Lipid biomarkers, carbon isotopes, and phylogenetic characterization of bacteria in California and Nevada hot springs

Abstract

Microbial mats were collected from hot springs in California (Eagleville) and Nevada (Paradise Valley and Crescent Valley) to determine bacterial community structure and pathways of carbon cycling in different geothermal environments of the western United States. Phospholipid fatty acids (PLFA) at Eagleville contained even-numbered fatty acids, with 16:0 being the most abundant (48.8%), followed by 18:1ω9c (17.2%), 16:1ω7c/t (6.3%), and 18:0 (6.2%), which are consistent with lipid profiles of cyanobacteria or other phototrophic bacteria. The PLFA profiles at Paradise Valley and Crescent Valley were dominated by similar even-numbered fatty acids; however, branched fatty acids such as iso- and anteiso- 15:0 and 17:0 were also abundant (up to 7.1% compared to 2.0% at Eagleville), suggesting greater relative abundance of heterotrophic bacteria in these springs. Analysis of neutral lipids was only performed on Eagleville and Paradise Valley springs, which revealed abundant bacterial hopanoids including the 2-methylbacteriohopane-32,33,34,35-tetrol (2-methylBHT) that is specific to cyanobacteria; however, the diversity of hopanoid compounds was significantly lower at Eagleville than at Paradise Valley. The carbon-isotope composition of individual PLFA averaged -30.7 ± 1.3%(n = 7) at Eagleville, -28.0 ± 1.8%(n = 3) at Crescent Valley, and -29.7 ± 3.1%(n = 12) at Paradise Valley. Carbon isotope fractionation between PLFA and CO2 was only available for Eagleville (-11.7%) and Paradise Valley (-21.7%), which indicated the predominance of the Calvin cycle for CO2 fixation in these hot springs. Bacterial 16S rRNA genes were extracted from environmental samples at Eagleville and Paradise Valley but not Crescent Valley. Clone libraries indicated the predominance of cyanobacteria (50-75%) at these locations, which is consistent with the lipid profiles. Phylogenetic tree of the 16S rRNA genes indicated that most of the cyanobacterial sequences are unknown and may be specific to the Nevada and California hot springs. Phototrophic green non-sulfur bacteria were also present at Eagleville (13%) and Paradise Valley (7%). The remaining sequences were related to α-, β -, and γ-Proteobacteria, Acidobacteria, Deinococcus/Thermus, Bacteroidetes, and Spirochaetes. However, not all of these sequences were present at each of the springs. Results of this study demonstrate the consistency among lipid profiles (phenotypes), carbon isotopes (biogeochemistry), and 16S rRNA genes (genotypes) of the bacterial community in these hot springs, which cumulatively suggest the importance of cyanobacteria in primary production of biomass under the environmental conditions examined.