Incubation procedure and nutrient analysis: Update

Preparing ammonium samples for HPLC

Preparing ammonium samples for HPLC

The past several weeks have been very exciting! As soon as we collected the seawater samples I was working in the lab and following protocol. The incubation and associated harvesting time points have all been on schedule and we even have some preliminary data. At each time point I have been tasked with filtering and collecting duplicate samples for the analysis of nutrient concentrations and bacterial abundance. One major aspect of nutrient analysis is learning how to accurately operate a high performance liquid chromatograph (HPLC). Using this machine allows me to separate compounds within a mixture and to determine the concentrations of those components. So far I have done tests to determine how concentrations of ammonium change with time and I will soon do the same for both nitrate and DON. One observation we made was that as the alanine was consumed by the bacteria, the levels of N15-ammonium began to increase. During the first week (i.e. the first six time points) the N15-ammonium accounted for approximately 44 percent of everything produced with the N15 label.

Exposing samples to ultra violet light

Exposing samples to ultra violet light

Another interesting aspect of the nutrient analysis process is the conversion of nitrate to ammonium through a process known as zinc reduction. By adding a zinc substrate along with a strong acid N15-ammonium can be produced and then measured by the HPLC. One of the most recent developments is that we started to use a very intense ultraviolet light to oxidize samples in order to test for total dissolved nitrogen (TDN). This process presents some hazards because it produces an excess amount of ozone. To ameliorate this problem the UV oxidation is performed on the rooftop of the main lab building. After a period of 24 hours all the nitrogen in the sample will have been transformed into nitrate, and like before the nitrate will be reduced to N15-ammonium and measured by the HPLC. As a side note, in about a week’s time I’ll have a chance to operate the flow cytometer in order to quantify how bacterial abundance changes with time. This will provide a good indication as to how the bacteria colony is responding to the N15-alanine. Overall I’m excited to be working here at UTMSI and I’m really looking forward to the next five weeks of the the program.

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