Phosphate limitation enhances malic acid production on nitrogen-rich molasses with Ustilago trichophora

Abstract

Background: An important step in replacing petrochemical products with sustainable, cost-effective alternatives is the use of feedstocks other than, e.g., pure glucose in the fermentative production of platform chemicals. Ustilaginaceae offer the advantages of a wide substrate spectrum and naturally produce a versatile range of value-added compounds under nitrogen limitation. A promising candidate is the dicarboxylic acid malic acid, which may be applied as an acidulant in the food industry, a chelating agent in pharmaceuticals, or in biobased polymer production. However, fermentable residue streams from the food and agricultural industry with high nitrogen content, e.g., sugar beet molasses, are unsuited for processes with Ustilaginaceae, as they result in low product yields due to high biomass and low product formation.

Results: This study uncovers challenges in evaluating complex feedstock applicability for microbial production processes, highlighting the role of secondary substrate limitations, internal storage molecules, and incomplete assimilation of these substrates. A microliter-scale screening method with online monitoring of microbial respiration was developed using malic acid production with Ustilago trichophora on molasses as an application example. Investigation into nitrogen, phosphate, sulphate, and magnesium limitations on a defined minimal medium demonstrated successful malic acid production under nitrogen and phosphate limitation. Furthermore, a reduction of nitrogen and phosphate in the elemental composition of U. trichophora was revealed under the respective secondary substrate limitation. These adaptive changes in combination with the intricate metabolic response hinder mathematical prediction of product formation and make the presented screening methodology for complex feedstocks imperative. In the next step, the screening was transferred to a molasses-based complex medium. It was determined that the organism assimilated only 25% and 50% of the elemental nitrogen and phosphorus present in molasses, respectively. Due to the overall low content of bioavailable phosphorus in molasses, the replacement of the state-of-the-art nitrogen limitation was shown to increase malic acid production by 65%.

Conclusion: The identification of phosphate as a superior secondary substrate limitation for enhanced malic acid production opens up new opportunities for the effective utilization of molasses as a more sustainable and cost-effective substrate than, e.g., pure glucose for biobased platform chemical production.

Keywords: Ustilago trichophora; Alternative feedstock; Bioavailability; Bioeconomy; Internal phosphate storage; Malic acid; Molasses; Online monitoring; Phosphate limitation; Platform chemical.

Fig. 1

Malic acid production on molasses, sucrose, fructose, and glucose. A Online data of OTR. For clarity, only every fifth data point over time is represented as a symbol. Lines are drawn through all measuring points. Shadows indicate the minimum and maximum values of biological duplicates. For fructose, only a unicate is shown. B Malic acid concentration and OD₆₀₀. Samples were drawn at the end of the cultivation. Error bars indicate the minimum and maximum values of biological duplicates. Cultivation conditions: U. trichophora, RAMOS device, 250 mL shake flasks, modified Verduyn medium (25 g/L sucrose, 26 g/L glucose, 26 g/L fructose, or molasses equivalent to 25 g/L sucrose), 0.8 g/L NH₄Cl, 0.3 M MES (pH 7.2), T = 30 °C, n = 350 rpm, d₀ = 50 mm, V_L = 20 mL, OD_600,start = 0.1 [–]

Fig. 2

Impact of secondary substrate limitations on fermentations with pure sucrose. Influence of decreasing concentrations of nitrogen, phosphorus, and magnesium on A–C OTR and D–F malic acid production. A–C For clarity, only every fifth data point over time is represented as a symbol. Lines are drawn through all measuring points. Shadows indicate standard deviation of biological triplicates. D–E Samples were drawn after the sugar was fully consumed, as indicated by a drop in the OTR. Error bars indicate standard deviation of biological triplicates. Corresponding yields and space–time yields are provided in Additional file 1: Figure S5. Cultivation conditions: U. trichophora, µTOM device, 96 round deep well plates, modified Verduyn medium [25 g/L sucrose, 0.3 M MES (pH 7.2)], T = 30 °C, n = 1000 rpm, d₀ = 3 mm, V_L = 100 µL, OD_600,start = 0.1 [–]. A, D 0.5–6 g/L NH₄Cl, 0–6.6 g/L NaCl, 2 g/L KH₂PO₄, 0.4 g/L MgSO₄ B, E 6 g/L NH₄Cl, 0.06–2 g/L KH₂PO₄, 0–1.1 g/L KCl, 0.4 g/L MgSO₄ C, F 6 g/L NH₄Cl, 2 g/L KH₂PO₄, 0.025–0.4 g/L MgSO₄, 0–0.23 g/L Na₂SO₄

Fig. 3

Impact of secondary substrate limitations on fermentations with the complex substrate molasses. Influence of decreasing additional nitrogen, phosphorus, and magnesium concentrations on A, B OTR and C, D malic acid production. A, B For clarity, only every ninth data point over time is represented as a symbol. Lines are drawn through all measuring points. Shadows indicate standard deviation of biological triplicates. C, D Samples were drawn after the sugar was fully consumed, as indicated by a drop in the OTR. Error bars indicate standard deviation of biological triplicates. C Open symbols indicate samples, which were taken from the experiment shown in A. Filled symbols indicate samples, which were taken from an additional experiment investigating different C/N ratios. Corresponding yields and space–time yields are provided in Additional file 1: Figure S7. The respective C/E ratios were calculated based on the total amount of secondary substrate in molasses [18], and the respective additional salts. Cultivation conditions: U. trichophora, µTOM device, 96 round deep well plates, modified Verduyn medium [molasses equivalent to 25 g/L sucrose, 0.3 M MES (pH 7.2)], T = 30 °C, n = 1000 rpm, d₀ = 3 mm, V_L = 100 µL, OD_600,start = 0.1 [–]. A, C 0–6 g/L NH₄Cl, 0–6.6 g/L NaCl, 2 g/L KH₂PO₄, 0.4 g/L MgSO₄ B, D 6 g/L NH₄Cl, 0–2 g/L KH₂PO₄, 0–1.1 g/L KCl, 0.4 g/L MgSO₄

Fig. 4

Comparison of malic acid production under nitrogen and phosphate limitation on sucrose and molasses. Complete data are shown in Fig. 2 and Fig. 3, the cultivation conditions are given in the respective figures. Error bars indicate standard deviation of biological triplicates. Open symbols indicate that sucrose was used as substrate. Filled symbols indicate that molasses was used as substrate and the respective C/N and C/P ratios were calculated based on the total amount of nitrogen and phosphorus in molasses, respectively (Helm et al., 2023), and the respective additional salts. A Half-filled symbols indicate a corrected C/N ratio based on the amount of bioavailable nitrogen, as determined in Additional file 1: Figure S9, and the respective additional salts B Half-filled symbols indicate a corrected C/P ratio based on the amount of bioavailable phosphate, as determined in Additional file 1: Figure S10, and the respective additional salts