The Effects of Ocean Acidification on Coastal Marine Phytoplankton

7/30/2019 The Effects of Ocean Acidification on Coastal Marine Phytoplankton

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The effects of ocean acidification on coastal marine

noncalcareous phytoplankton

Joshua Bergeron

Undergraduate, Department of Marine Science, The University of Southern Mississippi


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ABSTRACT

It has been suggested that nearly half of the CO2 emitted since human industrialization

began burning fossil fuels in the late eighteenth century has been absorbed into the global ocean

(Sabine et al. 2004). This is of concern to scientists due to the possible negative effects tomarine organisms. It has been predicted that the average surface water pH will decrease from

~8.2 to ~7.8 by the end of the century (Berge, T et al. 2010). This drop could affect the

intracellular pH, enzyme activity, and metabolism of common marine phytoplankton due to an

increase in [H+] (Giordano, M et al. 2005). There have been few studies concerning the affects of

lowered pH on noncalcareous coastal marine phytoplankton. It has been suggested that coastal

phytoplankton may be less sensitive to changes in pH when compared to true oceanic species

(Berge, T et al. 2010). If this can be assumed, then any small change in the growth rate of coastal

phytoplankton would be truly revealing. A comparison of the changes in the realized growth

rates at varying pH suggests that noncalcareous coastal marine phytoplankton are sensitive to

lowered ocean pH and the time allowed to acclimate to changes in pH. The growth rates werestatistically similar in the range of research; however, slight trends are evident. Despite the small

range of variation, I believe that this study reveals that noncalcareous marine phytoplanktons are

sensitive to extreme changes in pH. However, the changes in ocean pH suggested in consequence

of ocean acidification are not significant enough to affect their growth rate significantly. Natural

coastal fluctuations in pH provide an environment that promotes tolerance to veritable pH. If

other species of phytoplankton are more sensitive to pH, it could be that noncalcareous marine

phytoplankton will replace such species in the future oceans of the world.


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INTRODUCTION

Human industrialization began burning fossil fuels in the late eighteenth century. It has

been suggested that nearly half of the CO2 emitted in consequence has been absorbed into the

global ocean (Sabine et al. 2004). When CO2 gas dissolves in water it forms a balance of several

ionic and non-ionic carbon species collectively known as dissolved inorganic carbon or DIC. A

weak acid know as carbonic acid (H2CO3) forms and dissociates until equilibrium is reached.

This subsequently causes an increase in the hydrogen ion concentration ([H+]) and a decrease in

pH due to the relationship of pH =log [H+]. It has been calculated that an average drop in pH

of about 0.1 has occurred in the world ocean due to the absorption of fossil fuel CO2 since the

late eighteenth century (Matsumoto & McNeil 2013) This is of concern to scientists due to the

possible negative effects to marine organisms. A decrease in the concentration of carbonate

([CO3]) is especially important to calcifying organisms such as corals, shellfish, oysters, and

calcareous algae such as coccolithophorids. A drop in [CO3] increases the energy costs of

calcification. This in turn reduces their growth rate.

The pH level of seawater may also affect the growth rate of noncalcareous marine

phytoplankton. Marine phytoplanktons represent a significant function in the marine food web.

Even a small overall reduction in their growth within the world ocean could have potentially

profound impacts on future marine resource predictions. It has been predicted that the average

surface water pH will decrease from ~8.2 to ~7.8 by the end of the century (Berge, T et al. 2010).

This drop could affect the intracellular pH, enzyme activity, and metabolism of common marine

phytoplankton due to an increase in [H+] (Giordano, M et al. 2005). The drop could also affect

to some degree the chemical availability of critical micronutrients such as iron (Berge, T et al.

2010).


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There are few studies that directly target the effects of lowered pH on coastal

noncalcareous phytoplankton. Research has suggested that coastal phytoplankton may be less

sensitive to changes in pH when compared to true oceanic species (Berge, T et al. 2010). If this

can be assumed, then any change in the growth rate of coastal phytoplankton would be truly

revealing. The more evident effects to calcareous marine organisms tend to be the subject of

current research.

Furthermore, a direct study of the effects of pH to phytoplankton is very difficult due to

the many factors involved in manipulating the pH of seawater in a lab environment. The natural

buffering capacity of seawater is not only very high but also highly dynamic. The design of an

experimental system that accurately simulates the effects of ocean acidification on the growth of

marine phytoplankton must consider the varying ways in which individual species of

phytoplankton acquire and use CO2. Many species of phytoplankton use various forms of DIC to

satisfy the requirements of photosynthesis, respiration, and calcification. These metabolic

processes, themselves, alter the pH of seawater in varying ways.

The system is intended to mimic the absorption of anthropogenic carbon into the ocean.

However, the method selected to mimic this process will affect the concentrations of DIC and its

availability within the sample. In seawater, free CO2 (aq) is in equilibrium with a small

concentration of the carbonic acid species H2CO3. Both species are commonly considered to be

stoichiometrically equivalent with respect to their equilibrium reactions. This is represented by a

hypothetical species represented as H2CO3* where [H2CO3*] = [H2CO3] + [CO2 (aq)]. The

lowering of pH is an effect of the uptake of CO2 and an increase in [H+]. CO2 in the gas phase

equilibrates with H2CO3* in seawater. By using Henrys law of equilibrium it follows that:

where pCO2 is the partial pressure of CO2 (g) and KH is the Henrys law


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equilibrium constant. KH is a function of temperature and salinity. It then follows that at a given

temperature and salinity the pCO2 and [H2CO3*] are linearly related to each other. As [H2CO3*]

dissociates into bicarbonate (HCO3-) and then carbonate (CO3

2-), the [H

+] increases and pH

decreases. This relationship proves that it is physically impossible to vary systematically any one

factor while holding the rest constant (Hurd, C et al. 2009). Any changes made to the pH of

seawater by altering the [H2CO3*] will alter the subsequent concentrations of available carbon

species and force the system to strive for equilibrium concerning pCO2.

The bubbling of CO2 gas into a test solution works against equilibrium by matching the

equilibrium rate to the addition of CO2 gas or realistically the creation of DIC. This method

however is difficult due to the precision required by the process and again because the buffering

capacity of natural seawater can vary according to sample. Precise regulation of the gas mixture

and its flow into the solution is required and the method would have to be determined

experimentally. It has also been suggested that directly bubbling with CO2 may cause damage to

fragile phytoplankton such as green algae,cyanobacteria, and diatoms through the effects of

small-scale turbulence (Hurd et al. 2009, Berge et al. 2010).

The direct addition of a relatively low molar concentration of hydrochloric acid (HCl)

solution will change the pH without possibly harming the phytoplankton population; except

perhaps through localized concentrations at the time of the addition. However, this method has

very little to do with the absorption of CO2 gas naturally. Since natural ocean acidification

involves changes to the total amount of dissolved inorganic carbon (DICT) and the DICT affects

photosynthesis, respiration, and calcification by phytoplankton, then any accurate experiment

must include the addition of DIC.


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Therefore, I designed an experiment that uses equivalent low molar concentrations of

HCl and sodium bicarbonate (NaHCO3) (suggested by Hurd et al. 2009). The NaHCO3 exactly

neutralizes the HCl and supplies the increased DICT, mostly in the form of HCO3-, that would be

expected from CO2 gas bubbling.

I hope to answer two questions: 1) Are the growth rates of noncalcareous phytoplankton

affected by lowered pH? 2) Does the time involved in acidification have an affect experimentally

on the ability of the phytoplankton to acclimate to changes in their environment?

METHODS AND MATERIALS

The phytoplankton used in the experiment were collected directly from the coastal waters

of Long Beach, Mississippi using a rope and bucket. The seawater was poured through a 53 m

screen into a clean 10 liter sample container in order to eliminate large metazoan grazer

populations found within the sample. It was then transported to the lab for processing.

Temperature and salinity were recorded at the time

of sampling using a YSI 85 Oxygen, Conductivity,

Salinity & Temperature device. The 10 liter sample

was agitated and poured into 5 separate 1 liter

beakers. An enriched nutrient solution was

prepared in advance according to the specifications of

f/2 Medium with the exception of the f/2 vitamin

solution (Anderson 2005). This recipe was mixed into

each beaker in order to provide sufficient enough nutrients


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to promote growth.

Each sample was agitated and reduced by 200 mL. This produced 6 nutrient rich 800 mL

samples and one nutrient rich 200 mL sample. A sample splitter was used to separate each

800 mL into 400 mL and then

200 mL samples. The

200 mL samples were poured

into 250 mL Erlenmeyer

flasks and labeled according

to series A or B, target pH,

and sample A, B, or C. This

allowed for two experimental series. Series A involved the acidification of the samples in one

treatment on day 1. Series B involved the acidification of the samples in four treatments over the

course of four days. Each series targeted four levels of pH: The initial pH and lower in

increments of 0.7 pH. Each target pH had three identical samples in each series. Twelve samples

total in each series and 24 total in the experiment.

The additional 200 mL sample was used for

a spectrophotometric determination of chlorophyll a.

The sample was filtered through a paper filter using

a vacuum pump apparatus. The filter was folded and

placed into a sample tube containing 9 mL of acetone and 1 mL of dH2O. The sample tube a

corresponding blank solution of acetone were wrapped in foil and refrigerated overnight. The

sample tubes were then centrifuged at 4000 g for 5 minutes before a sub-sample was extracted

from the center of the sample. The sub-sample was analyzed using a Genesys 10 UV scanning


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spectrophotometer at the wavelengths required by the Jeffrey equation for the determination of

Chlorophyll a (Jeffrey et al. 2005).

The NaHCO3 solution was composed of 0.1 m HCl and 0.1 m NaHCO3 that was allowed

to equilibrate for 24 hours. The acidification procedure was determined experimentally on the

day the original seawater sample was collected. The NaHCO3 solution was titrated into a 200

mL sample of the seawater in 200 L increments. A Fisher pH/Ion 510 meter recorded the

changes in pH at each increment until a pH of 6.00 was reached. By this experiment, the

subsequent amounts of NaHCO3 solution needed to reach the various target pH levels was

defined.

Six samples, three in each series, were labeled with the initial pH level and were not

treated further. The A series samples were acidified according to the above procedure with the

amount determined for each calculated target pH. The B series was acidified with 25% of the

determined amount initially and then every 24 hours until 100% of the determined amount is

reached over the course of four days.

After the initial addition of the

NaHCO3 solution, all of the samples

were placed in a reflective chamber

illuminated with a 30W florecent bulb

on a timer set for 14 hours of light

and 10 hours of dark. Temperature was maintained at ~ 23

o

C. A 50 mL beaker was inverted

over each Erlenmeyer flask to prevent contamination but allow for gas exchange.


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A sub-sample of ~50 L was taken every two days from each sample in order to conduct

a cell count. A CELL-VU counting chamber slide was used to count individual phytoplankton

cells present within the gridded area of the slide.

The samples were allowed to grow for 12 days. Each sample was filtered according to

the above spectrophotometric method. The final pH and temperature was measured for each

corresponding filtered solution. The filters were placed in the same acetone solution described in

the earlier method, steeped for the same duration, and measured at the same wavelengths.


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y = -0.8389x + 9.6681

R = 0.9957

2.50

2.70

2.903.10

3.30

3.50

3.70

3.90

4.10

4.30

4.50

4.70

4.90

5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90

log

(mL

ofNaHCO

3

so

lutiona

dde

d)

pH

RESULTS

The initial filtered seawater sample was measured to have a pH of 8.17 and a salinity of

17.7 ppt at 21.9o

C. Target pH levels are selected at Initial, 7.47, 6.77, and 6.07. A pH curve

was established based on the addition of NaHCO3 solution to the natural seawater sample:

If the L of NaHCO3 solution added is changed to a log scale then a more linear trend is defined:

0.00

4.00

8.00

12.0016.00

20.00

24.00

28.00

32.00

36.00

40.00

5.90 6.10 6.30 6.50 6.70 6.90 7.10 7.30 7.50 7.70 7.90

mLo

fNaH

CO

3so

lutiona

dde

d

pH


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This established the varying amounts of NaHCO3 solution to be added to each sample as follows:

Target pHAddition of NaHCO3 solution

for Series A

Addition of NaHCO3 solution

for Series B

8.17 None None7.47 2.4 mL 4 @ 600 L

6.77 9.8 mL 4 @ 2,450 L

6.07 33.8 mL 4 @ 8,450 L

The final concentrations in the sample solutions based on the f/2 medium recipe are:

NaNO3 8.82 x 10-4

M

NaH2PO4 H2O 3.62 x 10-5

M

Na2SiO3 9H2O 1.06 x 10-4 M

FeCl3 6H2O 1.17 x 10-5

M

Na2EDTA 2H2O 1.17 x 10-5

M

MnCl2 4H2O 9.10 x 10-7

M

ZnSO4 7H2O 7.65 x 10-8

M

CoCl2 6H2O 4.20 x 10-8

M

CuSO4 5H2O 3.93 x 10-8

M

Na2MoO4 2H2O 2.60 x 10-8

M

Spectrophotometric absorbance was measured at 630 nm, 647 nm, and 664 nm for the followingequation for Chlorophyll a found in a mixed phytoplankton population; units g/L

(Jeffery et al. 2005):

( 11.85 E6641.54 E6470.08 E630 ) Ev

V1

Where Ev is the extractant volume of 90% acetone (10 mL) and V1 is the sample volume (0.2 L)

The realized growth rate was measured using the equation:

Pt= P0e( ( ln (Pt / P0)

tWhere Ptis the total experimental population at time t, P0 is the initial experimental population

at t = 0, is the realized growth rate, and is the grazing rate. It is assumed that is < 1 due tofiltering.

The realized growth rates were based on a pre and post determination:


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0.240

0.260

0.280

0.300

0.320

0.340

0.360

0.380

0.400

0.420

1 dose at start 4 doses over 4 days

Rea

lized

GrowthRateperDay

Realized Growth Rate of Mixed Phytoplankton

based on Spectrophotometric determination of

Chlorophyll a

pH 817

pH 747

pH 677

pH 607

0.240

0.260

0.280

0.300

0.320

0.340

0.360

0.380

0.400

0.420

1 dose at start 4 doses over 4 days

Rea

lize

dG

rowthRateperDay

Realized Growth Rate of Mixed Phytoplankton

based on Cell Counts by Sub-sampling

pH 817

pH 747

pH 677

pH 607


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-20

0

20

40

60

80

100

120

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Ce

llsperGri

d

Days

Cell Count

A 8.17

A 7.47

A 6.77

A 6.07

-20

0

20

40

60

80

100

120

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Ce

llsperGri

d

Days

Cell Count

B 8.17

B 7.47

B 6.77

B 6.07


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Cell Count Data

Counts were of counted cells per 1 mm x 1 mm grid divided into 100, 0.1 mm areas with a depth of 20 m. Data

below represents the average of the three samples per series. The counts were done twice per sample. Six values

were averaged per target pH level every 2 days.

Days A 8.17 A 7.47 A 6.77 A 6.07 B 8.17 B 7.47 B 6.77 B 6.07

2.00


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Chain Forming

Diatoms

4%

Centric

Diatoms

3%

Various Other

Diatoms

11%

Cylindrotheca

51%

Navicula

31%

Pennate

Diatoms

82%

Species Distribution

Pennate Chain Forming Centric

Cylindrotheca Navicula


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Various other observed Diatoms

Average Final pH and Temperature at time of filtering:

Sample pH Temperature

A8.17 9.60 20.9o

C

A7.47 9.80 21.2o

C

A6.77 9.76 20.9o

C

A6.07 9.86 21.6 o C

B8.17 9.69 22.2o

C

B7.47 9.87 21.6o

C

B6.77 9.69 21.7o

C

B6.07 9.86 21.9o

C


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DISCUSSION

The data from the experiment suggests that there was very similar growth in both series

A and B. The realized growth rate based on the spectrophotometric analysis (RGRS) suggests

that noncalcareous phytoplankton may have very slight sensitivity to the time allowed to

acclimate to changes in pH. The slight variation between the experimental controls (Series

A8.17 and B8.17), could define the natural variability in the growth rate under the conditions of

the experiment. The variability of ~ 0.010 in the controls and an overall variation of ~ 0.030 in

the RGRS suggest that they are almost the same statistically. However, the B series did achieve

higher growth rates comparatively. This could suggest that the greater time allowed for

acclimation, may have affected the overall growth rate in the samples. The high light

environment found within the experimentmay have caused a decrease in [Chl. a] due to the

natural responses of phytoplankton to high light environments. This would suggest that the

calculated realized growth based on chlorophyll a is an under-estimation of the true production.

This under-estimated slight variation in growth is significant in a marine species predicted to be

resistant to changes in pH.

The realized growth rate (RGRC) based on cell counts suggests that noncalcareous

phytoplankton may have very slight sensitivity to changes in pH. The variability in the controls

is twice as great at ~0.020. However, this is still a reasonable variation within the experiment.

The RGRC suggests that the growth rate was lower in the lower target pH samples. Chain

forming diatoms were counted as one cell. Since chains of diatoms are in fact a collection of

individual cells, the cell count is an underestimation of the true overall cell count. The sample

was thoroughly agitated to insure even distribution of cells. The same method and location of

sub-sampling was used and each count was repeated for each of the three samples per target pH.


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The variation in this data is greater but still small statistically. Production is again

underestimated and the existence of a trend does suggest some sensitivity to lower pH levels.

The experiment offers enough evidence to answer the initial questions. Based on the

realized growth rates of both the chlorophyll a analysis and the cell counts, it is evident that

noncalcareous coastal marine phytoplankton are minutely affected by extreme changes in pH.

The data also suggests that the amount of time the phytoplankton are allowed to acclimate has a

slight effect on their ability to adapt to the lower levels of pH. The changes in ocean pH

suggested by the end of the century by ocean acidification represent only a fraction of the levels

studied in this experiment. Furthermore, the measured affects to the growth rate of these species

are not great enough to significantly change the overall future population. Natural coastal

fluctuations in pH provide an environment that promotes tolerance to veritable pH. If other

species of phytoplankton are more sensitive to pH, it could be that noncalcareous marine

phytoplankton will replace such species in the future oceans of the world.

The pCO2 and the [CO2] gas are the driving factors behind ocean acidification. This

experiment allowed the samples to equilibrate with the atmosphere of the lab. A more accurate

experiment might control the atmosphere surrounding the sample. The samples could be

bubbled with their surrounding atmosphere and a new equilibrium could be reached. This is a

more complicated experiment and far harder to achieve. However, it would be interesting to see

if the data agrees with that of this experiment.


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REFERENCES

Anderson, R (2005) Algal Culturing Techniques. Elsevier Academic Press. 578 pp.

Berge T, Daugbjerg N, Balling Andersen B, Hansen PJ (2010) Effect of lowered pH on marine

phytoplankton growth rates. Mar Ecol Prog Ser 416:79-91

Giordano M, Beardall J, Raven JA (2005) CO2 concentrating mechanisms in algae: mechanisms,

environmental modulation, and evolution. Annu Rev Plant Biol 56:99131

Hurd, C. L., Hepburn, C. D., Currie, K. I., Raven, J. A. and Hunter, K. A. (2009), Testing the

effects of ocean acidification on algal metabolism: considerations for experimental

designs. Journal of Phycology, 45: 12361251. doi: 10.1111/j.1529-8817.2009.00768.x

Jeffrey, S.W., Mantoura, RFC, Wright, S.W. (2005) Phytoplankton pigments in oceanography:

guidelines to modern methods. UNESCO publishing. 667pp.

Matsumoto, K., & McNeil, B. (2013). Decoupled response of ocean acidification to variations in

climate sensitivity.Journal Of Climate, 26(5), 1764-1771. doi:10.1175/JCLI-D-12-

00290.1

Sabine, C. L., and Coauthors, 2004: The oceanic sink for anthropogenic CO2. Science, 305, 367

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The Effects of Ocean Acidification on Coastal Marine Phytoplankton