Quantifying nitrogen fixation by heterotrophic bacteria in sinking marine particles

The cell model

Growth rate of a cell

The growth rate of a bacteria cell depends on the acquisition of C (from the particle) and N (from the particle and through ({{rm{N}}}_{2}) fixation), as well as on metabolic expenses in terms of C.

Uptake of C and N

Bacteria get C from glucose and both C and N from amino acids. The total amount of C available for the cell from monomers is (units of C per time)

and the amount of N available from monomer is (N per time)

where ({J}_{rm{G}}) and ({J}_{rm{A}}) are uptake rates of glucose and amino acids, ({f}_{rm{G,C}}) is the fraction of C in glucose, and ({f}_{rm{A,C}}) and ({f}_{rm{A,N}}) are fractions of C and N in amino acids.

The rate of obtaining N through ({{rm{N}}}_{2}) fixation is:

where ({psi },(0 < {{psi }} < 1)) regulates ({{rm{N}}}_{2}) fixation rate and fixation can happen at a maximum rate ({M}_{{{rm{N}}}_{2}}). ({{rm{N}}}_{2}) fixation is only limited by the maximum ({{rm{N}}}_{2}) fixation rate as dissolved dinitrogen (({{rm{N}}}_{2})) gas in seawater is assumed to be unlimited⁷⁰.

The total uptake of C and N from different sources becomes

Costs

Respiratory costs of cellular processes together with ({{rm{N}}}_{2}) fixation and its associated ({{rm{O}}}_{2}) removal cost depend on the cellular ({{rm{O}}}_{2}) concentration. Two possible scenarios can be observed:

Case 1: When
({O}_{2})
concentration is sufficient to maintain aerobic respiration

Respiratory costs for bacterial cellular maintenance can be divided into two parts: one dependent on limiting substrates and the other one is independent of substrate concentration⁷¹. Here we consider only the basal respiratory cost ({R}_{rm{B}}{x}_{rm{B}}), which is independent of the limiting substrates and is assumed as proportional to the mass of the cell ({x}_{B}) (μg C). In order to solubilize particles, particle-attached bacteria produce ectoenzymes that cleave bonds to make molecules small enough to be transported across the bacterial cell membrane. Cleavage is represented by a biomass-specific ectoenzyme production cost ({R}_{rm{E}})⁷². The metabolic costs associated with the uptake of hydrolysis products and intracellular processing are assumed to be proportional to the uptake (({J}_{i})): ({R}_{{rm{G}}}{J}_{{rm{G}}}) and ({R}_{{rm{A}}}{J}_{{rm{A}}}) where the ({R}_{i})’s are costs per unit of resource uptake. In a similar way, the metabolic cost of ({{rm{N}}}_{2}) fixation is assumed as proportional to the ({{rm{N}}}_{2}) fixation rate: ({R}_{{{rm{N}}}_{2}}{rho }_{{rm{CN}},{rm{B}}}{J}_{{{rm{N}}}_{2}}), where ({rho }_{{rm{CN}},{rm{B}}}) is the bacterial C:N ratio. If we define all the above costs as direct costs, then the total direct respiratory cost becomes

Indirect costs related to ({{rm{N}}}_{2}) fixation arises from the removal of ({{rm{O}}}_{2}) from the cell and the production/replenishment of nitrogenase as the enzyme is damaged by ({{rm{O}}}_{2}). The cell can remove ({{rm{O}}}_{2}) either by increasing respiration⁷³ or by increasing the production of nitrogenase enzyme itself⁷⁴. Here we consider only the process of ({{rm{O}}}_{2}) removal by increasing respiration. To calculate this indirect cost, the concentration of ({{rm{O}}}_{2}) present in the cell needs to be estimated.

Since the time scale of ({{rm{O}}}_{2}) concentration inside a cell is short, we have assumed a pseudo steady state inside the cell; the ({{rm{O}}}_{2}) diffusion rate inside a cell is always balanced by the respiration rate¹⁴, which can be expressed as

Here ({rho }_{{rm{CO}}}) is the conversion factor of respiratory ({{rm{O}}}_{2}) to C equivalents and ({F}_{{{rm{O}}}_{2}}) is the actual ({{rm{O}}}_{2}) diffusion rate into a cell from the particle and can be calculated as

where ({r}_{{rm{B}}}) is the cell radius, ({X}_{{{rm{O}}}_{2}}) is the local ({{rm{O}}}_{2}) concentration inside the particle, ({X}_{rm{{O}}_{2},{rm{C}}}) is the cellular ({{rm{O}}}_{2}) concentration, and ({K}_{{{rm{O}}}_{2}}) is the effective diffusion coefficient of ({{rm{O}}}_{2}) over cell membrane layers. The effective diffusion coefficient can be calculated according to Inomura et al.¹⁴ in terms of diffusion coefficient inside particles (({bar{D}}_{{{rm{O}}}_{2}})), the diffusivity of cell membrane layers relative to water (({varepsilon }_{{rm{m}}})), the radius of cellular cytoplasm (({r}_{{rm{C}}})), and the thickness of cell membrane layers (({L}_{{rm{m}}})) as

The apparent diffusivity inside particles (({bar{D}}_{{{rm{O}}}_{2}})) is considered as a fraction ({f}_{{{rm{O}}}_{2}}) of the diffusion coefficient in seawater (({D}_{{{rm{O}}}_{2}}))

Combining (14) and (15) gives the cellular ({{rm{O}}}_{2}) concentration ({X}_{{rm{O}}_{2},{rm{C}}}) as

If there is excess ({{rm{O}}}_{2}) present in the cell after respiration (({X}_{{rm{O}}_{2},{rm{C}}} ,> , 0)), then the indirect cost of removing the excess ({{rm{O}}}_{2}) to be able to perform ({{rm{N}}}_{2}) fixation can be written as

where (H({{psi }})) is the Heaviside function:

Therefore, the total aerobic respiratory cost becomes:

Case 2: Anaerobic respiration

When available ({{rm{O}}}_{2}) is insufficient to maintain aerobic respiration (({R}_{{rm{tot}}}left({{psi }}right) ,> , {rho }_{{rm{CO}}}{F}_{{{rm{O}}}_{2},{{max }}})), cells use ({{{rm{NO}}}_{3}}^{-}) and ({{{rm{SO}}}_{4}}^{2-}) for respiration. The potential ({{{rm{NO}}}_{3}}^{-}) uptake, ({J}_{{{rm{NO}}}_{3},{rm{pot}}}), is

where ({M}_{{{rm{NO}}}_{3}}) and ({A}_{{{rm{NO}}}_{3}}) are maximum uptake rate and affinity for ({{{rm{NO}}}_{3}}^{-}) uptake, respectively. However, the actual rate of ({{{rm{NO}}}_{3}}^{-}) uptake, ({J}_{{{rm{NO}}}_{3}}), is determined by cellular respiration and can be written as

where ({rho }_{{rm{C}}{{rm{NO}}}_{3}}) is the conversion factor of respiratory ({{{rm{NO}}}_{3}}^{-}) to C equivalents and the maximum ({{rm{O}}}_{2}) diffusion rate into a cell ({F}_{{{rm{O}}}_{2},{{max }}}) can be obtained by making cellular ({{rm{O}}}_{2}) concentration ({X}_{{{rm{O}}}_{2},{rm{c}}}) zero in (15) as

Further, in the absence of sufficient ({{{rm{NO}}}_{3}}^{-}), the cell uses ({{{rm{SO}}}_{4}}^{2-}) as an electron acceptor for respiration. Since the average concentration of ({{{rm{SO}}}_{4}}^{2-}) in seawater is 29 mmol L^−1 75, ({{{rm{SO}}}_{4}}^{2-}) is a nonlimiting nutrient for cell growth and the potential uptake rate of ({{{rm{SO}}}_{4}}^{2-}) is mainly governed by the maximum uptake rate as

where ({M}_{{{rm{SO}}}_{4}}) is the maximum uptake rate for ({{{rm{SO}}}_{4}}^{2-}) uptake. The actual rate of ({{{rm{SO}}}_{4}}^{2-}) uptake, ({J}_{{{rm{SO}}}_{4}}), can be written as

where ({rho }_{{rm{C}}{{rm{SO}}}_{4}}) is the conversion factor of respiratory ({{{rm{SO}}}_{4}}^{2-}) to C equivalents.

According to formulations (23) and (26), ({{{rm{NO}}}_{3}}^{-}) and ({{{rm{SO}}}_{4}}^{2-}) uptake occurs only when the diffusive flux of ({{rm{O}}}_{2}), and both ({{rm{O}}}_{2}) and ({{{rm{NO}}}_{3}}^{-}) are insufficient to maintain respiration(.) Moreover, the uptake rates of ({{{rm{NO}}}_{3}}^{-}) and ({{{rm{SO}}}_{4}}^{2-}) are regulated according to the cells’ requirements.

Uptakes of ({{{rm{NO}}}_{3}}^{-}) and ({{{rm{SO}}}_{4}}^{2-}) incur extra metabolic costs ({R}_{{{rm{NO}}}_{3}}{rho }_{{rm{C}}{{rm{NO}}}_{3}}{J}_{{{rm{NO}}}_{3}}) and ({R}_{{{rm{SO}}}_{4}}{rho }_{{rm{C}}{{rm{SO}}}_{4}}{J}_{{{rm{SO}}}_{4}}), where ({R}_{{{rm{NO}}}_{3}}) and ({R}_{{{rm{SO}}}_{4}}) are costs per unit of ({{{rm{NO}}}_{3}}^{-}) and ({{{rm{SO}}}_{4}}^{2-}) uptake. The total respiratory cost can be written as

Synthesis and growth rate

The assimilated C and N are combined to synthesize new structure. The synthesis rate is constrained by the limiting resource (Liebig’s law of the minimum) and by available electron acceptors such that the total flux of C available for growth ({J}_{{rm{tot}}}) (μg C d⁻¹) is:

Here, the total available C for growth is ({J}_{{rm{C}}}-{R}_{{rm{tot}}}({{psi }})), the C required to synthesize biomass from N source is ({rho }_{{rm{CN}},B}{J}_{rm{N}}), and the C equivalent inflow rate of electron acceptors to the cell is ({rho }_{{rm{CO}}}{F}_{{rm{O}}_{2}}+{rho }_{{rm{C}}{{rm{NO}}}_{3}}{J}_{{{rm{NO}}}_{3}}+{rho }_{{rm{C}}{{rm{SO}}}_{4}}{J}_{{{rm{SO}}}_{4}}). We assume that excess C or N is released from the cell instantaneously.

Synthesis is not explicitly limited by a maximum synthesis capacity; synthesis is constrained by the C and N uptake in the functional responses (Eqs. 34 and 35). The division rate (mu) of the cell (d⁻¹) is the total flux of C available for growth divided by the C mass of the cell (({x}_{rm{B}})):

The resulting division rate, (mu), is a measure of the bacterial fitness and we assume that the cell regulates its ({{rm{N}}}_{2}) fixation rate depending on the environmental conditions to gain additional N while maximizing its growth rate. The optimal value of the parameter regulating ({{rm{N}}}_{2}) fixation ({{psi }}) ((0le {{psi }}le 1)) then becomes:

and the corresponding optimal division rate becomes

The particle model

We consider a sinking particle of radius ({r}_{{rm{P}}}) (cm) and volume ({V}_{{rm{P}}}) (cm³) (Supplementary Fig. S1). The particle contains facultative nitrogen-fixing bacterial population (B(r)) (cells L⁻¹), polysaccharides ({C}_{{rm{P}}}(r)) (μg G L⁻¹), and polypeptides ({P}_{{rm{P}}}(r)) (μg A L⁻¹) at a radial distance (r) (cm) from the center of the particle, where G and A stand for glucose and amino acids. We assume that only fractions ({f}_{{rm{C}}}) and ({f}_{{rm{P}}}) of these polymers are labile (({C}_{{rm{L}}}(r)={f}_{{rm{C}}}{C}_{{rm{P}}}(r),) ({P}_{{rm{L}}}(r)={f}_{{rm{P}}}{P}_{{rm{P}}}(r))), i.e., accessible by bacteria. Bacterial enzymatic hydrolysis converts the labile polysaccharides and polypeptides into monosaccharides (glucose) ((G) μg G L⁻¹) and amino acids ((A) μg A L⁻¹) that are efficiently taken up by bacteria. Moreover, the particle contains ({{rm{O}}}_{2}), ({{{rm{NO}}}_{3}}^{-}), and ({{{rm{SO}}}_{4}}^{2-}) with concentrations ({X}_{{{rm{O}}}_{2}}(r)) (μmol O₂ L⁻¹), ({X}_{{{rm{NO}}}_{3}}(r)) (μmol NO₃ L⁻¹), and ({X}_{{{rm{SO}}}_{4}}(r)) (μmol SO₄ L⁻¹). Glucose and amino acids diffuse out of the particle whereas ({{rm{O}}}_{2}) and ({{{rm{NO}}}_{3}}^{-}) diffuse into the particle from the surrounding environment. Due to the high concentration of ({{{rm{SO}}}_{4}}^{2-}) in ocean waters, we assume that ({{{rm{SO}}}_{4}}^{2-}) is not diffusion limited inside particles, its uptake is limited by the maximum uptake capacity due to physical constraint. The interactions between particle, cells, and the surrounding environment are explained in Supplementary Fig. S1 and equations are provided in Table 1 of the main text.

We assume that labile polysaccharide (({C}_{{rm{L}}})) and polypeptide (({P}_{{rm{L}}})) are hydrolyzed into glucose and amino acids at rates ({J}_{{rm{C}}}) and ({J}_{{rm{P}}}) with the following functional form

where ({h}_{{rm{C}}}) and ({h}_{{rm{P}}}) are maximum hydrolysis rates of the carbohydrate and peptide pool, and ({A}_{{rm{C}}}) and ({A}_{{rm{P}}}) are respective affinities. ({J}_{{rm{G}}}) and ({J}_{{rm{A}}}) represent uptake of glucose and amino acids:

where ({M}_{{rm{G}}}) and ({M}_{{rm{A}}}) are maximum uptake rates of glucose and amino acids, whereas ({A}_{{rm{G}}}) and ({A}_{{rm{A}}}) are corresponding affinities. Hydrolyzed monomers diffuse out of the particle at a rate ({D}_{{rm{M}}}).

({mu }^{ast }) is the optimal division rate of cells (Eq. 31) and ({m}_{rm{B}}) represents the mortality rate (including predation) of bacteria. ({F}_{{{rm{O}}}_{2}}) and ({J}_{{{rm{NO}}}_{3}}) represent the diffusive flux of ({{rm{O}}}_{2}) and the consumption rate of ({{{rm{NO}}}_{3}}^{-}), respectively, through the bacterial cell membrane. ({bar{D}}_{{{rm{O}}}_{2}}) and ({bar{D}}_{{{rm{NO}}}_{3}}) are diffusion coefficients of ({{rm{O}}}_{2}) and ({{{rm{NO}}}_{3}}^{-}) inside the particle.

At the center of the particle ((r=0)) the gradient of all quantities vanishes:

At the surface of the particle ((r={r}_{{rm{P}}})) concentrations are determined by the surrounding environment:

where ({G}_{infty },) ({A}_{infty ,}) ({X}_{{{rm{O}}}_{2},infty }) and ({X}_{{{rm{NO}}}_{3},infty }) are concentrations of glucose, amino acids, ({{rm{O}}}_{2}), and ({{{rm{NO}}}_{3}}^{-}) in the environment.

Calculation of total N₂ fixation rate

The total amount of fixed ({{rm{N}}}_{2}) in a specific size class of particle, ({{rm{N}}}_{{rm{fix}},{rm{P}}}) (({rm{mu }})g N particle⁻¹), is calculated as

where ({r}_{{rm{P}}}) (cm) is the particle radius and z (m) represents the water column depth.

({{rm{N}}}_{2}) fixation rate per unit volume of water, ({{rm{N}}}_{{rm{fix}},{rm{V}}}left(tright)) (({rm{mu }}{rm{mol}}) N m⁻³ d⁻¹), is calculated as

Here (x) (cm) represents the size range (radius) of particles, (rho) is the fraction of diazotrophs of the total heterotrophic bacteria, and (n(x)) (number of particles per unit volume of water per size increment) is the size spectrum of particles that is most commonly approximated by a power law distribution of the form

where ({n}_{0}) is a constant that controls total particle abundance and the slope (xi) represents the relative concentration of small to large particles: the steeper the slope, the greater the proportion of smaller particles and the flatter the slope, and the greater the proportion of larger particles³⁴.

Depth-integrated ({{rm{N}}}_{2}) fixation rate, ({{rm{N}}}_{{rm{fix}},{rm{D}}}) (({rm{mu }}{rm{mol}}) N m⁻² d⁻¹), can be obtained by

Assumptions and simplification in the modeling approach

According to our current model formulation, the particle size remains constant while sinking. However, in nature, particle size is dynamic due to processes like bacterial remineralization, aggregation, and disaggregation. We neglect these complications to keep the model simple and to focus on revealing the coupling between particle-associated environmental conditions and ({{rm{N}}}_{2}) fixation by heterotrophic bacteria. These factors can, however, possibly be incorporated by using in situ data or by using the relationship between carbon content and the diameter of particles⁴⁸ and including terms for aggregation and disaggregation⁵⁵.

Our model represents a population of facultative heterotrophic diazotrophs that grow at a rate similar to other heterotrophic bacteria but the whole community initiates ({{rm{N}}}_{2}) fixation when conditions become suitable. However, under natural conditions, diazotrophs may only constitute a fraction of the bacterial community, and their proliferation may be gradual²¹, presumably affected by multiple factors. In such case, our approach will overestimate diazotroph cell concentration and consequently the ({{rm{N}}}_{2}) fixation rate.

For simplicity, our approach includes only aerobic respiration, ({{{rm{NO}}}_{3}}^{-}) and ({{{rm{SO}}}_{4}}^{2-}) respiration, although many additional aerobic and anaerobic processes likely occur on particles (e.g Klawonn et al.¹⁹). To our knowledge, a complete picture of such processes, their interactions and effects on particle biochemistry is unavailable. For example, we have assumed that when ({{rm{O}}}_{2}) and ({{{rm{NO}}}_{3}}^{-}) are insufficient to maintain respiration, heterotrophic bacteria start reducing ({{{rm{SO}}}_{4}}^{2-}). However, ({{{rm{SO}}}_{4}}^{2-}) reduction has been detected only with a significant lag after the occurrence of anaerobic conditions, suggesting it as a slow adapted process⁷⁶, whereas we assume it to be instantaneous. On the other hand, the lag may not be real but due to a so called cryptic sulfur cycle, where ({{{rm{SO}}}_{4}}^{2-}) reduction is accompanied by concurrent sulfide oxidation effectively masking sulfide production⁷⁷. Hopefully, future insights into interactions between diverse aerobic and anaerobic microbial processes can refine our modelling approach and fine-tune predictions of biochemistry in marine particles.

Procedure of numerically obtaining optimal N₂ fixation rate

To avoid making the optimization in Eq. (30) at every time step during the simulation, a lookup table of ({mu }^{ast }) (Eq. 31) over realistic ranges of the four resources (glucose, amino acids, ({{rm{O}}}_{2}), and ({{{rm{NO}}}_{3}}^{-})) and the parameter determining ({{rm{N}}}_{2}) fixation rate (({{psi }})) was created at the beginning of the simulation.

The effects of temperature on N₂ fixation rate

To examine the role of temperature variation on ({{rm{N}}}_{2}) fixation rate in sinking particles, we consider hydrolysis of polysaccharide and polypeptide, uptake of glucose and amino acids, uptake of ({{{rm{NO}}}_{3}}^{-}), respiration, and diffusion dependent on temperature. Apart from diffusion, all other processes are multiplied by a factor ({Q}_{10}) that represents the factorial increase in rates with ({10}^{0})C temperature increase. The rate (R) at a given temperature (T) is then

Here the reference rate ({R}_{{rm{ref}}}) is defined as the rate at the reference temperature ({T}_{{rm{ref}}}.) We set the reference temperature ({T}_{{rm{ref}}}) at room temperature of 20 °C. The effect of temperature on the diffusion coefficient D for glucose, amino acids, ({{rm{O}}}_{2}), and ({{{rm{NO}}}_{3}}^{-}) is described by Walden’s rule:

where (eta) is the viscosity of water at the given temperature (T), and ({D}_{{rm{ref}}}) and ({eta }_{{rm{ref}}}) are diffusion coefficient and viscosity at ({T}_{{rm{ref}}}).

({Q}_{10}) values for different enzyme classes responsible for hydrolysis (({Q}_{10,{rm{h}}})) lie within the range 1.1–2.9⁷⁸. Here, we have chosen ({Q}_{10,{rm{h}}}=2) for hydrolysis from the middle of the prescribed range. The ({Q}_{10}) values for uptake affinities (({Q}_{10,{rm{A}}})) are taken as 1.5⁷⁹. ({Q}_{10,{rm{R}}}=2) is chosen for all parameters related to respiration (({R}_{{rm{B}}}), ({R}_{{rm{E}}}), ({R}_{{rm{G}}}), ({R}_{{rm{A}}}), ({R}_{{{rm{N}}}_{2}}), ({R}_{{{rm{NO}}}_{3}}), ({R}_{{{rm{SO}}}_{4}}))⁸⁰. ({R}_{{rm{ref}}}) and ({D}_{{rm{ref}}}) are the values of (R)’s and (D)’s provided in Table S1. The reference viscosity (({eta }_{{rm{ref}}})) and viscosities ((eta)) at different temperatures are taken from Jumars et al.⁸⁰.

Source: Ecology - nature.com