Consequences of ocean acidification for marine microorganisms

1. Consequences of ocean acidification for marine microorganisms Both bacteria and phytoplankton Ian Joint(irj@pml.ac.uk)Jack Gilbert, Kate Crawfurd & Glen Wheeler (PML)Declan Schroeder (MBA)

2. Questions Null hypothesis should be that ocean acidification will not affect marine microbes pH homeostasis Experimental approaches Long-term phytoplankton culture at high CO2 Mesocosm experiment on OA E huxleyistrain differences 16S tag sequencing – how did bacterioplankton respond? Presentation Outline

3. pH Homeostasis

4. 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)

5. 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?

6. Stomach pH is 1-3 Bacteria can pass through and survive this pH challenge (e.g. Campylobacter & pathogenicE. coli) Survival is possible because bacteria have proton pumps to remove H+ One mechanism is uptake of arginine and release of decarboxylationproduct (Fang et al, 2009). Maintain intracellular pH at 5 Many bacteria accommodate low pH

7. 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

8. Long-term diatom culture experiments

9. pH changes rapidly in culture Cell number pH Kate Crawfurd

10. 9.2 8.8 pH 8.4 8.0 7.6 0 2 4 6 8 10 12 Time (weeks) • T. pseudonana – maintained for >100 generations Kate Crawfurd

11. What changed after 100 generations? • No change in - • Cell size or morphology • Photosynthetic efficiency (Fv/Fm) • Functional cross section of PSII (σPSII) • RuBisCO expression (rbcS) • Change in - • C:N ratio - slightly decreased • Red fluoresence (= chlorophyll) - slightly increased Kate Crawfurd

12. T. pseudonana after 3 months Red fluorescence Fv/Fm C:N 760 µatmCO2 235±4 0.62±0.01 6.40±0.40* 380 µatm CO2 251±23 0.60±0.02 5.96±0.12* • One ∂-carbonic anhydrase(∂-CA4) was up-regulated in the high CO2 cultures (p=0.005). • Neither rbcS nor 3 other ∂-CAshad altered expression. Kate Crawfurd

13. Only CA4 expression different 2 1 0.5 Relative expression (high CO2 : present day CO2) 0.25 0.125 0.063 CA5 CA6 rbcS CA4 CA7 Kate Crawfurd

14. To 760 µatm CO2 To 380 µatm CO2 To 760 µatm CO2 To 380 µatm CO2 Evidence for acclimation or adaptation 3 months at 380 µatm CO2 3 months at 760 µatm CO2 Kate Crawfurd

15. 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

16. 8 6 4 2 0 HL LL HH LH C:N content C:N No significant differences between means of the four conditions. Global test ANOSIM (R=0.03) Kate Crawfurd

17. Phytoplankton laboratory experiments summary • We overcame changing pH by using low biomass cultures • No different detected in specific growth rate of T. pseudonanain CO2 treatments • Adaptation not detected after 100 generations • Some up-regulation of∂CA4 but not other CAs or rbcs • T. pseudonanaacclimates to 760 µatm CO2

18. Mesocosm Experiments

20. Microbial growth changes the environment pH Biomass Ian Joint

21. CO2 added CO2 added Nutrients added pH during experiment 8.4 8.2 760 µatmCO2 8 pH 380 µatm CO2 7.8 7.6 30-Apr 07-May 14-May 21-May 28-May Ian Joint

22. CO2 added CO2 added Chlorophyll fluorescence 760 µatmCO2 380 µatm CO2 Ian Joint

23. Primary Production } Present day CO2 } High CO2 Ian Joint

24. Coccolithophore number 760 µatmCO2 380 µatm CO2 Ian Joint

25. Different E huxleyistrains 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 huxleyihas different, distinguishable genotypes, although they all look the same. • They respond differently to pCO2 change • E huxleyiappeared to grow less well in this • experiment at high CO2 and was not replaced by any • other phytoplankton

26. Bacterial response to OA

27. Numbers of bacteria Pyrosequencing Pyrosequencing 1.6E+07 760 µatmCO2 380 µatm CO2 CO2 added CO2 added 1.2E+07 -1 8.0E+06 Cells ml 4.0E+06 0.0E+00 30-Apr 07-May 14-May 21-May 28-May Ian Joint

28. English Channel - High throughput sequencing 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 Jack Gilbert

29. Conclusions

30. Is “Null hypothesis” supported? T. pseudonanashowed acclimation to high CO2 but no adaptation after 100 generations E. huxleyiproduction 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)

31. NERC for funding the Aquatic Microbial Metagenomics consortium NERC Environmental Bioinformatics Centre – Dawn FieldRoyal Society Travel Grant Acknowledgements