Recently, a lot more of our clients have started using quantitative PCR (qPCR) to track the nitrifier populations in their systems. Several groups do this on a weekly basis – and we have seen some really interesting results that highlight how specific qPCR tests can be.
First off, for more than 95% of the systems we work with, the AOB population is dominated by Nitrosomonas. We will, on occasion, see a few other autotrophic ammonia oxidizers pop up, but not often. So, we usually focus on levels of Nitrosomonas in a system.
For tracking Nitrosomonas levels, most groups (us included), measure the levels of a specific gene, amoA encoding a subunit of the ammonia monooxygenase enzyme. The most commonly cited primers for tracking this gene are called amoA-1F and amoA-2R, first reported in 1997 (PUBMED). Unfortunately, these primers are very specific to Nitrosomonas europaea – and the primers fail to reliably detect other Nitrosomonas species. This strain may be easy to grow in a lab, but from all our microbial community analyses, it is rarely the dominant strain in industrial or municipal wastewater treatment systems.
So, we performed a meta-analysis from a few hundred DNA samples and identified the most common Nitrosomonas species present in a variety of different systems. Once we knew the strains we wanted to target, we developed a modified set of 8 primers that, in different combinations, cover 10 different species. And, it’s really paid off.
At one facility, we have tracked N. marina levels over the summer. After a brief upset, the facility recovered nitrification, but N. marina levels remained very, very low. Instead, we saw increasing levels of N. nitrosa. Without this primer set, we would not be able to track the nitrifiers in this system — and we certainly wouldn’t be able to see the change in species after the upset.