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Biological Nitrogen Fixation

(Snapp additions below, primarily from the TSBF Handbook of Methods, appendix G by Mark Peoples, pages 164-171.)

Biological nitrogen fixation is key to sustainability of farming systems, because ‘available’ nitrogen is needed in large quantities. Nitrogen is the nutrient that is most often the limiting factor for growth and production of food on smallholder farms. Legume plants and their symbiotic microorganism partners – and in particular legume food crops – can play a large role in providing much of the N through BNF. BNF provides about half of nitrogen inputs worldwide in agriculture, the Haber Bosch industrial process (manufactured fertilizer from fossil fuel feedstocks) provides the other half. There are many methods to measure symbiotic nitrogen fixation, and the most appropriate one will depend on research objectives and budget available, but this manual will focus on describing the two most important methods in wide use. This is the nitrogen difference method, and the natural 15N abundance method (Bremer, and van Kessel, 1990). If it is possible to source an iso-line of a legume species, one that doesn't have the ability to fix nitrogen, then this can be grown as the reference plant with very similar growth habit, which provides a superior reference for both methods (Kohl et al., 1980).

How to operationalize the metric

Measurement nitrogen difference method:

Growing a non-fixer reference crop on the same site adjacent to a legume crop is one way to estimate the amount of nitrogen fixation associated with the legume crop. Both plants are grown on the same soil and exposed to identical conditions, usually be growing on adjacent plots. The additional nitrogen yield (the nitrogen content associated with the total biomass) of the legume crop compared to the reference non-fixer is used as an estimate of symbiotically fixed nitrogen. This assumes that the pattern of soil inorganic nitrogen uptake is similar for the reference and the legume plant, and any additional nitrogen accrued can thus be attributed to N fixation.

Method of data collection and data needed to compute the method:

One or two reference plants that are not nitrogen fixing species are planted adjacent to the legume specie(s) of interest, and a destructive harvest of above ground biomass is conducted at about maximum biomass accrual. It is important to conduct a measurement before reproduction is advanced in plant species that senesce leaves mid-season, through natural processes (e.g., shrubs such as pigeonpea Cajanus cajan), or due to high susceptibility to leaf pathogens (e.g., common bean). Vegetation tissue samples are collected in a representative manner from the biomass collected, and these are analyzed for nitrogen concentration. The different plant tissues can be separated if nitrogen allocation patterns are of interest. The two widely used method for tissue N determination is dry combustion of C and N (using equipment such as a Costech or Carlo Erba), and kjehdahl hot acid digestion followed by colorimetric N determination. To ascertain biological N fixation, the total amount of nitrogen in the biomass of the reference species is subtracted from the legume species biomass.

Unit of analysis:

N concentration (kg N kg-1 biomass) multiplied by biomass mass kg ha-1, final units of kg N ha-1

Limitations regarding estimating and interpreting:

There can be large differences in root system architecture, growth habit and plant growth rates, and it is important to use a reference plant that has a similar plant growth pattern to that of the legume crop of interest, to try and meet the assumption that both sourced from similar soil inorganic nitrogen pools and that both accumulated N in a similar manner over the season. This method is more effective in low-N fertility sites. It can in many cases provide an underestimate, as reference plants such as cereals are often used and these may have rapid growth rates and acquire N in a more rapid manner than plants with a moderate growth rate which is typical of many legumes (particularly those with a perennial growth habit).

There is a small difference in the natural 15N enrichment of soil N compared to atmospheric N2 and this can be used to quantify biological N fixation. This method is a refinement on the N difference method, as the 15N to 14N signature of soil N varies from that of the atmosphere, providing an opportunity to assess the N pools sourced by the reference plant, and the N fixer plant. Similar to the N difference method, growing a non-fixer reference crop on the same site adjacent to a legume crop is important in this methodology. In the case of established perennial plants finding an adjacent non-fixing plant to act as a reference is feasible (thus this method can be applied to situations where the N difference method is not practical to employ), as it is not necessary to collect the entire plant biomass – rather, representative plant tissue samples can be used to assess 15N to 14N signature of the non-fixer, which is expected to be closely related to the soil 15N to 14N signature. How much of legume N is derived from the atmosphere (via biological N fixation), and how much form the soil is determined using a formula that requires information about the 15N to 14N signature expected from 100% reliance on biological N fixation, and 100% on soil N.

Method of data collection and data needed to compute the method:

Vegetation tissue samples are collected in a representative manner from reference specie(s) and N fixer species of interest.  These are analyzed for 15N to 14N signature using a mass spectrophotometer. The different plant tissues can be separated first if nitrogen allocation patterns are of interest. To ascertain biological N fixation (fNdfa), the following equation is used:

fNdfa = (δ15Nref – δ15Nfix)/(δ15Nref – δ15Nb)

where "ref" is the non-fixing plant and "fix" is the nitrogen-fixing plants grown under the same conditions, and "b" is the fixing plant grown with atmospheric N2 as the sole external nitrogen source (Oberson et al., 2007).

Unit of analysis:

Units for fNdfa: kg N kg biomass, and multiplied by biomass this is a metric for total amount of N fixation associated with this species per area: kg N ha-1

Limitations regarding estimating and interpreting:

Similar challenges and limitations as those observed with the N difference method except that overall growth habit differences are less important. Yet, the root system architecture does influence the ability of a reference plant to source from a similar soil inorganic nitrogen pools as the N fixer species, which is a key assumption of the natural abundance method. A mass spectrophotometer capable of precisely measuring differences of 0.00004 atom % 15N is necessary and sample preparation requires great care in order to avoid isotopic discrimination, and representative subsampling (e.g., a strategy to sample plant tissue in a representative manner, and to carry out fully homogenous sample preparation). If the soil has very low and heterogeneous soil N pools this poses a challenge; however, this is more of a problem with natural sites than most agricultural sites (Peoples, TSBF)

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