The Role of Microbes in the Precipitation of Microbialites in Cuatro Cienegas, Mexico: A Genomic and Stable Isotopic Perspective

Ancient biologically-mediated sedimentary carbonate deposits, including stromatolites and other microbialites, provide insight into conditions on early Earth. However, interpretation of the environmental and evolutionary significance of microbialites throughout the geological record is dependent upon an understanding of the complex linkages between biological and chemical processes, and isotopic properties associated with the formation of modern microbialites. Here we present the results of metagenomic and isotopic analyses two types of actively accreting freshwater microbialites in Cuatro Cienegas: oncolites and thrombolites. To determine the genetic capabilities of the microbial communities and to identify the dominant metabolic pathways present in the samples, total DNA was purified from microbialite surface samples and pyrosequenced. Genes identified in the metagenome included both autotrophic and heterotrophic processes. In addition, 16S rDNA sequences recovered from the metagenome included both cyanobacteria and heterotrophic bacteria. d13C of intra-crystalline organic matter (IC- OM) in the microbialites is -26°, consistent with the enzymatic fractionation associated with oxygenic photosynthesis. The microscopy-based occurrence of cyanobacteria on the microbialites, the visible presence of bubbling O2, and the metagenomic confirmation of genes attributed to photosynthesis confirm the photoautotrophic origin of IC-OM. Carbonate d13C values of the oncolite and thrombolite are depleted relative to equilibrium considerations indicating that a 13C-depeleted source of carbon is strongly influencing the DIC at the site of carbonate precipitation. Respiration of photoautotrophic biomass by heterotrophic organisms would release 13C-depleted CO2 to the DIC reservoir resulting in a localized negative shift in the 13C-DIC. Our results indicate that the microbialites are precipitating at sites in direct association with heterotrophic respiration where the remineralization of photoautotrophic carbon is important. Because aerobic respiration leads to carbonate dissolution, our results further suggest that anaerobic heterotrophic respiration processes may be critical for microbialite formation in Cuatro Cienegas. Metagenomic results confirm the presence of genes capable of assimilatory nitrate reduction, denitrification, and sulfate reduction in the microbialites. d15N values of IC-OM from the microbialites are 3-5° depleted relative to 15??DIN (+13 to +16°), consistent with enzymatic fractionation associated with nitrate assimilation by aquatic photoautotrophs where [NO3=] is not limited. Reduced organic sulfur from the thrombolite has a d34S value of - 25°, a value that can only be associated with dissimilatory sulfate reduction- an anaerobic, heterotrophic process. Together, the metagenomic and isotopic data suggest that the coupling of aerobic and anaerobic autotrophic and heterotrophic processes is a critical component to the formation of microbialites in the CCB.