Tracing the origin of marine sedimentary hopanoids using compound-specific multi-istope analysis and metagenomics
Final Report Abstract
Hopanoid lipids in the marine system may be sourced from primary producers or heterotrophic consumers. They may come from free-living, open ocean communities, or from shallow mats, tidal zone communities, or even be contributed primarily by terrigenous runoff. The relative importance of these sources is not yet known. Much of this ignorance is due to a paucity of isotope data for hopanoids, and multi-dimensional isotopic data supplemented by molecular genetic information may permit a better understanding of hopanoid sources, improving their potential as long-lived tracers of microbial biomass and microbial carbon processing. The main focus and biggest challenge of this project was the development of the LC purification scheme allowing for compound-specific isotope analysis of BHPs with purities >95%. The method development could only start 1.5 years after the DFG fellowship started when new instrumentation became available. Since then, I could establish a reverse phase method, which successfully separates BHPs that co-eluted using previously published methods and even separates a multitude of BHP isomers. Surprisingly, the development of the normal phase clean up method has been challenging and purities have not yet reached the level they need to have for compound-specific isotope analysis in general and 14C analysis in particular. However, I am confident that the project will succeed in the near future through the analytical possibilities made possible after May 2013 and the additional funds becoming available for this project through NSF allowing to purchase further LC columns needed to establish the normal phase clean up step. Due to financial considerations by Prof. Pearson, DNA sequencing has not yet been performed. However, the preliminary data show that DNA could be extracted and sqhC genes successfully amplified with previously used techiques. Nevertheless, after careful consideration, we decided to change the strategy from PCR-cloning-Sanger sequencing to 454 Pyrosequencing to improve the estimation of sqhC diversity. DNA sequencing will be performed once the isotope measurements are achieved.
