Review paper 9 pages

REVIEW

TESTING THE EFFECTS OF OCEAN ACIDIFICATION ON ALGAL METABOLISM:
CONSIDERATIONS FOR EXPERIMENTAL DESIGNS1

Catriona L. Hurd,2 Christopher D. Hepburn

Department of Botany, University of Otago, PO Box 56, Dunedin 9054, New Zealand

Kim I. Currie

National Institute for Water and Atmospheric Research Ltd., Centre of Excellence for Chemical and Physical Oceanography,

Department of Chemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand

John A. Raven

Division of Plant Sciences, Scottish Crop Research Institute, University of Dundee at SCRI, Invergowrie, Dundee DD2 5DA, UK

and Keith A. Hunter

Department of Chemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand

Ocean acidification describes changes in the car-
bonate chemistry of the ocean due to the increa-
sed absorption of anthropogenically released CO2.
Experiments to elucidate the biological effects of
ocean acidification on algae are not straightforward
because when pH is altered, the carbon speciation
in seawater is altered, which has implications for
photosynthesis and, for calcifying algae, calcifica-
tion. Furthermore, photosynthesis, respiration, and
calcification will themselves alter the pH of the sea-
water medium. In this review, algal physiologists
and seawater carbonate chemists combine their
knowledge to provide the fundamental information
on carbon physiology and seawater carbonate chem-
istry required to comprehend the complexities of
how ocean acidification might affect algae metabo-
lism. A wide range in responses of algae to ocean
acidification has been observed, which may be
explained by differences in algal physiology, time-
scales of the responses measured, study duration,
and the method employed to alter pH. Two meth-
ods have been widely used in a range of experimen-
tal systems: CO2 bubbling and HCl ⁄ NaOH
additions. These methods affect the speciation of
carbonate ions in the culture medium differently; we
discuss how this could influence the biological
responses of algae and suggest a third method based
on HCl ⁄ NaHCO3 additions. We then discuss eight
key points that should be considered prior to setting
up experiments, including which method of manipu-
lating pH to choose, monitoring during experiments,
techniques for adding acidified seawater, biological

side effects, and other environmental factors. Finally,
we consider incubation timescales and prior condi-
tioning of algae in terms of regulation, acclimation,
and adaptation to ocean acidification.

Key index words: algae; bicarbonate; calcium car-
bonate; carbon; carbon dioxide; climate change;
ocean acidification; phytoplankton; seawater car-
bonate system; seaweed

Abbreviations: AT, total alkalinity; CA, carbonic
anhydrase; CCM, carbon-concentrating mecha-
nism; CT, total inorganic carbon; pCO2, partial
pressure of CO2(g)

The term ‘‘ocean acidification’’ describes changes
in the carbonate chemistry of the ocean due to
increased CO2 absorption since the Industrial Revo-
lution (The Royal Society 2005, Doney et al. 2009).
Phytoplankton and macroalgae have key ecological
roles as primary producers in coastal and open
oceans, supplying fixed carbon to the entire marine
food web, recycling nutrients, and modifying global
climate (Smith 1981, Duggins et al. 1989, Field et al.
1998, Gattuso et al. 1998a, Zondervan 2007). Addi-
tionally, calcareous algae (e.g., planktonic cocco-
lithophores and benthic calcifying macroalgae) are
a major source of marine carbonates and sediments
(Gattuso et al. 1998b, Feely et al. 2004, Balch et al.
2007), and the coralline macroalgae fulfill impor-
tant ecological processes, including reef building
(Adey 1998, Chisholm 2003), and are the preferred
sites for settlement of invertebrate larvae (Roberts
2001).

A major focus of research into ocean acidification
has been the effects on calcifying organisms

1Received 28 November 2008. Accepted 6 May 2009.
2Author for correspondence: e-mail [email protected].

ac.nz.

J. Phycol. 45, 1236–1251 (2009)
� 2009 Phycological Society of America
DOI: 10.1111/j.1529-8817.2009.00768.x

1236

(animals and algae). Elevated seawater CO2 concen-
trations will lower carbonate saturation states, which
in turn may reduce the ability of calcifiers to main-
tain existing, and build new, carbonate skeletons
(Bijma et al. 1999, Riebesell et al. 2000, Orr et al.
2005, Shirayama and Thornton 2005, De’ath et al.
2009). These effects will occur in high latitudes first;
for example, in Southern Ocean surface waters,
undersaturation of aragonite is predicted between
2030 and 2038 (McNeil and Matear 2008). Such
reduced ability to calcify may decrease their compet-
itive fitness (Kuffner et al. 2007, McNeil and Matear
2008). In addition to influencing calcification, the
changed speciation of dissolved inorganic carbon in
seawater and decreased pH via the carbonate buffer
system, and the differing abilities of algae to utilize
CO2 and HCO3

), ocean acidification has the poten-
tial to affect the metabolism and growth rates of all
algae, both noncalcareous and calcareous.

Ocean acidification is an emerging field of
research. Experiments to elucidate the biological
effects of increased CO2 on marine organisms are
not straightforward because when pH is altered, the
carbon speciation in seawater is modified, which has
strong implications for photosynthesis, respiration,
and calcification. Furthermore, these same three
metabolic processes themselves alter the pH of sea-
water medium surrounding the algae. Therefore, an
understanding of both seawater carbonate chemistry
and physiological processes related to carbon metab-
olism and calcification is required to design experi-
ments without artifacts, which can be carefully
replicated and test the impacts of increased CO2
(i.e., lowered pH) on algae. This review is in two
sections. In the first section, we highlight key
aspects of seawater carbonate chemistry, algal car-
bon acquisition, and calcification and consider the
wide range of biological responses by different algae
to ocean acidification. On the basis of this appraisal,
we consider reasons for the observed broad range
of physiological responses, which include physiology
of different species, timescales of studies, and tech-
niques used to modify seawater pH (CO2 bubbling
vs. HCl ⁄ NaOH additions). In section two, we focus
on how bubbling with CO2 and adding HCl ⁄ NaOH
each modifies the carbonate chemistry of seawater
during incubation studies, discuss the probable bio-
logical responses of algae to each technique, and
recommend a series of steps that should be consid-
ered when designing experiments to test the effects
of ocean acidification on algae.

Carbon chemistry and biological responses to its manip-
ulation. Seawater carbonate chemistry: Since the Indus-
trial Revolution, levels of CO2 in the atmosphere
have increased at rates 100-fold greater than prein-
dustrial times and have caused a rise in atmospheric
CO2 levels from 280 to 384 ppmv (Solomon et al.
2007). CO2 in the atmosphere is increasing at rates
faster than that predicted as a ‘‘worst-case scenario’’

by the International Panel for Climate Change
(IPCC) in 2000 (Raupach et al. 2007). The world’s
oceans have absorbed up to 50% of anthropogeni-
cally derived CO2, and modeling studies suggest a
0.1 unit decline in surface seawater pH since 1750
(Caldiera and Wickett 2003).

In seawater, free CO2(aq) is in equilibrium with a
small concentration of the carbonic acid species
H2CO3, but it is conventional to regard both species
as stoichiometrically equivalent with respect to sub-
sequent acid-base reactions and denote this combi-
nation by a hypothetical species H2CO3

* where
[H2CO3

*] = [H2CO3] + [CO2(aq)]. The effects of
ocean acidification cannot be described in a simple
way using just the parameter pH, and it is necessary
to consider the effect of CO2 uptake on the entire
CO2 equilibrium system. CO2 in the gas phase equil-
ibrates with H2CO3

* in seawater through the well-
known Henry’s law equilibrium:

CO2ðgÞ$ H2CO3� KH ¼
½H2CO3��

pCO2
ð1Þ

where pCO2 is the partial pressure of CO2(g) and
KH, the Henry’s law equilibrium constant, is a func-
tion of temperature (T) and salinity (S). This rela-
tionship means that at a given T and S, pCO2 and
[H2CO3

*] are linearly related to each other. It is
most common to use pCO2 as a parameter because
this allows a simple comparison with the actual
atmospheric CO2 partial pressure when air and
water are not in equilibrium.

The acid dissociation reactions of H2CO3
* are as

follows:

H2CO3
�$HCO3�þHþ K1¼

½HCO3��½Hþ�
½H2CO3��

ð2Þ

HCO3
�$CO32�þHþ K2¼

½CO32��½Hþ�
½HCO3��

ð3Þ

where HCO3
) and CO3

2) are the bicarbonate and car-
bonate ions, respectively, and K1 and K2 are the first
and second acid dissociation constants of H2CO3

*.
Equations (2) and (3) show that the concentrations
of the three CO2 species and that of H

+ are inextrica-
bly linked, meaning that it is physically impossible to
vary systematically any one of these while at the same
time holding all of the others constant. Equation (1)
shows that this relationship also extends to pCO2.
This fact complicates understanding the underlying
chemistry affecting ocean acidification.

Concentrations of the individual CO2 species in
seawater cannot be directly measured. Instead,
changes in the speciation of the CO2 system in sea-
water are normally described, and measured, using
the following two parameters (Mackenzie and
Lerman 2006): (i) Total dissolved CO2 (usually
symbolized as CT for the total inorganic carbon in
solution or DIC for dissolved inorganic carbon),

O C E A N A C I D I F I C A T I O N A N D A L G A L M E T A B O L I S M 1237

which is the stoichiometric sum of all dissolved inor-
ganic carbon species

C T ¼ ½H2CO3��þ ½HCO3��þ ½CO32�� ð4Þ

(ii) Total alkalinity, AT, which is the total concentra-
tion of titratable weak bases in seawater relative to
the reference proton condition comprising pure
CO2 in seawater

AT¼½HCO3��þ2½CO32��þ½OH���½Hþ�þð…Þ ð5Þ
where (…) represents various minor acid-base spe-
cies, such as borate ion. Both these parameters have
the advantage of being independent of changes in
temperature and pressure and are conserved during
the mixing of different seawater masses. Useful soft-
ware programs for calculation of CO2 speciation in
seawater have been presented by Lewis and Wallace
(1998) and Hunter (2007).

For calcification, the removal of CO3
2) ions by

precipitation of calcium carbonate (CaCO3) causes
HCO3

) ions to dissociate to restore the CO3
2) ions

lost. The H+ ion released by this dissociation gener-
ates additional H2CO3

* by combining with a second
HCO3

) ion (Frankignoulle and Canon 1994). The
overall stoichiometric change is therefore:

Ca2þ þ 2HCO3� ! CaCO3ðsÞþ H2CO3� ð6Þ

At today’s pH (�8.07), 91% of CT is as bicarbon-
ate ions (2,200 lM), 1% as H2CO3

* (14 lM), and
8% as CO3

2). The predicted decrease to pH 7.65 by
2100 will result in a 300% increase in H2CO3

* con-
centration, a 9% increase in that of HCO3

), and a
56% decrease in that of CO3

2) (from table 1 of The
Royal Society 2005). These changes will affect the
ability of algae to acquire carbon and ⁄ or produce
and maintain calcium carbonate structures.

Physiological basis for algal carbon acquisition and
calcification: Most marine algae can acquire the
CO2 required as a substrate for RUBISCO via active
uptake from seawater of CO2 and ⁄ or bicarbonate;
the active transport of either of these species, or in
some cases of protons, constitutes a carbon-concen-
trating mechanism (CCM; Giordano et al. 2005).
The photosynthetic rates of algae that have CCMs
are not generally carbon limited under most envi-
ronmental conditions (Giordano et al. 2005). Some
bicarbonate-using algae convert HCO3

) to CO2
using extracellular carbonic anhydrase (CA); the
CO2 then enters the cell by active transport or by
diffusion (if there are zones of surface acidification
where the steady-state CO2 concentration exceeds
that in the medium). Other bicarbonate-using algae
with CCMs actively take up the HCO3

) ion across
the cell membrane, and CA acts intracellularly.
Some algae (e.g., some dinoflagellates) have little or
no capacity to use bicarbonate, and their CCM relies
on active CO2 uptake (Dason et al. 2004). CA syn-
thesis and CCM activities in eukaryotes are con-

trolled by the concentration of H2CO3
*, in the few

cases examined (Giordano et al. 2005).
Energy and nutrients are required to operate

active transport and to make the CCMs (generally
including CAs), whereas the alternative of diffusive
H2CO3

* has energy and nutrient costs of operating
photorespiration and making the relevant enzymes
and additional RUBISCO (Raven et al. 2000). Some
algae adapted to low light levels lack CCMs and rely
on diffusive H2CO3

* entry (e.g., the red seaweed
Lomentaria, Kübler et al. 1999). Under low irradianc-
es, energy limitation outweighs limitation by CO2,
and the use of diffusive H2CO3

* entry has energetic
advantages (see Raven et al. 2000, 2005). Growth at
low irradiances cannot explain all cases of the
absence of detectable CCMs; for example, some
strains of coccolithophores rely on diffusive uptake
of H2CO3

* and cannot utilize HCO3
) or carry out

active CO2 transport (Nimer and Merrett 1993).
Algae that rely on H2CO3

* diffusion alone are gen-
erally carbon-limited under today’s seawater concen-
trations (Kübler et al. 1999).

Calcification is the biogenic formation of calcium
carbonate (Borowitzka 1987). The most common
forms of calcium carbonate (CaCO3) synthesized by
algae are calcite, aragonite, or high-magnesium cal-
cite (Adey 1998). High magnesium calcite is the
most soluble form of these three (Chave et al.
1962), and therefore algae with high-magnesium
calcite are most susceptible to predicted decreases
in pH due to ocean acidification (The Royal Society
2005). Algae have a range of mechanisms for calcify-
ing. For example, coccolithophores produce calcite
coccoliths intracellularly and extrude them to the
cell’s surface, whereas in the tropical green seaweed
Halimeda, aragonite mineralizes in the intercellular
space between tightly appressed utricles, and red
coralline seaweeds deposit high-magnesium calcite
into their cell walls (Borowitzka 1987).

Algae themselves modify the pH of seawater.
When algae photosynthesize, the removal of CO2 in
assimilation by RUBISCO usually occurs faster than
CO2 can be resupplied from the atmosphere or dee-
per water, so that there is a reequilibration among
the inorganic species that yields a decrease in
HCO3

) and an increase in CO3
2) and pH. In nat-

ure, this results in significant pH increases most
especially in isolated habitats like high-intertidal
rock pools (Midelboe and Hansen 2007) but also in
coastal waters (Hinga 2002). Indeed, such effects
are the principle underpinning pH-drift experi-
ments where a rapid increase in pH as a result of
photosynthesis is used to elucidate mechanisms of
carbon acquisition by seaweeds and microalgae
(Maberly 1990, Chen et al. 2006). Calcification and
respiration alter the seawater carbonate system in
ways that decrease the pH of the culture medium.
Respiration results in CO2 being released into the
surrounding medium, and when algae are in the
dark, the pH of the culture medium will decline.

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O C E A N A C I D I F I C A T I O N A N D A L G A L M E T A B O L I S M 1241

Calcification also results in CO2 production (see
eq. 6).

Biological responses of algae to pH manipulation: The
goal of most experiments investigating ocean acidifi-
cation is to increase the concentration of H2CO3

*

and decrease the pH in seawater to mimic the
increase in H2CO3

* predicted to occur as the ocean
takes up anthropogenic carbon and examine the
effects (physiological, ecological, biogeochemical)
of such manipulations on algae. Two techniques
have been used to manipulate seawater pH in the
majority of biological perturbation experiments:
CO2 bubbling and HCl ⁄ NaOH additions. These
have been used in a variety of experimental setups
over various incubation timescales (Tables 1 and 2).

Across the range of calcareous and noncalcareous
algae tested, there are no clear patterns regarding
the responses of primary production, growth, or cal-
cification rates to ocean acidification (Tables 1 and
2). The growth rate of some species is unchanged
by altered CO2 treatments—for example, Thalassios-
ira pseudonana (Pruder and Bolton 1980), four dia-
toms and one dinoflagellate (Burkhardt et al.
1999), the coccolithophores Calcidiscus leptoporus
and Coccolithus pelagica (Langer et al. 2006), and
Emiliania huxleyi (Feng et al. 2008). For T. pseudo-
nana, the lack of change in growth rate following
CO2 treatment is consistent with inorganic carbon
concentrations being saturating for growth (Clark
and Flynn 2000). Species that responded to
CO2 ⁄ pH treatments include E. huxleyi, in which
increased H2CO3

* concentrations resulted in
increased organic carbon content per cell, but no
increase in the number of cells (Leonardos and
Geider 2005, Iglesias-Rodriguez et al. 2008a).
Growth rates of Antarctic phytoplankton assem-
blages were also affected by pCO2, but the response
to treatments varied depending on the time of year
when the experiment was conducted (Tortell et al.
2008). The marine diazotrophic cyanobacterium
Trichodesmium also demonstrated significant
increases in the rate of carbon assimilation (and of
diazotrophic nitrogen assimilation) with substantial
increases in CO2 concentration, in each of three
studies (Barcelos e Ramos et al. 2007, Hutchins
et al. 2007, Levitan et al. 2007). Similar results were
reported for the unicellular marine diazotrophic
cyanobacterium Crocosphaera under iron-sufficient,
but not iron-limiting conditions (Fu et al. 2008).

For calcareous E. huxleyi and Ca. leptoporus, there
was a decrease in rates of calcification and ⁄ or mal-
formed coccoliths at pCO2 values greater (or lower)
than the present day; however, for Co. pelagica, there
was no effect of pCO2 treatment on lith formation
(Riebesell et al. 2000, Langer et al. 2006). Interest-
ingly, when nanofossil records from cores from the
last glacial maximum (�18,000 years ago, atmo-
spheric CO2 180–200 lmol Æ mol

)1 total gas) were
examined, there was no evidence of malformed or
incomplete liths for Ca. leptoporus or Co. pelagica;

Langer et al. (2006) suggest adaptation (see below
for definition) by these species to the pCO2 environ-
ment they inhabit. Iglesias-Rodriguez et al. (2008a)
found no decrease in calcification or lith malforma-
tion in E. huxleyi grown at pCO2 higher than the
present day.

Relatively few studies have determined the likely
impact of elevated CO2 on calcareous and noncal-
careous macroalgae. Earlier works (unrelated to
ocean acidification) used manipulations of seawater
pH and carbon chemistry to unravel mechanisms of
carbon acquisition and calcification (Smith and
Roth 1979, Borowitzka 1981, Gao et al. 1993). As for
phytoplankton, there are a wide range of responses
by macroalgae to elevated CO2 concentration. A
52% increase in growth in response to a doubling
of pCO2 was observed for Lomentaria, a species that
uses only CO2 (Kübler et al. 1999). This increase in
growth rate is consistent with the idea that algae
without CCMs are likely to respond to increased
pCO2. Some macroalgal studies have suggested a
negative effect of acidification on particular species,
while other species show positive or no response to
elevated CO2 (Israel et al. 1999, Israel and Hophy
2002, Swanson and Fox 2007). Tropical macroalgal
assemblages have shown positive influences of ele-
vated CO2 on recruitment of noncalcifying macroal-
gae, while inhibiting recruitment of corallines
(Kuffner et al. 2008). It is not clear if these differ-
ences in response were due to reduced survivorship
or competitive ability of calcifying recruits, and ⁄ or
increased competitive ability of noncalcifying algae,
or other factor(s) (Kuffner et al. 2008). Hall-Spen-
cer et al. (2008) demonstrated that within 120 m of
cold CO2 vents (average pH 7.83), macroalgal com-
munities are dominated by fleshy seaweeds, whereas
calcareous seaweeds dominated farther from the
vent (average pH 8.14).

Methods: an appraisal. There are clearly a range
of biological responses to pH manipulation treat-
ments. …