Cooperating scientist: Rene Diocares , Griffith University, Brisbane Qld, Australia

Dedication: This website is dedicated to the memory of the late Mexican mangrove researcher
Dr. Gina Holguin who participated in the development of this technique.

A team from the group of Environmental Microbiology, the Northwestern Center for Biological Research, Mexico evaluates nitrogen fixation of mangroves in the field

Why do we need this technique?
The heavy nitrogen non-radioactive isotope (¹⁵N₂) is often used as a label for nitrogen fixation experiments, in which it is desirable to monitor directly the rate of nitrogen fixation and the destination of the resulting metabolites in an organism or ecosystem. This is one of the most acceptable and reliable techniques for this purpose, and it has been used for decades.

Heavy nitrogen gas can be purchased in a pressurized cylinder, or produced in the laboratory with specialized equipment. However , both methods of obtaining the gas are expensive, and for some laboratories, particularly those in developing countries, this represents an insurmountable obstacle to their research. These methods of obtaining the gas also introduce a complication, because a typical nitrogen fixation experiment requires samples of gas at atmospheric pressure. The pressure must be reduced with a pressure regulator in the case of gas in a pressurized cylinder, and traditional laboratory methods produce heavy nitrogen at pressures well below atmospheric that are not generally suitable for direct use in nitrogen fixation experiments. This is because there is no easy way in which to equalize the gas to atmospheric pressure for subsequent injection into a sample bottle.

Previously, a simple technique was developed in Australia by Wood and Kennedy (2001) to produce ¹⁵N₂ gas at a research laboratory involved in biological nitrogen fixation research (not a specialized chemical production facility). This technique, and the modification to it described here, are both limited by the volume of available plastic syringes, but the procedure here does not require the special apparatus described by Wood and Kennedy (2001). The technique produces small quantities of heavy nitrogen at atmospheric pressure with readily available analytical chemicals, and inexpensive laboratory supplies and equipment.

Chemical principle
The technique produces heavy nitrogen through the reaction of ¹⁵N-labeled ammonium sulfate [(¹⁵NH₄)₂SO₄] with lithium hypobromite (LiOBr), and the gas subsequently is sparged first with acidified potassium permanganate (KMnO₄) and then with acidified water to remove contaminating oxides that otherwise would be taken up by the organisms and thus produce errors in nitrogen fixation experiments.

The advantages of the technique are:

1. It has the primary advantage over traditional methods of producing ¹⁵N gas at atmospheric pressure, so the technique is usable directly for nitrogen fixation experiments.
2. The equipment is easily constructed from a few common laboratory materials, including standard tubing, connectors, and disposable syringes; therefore it is inexpensive.
3. The ¹⁵N-labelled salt is relatively inexpensive, and can be shipped cheaply from the manufacturer because it is solid and not radioactive.
4. The amount of gas that can be generated per batch using this method is limited only by the volume of the syringes, and larger syringes can be adapted to the method if they are available.
5. The technique does not require any special equipment such as pressure regulators, vacuum equipment, or glassware.
6. Once all necessary materials have been collected, any person with training in chemical techniques can produce nearly pure ¹⁵N₂ gas at atmospheric pressure in about four hours, after about four hours to study the procedure.