WP1 : Integrated field campaigns

Last update : December 1st 2020

The two field campaigns represented a major collaborative effort and generated a wealth of detailed data sets on microbial biodiversity and ecosystem functioning (EF). Data analysis (especially the SIP data) for the 2017 campaign took longer than foreseen, and joint papers are being prepared. Most data for the 2018 campaign are ready, but analysis and data preparation are still ongoing.

In the 2017 campaign, major differences were observed in biodiversity and EF of the silty and sandy site. Spectral reflectance and pigment analyses showed that benthic microalgae (BMA) biomass (~ NDVI, chl a) was higher at the silty site. rDNA and rRNA amplicon sequencing revealed higher pro- and eukaryotic microbial diversity in the sandy site, with different diatom communities dominating BMA activity at both sites (confirmed by the reflectance and pigment data). Prokaryotic activity at the silty site was, surprisingly, dominated by the cyanobacterium Planktothricoides sp., while at the sandy site alpha- and gammaproteobacteria were codominant. Silty sediments were more productive than sandy ones (based on CO2 fluxes). Variation in NDVI values during low tide was higher at the muddy site, revealing the importance of BMA vertical migration. Pronounced changes in prokaryotic activity took place during tidal emersion, with an increasing importance of heterotrophic activity (possibly related to BMA extracellular polymeric substances (EPS) production).

The 13C pulse-chase experiment was, apart from the RNA-SIP, successful. The inorganic 13C was rapidly taken up at both the sandy and the muddy site and assimilated by the different benthic organisms inhabiting the sediment. The sandy site incorporated less 13C than the silty one, but due to the lower C standing stock, proportional enrichment was much higher. Based on 13C incorporation of group specific fatty acids, C fixation at both sites was largely due to the diatoms and cyanobacteria, confirming the rRNA data. C fixation by chemoautotrophic bacteria was also detected at both sites. The C fixed by phototrophs was rapidly transferred to the other trophic levels and to the EPS. Within one hour after labelling, the colloidal and bound factions of the EPS in the sandy site were respectively composed of 12% and 2% 13C, at the muddy site 8% and 3.5% 13C. Nematode species diversity in both sediments was low, with no more than five species together accounting for > 75% of abundance. Nematode abundance was one order of magnitude higher in the silty sediment. Specific 13C uptake was highest in deposit-feeding and epistratum-feeding nematodes, which both use BMA. However, there was also clear 13C enrichment in ciliate feeders and predators of other nematodes, suggesting a rapid trophic transfer of fresh BMA production. In addition, the nematode Terschellingia longicaudata, which derives most of its C from chemoautotrophy, also took up 13C, confirming that chemoautotrophic production in the silty sediment was significant. Towards the end of the low tide, specific uptake in nematodes was similar in the silty and sandy sediment. By the next low tide however, specific uptake further increased in silty sediment nematodes, whereas it decreased in the sandy ones. This most probably reflects erosion/resuspension of labelled BMA and loss of unincorporated label during high tide in the sandy site (Middelburg et al., 2000). Strikingly, specific uptake of deposit-feeding and epistrate-feeding nematodes was up to one order of magnitude higher than that of macrobenthos.

Unfortunately, the RNA-SIP experiment did not yield usable results, as labeling [despite using high amounts of NaH13CO3 (10g per m2)] resulted in insufficient enrichment and no clear heavy C signal could be obtained for most time points with the density gradient ultracentrifugations. As a result, we could not infer C flows at very high taxonomic resolution. Only a single potential 13C photosynthetic incorporator, the diatom Synedra spp. (silty site only) could be detected based on the RNA-SIP results. The low 13C incorporation of the RNA likely results from a diffuse C flow within both sediment types: C originating from remineralisation, internally stored C, cross-feeding and mixotropy dilute the signature of the 13C pulse. For this reason, we decided to abandon the very labour-intensive and costly SIP approach in the 2018 campaign, and opted for a detailed omics approach instead.

For the 2018 campaign, we used a metatranscriptomic and metabolomic approach to study, at high taxonomic resolution, transcriptional activity of microbial pro- and eukaryotes in relation to environmental and functional changes in a mudflat BMA community throughout two consecutive low tides (day vs night). Amplicon sequencing revealed that prokaryotic activity (rRNA), both during day and night, was mainly dominated by alphaproteobacteria and cyanobacteria, while day and night eukaryotic activity was almost completely dominated by diatoms. Reflectance data showed that BMA biomass was high during the first 3hrs of low tide (NDVI up to 0.5), and was largely dominated by diatoms (IDiatom 2.3 x IEuglenid), after which it decreased (NDVI ~ 0.2) with a lower dominance of diatoms (IDiatom 1.2 x IEuglenid). Preliminary metatranscriptome analyses of a selection of samples revealed that day time emersion, photosystem II related genes dominated gene expression, while at night expression of C fixation related genes (esp. Rubisco) was significantly higher. The full metatranscriptome data set is currently being analyzed and will be integrated with the diversity, metabolomic and functioning data in a joint manuscript. The untargeted metabolomics approach developed by P4 (M11) revealed a surprisingly high diversity of metabolites (including phytyl esters, lactones, specific alkanes and fatty acids) whose dynamics were significantly affected by light exposure as well as day night and tidal cycles. These molecules are known markers of stress, chloroplast senescence and/or cell-cell communication and may reflect the consumption of energy reserves, and their synthesis influences biofilm EF. These data will be compared with the amplicon and metatranscriptome sequencing data in order to assess how metabolite and taxonomic diversity and functioning are related, and will an unprecedented insight into BEF relations in BMA biofilms. Natural stable isotope data of EPS were analysed and a short communication about the origin of bound and colloidal EPS is being prepared.

The results of the seasonal campaigns (La Coupelasse) show how seasonal changes in BMA biodiversity affect PP, pigment and EPS diversity. Three manuscripts are currently being prepared. In a first paper, changes in epipsammic and epipelic diatom communities are compared over the course of two years, focusing on the role of biodiversity and the impact on PP patterns. In a second paper, the effect of daily vertical migrations of the BMA biofilms on PP estimates, and how this changes with season (and associated shifts in BMA diversity) will be discussed. In a third manuscript, spatial and temporal variability in benthic kleptoplastidic foraminifera and its relationship with BMA diversity is analyzed. The seasonal field work also led to a follow-up experiment in which the impact of sediment type and light climate on BMA PP and photo-regulation was investigated (M13). Furthermore, a manuscript is close to submission on the relationship between benthic diatoms and kleptoplastidic foraminiferaand another on the impact of epipelic diatom biodiversity on their photo-regulation mechanisms.