How we evaluate nitrogen fixation at Insight Plant Health

Nitrogen (N) fixation is the conversion of atmospheric nitrogen into ammonia, and is pivotal in sustaining ecosystems and in global food production. While most of the fertilizer N used for agriculture is derived through the energy-intensive Haber-Bosch process, N fixation can also be carried out naturally by certain bacteria and archaea. These N-fixing bacteria can be classified according to the strength of their associations with plants, which range from free-living, to associative, to symbiotic. Rhizobia are symbiotic N fixers that work with the plant to form specialized N-fixing organs called nodules on the roots of legumes. These symbioses are usually quite specific, where only a certain species of rhizobial bacteria can interact with a certain species of plant (e.g. Bradyrhizobium japonicum and soybeans or Mesorhizobium ciceri and chickpeas). Symbiotic N fixation has long been recognized as an agricultural tool, and rhizobial inoculants have been sold for over 100 years.

Rhizobial inoculant ad from 1925.

More recently, free-living associative N fixing bacteria have been introduced for use in agriculture (e.g. Azospirillum brasilense, Methylobacterium symbioticum, and Gluconacetobacter diazotrophicus). These associative bacteria form looser relationships with plants and are able to interact with a range of different plant hosts. The free-living N fixers don’t provide the same level of N to their associated plants as symbiotic rhizobia do, but they can provide some N, and can often also promote plant growth through other biostimulant activities.

Identifying and using the best-performing N-fixing strains in agriculture will lead to more fixed N being transferred to the crop. Similarly, selecting crop varieties with improved N fixation traits or incorporating high performance N-fixing species into a cropping system will also lead to more fixed N into the operation. At Insight Plant Health, we’ve helped several customers identify and characterize novel N-fixing bacteria to make new and improved inoculant products. We’re also working with the Saskatchewan Ministry of Agriculture and Saskatchewan Pulse Growers to identify high-performing rhizobia for fenugreek in Saskatchewan.

Using N isotopes to measure N fixation

Nitrogen makes up 78% of the atmosphere and exists in two stable isotopic forms, 14N and 15N. The isotopic composition of N is 99.634% 14N and 0.366% 15N. The constant ratio of 14N/15N in the atmosphere and other natural compounds provides a basis for using 15N2 or 15N-enriched fertilizers for quantifying the amount of N fixed by bacteria and transferred to plants. Neither 14N nor 15N are radioactive and therefore pose no health risks. Their stable nature allows experiments involving these isotopes to be run over long periods.

Incorporation and measurement of 15N into plant biomass

To evaluate the N-fixing ability of a particular plant-bacteria association, we use an 15N dilution protocol. We add a small amount of 15N-labelled fertilizer (15N-NH4NO3) to pots containing the test plants and to non-N-fixing reference plants (e.g. wheat). The plants are grown to just after the peak N-fixing stage (usually the pod-filling stage) and analyzed for N isotopes by mass spectrometry. IPH collaborates with University of Saskatchewan’s and University of California -Davis’s isotope labs for mass spectrometry analyses. The non-N-fixing plants have soil N  and fertilizer N available to them. The fixing plants have soil N, fertilizer N and atmospheric N available to them. Because we added 15N-labelled fertilizer to the soil, the atmospheric N has a lower 15N signal than the soil and fertilizer N sources, and plants that fix the most atmospheric N have the lowest 15N signal. We calculate the percent N derived from atmosphere (%Ndfa) using an equation based on the 15N dilution in the N-fixing plants.

Initial screening of symbiotic bacteria for their ability to fix N in Leonard’s jar system. The left jar contains plants that were grown in no N growth medium, and seeds were not inoculated with the symbiotic bacteria (negative control). The middle plants were grown in a medium supplemented with N, and seeds were not inoculated. The right jar contains plants that were grown in no N medium, and seeds were inoculated with a novel symbiotic rhizobium.

Variable nodule qualities produced by different symbiotic rhizobia.

Evaluating N fixation in free-living bacteria using 15N

To show that a prospective inoculant or biostimulant product is able to fix N, we use a similar approach to evaluating symbiotic N fixation, but we grow the bacteria in pure culture in the lab instead of in soil in the greenhouse. To evaluate free-living N-fixers, the bacteria are first grown in an N-free medium with several media changes to ensure that all of the N in the culture is from the bacteria themselves. Once the culture has stopped growing, we incubate them them in a synthetic sterile environment enriched with 15N2. After incubating for two weeks in this 15N2-enriched environment, we collect the cells and analyze the 14N/15N isotope ratio by mass spectrometry. Known positive controls (e.g. Azospirillum brasilense) and negative controls e.g. Escherichia coli) are used to compare the novel bacteria. 

Experimental setup for evaluating free-living bacteria (left) and enriching the environment with 15N2 gas (right).

Insight Plant Health has experience testing N fixation ability and efficiency in rhizobia, free-living bacteria, and legume plants. We can help support your product development, registration, and sales by providing concrete numbers to back up product claims. 

Contact us today to provide your sales team and customers reason to believe!