1. © Ian Joint Plymouth Marine Laboratory 2011 Ian Joint (irj@pml.ac.uk) Jack Gilbert, Kate Crawfurd & Glen Wheeler (PML) Declan Schroeder (MBA) Consequences of ocean acidification for marine microorganisms Both bacteria and phytoplankton

2. © Ian Joint Plymouth Marine Laboratory 2011 Presentation Outline Q UESTIONS  Null hypothesis should be that ocean acidification will not affect marine microbes  pH homeostasis E XPERIMENTAL APPROACHES  Long-term phytoplankton culture at high CO 2  Mesocosm experiment on OA  E huxleyi strain differences  16S tag sequencing – how did bacterioplankton respond?

3. © Ian Joint Plymouth Marine Laboratory 2011 pH Homeostasis

4. © Ian Joint Plymouth Marine Laboratory 2011 pH Homeostasis P H OF SEAWATER IS NOT CONSTANT  Phytoplankton blooms may increase pH by >0.4 pH units  Freshwater lakes are poorly buffered B ACTERIA & PHYTOPLANKTON REGULATE INTERNAL P H  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. © Ian Joint Plymouth Marine Laboratory 2011 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. © Ian Joint Plymouth Marine Laboratory 2011 Many bacteria accommodate low pH  Stomach 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

7. © Ian Joint Plymouth Marine Laboratory 2011 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. © Ian Joint Plymouth Marine Laboratory 2011 Long-term diatom culture experiments

9. © Ian Joint Plymouth Marine Laboratory 2011 Cell number pH pH changes rapidly in culture Kate Crawfurd

10. © Ian Joint Plymouth Marine Laboratory 2011 T. pseudonana – maintained for >100 generations Kate Crawfurd

11. © Ian Joint Plymouth Marine Laboratory 2011 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

12. © Ian Joint Plymouth Marine Laboratory 2011  One ∂-carbonic anhydrase (∂-CA4) was up- regulated in the high CO 2 cultures (p=0.005).  Neither rbcS nor 3 other ∂-CAs had altered expression. T. pseudonana after 3 months Red fluorescenceFv/FmC:N 760 µatm CO 2 235±40.62±0.016.40±0.40* 380 µatm CO 2 251±230.60±0.025.96±0.12* Kate Crawfurd

13. © Ian Joint Plymouth Marine Laboratory 2011 Only CA4 expression different 2 1 0.5 0.25 0.125 0.063 CA4 CA5CA6 CA7 rbcS Relative expression (high CO 2 : present day CO 2 ) Kate Crawfurd

14. © Ian Joint Plymouth Marine Laboratory 2011 Evidence for acclimation or adaptation 3 months at 760 µatm CO 2 To 760 µatm CO 2 To 760 µatm CO 2 To 380 µatm CO 2 To 380 µatm CO 2 3 months at 380 µatm CO 2 Kate Crawfurd

15. © Ian Joint Plymouth Marine Laboratory 2011 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. © Ian Joint Plymouth Marine Laboratory 2011 C:N content No significant differences between means of the four conditions. Global test ANOSIM (R=0.03) 0 2 4 6 8 HLLLHHLH C:N Kate Crawfurd

17. © Ian Joint Plymouth Marine Laboratory 2011 Phytoplankton laboratory experiments summary  We overcame changing pH by using low biomass cultures  No different detected in specific growth rate of T. pseudonana in CO 2 treatments  Adaptation not detected after 100 generations  Some up-regulation of ∂CA4 but not other CAs or rbcs  T. pseudonana acclimates to 760 µatm CO 2

18. © Ian Joint Plymouth Marine Laboratory 2011 Mesocosm Experiments

19. © Ian Joint Plymouth Marine Laboratory 2011

20. Microbial growth changes the environment pH Biomass Ian Joint

21. © Ian Joint Plymouth Marine Laboratory 2011 Nutrients added CO 2 added pH during experiment 7.6 7.8 8 8.2 8.4 30-Apr07-May14-May21-May28-May pH 760 µatm CO 2 380 µatm CO 2 Ian Joint

22. © Ian Joint Plymouth Marine Laboratory 2011 Chlorophyll fluorescence CO 2 added 760 µatm CO 2 380 µatm CO 2 Ian Joint

23. © Ian Joint Plymouth Marine Laboratory 2011 Primary Production High CO 2 Present day CO 2 } } Ian Joint

24. © Ian Joint Plymouth Marine Laboratory 2011 Coccolithophore number 760 µatm CO 2 380 µatm CO 2 Ian Joint

25. © Ian Joint Plymouth Marine Laboratory 2011 Different E huxleyi strains were present  Genotype ‘D’ reduces in abundance during bloom at 760 µatm CO 2  No significant change in genotype ‘B’ throughout bloom at 380 µatm CO 2  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 pCO 2 change E huxleyi appeared to grow less well in this experiment at high CO 2 and WAS NOT REPLACED BY ANY OTHER PHYTOPLANKTON

26. © Ian Joint Plymouth Marine Laboratory 2011 Bacterial response to OA

27. © Ian Joint Plymouth Marine Laboratory 2011 Numbers of bacteria CO 2 added Pyrosequencing 0.0E+00 4.0E+06 8.0E+06 1.2E+07 1.6E+07 30-Apr07-May14-May21-May28-May Cells ml 760 µatm CO 2 380 µatm CO 2 CO 2 added Pyrosequencing Ian Joint

28. © Ian Joint Plymouth Marine Laboratory 2011 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

29. © Ian Joint Plymouth Marine Laboratory 2011 Conclusions

30. © Ian Joint Plymouth Marine Laboratory 2011 Is “Null hypothesis” supported?  T. pseudonana showed acclimation to high CO 2 but no adaptation after 100 generations  E. huxleyi production lower under high CO 2 but we have demonstrated that there are different genotypes that dominate during a bloom  10 million bacterial 16S sequences revealed no effect of CO 2 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. © Ian Joint Plymouth Marine Laboratory 2011 NERC for funding the Aquatic Microbial Metagenomics consortium NERC Environmental Bioinformatics Centre – Dawn Field Royal Society Travel Grant Acknowledgements