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Hurd2009.pdf
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.
1238 C A T R I O N A L . H U R D E T A L .
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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 1239
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o n
, 2
-w e e k
e x
p e ri
m e n
t. p
H =
6 .0
, 6
.5 ,
7 .0
, 8
.7 N
o t
cl e a r
N o
cl e a r
d if
fe re
n ce
s w
it h
in re
a li
st ic
ra n
g e
(7 .5
– 8
).
Is ra
e l
e t
a l.
(1 9
9 9
)
C o
n st
a n
t a e ra
ti o
n w
it h
C O
2 ⁄a
ir m
ix ,
4 0
L cu
lt u
re ta
n k
s, o
th e r
co n
d it
io n
s n
o t
st a te
d .
3 w
e e k
s C
o n
tr o
l =
3 6
0 m
g Æ
L )
1
T re
a tm
e n
t =
7 5
0 m
g Æ
L )
1
n =
3 5
0 %
re d
u ct
io n
in g
ro w
th a t
h ig
h C
O 2 .
1240 C A T R I O N A L . H U R D E T A L .
T a
b l e
1 .
C o
n ti
n u
e d
.
S p
e ci
e s
⁄s ys
te m
M e th
o d
s T
im e
fr a m
e p
H ⁄p
C O
2
tr e a tm
e n
ts R
e p
li ca
ti o
n R
e su
lt s
A u
th o
rs
1 4
g re
e n
, re
d ,
a n
d b
ro w
n m
a cr
o a lg
a e
C o
n st
a n
t a e ra
ti o
n w
it h
C O
2 ⁄a
ir m
ix ,
4 0
L cu
lt u
re ta
n k
s, 0
.2 m
M p
h o
sp h
a te
, 2
m M
a m
m o
n iu
m .
4 – 1
5 w
e e k
s C
o n
tr o
l =
3 6
0 l
m o
l Æ
m o
l) 1
T re
a tm
e n
t =
7 5
0 l
m o
l Æ
m o
l) 1
N o
t cl
e a r
N o
cl e a r
e ff
e ct
o f
e le
va te
d C
O 2
o n
g ro
w th
o r
p h
o to
sy n
th e si
s. L
o w
p H
m a y
h a ve
d a m
a g
e d
so m
e sp
e ci
e s.
Is ra
e l
a n
d H
o p
h y
(2 0
0 2
)
P u
re C
O 2
in je
ct e d
a t
th e
b a se
m e sh
b a g
s co
n ta
in in
g se
a w
e e d
s in
si tu
, a ve
ra g
e 1
,4 9
0 l
m o
l p
h o
to n
s Æ
m )
2 Æ
s) 1 ,
2 8
�C ,
a m
b ie
n t
n u
tr ie
n ts
.
5 d
C o
n tr
o l
p H
= 8
.1 8
– 8
.1 4
T re
a tm
e n
t p
H =
7 .4
– 7
.6 3
N o
t cl
e a r
N o
cl e a r
e ff
e ct
s o
f e le
va te
d C
O 2 .
S a cc
h a ri
n a
la ti
ss im
a a n
d N
er eo
cy st
is lu
et ke
a n
a
C o
n st
a n
t a e ra
ti o
n w
it h
C O
2 ⁄a
ir m
ix (3
,0 0
0 p
p m
) d
ir e ct
ly in
o u
td o
o r
7 5
0 L
ta n
k s,
m a x
. 2
,2 0
0 l
m o
l p
h o
to n
s Æ
m )
2 Æ
s) 1 ,
1 1
�C – 1
5 �C
, a m
b ie
n t
n u
tr ie
n ts
.
5 5
d C
o n
tr o
l p
H �
8 .0
2 – 8
.1 6
, tr
e a tm
e n
t �
7 .1
7 – 7
.9 9
a t
m id
d a y.
p H
co u
ld b
e a s
m u
ch a s �
0 .8
u n
it s
lo w
e r
a t
d a w
n .
P se
u d
o -r
e p
li ca
te d
3 ·
in cr
e a se
d g
ro w
th in
S .
la tt
is m
a ,
4 ·
d e cr
e a se
in N
. lu
et ke
a n
a .
S w
a n
so n
a n
d F
o x
(2 0
0 7
)
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. This observed spectrum of responses may be due to inherent differences in algal physiology, the environment in which the algae have grown prior to experiments (e.g., light climate), the timescale of the physiological response measured (e.g., short- term estimates of photosynthesis vs. integrated growth), duration of the study (days vs. months), or time of year (Tortell et al. 2008). Another key influ- ential factor could be the method of pH manipula- tion because CO2 bubbling and HCl ⁄ NaOH affect carbonate chemistry differently (see below); there have been heated discussions on which method is most suitable (Iglesias-Rodriguez et al. 2008a,b, Riebesell et al. 2008). Critically, it is difficult to tease apart the relative importance of each of these potentially influential factors on the outcome of experiments. Here, we focus on the different ways in which carbonate chemistry is altered during pH manipulation experiments and how this might
1242 C A T R I O N A L . H U R D E T A L .
T a
b l e
2 .
C a lc
a re
o u
s a lg
a e .
E x
a m
p le
s o
f e x
p e ri
m e n
ts co
n d
u ct
e d
o n
e ff
e ct
s o
f C
O 2
co n
ce n
tr a ti
o n
o n
a lg
a l
g ro
w th
, ca
lc ifi
ca ti
o n
, o
r m
e ta
b o
li c
ra te
s. U
n it
s fr
o m
th e
o ri
g in
a l
p a p
e rs
a re
re p
o rt
e d
.
S p
e ci
e s
⁄s ys
te m
M e th
o d
s T
im e
fr a m
e p
H ⁄p
C O
2
tr e a tm
e n
ts R
e p
li ca
ti o
n R
e su
lt s
A u
th o
rs
P h
yt o
p la
n k
to n
C o
cc o
li th
o p
h o
re s
E m
il ia
n ia
h u
xl ey
i a n
d G
ep h yr
oc a p sa
oc ea
n ic
a
H C
l⁄ N
a O
H ,
d il
u te
b a tc
h cu
lt u
re ,
3 0
– 1
5 0
l m
o l
p h
o to
n s
Æ m
) 2
Æ s)
1 ,
1 6
:8 o
r 2
4 :0
L :D
, 1
5 �C
, 1
0 0
l M
n it
ra te
, 6
.5 l
M p
h o
sp h
a te
, f ⁄2
tr a ce
e le
m e n
ts .
A cc
li m
a ti
o n
fo r
7 – 9
g e n
e ra
ti o
n s
th e
8 ce
ll d
iv is
io n
s d
u ri
n g
e x
p e ri
m e n
t.
p C
O 2
= 2
8 0
– 7
5 0
. n
= 3
R e d
u ce
d ca
lc ifi
ca ti
o n
o f
1 5
.7 %
in E
. h u
xl ey
i a n
d 4
4 .7
% in
G .
oc ea
n ic
a .
M a lf
o rm
e d
co cc
o li
th s,
sl ig
h t
in cr
e a se
in p
h o
to sy
n th
e si
s in
b o
th .
R ie
b e se
ll e t
a l.
(2 0
0 0
)
N o
rt h
P a ci
fi c
p h
yt o
p la
n k
to n
a ss
e m
b la
g e s
B u
b b
li n
g w
it h
C O
2 a ir
m ix
tu re
s o
r H
C l⁄
N a O
H ,
sh ip
b o
a rd
, 3
0 %
in ci
d e n
t ir
ra d
ia n
ce ,
1 3
�C ,
n u
tr ie
n t
co n
ce n
tr a ti
o n
s n
o t
re p
o rt
e d
.
1 .5
– 9
d p C
O 2
= 2
5 0
, p
H =
8 .2
a n
d p C
O 2
= 8
0 0
, p
H 7
.7 5
n =
3 S
ig n
ifi ca
n t
re d
u ct
io n
s (3
6 %
– 8
3 %
) in
ca lc
ifi ca
ti o
n in
4 in
d e p
e n
d e n
t e x
p e ri
m e n
ts .
N o
rt h
A tl
a n
ti c
p h
yt o
p la
n k
to n
a ss
e m
b la
g e s
fr o
m su
rf a ce
a n
d d
e p
th
H C
l⁄ N
a O
H ,
sh ip
b o
a rd
, 3
5 0
a n
d 5
0 l
m o
l p
h o
to n
s Æ
m )
2 Æ
s) 1 ,
a m
b ie
n t
te m
p e ra
tu re
, n
u tr
ie n
t co
n ce
n tr
a ti
o n
s n
o t
re p
o rt
e d
.
2 h
in cu
b a ti
o n
3 ,
1 0
, 3
6 ,
a n
d 9
1 l
M C
O 2
n =
3 S
ig n
ifi ca
n t
in cr
e a se
in 1 4 C
in co
rp o
ra ti
o n
w it
h 3
6 a n
d 9
1 l
M tr
e a tm
e n
t co
m p
a re
d w
it h
1 0
l M
. D
e cr
e a se
fo r
3 l
M tr
e a tm
e n
t.
H e in
a n
d S
a n
d -J
e n
se n
(1 9
9 7
)
E m
il ia
n ia
h u
xl ey
i (c
a lc
if yi
n g
st ra
in )
C O
2 b
u b
b li
n g
, se
m ic
o n
ti n
u o
u s
cu lt
u re
, 5
0 a n
d 4
0 0
l m
o l
p h
o to
n s
Æ m
) 2
Æ s)
1 ,
2 0
�C a n
d 2
4 �C
, f ⁄2
n u
tr ie
n ts
.
A cc
li m
a ti
o n
in tr
e a tm
e n
t co
n d
it io
n s
fo r
7 g
e n
e ra
ti o
n s.
p C
O 2
= 3
5 7
a n
d 7
5 0
p p
m n
= 3
N o
e ff
e ct
o f
p C
O 2
o n
g ro
w th
ra te
o r
o rg
a n
ic C
p e r
ce ll
a t
g iv
e n
ir ra
d ia
n ce
a n
d te
m p
e ra
tu re
tr e a tm
e n
t. P
IC w
a s
re d
u ce
d in
th e
h ig
h li
g h
t ⁄7
5 0
p p
m tr
e a tm
e n
t.
F e n
g e t
a l.
(2 0
0 8
)
C a lc
id is
cu s
le p to
p or
u s
a n
d C
oc cl
it h u
s p el
a gi
cu s
H C
l⁄ N
a O
H ,
d il
u te
b a tc
h cu
lt u
re s,
3 5
0 l
m o
l p
h o
to n
s Æ
m )
2 Æ
s) 1 ,
1 6
:8 L
:D ,
2 0
�C fo
r C
. le
p to
p or
u s,
1 7
�C fo
r C
. p el
a gi
ca ,
1 0
0 l
M n
it ra
te ,
6 .5
l M
p h
o sp
h a te
, f ⁄2
tr a ce
e le
m e n
ts .
A cc
li m
a ti
o n
in tr
e a tm
e n
t co
n d
it io
n s
fo r
1 0
g e n
e ra
ti o
n s.
C .
le p to
p or
u s:
6 p C
O 2
tr e a tm
e n
ts fr
o m
9 8
– 9
2 0
l a tm
. C
. p el
a gi
ca :
3 tr
e a tm
e n
ts 1
4 9
, 3
4 5
, a n
d 9
1 5
l a tm
.
N o
t cl
e a r
C .
le p to
p or
u s:
m a lf
o rm
e d
co cc
o li
th s
a t
lo w
a n
d h
ig h
p C
O 2
tr e a tm
e n
ts co
m p
a re
d to
co n
tr o
l (3
4 5
p p
m ).
C .
p el
a gi
cu s:
n o
ch a n
g e
in li
th m
o rp
h o
lo g
y. B
o th
sp e ci
e s:
n o
e ff
e ct
o f
C O
2 o
n p
o p
u la
ti o
n g
ro w
th ra
te b
u t
in cr
e a se
d o
rg a n
ic co
n te
n t
o f
ce ll
s u
n d
e r
h ig
h C
O 2
tr e a tm
e n
ts .
L a n
g e r
e t
a l.
(2 0
0 6
)
E m
il ia
n ia
h u
xl ey
i C
O 2
b u
b b
li n
g ,
d il
u te
b a tc
h cu
lt u
re s,
1 5
0 l
m o
l p
h o
to n
s Æ
m )
2 Æ
s) 1 ,
1 2
:1 2
L :D
, 1
9 �C
, f ⁄2
m e d
iu m
.
A lg
a e
a cc
li m
a te
d a t
e x
p e ri
m e n
ta l
co n
d it
io n
s fo
r 9
g e n
e ra
ti o
n s.
C O
2 =
2 8
0 ,
3 0
0 ,
4 9
0 ,
6 0
0 ,
7 5
0 p
p m
v
n =
6 D
o u
b li
n g
in p
a rt
ic u
la te
in o
rg a n
ic ca
rb o
n a n
d p
a rt
ic u
la te
o rg
a n
ic ca
rb o
n ,
a n
d d
e cr
e a se
in g
ro w
th ra
te s
a t
7 5
0 co
m p
a re
d to
2 8
0 p
p m
v. N
o e ff
e ct
o f
d o
u b
le d
C O
2 o
n li
th m
o rp
h o
lo g
y.
Ig le
si a s-
R o
d ri
g u
e z
e t
a l.
(2 0
0 8
a )
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 1243
T a
b l e
2 .
C o
n ti
n u
e d
.
S p
e ci
e s
⁄s ys
te m
M e th
o d
s T
im e
fr a m
e p
H ⁄p
C O
2
tr e a tm
e n
ts R
e p
li ca
ti o
n R
e su
lt s
A u
th o
rs
M a c ro
a lg
a e
C or
a ll
in a
p il
u li
fe ra
C o
n st
a n
t a e ra
ti o
n w
it h
C O
2 ⁄a
ir m
ix ,
1 5
cm h
ig h
w it
h 1
4 cm
d ia
m e te
r 2
0 �C
, 3
0 l
m o
l p
h o
to n
s Æ
m )
2 Æ
s) 1 .
1 2
h p C
O 2
= 3
5 0
, p
H 8
.2 a n
d p C
O 2
1 ,5
0 0
a n
d p
H 7
.6
P se
u d
o -
re p
li ca
te d
4 4
% re
d u
ct io
n in
ca lc
ifi ca
ti o
n .
G a o
e t
a l.
(1 9
9 3
)
C ru
st o
se C
or a ll
in e
re cr
u it
s
In je
ct io
n o
f 1
0 %
H C
l⁄ fr
e sh
w a te
r m
ix a t
in fl
o w
to 5
0 0
L o
u td
o o
r m
e so
co sm
s, a m
b ie
n t
ir ra
d ia
n ce
, se
a w
a te
r te
m p
e ra
tu re
, a n
d n
u tr
ie n
ts .
7 w
e e k
s C
o n
tr o
l p C
O 2
= 4
0 0
l a tm
, p
H 8
.1 7
a n
d tr
e a tm
e n
t p C
O 2
= 7
6 5
l a tm
, p
H 7
.9 1
a t
m id
d a y.
D iu
rn a l
fl u
ct u
a ti
o n
s o
f p
H o
f �
0 .5
u n
it s.
n =
3 M
a rk
e d
re d
u ct
io n
in re
cr u
it m
e n
t a n
d %
co ve
r o
f cr
u st
o se
co ra
ll in
e s
in h
ig h
- C
O 2
tr e a tm
e n
t. In
cr e a se
in n
o n
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1244 C A T R I O N A L . H U R D E T A L .
impact the physiological response of algae. We then consider the steps required to design and imple- ment improved experiments on ocean acidification, and finally, we discuss the implications of a lack of standardization in the other environmental factors on making a direct comparison between the results from different published studies.
Comparison of HCl additions versus CO2 bubbling on seawater carbonate chemistry: The most commonly used means to simulate the effects of acidification involves an initial adjustment of the CO2 speciation of a seawater culture medium to achieve the desired degree of acidification and then maintaining those conditions as growth of the alga(e) proceeds. There are two methods of decreasing seawater pH, by bub- bling the seawater with CO2 gas or by the addition of acid (commonly HCl).
Each method produces a specific target CO2 spe- ciation, but they achieve this in different ways (Langdon 2003). The equilibration of CO2 gas with seawater fixes the equilibrium pCO2 to that of the control gas, without any change in total alkalinity, AT. As a result, CT will increase from its initial value. In contrast, addition of HCl to seawater involves a known decrease in the original AT, but no change in CT.
Figure 1 shows simulated values of pCO2, [HCO3
)], [CO3 2)], and calcite saturation (X)
achieved by acidifying a seawater sample of typical surface-water composition using each method. There is very little difference in the two methods for a given pH with respect to [CO3
2)] and X, but at pH 7.5, pCO2 and HCO3
) are 23% and 22% lower, respectively, using HCl additions compared to CO2 bubbling. This is because when seawater is acidified with HCl, CT remains constant (provided
CO2 does not escape into the gas phase), and so [HCO3
)] increases slightly as a small amount of CO3
2) is converted to HCO3 ) by reaction with H+
ions. However, when CO2(g) is added, CT is increased, mainly in the form of HCO3
). CO2 bubbling is arguably much closer to the
actual ocean acidification that is currently affecting the surface ocean. Here, we suggest a third method that will achieve the same effect without bubbling with CO2, which is by adding, separately, equivalent concentrations of HCl and sodium bicarbonate solu- tions. In this case, the NaHCO3 exactly neutralizes the alkalinity decrease caused by the HCl and sup- plies the increased CT (mostly as HCO3
)) that would be the result of CO2 gas bubbling. While this method has not been used in published biological experiments to date, the use of separate HCl and NaHCO3 additions has the advantage that it avoids any possible physiological effects that bubbling itself might cause (see later). The changes in CO2 specia- tion induced by this method are identical to those achieved by bubbling with CO2 gas, as shown in Figure 1.
How might the method of pH manipulation affect algal physiology? The question is whether the different methods that have been used to manipulate seawa- ter pH could affect the outcome of experiments. For algae without CCMs that rely on diffusive uptake of CO2, the 23% difference in H2CO3
*
between the CO2-bubbling and the HCl-addition methods at pH 7.5 (and indeed a 17% difference at pH 7.75; Fig. 1) could cause significantly lower rates of photosynthesis and growth with the HCl method. For noncalcifying algae with CCMs, the concentra- tion of CO2 at the alga’s surface controls the activity of the CCM and carbonic anhydrase (Giordano et al. 2005); the 23% higher H2CO3
* concentration (with CO2 bubbling) may be sufficient to cause a down-regulation of the CCM and CA relative to the HCl method. Such a down-regulation in the CCM and the CA could result in the reallocation of cellu- lar energy to, for example, more rapid growth.
At the current oceanic pH of �8.1, HCO3) concentrations in seawater are sufficient to saturate photosynthesis in most organisms with CCMs (Giordano et al. 2005). Therefore, for algae with CCMs that utilize bicarbonate, the 22% difference that results from the two methods of pH manipula- tion should not affect photosynthesis per se because HCO3
) is not limiting. The saturation state of cal- cite (X) is key in determining if organisms can lay down calcium carbonate, and this is similar for both methods of pH manipulation (Fig. 1). Whether the 22% difference in HCO3
) concentration between the two methods might influence calcification is not clear.
A direct comparison of the two methods (CO2 bubbling or HCl ⁄ NaOH additions) of CO2 manipu- lation on the responses of algae has not been made. However, the responses of calcifying strains of coc-
Fig. 1. Calculated equilibrium CO2 parameters as a function of seawater pH for a seawater sample having initial CT = 1,900 lmol Æ kg
)1 and AT = 2,300 lmol Æ kg )1, salinity 35
and temperature 10�C, that has been acidified either with strong acid HCl or CO2 gas: (a) equilibrium CO2 partial pressure pCO2, (b) bicarbonate ion concentration [HCO3
)], (c) carbonate ion concentration [CO3
2)], and (d) saturation ratio X for calcite. It is assumed that the HCl addition involves insignificant dilution of the seawater.
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 1245
colithophores to ocean acidification have been tested (by different research groups and often, when the same species was employed, using differ- ent strains) using the two methods. Feng et al. (2008) used the CO2-bubbling method to show that the particulate inorganic carbon (PIC) production rate of E. huxleyi remains unaffected by high CO2 concentration at low irradiances for growth, but it is decreased by high CO2 concentration at high growth irradiances. The particulate organic carbon (POC) per cell increased in high CO2(aq) at the higher growth irradiances. Iglesias-Rodriguez et al. (2008a) also used the CO2-bubbling method on E. huxleyi, working at light saturation, and deter- mined that while increased CO2 concentrations increased the POC and PIC per cell, there was no change in the PIC:POC ratio with increasing CO2, and a significant decrease in the specific growth rate of the cells at the highest CO2 concentration used.
Using the HCl ⁄ NaOH method of CO2 manipula- tion, Riebesell et al. (2000) showed that high CO2 concentration decreased the rate of calcification (on a per cell basis) in E. huxleyi and Gephyrocapsa oceanica, with increased calcification at lower than present CO2 levels. Again, using the HCl ⁄ NaOH technique, Langer et al. (2006) observed no increase in inorganic carbon precipitation by Co. pelagicus by increasing CO2 concentrations from that occurring during the last glacial maximum level to more than twice present-day CO2 levels, while Ca. leptoporus had the highest rate of calcification at the present CO2 level, with lower rates at more than twice the present-day level and an even greater decrease at the last glacial maximum level. Clearly, there are intergeneric variations in the response of calcification to CO2 concentrations in all experi- ments using the HCl ⁄ NaOH method. Whether the difference in response of E. huxleyi to CO2 variations reported by Riebesell et al. (2000), Feng et al. (2008), and Iglesias-Rodriguez et al. (2008a) is a function of the different methods of changing CO2 or of other experimental differences or the use of dif- ferent algal strains awaits further experimentation.
Methodological considerations: Here we discuss eight points that we consider essential in designing experiments that examine the impacts of ocean acidification on algae. Most considerations are rele- vant to studies involving either macroalgae or micro- algae.
1. Which method of manipulating pH to choose? This will depend on the research question being asked. If the question is related to carbon acquisition and rates of photosynthesis and growth, then adding CO2 to shift seawater pH most closely mimics the changes in seawater carbon speciation predicted in the future due to climate change (changing CT, constant alkalinity). For phytoplankton, however, there may be side effects of directly bubbling with CO2, such as damage to fragile phytoplankton through the effects of small-scale turbulence with
increasing sensitivity in the order green algae > cyanobacteria > diatoms > dinoflagellates (Thomas and Gibson 1990). The HCl method is relatively simple to implement and can be used for investigat- ing the effects of changes in pH, H2CO3
*, or CO3 2)
as long as the resulting changes in AT (and lack of change in CT) are of no concern. In addition, the scale of experiment might affect the pH manipula- tion used. For small-scale laboratory cultures and shipboard experiments, pH can be manipulated using CO2 bubbling, but the HCl-addition tech- nique may be more practicable for large-scale meso- cosm experiments (Kuffner et al. 2008). Whichever method is chosen, clear interpretation of physiologi- cal responses of algae to pH manipulation will be facilitated by a thorough understanding of the entire carbonate system in the culture medium. To this end, it is essential to conduct regular monitor- ing of at least two of the carbonate analytical param- eters (see below).
2. Methods of pH control. For CO2 bubbling, pertur- bations can take place using pure CO2 gas (Leclercq et al. 2000, Israel and Hophy 2002), CO2 ⁄ air mix- tures (Gao et al. 1993, Riebesell et al. 2000, Engel et al. 2005), or CO2 mixed with other gases (O2, N2) (the latter to permit an analysis of CO2 ⁄ O2 interaction effects on growth, Kübler et al. 1999). CO2 ⁄ air and gas mixtures at certified concentrations are expensive, and using gas mixers to produce appropriate CO2 ⁄ gas mixtures is an option (Kübler et al. 1999, Engel et al. 2005). Parsons et al. (1992) describe how to build an inexpensive gas-mixing sys- tem that was used effectively to grow a macroalga at a range of pCO2 (Kübler et al. 1999). Careful bub- bling of small amounts of inexpensive food-grade 100% CO2 while monitoring pH is also effective in providing required pH ⁄ CO2 concentration (C. D. Hepburn, K. Currie, and C. L. Hurd, unpublished data).
When using HCl, it is important not to introduce any other perturbation to the system. The HCl should be made up in NaCl solution such that the total ionic strength is similar to that of the seawater, that is, 0.7 M (e.g., 0.1 M HCl and 0.6 M NaCl). The HCl solution should not be contaminated with trace metals if this is important (e.g., in iron-limited waters).
Higher pH and reduced pCO2 (i.e., mimicking the preindustrial era) can also be achieved through bubbling seawater with another gas (e.g., N2), CO2- depleted air (produced by pumping ambient air through Na2CO3 traps) (Leclercq et al. 2000, Engel et al. 2005), or CO2-depleted gas mixtures (Kübler et al. 1999). Due to seawater’s strong buffering capacity, bubbling with CO2-free or CO2-depleted gases can take some time and a significant amount of depleted gas before the required pCO2 is reached. Shifting pH using concentrated NaOH is a faster and straightforward method, but, as for HCl, the resulting carbonate speciation is different from
1246 C A T R I O N A L . H U R D E T A L .
that which occurs in situ in response to changing pCO2.
3. Monitoring pH and carbonate analytical parameters during experiments. Algal metabolism will alter the pH (and thus carbon speciation) of the incubation medium. It is essential to measure at least daily pH and one other carbonate analytical parameter of the incubation medium. Measurement of two of the car- bonate analytical parameters (pH, CT, alkalinity, or pCO2) allows determination of the other two param- eters and the concentrations of the species when combined with knowledge of the carbonate equilib- rium constants (Lewis and Wallace 1998, Hunter 2007). pH measurements can be made either poten- tiometrically, using high precision and carefully maintained electrode ⁄ meter combinations, or opti- cally, using spectrophotometric measurement of a dye ⁄ seawater mixture (Tapp et al. 2000, Ohline et al. 2007). Care must be taken not to lose (or gain) CO2 by exchange with the atmosphere during the subsampling and measurement process. Careful calibration of the pH measurement system is required using appropriate buffer solutions made up in synthetic seawater (Dickson 1993a,b, Dickson et al. 2007). The defined pH value of the buffer is temperature-dependent, so careful temperature con- trol is required. The measurement of other carbon- ate analytical parameters is described by Dickson et al. (2007), and pCO2 can be measured directly using membrane inlet mass spectrometry (MIMS; Gueguen and Tortell 2008).
4. Modification of seawater pH ⁄ pCO2 within an experi- mental system. Modification of pH ⁄ pCO2 should occur in separate (mixing) containers, not directly in seawater in contact with the study organisms. This can be achieved in flow-through systems by adding the amendment solution or gas to indepen- dent header tanks (e.g., collapsible bags to prevent gas exchange with headspace), to tubing (Kuffner et al. 2007), or in mixing chambers (Leclercq et al. 2000) upstream of culture containers in flow- through systems. The bubbling of CO2 ⁄ air mixtures directly into culture containers is also acceptable for macroalgae (as long as it is at the correct pCO2 for the treatment) but could damage delicate phyto- plankton (Thomas and Gibson 1990, Berdalet et al. 2007). Direct addition of acid or concentrated CO2 into the culture medium surrounding experimental subjects makes it difficult to separate damage due to direct shock to the organisms from localized spikes of pH from the cumulative effect of altered pH ⁄ pCO2 due to the treatment.
5. Biological side effects and pH range during experi- ments. Photosynthesis (increasing pH), respiration, and calcification (lowering pH) by algae can strongly modify the pH and carbonate chemistry of seawater in culture containers (Israel and Hophy 2002). Such effects are particularly important when using macroalgae that are often large and have rapid metabolic rates, and in mesocosms where bio-
mass levels, and hence biological activity, can be high. For experiments that require a constant pH, variation due to photosynthesis or respiration can be reduced by high seawater to macroalgal tissue ratios and ⁄ or systems that have seawater flow from reservoirs with fixed pH levels, and for microalgae, continuous and semicontinuous culture systems can be advantageous over batch cultures because of the constant replenishment with fresh media. Critically, care must be taken not to mistake the effects of short periods of unrealistic pH resulting from meta- bolic processes to the effect of different pCO2 and pH treatments that simulate acidification.
Seawater pH naturally varies on timescales from diurnal to seasonal. An understanding of this natu- ral variability is important when designing an experiment and interpreting results. Culture cham- bers with inflow from surrounding coastal waters can exhibit pH fluctuations of up to 0.6 or 1 pH unit due to natural diurnal variations in the seawa- ter source (Swanson and Fox 2007, Anthony et al. 2008). Prior to experiments that use seawater pumped from coastal waters, seawater pH should be monitored at least over a daily cycle so that nat- ural fluctuations experienced by the algae are known. In experiments to examine community-level effects of pH, the goal may be to achieve a variable pH that reflects that of the natural environment. For example, a diurnal cycle of �0.6 pH units was evident for mesocosm cultures of coralline algae (Kuffner et al. 2007), while seawater pH increased by 0.2–0.3 units gradually over the first 11 d of the 21 d long PeECE III mesocosm experiments (Bell- erby et al. 2008).
6. Overcoming chemical artifacts. In both methods for simulating acidification, secondary changes in CO2 speciation are possible through the following processes. For calcifying algae, the most important will be dissolution of biogenic CaCO3 in the experi- mental chamber as a result of acidification. This phenomenon will increase both CT and AT. How- ever, in a realistic experimental setup, one would want to know about dissolution of biogenic CaCO3 as an outcome, so it is likely that this would be mon- itored, either by measuring weight loss of the CaCO3 or by parallel measurements of any two of the CO2 system parameters. For example, one could monitor pH or pCO2 continuously during the cul- ture experiment and also take samples for CT and ⁄ or AT measurements. This approach would enable any changes arising from CaCO3 dissolution to be corrected for.
Another secondary effect is loss of CO2 gas because the equilibrium of pCO2 in the chamber is greater than that of the ambient atmosphere. For culture experiments controlled by addition of CO2 gas, this can be minimized by partly enclosing the ambient air so that both air and water phases remain in equilibrium. The disadvantage of this approach is that each chamber requiring a different
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 1247
pCO2 condition must be supplied with its own stan- dard air-CO2 mixture.
For a system maintained by periodic additions of HCl, the extent of CO2 loss can be monitored by measurement of CT before and after. In practice, the loss of CO2 under realistic pH conditions (pH > 7.5) does not appear to be very large if the culture chamber is fitted with an inflated plastic bag that has a volume similar to, or smaller than, the volume of water in the chamber. This is because the quantity of CO2 in a head space is extremely small compared to that contained in an equivalent vol- ume of seawater.
7. Replication. A problem with some studies on ocean acidification is that they exhibit low levels of replication and ⁄ or that replicates are not truly inde- pendent of each other; this is especially the case for macroalgae (Tables 1 and 2). Care must be taken to provide independent replicates required for the cor- rect application of statistical tests (i.e., each repli- cate culture tank should have its seawater modified to the appropriate pH independently, not in one header tank per treatment). Psuedoreplication (i.e., growing ‘‘replicate’’ algae in the same treatment container) must be avoided (see Hurlbert 1984). Obtaining appropriate levels of independent repli- cation is especially difficult for the larger macroal- gae (e.g., Fucales and Laminariales), which can be problematic to maintain in culture long-term, and enclosing individuals or populations for field manip- ulations is extremely difficult. Experiments using macroalgae naturally suffer from high levels of stan- dard deviation between replicates because each rep- licate comprises one individual (compared to a phytoplankton culture of millions of cells); a popu- lation response of macroalgae to a treatment is therefore difficult to achieve.
8. Other environmental factors. A preoccupation with mimicry of seawater carbonate chemistry may result in overlooking other factors important for algal growth (e.g., temperature, UV radiation, light climate [photon flux density, light:dark cycle], nutri- ent concentrations, water motion). It is evident from Tables 1 and 2 that these factors vary widely across the range of studies considered. Rates of calcification and carbon acquisition are energy (i.e., light) dependent. Light limitation may result in algae taking up CO2 in preference to HCO3
); light levels that are too high might induce photoinhibi- tion and cause redirection of energy to cellular repair mechanisms and away from growth. Suitable irradiances can be determined from the results of photosynthesis versus irradiance curves. Tempera- ture influences all aspects of algal growth and physi- ological rates. Inadequate water motion will cause diffusion-limited growth, particularly for macroalgae (Hurd 2000). Ideally, standardization between studies would be valuable and would permit direct comparison of the results from different studies. There is also a need for experiments that test the
interactive effects of ocean acidification and other predicted changes in climate (Feng et al. 2008). We recommend providing experimental conditions that are as similar to those in the natural environment as possible. Nevertheless, it is essential to report all the experimental growth conditions listed above to permit critical evaluation of the results.
Incubation timescales and prior conditioning of organ- isms: Several important issues are apparent from the CO2-manipulation experiments conducted to date. A major issue is the degree of physiological response to altered environmental conditions, and this is strongly influenced by the timescale of exper- iments. The outcomes of these studies have mainly been at the level of acclimation, as defined by Raven and Geider (2003), that is, studies of organisms that have had time to show qualitative or quantitative changes in gene expression during growth in response to the experimental treatments. An impli- cit or explicit assumption in interpreting the results is that the experiments did not last long enough to permit adaptation (genetic change) of the a strain in unialgal cultures of the kind investigated over 1,000 generations of the freshwater alga Chlamydo- monas reinhardtii by Collins and Bell (2004) using the CO2-enrichment method. ‘‘Natural laborato- ries’’ such as underwater CO2 vents also provide opportunities to study adaptation (Hall-Spencer et al. 2008). Very short-term experiments (e.g., mea- surements of short-term inorganic 14C assimilation, net oxygen exchange, or chlorophyll-fluorescence- derived electron transport rates), involving exposure of organisms grown at the present day, or some other CO2 concentration to step changes in CO2, only permit the operation of cellular mechanisms termed regulation (preexisting metabolic machin- ery; Raven and Geider 2003) but are essential in defining the kinetic properties of the inorganic car- bon acquisition mechanisms under a given set of growth conditions. Short-term (2 h) photosynthesis experiments on natural marine phytoplankton assemblages do not give time for complete acclima- tion to new experimental conditions and may (as acknowledged by Hein and Sand-Jensen 1997, see also Schippers et al. 2004) overestimate the longer- term (days) effect of the increased CO2 on meta- bolic rates.
Research using laboratory cultures suffers from selection of genotypes favored by the maintenance conditions for the isolate. These conditions include the absence of UV radiation, low PAR fluxes, and unnatural nutrient solute concentrations (including inorganic carbon) in the medium if the isolates have been in culture for a long time (months to years). Against this, there is the possibility of using data from other experiments involving the same algal strain in planning and interpreting experiments for the organ- isms that were isolated and cultured a long time ago. In some cases, laboratory experiments used recently isolated strains when these were available: Burkhardt
1248 C A T R I O N A L . H U R D E T A L .
et al. (1999) used Asterionella glacialis, Coscinodiscus wailesii, Thalassiosira punctigera, and Scrippsiella trochoidea strains that had recently been isolated from the North Sea but obtained Phaeodactylum tricornutom from a culture collection. For mesocosms, there is the advantage that the algae examined have not spent a long period in culture, but there is the problem of separating the main species contributing to the algal biomass from other species if more than cell counts are needed, and the requirement to check by molecu- lar phylogenetic means that the same genotype is involved not only in the various CO2 treatments but also within replicates of a given treatment. This prob- lem of intraspecific genotypic variability also applies to the use of macroalgae and seagrasses from natural populations for CO2 manipulations in the field or in the laboratory. For natural vents, the presence of chemicals in addition to CO2 in the vent fluids, plus the problem of advection of parcels of water and their contained biota to and away from the vent site, are complicating factors for planktonic organisms (Dando et al. 2000), and of genotypic selection of adjacent macroalgae and seagrasses.
In conclusion, there is a need to run experi- ments using both approaches to altering CO2 chemistry, so that any differences in approaches as a contributing factor to the wide range of responses reported for both micro- and macroalgae upon alteration of CO2 chemistry can be accounted for. It is essential that the carbon specia- tion within culture vessels is carefully monitored at least daily during incubation experiments by mea- suring pH and one other analytical parameter (CT, alkalinity or pCO2) of the seawater carbonate sys- tem. There is also a need to better standardize across the scientific community the timescales of preconditioning of samples (e.g., natural communi- ties vs. laboratory cultures) and of incubations dur- ing experiments (from 2 h, Schippers et al. 2004, to 2 years, Collins and Bell 2004, 2006). Finally, independent replication of experimental pCO2 treatments is an essential prerequisite for statisti- cally meaningful results, and other incubation con- ditions should mimic natural environmental conditions (e.g., light climate, inorganic nutrient concentrations) wherever possible.
This work was funded by University of Otago Research Grants to C. L. H., C. D. H., and K. A. H., and a Royal Society of New Zealand ISAT-linkages grant to C. L. H. J. A. R.’s work on calcified algae is supported by the Natural Environment Council (UK). The University of Dundee is a registered Scot- tish charity, No. SC015096. We thank Philip Boyd for his insightful comments, and Daniel Pritchard and Christopher Cornwall for helpful discussions. We are grateful to three anonymous reviewers for their perceptive and generous reviews. This manuscript is dedicated to our colleague and mentor Prof. Peter Bannister.
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