Characterization of Bacterial Enrichments and Natural MgSO4 Salts Containing Halophilic Archaea and Bacteria Using Reflectance IR, Protein Assays, and Confocal Microscopy: Implications for Identifying Biomarkers on Mars and the Jovian Satellites
Magnesium sulfate minerals have been identified at many sites on Mars and in Martian meteorites, suggesting that Mars once possessed brines rich in magnesium and sulfate ions. Furthermore, magnesium sulfate minerals and brines are thought to occur on Europa, Ganymede and Callisto. Therefore, terrestrial sites with magnesium sulfate-rich mineralogy and chemistry provide relevant analogues for planetary missions. We specifically examined the distribution and concentration of biomass in terrestrial magnesium sulfate salts, using confocal microscopy, a modified Lowry protein assay and Diffuse Reflectance Infrared Fourier Transform Spectroscopy (reflectance IR) analysis. Our results may be applied to searching for biomass on other planetary bodies. The Basque Lakes of British Columbia, Canada, are an ephemeral system of 6 lakes/ponds with brines and mineral deposits dominated by magnesium sulfate, and support the growth of halophilic Archaea and Bacteria in surface and near-subsurface environments. Imaging of magnesium sulfate crystals collected from the Basque Lakes using confocal microscopy revealed halophilic organisms trapped primarily within fluid inclusions and void spaces between intergrown crystals. Diffuse biomass containing fluorescent pigments was also identified separate from cells, suggesting cell lysis might have occurred after or during entrapment in the sulfate salt matrix. A modified Lowry protein assay performed on these salt crystals demonstrated that biomass was preserved in every crystal analyzed, and protein contents ranged from 0.009 to 4.60 mgbiomass/gsalt. Reflectance IR analysis of enrichment cultures containing cyanobacteria, Archaea, and dissimilatory sulfate reducing bacteria (SRB), grown from material sampled at the Basque Lakes, allowed us to identify biomarker absorption features between 600-1600 and 2000-3000 cm-1. Distinctive spectral features were identified for cyanobacteria and Archaea at ∼1075 cm-1 and ∼1550 cm-1 corresponding to C-N, N-H, PO4-3, and C-N, N-H, COOH respectively, while samples containing SRBs and Microbial mats possessed unique features at ∼910 cm-1 corresponding to C-CH2, and ∼1370 cm-1 corresponding to C-CH3. Spectra obtained from natural salt samples were also found to contain biomarkers in these regions, demonstrating that we can detect biomass within sulfate salt crystals. The most sensitive biomarkers observed in this study were the 1550 +/- 20 cm-1, C-N, N-H, COOH absorption and the 630 +/- 10 cm-1 C-N, C-S, C-H, N-H, and -OH bond features, and the limit of detection of biomass within a sulfate crystal was in a sample that possessed 0.78 mgbiomass/gsalt. Using a 0.5 x 1 micron size cell model, the biomass value of this sample corresponds to 7.8 x 108 cells/gsalt. Based on the ability of reflectance IR to detect small quantities of biomass within natural sulfate salts, and to distinguish between different groups of organisms, we propose that it is an effective tool for identifying outcrops and surficial sedimentary deposits containing enrichments of organic molecules on other planetary bodies.