Biofilms are dense bacterial structures with high growth and conversion rates, therefore, ideal systems to link populations to activities. Since a few years microsensor measurements, quantitative microautoradiographic techniques (beta-microimaging) and fluorescence in situ hybridization are successfully combined to investigate activities and community structure of biofilms in situ. Two original research lines were followed: (I) nitrification at very low pH and (II) marine biofilms.
Structure and function of a highly active nitrifying biofilm at very low pH.
In a cooperation with Michal Green and Sheldon Tarre (Technion, Haifa, IL) started in June 2002, nitrifying biofilms from a fluidized-bed reactor system with chalk as the carrier material was investigated. Textbook inform us that nitrification is optimal between pH 7-8, and stops below pH 6. However, high nitrification activity was detected at pH 3.5. Two hypotheses were tested: (a) a local less acidic microenvironment within the biofilm (b) presence of new acid-tolerant nitrifiers. During the studies both assumptions were rejected: we showed that nitrification occurs by a "normal" microbial community at a very low pH. Why was this not observed before and how do nitrifiers adapt to low pH? Biochemical and proteomics approaches are needed to go further with this matter.
Key organisms and their interactions in a zero-discharge mariculture system.
In 2002, we started a project with Jaap van Rijn (Faculty of Agriculture, Hebrew Univ. of Jerusalem (IL)), Dror Minz (Volcani Research Center, Bet Dagan (IL)) and Andreas Schramm (Institute for Ecological Microbiology, Univ. Bayreuth) to study biofilms in a zero-discharge mariculture system. This system has complete water recycling, which couples nitrification with sludge digestion and denitrification. Two joint measurement campaigns focused on in situ kinetics and community structure of the nitrifying biofilm, and substrate and electron acceptor dependence of local activities in flocs from the anaerobic treatment system. Measurements with nitrate biomicrosensor allowed us to calculate in situ kinetics of nitrification in a marine biofilm. The ammonium affinity of >100 µM is higher than of freshwater nitrifying biofilms. FISH and amoA sequence analyses revealed a variety of AOB (Nitrosospira marina, Nitrosomonas marina, N. europaea and N. nitrosa) as well as Nitrospira marina-related NOB in the biofilm. The spatial organization of denitrification and sulfate reduction is studied in flocs from the anaerobic digester, by microsensor analysis, characterization of isolated bacteria, molecular analysis of functional genes (nosZ) and microautoradiography of single flocs. Data analysis is in progress.

Helle Ploug, Armin Gieseke, Carsten Schwermer

Flocs

Heterogeneity of nutrients in the ocean is of significant importance for nutrient remineralization within the euphotic zone and, hence, also export of organic carbon to the ocean interior. Sinking aggregates add greatly to this heterogeneity of nutrients as well as to the vertical carbon fluxes in the ocean. The heterogeneity of dissolved nutrients associated with sinking aggregates depends on the relative importance of diffusion and flow on a mm-scale and on remineralization rates within aggregates. The aim of the project is to quantify and understand flow and diffusion within and around organic aggregates composed of phytoplankton, detritus, and microorganisms, and to quantify the colonization and remineralization of particles by bacteria.
1) The diffusivity in diatom (Skeletonema costatum) aggregates formed in roller tanks has been directly measured using a diffusivity microsensor (Revsbech et al., 1998). The same aggregates were afterwards sampled for porosity and TEP measurements. The apparent diffusivity varied between 94.7% and 99.9 % (average: 98.5%) of the free diffusion coefficient in sea water, and it varied with aggregate age. Measurements performed with and without flow suggested that fluid flow was insignificant within these diatom aggregates. Porosity is the main variable determining diffusivity in aggregates whereas TEP is of minor importance. Particle image velocimetry (PIV) has been used to describe and quantify the fluid velocity within and around porous and solid spheres. Fluid flow within porous spheres could be detected by elevated fluid velocities up to 1 radius downstream from the sphere surface. So far, no model describing the fluid velocity within and around porous aggregates with 1<Re<10 exists. Similar measurements will be performed for diatom aggregates with different species and variable porosity.
2) Bacterial abundance on aggregates in the field is dependent on the rate of attachment, detachment, growth and mortality as well as fluid motion and complex inter- and intra-specific interactions between the organisms. Organic aggregates are physically and chemically complex microenvironments. In the present project, quantification of the different factors have been performed on agar spheres, which are simple in structure and allow "clean" rate measurements of each of the above mentioned parameters for model development. Using the experimentally estimated process rates and integrating the component processes in a simple model reproduced the main features of the observed microbial population dynamics when agar spheres were colonized by raw sea water. Differences between observed and predicted population dynamics suggest, however, that other factors, e.g. antagonistic interactions between bacteria are of importance in shaping marine snow microbial communities.
3) The majority of silica dissolution occurs within the upper 200 m of the ocean, and sedimentation rates of diatom frustules generally do not decrease significantly with depth, suggesting reduced dissolution rates of diatoms embedded within sinking aggregates. Silica dissolution rates of aggregated diatom cells were compared to those of cells dispersed in the surrounding seawater during conditions mimicking sedimentation below the euphotic zone. A detailed analysis of fluxes of silica between the different pools could not falsify or strengthen the hypothesis. Possibly increased sinking velocity of aggregated cells rather than reduced dissolution rates of aggregated cells explain low dissolution rates of diatoms within the deep ocean.
4) Coccolithophorids like Emiliana huxleiy (Ehux) are calcareous micro-alga which form blooms in the ocean. We currently study the fate of CaCO3 and POC by use of pH, Ca- and O2-microsensors in fecal pellets from zooplankton which have been feed on Ehux. Helle was able to measure beautiful, highly reproducible, profiles on aggregates as small as 50x100 µm. These measurements will be incorporated in a model of vertical carbon flux which is currently being developed at Max-Planck Institute for Biogeochemistry in Jena.

Helle Ploug, Armin Gieseke, Carsten Schwermer