Time to get Technical

The following are the methods that have been and will be used in this project:

Our Aim: To identify a specific group of microbes found in natural and restored eelgrass beds, positively correlated with transplant success.

Four native eelgrass beds, 3 restored beds, and 3 nonvegetated tidal flats (two of which are previously attempted restoration sites and one of which is adjacent to a successfully restored site) were extensively sampled in June to taxonomically characterize the microbial communities during the early summer period typical of restoration due to increased eelgrass growth.

By looking at these ten different sites, I will be able to rule out some of the natural community variation between sites, and more easily elucidate which microbes are tied to eelgrass survival.

 

To assess the microbial community composition, sediment cores were taken using sterile 10 ml syringes with removed tips, to a depth of approximately 7 cm. Twenty cores were taken within each established bed, no closer than 2 meters apart, ten of which were taken adjacent to eelgrass rhizomes and ten cores taken at least 15 cm from rhizomes, in order to characterize the two distinct microbial communities within vegetated beds (Jensen 2007)

 

All cores were transported on ice and stored at -80°C within 2 hours of collection to cryopreserve the bacterial communities, and are currently in -80°C storage.

From each sampled core, 0.25 g of sediment will be collected from a depth of 4-5 cm, to characterize the community at the approximate rhizome depth. Powersoil extraction kits (Mo-Bio, Carlsbad, California) will be used to extract total community genomic DNA.

 

Bacterial primers 341f and 805r, which have been shown to perform well in targeting bacteria and archaea, will be used to select for the V3-4 region of the 16s rRNA gene (Klindworth 2013)

QIIME (Quantitative Insights into Microbial Ecology) will be used to analyze the sequencing reads (Caporaso 2012)

A heat map will be created in order to visually interpret the differences in microbial composition by treatment using the phyloseq package in R (McMurdie 2013)

 

Example from Seekatz 2014. Notice that some species are found under some "treatments" (like the blue and green) but not others (like the red)? In my samples, I expect to see some species found where eelgrass is, but missing where transplanted eelgrass won't grow.

Metadata was collected in the field at the time of microbial community sampling to provide context for the collected microbial samples, including: •Sediment temperature and pH •Redox potential •Water column salinity and dissolved oxygen •Depth (elevation) below MLLW •Sediment porosity and bulk density •Total carbon, total nitrogen, and organic matter content •Light attenuation •Sediment grain size composition •Eelgrass shoot density

In addition, a standard acetylene reduction assay was used to assess nitrogen fixation activity at each site. This tests one of many possible microbial mechanisms that may be keeping the eelgrass alive.. but that's for another note...!

 

Above, a colleague takes redox potential measurements as a restored site during a sunrise low tide.