Cellular Kinetics of Ammonia-oxidizing Microorganisms

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In this study we used a diverse set of ammonia-oxidizing microorganisms (AOM) to investigate how cellular kinetic characteristics differ between AOM and how these differences affect substrate competition / niche differentiation between AOM. In this article, our goal is to shed more light on how multidimensional the topics of niche differentiation and substrate competition between AOM are. 

Background: Why do we care about nitrification and nitrifying microorganisms?

Nitrification, the microbially mediated oxidation of ammonia (NH3) to nitrate (NO3-), plays an essential role in global nitrogen cycling. From the time nitrification was first described in soils (~1877), researchers have been working to characterize the responsible nitrifying microorganisms and regulate nitrification activity in both environmental (agriculture) and engineered (water treatment) ecosystems. In agricultural settings, nitrification is problematic because it enhances nitrogen loss from soils through the leaching of NO3-, which promotes the eutrophication of aquatic ecosystems and the loss of the potent greenhouse gas nitrous oxide (N2O). In contrast, nitrification is needed for effective wastewater and drinking water treatment, helping to remove nitrogenous compounds from water before it is used or returned to natural waterways. In order to work towards solutions in both directions (i.e., preventing nitrification in fertilized soils and promoting nitrification during waste- and drinking water treatment), an understanding of the physiology and physiological limits of AOM is key.

The process of nitrification is driven by three distinct groups of autotrophic AOM: ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and complete ammonia-oxidizers (comammox). Interestingly, all three types of ammonia-oxidizers often co-occur in the environment, which has left us with quite a puzzle:

How do these three distinct groups of microorganisms, that all rely on the same primary substrate (NH3), compete and coexist with each other in the environment?

Our study: The ammonia oxidation activity kinetics of AOM

We chose to address this puzzle by looking at the ammonia oxidation activity kinetics of individual AOM species. We determined the cellular kinetic properties of 12 AOA, representing AOA from, and adapted to, a wide variety of environments. By characterizing each AOA individually under their optimum growth/activity conditions, we determined the following characteristics for each AOA:

  • Maximal substrate turnover (Vmax), maximal rate of NH3 oxidation
  • Substrate affinity (Km), NH3 concentration, at 50% of Vmax
  • Substrate specific affinity (ao), the ability to scavenge NH3

We discovered that AOA have a huge range of affinities for their primary substrate (NH3), which overlap with the affinities of AOB and comammox microorganisms. So, although we previously thought that there was a hard dichotomy between AOA (all having a high affinity for NH3) and AOB (all having a lower affinity for NH3), this isn’t the case. In fact, there appears to be some phylogenetic link between the four groups of AOA characterized here and their affinity for NH3 (Figure 1).

Figure 1. Here you can see the NH3 substrate affinities (Km) for AOM that have been determined, in this study or in previous studies. The Km numerical value is an inverse metric, where a low numeric Km represents a high substrate affinity.

In addition, we looked at which AOM are the best competitors for NH3 based on their substrate affinity (Km) and specific substrate affinity (ao) (Figure 2).

 

Figure 2. Here you can see that the ‘Ca. Nitrosotaleales’ AOA found in the top right of the graph are the best competitors for NH3, having both a high substrate affinity (Km) and a high substrate specific affinity (ao). In comparison, the Nitrososphaerales AOA and the AOB are among the worst competitors for NH3.

It’s important to note that our kinetics data and comparisons are based on cellular activity kinetics in the absence of growth and under species specific optimum conditions (which differ widely between species). How these two important experimental details affect substrate competition and niche differentiation is discussed below.

Substrate competition and niche differentiation: A multidimensional problem

Just by looking at Figure 2, one could hypothesize that: the ‘Ca. Nitrosotaleales’ AOA are the best competitors for NH3 and will always outcompete other AOM. However, this assumption can be a bit misleading. This is because there is a wide array of factors that affect the niche differentiation in a given environment. For instance, the ‘Ca. Nitrosotaleales’ AOA are the most competitive AOM for NH3, but they are strict acidophiles and not active at a pH of 7-8 where most other AOM are the most active. Similarly, AOA from marine environments have a very high affinity for NH3, but this won’t give them a competitive advantage unless their environment has a high enough salinity. But, even though the ‘Ca. Nitrosotaleales’ and Nitrosopumilales AOA may never directly compete for niche space (or NH3) in nature, understanding their physiological limitations allows us to ask new and exciting questions about:

  • Why are some AOM more competitive for substrate than others?

        (Enzyme active site modification?)

  • What effect do these kinetic properties have under different modes of growth?

        (Enrichment versus pure culture? Batch versus continuous culture?)

  • How predictive are these cellular kinetic limitations?

        (Can we model laboratory AOM competitions based on these properties?)

In this study, we investigated the physiological limit of how successfully each AOM can compete for substrate under their specific optimal conditions (pH and temperature), when grow doesn’t factor in. Understanding these limitations of physiological activity allows us to define what each AOM is capable of, in their own perfect world and we are able to start looking at niche differentiation as the multidimensional space it is.

Moving forward: More hands make less work

AOA (and nitrifiers in general) are notoriously hard to isolate and cultivate in the lab. We often joke that we “grow water slowly” because most AOA double about once a day and the cultures do not produce much, if any, turbidity when in pure culture. In the field of nitrification, new cultures come slowly. This article highlights why large comparative studies, where many people have come together and shared their hard to isolate and culture microorganisms, are essential for moving forward with nitrifier physiology research questions.

 

For a more in-depth look, visit our open access article:
https://www.nature.com/articles/s41396-021-01064-z
or some of our previous work on nitrifier cellular kinetics: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600814/

Christopher Sedlacek

Postdoctoral researcher, University of Vienna