A surprise from a smelly bucket: a novel acid loving ammonia-oxidizing bacterium

Ammonia is a compound contained in animal waste and its concentrations in the environment are strictly regulated. For this reason, waste waters and polluted air are treated to reduce the concentration of this molecule before releasing it to the environment.

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Ammonia is a compound contained in animal waste and its concentrations in the environment are strictly regulated. For this reason, waste waters and polluted air are treated with systems that make use of ammonia-oxidizing microorganisms that reduce the concentration of this molecule before releasing it to the environment.

Ammonia oxidation is a process that usually happens at neutral pH. One day our lab was contacted by a company that operated an ammonia-removing biofilter to clean the air from a pig farm. The biofilter showed good and stable performance at pH 2. They could not explain how this was possible and asked us to investigate it. We were quite excited about this, since the system was working extremely efficiently despite the acidic conditions. Could there be a new microbe present able to perform ammonia oxidation at such low pH?

The samples arrived in closed buckets containing water from the biofilter tank and biofilm scraped-off from the carrier material. When opening the lids, the first thing we noticed was an intense chemical smell. Was it chlorine? We couldn’t tell. Further analysis revealed the very low pH value together with extremely high concentrations of ammonium and nitrate in the samples, but no nitrite. In preliminary experiments, we took a sample of the biomass for microscopic analysis using fluorescent probes. We found out that there were many different bacteria in the biofilm, but one recurrent type showed a quite unique morphology. They were visibly bigger than the rest and they had the tendency to form aggregates (Figure 1). When we used a more specific probe that recognizes ammonia oxidizers, the bigger cells were the only ones to show a signal.

Figure 1. Fluorescent in situ hybridization (FISH) microscopic photographs of the biomass from the biofilter. For the left photo a more general FISH probe was used while for the right photo a more specific FISH probe was used.

DNA sequencing revealed the presence of only one microorganism encoding the ammonia monooxygenase enzyme (AMO), which was distantly related to known nitrifying bacteria. Our suspicion was right: the biofilter somehow enriched a novel acidophilic ammonia oxidizer.

The next challenge was to cultivate this microorganism at the low pH value, which turned out to be everything but straight-forward. It took us almost a year of trial and error to figure out that our “pet bug” liked very high gas flow rates and stirring speed and more phosphate than we were initially supplying. To achieve all of this, we set up a bioreactor (see picture below) that soon enough showed the presence of high nitrate and that funny smell we first noticed when the samples arrived. It turned out that the smelly compound was nitric oxide. Apparently, the product of ammonia oxidation, nitrite, is chemically converted to nitrate and nitric oxide at low pH.

 

Figure 2. The bioreactor with bacteria performing ammonia oxidation at pH 2.5. The pH was controlled using a sterile 1 M KHCO3 solution. The gas supplied to the reactor contained air (290 mL/min) CO2 (4 mL/min). Biomass was removed at 0.1 D-1.

 Once we obtained a stable enrichment, we had many questions regarding the metabolism of this bacterium. One of the main challenges of ammonia oxidation at acidic pH levels is the decreased substrate availability, since ammonia and not ammonium is considered to be the true substrate of the AMO enzyme. How could this strain cope with such low pH? In our study we characterized this new species at genomic, physiological and morphological level. Click here to read everything we found out (https://doi.org/10.1038/s41396-020-00840-7).

Nunzia Picone

PhD student, Radboud University Nijmegen

Huub J.M. Op den Camp

Professor, Department of Microbiology, Faculty of Science, Radboud University Nijmegen

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