Utilization of lysed and dried bacterial biomass from the marine purple photosynthetic bacterium *Rhodovulum sulfidophilum* as a sustainable nitrogen fertilizer for plant production

The study addresses the urgent need to increase crop productivity while reducing the environmental impacts of synthetic nitrogen fertilizers produced via the energy-intensive Haber–Bosch process. Fertilizer production and use contribute substantially to greenhouse gas emissions, and excessive mineral N use leads to N losses (runoff, leaching, N2O emissions) and soil organic carbon depletion. Conventional organic fertilizers (manure, compost) often have low N content, high application requirements, salinity risks, and can increase N2O emissions when C:N >10. Microbial biomass has emerged as a potential slow-release, high-N fertilizer. Purple non-sulfur bacteria (PNSB), including Rhodovulum sulfidophilum, can fix nitrogen and accumulate metabolites beneficial to plants, but direct evidence of plant uptake of N from PNSB biomass and its ability to replace mineral fertilizers is lacking. This study tests whether lysed and dried biomass from R. sulfidophilum (Processed Biomass, PB) can function as a sustainable N fertilizer for komatsuna, matching growth achieved with mineral fertilizer under different temperature regimes and nutrient contexts, and evaluates plant uptake of PB-derived N.

Background literature indicates: (1) global agriculture must increase production while mitigating environmental impacts; synthetic N fertilizer production via Haber–Bosch consumes 1–2% of global energy and contributes ~1.8% of CO2 emissions. Agrifood systems generate ~30% of global GHGs, with significant contributions from N2O linked to fertilizer use. (2) Organic amendments’ C:N ratio governs N mineralization vs. immobilization; many common organic fertilizers have low N content, require high application rates, raise soil salinity, and can increase N2O when C:N >10. (3) Microbial biomass, including PNSB, is promising as slow-release fertilizer due to higher N content and additional metabolites (carotenoids, vitamins, biostimulants). PNSB inoculants can promote plant growth and stress tolerance, but evidence for plant uptake of PNSB-derived N and equivalence to mineral fertilizer is limited. (4) Plants possess amino acid and peptide transporters enabling uptake of organic N forms; prior work shows direct root uptake of several amino acids, suggesting potential benefits of protein-rich microbial biomass as amino acid sources. These gaps motivate testing PNSB biomass as an N fertilizer and quantifying plant N uptake from it.

Processed Biomass (PB) preparation: Rhodovulum sulfidophilum (DSM 1374; ATCC 35886) was cultured in marine broth in 10-L bottles at ~26 °C under broad-spectrum light (80 W m−2) with mixing (350 rpm) for 5 days. Cells were harvested (14,000 × g), washed in water to remove salts, pelleted (14,000 × g), and stored at −80 °C. Lysis was performed by high-pressure homogenization (1000 bar, 7 cycles) or ultrasonication (19.2 kHz, 60% amplitude, 3 cycles). Complete lysis was confirmed by absence of growth on marine agar. Lysates were freeze-dried to obtain PB. Characterization: Elemental composition (total N and C by dry combustion; P2O5 by absorptiometry after nitric acid decomposition; K2O by atomic absorption spectroscopy). pH measured. Amino acid profiling: PB prepared from cultures in seawater with yeast extract and peptone; free amino acids extracted with 0.2 M perchloric acid; total amino acids quantified after 4 M methanesulfonic acid hydrolysis (cysteine excluded; ~20% tryptophan loss noted). Analysis via Hitachi L-8900 with post-column ninhydrin derivatization. Soil and treatments: Field topsoil (lowland soil) was air-dried, sieved (2 mm); 2.5 L batches were mixed with water and fertilizers and subsampled into pots (550 g per 12-cm pot). Treatments: NF (no fertilizer), NC (no-N control with P and K), mineral fertilizer controls C1 (1x NPK; fast-acting 8:8:8 at 1 g/L) and C2 (2x NPK; 2 g/L), PB1/PB2/PB4 supplying PB-derived N equivalent to 1x/2x/4x the N in C1; dose-range test also included PB8/PB16/PB32. For P and K supplementation, PB1 and PB2 were supplemented to match C1 levels using superphosphate and KCl (PB1P, PB1K, PB1PK; PB2P, PB2K, PB2PK). Initial soil pH and EC measured for soil–fertilizer mixes. Cultivation conditions: Three experiments: (i) Spring cultivation (Feb–Mar 2022) under natural light with minimum 15 °C; top-watering daily; insecticide and fungicide applied at 18 DAS. (ii) Summer cultivation (Aug–Sep 2022) to define maximum safe PB application rate; bottom-watering via wick and water furrow twice daily. (iii) Temperature-controlled cultivation (May–Jun 2023) in two greenhouses: cool (15–25 °C) and warm (22–32 °C), bottom-watering schedule standardized; care to avoid upstream contamination from high-N pots; plant protection applied at 16 DAS. Planting and measurements: Komatsuna (Brassica rapa var. perviridis, cv. Natsu Rakuten) seeds sown (5 positions/pot, 4 seeds per position). Germination assessed at 7 DAS; seedlings thinned to 5 plants/pot. Maximum leaf length measured at 14, 17, 21, 24, 28, 31, 35 DAS. SPAD chlorophyll readings at 14, 21, 28, 35 DAS on largest leaf (three positions when possible). Harvest at 35 DAS for fresh weight (FW) and dry weight (DW; oven-dried at 60 °C for one week). Soil and plant analyses: Soil pH and EC measured (1:5 soil:water suspensions). Exchangeable cations, CEC, available P (Truog method), ammonium-N and nitrate-N (KCl extraction), humic acids per standard protocols. Soil organic carbon (SOC) measured by Walkley–Black before and after cultivation. Total soil N by Kjeldahl. Plant total N determined by combustion (Microcorder MT-5), expressed as g/100 g dry weight and multiplied by DW for total N per plant. Statistics: One-way ANOVA with Tukey’s HSD for treatment comparisons; two-way ANOVA for temperature × treatment effects on DW; Pearson correlations for N input vs. plant DW and total plant N. Significance thresholds: p ≤ 0.05, 0.01, 0.001, 0.0001. Replication: four pots per treatment (n = 4), five plants per pot.

- PB composition: Total N 11.0 ± 0.1% w/w; total C 51.7 ± 0.5% w/w; pH 6.70; P2O5 2.95 ± 0.67% w/w; K2O 0.51 ± 0.06% w/w; C:N ratio 4.7. Estimated protein ~69% (w/w) (N-to-protein 6.25). Free amino acids dominated by Asp, Glu, Lys, Ala, Leu; total amino acids rich in Arg, Asx, Glx, Ala, Val, Gly, Leu. - Dose-range (summer) showed soil EC comparable to mineral control up to PB4; EC increased markedly at PB8–PB32, with significant germination reduction at PB32 (p < 0.0001) and visible growth defects at PB8–PB32. PB1, PB2, PB4 were deemed safe application rates. - Growth performance (temperature-controlled): SPAD values at 28 DAS increased with PB rate (PB4 > PB2 > PB1) under both cool and warm regimes; PB4 significantly higher than PB1 (p = 0.0002 cool; p < 0.0001 warm) and PB2 (p = 0.0252 cool; p < 0.0001 warm), and higher than C2 in warm (p = 0.0297). Maximum leaf length: PB2 ≈ C1; PB4 ≈ C2 across temperatures. Biomass: PB2 FW and DW ≈ C1; PB4 FW and DW ≈ C2 under cool and warm conditions. In warm conditions, DW: PB4 > PB2 (p < 0.0001) > PB1 (p = 0.0071); in cool conditions PB4 > PB2 (p = 0.0023) ≥ PB1. - P and K supplementation to PB1/PB2 did not significantly alter SPAD, leaf length, FW, or DW under cool, warm, or spring conditions, consistent with sufficient soil P and K reserves. - Temperature effects: Single-plant DW tended to be higher in warm vs. cool for all treatments; significant for C1 (p = 0.0457), PB2 (p = 0.0042), PB4 (p = 0.0204). Increases in DW relative to C1: in cool, PB1 −25%, PB2 −1%, PB4 +90%; in warm, PB1 −33%, PB2 +11%, PB4 +71%. - N uptake evidence: Strong positive correlation between N input per pot and single-plant DW across experiments: r = 0.985 (spring, p < 0.0001), r = 0.977 (cool, p < 0.0001), r = 0.966 (warm, p < 0.0001). Total plant N correlated with N input: r = 0.945 (cool, p = 0.0045), r = 0.980 (warm, p = 0.0006). These confirm plant uptake of N from PB. - SOC effects: Percent increases in SOC during cultivation with PB1–PB4 were comparable to mineral fertilizer control C1, suggesting PB did not drastically alter SOC under the tested conditions. - Equivalency to mineral fertilizer: Approximately double the PB-derived N was needed to match plant growth with mineral N (PB2 ≈ C1; PB4 ≈ C2), aligning with an estimated PB N mineralization rate of ~62% inferred from its low C:N ratio.

The study demonstrates that lysed and dried biomass of the PNSB Rhodovulum sulfidophilum can supply plant-available nitrogen and achieve komatsuna growth comparable to mineral fertilizers when applied at roughly twice the mineral N rate. This aligns with expectations for slow-release organic N sources, where a fraction of total N is mineralized for plant uptake across the growth period. The low C:N ratio (4.7) of PB favors mineralization, while PB’s protein-rich composition may also contribute organic N forms (amino acids/peptides) that plants can directly absorb, consistent with known amino acid and peptide transporters in roots. PB performed robustly under both cool (15–25 °C) and warm (22–32 °C) regimes, with warm conditions generally enhancing DW irrespective of treatment, possibly reflecting temperature effects on mineralization rates and plant growth. PB did not require supplemental P or K in a PK-abundant soil, and soil EC remained acceptable at PB1–PB4, though excessive PB (≥PB8) increased EC and impaired germination and early growth. Strong correlations between N input and plant DW/total plant N across conditions provide direct evidence for plant uptake of PB-derived N. Environmentally, PB offers a pathway to reduce reliance on synthetic fertilizers and associated GHG emissions, with SOC responses comparable to mineral fertilizer in this study, suggesting limited risk of SOC-driven N2O increases typical of high C:N manures. Overall, PB from PNSB is a promising, potentially lower-footprint, slow-release N fertilizer with added biostimulant and amino acid value.

Lysed and dried biomass (PB) from the marine purple photosynthetic bacterium Rhodovulum sulfidophilum is an effective nitrogen fertilizer for komatsuna. Safe application rates up to fourfold of mineral N caused no germination or growth penalties; PB applied at approximately double the mineral N rate achieved comparable chlorophyll, leaf growth, and biomass to mineral fertilizer under both cool and warm conditions. PB’s favorable composition (11% N, C:N 4.7, ~69% protein) supports mineralization and potential direct amino acid uptake. In PK-abundant soils, PB can be used without P/K supplementation. The approach could reduce environmental impacts of fertilizer use and production, especially if PB is produced autotrophically with CO2 fixation. Future work should include: (1) direct quantification of N mineralization kinetics across soils, moisture, and temperature; (2) isotopic tracing to resolve mineral vs. organic N uptake pathways; (3) multi-crop, multi-soil field trials; (4) optimization of application timing and blends (e.g., with carbonaceous residues) for tailored release; (5) comprehensive life cycle assessment and techno-economic analysis; and (6) evaluation of storage stability, shelf life, and quality control for scaled production.

- The equivalence of PB to mineral fertilizer was inferred from growth responses and correlations, with the PB mineralization rate (~62%) estimated from literature-based C:N relationships rather than directly measured mineralization kinetics. - Experiments were conducted in pots and greenhouses with one soil type; field-scale variability across soils, climates, and management was not assessed. - Differences in irrigation regimes and seasonal light conditions between experiments may have confounded comparisons (e.g., spring vs. cool conditions). - Excessive PB increased soil EC and reduced germination at very high rates (≥PB8), indicating salinity constraints in some contexts. - P and K sufficiency in the test soil means results on PK supplementation may not generalize to PK-deficient soils. - Scale-up challenges were acknowledged: potential contamination risks, energy demands for cultivation, lysis and drying, storage constraints, and cost competitiveness relative to mineral fertilizers; shelf-life and performance variability under different environmental conditions were not fully evaluated. - A full life cycle assessment was not performed; environmental footprint across production, transport, application, and disposal remains to be quantified.