Insights into IAA Production by the Halotolerant Bacterium Vreelandella titanicae

Abstract

The excessive use of chemical fertilizers raised concerns regarding environmental sustainability and soil degradation, prompting increasing interest in biofertilizers as eco-friendly alternatives. Among these, a compound that is effective in stimulating root and plant growth is indole-3-acetic acid (IAA). In our study, we evaluated IAA production by the halotolerant bacterium Vreelandella titanicae under different and varying nutritional conditions, such as tryptophan availability, temperature, pH, salinity, etc. The bacterium showed significant IAA production under a broad range of conditions and a dependence on the presence of tryptophan for IAA biosynthesis. High salinity (1.0 M NaCl), slightly alkaline pH (8.0–9.0), and temperatures of 34 °C increased IAA production, while optimal growth occurred in the absence of NaCl at a range of temperatures of 25–28 °C, suggesting a stress-responsive regulation of its biosynthesis. Easily metabolizable carbon sources, such as glucose and mannitol, enhanced IAA yield again, whereas additions of 1.0 g L⁻¹ NH₄NO₃ and KH₂PO₄ in the basal medium, poor in these salts, inhibited both the growth of the bacterium and IAA production. Notably, V. titanicae produced relevant amounts of IAA in seawater (24.57 ± 11.28 μg⋅mL⁻¹) when used as growth medium and dairy whey (15.68 ± 2.42 μg⋅mL⁻¹), highlighting its suitability for low-cost and circular bioprocessing strategies. In conclusion, V. titanicae is a promising Plant Growth-Promoting Rhizobacterium (PGPR) candidate for sustainable IAA production and potential application in saline or marginal agricultural soils. Its ability to synthesize IAA in different growth media could allow its exploitation in environmentally friendly bioprocesses.

1. Introduction

Chemical fertilizers have been widely used in agriculture to enhance crop productivity since the beginning of the green revolution during the seventies of the last century [1]. Nevertheless, their intensive and repeated application over time has negatively affected soil quality, leading to acidification and accumulation of toxic compounds, accelerating its degradation [2]. To address these issues, it is essential to limit the use of chemical fertilizers and promote alternative and sustainable strategies. About those, biofertilizers represent an ecological solution.

Several biofertilizers are microbial inoculants that improve plant growth by mobilizing essential nutrients in the soil, stimulating plant physiological processes, and reducing stress [3]. Plant Growth-Promoting Rhizobacteria (PGPR) colonize the rhizosphere and influence plant growth and its health through the production of phytohormones, regulating hormonal balance and undergoing different environmental conditions [4].

Indole-3-acetic acid (IAA) is a major auxin, a class of phytohormone essential for plant development. In fact, IAA controls various developmental processes, such as cell division, differentiation, root elongation, stress tolerance, etc. [5]. Many microorganisms living in tight association with plants are able to synthesize this hormone that is able to promote rhizosphere colonization and increase nutrient and water uptake, consequently favoring both biomass and crop yields [6,7,8].

Bacterial IAA biosynthesis is primarily tryptophan-dependent, proceeding through several pathways, such as via indole-3-pyruvate (IPyA), indole-3-acetamide (IAM), tryptamine (TAM), and indole-3-acetonitrile (IAN) [9]. Flux through these pathways is sensitive to precursor availability (L-tryptophan), carbon and nitrogen sources, and environmental conditions (pH, temperature, salinity) [10]. Accumulating evidences suggest that IAA production in the plant-associated microorganisms (e.g., PGPR) may act as a stress-responsive trait, being enhanced under suboptimal conditions such as temperature, pH, nutrient limitation, or osmotic stress [11]. For many PGPR, this stress-induced auxin biosynthesis facilitates plant–microbe interactions and may decouple biomass accumulation from hormone production [12]. Consequently, medium composition and process parameters can decouple biomass accumulation from auxin synthesis, adopting specific and elective conditions useful for IAA production [13].

Generally, IAA industrial production is obtained through chemical synthesis, even using toxic solvents. These industrial processes allow for obtaining significant amounts of IAA; however, they are economically expensive and environmentally unsafe, limiting their feasibility for large-scale agricultural use. In this regard, a sustainable alternative could be the IAA biosynthesis by means of selected PGPR strains for this purpose, especially the halotolerant ones, offering additional advantages for biotechnological applications [14].

These microorganisms tolerate high salt concentration and, in some cases, they also tolerate a wide range of pH, growing in environments that are often unsuitable [15]. About that, the growth in saline media allows the use of marine water or industrial brines, thus reducing the freshwater demand. However, although there is increasing interest in halotolerant PGPR as biofertilizers, the optimization of IAA production in these bacteria is still quite limited. In particular, the combined effects of salinity, nutrient availability, and growth conditions regarding its production remain poorly studied and understood, as well as the use of low-cost and alternative culture media.

Therefore, they represent interesting candidates for the sustainable production of large amounts of IAA. In our previous study, we demonstrated that Vreelandella titanicae, a Gram-negative and moderately halophilic bacterium, first isolated from the Titanic wreck, exhibited several Plant Growth-Promoting (PGP) features, and among them, an abundant IAA production, thus representing the ideal candidate [16,17] for our studies. Its biological characterization showed its ability to grow optimally in a temperature range between 28 and 37 °C, a pH range of 7.0–8.0, and NaCl concentrations between 2 and 8% w/v. To date, IAA production by V. titanicae has been little explored, and optimized growth conditions are far from having been defined. In this context, its physiology related to the halotolerance together with its previously identified PGP traits makes this bacterium an attractive candidate for the development of sustainable and low-input bioprocesses for IAA production. While most studies on PGPR focus on maximizing IAA yield under common growth conditions, its production under stress conditions, in saline environments, or in alternative culture media, as well as that coming from agro-industrial waste, remains little explored.

Therefore, in this study, we investigated the potential of V. titanicae strain QH24 for IAA production by evaluating the effects of nutrients and environmental factors on auxin biosynthesis. Furthermore, its halotolerance physiology was exploited to test low-cost and alternative culture media, including seawater and dairy whey, in a sustainability scenario.

2. Materials and Methods

2.1. Growth Conditions

The V. titanicae strain QH24 (BioSample SAMN51242625, consultable at the National Centre for Biotechnology Information – NCBI database, https://www.ncbi.nlm.nih.gov, 22 December 2025) was cultivated in batch in modified Marine Broth (MB) (in gL⁻¹: MgCl₂⋅6H₂O 5.9, yeast extract 0.1, Na₂SO₄ 3.24, CaCl₂⋅2H₂O 1.8, NaHCO₃ 0.16, KCl 0.55, glucose 1.0, FeSO₄⋅6H₂O 0.001, KBr 0.08, H₃BO₃ 0.022, Na₂HPO₄ 0.008, (NH₄)NO₃ 0.0016, SrCl 0.034; all the reagents are provided by Sigma-Aldrich S.r.l., Milan, Italy), adding 0.5 gL⁻¹ tryptophan, and it was prepared in Milli-Q water and sterilized by autoclaving (121 °C, 20 min). The best tryptophan concentration was determined, as presented below.

Pre-cultures were prepared by inoculating a single colony into 5.0 mL of Marine Broth (MB) and incubating overnight at 28 °C in an orbital shaker (New Brunswick™ Innova^® 43 Shaker, Eppendorf, Inc. – Enfield, CT, USA) at 220 rpm. All the experiments were started by diluting the pre-culture in 50 mL fresh medium to an initial Optical Density (OD₆₀₀) of 0.01 in 250 mL Erlenmeyer flasks and incubating in the above-described conditions. Since these represented the optimal growth conditions for this strain, all the following experiments were consequently conducted. For the assessment of IAA production, each parameter (temperature, pH, carbon source, etc.) was singularly varied, while the remaining conditions were kept constant at their optimal conditions.

2.2. Nutrients and Environmental Factors

IAA production was performed in MB under different nutritional or environmental conditions. The parameters evaluated were the temperatures (22, 25, 28, 31, 34 °C), pH (from 4.0 to 12.0), carbon source (1.0 g·L⁻¹: glucose, mannitol, sucrose, starch), nitrogen and phosphorus (adding 1.0 g·L⁻¹ of NH₄NO₃ or KH₂PO₄ at culture medium), and NaCl (0, 0.5, 1.0 M). Moreover, alternative media were also tested, including Luria–Bertani (LB) (in g⋅L⁻¹: tryptone 10.0, yeast extract 5.0, NaCl 5.0) supplemented with tryptophan 0.5 g·L⁻¹; dairy whey, clarified and sterilized by autoclaving (121 °C, 15 min), either supplemented or not with tryptophan 0.5 g·L⁻¹; and costal Tyrrhenian seawater sampled in the Sapri bay (Italy, 40.072562, 15.628943), sterilized by 0.22 µm filtration and supplemented with both tryptophan (0.5 g·L⁻¹) and glucose (2.0 g·L⁻¹). The QH24 strain, grown either in the LB medium or dairy whey, was incubated for 48 h at 28 °C. In the case of seawater, the strain was incubated for 6 days since it requires longer incubation to allow for proper growth and detectable IAA levels.

2.3. UHPLC-PDA-ESI/MS Setup for IAA Determination

UHPLC-PDA-ESI/MS analysis was employed as a confirmatory method to verify the identity of the compound measured by the Salkowski colorimetric assay, ensuring that the detected product corresponded to IAA. Specifically, UHPLC-PDA-ESI/MS analysis was conducted on two representative culture supernatants (samples cultivated in MB at 28 °C, tryptophan 0.5 g L⁻¹, pH 8.0, NaCl 0 M and 1.0 M) to confirm that the product was truly IAA. Following the IAA confirmation, its quantification for all experimental conditions was carried out using the Salkowski colorimetric assay [18].

The analyses were performed using a Nexera XR UHPLC system (Shimadzu, Kyoto - Japan) equipped with a photodiode array (PDA) detector and coupled to a single-quadrupole mass spectrometer (LCMS-2050, Shimadzu Europe GmbH, Duisburg, Germany). Ionization was achieved using an electrospray ionization (ESI) source operated in positive-ion mode. Chromatographic separation was carried out on a C18 reversed-phase column, and all mass-spectrometric interface parameters were optimized to ensure maximal sensitivity and reproducibility (

Table 1. Instrumental conditions.

UHPLC system: Nexera XR
Column	Shim-pack GIST-HP C18 (100 × 2.1 mm I.D., 2 μm)
Detection	PDA, 190–800 nm
Column temperature	40 °C
Injection volume	5 μL
Mobile phases	A (0.1% formic acid in water): B (methanol)=30:70
Flow rate	0.30 mL/min
Elution mode	Isocratic
Mass spectrometer: LCMS-2050 (ESI positive-ion mode)
Acquisition mode	SIM (selected-ion monitoring)
Interface voltage	2.0 kV
Nebulizing gas	3 L/min
Drying gas	7 L/min
Heating gas	5 L/min
Desolvation line temperature	200 °C
Desolvation temperature	450 °C

). The results obtained are reported in the Supplementary Materials.

2.4. Bacterial Growth and IAA Production Estimation

For all experiments, the bacterial growth was monitored by measuring the OD₆₀₀ at 24 h and 48 h (start of the stationary phase), except in the case of different NaCl concentrations, where the growth was assayed in a kinetic manner. In the case of kinetic curves, the average growth rate (μ) was calculated as reported in Equation (1).

$$\mathsf{\mu }={\displaystyle \frac{{\mathrm{Log}\left({\mathrm{OD}}_{600}\right)}_{t2}-{\mathrm{L}\mathrm{o}\mathrm{g}\left({\mathrm{OD}}_{600}\right)}_{t1}}{\mathrm{Log}\left(2\right)}}$$

(1)

Instead, Salkowski’s method was employed to estimate IAA production under the same conditions described above. Briefly, at the end of the incubation period, the bacterial growth solutions were centrifuged at 6000 rpm for 20 min, and 1.0 mL of supernatant was collected and mixed with 2.0 mL of Salkowski’s reagent (HClO₄ 35% with FeCl₃ 0.01 M). Each sample was incubated for 2 h in the dark at room temperature, and then their absorbances were estimated spectrophotometrically at 530 nm.

Moreover, the IAA production rate across different intervals of time (7–9; 9–15; 15–25; 25–35; and 35–48 h) was calculated by subtracting the IAA production of the previous interval. Finally, we defined the average biomass-specific production as the ratio between IAA production and biomass at 24 and 48 h.

2.5. Economic Assessment

A screening economic assessment was conducted to provide an evaluation of the economic feasibility of IAA bioproduction and the limits of the processes. For the cost analysis, we used a simplified approach, considering only the costs of raw materials (culture medium, glucose, L-tryptophan) and electricity consumption. We have not considered the costs related to maintenance, labor, transportation, purification of the final product, etc.

First of all, the costs of the MB medium from various suppliers (Merck, Thermo Fisher, BD Difco, Condalab) were evaluated, and the cheapest one was chosen (Condalab, 180 EUR kg⁻¹, 6.75 EUR L⁻¹). Industrial feed-grade prices were used for L-tryptophan (~ 8 EUR kg⁻¹) and glucose (~ 0.5 EUR kg⁻¹) based on publicly available market data (https://www.imarcgroup.com/tryptophan-pricing-report, accessed 8 January 2026; international trade statistics for glucose (HS 170230)). Electricity consumption was estimated assuming operation at 28 °C with constant agitation at 220 rpm using an average specific power demand reported for stirred-tank bioreactors (0.43 kW m⁻³) [19] and applying the non-household electricity price (0.24 EUR kWh⁻¹).

The analysis was normalized to a functional unit of 1.0 g of IAA produced and was based on the experimental data collected. For each cultivation medium, the required fermentation broth volume for a unit of IAA was calculated from the measured IAA concentration.

2.6. Data Analysis and Statistics

All the experiments were performed with at least three independent biological replicates. Data are presented as mean ± standard deviation (SD), and more details are reported in the attached Supplementary Materials. Statistical analyses were performed using PAST software v. 4.13. Differences between multiple groups were assessed by one-way ANOVA followed by Tukey’s post hoc test for pairwise comparisons. A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Optimal Tryptophan Concentration

Cell growth and IAA production by V. titanicae were assessed at different tryptophan concentrations (0.05–0.50 g L⁻¹) after 24 and 48 h of incubation (Figure 1). At 24 h, cell growth (OD₆₀₀) increased slightly with rising tryptophan concentrations. After 48 h, biomass doubled but remained nearly constant for all tryptophan levels. In both cases, no significant statistical differences were observed with amino acid variation. The average production of IAA showed a clear dependence on tryptophan concentration. At 24 h, IAA levels increased significantly, passing from 2.4 ± 0.38 µg mL⁻¹ with 0.05 g L⁻¹ of the amino acid to 7.0 ± 1.26 µg mL⁻¹ with 0.50 g L⁻¹. At the latter concentration, average production per unit biomass (average biomass-specific production, mg mL⁻¹ OD₆₀₀⁻¹) increased almost threefold, from 4.5± 0.8 to 12.6 ± 1.97 (Figure S1A). After 48 h of growth, at higher biomass values than at 24 h, IAA production was substantially higher, rising statistically significantly from 14.2 ± 0.54 µg mL⁻¹ at 0.05 g L⁻¹ of the amino acid to approximately 21.5 ± 0.39 µg mL⁻¹ at 0.50 g L⁻¹. Biomass-specific production at 48 h increased 1.5-fold, from 13.1 ± 0.2 to 19.4 ± 0.8 mg mL⁻¹ OD₆₀₀⁻¹, respectively. Overall, in this strain, at tested concentrations, tryptophan regulates specifically IAA production and does not affect biomass.

3.2. Effect of Temperature and pH

The strain V. titanicae QH24 grew and produced IAA in the range of 22–34 °C (Figure 2). Optimal growth temperature was in the range 25–28 °C, resulting in higher biomass production (OD₆₀₀ 0.63 ± 0.002 and 0.68 ± 0.001 OD₆₀₀ at 24 h, and OD₆₀₀ 1.17 ± 0.002 and 1.12 ± 0.001 OD₆₀₀ at 48 h, 25 °C and 28 °C, respectively) (p < 0.05).

At 24 h, the average amount of IAA recorded in the liquid media reached its highest values in the range of 25–34 °C, and then it remained constant (on average, 10.25 mg mL⁻¹). However, the average biomass-specific production increased steadily from 22 °C to 34 °C (from 5.3 ± 0.5 to 27.2 ± 9.6 mg mL⁻¹ OD₆₀₀⁻¹; Figure S1B). At 48 h, both the IAA production in the liquid media and the biomass-specific production resulted in higher and increased steadily with the temperature increase. For both, maximum values were reached in the range of 31–34 °C (39.3 ± 2.4 and 40.8 ± 1.8 mg mL⁻¹, and 39.3 ± 2.5 and 62.7 ± 6.7 mg mL⁻¹ OD₆₀₀⁻¹, at 31 and 34 °C, respectively, as shown in Figure S1B and Figure 2). At the optimal growth temperature range, the highest values of the average production and biomass-specific production were reached at 28 °C (29.7 ± 3.4 mg mL⁻¹ and 26.5 ± 3.7 mg mL⁻¹ OD₆₀₀⁻¹); however, they resulted in lower values of 1.4 and 2.4 times, respectively, than those obtained at 34 °C.

The pH influenced the bacterial growth and IAA production; unlike tryptophan and temperature, a greater correspondence between biomass and IAA amount (Figure 3) occurred. The bacterial strain was able to grow and produce IAA over a wide range of pH (5.0 < pH < 12). Optimal growth was achieved in the range between 6.0 and 9.0, with a slight increase for both biomass and hormone at alkaline values (8.0–9.0). In pH range 6.0–9.0, at 48 h of growth, the highest IAA production was obtained at pH 9.0 (33.22 ± 1.06 μg⋅mL⁻¹) (p < 0.05), while biomass-specific production underwent slight variation (21.2 ± 4.6 mg mL⁻¹ OD₆₀₀⁻¹ at pH 6.0, and 26.8 ± 1.3 mg mL⁻¹ OD₆₀₀⁻¹ at pH 9.0; Figure S1C), with a mean value of 24.6 mg mL⁻¹ OD₆₀₀⁻¹. Thus, pH regulated both biomass and IAA production similarly, and in the range of 6.0–9.0, its variation had little effect on either.

3.3. Effect of Carbon and Inorganic Nitrogen and Phosphorus Sources

Different carbon sources showed variable effects on bacterial growth and the amount of IAA (Figure 4). Glucose addition to culture media favored the highest biomass (1.12 ± 0.01 OD₆₀₀) and IAA production (28.86 ± 0.28 μg⋅mL⁻¹) after 48 h, followed by mannitol (0.90 ± 0.04 OD₆₀₀ and 14.6 ± 1.1 μg⋅mL⁻¹). Starch had a higher negative effect on biomass (0.63 ± 0.01 OD₆₀₀, 13.1 ± 0.6 μg⋅mL⁻¹), whereas sucrose reduced the biomass only slightly but had more significant effects on IAA production (0.97 ± 0.03 OD₆₀₀, 9.8 ± 0.5 μg⋅mL⁻¹) (p < 0.05). Biomass-specific production at 48 h was highest in the presence of glucose (25.7 ± 0.3 mg mL⁻¹ OD₆₀₀⁻¹), followed by starch (20.8 ± 1.0 mg mL⁻¹ OD₆₀₀⁻¹), mannitol (16.3 ± 1.4 mg mL⁻¹ OD₆₀₀⁻¹), and sucrose (10.6 ± 0.6 mg mL⁻¹ OD₆₀₀⁻¹; Figure S1D). The increase in nitrogen and phosphorus amounts in the MB culture medium (poor in these nutrients in its basic formulation) also influenced the growth and IAA production, both at 24h and 48 h. Supplementation with NH₄NO₃ and KH₂PO₄ at concentrations comparable to those commonly used in mineral medium (1.0 g L⁻¹) negatively affected bacterial growth and IAA production (Figure 5). Specifically, after 48 h, NH₄NO₃ reduced biomass and average hormone concentration by 34% and 82%, respectively, while KH₂PO₄ supplementation resulted in reductions of 68% and 35%, respectively. Average production for unit biomass (Figure S1E) in the medium without or with the addition of NH₄NO₃ was 19.6 ± 0.3 mg mL⁻¹ OD₆₀₀⁻¹ and 5.2 ± 2.7 mg mL⁻¹ OD₆₀₀⁻¹, respectively, while with the supplementation of KH₂PO₄, it was 39.3 ± 3.7 mg mL⁻¹ OD₆₀₀⁻¹, which is a twofold increase when compared to the one obtained with the MB culture medium. Our data showed that NH₄NO₃ and KH₂PO₄ influenced IAA production and growth differently: NH₄NO₃ strongly inhibited the cellular biosynthesis of the hormone, while KH₂PO₄ had an activating effect. Furthermore, both exerted an inhibitory effect on growth, which was particularly pronounced in the case of KH₂PO₄ supplementation. The latter effect could explain the reduction in IAA production (μg mL⁻¹) observed with phosphorus supplementation.

3.4. Effect of NaCl Concentration

The kinetics of growth and IAA production by V. titanicae were markedly affected by the salinity of the culture media (Figure 6A,B). The lag phase in the absence of NaCl was less than 1.0 h and increased significantly with values of less than 7.0 h and greater than 7.0 h in the presence of 0.5 M and 1.0 M NaCl, respectively. The average growth rate was similar at every NaCl concentration of 0.064 h⁻¹ without NaCl and 0.060 and 0.065 h⁻¹ at 0.5 and 1.0 M NaCl, respectively. The cell density was reduced by salt, and at 48 h of growth (beginning of the stationary phase), the measured values were 1.123 ± 0.001 OD₆₀₀, 0.79 ± 0.01 OD₆₀₀, and 0.65 ± 0.01 OD₆₀₀ at 0.0M, 0.5 M, and 1.0 M NaCl, respectively (30% and 42% of biomass reduction, respectively). For all bacterial culture conditions, IAA production became measurable a few hours after entering the exponential growth phase, in agreement with the salt concentration-dependent lengthening of the lag phase. In the absence of NaCl, IAA was undetectable up to 7 h, and its increase proceeded slowly, reaching 11.0 ± 0.3 μg⋅mL⁻¹ at 24 h and 28.22 ± 0.27 μg⋅mL⁻¹ at 48 h. At 0.5 M NaCl, IAA production became measurable after 9 h, reaching a value of 20.38 ± 0.27 μg⋅mL⁻¹ at 24 h and 37.80 ± 0.64 μg⋅mL⁻¹ at 48 h. Finally, at the concentration 1.0 M NaCl, the hormone was measurable after 10 h, reaching at 24 h a concentration similar to that obtained with 0.5 M NaCl, and, at 48 h, it reached a value of 49.88 ± 0.49 μg⋅mL⁻¹, significantly higher than that obtained under all the previous growth conditions. The highest production per unit biomass was 25.1 ± 0.3 mg mL⁻¹ OD₆₀₀⁻¹ in the absence of NaCl, 47.8 ± 1.1 mg mL⁻¹ OD₆₀₀⁻¹ at 0.5 M, and 73.4 ± 1.2 mg mL⁻¹ OD₆₀₀⁻¹ at 1.0 M (Figure S1F).

Table S1 illustrates the average production rate (mg mL⁻¹ h⁻¹) of IAA in the absence of NaCl and under the two different NaCl concentrations, across the indicated intervals of time. At the early stage (7–9 h), the production rate was negligible under all growth conditions. Between 9 and 15 h, an increase was observed, with slightly higher values in the absence of salt. From 15 to 25 h, a sharp rise was recorded in the presence of NaCl, in particular with 0.5 M NaCl concentration, where the production rate reached its maximum value (1.90 µg mL⁻¹ h⁻¹). Unlike the latter, with 1.0 M NaCl, the production rate continued to increase, reaching higher values than the other culture conditions (2.25 µg mL⁻¹ h⁻¹) in the interval between 25 and 35 h. In the medium in the absence of NaCl, the increase in the IAA production rate was more gradual and lower than in the other two growth conditions, reaching a maximum of 0.83 µg mL⁻¹ h⁻¹ (at 35–48 h). Overall, as expected for a halotolerant bacterium, the NaCl increase reduced growth and biomass; however, in V. titanicae strain QH24, the IAA biosynthetic pathway was strongly stimulated to the point that, despite the negative effect on growth, its production increased.

3.5. Alternative Growth Media

Table 2. Effect on growth and IAA production in V. titanicae by alternative culture media (LB and dairy whey after 48 h of incubation, while in the case of seawater after 144 h of incubation).

Medium	pH	E.C. (uS⋅cm−2)	OD600	IAA (μg⋅mL−1)
LB (Lennox) *	7.2	9000	1.71 ± 0.01	3.26 ± 0.92
Dairy whey	4.94	10,870	0.05 ± 0.01	11.30 ± 4.26
Dairy whey *	4.94	10,870	0.10 ± 0.04	15.68 ± 2.42
Seawater **	7.2	68,000	0.13 ± 0.01	24.57 ± 11.28

* With tryptophan 0.5 g⋅L⁻¹; ** with tryptophan 0.5 g⋅L⁻¹ and glucose 2.0 g⋅L^−1.

illustrates alternative, cost-effective media evaluated for IAA production by means of V. titanicae, including dairy whey, natural seawater, and LB (Lennox) formulated with minimal supplementation to reduce input costs and leverage waste-derived or saline resources. LB (Lennox) supplemented with tryptophan supported the highest cell growth (OD₆₀₀ = 1.71 ± 0.01) but the lowest IAA production (3.26 ± 0.92 μg⋅mL⁻¹). Dairy whey sustained minimal growth (OD₆₀₀ = 0.05 ± 0.01) while producing higher IAA (11.30 ± 4.26 μg⋅mL⁻¹), which increased with tryptophan supplementation (OD₆₀₀ = 0.10 ± 0.04; IAA = 15.68 ± 2.42 μg⋅mL⁻¹). Finally, natural seawater supplemented with glucose and tryptophan showed a limited biomass (OD₆₀₀ = 0.13 ± 0.01) but the highest IAA levels among the tested media (24.57 ± 11.28 μg⋅mL⁻¹). These data indicate that a saline environment with precursor supplementation and a carbon source can favor IAA biosynthesis.

3.6. Economic Assessment

An economic assessment of the IAA production cost was conducted to evaluate the process’s feasibility and its limits (

Table 3. Screening economic assessment of IAA production.

Medium	IAA Yield (mg L−1)	Incubation Time (h)	Medium Requirement (L) for 1.0 g IAA	Medium Cost (EUR g−1)	Trp Cost (EUR g−1)	Glucose Cost (EUR g−1)	Energy Cost (EUR g−1)	Total Cost (EUR g−1 IAA)
Marine Broth (MB)	49.88	48	20.05	135.32	0.07	0.01	0.10	135.50
Dairy whey	11.30	48	88.50	0	0	0	0.43	0.43
Dairy whey *	15.68	48	63.78	0	0.23	0	0.31	0.54
Seawater **	24.57	144	40.70	0	0.15	0.03	0.59	0.78

* With tryptophan 0.5 g⋅L⁻¹; ** with tryptophan 0.5 g⋅L⁻¹ and glucose 2.0 g⋅L^−1.

). The best IAA production was recorded in the case of the MB medium, but the estimated cost exceeded 130 EUR g⁻¹ IAA due to the high cost of this culture medium. For the seawater, the estimated cost was approximately 0.8 EUR g⁻¹ IAA, while the cultivation in dairy whey supplemented with L-tryptophan resulted in a cost of ~0.5 EUR g⁻¹ IAA. Finally, the dairy whey in the absence of precursor supplementation showed the lowest estimated cost (0.4 EUR g⁻¹ IAA). The LB medium was not included in the cost assessment because of the very limited IAA production.

4. Discussion

The excessive use of chemical fertilizers and pesticides leaded and lead, also at present, to several environmental problems, directly affecting crop productivity and human health [20]. The use of PGPB and/or their useful by-product as biofertilizers is considered an alternative to traditional and not eco-friendly chemical fertilizers. In our study, we investigated either IAA production by V. titanicae under different physical and nutritional conditions or inexpensive culture media, with the aim of highlighting the correlation between IAA production and bacterial growth and identifying cost-effective conditions suitable for an eco-friendly bioprocess.

Microbial IAA biosynthesis occurs mainly through tryptophan-dependent pathways; in fact, the indole-3-pyruvate (IPyA), indole-3-acetamide (IAM), tryptamine (TAM), and indole-3-acetonitrile (IAN) pathways are known and share tryptophan as a common precursor [21]. Although a tryptophan-independent pathway has also been proposed, its actual existence in bacteria has not yet been demonstrated [9]. Both biosynthetic pathways can generate by-products following the accumulation of precursors and their derivatives and the modification/degradation of IAA itself (e.g., indole-3-lactate, indole-3-pyruvate, indole-3-acetonitrile, dioxindole-3-acetic acid, tryptophol, etc.) [6,9]. However, the regulation of their production is highly complex and depends on many factors, such as the bacterial strain and the biosynthetic pathway used, stressors, nutritional imbalance, and physical conditions. In this study, we focused on IAA production, as it is the active hormonal component in plant growth regulation [22,23].

First, we investigated the role of the amino acid tryptophan. We measured IAA production by V. titanicae at increasing tryptophan concentrations, highlighting the best production at 0.25 and 0.50 g L⁻¹. Several studies have reported similar results, showing the best tryptophan concentration between 0.1 and 0.5 g L⁻¹ [24,25,26]. We showed that in V. titanicae, the amino acid addition has a specific effect on production, while at all concentrations tested, the effects on biomass are negligible. This latter aspect highlights that the used medium supports the tryptophan requirement for its metabolism and/or that the microorganism is not limited in its biosynthesis. Therefore, in V. titanicae, the control of tryptophan concentration is essential for specifically improving IAA production efficiency. Different studies have shown a dependence of hormone production on temperature and that optimal temperature conditions for growth and IAA production may not correspond; furthermore, for some microorganisms, higher temperatures can enhance hormone production. Sun et al. (2014) evidenced that in twelve different yeast strains, all with optimal growth at 30 °C, the optimal production varied between 16 °C and 37 °C [27]. Scarcella et al. (2017) demonstrated that in Rhodotorula mucilaginosa and Trichosporon asahiiand, a temperature increase of 5.0 °C above the optimal one for growth delayed the onset of IAA biosynthesis but increased its production, and similar results were also reported for Bacillus altitudinis [28,29]. Therefore, a temperature value that may represent a growth stressor could enhance IAA production. In V. titanicae, we observed that the optimal temperature for growth and hormone production does not correspond; the first one is in the range of 25–28 °C, and the latter is 34 °C. Temperature positively regulates IAA production; however, until the optimal growth temperature is reached, variations in IAA levels and biomass appear positively correlated, while for higher values, they proceed in the opposite direction. At 34 °C and at 48 h, a marked reduction in biomass is observed, but the significant increase in average production per unit biomass (2.4 times) outweighs this reduction, increasing average production levels in the medium by more than 1.4-fold compared to that obtained at 28 °C.

V. titanicae tolerates a wide pH range. Biomass and IAA production appear correlated, and maximum values for both are reached and maintained almost constantly in a range between slightly acidic and slightly alkaline (6.0–9.0). Similar characteristics highlighted the versatility of V. titanicae, making it suitable for cultivation in saline/marine environments. The preference for neutral to slightly alkaline pH by V. titanicae is in accord with the scientific literature. About that, Veerasamy et al. (2021) have shown that different Bacillus spp. maintains or enhances IAA production under both neutral to alkaline conditions (pH 7.0–10.0) [30]. Similarly, Ait Bessai et al. (2022) reported that two Pseudomonas spp. strains can maintain their IAA production constant up to pH 9.0 [31]. In contrast, other research highlighted a severe inhibition in IAA production in different bacterial strains already at pH 7.0–7.5 [25,32]. These differences suggest that alkaline pH can either act as a stimulatory mild stress or as an inhibitory factor for IAA biosynthesis, depending on the physiological adaptability of a specific bacterial strain. In this context, the capability of V. titanicae to sustain IAA production under alkaline conditions reflects an adaptive trait commonly found in halotolerant bacteria [33].

Carbon source plays an important role in energy and biosynthetic metabolism, influencing both growth and production of secondary metabolites. It is well known in the literature that the optimal source for IAA production can vary from one microbial strain to another. Bhutani et al. (2018) showed that some endophytic strains of Bacillus spp. exhibited increased IAA production in the presence of sucrose, others in the presence of mannitol, and still others in the presence of glucose [34]. Bharucha et al. (2013) indicated sucrose as an optimal source for IAA production in Pseudomonas putida strain UBI [25]. In our experiment with V. titanicae, glucose supported the highest production, followed by mannitol. This trend is consistent with the one described by Mohite (2013), who reported that easily metabolizable carbon sources, such as glucose or mannitol, enhance IAA biosynthesis in several rhizosphere bacteria [35]. We also demonstrated that in V. titanicae, different carbon sources can control production by acting to varying degrees on growth and cellular biosynthesis of IAA (biomass-specific production). Starch slightly reduced biomass-specific production compared to glucose, but IAA production in the medium resulted in the lowest compared to other carbon sources tested due to its strong inhibitory effect on biomass production (42% reduction). For sucrose, the reduction in hormone production was almost exclusively due to the strong inhibition of the IAA biosynthetic pathway (66% reduction in biomass-specific production), confirming that the regulation of IAA production is not necessarily correlated with that of biomass.

The addition of inorganic nitrogen and phosphorus negatively affected both growth and average production of IAA, suggesting that nutrient-rich conditions may repress secondary metabolism or trigger catabolic pathways, as reported for other auxin-producing bacteria [36]. Unlike nitrogen, increasing phosphorus led to a 2-fold increase in production per unit biomass; however, due to a strong inhibitory effect on cellular biomass, this did not result in increased production compared to growing conditions without its addition. A relationship between the regulation of phosphate and IAA levels has been documented in some PGPR. It has been reported that adding IAA to the growth medium or tryptophan to induce production increases the ability of these microorganisms to solubilize phosphorus from insoluble complexes, such as Ca₃(PO₄)₂ or rock phosphate, making it available to plants [37,38]. IAA appears to modulate bacterial synthesis of carbamates, which are thought to be primarily responsible for the ability of PGPR to solubilize phosphate. With this mechanism, IAA promotes the absorption of phosphorus by stimulating root development and increasing its availability in the surrounding environment. However, the role of phosphate in IAA regulation has not yet been described, and the existence of a possible positive feedback mechanism in which phosphorus released from stores could increase cellular production of IAA, thus further enhancing its solubilization process, cannot be excluded.

V. titanicae is a moderate halotolerant bacterium and can grow up to ~ 15 gL⁻¹ NaCl [17]. About that, the growth kinetics confirmed the halotolerant nature of V. titanicae and showed the optimal growth when NaCl was not present in the culture medium. Contrarily, an opposite behavior was observed for IAA production, highlighting the higher amount of IAA at 1.0 M NaCl and indicating that osmotic conditions compatible with halophilic physiology may induce auxin biosynthesis, possibly through stress-responsive regulatory systems. Although NaCl generally inhibits IAA production in many bacterial species [39,40], some studies have described the opposite effect. About that, Sharma et al. (2023) observed an increase in IAA production in saline conditions in Virgibacillus halodenitrificans ASH15 (54.79 μg mL⁻¹ without NaCl, 76.19 μg mL⁻¹ at 1.0 M NaCl, and 82.8 μg mL⁻¹ at 2.0 M NaCl) [41]. Moreover, Yousef (2018) observed an increase in IAA production in Bacillus subtilis CW-2 in relation to NaCl amounts added to the culture medium, albeit adopting less severe saline conditions (0.1–0.2 M) [42].

A comparison among V. titanicae and other Halomonas/Vreelandella species (

Table 4. Comparison of IAA production by halotolerant PGPR reported in the literature. “na” indicates data not available.

Bacterial Strain	Culture Medium	L-trp [g L−1]	NaCl	Incubation Time	IAA Quantification Method	IAA [μg mL−1]	Reference
V. titanicae QH24	MB	0.5	1.0 M	48 h	Salkowsky	49.9 ± 0.5	This study *
H. acquamarina	na	na	na	na	Salkowsky	9.0	[43]
H. alkaliantarcticae	Nutrient broth	0.1	1%	48 h	Salkowsky	30.0 ± 1.5	[44]
Halomonas sp. QSLA2	Nutrient agar	10.0	0%	7 days	Salkowsky	15.45 ± 0.91	[45]
H. kashgarensis	LB	na	7.5%	11 days	Salkowsky	57.15	[46]
Halomonas sp. MAN5	Basal medium	2.5	9.0%	7 days	Gordon & Weber	144.5	[47]
B. megaterium S3	LB	0.5	0.3 M	4 days	Gordon & Weber	70.0	[48]
P. fluorescens S1	LB	0.5	0.3 M	4 days	Gordon & Weber	37.0	[48]
A. tumefaciens	N-free NFb	0.5	4.0%	48 h	Salkowsky	16.9 ± 0.1	[49]
B. safensis	LB	1.0	0%	7 days	Salkowsky	116.5 ± 0.1	[50]

* pH = 8; temperature = 28 °C.

), cultivated in the optimal conditions for IAA production, highlighted that our bacterial strain produced a higher amount of IAA (~50 μg mL⁻¹) when compared with Halomonas acquamarina, Halomonas alkaliantarcticae, and Halomonas sp. QSLA2 (~9.0, ~15.0, and ~30.0 μg mL⁻¹, respectively) [43,44,45]. At the same time, other bacterial strains or species, such as Halomonas kashgarensis and Halomonas sp. MAN5, showed higher IAA production (~57.0 and ~130.0 μg mL⁻¹, respectively), but they took more than 7 days of optimal growth conditions to reach such an amount [46,47]. Finally, in Table 4, IAA production in some different PGPR genera is also reported.

In a circular economy and life-cycle perspective, this study highlighted that V. titanicae can be cultivated in alternative and low-cost media, such as seawater or dairy whey, providing viable substitutes for more expensive culture media (as Marine Broth), with the aim of increase resource efficiency and waste valorization. V. titanicae maintained measurable IAA production in both media, supporting high IAA levels despite low biomass. The use of seawater in fermentation processes enables a significant reduction in freshwater consumption and use of inorganic salts, reducing also the economic costs. Dairy whey is an agro-industrial by-product that is typically managed as wastewater that requires treatment before disposal. Its reuse as a substrate for the fermentation process transforms a waste management problem into a new resource, reducing environmental pollution and treatment costs.

Finally,

Table 5. Comparative assessment of IAA production at tested cultivation conditions.

Cultivation Medium	Resource Input	Advantages	Main Limitations
Marine Broth (MB)	Synthetic medium, tryptophan, freshwater	Greater IAA yield (laboratory benchmark)	High medium cost
Dairy whey	Agro-industrial by-product only	Minimal inputs, resource efficiency, minimal costs	Limited IAA yield
Dairy whey *	Agro-industrial by-product, tryptophan	By-product valorization, reduced medium cost	Limited IAA yield
Seawater **	Seawater, glucose, tryptophan	Freshwater saving, no synthetic salt addition	Long cultivation time, lower IAA production

* With tryptophan 0.5 g⋅L⁻¹; ** with tryptophan 0.5 g⋅L⁻¹ and glucose 2.0 g⋅L^−1.

summarizes the cultivation media investigated in this study and highlights the trade-offs between IAA productivity, resource use, and sustainability-related aspects. Specifically, the MB medium has the highest IAA production, but its main limitation is the high cost. In contrast, the other culture media have significantly lower costs, either by recycling waste products from industrial processes, like dairy production, or by reducing the use of freshwater, as is the case with seawater. However, these media are limited by lower yields and, in the case of seawater, also by longer incubation times.

5. Conclusions

Using a batch culture system, we evaluated the effects of various nutrients and physical factors on IAA production by the halotolerant PGPR strain V. titanicae, defining their optimal values and the relationship between production and growth. We found that for pH, organic carbon source, and inorganic nitrogen and phosphorus sources, IAA and biomass production are positively correlated, while for IAA precursor tryptophan, NaCl concentrations, and temperature, they appear independent. In this case, an activation of cellular biosynthesis (biomass-specific production) compensates for the reduction in biomass by increasing its production in the medium.

Overall, V. titanicae is a promising candidate for sustainable IAA production and future development as biofertilizers in the optic of a more sustainable agriculture. The capability of this strain to maintain IAA synthesis under osmotic and thermal stress, together with its capacity to grow in inexpensive media, such as seawater or dairy whey, suggests a good potential for its integration into circular and low-cost bioprocesses. On the other hand, MB enabled higher IAA production, but its application is limited by economic constraints. Contrarily, seawater and dairy whey represent more sustainable cultivation strategies by reducing freshwater consumption and valorizing agro-industrial by-products, but their use currently requires further optimization to improve IAA productivity and process performance.

Therefore, future research should focus on increasing IAA productivity, optimizing the fermentation process in a bioreactor, and setting the best parameters for growth and IAA production. Moreover, testing alternative low-cost culture media and tryptophan sources represents a further opportunity to reduce costs and environmental impacts.