Effect of high pH on the growth and survival of marine phytoplankton: Implications for species succession

Abstract

Ten years of pH measurements (1990 to 1999) in the surface waters of the eutrophic Mariager Fjord, Denmark, revealed profound seasonal variation. Typically, pH was relatively constant around 8 from January to March, increased during spring, reached maximum levels in July to August (9 to 9.7), and declined during autumn to about 8 in October. The influence of pH on the growth rate of phytoplankton was tested on 3 species (Ceratium lineatum, Heterocapsa triquetra and Prorocentrum minimum) in laboratory experiments. The growth rate was highest at pH 7.5 to 8.0 in all species. The growth rate of C. lineatum declined by ∼20% at pH 8.3 to 8.5, while a similar reduction in the growth rate in H. triquetra and P. minimum was observed at pH 8.8 to 8.9. C. lineatum stopped growing above pH 8.8, while growth ceased at about pH 9.45 in H. triquetra and 9.6 in P. minimum. Compilation of literature data on pH and phytoplankton growth suggested that while some species cannot grow at pH 8.4, others are able to grow up to pH 10. However, none of the species studied can attain their maximum growth rate above pH 9. Competition experiments using a mixture of C. lineatum, H. triquetra and P. minimum always resulted in the species with the highest pH tolerance (P. minimum) outcompeting the other species, irrespective of the initial pH value. The role of high pH in the succession of marine phytoplankton in nature is discussed.

Full text

AQUATIC MICROBIAL ECOLOGY
Aquat Microb Ecol
Vol. 28: 279–288, 2002 Published July 25
INTRODUCTION
Uptake of inorganic carbon by phytoplankton during
photosynthesis has the potential to increase pH in the
surrounding water. However, while pH can rise above
10 in freshwater (e.g. Talling 1976), it is usually quite
constant around 8.2 in surface seawaters. This is
because seawater contains high concentrations (2 mM)
of inorganic carbon, which buffers pH. Apart from
some reports from natural environments (e.g. Hinga
1992, Macedo et al. 2001), high pH has usually been
observed in marine enclosures after the addition of
nutrients or in laboratory cultures of phytoplankton
(Goldman 1982a,b, Hinga 1992). Although the occur-
rence of high pH in marine waters may not be uncom-
mon, pH has generally not been considered an impor-
tant determinant of pelagic processes, and papers
dealing with the possible effect of pH on the growth
and succession of marine phytoplankton are sparse.
However, a few studies that have been carried out
indicate that pH indeed may have an effect on phyto-
plankton growth and that this effect drives species
succession (e.g. Goldman et al. 1982a,b, Schmidt &
Hansen 2001).
The present study was initiated in response to the
observation of very high pH (up to 9.75) in the surface
waters of Mariager Fjord, Denmark, during a summer
© Inter-Research 2002 · www.int-res.com
*E-mail: pjhansen@zi.ku.dk
Effect of high pH on the growth and survival of
marine phytoplankton: implications for species
succession
P. J. Hansen*
Marine Biological Laboratory, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark
ABSTRACT: Ten years of pH measurements (1990 to 1999) in the surface waters of the
eutrophic Mariager Fjord, Denmark, revealed profound seasonal variation. Typically, pH
was relatively constant around 8 from January to March, increased during spring, reached
maximum levels in July to August (9 to 9.7), and declined during autumn to about 8 in Octo-
ber. The influence of pH on the growth rate of phytoplankton was tested on 3 species (Cer-
atium lineatum, Heterocapsa triquetra and Prorocentrum minimum) in laboratory experi-
ments. The growth rate was highest at pH 7.5 to 8.0 in all species. The growth rate of C.
lineatum declined by ~20% at pH 8.3 to 8.5, while a similar reduction in the growth rate in
H. triquetra and P. minimum was observed at pH 8.8 to 8.9. C. lineatum stopped growing
above pH 8.8, while growth ceased at about pH 9.45 in H. triquetra and 9.6 in P. minimum.
Compilation of literature data on pH and phytoplankton growth suggested that while some
species cannot grow at pH 8.4, others are able to grow up to pH 10. However, none of the
species studied can attain their maximum growth rate above pH 9. Competition experi-
ments using a mixture of C. lineatum, H. triquetra and P. minimum always resulted in the
species with the highest pH tolerance (P. minimum) outcompeting the other species, irre-
spective of the initial pH value. The role of high pH in the succession of marine phyto-
plankton in nature is discussed.
KEY WORDS: pH · Species succession · Competition · Marine · Phytoplankton · Growth ·
Inorganic carbon · DIC
Resale or republication not permitted without written consent of the publisher

Aquat Microb Ecol 28: 279 –288, 2002
bloom of the dinoflagellate Prorocentrum minimum in
1997 and to some recent results which have pointed to
the fact that pH may be responsible for species succes-
sion (Schmidt & Hansen 2001).
The first phase of this study was to collect data on
the seasonal change in pH in the highly productive
Mariager Fjord in order to describe the natural range
of pH in a marine environment. The second phase
aimed to study (1) the role of pH in limiting growth in
laboratory cultures of marine phytoplankton and (2)
the role of pH in the succession of species in mixed
phytoplankton cultures in the laboratory.
MATERIALS AND METHODS
Seasonal and inter-annual variation in pH in a
eutrophic fjord in Denmark. pH has been measured
regularly during the past 20 yr at a fixed station in
Mariager Fjord, Denmark, by the counties of Aarhus
and North Jutland as part of their monitoring program.
The station is located at 56° 39’ 80’’ N, 9° 58’69’’ E close
to the center of the deep basin, which has a water
depth of 28 m. The salinity in the surface water at this
station varies from 16 to 17 ppt. For the past 10 yr mea-
surements have been carried out weekly to bi-weekly,
and these data were kindly provided by the counties.
Data on pH are from the top part of the water column.
Water, subsequently used for primary production, was
gently poured into polyethylene bottles (2.5 l) to capac-
ity to avoid aeration of the samples. The bottles were
stored in the dark in a thermo-container until the
measurement of pH shortly after, using a standard
Radiometer®glass pH electrode (sensitivity 0.01). The
pH sensor was calibrated (2 point) using buffers of
pH 7 and 10.
Isolation and culture of phytoplankton. Information
on isolation date, isolation place and clone designation
of the 3 species of dinoflagellates used in the present
study is listed in Table 1. The Scandinavian Culture
Collection of Algae and Protozoa, Botanical Institute,
University of Copenhagen, provided Heterocapsa tri-
quetra and Prorocentrum minimum, while the Marine
Biological Laboratory, University of Copenhagen, pro-
vided Ceratium lineatum. These species were chosen
based on previous experience concerning their differ-
ent tolerance to pH in batch cultures. According to
Schmidt & Hansen (2001), C. lineatum should have a
pH limit for growth at pH 8.79, while the pH limits for
growth of H. triquetra and P. minimum should be 9.43
and 9.62, respectively. The algae were grown as non-
axenic cultures in f/2 medium (Guillard 1983) based on
seawater (30 psu) at 15 ± 1°C following a light:dark
cycle of 16:8 h. Illumination was provided by cool
white fluorescent lamps, and cultures were kept at an
irradiance of 25 µmol photons m–2 s–1. Irradiance was
measured using a LI-COR LI-1000 radiation sensor
equipped with a spherical probe.
Experimental conditions. All experiments were car-
ried out at an irradiance of 60 µmol photons m–2 s–1.
The dinoflagellates were adapted to this irradiance for
at least 14 d prior to each experiment. Only cells from
exponentially growing cultures were used for inocula-
tion. For enumeration of cells, subsamples were fixed
in acidic Lugol’s iodine (2.5% final concentration).
Cells were counted in a Sedgewick-Rafter chamber or
a multidish well (Nunclon®). Growth rates (
µ
)were
measured as increase in cell number and were calcu-
lated assuming exponential growth:
N0and N1are number of cells at time t0and t1, and tis
the difference in time (d) between t0and t1samples. All
experiments were carried out in triplicate, and data
from each triplicate were the mean of at least 3 growth
rates (= 4 sampling dates). pH was measured using a
Sentron®pH-meter (model 2001) equipped with Red-
line probe, which is an ISFET®sensor (Semi-conductor
Ion Field Effect Transistor) with a detection limit of
0.01. The pH sensor was calibrated (2 point) using Sen-
tron buffers of pH 7 and 10.
Effect of pH on phytoplankton growth rate. The
growth rates of the 3 dinoflagellates were measured at
different pH within the range of 7.5 to 10. The pH was
adjusted by the addition of 0.1 M NaOH or HCl. All
experiments were carried out in Nunclon®tissue cul-
ture flasks (250 ml), which were mounted on a plank-
ton wheel (1 rpm) in order to keep the phytoplankton
in suspension. Each experiment was initiated by the
inoculation of between 20 and 100 cells
ml–1 and allowed to run for 7 d. Daily,
pH of the culture media was measured,
and subsamples (6 ml) were taken for
enumeration of phytoplankton cells.
After subsampling, the bottles were
refilled to capacity with f/2 growth
medium, which had the same pH as the
respective experimental bottle. If the
pH differed more than 0.03 from the
µ
()
(ln ln )
d−=−
110
NN
t
280
Species Clone Isolation place and time
Ceratium lineatum Øresund, Denmark, 1995
Heterocapsa triquetra K-0481 Øresund, Denmark, 1988
Prorocentrum minimum K-0295 Kattegat, Denmark, 1989
Table 1. List of dinoflagellate species used in the experiments, their clone desig-
nation and isolation place and time

Hansen: Effect of pH on phytoplankton growth and survival
set point, it was adjusted by addition of small amounts
of 0.1 M NaOH or HCl. The first 2 d of the experiments
were considered an acclimation period; therefore cell
counts from these samplings were not included in the
calculations of growth rates. Also, the daily dilution (of
6 ml) was adjusted for in the calculations of algal
growth rates.
Growth experiments with mixed phytoplankton
cultures. Two types of succession experiments were
carried out using the 3 dinoflagellates in mixture. In
the first type of experiments, mixed cultures were initi-
ated at pH levels of 8, 8.5, and 9.0. The pH was
adjusted by the addition of 0.1 M NaOH or HCl. All
experiments were carried out in Nunclon®tissue cul-
ture flasks (250 ml), which were mounted on a plank-
ton wheel (1 rpm) in order to keep the phytoplankton
in suspension. The species were inoculated together at
an initial concentration of 100 cells ml–1 of each species
and allowed to grow. Every 2 to 3 d, pH of the culture
media was measured, and subsamples (6 ml) were
retrieved for enumeration of phytoplankton cells.
Immediately after sampling, the bottles were refilled to
capacity with f/2 growth medium, which had the same
pH as the sampled medium.
Because the species Heterocapsa triquetra and Pro-
rocentrum minimum have an almost similar tolerance
to high pH, a second type of experiment was carried
out in order to exclude other possible effects, such as
nutrient/vitamin limitation or effects of allelochemicals
(toxins). This experiment was carried out in a 1 l Pyrex
flask containing 500 ml of f/2 medium in triplicate.
Atmospheric air was applied by bubbling in order to
supply CO2to the medium and thereby reduce the
elevation of pH, when cultures become dense. The
species were inoculated together at an initial concen-
tration of 100 cells ml–1 of each species and allowed to
grow. Every 2 to 3 d, pH of the culture media was
measured, and subsamples (6 ml) were retrieved for
enumeration of phytoplankton cells.
RESULTS
pH in Mariager Fjord
Median, minimum, and maximum pH values from
10 yr of pH measurements (1990 to 1999) of surface
water from the deep basin are shown in Fig. 1. The
seasonal variation in pH was bell-shaped. From Octo-
ber to March, pH varied around 7.7 to 8.2. In April and
May, pH increased rapidly to 8.7. The highest pH was
found in July and August (pH 9). By August pH
decreased again to reach pH 8 in late September. The
data also show that pH above 9 (max. 9.75) could be
found at any time from May to August.
pH limits for phytoplankton growth/survival in
batch cultures
The effect of pH on the growth rate was very pro-
found in the 3 phytoplankton species Ceratium linea-
tum, Heterocapsa triquetra and Prorocentrum mini-
mum (Fig. 2). The growth rate was highest at pH 7.5 to
8.0 in all species. The growth rate of C. lineatum
declined by ~20% at pH 8.3 to 8.5, while a similar
reduction in the growth rate in H. triquetra and P. mi n-
imum was achieved at pH 8.8 to 8.9. C. lineatum
stopped growing above pH 8.8, while growth ceased at
about pH 9.45 and 9.6 in H. triquetra and P. minimum,
respectively
Succession experiments in mixed phytoplankton
batch cultures
The importance of pH in the succession of phyto-
plankton species was studied using mixed cultures of
the 3 species at 3 different initial pH levels: 8, 8.5, and
9 (Fig. 3). The experiments initiated at pH 8 and 8.5
were quite similar. In both cases all species grew until
pH reached 8.70 to 9 on Day 7. At Day 10 the pH had
increased to above pH 9.4, and the entire population of
Ceratium lineatum had died out. At Day 10 the growth
of Heterocapsa triquetra also stopped, and during the
following 2 wk the concentration of H. triquetra
declined. At Day 24 the entire population of H. trique-
tra had died out. The degradation of the H. triquetra
population caused pH to decrease to 9 at Day 24.
Prorocentrum minimum was able to keep a positive
growth rate until Day 17. During the period from
281
Fig. 1. Seasonal distribution of median pH (d) in surface
waters of Mariager Fjord, Denmark, for a 10 yr period
(1990–1999). Maximum and minimum values are indicated
with a solid grey line

Aquat Microb Ecol 28: 279 –288, 2002
Days 17 to 21, the P. minimum population just main-
tained itself, but after Day 21 it regained positive
growth, resulting in an increase in pH.
In the experiment initiated at pH 9, the Ceratium lin-
eatum population died within the first 3 d of incubation,
while Heterocapsa triquetra and Prorocentrum mini-
mum grew until Days 14 and 19, respectively, when pH
had increased to above 9.4 (Fig. 3E,F). The H. triquetra
population declined from Day 16, and after 31 d of incu-
bation had completely died out. In that period, pH de-
creased to below 9. The P. minimum population main-
tained itself from Day 19 to Day 31, whereupon it
increased again, resulting in an increase in pH.
A control experiment was carried out in which bub-
bling with atmospheric air was applied to reduce the
elevation of pH in a mixed culture of Heterocapsa tri-
quetra and Prorocentrum minimum (Fig. 4). The pH in
this experiment stayed fairly constant around 7.75 to 8
for the first 12 d, whereupon it increased to reach
about 9 on Day 17 and stayed at this level until the
experiment was terminated. At this time the cell con-
centrations of H. triquetra and P. minimum had
reached 185 000 and 18 500 cells ml–1, respectively. In
terms of total cell concentration, this was about
10 times higher than the cell concentration found in the
experiment without bubbling initiated at pH 8, and
20 times the cell concentration reached at the experi-
ment initiated at pH 9.
DISCUSSION
High pH in marine waters
A number of biological and physical processes may
influence the pH of marine surface waters. Uptake of
inorganic carbon by phytoplankton during photosyn-
thesis may increase pH, while release of CO2through
respiration processes will decrease pH. Excursions of
pH in marine waters are counteracted by the physical
exchange of CO2 between the atmosphere and the sur-
face water. Thus, the exchange of CO2at the air-water
transition is highly dependent on the vertical mixing of
the water column, and thereby very much dependent
on the strength of the wind stress and the stability of
the water column.
In Mariager Fjord, the data from the past 10 yr
show a median pH of close to 9 during the summer
(May to August). The maximum pH recorded within
the past 10 yr was 9.75, which is close to the maxi-
mum pH that can be achieved in seawater. The
development of such high pH values in Mariager
Fjord is largely due to its high primary production
and to its morphology and hydrography. The fjord is
highly productive, and summer chlorophyll aconcen-
trations may reach >50 µg l–1 (Fenchel et al. 1995,
Fallesen et al. 2000, Olesen 2001). The high produc-
tivity of the fjord is supported by inorganic nutrients
draining from the surrounding farmland (Fallesen et
al. 2000). The fjord has a sill and is permanently strat-
ified with a salinity of 16 to 17 psu in the top 10 m.
The water exchange of Mariager Fjord is relatively
small, and the average residence time of water in the
central part (above the 16 m) is about 8 mo (Fenchel
et al. 1995, Fallesen et al. 2000). Thus, the combina-
tion of a stable water column, a low residence time of
surface waters, high solar radiation during summer,
and sufficient supply of nutrients form the physical
basis for the development of phytoplankton blooms
and high pH levels in Mariager Fjord.
Although the pH can become extremely high in
Mariager Fjord, the fjord is not unique. In the
282
Fig. 2. Growth rate as a function of pH in laboratory cultures
of (A) Ceratium lineatum, (B) Heterocapsa triquetra, and
(C) Prorocentrum minimum. Data points refer to treatment
means ± SE (n = 3)

Hansen: Effect of pH on phytoplankton growth and survival
German Bight and in the Chesapeake Bay system,
maximum pH of between 8.7 and 9.25 has been
observed during the summer (e.g. Pegler & Kempe
1988, Hinga 1992 and references therein). In en-
closed lagoons that are only temporarily flooded, pH
can reach values as high as 9.5 to 9.6 (Macedo et al.
2001). Thus, elevated pH may be a common phenom-
enon in the pelagial of many highly productive
coastal waters and is a parameter that we should con-
sider when dealing with phytoplankton production
and species succession.
Importance of pH for the growth of marine
phytoplankton
Chen & Durbin (1994) studied the growth of
2 diatoms (Thalassiosira spp.) as a function of pH in the
culture medium. Both diatoms were able to maintain
their maximum growth rate up to pH 8.6. Above this
pH, the growth rate of the diatoms decreased to only
ca. 10% of the maximum growth rate at pH 9.4, resem-
bling the pH dependency of the dinoflagellates
Heterocapsa triquetra and Prorocentrum minimum in
283
Fig. 3. Succession experiment. (A,C,E) Changes in cell concentration of the species Ceratium lineatum, Heterocapsa triquetra
and Prorocentrum minimum as a function of time (d) from inoculation at 3 initial pH levels. (B,D,F) pH as a function of time from
inoculation. Please note that the cell concentration of C. lineatum is shown on the left y-axes, while the cell concentration of the
2 other species is shown on the right y-axes

Aquat Microb Ecol 28: 279 –288, 2002
the present study. Apart from the work by Chen &
Durbin (1994) and the present study, information on
pH limits for the growth of marine phytoplankton
comes from studies of batch cultures in which pH has
not been kept constant.
The work of Schmidt & Hansen (2001) suggested
that marine phytoplankton cultures grown in a stan-
dard phytoplankton growth medium (like the f/2
growth medium) are limited by high pH rather than
inorganic nutrients such as nitrogen and phosphorus.
My data support this conclusion.
First, a simple calculation using the maximum con-
centrations of Ceratium lineatum, Heterocapsa trique-
tra and Prorocentrum minimum reached in f/2 medium
(Schmidt & Hansen 2001) and their cellular carbon
content clearly suggests that such cultures are not
nutrient-limited. In fact, the estimated uptake of nitro-
gen and phosphorus only accounted for about 5 to 11
and 7 to 17% of the available nitrogen and phospho-
rus, respectively (Table 2).
Second, the pH at which the 3 selected dinoflagel-
lates just maintained themselves in the constant pH
experiment at low cell concentrations in the present
study was similar to the pH obtained in the stationary
growth phase of the same species in enriched batch
cultures (cf. Table 3 with Fig. 2). Likewise, the pH
causing a reduction in growth rates of the 3 studied
species by 20% in the present study was similar to the
pH causing a ca. 20% reduction of the growth rate in
ordinary batch cultures (cf. Table 3 with Fig. 2).
Thus, in the absence of data from experiments
carried out at constant pH levels, data on pH limits for
maximum growth rate (reduction ca. 20%) as well as
the pH limits for growth for marine phytoplankton can
be obtained from ordinary batch culture experiments
(Table 3). Comparison of literature data on 35 species
of marine phytoplankton suggested a great variation in
their tolerance to high pH. Some species (Ceratium
tripos, C. furca) stopped growing at a pH above 8.3 to
8.4, while others were able to grow at a pH close to
10 (e.g. Phaeodactylum tricornutum,
Rhodomonas salina). It is notable that
within the same family some species
were sensitive while others were
insensitive to high pH (e.g. dinoflagel-
lates and diatoms). When the number
of phytoplankton species capable of
growing at a specific pH was plotted as
a function of pH, the data fitted a 1-
tailed normal distribution (Fig. 5). Only
half of the species investigated could
sustain growth at pH above 9.2.
For 17 species, data were also avail-
able on the pH at which maximum
growth was reduced by 20%. While
284
Species Maximum cell C uptake N uptake P uptake
concentration (µM) (µM) (µM)
(cells ml–1)
Ceratium lineatum 4.0 ×103280 42.3 2.64
Heterocapsa triquetra 4.0 ×104639 96.6 6.03
Prorocentrum minimum 5.5 ×104652 98.3 6.15
Table 2. Estimated uptake of C, N and P in Ceratium lineatum, Heterocapsa tri-
quetra and Prorocentrum minimum cultures that have reached maximum cell
concentrations in f/2 medium. Addition of N and P to the seawater in the f/2
medium was 883 and 36 µM, respectively. Estimates were based on a red field
ratio of 106 C:16 N: 1 P (by mol). The carbon contents of the species were calcu-
lated from the carbon to cell volume equation published by Strathmann (1967)
Fig. 4. Succession experiment — control experiment. (A) Changes in cell concentration of Heterocapsa triquetra and Prorocen-
trum minimum as a function of time from inoculation in mixed cultures, which were bubbled with atmospheric air to buffer pH.
(B) pH as a function of time from inoculation. Please note that the cell concentration of H. triquetra is shown on the left y-axes,
while the cell concentration of P. minimum is shown on the right y-axes

Hansen: Effect of pH on phytoplankton growth and survival
the growth of some species was affected at pH below
8.4, other species could maintain their maximum
growth at pH 9. However, none of the tested species
could grow at their maximum growth rate much above
pH 9. Thus, although Rhodomonas salina or Prorocen-
trum minimum could grow at pH close to 10, their
growth rates were already affected at a pH of 9 to 9.1.
It is also noteworthy that only half of the species inves-
tigated reached maximum growth rates at pH between
8.6 and 8.8. Therefore pH should be taken into account
when dealing with phytoplankton growth in the labo-
ratory and in natural environments.
Several reasons for the effects of high pH on the
growth of phytoplankton may be suggested. Changes
of pH in seawater influence the inter-speciation of
inorganic carbon (as CO2aq., HCO3–, CO32–). In sea-
water at pH 8, ca. 1% of DIC is present as CO2, while
at pH 9 only ca. 0.1 % of the DIC is available in this
form. Limitation in the supply of CO2caused by ele-
vated pH may therefore potentially restrict photosyn-
thesis and growth in marine phytoplankton. However,
at least some phytoplankton species have active trans-
port systems by which they can take up HCO3–in order
to avoid DIC limitation at elevated pH (e.g. Colman &
Gehl 1983, Dixon & Merrett 1988, Colman & Rotatore
1995, Korb et al. 1997, Huertas et al. 2000). Thus, ele-
vated pH should favour species which can utilize
HCO3–as an inorganic carbon source. Alternatively,
high extracellular pH may cause gross alterations in
the membrane transport processes and metabolic
285
Species Taxon pH limits for pH limits for growth Source
exponential growth* (
µ
= 0)
Phaeodactylum tricornutum Diatom >10; 10.3;10.4 1,2,3
Amphidinium carterae Dino. >10 1
Nanochloropsis sp. Eumast. 10.0804
Rhodomonas marina Crypt. 8.74 9.93 5
Prorocentrum micans Dino. 8.75 9.92 5
Thalassiosira pseudonana Diatom 9.77 4
Prorocentrum minimum Dino 9.20 9.62 5
Dunaliella tertiolecta Chloro. 8.69; 9.5 4,1
Nitzschia closterium Diatom 9.501
Nitzschia sp. Diatom 9.501
Heterocapsa triquetra Dino 8.80; 8.90 9.43 5
Synechococcus sp. Cyano. 9.40 4
Chroomonas sp. Crypto. 9.301
Emiliania huxleyi (heavy coccoliths) Prym. 9.29 4
Chrysochromulina simplex Prym. 9.20 9.25 5
Eutrieptiella gymnastica Eugl. 9.00 9.22 5
Skeletonema costatum Diatom 8.49 9.21 5
Chrysochromulina polylepis Prym. 8.70 9.20 5
Heterosigma akashiwo Raphid. 8.52 9.15 5
Phaeocystis globosa Prym. 9.14 4
Biddulphia aurita Diatom 9 1
Chaetoceros didymus Diatom 9 1
Gymnodinium splendens Dino. 9 1
Monochrysis lutheri Prym. 9 1
Gymnodinium mikimotoi Dino. 8.72 9.00 5
Alexandrium ostenfeldii (2 clones) Dino. 8.66 8.90 5
Thalassiosira punctigera Diatom 8.90 4
Alexandrium tamarense Dino. 8.66 8.85 5
Dictyocha speculum Silicofl. 8.30 8.81 5
Emiliana huxleyi (weak coccoliths) Prym. 8.80 4
Pyramimonas propulsa Prasino. 8.55 8.80 5
Ceratium lineatum Dino. 8.30 8.79 5
Cylindrotheca closterium Diatom 8.501
Ceratium furca Dino. 8.29 8.40 5
Ceratium tripos Dino. 8.24 8.30 5
1: Humphrey (1975), 2: Goldmann et al. (1982), 3: Nimer et al. (1997), 4: Elzenga et al. (2000), 5: Schmidt & Hansen (2001)
Table 3. pH limits for exponential growth and for positive growth (
µ
= 0) in highly enriched batch cultures for phytoplankton
belonging to a variety of taxa. Salinity was in all cases between 30 and 34 psu. Taxa abbreviations refer to eumastigohytes
(Eumast.), cryptophytes (Cryp.), dinoflagellates (Dino.), prymnesiophytes (Prym.), raphidophytes (Raphid.), silicoflagellates (Sil-
ico.), prasinophytes (Prasino.), chlorophytes (Chloro.). *Criterion: growth is affected when growth rate is reduced by > 20%

Aquat Microb Ecol 28: 279 –288, 2002
functions involved in internal pH regulation (Smith &
Raven 1979, Raven 1980, 1993) or cause changes in
cellular content of amino acids and their relative
composition, possibly affecting cellular growth (e.g.
Taraldsvik & Myklestad 2000).
Species succession
Although there are many studies on species succes-
sion in multi-species phytoplankton batch cultures,
only a few have taken pH into account (see reviews by
Maestrini & Bonin 1982a,b). The possible role of high
pH in the succession of phytoplankton species was first
addressed by Goldman et al. (1982a,b). They observed
that the diatom Phaeodactylum tricornutum often suc-
cessfully invaded other phytoplankton cultures in the
laboratory. Through a set of competition experiments
they demonstrated that P. tricornutum out-competed
Dunaliella tertiolecta in continuous cultures when pH
exceeded 9.2.
The role of high pH in species succession among
marine phytoplankton was studied here by exposing
3 species in mixture to different initial pH values
between 8 and 9 (Fig. 3). Irrespective of the initial pH,
Prorocentrum minimum always outcompeted the
2 other species. Ceratium lineatum died out soon after
the pH had exceeded 8.7, independently of the initial
pH, which is in accordance with the results obtained
for this species in the monoculture experiments at
fixed pH levels (Fig. 2A). Heterocapsa triquetra grew
in the mixture experiments until pH exceeded 9.4
(Fig. 3), which again is in accordance with the results
obtained in the monoculture experiments at fixed pH
levels (Fig. 2B).
Prorocentrum minimum has a pH limit for growth,
which is only slightly higher than that of Heterocapsa
triquetra, and thus other factors such as nutrient/vita-
min limitation or production of allelochemicals (toxins)
may have been involved in the interaction between
these 2 species in the mixture experiment.
Earlier in this section (when discussing the im-
portance of pH for growth of marine phytoplankton)
a rough calculation suggested that nitrogen and
phosphorus are not limiting the growth of either
Heterocapsa triquetra or Prorocentrum minimum in
f/2 growth medium. The results obtained in the aer-
ated mixture experiment support that suggestion and
also exclude vitamin limitation, because total cell
yield in the aerated mixture experiment was at least
10 times higher than in the non-aerated mixture
experiment (Figs. 3 & 4). Also, the maximum cell yield
in the non-aerated mixture experiment decreased
when the initial pH set point was elevated. Thus, any
kind of nutrient/vitamin limitation can be ruled out as
the cause of the species succession in these experi-
ments.
A few studies have indicated that some isolates of
Prorocentrum minimum can produce toxic substances
(Trick et al. 1981, Grzebyk et al. 1997). In laboratory
cultures, these toxins appear to be produced only
during the stationary growth phase. In the present
study no clear indications of negative effects due to P.
minimum toxins on the other algae could be found.
However, because P. minimum has a pH limit for
growth which is only slightly higher than that of
Heterocapsa triquetra, it cannot be completely ruled
out that toxins exuded by P. minimum may have
contributed to the decline of the H. triquetra popula-
tion in the non-buffered mixture experiments (Fig. 3).
Nevertheless, it seems justified to suggest that
286
Fig. 5. pH limits of marine phytoplankton obtained in batch
cultures. (A) pH at which the growth rate is reduced by
>20%. (B) pH obtained in stationary growth phase. Data
collected from the literature (Table 3). The black bars refer to
data from Schmidt & Hansen (2001)

Hansen: Effect of pH on phytoplankton growth and survival
elevated pH alone can drive a species succession
among marine phytoplankton.
Very few data are available on the occurrence of the
3 studied species in relation to high pH in nature. How-
ever, in Mariager Fjord the dinoflagellates Hetero-
capsa triquetra and Prorocentrum minimum usually
form almost mono-specific blooms during summer
periods (Fenchel 1995, Fallesen et al. 2000, Olesen
2001), in which pH is extremely high (pH > 9.2, this
data set). Similar observations have been made in the
coastal Santo André Lagoon, SW Portugal (Macedo et
al. 2001), where almost mono-specific blooms of P.
minimum co-occur with pH of 9.5 to 9.6. It is note-
worthy that the pH-sensitive Ceratium species are
completely lacking in both Mariager Fjord and Santo
André Lagoon (Fenchel et al. 1995, Macedo et al. 2001,
Olesen 2001), although Ceratium spp. are common in
waters just outside these areas (e.g. Taylor &
Pollingher 1987).
It is evident from the compiled data on pH limits for
marine phytoplankton growth that the ability to toler-
ate high pH is not related to any particular algal
groups, but rather is species-specific (Table 3). A few
studies have indicated that pH in nature in fact may be
associated with certain groups of phytoplankton. Yoo
et al. (1991) performed a correlation analysis between
dinoflagellate abundance and environmental para-
meters in Masan Bay, Korea, which suggested that pH
was the main factor influencing dinoflagellate abun-
dance. Hinga (1992) found that high abundance of
dinoflagellates was strongly correlated with high pH,
whereas high abundance of diatoms was not. Similarly,
in the Santo André Lagoon, blooms of dinoflagellates
(Prorocentrum) have co-occurred with pH of 9.5 to 9.6
(Macedo et al. 2001). At slightly lower pH (<9.1 to 9.4),
the phytoplankton community in this lagoon was more
diverse, and other phytoplankton groups co-domi-
nated. Thus, although a few reports suggest that
mainly dinoflagellates are associated with extremely
high pH in nature, our knowledge on the topic is still
very limited, and other groups of algae, especially
bloom-forming species, may in the future indeed be
found to be associated with high pH.
CONCLUSIONS
pH can rise to very high levels in eutrophic estuaries,
lagoons and embayments during summers with high
insolation and calm weather. In such environments, pH
changes may drive species succession of phytoplank-
ton and impede primary production, because many
species are quite sensitive to high pH. The reason for
the growth reductions of phytoplankton at high pH
needs yet to be assessed.
Acknowledgements. I thank the counties of Northern Jutland
and Aarhus for allowing me to use their data on pH from
Mariager Fjord. I am indebted Marina Madsen, Bo Sørensen
and Lena Malmberg for technical assistance. I would like to
thank Kaj Sand-Jensen for his constructive criticism of and
suggestions for the manuscript. The work was funded by both
the Danish Natural Research Council project no. 9801391 and
the European Commissions Environment & Sustainable
Development (ESD), (FP-V, Research into the Development of
Sustainable Marine Ecosystems, Key Action 3) under contract
EVK3-CT-1999-00015 BIOHAB (Biological Control of Harm-
ful Algal Blooms in European Coastal Waters).
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288
Editorial responsibility: David Caron,
Los Angeles, California, USA
Submitted: January 2, 2002; Accepted: April 22, 2002
Proofs received from author(s): June 27, 2002