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We were able to match part of the money of the PRF grant with internal UConn funding in order to hire two grad students, Lexy Fowler and Natalie Stork. We were thus able to save money for the second year. No student calendar salary money was used during this first year. This maneuver will allow for paying both graduated students for the second year, and, thus, be more productive.

The proposal is composed of two parts: (1) Laboratory experiments/manipulations and (2) Comparison with natural systems. Progresses were made on both fronts.

On the laboratory side, this first year was mainly spent developing, building and testing the ‘controlled organomineralization device’ (COD) in my laboratory. The two new grad students, worked in close relationship with Kim Gallagher, a finishing PhD student from Prof. Visscher lab. Kim is working on the role of sulfate reducing bacteria (SRB) in the precipitation of calcium carbonate in batch culture. Her expertise in culturing SRB allowed for a better comparison with the natural environments. The SRB taxa used in experiments were isolated from modern marine stromatolites.

Principal progresses are as followed and related to the two principal hypotheses of the proposal:

A. Laboratory experiments/manipulations

1. Hypothesis I: EPS alteration and control on morphology and mineralogy

We decided to compare the control of two types of extracellular polymeric substances (EPS) matrix on the morphology and the mineralogy of carbonate precipitates. EPS resulting from SRB cultures and industrial model EPS were tested. The goal was to produce precipitates in vitro that show similar mineralogical and morphological features than the natural microbialites.

1.1 Batch cultures from hypersaline microbial mat

The study started with batch cultures of SRB with various combination of starting pH, Ca and Mg content. The SRB were used because of the increasing evidence of their seminal role in calcium carbonate precipitation during early diagenesis. Visscher laboratory has also a long experience in the culturing of such bacteria. Time series of usual parameters were performed: pH, OD600, calcium, magnesium, sulfide, protein, etc. Organonimeralization experiments were done using extrinsic alkalinization (forced precipitation) but results were not satisfying. We were not able to produce precipitates showing similar features as the natural environments. This is mainly due to the batch culture format that is not mimicking the real natural conditions.

1.2 Transfer to flow cell (COD) and creation of a biofilm

We were able to build a COD following a flow cell setup. This setup is highly flexible and allows for testing organomineralization with and without bacteria (only EPS matrix). The flowcells (COD) were inoculated with cultures of SRB in order to grow biofilm. After long months of fine-tuning and design reassessment, we were successful establishing SRB biofilms in steady state on various physical substrates, i.e., glass coverslip, Aclar. We specially designed flowcell for in situ observation of biofilms and potential precipitates using confocal laser scanning microscopy (CLSM). After obtaining a steady biofilm, we rinsed it to remove the excess of Ca and Mg and alkalinized. This process insures that the precipitation of calcium carbonate is related to the calcium that was bound to the EPS. These experiments have produced nanometer-scale spheroids of high-Mg calcite crystals (nanospherulites) similar to the one observed in the natural hypersaline microbialites (San Salvador and Eleuthera Island, Bahamas). These experiments documents for the first time the precipitation of very-high Mg calcite to Ca-dolomite in organomineralization experiments without living bacteria. It emphasizes the important control of EPS on the morphology and mineralogy of the mineral precipitate in microbial mat. In this case, the metabolism is producing the EPS, but is not involved in shaping the final mineral product as suggested by several authors in the literature. This work is in preparation for publication.

We started to use model EPS in similar experiments. So far, the various recipes of EPS and amino acids composition did not produced similar results. Extracted EPS did not produce the projected results. It looks like the natural EPS produced by the bacteria show the specific sterical conformation needed to obtain suitable results. However, clearly the first hypothesis of the proposal seems verified, especially the questions related to the relationship between crystal morphology and alkalinization processes. These experiments also address the overcoming of kinetic dolomite inhibition by nucleation on EPS. The dynamic of the magnesium within the EPS matrix has thus to be investigated using cation competition experiments. In addition, experiments to test oversaturation versus enzymatic/physicochemical alteration of the EPS are on the way.

2. Hypothesis II: Geochemical biosignature

This part of the project will be addressed during this second year. Now that we have a successful set-up, a methodology to grow biofilm, and analytical capability, the dynamic of REE will be studied using SRB biofilm as well as model EPS (xanthan and alginate).

B/ Comparison with the natural systems

Lexy Fowley and Natalie Stork went to two field sites: open marine stromatolite (Highborne Cay, Bahamas) and hypersaline microbialite (San Salvador, Bahamas) in order to collect samples of modern microbialites for culturing and EPS extraction. Those samples have been used to isolate bacteria (cyanobacteria and SRB) for culturing in flowcell experiments. In addition, the mineral products have been characterized, using light and SEM microscopes, confocal, and geochemical analyses (X-ray diffraction and isotopic composition). We acquired a new high precision saw and an embedding system (Cytovac) that allow for amazing serial thin sectioning. Natalie Stork is culturing cyanobacteria with great results. Cyanobacteria as well as extracted EPS from cyanobacteria have not been used in organomineralization experiments yet. Those experiments with pure cultures will allow to address the specific role of each bacterial EPS in the faith of the mineral product.

Publications

On this project, we have already one publication: Gallagher et al. in press. In addition, three publications are in preparation: Gallagher et al. on nannospherulites in flowcell, (2) Fowler et al. on microbialites development in San Salvador, (3) Glunk et al. on the dynamic of magnesium in natural EPS (Eleuthera Island, Bahamas) and laboratory experiments. All three publications have benefited from the PRF money.