# Dinitrogen fixation by open purple non-sulfur bacteria cultures for protein production: Diazotrophy boosts photoheterotrophic uptake rates

María Del Rosario Rodero, Jean-Philippe Steyer, María Fernanda Pérez-Bernal, Willy Verstraete, Renaud Escudié, Gabriel Capson-Tojo

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María Del Rosario Rodero, Jean-Philippe Steyer, María Fernanda Pérez-Bernal, Willy Verstraete, Renaud Escudié, et al., Dinitrogen fixation by open purple non-sulfur bacteria cultures for protein production: Diazotrophy boosts photoheterotrophic uptake rates. Bioresource Technology, 2025, 430, pp.132554. 10.1016/j.biortech.2025.132554. hal-05038910

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Journal Pre-proofs

Dinitrogen fixation by open purple non-sulfur bacteria cultures for protein production: Diazotrophy boosts photoheterotrophic uptake rates

María del Rosario Rodero, Jean-Philippe Steyer, María Fernanda Pérez-Bernal, Willy Verstraete, Renaud Escudié, Gabriel Capson-Tojo

PII: S0960-8524(25)00520-6

DOI: https://doi.org/10.1016/j.biortech.2025.132554

Reference: BITE 132554

To appear in: *Bioresource Technology*

Received Date: 14 January 2025

Revised Date: 24 March 2025

Accepted Date: 15 April 2025

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Please cite this article as: Rosario Rodero, M.d., Steyer, J-P., Pérez-Bernal, M.F., Verstraete, W., Escudié, R., Capson-Tojo, G., Dinitrogen fixation by open purple non-sulfur bacteria cultures for protein production: Diazotrophy boosts photoheterotrophic uptake rates, *Bioresource Technology* (2025), doi: https://doi.org/10.1016/j.biortech.2025.132554

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Dinitrogen fixation by open purple non-sulfur bacteria cultures for protein
production: diazotrophy boosts photoheterotrophic uptake rates

María del Rosario Rodero a,b,c, Jean-Philippe Steyer a, María Fernanda Pérez-Bernal a,
Willy Verstraete d, Renaud Escudié a, Gabriel Capson-Tojo a,*

a INRAE, Univ Montpellier, LBE, 102 avenue des Étangs, 11100 Narbonne, France

b Institute of Sustainable Processes, University of Valladolid, 47011, Valladolid, Spain

c Department of Chemical Engineering and Environmental Technology, University of Valladolid, Dr. Mergelina s/n., Valladolid 47011, Spain.

d Center for Microbial Ecology and Technology (CMET), Ghent University, Coupure Links 653, Gent B-9000, Belgium

* Corresponding author: gabriel.capson-tojo@inrae.fr

Acknowledgements

María del Rosario Rodero acknowledges the NextGenerationEU Margarita Salas program
from the European Union for her research contract. We also acknowledge the INRAE
Bio2E Facility (Bio2E, INRAE, 2018. Environmental Biotechnology and Biorefinery
Facility; https://doi.org/10.15454/1.557234103446854E12), where the experiments were
conducted.

Declaration of Interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
**Abstract**

Purple non-sulfur bacteria (PNSB) offer a sustainable alternative to current inefficient protein production systems thanks to their high yields. This study explored the potential of specialized diazotrophic PNSB open cultures for protein production, benchmarking their performance against ammonium-grown PNSB and other diazotrophs. While diazotrophic yields (0.85-0.93 gCOD~biomass~·gCOD~substrate~⁻¹; COD being chemical oxygen demand) were slightly lower than non-diazotrophic (~1.0), they were over double those of heterotrophic-diazotrophic rhizobacteria, with full N recovery as biomass (~1.0 gN~biomass~·gN~fixed~⁻¹). Photoheterotrophic-diazotrophic uptake rates were the fastest ever reported for PNSB and any other diazotroph (e.g., 5.20±0.83 vs. 2.64±0.34 gCOD~substrate~·gCOD~biomass~⁻¹·d⁻¹ for PNSB on NH₄⁺). Optimal rates required high light intensities, aligning with diazotrophic energy demands. Photoheterotrophic-diazotrophic conditions were highly selective, enriching a specialized *Rhodopseudomonas palustris* strain. Biomass protein contents and essential amino acid metrics confirmed nutritional suitability for humans. This work lays the background to exploit PNSB's potential to address global protein demands through sustainable nitrogen fixation.

**Keywords:** N₂ fixation; Phototrophic bacteria; Anoxygenic photosynthesis; Mechanistic modelling; Microbial protein; Novel foods.

**1. Introduction**

The current food production system is heavily dependent on ammonia produced via the Haber-Bosch process, which synthesizes ammonia by reacting molecular hydrogen (H₂) and dinitrogen (N₂) at high pressures (>100 bar) and elevated temperatures (~500 °C).

This process is highly energy-intensive, consuming 2-4% of the total global energy and emitting substantial CO₂ (2.9 tons equivalent per ton of ammonia produced), with an energy requirement of 500 kJ per mole of N fixed (Hu et al., 2020). Furthermore, the
use of ammonia-based fertilizers is extremely inefficient, with roughly 50-70% of the N applied as crop fertilizer being lost through mechanisms such as runoff, volatilization, denitrification, and leaching, which results in low N use efficiency (NUE) (Pikaar et al., 2017). In animal agriculture, NUEs are even lower, with N losses reaching up to 90% due to inefficiencies inherent to the feed chain (Pikaar et al., 2017). These N losses contribute to severe environmental problems, e.g., eutrophication, soil acidification, biodiversity loss, and greenhouse gas emissions (Matassa et al., 2016a). As demand for food, especially protein, increases, the need for sustainable and efficient nitrogen-fixation methods for protein production has become urgent (Matassa et al., 2016a).

Producing microbial protein (MP) from microorganisms offers advantages over traditional plant- and animal-based sources of food and feed. MP offers higher NUE, as it reduces N losses, improves protein productivity, and lessens environmental footprints (Pikaar et al., 2017). Additionally, utilizing microorganisms that fix N₂ -known as diazotrophs- can bypass the need for chemically-fixed N, avoiding the financial and environmental costs of the Haber-Bosch process. Biological nitrogen fixation (BNF) carried out by diazotrophs occurs under mild conditions, reducing energy consumption.

A variety of bacteria can fix N₂ under autotrophic or heterotrophic conditions.

Diazotrophs possess nitrogenase enzymes, which are metalloprotein complexes (either containing Mo, Fe, or V) that catalyze N₂ reduction when NH₄⁺ is not available. Mo-nitrogenase is the most efficient and widely occurring form (Masepohl, 2017). A limitation of BNF is its high energetic requirement. A minimum of 16 mol of adenosine triphosphate (ATP) are consumed to reduce 1 mol of N₂ to 2 mol of NH₄⁺ (Soundararajan et al., 2019). This high energy demand results in low conversion yields in chemoheterotrophs and slow growth rates in autotrophs due to the energy-intensive simultaneous N and C fixation (Hu et al., 2020; Trentin et al., 2023).
Purple non-sulfur bacteria (PNSB) are diazotrophs with an extremely unique capability: they can fix N₂ while deriving the required energy from light and the electrons from organic compounds (as well as the C for growth). PNSB can achieve biomass yields as high as 1 g per g of chemical oxygen demand (COD) consumed, leveraging their capacity for anoxygenic photosynthesis (Capson-Tojo et al., 2020). These yields are considerably higher than those of non-photosynthetic microorganisms, such as aerobic heterotrophs (~0.5 g COD_biomass·g⁻¹ COD_consumed) and anaerobic fermenters (<0.2 g COD_biomass·g⁻¹ COD_consumed) (Batstone et al., 2002). PNSB exhibit faster growth rates under photoheterotrophic conditions than CO₂-fixing organisms (Capson-Tojo et al., 2023). By utilizing organic compounds and light for growth (i.e., photoheterotrophy), PNSB may perform BNF more efficiently than chemoheterotrophs (e.g., Rhizobium sp.) and faster than autotrophs (e.g., cyanobacteria). PNSB also have high protein content, with values over 50% of their biomass by weight, as well as amino acid profiles suitable for feed and potentially human food applications (Delamare-Deboutteville et al., 2019).

The photoheterotrophic capabilities of PNSB have attracted substantial interest for biotechnology, yet most studies have cultivated PNSB using ammonia as N source, neglecting their BNF potential (Capson-Tojo et al., 2020). Although PNSB's ability to fix N₂ is well-documented (Madigan and Govindjee, 1995), biotechnological research has focused on optimizing H₂ production, a necessary by-product of BNF, rather than on N₂ fixation itself. Most optimization efforts have been aimed at enhancing H₂ yields and production rates, without considering biomass production or the amount of N₂ fixed (Capson-Tojo et al., 2020). Key aspects of N₂ fixation in PNSB remain mostly unknown, including fixation rates, biomass yields, or optimal BNF conditions. No PNSB species have been selected or enriched based on their BNF capabilities. Studying such strains could help understanding BNF in PNSB and enhance performance.
This study provides the first evaluation of the BNF performance of PNSB open cultures enriched based on their diazotrophic capabilities, using biomass yields and N₂ uptake rates as key indicators. These parameters were determined under various conditions to ensure unbiased estimates, focusing on key factors affecting PNSB growth. The effects of C substrates (i.e., acetate, butyrate, propionate, and CO₂), incident light intensity, and pH were assessed. Additionally, the protein and amino acid contents of the biomass were analyzed, and the microbial community composition was assessed. The results have been benchmarked against other options for BNF and MP production.

## 2. Materials and methods

### 2.1. Growth media composition

The medium for diazotrophic photoheterotrophic cultivation was based on Ormerod et al. (1961), but excluding the N source (NH₄Cl) to enable BNF. The medium contained (g·L⁻¹): 0.9 KH₂PO₄, 0.6 KH₂PO₄, 0.2 MgSO₄·7H₂O, 0.075 CaCl₂·2H₂O, 0.02 EDTA, 0.012 FeSO₄·7H₂O, 0.02 yeast extract (minimal to avoid significant N and COD contributions), 1 mL of trace element solution, and 550 mg COD·L⁻¹ of volatile fatty acid (VFA; i.e., acetate, propionate, or butyrate). For diazotrophic photoautotrophic tests, a modified Ormerod medium with 4.2 g·L⁻¹ NaHCO₃ as the sole C source was used, following Rodero et al. (2024), but without NH₄Cl. All cultures were grown with N₂ as the N source. H₂ was the electron donor for autotrophic growth. Non-diazotrophic controls were grown using regular Ormerod medium (Capson-Tojo et al., 2021).

### 2.2. Inoculum and diazotrophic enrichments

Sequential heterotrophic (with acetate as substrate) and autotrophic (H₂ and CO₂) enrichments were conducted to obtain PNSB open consortia capable of efficient N₂ fixation. Enrichments were performed in 0.5 L flasks, with a working volume of 0.2 L, at 25-28 °C, and under continuous stirring (600 rpm) and illumination using near-
infrared (NIR) light-emitting diodes (LEDs; INSTAR IN-905) at an incident intensity of
50-60 W·m⁻² (emission peak at 850 nm; 99% of the spectrum within 800-900 nm).

Flasks were heated and agitated with magnetic plates (AM4, VELP SCIENTIFICA). A
foil absorbing ultraviolet-visible light without impacting NIR wavelengths (Lee filter
ND 1.2 299) limited undesirable light inputs (Capson-Tojo et al., 2021).

The inocula used to start heterotrophic and autotrophic enrichments were biomass from
non-diazotrophic photoheterotrophic and photoautotrophic PNSB enriched reactors,
respectively (Rodero et al., 2024). Sequential diazotrophic enrichments were then
inoculated with biomass from the preceding batch, using 10 mL (5% of the working
volume), resulting in an initial biomass concentration of 6-8 mg COD·L⁻¹. Flasks were
sealed with a rubber septum and the headspace was flushed with the appropriate gas for
3 min. Photoheterotrophic tests were filled with 100% N₂ at a pressure of 1.3 bar. For
autotrophic tests, a H₂/N₂ mixture of 80/20% v·v⁻¹ was used (up to 1.3 bar). Seven
identical sequential enrichments were conducted, each lasting 3-7 d (time to reach a
biomass concentration of around 400 mg COD·L⁻¹, when no substrate was left for
heterotrophic growth), until stable results were achieved (e.g., the average biomass yield
of the last three heterotrophic enrichments was 0.88±0.01 g COD_biomass·g COD_consumed⁻¹;
the total relative abundance of 16S rRNA gene copies for PNSB was 0.69-0.79). All the
experiments were performed at the Bio2E platform (Bio2E INRAE, 2018).

**2.3. Determination of diazotrophic biomass yields**

Batch tests were performed agitating at 600 rpm, at 25-28 °C, and under continuous
illumination. The effects of the C source (e.g., acetate, propionate, butyrate, or
NaHCO₃, and each of these VFAs further combined with 100 mg·L⁻¹ NaHCO₃),
incident light intensities (14, 32, 54, and 103 W·m⁻²), and initial pH (6, 7, and 8) on
biomass COD and N yields were evaluated. The COD required for N₂ fixation was also
determined. Batch assays were conducted in triplicate, with acetate, a light intensity of 50-60 W·m⁻², and an initial pH of 7, unless otherwise specified (see supplementary material for a detailed description of operational conditions). To allow for unbiased comparisons, only one factor was modified in each test (i.e., tests at different pH values or light intensities were performed with acetate as substrate, and tests with different C sources were performed at the same initial COD, initial pH, and incident light intensity).

The working volumes were 400 and 200 mL for heterotrophic and autotrophic tests, respectively, with the inoculum representing 5% of the volume. The exact working and total volumes were determined by measuring the difference in the flask weight with and without liquid. The headspace volumes were estimated by difference between the working and total volumes. Batch tests followed the same procedure as the enrichments, with variations in the C source, light intensity, or initial pH when required. The batch duration was 3 and 4 days for the heterotrophic and autotrophic tests, respectively.

Gas composition and pressure were analyzed at the start and end of each batch to calculate the amount of N₂ consumed, needed for the N yields. H₂ consumption was determined for autotrophic tests. Initial and final samples from the liquid were collected to measure concentrations of N (total and soluble), NH₄⁺, COD (total and soluble), volatile suspended solids (VSS), VFAs, and dissolved inorganic carbon (IC). These data were used to determine yields and perform mass balances. Gas pressure measurements were performed daily to ensure that no leaks existed and to minimize errors in N yields.

## 2.4. Estimating diazotrophic rates

Dedicated batch tests were conducted in 2 L flasks with a working volume of 400-500 mL to estimate uptake rates for various C sources (e.g., acetate, propionate, butyrate, and NaHCO₃) and incident light intensities (15, 30, 53, and 96 W·m⁻²). This experimental setup, with a larger gas headspace compared to the previous tests, was
designed to prevent limitations in gas mass transfer while maintaining a sufficient liquid volume for periodic sampling. All batch assays were carried out in triplicate under similar stirring (600 rpm) and an initial pH of 7, which was found to be optimal. The inoculation ratio was the same used for the enrichments, to ensure that rates were not limited by gas mass transfer. Acetate was used as the C source, and the light intensity was set at 50-53 W·m⁻², unless otherwise specified (see supplementary material).

After measuring the gas pressure, initial samples of gas and liquid were collected to determine gas composition, pH, and concentrations of N (soluble and total), Total Kjeldahl N (TKN; soluble and total), NH₄⁺, COD (soluble and total), VFAs, and VSS.

Gas pressure and composition, total and soluble COD concentrations, pH, and temperature were monitored every 2-5 hours. Gas pressure was firstly measured and later gas composition was analyzed. After, 5 mL of liquid (precisely) were drawn using a syringe. Final gas and liquid samples were fully characterized, as the initial samples.

Crude protein (CP) contents were estimated from TKN (Eding et al., 2006).

Dedicated tests under optimal conditions served to compare diazotrophic and non-diazotrophic uptakes rates. These tests were performed as above, but with the non-diazotrophic assay using ammonium as the N source, as in Capson-Tojo et al. (2022).

## 2.5. Analytical procedures

Temperature was monitored using a sensor PT 100 (Jumo). Incident light intensity was measured using an HDX spectrometer (Ocean Optics), and the headspace pressure using a manual pressure sensor (LEO2, Keller). Gas composition, including N₂, H₂, CO₂, CH₄, and O₂, was analyzed using a Perkin Clarus 580 gas chromatograph (Rodero et al., 2024). The pH was measured using a FiveGo F2 pHmeter (Mettler Toledo).

Filtered samples (0.22 µm pore size) were used to measure concentrations of soluble components. N-NH₄⁺ concentrations were measured with a Gallery Plus sequential
analyzer (Thermo Fisher), and total and soluble COD concentrations with Aqualytic
420721 COD Vario Tube Test LR (0-150 mg·L⁻¹) and MR (0-1,500 mg·L⁻¹). TKN
concentrations were measured using a Nitrogen (Total) TNTplus Vial Test, LR (5-40
mg N·L⁻¹), HR (5-40 mg N·L⁻¹) and UHR (20-100 mg N·L⁻¹). VFAs were quantified
using a Clarus 580 gas chromatograph, and IC concentrations with a TOC-L analyzer
(Shimadzu) (Rodero et al., 2024). VSS contents were measured following Standard
Methods. The amino acid composition of biomass from the tests used to estimate uptake
rates with various C sources was analyzed by the Biological Research Centre of the
Spanish National Research Council (Madrid, Spain) (Rodero et al., 2024).

**2.6. Analysis of microbial communities, strain isolation, and genome sequencing**

Samples taken at the end of the C source uptake rate tests were used to assess the
composition of the bacterial community via 16S rRNA gene sequencing. Duplicate
samples were collected, centrifuged, and the DNA was extracted and sequenced, as
described in Rodero et al. (2024). For strain isolation, liquid serial dilutions along with
roll-tube dilutions were performed using a sample from the diazotrophic-heterotrophic
enrichment. The medium utilized for the enrichments was used for isolation, but the
preparation was slightly modified following techniques to ensure strictly anaerobic
conditions (Miller and Wolin, 1974). Agar (2%) was added to perform roll-tube
dilutions. This allowed working anaerobically and recovering colonies, while
maximizing gas exchange due to the thin coated layer. The initial gas phase was 100%
N₂ at a pressure of 1.2-1.3 bar. The tubes were incubated under continuous illumination
as above. After 1.0-1.5 weeks, a pigmented colony was picked-up and inoculated in
liquid medium. Once growth was observed, a new liquid and roll-tube serial dilution
(10⁻¹-10⁻¹⁰) was performed for strain isolation. This iterative procedure was repeated
three times. Bacterial genome sequencing and bioinformatic analyses were carried out
by AllGenetics & Biology SL, following standard procedures. The resulting genome has
been deposited at DDBJ/ENA/GenBank under the accession JBJOVB000000000.

**2.7. Data treatment and statistical analyses**

N yields were calculated considering the increase in particulate N in the liquid and the
amount of N₂ consumed in the headspace, which was determined using the ideal gas
law. COD yields were calculated similarly, as in Rodero et al. (2024). The essential
amino acid (EAA) contents were used to calculate the dietary match for humans and
essential amino acids index (EAAI), considering both total amino acid (TAA) and CP
contents. This was done as in Spanoghe et al. (2021). Results are presented as average
values of triplicate measurements (unless specified otherwise), showing standard
deviations for measured values and 95% confidence intervals for calculated parameters.

T-tests or one-way ANOVAs were used to compare means (significance level of 5%).

Specific COD uptake rates were calculated using a mechanistic model tailored for
diazotrophic growth, building on previous PNSB models (Capson-Tojo et al., 2023;
Rodero et al., 2024). This approach prevented biased estimations of rates due to gas
mass transfer limitations. The model incorporated heterotrophic and autotrophic PNSB
growth, and biomass decay. Gas-liquid mass transfer kinetics were considered (Rodero
et al., 2024). See supplementary material for the model structure and parameter values.

Simulations were executed in MATLAB R2021a (The MathWorks Inc.). Specific
uptake rates and half-saturation constants (Ks) were estimated as in Rodero et al.

(2024). The code is available at: https://github.com/GabrielCapson/PPBN2rates/. The
impact of the average light intensity on the rates was considered as in Capson-Tojo et
al. (2023). Average intensities through the cultures during the exponential growth phase
were estimated using empirical equations for PNSB cultures (Capson-Tojo et al., 2022).

**3. Results and discussion**
**3.1. Diazotrophic biomass yields are slightly lower than in ammonia-based growth**

Biomass yields under photoheterotrophic conditions were similar across different substrates, with average values of 0.88±0.02, 0.85±0.04, and 0.87±0.01 g COD<sub>biomass</sub>·g COD<sub>consumed</sub><sup>-1</sup> for acetate, propionate, and butyrate, respectively (Figure 1A). Due to the production of H<sub>2</sub> concomitant to N<sub>2</sub> fixation, diazotrophic yields were slightly lower than non-diazotrophic values (~1 g COD<sub>biomass</sub>·g COD<sub>consumed</sub><sup>-1</sup>) (Alloul et al., 2019).

The volumes of H₂ produced were below detection limits, but COD balances could be closed assuming typical H₂ production ratios of 1-7 mol H₂<sub>produced</sub>·mol N<sub>2,fixed</sub><sup>-1</sup> (Alloul et al., 2023). When assuming a ratio of 7 mol H₂<sub>produced</sub>·mol N<sub>2,fixed</sub><sup>-1</sup>, balances closed at 98-100%, whereas a ratio of 1 mol H₂<sub>produced</sub>·mol N<sub>2,fixed</sub><sup>-1</sup> resulted in 89-92% closure.

The latter corresponds to BNF by Mo-nitrogenase, the most common and efficient N<sub>2</sub> fixation enzyme in PNSB (Masepohl, 2017), which was expected to dominate. The COD gap of 8-11% when assuming a ratio of 1 mol H<sub>2,produced</sub>·mol N<sub>2,fixed</sub><sup>-1</sup> may be explained by the growth of other bacteria, such as heterotrophs, which were present in the cultures (Section 3.4). The lower biomass yields of these bacteria compared to PNSB could account for the lost COD. The presence of other bacteria could also explain why COD yields were slightly higher for the photoautotrophic test compared to photoheterotrophic values (0.88±0.02 vs. 0.93±0.02 g COD<sub>biomass</sub>·g COD<sub>consumed</sub><sup>-1</sup>), as diazotrophic-autotrophic conditions led to higher PNSB abundance (Section 3.4).

The average C oxidation state of the substrate did not affect the biomass yields, as more reduced substrates like propionate or butyrate produced yields similar to acetate.

NaHCO₃ addition did not enhance yields either (average values for photoheterotrophic tests of 0.88±0.04 g COD<sub>biomass</sub>·g COD<sub>consumed</sub><sup>-1</sup> with NaHCO₃ vs. 0.87±0.02 g COD<sub>biomass</sub>·g COD<sub>consumed</sub><sup>-1</sup> without it), proving that extra IC did not benefit growth and suggesting that IC addition did not boost the role of IC fixation as redox homeostasis
mechanism. The high yields confirmed that the COD was not utilized to produce ATP for N₂ fixation, with the energy for nitrogenases being derived from light via photophosphorylation. The COD in the substrate was directly used for biomass synthesis, with a concomitant electron loss as H₂ due to N₂ fixation. The added NaHCO₃ was not used to re-consume partially the H₂ produced. Energetically, it would be impossible to grow biomass using the produced H₂ from N₂ fixation alone (H₂ is a by-product of diazotrophic growth), but H₂ could be potentially consumed for producing internal storage compounds that do not require N, such as polyhydroxyalkanoates (PHAs) (Abdul Rahman et al., 2024). PHA measurements confirmed that the enriched PNSB cultures did not synthesize PHA from H₂ (results not shown). COD/N ratios of 14-17 g CODconsumed·g Nfixed⁻¹ were consistent across all conditions, further suggesting that the C oxidation state of the substrate did not impact N₂ fixation. The similar COD/N ratios suggest that N availability was the limiting factor for growth, as extra COD or C did not enhance yields.

Light intensity did not significantly affect the COD yields, which averaged 0.89±0.01 g CODbiomass·g CODconsumed⁻¹ across all tested intensities (Figure 1B). This is consistent with the premise that light availability does not influence the amount of N₂ fixed per COD consumed (similar ratios of 16.3±1.2 g CODconsumed·g N-N₂fixed⁻¹). While light availability affected ATP production and fixation rates, it did not have any effect on the overall growth yields (Figure 1B). Biomass COD yields remained consistent across all tested initial pH values, averaging 0.88±0.02 g CODbiomass·g CODconsumed⁻¹ (Figure 1C). The N yields, with an average of 0.99±0.06 g Nbiomass·g Nconsumed⁻¹, were unaffected by any of the parameters tested, confirming that the fixed N was completely used for the synthesis of biomass components. No reduced N was secreted by the cells. Biomass N yields did not differ between substrates (Figure 1A), with average values of 0.95±0.14,
1.01±0.02, and 0.98±0.17 g N<sub>biomass</sub>·g N<sub>consumed</sub><sup>-1</sup> for acetate, propionate and butyrate, respectively. NaHCO<sub>3</sub> supplementation did not affect the N yields. As a result, COD/N-N<sub>2</sub> ratios were similar, with values of 16.0±1.6 g COD<sub>consumed</sub>·g N<sub>fixed</sub><sup>-1</sup> under heterotrophic conditions and 15.0±0.9 g COD<sub>consumed</sub>·g<sup>-1</sup> N-N<sub>2fixed</sub> under autotrophic conditions. Autotrophic N yields could not be estimated due to the low amounts of N<sub>2</sub> consumed in the headspace, which could not be accurately quantified. Operational conditions (e.g., pH and light intensity) did not affect the COD/N ratios, except at pH 8, where an unexplained high value was obtained. Similar COD/N ratios were expected, since COD and N yields were consistent across conditions. Closed N and COD balances confirmed that yeast extract did not contribute considerably to these components.

Open PNSB cultures demonstrated higher diazotrophic COD yields (0.89 COD<sub>biomass</sub>·g COD<sub>consumed</sub><sup>-1</sup>) compared to other diazotrophs, such as hydrogen-oxidizing bacteria (0.14 COD<sub>biomass</sub>·g COD<sub>consumed</sub><sup>-1</sup>) or rhizobacteria (0.27-0.40 COD<sub>biomass</sub>·g COD<sub>consumed</sub><sup>-1</sup>) (Blagodatskaya et al., 2014; Hu et al., 2020). As for PNSB, the diazotrophic COD yields are also lower than those obtained with NH<sub>4</sub><sup>+</sup> for other diazotrophs. However, unlike PNSB, these non-phototrophic organisms rely on substrate-derived energy for ATP generation, reducing their COD yields. Additionally, PNSB did not excrete any of the fixed N<sub>2</sub>, retaining it in the biomass. In contrast, diazotrophic cyanobacteria may release up to 50% of their newly fixed N<sub>2</sub> to stimulate surrounding organisms (Hieronymus et al., 2021), and diazotrophic rhizobacteria excrete NH<sub>3</sub> to promote plant growth, releasing 5-20% of the total N<sub>2</sub> fixed (Haskett et al., 2022). The higher COD and N yields of PNSB are a clear advantage for MP production, as most of the provided COD is recovered as biomass, with no N losses.

## 3.2. Dinitrogen fixation results in higher specific uptakes rates in purple bacteria

The growth curves for the tests conducted with various C sources and incident light
intensities are shown in Figure 2. By 1.0-1.5 days, soluble COD was consumed in all
heterotrophic tests and a biomass plateau was reached, confirming that the tests
concluded successfully. The autotrophic test was stopped after 3.9 days, as sufficient
data points had been obtained for rate estimation. Residual O₂ in the reactor headspace
(initial maximum concentration of ~1% v·v⁻¹) could not be fully eliminated, explaining
the observed lag phase of up to 0.5 days in the diazotrophic kinetic curves (Figure 2), as
nitrogenase activity is highly sensitive to oxygen inhibition (Masepohl, 2017).

The highest specific uptake rate was obtained with acetate as substrate, with a value of 5.20±0.83 g COD<sub>substrate</sub>·g COD<sub>biomass</sub><sup>-1</sup>·d<sup>-1</sup>. Both propionate and butyrate resulted in lower specific rates (4.29±0.13 and 4.38±0.13 g COD<sub>substrate</sub>·g COD<sub>biomass</sub><sup>-1</sup>·d<sup>-1</sup>, respectively). Interestingly, these photoheterotrophic rates exceeded considerably those obtained for equivalent non-diazotrophic conditions (Alloul et al., 2019; Capson-Tojo et al., 2023; Puyol et al., 2017). A comparative test using acetate as substrate confirmed this, as it showed significantly higher diazotrophic uptake rates than a non-diazotrophic control (5.25±1.16 vs. 2.64±0.34 g COD<sub>substrate</sub>·g COD<sub>biomass</sub><sup>-1</sup>·d<sup>-1</sup>) (Table 1).

The rates in Table 1 are comparable because they were obtained using equivalent
experimental setups (i.e., reactor geometry, light path, incident light spectrum, C source,
and final biomass concentrations). This is particularly relevant because the rates might
be slightly underestimated, as the model used does not account for light attenuation
through the culture. Further research should consider light attenuation and distribution
in the reactors, estimating accurately rates and light half-saturation constants.

Photoheterotrophy in PNSB can induce redox imbalance, making redox homeostasis a
crucial mechanism regulating growth. To mitigate excess electrons, these bacteria
employ electron-sinking strategies, including CO₂ fixation, H₂ production, or PHA
synthesis (Alloul et al., 2023). N₂ fixation acts as an additional electron sink, consuming
8 mol of electrons per mol of N₂ fixed. This reduces reliance on other electron-sinking pathways and can explain the observed faster uptake rates. Non-diazotrophic growth relies on reduced nitrogen forms (e.g., NH₄⁺), which act as electron carriers and further jeopardize effective redox homeostasis. The hypothesis of a faster growth under diazotrophy by avoiding other electron sinks (e.g., PHA synthesis, H₂ production, or CO₂ fixation) remains to be verified. Redox homeostasis considerations can also explain why the specific rates were faster for acetate than for propionate or butyrate, as growth on acetate (with less electron available per C atom) reduces redox stress compared to longer VFAs (Alloul et al., 2023). Slower non-diazotrophic kinetics with more reduced VFAs have been previously reported (Capson-Tojo et al., 2023; Puyol et al., 2017).

The photoautotrophic specific uptake rate (1.02±0.33 g COD<sub>substrate</sub>·g COD<sub>biomass</sub><sup>-1</sup>·d<sup>-1</sup>) was lower than heterotrophic values. This lower rate is likely due to electron competition between CO₂ and N₂ fixation. The diazotrophic-autotrophic specific rate was also lower than reported values on NH₄⁺ (up to 2.00±0.14 g COD<sub>substrate</sub>·g COD<sub>biomass</sub><sup>-1</sup>·d<sup>-1</sup>) (Rodero et al., 2024). While photoheterotrophic growth must deal with an excess of electrons, the opposite happens under autotrophic conditions, where the need for extra electrons for N₂ fixation limits growth. The latter has also been observed for diazotrophic cyanobacteria, which need readily available N (i.e., NH₄⁺) to reach maximum growth rates, as BNF induces a strict electron tradeoff (Osburn et al., 2021).

BNF is not only electron-intensive but also energy-demanding. PNSB derive this energy from light via cyclic photophosphorylation. Specific uptake rates increased with light intensity (Table 1), plateauing at 53 W·m⁻² (corresponding to an average light intensity through the culture of 34 W·m⁻² during exponential growth). Lower light intensities (i.e., incident light of 15 and 30 W·m⁻², corresponding to average values of 9.5 and 19 W·m⁻²) resulted in lower rates (3.26±0.14 and 4.19±0.41 g COD<sub>substrate</sub>·g COD<sub>biomass</sub><sup>-1</sup>·d<sup>-1</sup>·
¹, respectively), suggesting insufficient energy for diazotrophic growth. Conversely, incident intensities above 53 W·m⁻² did not yield higher rates, indicating saturation (rates of 5.20±0.83 and 5.38±0.66 g CODₐsubstrate·g CODₐbiomass⁻¹·d⁻¹ at incident intensities of 57 and 96 W·m⁻² (34 and 61 W·m⁻² in average), respectively). As for previous non-diazotrophic tests, no photoinhibition was observed (Capson-Tojo et al., 2022).

The average light intensity required to reach optimal uptake rates was estimated using a Monod function (Figure 3A; R² of 0.995). This fit yielded a light half-saturation constant (K₅₁) of 6.39±1.58 W·m⁻², slightly higher than reported values for non-diazotrophic PNSB growth (4.58 W·m⁻² (Capson-Tojo et al., 2022)). This suggests that diazotrophy requires higher light intensities to achieve maximum uptake rates, due to the additional energy demand for N₂ fixation. These K₅₁ values are comparable because both considered the actual light intensity. The diazotrophic value considered the average light intensities throughout the culture during the exponential growth phase, and the non-diazotrophic value was estimated using an experimental setup where light attenuation could be neglected (Capson-Tojo et al., 2022).

As an example of model application for rate estimation, experimental and modelling results are shown in Figure 3B for the test conducted with acetate at 53 W·m⁻². The model reproduced accurately the exponential growth phase (R² of 0.98 and 0.99 for biomass and soluble COD, respectively), confirming the reliability of the estimated rates. The discrepancies between modelled and predicted values can be a result of the calibrated Kₛ for each substrate, which were kept at low values (0.1 g COD·L⁻¹ as upper calibration boundary; close to non-diazotrophic values (Capson-Tojo et al., 2023)). This ensured that the specific uptake rates were not being overestimated due to correlations between these parameters. Also aiming to avoid rate overestimation, it was assumed that all particulate COD was active biomass. Therefore, the uptake rates presented here
might be underestimated, highlighting the need for further research to determine K<sub>S</sub> and
uptakes rates. Despite these uncertainties, the non-diazotrophic specific uptake rates
were consistent with literature values (e.g., 2.64±0.34 vs. 2.37±0.37 g COD<sub>substrate</sub>·g
COD<sub>biomass</sub><sup>-1</sup>·d<sup>-1</sup> (Puyol et al., 2017)), confirming the accuracy of the used approach.

**3.3. Protein contents and amino acid profiles are promising for human food**

CP and TAA contents in the biomass were consistent across different conditions,
ranging from 43-54% and 38-43%, respectively (Table 1). The protein contents align
with reported values for heterotrophic and autotrophic biomass from open PNSB
cultures grown under non-diazotrophic conditions, confirming that diazotrophy does not
compromise protein yields (Rodero et al., 2024). Protein contents are also comparable
to those of other MP candidates, such as methanotrophs, cyanobacteria, or hydrogen
oxidizing bacteria (Matassa et al., 2016b, 2016a). The EAAs content is key for food
products, as they cannot be synthesized by the human body. The EAA proportion in the
biomass was consistent across all conditions, ranging from 42-46% of the TAA (Table
1). All samples contained the full spectrum of EAAs (see supplementary material;
tryptophan not measured), with similar amino acid profiles. The TAA and EAA
contents align with those in key protein sources such as soybean meal, as well as with
non-diazotrophic PNSB biomass (Rodero et al., 2024; Spanoghe et al., 2021).

Given that CP and TAA values either overestimate or underestimate protein contents,
protein quality metrics were calculated using both values for normalization. Regardless
of the contents considered, EAAIs were equal to or greater than 1 across all samples
(see supplementary material), demonstrating the high quality of PNSB-enriched
biomass for human food consumption. Dietary match values confirmed that
diazotrophic PNSB biomass can fulfill human dietary requirements, with all samples
exceeding 100% match values for all amino acids when normalized by TAA (results not
shown). Photoheterotrophic samples from reactors fed with propionate or butyrate occasionally showed dietary match values slightly below 100% when normalized by CP, likely due to overestimation of the CP contents. Importantly, diazotrophic samples grown on acetate or H₂ exhibited dietary match values equal to or greater than those grown on NH₄⁺, highlighting that diazotrophy did not negatively affect protein quality.

**3.4. Anaerobic diazotrophic-phototrophic conditions are highly selective**

The relative abundances of 16S rRNA gene copies exceeded 0.7 under both heterotrophic and autotrophic conditions at the end of the tests, confirming the development of diazotrophic PNSB communities (Figure 4). Regardless of the C source, the predominant operational taxonomical unit (OTU) under heterotrophic conditions belonged to the family *Rhodobacteraceae* (genera level could not be initially assigned, but BLAST against the NCBI database found 99% similarity to a *Rhodobacter capsulatus* strain). The relative abundance of this OTU was 0.73, 0.67 and 0.71 in tests fed with acetate, propionate and butyrate. *R. capsulatus* is a known PNSB capable of BNF (Maeda, 2022). Genera such as *Rhodopseudomonas* sp. and *Rhodocista* sp., common photoheterotrophic PNSB, were detected at lower abundances (below 0.02). While microbial communities across different VFA were remarkably similar, under autotrophic conditions a combination of *Rhodopseudomonas* sp. (0.47) and *Rhodobacter* sp. (0.32) was dominant (i.e., most abundant OTUs). Specifically, the predominant species were *Rhodopseudomonas palustris* and *Cereibacter sphaeroides* (both with 99% similarities), which are common in non-diazotrophic enrichments under both autotrophic or heterotrophic conditions (Alloul et al., 2019; Capson-Tojo et al., 2021; Rodero et al., 2024). The non-diazotrophic photoheterotrophic enrichments were clearly dominated by *Rhodobacter* sp. (0.81; 0.02 for *Rhodopseudomonas* sp.), as in the diazotrophic photoheterotrophic reactors. Thus, the diazotrophic selective pressure was
not key to shaping the PNSB community. This suggests that the electron source is a
critical factor shaping the microbial community under diazotrophic conditions. The C
source, rather than the N source, determined the PNSB community.

Non-PNSB bacteria were also detected, including aerobic bacteria and anaerobic fermenters. *Pseudomonas* sp. was the most abundant non-PNSB genus under heterotrophic conditions, with relative abundances up to 0.21. The ability of some species to fix N₂ has been documented (Bentzon-Tilia et al., 2015). Other aerobic bacteria, including *Chryseobacterium* sp., *Stenotrophomonas* sp. and *Flavobacterium* sp., were also detected in lower proportions. The presence of these aerobes can be attributed to the residual oxygen in the reactor headspace, as these aerobic bacteria outcompete PNSB due to their faster growth rates when oxygen is present (Capson-Tojo et al., 2021). Given the low amounts of residual oxygen (below 1% v·v⁻¹ initially), it was assumed that the presence of these bacteria did not affect the obtained results.

Continuous anaerobic operation will likely wash out these aerobes and ensure stable conditions and performance. Anaerobic fermenters were also present at low proportions (e.g., *Cloacibacterium* sp. in all conditions (<0.12), or *Paludibacter* sp. in autotrophic tests (<0.07%)). The presence of anaerobic fermenters likely stems from the decay of PNSB biomass and their subsequent degradation, especially under autotrophic conditions due to the lower growth rates. Additionally, acetogens may have partially consumed propionate and butyrate, explaining the slightly reduced PNSB abundances in these tests compared with acetate-fed reactors. These genera were also observed in non-diazotrophic mixed PNSB cultures (Alloul et al., 2019; Rodero et al., 2024).

Given the clear dominance of a specific strain in the diazotrophic experiments, isolation and further identification was pursued. Using diazotrophic-photoheterotrophic sequential plating, isolation of the dominant species was performed and its genome was
sequenced. The genome assembly achieved >99% completeness, meeting validation
requirements. However, contamination was detected, with a clade separation score of
0.96 (0 = uncontaminated; 1 = mixed taxonomy in contigs), indicating that the strain
could not be isolated. Additionally, 96% of the mapped assembled contigs corresponded
to a strain identified as *Rhodopseudomonas palustris* (family *Nitrobacteraceae*). ANI
results confirmed 97% similarity to *R. palustris*. This was extremely surprising, as the
dominant strain in the diazotrophic photoheterotrophic enrichment corresponded to a
*Rhodobacteraceae*. Further work will aim at isolating this strain.

R. palustris is a common PNSB species found in non-diazotrophic enrichments (Alloul et al., 2019; Rodero et al., 2024), and its diazotrophic capabilities are well-documented (Masepohl, 2017). Automatic genome annotation of the isolated strain showed that, while genes associated with BNF comprised only 1% of the strain’s genome, they accounted for 80% of the genes related to N metabolism (0.8% of the total genome). The presence of genes for all three known nitrogenases highlighted its exceptional suitability for BNF. Comparison with the seven R. palustris genomes available in the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that only one of these strains (R. palustris CGA009) had comparable proportions of BNF-related genes (1.1% of its genome). The other genomes contained BNF genes at roughly half this proportion (~0.5%) and lacked genes for one of the nitrogenases (V-). Its specialization in BNF likely explains its presence under diazotrophic conditions. A Krona plot for genome annotation is available at https://doi.org/10.6084/m9 figshare.27871725.v1.

**3.5. Diazotrophic yields and uptake rates show great potential for food production**

Benchmarking the performance of the diazotrophic open PNSB cultures in terms of
yields and uptake rates with values for PNSB grown under non-diazotrophic conditions
and with those of other diazotrophic organisms (i.e., cyanobacteria and rhizobacteria),
the potential of diazotrophic PNSB growth for food production appears evident,
exhibiting the fastest uptake rates and near-optimal biomass yields (Figure 5; data from
this study and Alloul et al. 2019; Blagodatskaya et al. 2014; Capson-Tojo et al. 2023;
Kramer et al. 2022; Rezasoltani and Champagne, 2023; Rodero et al. 2024;
Soundararajan et al. 2019; Trentin et al. 2023; Turk-Kubo et al. 2018; Velu et al. 2019).

While diazotrophic PNSB growth results in slightly lower yields compared to growth on
NH₄⁺, the specific uptake rates are significantly faster. The specific uptake rates for
diazotrophic cyanobacteria are within the range of PNSB autotrophic rates, showing
that the limitation of electrons for simultaneous CO₂ fixation and BNF affects both
microbial clades. Nevertheless, unlike cyanobacteria, PNSB do not face the paradox
where oxygen produced during photosynthesis inhibits the nitrogenase (Paerl, 2017).

Consequently, PNSB avoid common adaptations found in cyanobacteria, like night-time
only N₂ fixation or heterocyst formation. Moreover, PNSB retain the fixed N in the
biomass, unlike cyanobacteria, which may release up to 50% of it (Hieronymus et al.,
2021). The NUE of PNSB is thus much higher if MP is the desired product.

Heterotrophic rhizobacteria and soil organisms inherently achieve lower biomass yields
than phototrophs. Their uptake rates are surprisingly low compared to diazotrophic open
PNSB cultures, and these values should be interpreted with caution due to the extremely
limited data available. Most studies on rhizobacteria focus on overall BNF rates rather
than growth rates or biomass yields, often assessing their effects on plant growth (Ladha
et al., 2022). Estimating uptake rates from these values requires too many assumptions.

The resulting overall protein productivities are up to 312±23 mgprotein·L⁻¹·d⁻¹ under diazotrophic conditions (Table 1). This productivity surpasses values for non-diazotrophic autotrophic PNSB systems (90-180 mgprotein·L⁻¹·d⁻¹ (Rodero et al., 2024; Spanoghe et al., 2021)) and hydrogen oxidizing bacteria (270 mgprotein·L⁻¹·d⁻¹ (Matassa
et al., 2016b)) and, while they are slightly lower than some heterotrophic PNSB
consortia on NH₄⁺ (290–640 mgprotein·L⁻¹·d⁻¹ (Alloul et al., 2019; Hülsen et al., 2022)),
the highest values of the latter were achieved at much higher biomass concentrations
(more than four times higher than the final concentrations in this study) and using
synthetic media. Assuming that higher biomass concentrations can also be maintained
in diazotrophic PNSB cultures, reasonable protein productivities of, at least, 1.2-1.5
gprotein·L⁻¹·d⁻¹ could be achieved. Research is needed to prove this.

Biomass productivities for N₂-fixing cyanobacteria in open systems range between 2.0-23 g_biomass·m⁻²·d⁻¹. Assuming a typical protein content of 54% and a surface-to-volume ratio of 7 m⁻¹ (common in upscaled systems for cyanobacteria cultivation), this equates to a modest 0.03 g_protein·L⁻¹·d⁻¹ (Nascimento et al., 2019; Razon, 2012). These productivities are much lower than the expected values for PNSB for various reasons, including slower growth rates, and, crucially, extremely diluted media with low gas transfer rates due to the use of open ponds. Using closed anaerobic photobioreactors could allow efficient mass transfer and high biomass concentrations. Similarly, BNF by crop-related microorganisms typically focuses on on-site fertilizer production, measuring BNF rates as N fixed per hectare per crop cycle. Common BNF rates in this context reach up to 290 kg N·ha⁻¹·crop⁻¹ (Ladha et al., 2022). In contrast, a 100 m³ PNSB reactor could fix the same N within 12 days requiring minimal land area, assuming the N fixation rates obtained here and reasonable biomass concentrations.

Further research needs to be performed in scaled systems to verify these numbers.

**3.6. Implications for implementation and further research**

This study demonstrates the remarkable potential of PNSB as efficient N₂-fixers for
sustainable protein production. By showing that diazotrophy can enhance
photoheterotrophic uptake rates, this work opens new possibilities for exploiting the
metabolic capabilities of PNSB in biotechnological applications. The unprecedentedly
high diazotrophic yields and uptake rates of PNSB position them as a competitive
option compared to existing alternatives. Additionally, the productivity numbers
achieved here are likely far from optimal values. Higher values can be expected in
continuous reactors under optimal conditions, retention times, and feeding strategies.
While this research advances understanding of PNSB's diazotrophic capabilities, the
specific reasons driving faster specific rates for diazotrophic PNSB remain unknown.
The hypothesis that diazotrophy bypasses other electron sinks (e.g., PHA synthesis, H₂
production, or CO₂ fixation) needs further validation. Further research should elucidate
if BNF alleviates redox stress when ATP is sufficient, if certain electron-sinks are
favored during diazotrophy, or if redox balancing mechanisms are conserved across
diazotrophic PNSB. Concerning application, strategies should be developed to further
enhance BNF rates, also optimizing biomass concentrations to maximize productivity
without compromising efficient gas-liquid mass transfer and light distribution.

Mechanistic and metabolic models should guide performance optimization, simulate
implementation scenarios, and formalize knowledge. Upscaled experiments using
continuous gas-fed reactors will be essential to provide key information to support
techno-economic and life cycle assessments, also clarifying contamination resistance
and producing sufficient biomass for detailed product characterization. From an
environmental perspective, BNF using PNSB cultures could facilitate the complete
treatment of waste streams constrained by reactive N availability for full COD or P
removal, addressing the critical challenge of COD:N:P imbalances.

The generated MP could be used as a substitute for feed in aquaculture (Delamare-
Deboutteville et al., 2019). However, the relatively low price of fishmeal (~2 €·kg<sub>protein</sub><sup>-1</sup>) presents a challenge for cost-effectiveness. While feed production is subjected to
simpler regulations, targeting human-grade nutraceuticals and food products represents a more ambitious and impactful direction. This approach might allow solving the primary challenge for implementation: generating a product with sufficient market value. The results presented here underscore the nutritional potential of PNSB biomass, which, combined with the high value of food products, the selective anaerobic diazotrophic-phototrophic conditions, the high productivity, and the extremely efficient N and COD usages, present a compelling case for producing MP for human consumption. The existing market for comparable products, such as spirulina (*Arthrospira* sp.) or unicellular fungi-based foods, further supports this approach, suggesting also an increasing public openness to novel dietary options.

The potential of MP as human food warrants a discussion on implementing pragmatic regulatory quality control measures to support the development of these novel foods.

Despite a considerable body of evidence demonstrating that MP derived from precision symbiotic fermentations (using novel species or microbial consortia) can be adequately produced and processed (Javourez et al., 2024), current regulations strongly hinder bringing MP products to the market, especially in the EU. It stems to reason that by implementing control methods already in place for conventional mixed culture-based foods (e.g., beer, cheese, processed meat, or choucroute), it should be possible to ensure the safety of novel fermentation products. Further research should test if existing safety and quality control methods can ensure the safety of novel foods and, if needed, new methodologies should be developed to assess nutritional and toxicological properties.

Regulatory frameworks ensuring product safety and consistency will be critical for gaining public trust and encouraging adoption. With appropriate photobioreactor development (based on years of research for microalgal biotechnology), supportive policies, and public engagement, scaling up PNSB MP production could be achievable.
**4. Conclusions**

PNSB diazotrophic yields (0.85-0.93 gCOD<sub>biomass</sub>·gCOD<sub>substrate</sub><sup>-1</sup>) were slightly lower than non-diazotrophic values but were over double those of heterotrophic-diazotrophic rhizobacteria. Diazotrophic PNSB provided full N recovery. The specific uptake rate obtained for acetate-fed photoheterotrophic-diazotrophic conditions (5.20±0.83 gCOD<sub>substrate</sub>·gCOD<sub>biomass</sub><sup>-1</sup>·d<sup>-1</sup>) was the fastest ever reported for any diazotroph or PNSB, being also faster than values for equivalent non-diazotrophic conditions.

Optimal diazotrophic uptake rates require slightly higher light intensities that non-diazotrophic conditions. Photoheterotrophic-diazotrophic conditions were highly selective. Biomass protein contents (44-54% CP) and essential amino acid metrics confirmed nutritional values. This work demonstrates the potential of specialized PNSB for MP production via sustainable N<sub>2</sub> fixation.

**Supplementary material**

E-supplementary data for this work can be found in the e-version of this paper online.

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Author contributions

**María del Rosario Rodero**: Conceptualization, Methodology, Investigation, Writing - Original Draft, Visualization. **Jean-Philippe Steyer**: Conceptualization, Resources, Writing - Reviewing and Editing, Project administration, Supervision. **María Fernanda Pérez-Bernal**: Methodology, Investigation, Writing - Reviewing and Editing. **Willy Verstraete**: Conceptualization, Writing - Reviewing and Editing. **Renaud Escudié**: Conceptualization, Resources, Writing - Reviewing and Editing, Supervision, Funding acquisition. **Gabriel Capson-Tojo**: Conceptualization, Methodology, Investigation, Resources, Writing - Original Draft, Writing - Reviewing and Editing, Visualization, Supervision, Project administration, Funding acquisition.
**Declaration of interests**

[x] The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[ ] The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
<figure><img src="image_4.png" /></figure>

**Figure 1.** COD (red bars) and N (blue bars) biomass yields for: (A) various substrates (incident light intensity of 50-53 W·m⁻² and initial pH of 7); (B) various light intensities (acetate as substrate and initial pH of 7); and (C) various initial pH values (acetate as substrate and incident light intensity of 50-52 W·m⁻²). The ratios of COD consumed per N₂ fixed are also shown (grey points). Each bar/point represents the average with 95% confidence intervals (n = 3). Groups sharing the same letters indicate no significant differences (p>0.05).
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Growth curves for diazotrophic batch tests with: (A) various substrates at an incident light intensity of 50-53 W·m⁻²; and (B) different incident light intensities using acetate as the substrate. Each point represents the average with 95% confidence intervals (n = 3).</figcaption></figure>

<figure><img src="image_6.png" /></figure>
**Figure 3.** (A) Specific photoheterotrophic-diazotrophic uptake rates vs. average light intensities, including modelling results using a Monod function; and (B) Experimental and modelled data for the batch test with acetate at 53 W·m⁻². Average light intensities were estimated considering attenuation, as in Capson-Tojo et al. (2022). Each point represents the average with 95% confidence intervals (n = 3). Exp stands for experimental, Model for modelled, SCOD for soluble chemical oxygen demand, and PCOD for particulate chemical oxygen demand.
<figure><img src="image_7.png" /><figcaption>**Figure 4.** Relative abundance of 16S rRNA gene copies in diazotrophic batch tests with different substrates (genus level). Each bar represents the average with standard deviation (n = 2). HET stands for heterotrophic, ACET for acetate, PROP for propionate, BUT for butyrate, and AUT for autotrophic (growth on H₂ and CO₂).</figcaption></figure>
<figure><img src="image_8.png" /></figure>

**Figure 5.** Comparative performance of diazotrophic PNSB vs. other growth strategies and diazotrophic organisms. The numbers in the legend correspond to the temperatures at which the rates were estimated. Cyanobacteria yields, lacking physical significance due to their use of H₂O as an electron donor, are shown outside the numerical y-axis. Uptake rates for cyanobacteria reflect growth rates. Data were taken from this study and from literature (Alloul et al., 2019; Blagodatskaya et al., 2014; Capson-Tojo et al., 2023; Kramer et al., 2022; Rezasoltani and Champagne, 2023; Rodero et al., 2024; Soundararajan et al., 2019; Trentin et al., 2023; Turk-Kubo et al., 2018; Velu et al., 2019).

* Denotes *In situ* marine temperature.
## Highlights

* Heterotrophic diazotrophic uptake rates are the fastest ever reported for PNSB

* Diazotrophic COD yields are slightly lower than on $NH_4^+$

* PNSB diazotrophic growth achieves 100% conversion of fixed $N_2$ into biomass

* Diazotrophic protein yields and quality show potential for high-value applications

* Diazotrophic-photoheterotrophic conditions are extremely selective
Graphical abstract

What are the key performance indicators of specialized diazotrophic PNSB cultures?

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

VFA
H₂ + CO₂

Sequential enrichment

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

Yield and rate estimation

Light

CO₂

pH

<figure><img src="image_13.png" /></figure>

Protein and
microbial
analysis

<figure><img src="image_14.png" /></figure>

Performance
benchmarking

* Biomass COD yields: 0.85-0.93 g·g⁻¹

* Biomass N yields: ~1 g N·g⁻¹

* Diazotrophic-heterotrophic conditions showed the fastest specific uptake rates: up to 5.20±0.83 g COD·g⁻¹·d⁻¹

* A specialized *R. palustris* strain dominated

* Protein contents of 43-54%

* Adequate essential amino acid metrics

<figure><img src="image_15.png" /></figure>

Diazotrophic PNSB
from this study

Conclusion:

Diazotrophic-heterotrophic
uptake rates are the fastest
reported for any PNSB or
diazotroph. Clear potential
for protein production: high
yields, fast rates, selective
conditions, nutritional quality.
<table><caption>**Table 1.** Specific substrate uptake rates, final biomass concentrations, contents in the biomass of COD, crude protein, and total amino acids, and overall protein productivities for the batch tests used for rate estimation (n = 3; with standard deviations or 95% confidence intervals).</caption><thead><tr><th rowspan="2">Objective</th><th colspan="4">Assess impact of C source</th><th colspan="4">Assess impact of light intensity</th><th colspan="2">Comparison N<sub>2</sub> vs. NH<sub>4</sub><sup>+</sup></th></tr><tr><th>Acet</th><th>Prop</th><th>But</th><th>Auto</th><th colspan="4">Acet</th><th>Acet</th></tr></thead><tbody><tr><td>Carbon and electron source</td><td>N<sub>2</sub></td><td>N<sub>2</sub></td><td>N<sub>2</sub></td><td>N<sub>2</sub></td><td>N<sub>2</sub></td><td>N<sub>2</sub></td><td>N<sub>2</sub></td><td>N<sub>2</sub></td><td>N<sub>2</sub></td><td>NH<sub>4</sub><sup>+</sup></td></tr><tr><td>Nitrogen source</td><td>53</td><td>50</td><td>52</td><td>53</td><td>15</td><td>30</td><td>53</td><td>96</td><td>69</td><td>69</td></tr><tr><td>Incident light intensity (W·m<sup>-2</sup>)</td><td>5.20<br/>±0.83</td><td>4.29<br/>±0.13</td><td>4.38<br/>±0.13</td><td>1.02<br/>±0.33</td><td>3.26<br/>±0.14</td><td>4.19<br/>±0.41</td><td>5.20<br/>±0.83</td><td>5.38<br/>±0.66</td><td>5.25<br/>±1.16</td><td>2.64<br/>±0.34</td></tr><tr><td>Specific uptake rates (g COD·g COD<sup>-1</sup>·d<sup>-1</sup>)</td><td>0.44<br/>±0.01</td><td>0.36<br/>±0.06</td><td>0.41<br/>±0.01</td><td>0.37<br/>±0.05</td><td>0.43<br/>±0.02</td><td>0.39<br/>±0.02</td><td>0.44<br/>±0.01</td><td>0.40<br/>±0.02</td><td>0.50<br/>±0.06</td><td>0.57<br/>±0.04</td></tr><tr><td>Final biomass concentration (g VSS·L<sup>-1</sup>)</td><td>1.1<br/>±0.1</td><td>1.4<br/>±0.3</td><td>1.1<br/>±0.3</td><td>1.2<br/>±0.1</td><td>1.1<br/>±0.2</td><td>1.2<br/>±0.1</td><td>1.1<br/>±0.1</td><td>1.2<br/>±0.1</td><td>-</td><td>-</td></tr><tr><td>COD content (g COD·g VSS<sup>-1</sup>)</td><td>43<br/>±2</td><td>54<br/>±14</td><td>44<br/>±10</td><td>48<br/>±5</td><td>43<br/>±6</td><td>48<br/>±1</td><td>43<br/>±2</td><td>47<br/>±1</td><td>-</td><td>-</td></tr><tr><td>Crude protein content (%)</td><td>40<br/>±0</td><td>38<br/>±0</td><td>38<br/>±0</td><td>43<br/>±0</td><td>-</td><td>-</td><td>40<br/>±0</td><td>-</td><td>-</td><td>-</td></tr><tr><td>TAA (%)</td><td>44<br/>±1</td><td>45<br/>±0</td><td>42<br/>±2</td><td>46<br/>±0</td><td>-</td><td>-</td><td>44<br/>±1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>EAA (%TAA)</td><td>312<br/>±23</td><td>201<br/>±16</td><td>260<br/>±28</td><td>55<br/>±5</td><td>-</td><td>-</td><td>312<br/>±23</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Overall protein productivities (mg·L<sup>-1</sup>·d<sup>-1</sup>)<sup>a</sup></td><td>312<br/>±23</td><td>201<br/>±16</td><td>260<br/>±28</td><td>55<br/>±5</td><td>-</td><td>-</td><td>312<br/>±23</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

COD stands for chemical oxygen demand, VSS for volatile suspended solids, TAA for total amino acids, EAA for essential amino acids. Empty cells imply that the value was not measured.

a. Calculated considering the exponential growth phase.