at the center; and Little Egg Inlet
at the southern end (which also connects to the Great Bay-Mullica River
estuary). Tidal exchange is relatively restricted in the central and
northern portions and is greater in the southern portion. Primary exchange
of ocean and bay water in Barnegat Bay occursthrough the Barnegat Inlet.
Wind has a dominant role in circulation, which has a complex pattern.
Periods of complete vertical mixing occur, particularly when wind velocities
are high, but atendency toward two-layered circulation exists in areas
deeper than 1.5 m. Several major freshwater tributaries, the largest
of which is Toms River at northern Barnegat Bay, discharge into the system
along the northwestern perimeter, whereas only a few smaller creeks discharge
along the southwestern perimeter. Numerous storm drains contribute freshwater
runoff, either directly or through lagoons and tributaries. Freshwater
input from groundwater seepage is also considerable. The proxmity of
the ocean causes a moderation of summer and winter termperatures.July
and August are the warmest months, with a water temperature high of ~28°C,
and Januaryand February are the coldest, with a low of ~1°C. Precipitation
is well distributed. Extratropicalstorms, especially from the northeast,
may occur from September to March.
A. anophagefferens temperature and salinity tolerances are of
particular interest because of known or potential bloom regulatory importance
of these factors in the respective epicenters. Water temperature >25°C
truncated blooms of the species in various eastern Long Island embayments (Nuzzi and Waters, 2004), and truncated a major
bloom in the New Jersey Barnegat Bay-Little Egg Harbor system (personal
observation). Salinity is not considered a primary regulator of A.
anophagefferens blooms in the Long Island bays
because salinity of those waters rarely falls below levels favorable
for growth of the picoplankter (LaRoche et al., 1997). Based on
a two-year survey (unpublished data), salinity can have primary importance
in A. anophagefferens bloom regulation in the Barnegat Bay-Little
Egg Harbor system. This is particularly true in the northern half,
given its restricted tidal exchange and greater freshwater input. Temperature
and salinity tolerances of New York and New Jersey A. anophagefferens strains (CCMP
1784 and CCMP 1794) are compared to assess physiological differences.
Temperature Tolerances of New York and New
Jersey Isolates
Cosper et al. (1989) reported growth rates of Long
Island A. anophagefferens (presumably BT-1 = CCMP 1784) in a temperature
range of 5 to 25°C, tested with 5°C increments, to be greatest
at 20 and 25°C. Data in Figure 9 of Cosper et al. (1989) indicates
that growth rate at 25°C, although one of the highest, was reduced
from the optimum at 20°C, so the optimum temperatures may be ~20<25°C. In
this laboratory, CCMP 1784 and CCMP 1794, New York
and New Jersey isolates respectively,
had similar growth and maximum cell abundance at routine incubation temperature,
18°C. Estimating from the Cosper et al. Figure 9, temperature
decrease of 18°C to 10°C resulted in approximate halving
of BT-1 growth rate, declining from ~0.7 to ~0.3 doublings day-1. Strain
CCMP 1794 yield at 10°C in this laboratory was considerably
reduced below that with 18°C, requiring 16 to 18 days versus 10 to
13 days with the higher temperature to achieve a comparable level. Fluorescence
measurements may have lessened accuracy in indicating cell population
in this type of test (Brand et al., 1981); e.g., if cells incubated at
10°C had less pigment than cells grown at the higher temperature,
the apparent yield difference would shrink. CCMP 1784 and CCMP
1794 grew comparably well at 5°C in ASP12. Growth
with this temperature was consistent but slow, 8 to 10 weeks being required
to achieve yields comparable to those reached in ~2 weeks with 18°C. Data
in Figure 9 of Cosper et al. (1989) shows that BT-1 grew at ~0.1 doublings
day-1 with 5°C. Quantitative comparison of data
from the Cosper study and this one is not possible. However, accepting
that the BT-1 culture grew seven times faster at 18°C than at 5°C,
and considering difference in time to achieve comparable yield with these
temperatures in the present study having a factor of ~4 to 5, some similarity
of growth response over the temperature range is suggested. Neither
Cosper et al. (1989) nor the present study tested A. anophagefferens upper
temperature tolerance. From field observations (Nuzzi and Waters,
2004; Mahoney, unpublished data) this appears to be >25°C in
both epicenters, suggesting similar tolerance of multiple strains.
CCMP 1784 and CCMP 1794 Salinity Tolerances
A. anophagefferens was found between Portsmouth, New Hampshire and Chesapeake
Bay in the salinity range 18 to 32 PSU (Anderson et al., 1993). It
was found in a similar salinity range (18.5 to 34.5 PSU) in western New
York Bight coastal waters (Mahoney et al., 2003b). Salinity range
for optimal growth and bloom development apparently is considerably narrower,
however. Cosper et al. (1989) determined that maximal growth of A.
anophagefferens BT-1 (=CCMP 1784) was with salinity of 30 PSU. Major Long
Island blooms of the species were associated with salinities ≥27
PSU (Bricelj and Lonsdale, 1997). Salinity tolerances of strains
CCMP 1784 and CCMP 1794 in this laboratory were determined in ASP12 by
varying concentrations of Na, Mg, Ca, and K, while the ratio of these,
and the concentrations of other constituents, were maintained (after
Pintner and Provasoli, 1963). Test inocula were acclimated to 27
and 21 PSU, respectively, through five serial transfers over a two month
period, prior to parallel salinity tests in the range 17 to 38 PSU, with
salinity increments of 1.9 PSU. Salinity values other than whole
numbers are an artifact of test design. It is assumed that relatively
high increase of yield in the first week after inoculation reflects medium
compatibility with the state of A. anophagefferens physiology
achieved during inoculum incubation. Results for CCMP 1784 are
compared with those reported by Cosper et al. (1989) for BT-1 (= CCMP
1784); the latter study employed Instant Ocean Sea Salts medium with ‘f/2’ medium
nutrient enrichment. Cosper et al. (1989) reported adaptability
of A. anophagefferens BT-1 (=CCMP 1784) to relatively low and
high salinities; BT-1 tolerated from 22 to 35 PSU if given time to acclimate.
CCMP 1784, when high salinity-adapted (27 PSU), had greatest cell abundance
in the first week of test incubation in the salinity range 24.7 to 28.5
PSU. Low salinity intolerance following high salinity adaptation,
such as severe BT-1 growth inhibition below 28 PSU when adapted to 30
PSU (Cosper et al., 1989), was not found in the present study. The
difference in high salinity acclimation levels (27 versus 30 PSU) possibly
was a factor in this. Growth of CCMP 1784 lagged temporarily with
initially less suitable salinities; Cosper et al. (1989) noted similar
growth response. After two weeks, comparably high cell abundance
was evident at an expanded range, 22.8 to 34.2 PSU. With another
week of incubation, the salinity range for comparably high cell abundance
expanded to include 20.9 and 36.1 PSU, and included 38 PSU in week four. The
strain did not grow below 20.9 PSU. Growth to comparably high yield
in these tests was in a broader salinity range than the range for high
growth rates reported by Cosper et al. (1989).
When low salinity adapted (21 PSU), greatest cell abundance of CCMP
1784 in the first week of incubation was in the salinity range 20.9 to
28.5 PSU. Therefore, acclimated at the lower salinity, CCMP 1784
grew optimally without lag at a lower salinity than when acclimated at
the higher salinity. As occurred when high salinity adapted, growth
lagged temporarily with initially less suitable salinities. After
two weeks, comparable high cell abundance was obtained in the range 20.9
to 34.2 PSU, and this level was obtained with 36.1 PSU in week three. With
38 and 19 PSU, at least moderate growth was achieved after two or more
additional weeks of incubation; reduced uniformity of cell abundance
among the four replicates suggested culture stress, however.
High salinity adapted (27 PSU) Barnegat Bay isolate CCMP 1794 had greatest
cell abundance in the first week of incubation in the salinity range
26.6 to 32.3 PSU, a slightly higher range than the similarly adapted
New York strain. As with the New York strain, growth lagged temporarily
with initially less suitable salinities After two weeks, the salinity
range for comparably high cell abundance was expanded to 22.8 to 34.2
PSU, the same range for post two week incubation of high salinity adapted
CCMP 1784. Cell abundance of the New Jersey isolate comparable
to that with the above salinities was reached in weeks three and four,
respectively, with 36.1 and 38 PSU, respectively, the same response as
CCMP 1784 when tested similarly. Growth of CCMP 1794 was moderate
with 20.9 PSU, and still lower although considerable with 19 PSU. The New York strain, CCMP 1784, similarly adapted, would not grow with
salinity level at 19 PSU, even after weeks of incubation.
Low salinity acclimated (21 PSU) CCMP 1794 had greatest cell abundance
in the first week of incubation in the salinity range, 20.9 to 32.3 PSU. As
the New York strain did when acclimated at the lower
salinity, the Barnegat Bay strain grew optimally without
lag at a lower salinity than when acclimated at the higher salinity. Additionally,
it had slightly greater high salinity tolerance than the New York strain under the same conditions. Comparably high cell
abundance was evident with 20.9 to 36.1 PSU in week two. Comparable
cell abundance was obtained with 38 PSU in week three. Again, cell
abundance was lower than optimal but considerable with 19 PSU.
The lag in CCMP 1784 and CCMP 1794 growth with salinities higher or
lower than those initially favorable may reflect need for an adaptation
period, possibly recovery from transfer shock, or both. The test
inoculations represented sudden considerable salinity changes, e.g.,
from 27 to 17 PSU, and from 21 to 38 PSU. The observed lag periods
might be shortened or eliminated if the change was gradual, but this
was not tested. The ability of the species to withstand much of
this change demonstrates robustness. This may or may not have environmental
relevance; salinity change of ~10 PSU can occur in the Barnegat Bay-Little
Egg Harbor system over days or weeks, but not instantaneously.
Salinity tolerances of CCMP 1784 and CCMP 1794 A. anophagefferens strains
are basically similar. Both flourish with neretic salinities and
have considerable ability to adapt to open ocean salinities or higher,
and to lower salinities generally present in coastal bays in the northeast
region in late winter and spring. Evidencing the latter in the
tests, when both isolates were acclimated to 21 PSU, cell abundance with
this salinity was comparable during week one to cell abundance reached
with consistently suitable higher salinities. Both strains likewise
grew moderately well with 19 PSU when low salinity (21 PSU) adapted. Both
strains have unchanged, although different for each, high salinity preference
whether acclimated in low or high salinity. Cosper et al. (1989)
also found that A. anophagefferens BT-1 (=CCMP 1784) has the same
high salinity preference whether acclimated in low or high salinity. Comparison
of ranges of salinities suitable for growth without lag following acclimation
to 21 or 27 PSU, suggests that CCMP 1794 has a slightly broader salinity
tolerance than CCMP 1784, with a preference for higher salinity. This
distinction disappeared later in incubation, however.
CCMP 1784 and CCMP 1794 Light Requirements
Phytoplankton laboratory light bank illumination commonly is 5 to 10
percent of maximum daylight intensity, e.g., 170 µE m-2 sec-1 (Guillard
and Keller, 1984). Cosper et al. (1989) used 100 µE m-2 sec-1 for A.
anophagefferens culture. Illumination at 50 µE m-2 sec-1 routinely
was employed in this study, and this level apparently is adequate for A.
anophagefferens. The species is well adapted to growth with relatively
low light intensities when not nutrient-limited (Milligan, 1992). Growth
with lighting levels lower than 50 µE -2 m-2 sec-1 was
tested by light attenuation with Kodak neutral density filters variously
having percent transmissions of 50, 25, and 10. Culture tubes were
incubated atop the filters (filters were sandwiched between two squares
of thin glass), on the light bank shelf, in sections of 3-inch ID PVC
pipe, open on the bottom (lighting is from below) and light-sealed on
top. With
the lowest light level, cell abundance of A. anophagefferens CCMP
1784 was slightly less than yields with lighting from 25% to full light,
which was comparable until day 12 of incubation, when cell abundance
with all lighting levels was comparable. Ability to grow under
low light conditions can be particularly advantageous during major blooms
of the species. During an intense A. anophagefferens bloom
in 1999 in the Barnegat Bay-Little Egg Harbor system, Secchi depths in
various locales where it was most prevalent ranged only 0.2 to 0.4 m
(unpublished data).
RESULTS
SUMMARY AND COMMENTS
Modification of ASP12 for A. anophagefferens entailed
increase of concentrations of most original constituents, decrease of
concentrations of others, and adoption of additional nutrients found
beneficial. The most critical medium modification is the addition
of selenium. Changes in concentrations of major ions are relatively
modest, with the largest being reduction of magnesium and sulfur levels,
and introduction of fluoride. A change of magnesium, calcium, and
potassium molar ratio from 5.1 : 1.06 : 1 in ASP12 to 3.63
: 1.13 : 1 in ASP12A is due almost entirely to magnesium reduction
in the latter. A wide range of magnesium, sulfur, and potassium
concentrations support flourishing growth of A. anophagefferens;
absence of exacting requirement for magnesium and potassium is shared
by many phytoplankton species (Provasoli et al., 1957).
Changes in original recipe minor constituents include: considerable
reduction of silicon concentration (requirement by A. anophagefferens for
silicon was found); slight increase of nitrate nitrogen (ammonium is
used but is not sufficiently beneficial to warrant inclusion); moderate
reduction of inorganic and organic phosphorus constituents (flourishing
growth in medium with inorganic phosphorus, K3PO4,
as sole phosphorus source was obtained); increase of all constituents
of PII metals mix and one constituent of SII metals mix; selenium, arsenic,
vanadium, and aluminum are newly introduced.
EDTA chelator level is greatly increased, or original level raised only
to account for PII trace metals increases, if NTA is provided as second
chelator. The two chelator regimes are conducive to excellent growth
of A. anophagefferens. More study is needed to determine
if they can be improved. This could include evaluation of untested
EDTA : metals ratios between 3 : 1 and 10 : 1, and simultaneous chelator
: trace metals testing. NTA, a less effective chelator than EDTA
(Provasoli et al., 1957; McLachlan, 1973), is found basically suitable
but less so than EDTA for culturing A. anophagefferens in ASP12A. This
is in contrast to the findings of Dzurica et al. (1989), who reported
that NTA and citric acid were suitable chelators for A. anophagefferens and
growth was slight at best with EDTA. Underchelation is a possible
explanation for this because Dzurica et al. (1989) used medium ‘f’ or ‘f/2’ chelator
to trace metals ratio of 0.9 : 1. An explanation of the seemingly
contradictory situation of ASP12 being found under-chelated
for A. anophagefferens, but metals tests suggesting potential
deficiencies of some, which resulted in largely precautionary increases
to defer limitation, is the emphasis on long-term culture survival. The
effects on chelator and metals balance of lowered salinity, increased
calcium concentration, and lowering by half of TRIS pH buffer concentration
in ASP12A medium, are undetermined. Requirement for
vitamin B12 and thiamine is established. Vitamin B12 concentration
five times original recipe level (after Iwasaki, 1967) is adopted as
a precautionary measure; thiamine level is unchanged but doubling the
concentration may be beneficial and certainly is not harmful. Biotin
apparently is not needed by A. anophagefferens but is retained. Effect,
if any, on A. anophagefferens growth of three plant hormones tested
was not apparent.
The final version of the medium ASP12A as prepared in this
laboratory is autoclavable, does not precipitate, and retains utility
long term; e.g., with six months’ refrigerator storage. Batch-to-batch
quality of ASP12A is not perfectly consistent, but it is consistently
superior to enriched sea water media prepared in this laboratory. Growth
of A. anophagefferens with microwave-sterilized or autoclave-sterilized
batches is comparable. ASP12A supports A. anophagefferens growth
over at least a 5 to 18°C range of temperatures, and with relatively
low light levels. With the culture conditions in this laboratory,
ASP12A routinely provides maximum A. anophagefferens yields
of 2.0 to 3.0 x 107 cells ml-1. Because prolonged
culture survival was a primary development criterion for ASP12A,
it is a conservation medium. In one possibly extreme instance a
CCMP 1984 six-month-old ASP12A batch culture was successfully
transferred. This is not to say that the medium is sufficient to
support, routinely, viability of A. anophagefferens for this duration
in batch culture, but at least it did not prevent whatever was operative. Acclimation
of certain strains of A. anophagefferens to ASP12A
medium may be required. When transferred into final version of
the medium from an earlier version, CCMP 1794 growth was partly inhibited
in week one; such lag was not seen when the same strain was transferred
from final version to final version. In contrast, axenic CCMP 1984,
previously cultured in L1 medium in the NMFS Milford Laboratory, on initial
transfer to ASP12A, grew without lag and flourished. ASP12A
medium may be especially beneficial for bacterized A. anophagefferens cultures
by delaying microbial contaminant ascendancy or ameliorating stress of
such ascendancy to the picoplankter. Multiple laboratories (~10)
experienced loss of one A. anophagefferens strain (CCMP 1794)
at about the same time (Andersen, personal communication). What
caused these losses is undetermined, but culturing in ASP12A
may be an explanation for the strain’s survival solely in this
laboratory.
Because ASP12A is a conservation medium for A. anophagefferens the
concentrations of various medium constituents likely could be lowered
without major detriment to at least A. anophagefferens’ short-term
growth if a more dilute medium is required. Shown by the salinity
tolerance tests, proportionate reduction of magnesium, potassium and
calcium and sodium chloride to provide a salinity of 25 or 27 PSU does
not diminish the medium’s suitability. Nitrogen likely could
be lowered safely to about original recipe level and phosphorus concentration
could be reduced drastically. A moderate reduction of metals and
chelator likely would be feasible.
Sensitivity of A. anophagefferens to silver and copper has been
reported to be relatively high; these metals at ~10 ppb (9.27 x 10-8 M
and 1.57 nM, respectively) inhibited A. anophagefferens growth
by 50% (Steele et al., 1989). However, the concentration ranges
of silver and copper in sea water are only 0.5 to 35 pM and 0.5 to 4
nM, respectively (Bruland, 1983), so it is unlikely that the species
normally would encounter these inhibitory levels in nature. Moreover,
tested in this laboratory, silver was not inhibitory in concentrations
to 2.78 x 10-8 M,
and the species tolerated three times the ‘K’ medium level
of copper, 3 x 10-8 M, the latter being the highest concentration
tested. Also, comparison of metals enrichments of three media suitable
for culturing the species, including ASP12A, ‘K’ medium
(Keller et al., 1987), and Aquil medium (Morel et al., 1979) modified
by Cosper et al. (1993) to include selenium, shows these have widely
disparate concentrations and proportions of metals enrichments. Total
level of chelatable trace metals in unmodified Aquil is only 4.8 x 10-7 M
in contrast to 1.27 x 10-5 M in ‘K’ medium (mostly
iron). Comparison of metals proportions reveals ‘K’ medium
has 3.2 times more iron than ASP12A, but the latter has 10
and 13.3 times more manganese and zinc, respectively, than ‘K’ medium. This
suggests A. anophagefferens may not be especially intolerant of
at least some toxic metals and relatively high levels of essential metals
and can flourish with a wide concentration range of many required metals. During
culture testing it has grown after a lag of several weeks, suggesting
acclimation to an inhibitory condition or at least survival without cell
division until the inhibitor concentration lessened.
ASP12A suitability for a multiplicity of A. anophagefferens isolates
is certain. A group of 11 A. anophagefferens Long
Island isolates, routinely cultured in L1 NW medium (Guillard and Morton,
2003), were evaluated for growth in ASP12A by R. Andersen
at CCMP, Bigelow Laboratory. Cultures included CCMP 1785, a 1987
isolate from Great South Bay; CCMP 1706 and CCMP 1707, isolates from
West Neck Bay in 1995; CCMP 1790 from Great South Bay in 1995; CCMP 1847,
1848, 1849, 1851, 1852, and 1853, isolates from the same 1998 collection
time and site in Great South Bay; and CCMP 1850, an isolate from another
Great South Bay site in 1998. All isolates grew well in ASP12A
through an initial transfer followed by two serial transfers, which together
represented a ~1 : 16,000 dilution of L1. Viability of all strains
in ASP12A was supported for at least three months; most strains
apparently were viable after 4.5 months. Overall ASP12A
suitability for these A. anophagefferens cultures was ~equal
to that of L1 medium (Andersen, personal communication). Although “tailored” for A.
anophagefferens ASP12A retains general utility. Suitability
of ASP12A for 28 other phytoplankton species in a wide range
of classes was tested at CCMP (Andersen, personal communication). Increase
of silicon concentration of ASP12A would be necessary for
species that require more than the low level in this medium. The
medium supported excellent growth of the following CCMP cultures: 732,
736, 1168, 1175, 1278, 1283, 1320, 1333, 1420, 1485, 1493, 1674, 1675,
1768, 1954, 1994, 2057, 2064, 2070, 2112, 2199, 2424, 2495, and 2579. These
included, for example, the diatom Skeletonema costatum; a chlorophyte, Dunaliella
tertiolecta; the dinoflagellates Alexandrium tamarense and Prorocentrum
lima; a haptophyte, Emiliania huxleyi; a pelegophyte, Pelegomonas
calciolata; and a raphidophyte, Heterosigma akashiwo. CCMP
1722 grew weakly in the medium, and CCMP 1153, 1475, and 2164 did not
grow in it. Trial of ASP12A at the NMFS Milford
Laboratory (Wikfors and Alix, personal communication) showed the medium
is suitable for a diatom, Nitzschia sp. (with 1.06 x10-4 M
silicon added); a dinoflagellate, Prorocentrum minimum; a cryptophyte, Rhodomonas sp.;
a prasinophyte, Tetraselmis chui; a prymnesiophyte, Pyymnesium
parvum; and a rhodophyte, Porphyridium cruentum. For
decades, ASP12 medium has had proven success for a wide variety
of algae. The suitability of ASP12A for the above group
of phytoplankton indicates that its “tailoring” for A.
anophagefferens has not interfered, at least not greatly, with its
general utility.
Information on the relative physiology of A. anophagefferens strains
which bloom in New York and New Jersey locales -- perhaps multiple strains
in each region -- has not been available. Consequently, application
of aspects of the information on Long Island blooms to New
Jersey blooms, the latter having relative paucity of information developed,
may or may not be sound. Some growth features of a Long Island
isolate and the only New Jersey A. anophagefferens culture isolated
are compared, despite uncertainty that the A. anophagefferens cultures
employed in this study were consistently representative of the natural
populations. Comparison of growth responses of the New
York and New Jersey strains
in this study to most of the medium constituents is not possible, because
too few tests were done for both. Rather than offering conclusions,
perhaps the main benefit of this limited comparison is to highlight the
subject.
The two isolates appear physiologically similar in some respects, but
differences in tolerance to some of the medium constituents tested are
apparent. Requirement for selenium is shared; the New York isolate
and its axenic version appear to tolerate higher concentration of selenium
than the New Jersey strain. All isolates benefited from fluoride
with no tolerance difference in the range tested. NH4Cl
is utilized as sole nitrogen source by New York and New Jersey isolates;
in higher concentrations tested, NH4Cl is more toxic to the
latter. The axenic New York isolate was not tested for NH4Cl
toxicity, so possible amelioration by bacterial contaminants rather than
higher New York strain tolerance cannot be ruled out. All
isolates utilize inorganic or organic phosphorus equally well. Growth
lag before use of inorganic phosphorus solely (when previously grown
with combined inorganic and organic phosphorus) was greater with the
New Jersey isolate and the axenic New York culture than with the non-axenic
New York culture. New York and New
Jersey strains require the same vitamins. Bacterized New
York and New Jersey strains had positive growth response to iron in the
highest level tested. This level was slightly inhibitory to the
axenic New York isolate, which benefited from a lower level increase. All
isolates had positive growth response to increases of boron and manganese,
with no apparent inhibition with the levels tested. Salinity tolerances
of the New York (CCMP 1784) and New
Jersey (CCMP 1794) strains basically are similar. The finding by
Cosper et al. (1989) that A. anophagefferens retains the same
high salinity preference, whether pre-test acclimated in low or high
salinity, is confirmed for CCMP 1784 and also found for CCMP 1794 in
this study. Both
CCMP 1784 and CCMP 1794 grew at 19 PSU, and at the highest salinity tested,
38 PSU. Comparison of ranges of salinities suitable for early incubation
growth following pre-test acclimation to 21 or 27 PSU suggests that CCMP
1794 high salinity preference may be slightly greater than that of CCMP
1784. Direct comparison of published data on temperature tolerance
of CCMP 1784 (Cosper et al., 1989) with that determined for CCMP 1794
is not possible due to test design difference. However, in this
laboratory both grew comparably well at the normal incubation temperature
18°C, which is close to the reported most favorable temperature range,
20 to 25°C (Cosper et al., 1989). Ability to grow at winter
water temperature, 5°C, considered a potentially important factor
in eastern Long Island A. anophagefferens bloom occurrence, shown
for the New York isolate (Cosper et al., 1989), was shown for the New
Jersey isolate as well. The latter steadily increased in cell abundance
throughout over two months’ incubation at 5°C. These
limited and somewhat mixed results are unsatisfying but serve to underscore
the need for physiological comparison of A. anophagefferens bloom
dominants from different regions.
ACKNOWLEDGMENTS
This study benefited from advice from S.H. Hutner, Haskins Laboratories
at Pace University, New York; R.R.L. Guillard, Bigelow
Laboratory for Ocean Science, West Boothbay Harbor, Maine;
and K.I. Keating, Department of Environmental Sciences, Rutgers University,
New Jersey. The author
is indebted to R. Andersen, Bigelow Laboratory for Ocean Science, for
evaluating suitability of ASP12A for multiple A. anophagefferens isolates
and a variety of other phytoplankton, and to G. Wikfors and J. Alix,
NMFS, Milford Laboratory, who also evaluated the medium with a variety
of phytoplankton. Thanks are due Dianne Kapareiko, NMFS Milford
Laboratory, for assessing CCMP 1784 culture contaminants. John
Sibunka, NMFS, James J. Howard Marine Sciences Laboratory, provided sea
water for media preparation on numerous occasions from Gulf
of Maine and offshore Middle Atlantic Bight
locales. The author thanks L. Garner, NMFS, Woods Hole Laboratory, for
editing the manuscript.
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