Sometimes, the same question pops up a few times in one week. This week, we were asked about tracking nitrogen fixation in cases of suspected nutrient limitation.
Some wastewater influents are low in nitrogen (ammonia & TKN). For example, pulp & paper processing usually doesn't contain appreciable amounts of TKN - and the same is true for some chemical plant influents. For macro nutrient requirements, operators add supplemental N in the form of ammonia or urea. But are the operators adding enough... or too much?
How do you answer the question "Is nitrogen fixation occurring in my MLSS sample?". The most direct method of measuring nitrogen fixation is the acetylene reduction assay. But that requires a healthy sample, a tank of acetylene, and a gas chromatograph. It may be a simple assay, but there's a good bit of equipment required.
An alternative is to track the genes that encode the nitrogenase enzyme complex (the enzyme that converts dinitrogen gas to ammonia/ammonium). A good example is a paper by Gauthier et al., from 2000. In the paper, they did use the acetylene reduction assay to quantify nitrogen fixation activity, but they also used quantitative nitrogenase (nifH) gene probing.
Today, we can use qPCR in place of the older gene probing technique for quantifying nifH in an activated sludge sample. But we need to remember that qPCR (and gene probing) are both indirect assays. They detect the presence of the gene (DNA). Unfortunately, this gives us an idea about the genotype(s) present in the sludge - the potential metabolic capabilities of an organism - but it can't necessarily tell us if that capability is in use as many secondary metabolic pathways are only turned on under certain situations. The above reference points this out in the abstract: "only 13 (27%) [of the isolates] showed inducible N2-fixing activity".
Regardless, qPCR can be helpful in tracking nitrogen fixation in activated sludge when it is combined with other information such as the residual levels of nitrogenous compounds in the effluent. This is particularly true when supplementing with ammonia or urea. If there's no residual nitrate, nitrite or ammonia and lots of nifH by qPCR, it's reasonable to assume there is nitrogen fixation.
