Metabolic biochemical models of N2 fixation for sulfide oxidizers, methanogens, and methanotrophs

Abstract

Dinitrogen (N₂) fixation provides bioavailable nitrogen to the biosphere. However, in some habitats (e.g., sediments), the metabolic pathways of organisms carrying out N₂ fixation are unclear. We present metabolic models representing various chemotrophic N₂ fixers, which simulate potential pathways of electron transport and energy flow, resulting in predictions of whole-cell stoichiometries. By balancing mass, electrons, and energy for metabolic half-reactions, we quantify the electron usage for nine N₂ fixers. Our results demonstrate that all modeled organisms fix sufficient N₂ for growth. Aerobic organisms allocate more electrons to N₂ fixation and growth, yielding more biomass and fixing more N₂, while methanogens using acetate and organisms using sulfate allocate fewer electrons. This work can be applied to investigate the depth distribution of N₂ fixers based on nutrient availability, complementing field measurements of biogeochemical processes and microbial communities.IMPORTANCEN₂ fixation is an important process in the global N cycle. Researchers have developed models for heterotrophic and photoautotrophic N₂ fixers, but there is a lack of modeling studies on chemoautotrophic N₂ fixers. Here, we built nine biochemical models for different chemoautotrophic N₂ fixers by combining different types of half-chemical reactions. We include three sulfide oxidizers using different electron acceptors (O₂, NO₃^-, and Fe³⁺), contributing to the sulfur, nitrogen, and iron cycles in the sediment. We have two methanogens using different substrates (H₂ and acetate) and four methanotrophs using different electron acceptors (O₂, NO₃^-, Fe³⁺, and SO₄^2-). By modeling these methane producers and users in the sediment and their N₂-fixing metabolic pathways, our work can provide insight for future carbon cycle studies. This study outlines various metabolic pathways that can facilitate N₂ fixation, with implications for where in the environment they might occur.

Keywords: CFM-CNF; N2 fixation; biochemical model; electron allocation; energy; methanogens; methanotrophs; sediment; sulfide oxidizers.

Fig 1

Model and simulation. (a) CFM-CNF schematic. Black arrows mean four main reaction types: Ra (electron acceptance), Rd (electron donation), Rn (N₂ fixation), and Rc (biosynthesis via C fixation). Blue arrows mean electron flow from Rd to others. Red dash arrows mean energy flow. (b) Workflow to do the simulation. For each of the models, we listed the potential chemical reactions included, balanced the electrons and mass, and calculated the electron allocation. We added all the half reactions and calculated the overall reactions. Based on overall reactions, we calculate the biomass and N₂ fixation yield and do the comparison.

Fig 2

Electron allocation for different organisms. SO: sulfide oxidizers (top panel). MG: methanogens (middle panel). MT: methanotroph (bottom panel). The length of the bars represents electron usage in different reactions. Pink bars are electrons used for Ra (electron acceptance), blue bars are electrons used in Rc (biosynthesis via C fixation), and yellow bars are electrons used for Rn (N₂ fixation). The parentheses mean different electron acceptors or donors for different models.

Fig 3

Biomass formation (a) and N₂ fixation (b) comparison. Yellow bars represent sulfide oxidizers (SO) results, blue bars are the methanogens (MG) results, and pink bars are methanotrophs (MT) results.

Fig 4

The effect of pH and temperature on N₂ fixation yield (unit: mmol N₂/mol e⁻) for different N₂ fixers (sulfide oxidizers [SO] using O₂ (a), NO₃⁻ (b), and Fe³⁺ (c); methanogens [MG] using acetate (d) and H₂ (e); Methanotrophs [MT] using O₂ (f), NO₃⁻ (g), Fe³⁺ (h), and SO₄²⁻ (i)). Color means the N₂ fixation yield (mol N₂/mol e⁻).