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From the laboratory point of view, this second year was mainly spent creating a cyanobacteria culture collection for extracellular organic matter (EOM) extraction, characterizing model extracellular polymeric substances (EPS) and continuing testing organomineralization experiments with the 'controlled organomineralization device' (COD). Natalie Stork, a graduate students associated to the project, was able to isolate a large variety of cyanobacteria from various field sites for EOM extraction. Natalie Stork also continued working in close relationship with Kim Gallagher, a PhD student from Dr. Visscher lab. Kim is working on the relationship between sulfate reducing bacteria (SRB) and precipitation of calcium carbonate. Comparison between EOM from cyanobacteria and SRB are in progress.

From the 'natural system' point of view, Lexy Fowler, another graduate student, finished her master thesis on the formation of microbialites in Storrs Lake, Bahamas. The results are very exciting and a paper will be submitted before the end of the year. Cyanobacteria from Storr's lake have also been isolated by Natalie Stork and will be used with the COD.

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

A. Laboratory experiments/manipulations

1. Hypothesis I: EOM alteration and control on morphology/mineralogy

We continued our assessment of the impact of various EOM on morphology and mineralogy of carbonate precipitates. We believe that although metabolisms are important in organomineralization by creating the needed carbonate alkalinity and producing EOM, the characteristics of the EOM are 'shaping' the final mineral product. In addition to SRB and industrial model EOM, we also characterized cyanobacterial EOM. The goal is to produce in vitro precipitates that show mineralogical and morphological features similar to natural microbialites.

1.1 Model EPS and calcium binding

Xanthan as a model EPS for organomineralization has produced relatively inconsistent results so far. One issue was that the 'out of the box' Xanthan was not pure and present a large and inhomogeneous initial Ca²⁺ content. A part of this calcium is strongly bound to EPS as it is still present after several dialyses. We performed thorough analyses of commercial Xanthan to determine how much calcium and magnesium can be bound to this initial model EOM and how much and how fast bound calcium can be removed by dialyze. This is an important step to build accurate Ca and C budgets during organomineralization experiments.

1.2 Cyanobacteria cultures and EOM extraction

Isolating and culturing cyanobacteria is a very delicate and time-consuming task. Natalie Stork was able to create and maintain cultures of a wide diversity of cyanobacteria from various field sites.

During natural EPS extraction, a key problem was the amount of phosphate that can precipitate as apatite during the process. Phosphate levels in cultures are much higher than natural systems. We performed chemical modeling that predicts oversaturation with respect to hydroxyapatite. This oversaturation has of course a strong impact on other mineral saturation indexes when allowed to precipitate. This phenomenon is creating an artifact in organomineralization experiments in laboratory that is rarely taking into account in the published literature. We submitted a paper on this subject.  This artifact is not present for EOM extracted from natural microbial mats. The challenge was to balance the need to growth culture, which require phosphate and the negative effects of phosphate resulting in artifacts. Although the problem has not been fully fixed for SRB cultures yet, we were able to grow cyanobacterial cultures with very low phosphate levels in order to minimizing artifacts. Our results reinforce the well known adage that extrapolation of culture results to natural systems should be done very cautiously.

1.3 Organomineralization experiments

Experiments were put on hold for a large part of the year because of the problems described in 1.2. We recently restarted our investigation of EOM degradation to produce minerals by using degrading agent such as H₂0₂. The goal of such experiments is to induce carbonate precipitation with carbon and bound cations provided by the EOM itself. First results are encouraging with production of dumbbells of calcite and aragonite. The next step is to investigate the dynamic of the magnesium within the EOM matrix using cation competition experiments. Preliminary experiments using Ca²⁺ electrode and diffusion cells indicate that certain EOM have a possible preference for Mg (compare to other cations). Different pools of EOM, i.e., extracellular polymeric substances (EPS) and low molecular weight organic carbon (LMW-OC), seem to have different behaviors (see model of organomineralization in 'Nugget'). More work is certainly needed to confirm these initial results before publication.

2. Hypothesis II: Geochemical biosignatures

Dr. Robert Mason, who is in charge of the rare earth elements (REE) analysis, took a 2-year leave of absence as a rotator at NSF. Together with the issues described in 1.2, this has delayed the investigation of REE fractionation during EOM mineralization. Rob is now back at UConn for one day a week and we are working out a schedule to perform experiments this year (no-cost extension). We also started collaborating with Pascal Philippot (IGPG Paris) to tests in situ analysis of trace metals in organominerals using synchrotron techniques to see if we could map the distribution and speciation of metals (enzymatic co-factor and substrate) at nanometer scale. We provided samples from natural microbialites. The analyses has been performed this summer, we are waiting for the results. This approach is highly complementary to the REE work.

B/ Comparison with the natural systems

Lexy Fowley finished her master thesis in July on hypersaline microbialites (San Salvador, Bahamas). Amount the relevant results to this proposal, it showed that precipitation of high mg calcite is taking place within the EOM matrix and is not related to cyanobacterial photosynthetic CO₂ uptake. SEM study demonstrated that the onset of organomineralization within EOM is characterized by nanometer-sized carbonate spherulites, replacing organic matrix. These nanospherulites then serve as nuclei for further precipitation of microsparite and sparite, creating micropeloidal microstructure. This is highly relevant for fossil microbialites that often show similar texture (e.g., microbialite in reefs and mud mounds). A paper will be submitted before the end of the year.