Microbial Sampling for the TRACERS Project on the NBP

Working on behalf of Gerhard Herndl’s lab at the University of Vienna in Austria, Santiago Gonzalez and I are sampling the microbes in the water column as part of the TRACERS project.  Prokaryotic microbes (specifically bacteria and archaea) are present all throughout the water column in all the world’s oceans, with as many as millions of cells in a single milliliter of seawater at the surface and tens or hundreds of thousands at depth.  So there are lots and lots of microbes present, and we’re studying their role in the cycling of organic matter and nutrients.  As the phytoplankton responsible for photosynthesis in surface waters sink down into the water column, it’s the microbes that work to degrade and recycle this organic material and we expect the high flux of sinking particles following an algal bloom to provide lots of ‘food’ for the microbes down below.

Sampling seawater from the rosette, which can collect water at twenty different depths throughout the water column. From left to right: Dave, Cassandra, Sarah and Roberta collecting water, with Amanda and Christina in the background. Photo credit: A. Margolin.

When sampling from the Niskin bottles on the CTD, I first take small samples for determining the abundance of microbes in the water -- just a few milliliters of seawater are fixed with glutaraldehyde and then frozen.  We’ll ship these back to Vienna and count the prokaryotes and viruses in these samples using flow cytometry.  I then collect a much larger amount of seawater (usually ~10L) and filter it through a 0.2µm filter to collect the microbes in the water.  I store these filters in small tubes and freeze them, and will also send these back to Vienna.  Back in the lab there, we’ll extract the DNA from the microbes collected on the filter and use that DNA for various analyses like PCR, T-RFLP to determine community composition, cloning and sequencing of 16S rRNA genes, and qPCR of particular functional genes of interest.  This will give us an idea of what organisms are present in our samples, how the microbial communities differ at different depths and stations, and to some extent what metabolic capabilities these microbes have.  For all these analyses, I am just collecting the samples here at sea but most of the work will be done back home in Vienna.

The filtration setup in the Bio Lab that Roberta uses to collect particles from seawater samples, for the extraction of microbe DNA back in Vienna. Photo credit: R. Hansman.

On board, Santiago is working to make some metabolic rate measurements.  He collects seawater and then adds two different radioactively-labeled substrates (³H-leucine and ¹⁴C-bicarbonate) to separate samples that the microbes use in two different ways, either as heterotrophs (taking up ³H-leucine) or autotrophs (fixing ¹⁴C-bicarbonate).  After adding the substrates to a set volume of seawater and incubating the microbes in the seawater for a fixed amount of time at in situ temperature, he then filters these samples to collect the microbes on 0.2µm filters.  Any of the labeled substrates that were incorporated by the microbes are now part of cellular material that is collected on the filter.  By using a scintillation counter to determine the amount of radioisotope on each filter, he can calculate how much substrate was utilized.  And using the set volume and fixed incubation time, he can further calculate the rate of heterotrophic production (for the ³H-leucine samples) or autotrophy (for the ¹⁴C-bicarbonate samples).  Because the radioisotopes are considered hazardous, Santiago has special training to work with these materials and does all his work in an isolated container called the rad van located up on the helo deck of the NBP.

Santiago enjoying his work in the rad van. Photo credit: R. Hansman.

The incubators on the helo deck that Santiago uses. Photo credit: A. Margolin.

The helo deck, with the rad van and incubators. Photo credit: A. Margolin.

After the cruise, we’ll combine our data from sampling the microbes with the many other types of measurements also being done by other groups on board to help get a better picture of what’s happening in the Ross Sea.

--Roberta

P.S. Hello to Mrs. Hansman’s 3^rd grade class at Glen Arden Elementary!