1 Department of Biological Sciences, Microbiology, Faculty of Science, Aarhus University, Aarhus University2 Department of Bioscience - Microbiology, Department of Bioscience, Science and Technology, Aarhus University3 Dustaffnage Marine Laboratory, Scottish Association for Marine Science, Oban, Scotland.4 Department of Bioscience - Microbiology, Department of Bioscience, Science and Technology, Aarhus University
When a bacteria-based nitrate biosensor with tip diameter down to 20 µm was invented about 12 years ago it became possible to measure detailed nitrate profiles in marine sediments, but functional tip membranes in the sensors were difficult to make, and the sensors did not work at temperatures below about 8°C. Large resources are being spent on exploration of the deep sea using sensor-equipped benthic landers, and it would be of significant value to add nitrate to the array of chemical sensors for in situ use, but it is then necessary to construct more robust sensors that work at temperatures around 2°C. By isolation of psychrotrophic nitrate-reducing and N2O producing bacteria from arctic environments and by application of a new procedure for making microscale ion-permeable membranes we have now succeeded in making biosensors that function reproducibly at low temperatures. It has thus been possible to analyze detailed nitrate microprofiles with < 1 µM resolution in sediments off the Japanese coast at 1500 m water depth where the temperature was 2.5°C. Many of the profiles exhibited near-surface nitrate peaks due to nitrification, and subsequent nitrate consumption due to nitrate respiration in anoxic layers led to total depletion at 10-15 mm depth. A result from comparison of shallow (75 m) water sediment profiles recorded both in situ and on recovered sediment cores is that there was more variability in situ than in the laboratory, probably due to decreased animal activity during laboratory conditions. It is difficult to work strictly aseptically when producing the sensors, and there is thus a risk that the bacterial culture may be contaminated with N2O reducing bacteria that will make the sensor insensitive to nitrate. However, when used continuously at room temperature they usually work well for several days, and the shelf-life is up to several weeks if they are stored at 4-6°C.