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Ian Joint (irj@pml.ac.uk)

Jack Gilbert, Kate Crawfurd & Glen Wheeler (PML)Declan Schroeder (MBA)

Consequences of ocean acidificationfor marine microorganisms

Both bacteria and phytoplankton

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Presentation Outline

QUESTIONS Null hypothesis should be that ocean

acidification will not affect marine microbes pH homeostasisEXPERIMENTAL APPROACHES Long-term phytoplankton culture at high

CO2

Mesocosm experiment on OA E huxleyi strain differences 16S tag sequencing – how did

bacterioplankton respond?

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pH Homeostasis

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pH Homeostasis

PH OF SEAWATER IS NOT CONSTANT Phytoplankton blooms may increase pH by >0.4 pH units

Freshwater lakes are poorly buffered

BACTERIA & PHYTOPLANKTON REGULATE INTERNAL PH

This explains how pathogenic bacteria can survive stomach pH of <1.

Acidophilic Chlamydomonas – energetics of growth at pH 2 rather than pH 7

A 7% increase in ATP requirement

(Messerli et al. 2005. J Exp Biol, 208, 2569-2579)

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pH of freshwater lakes

Lakes are much less buffered than the oceans

They experience large daily variations in pH - as much as 2-3 pH units (e.g. Maberly et al., 1996).

Variations in pH also occur over very small distances. Talling (2006) showed that in some English lakes, pH could change by > 2.5 pH units over 14 m depth

Yet phytoplankton, bacteria and archaea are all present in lakes, and appear to be able to accommodate large daily and seasonal changes in pH.

Are marine microbes different from freshwater, with less ability to acclimate and adapt?

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Many bacteria accommodate low pHStomach pH is 1-3

Bacteria can pass through and survive this pH challenge (e.g. Campylobacter & pathogenic E. coli)

Survival is possible because bacteria have proton pumps to remove H+

One mechanism is uptake of arginine and release of decarboxylation product (Fang et al, 2009).

Maintain intracellular pH at 5

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Null hypothesis

I suggest that the Null hypothesis should be – non-calcifying microbes will not be affected by OA

Joint, I, Doney S.C., Karl, D.M. (2011) Will ocean acidification affect marine microbes? ISME Journal. 5, 1-7

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Long-term diatom culture experiments

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0 1 2 3 4 57.5

8.0

8.5

9.0

9.5

0

1

2

3

4

Time (d)

pH

Cel

ls x

106

ml-1

Cell number

pH

pH changes rapidly in culture

Kate Crawfurd

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T. pseudonana – maintained for >100 generations

7.6

8.0

8.4

8.8

9.2

0 2 4 6 8 10 12Time (weeks)

pH

Kate Crawfurd

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What changed after 100 generations?

Change in - C:N ratio - slightly decreased

Red fluoresence (= chlorophyll) - slightly increased

No change in - Cell size or morphology

Photosynthetic efficiency (Fv/Fm)

Functional cross section of PSII (σPSII)

RuBisCO expression (rbcS)

Kate Crawfurd

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One ∂-carbonic anhydrase (∂-CA4) was up-regulated in the high CO2 cultures (p=0.005).

Neither rbcS nor 3 other ∂-CAs had altered expression.

T. pseudonana after 3 months

Red fluorescence Fv/Fm C:N

760 µatm CO2 235±4 0.62±0.01 6.40±0.40*

380 µatm CO2 251±23 0.60±0.02 5.96±0.12*

Kate Crawfurd

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Only CA4 expression different

2

1

0.5

0.25

0.125

0.063

CA4 CA5 CA6 CA7 rbcS

Rel

ativ

e ex

pres

sion

(hig

h C

O2 :

pre

sent

day

CO

2)

Kate Crawfurd

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Evidence for acclimation or adaptation

3 months at 760 µatm CO2

To 760 µatm CO2

To 760 µatm CO2

To 380 µatm CO2

To 380 µatm CO2

3 months at 380 µatm CO2

Kate Crawfurd

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Acclimation or adaptation?

No statistically significant change in - Cell size or morphology

C:N ratio

Red fluorescence

Photosynthetic efficiency (Fv/Fm)

Functional cross section of PSII (σPSII)

RuBisCO expression (rbcS)

CA expression (CA4, CA5, CA6 or CA7)

Kate Crawfurd

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C:N content

No significant differences between means of the four conditions. Global test ANOSIM (R=0.03)

0

2

4

6

8

HL LL HH LH

C:N

Kate Crawfurd

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Phytoplankton laboratory experiments summary

We overcame changing pH by using low biomass cultures

No different detected in specific growth rate of T. pseudonana in CO2 treatments

Adaptation not detected after 100 generations

Some up-regulation of ∂CA4 but not other CAs or rbcs

T. pseudonana acclimates to 760 µatm CO2

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Mesocosm Experiments

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0

4

8

12

16

20

09-May 10-May 11-May 12-May 13-May 14-May 15-May 16-May

Fluo

resc

ence

(arb

itrar

y un

its)

7.7

7.8

7.9

8

8.1

pH

Microbial growth changes the environment

pHBiomass

Ian Joint

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Nutrients added

CO2 added

CO2 added

pH during experiment

7.6

7.8

8

8.2

8.4

30-Apr 07-May 14-May 21-May 28-May

pH 760 µatm CO2

380 µatm CO2

Ian Joint

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Chlorophyll fluorescence

0

5

10

15

20

25

30-Apr 07-May 14-May 21-May 28-May

Arbi

trary

uni

ts

High CO2

Present day

CO2 added

CO2 added

760 µatm CO2

380 µatm CO2

Ian Joint

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Primary Production

8-May 9-May 10-May 11-May 12-May 13-May 14-May 15-May0

200

400

600

800

1000

1200

mg

C m

-2 d

-1

High CO2

Present day CO2

}}

Ian Joint

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0.0E+00

1.0E+03

2.0E+03

3.0E+03

4.0E+03

30-Apr 07-May 14-May 21-May 28-May

Cel

ls m

l-1

High CO2Present day

Coccolithophore number

760 µatm CO2

380 µatm CO2

Ian Joint

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Different E huxleyi strains were present

Genotype ‘D’ reduces in abundance during bloom at 760 µatm CO2

No significant change in genotype ‘B’ throughout bloom at 380 µatm CO2

Genotype ‘C’ did not change in either treatment Genotype ‘A’ slight positive selection BUT it’s not

significant.

E huxleyi has different, distinguishable genotypes, although they all look the same.

They respond differently to pCO2 change

E huxleyi appeared to grow less well in this experiment at high CO2 and WAS NOT REPLACED BY ANY OTHER PHYTOPLANKTON

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Bacterial response to OA

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Numbers of bacteria

CO2 added

Pyrosequencing

0.0E+00

4.0E+06

8.0E+06

1.2E+07

1.6E+07

30-Apr 07-May 14-May 21-May 28-May

Cel

ls m

l-1

760 µatm CO2

380 µatm CO2

CO2 added

Pyrosequencing

Ian Joint

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English Channel - High throughput sequencing

Jack Gilbert

Bacterial diversity determined using 16S rDNA V6 tag pyrosequencing (Sogin et al., 2006)

Over 10 million sequences Over 20,000 genotypes detected Small number of taxa dominated The most abundant organisms were a strain of

SAR11 (Rickettsiales) and Rhodobacteriales

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Conclusions

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Is “Null hypothesis” supported?

T. pseudonana showed acclimation to high CO2 but no adaptation after 100 generations

E. huxleyi production lower under high CO2 but we have demonstrated that there are different genotypes that dominate during a bloom

10 million bacterial 16S sequences revealed no effect of CO2 treatment throughout a 3 week mesocosm experiment

Both 16S tag sequencing and metatranscriptomics study revealed that the largest differences were with time (bloom effect) rather than with treatment (ocean acidification)

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NERC for funding the Aquatic Microbial Metagenomics consortium

NERC Environmental Bioinformatics Centre – Dawn Field

Royal Society Travel Grant

Acknowledgements