For three billion years, before the Cambrian diversification of life, laminated carbonate build-ups called stromatolites were widespread in shallow marine seas. These ancient structures are generally thought to be microbial in origin and potentially preserve evidence of the Earth's earliest biosphere. Despite their evolutionary significance, little is known about stromatolite formation, especially the relative roles of microbial and environmental factors in stromatolite accretion. Here we show that growth of modern marine stromatolites represents a dynamic balance between sedimentation and intermittent lithification of cyanobacterial mats. Periods of rapid sediment accretion, during which stromatolite surfaces are dominated by pioneer communities of gliding filamentous cyanobacteria, alternate with hiatal intervals. These discontinuities in sedimentation are characterized by development of surface films of exopolymer and subsequent heterotrophic bacterial decomposition, forming thin crusts of microcrystalline carbonate. During prolonged hiatal periods, climax communities develop, which include endolithic coccoid cyanobacteria. These coccoids modify the sediment, forming thicker lithified laminae. Preservation of lithified layers at depth creates millimetre-scale lamination. This simple model of modern marine stromatolite growth may be applicable to ancient stromatolites.

N2 fixation is a key process in photosynthetic microbial mats to support the nitrogen demands associated with primary production. Despite its importance, groups that actively fix N2 and contribute to the input of organic N in these ecosystems still remain largely unclear. To investigate the active diazotrophic community in microbial mats from the Elkhorn Slough estuary, Monterey Bay, CA, USA, we conducted an extensive combined approach, including biogeochemical, molecular and high-resolution secondary ion mass spectrometry (NanoSIMS) analyses. Detailed analysis of dinitrogenase reductase (nifH) transcript clone libraries from mat samples that fixed N2 at night indicated that cyanobacterial nifH transcripts were abundant and formed a novel monophyletic lineage. Independent NanoSIMS analysis of 15N2-incubated samples revealed significant incorporation of 15N into small, non-heterocystous cyanobacterial filaments. Mat-derived enrichment cultures yielded a unicyanobacterial culture with similar filaments (named Elkhorn Slough Filamentous Cyanobacterium-1 (ESFC-1)) that contained nifH gene sequences grouping with the novel cyanobacterial lineage identified in the transcript clone libraries, displaying up to 100% amino-acid sequence identity. The 16S rRNA gene sequence recovered from this enrichment allowed for the identification of related sequences from Elkhorn Slough mats and revealed great sequence diversity in this cluster. Furthermore, by combining 15N2 tracer experiments, fluorescence in situ hybridization and NanoSIMS, in situ N2 fixation activity by the novel ESFC-1 group was demonstrated, suggesting that this group may be the most active cyanobacterial diazotroph in the Elkhorn Slough mat. Pyrotag sequences affiliated with ESFC-1 were recovered from mat samples throughout 2009, demonstrating the prevalence of this group. This work illustrates that combining standard and single-cell analyses can link phylogeny and function to identify previously unknown key functional groups in complex ecosystems.

We used micromanipulation to isolate from their environment representative samples of seven geographically distant field populations fitting the description of Microcoleus chthonoplastes (a cyanobacterium) and obtained seven corresponding cultured strains. Samples of both field populations and cultures were phenotypically characterized by microscale techniques, and their partial 16S rRNA gene sequences were compared by denaturing gradient gel electrophoresis and in some cases by sequencing. All field populations and strains were phenotypically extremely coherent, and their 16S rRNA sequences were indistinguishable by DGGE. The sequences determined were identical or virtually identical. Thus, M. chthonoplastes represents a single, welldelimited taxon with a truly cosmopolitan distribution. Comparison with three culture collection strains originally assigned to M. chthonoplastes revealed that strain PCC 7420 belongs to the same tightly delimited group, both phenotypically and in 16S rRNA gene sequence, but that strains SAG 3192 and 10mfx do not.

Physiological studies of Trichodesmium species have been hindered by difficulties in maintaining actively growing, nitrogen-fixing cultures. Previous cultivation successes have not been widely duplicated. We present here a simple modified seawater medium and handling techniques which have been used to maintain actively growing Trichodesmium thiebautii in laboratory culture for over 1 year. The cultured population, isolated from coastal Atlantic waters, has a growth rate of 0.23 division day-l and exhibits light-dependent nitrogen fixation during exponential growth. Various morphologies, including solitary trichomes, and aggregates (spherical puffs and fusiform tufts) are present during growth. Spectral and scalar irradiance were measured within naturally occurring (coastal Atlantic) aggregates with small (diameter, 50 to 70 im) spherical fiber-optic sensors. In contrast to naturally occurring puffs, cultivated Trichodesmium aggregates exhibited spectral properties consistent with low-light adaptation. Cultivated puff-type aggregates were also examined by using oxygen microelectrodes. The simple medium and approach used for cultivation should be easily reproducible and amenable to further manipulations and modifications useful for physiological studies of Trichodesmium spp. in culture.

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