1. WP 7: Microbial diversity and activity, in particular N cycling Objectives 1.To determine the impact of changing pH on prokaryotes: production and respiration, diversity and gene expression 2.To quantify the direct and indirect effects of pH on benthic microbes and nitrogen cycling due to changes in faunal community structure and function Who? Partner 1.1 (LOV): Markus Weinbauer c.s. Partner 8 (PML): Karen Tait, Martin Mühling, Stephen Widdicombe c.s. Partner 15 (NIOO-KNAW): Jack Middelburg, Frederic Gazeau c.s.

2. What will be done? 1.Study the direct and indirect effects of ocean acidification on prokaryote- mediated carbon flow (production and respiration), prokaryotic diversity and their linkages. 2.Study OA induced differences in gene expression of prokaryotic assemblages using molecular and metagenomic approaches 3.Study the responses of benthic animals and their bioturbation activity to enhanced CO 2 levels in (benthic) mesocosm experiments and their consequences for microbial diversity and benthic nitrogen recycling (sediment-water exchange rates and denitrification)

3. Rationale for water column studies The direct effect of OA on primary production and calcification has been studied to some extent. Less emphasis on direct effect of OA on heterotrophic and chemoautotrophic microbes: How does OA affect bacterial production, respiration, hence growth efficiency? How does OA impact microbial diversity? How does OA affect nitrifiers (CO 2 fixing microbes (bacteria and archaea oxidizing ammonium to nitrite/nitrate)? More CO 2 and less NH 3. Primary producers may exhibit many changes (e.g. stoichiometry, TEP production,…). How does this indirectly influence microbial production, respiration and diversity?

4. Rationale for benthic studies Benthic animals govern carbon flow to sediments (biodeposition, e.g. by suspension feeders) and transport processes in sediments (bioturbation and bioirrigation). OA may affect benthic calcifiers in multiple ways. As a consequence, organic matter delivery and transport of particles and solutes in sediments will be modified. This in turn has significant consequences for sediment biogeochemistry and diversity of microbes, including sediment-water exchange of nutrients and fixed nitrogen removal by denitrification s.l., one of the major ecosystem functions and services of sediments.

5. DNA RNA Analysis of changes to microbial diversity and nitrifying community +PCR DGGE to analyse changes to nitrifying community Clone libraries constructed for those samples showing key differences for fine- scale analysis AOA AOB 16S restriction digest TRFLP to analyse changes to microbial diversity Fragment length (nucleotides) Fluorescence intensity Source: Karen Tait (PML)

6. With Animals Without Animals Bioturbation and bioirrigation, hence animals, enhance biogeochemical cycling by creating niches with large gradients Animals enhance nitrification and denitrification

7. Implementation Direct effects: small scale laboratory (microcosms, culture) incubations Conceptual appealing and elegant, statistical significant results Indirect effects: pelagic and benthic mesocoms experiment in Svalbard Ecological significant results but often less conclusive