Optimization of a food industry-waste-based medium for the production of the plant growth promoting microorganism Pseudomonas oryzihabitans PGP01 based on agro-food industries by-products

Abstract

In this study, three wastes based on potato peels and pulps, tomato seeds and wheat bran were used as basis for the preparation of a cheap medium to produce the bacterium P. oryzihabitans PGP01. In flasks experiments, P. oryzihabitans PGP01 growth at 25 °C in a medium based on frozen potato peels and pulp (FPP) with tryptone as a nitrogen source resulted in the maximum production compared to the commercial TSB medium. In the scale-up to 2 L bioreactors, FPP supplemented with tryptone, molasses, NaCl and K₂HPO₄ allowed to reach similar biomass production than in the TSB medium. A maximum growth of 4.4 × 10⁹ CFU mL^-1 after setting the agitation and the air flux conditions at 400 rpm and 0.75 vvm. Finally, P. oryzihabitans PGP01 growing in this optimized medium conserved its biological activity showing the expected effect in root development previously reported for this microorganism.

Keywords: Agro-food by-products; In vitro micropropagated plants; Low-cost production; Molasses; Plant growth-promoting microorganisms.

Fig. 1

- Growth in flasks of P. oryzihabitans PGP01 after 24 h (dark bars) and 48 h (grey bars) at different temperatures in the commercial medium tryptone soy broth (TSB) (A) and using media based on tomato seeds after flotation (TS), frozen potato peels and pulps (FPP) and wheat bran (WB) as agro-food wastes at 25°C (B). Media based on by-products were prepared by dissolving TS, FPP and WB at a concentration of 100, 300 and 20 g L⁻¹ in 50 mL of distilled water using 250 mL conical flasks. Initial pH of all media based on agro-food industry wastes were adjusted to 7 by the addition of K₂HPO₄ 0.2 M, and the growth of the microorganism P. oryzihabitans PGP01 in the different media was compared to the growth of the microorganism in the commercial medium TSB. Data represents mean ± Standard Error (SE) of three independent replicates. In both cases, different lower case letters represents significant differences amongst treatments at 24 h of culture, and different capital letters indicate significant differences amongst treatments at 48 h of culture according to Tukey HSD test (P < 0.05).

Fig. 2

- Growth in flasks of P. oryzihabitans PGP01 after 24 (A) and 48 h (B) using potato wastes (FPP = Frozen potato peels and pulps) supplemented with several nitrogen sources at low () and high () concentration. P. oryzihabitans PGP01 cells were cultured at 25°C and 150 rpm. PEP = Peptone, PS = PROSTAR 510A, YE = Yeast extract, ME = Meat extract, PP = Pea protein, MP = Maize protein, TRP = Tryptone. Initial pH of all media were adjusted to 7 by the addition of K₂HPO₄ 0.2 M. Data represent mean ± SE of three independent replicates, and different letters indicate significant differences between TSB and different media according to Tukey HSD test (P < 0.05).

Fig. 3

- P. oryzihabitans PGP01 growing in the commercial medium tryptone soy broth (TSB) () and in media based on potato wastes (FPP) supplemented with 10 g L⁻¹ () or 20 g L⁻¹ of TRP () in a 2 L bioreactor at 25°C and 400 rpm. Data represent means ± SE of three independent replicates, and asterisks symbols (*) denote significant differences between commercial and by-products optimized media according to Tukey HSD test (P < 0.05).

Fig. 4

- P. oryzihabitans PGP01 growing in the commercial medium tryptone soy broth (TSB) () and in media based on frozen potato peels and pulps and 10 g L⁻¹ of TRP (FPP + TRP10) supplemented with 5 g L⁻¹ of NaCl (), 5 g L⁻¹ of NaCl, 5 g L⁻¹ of GLU () and 5 g L⁻¹ of NaCl, 10 g L⁻¹ of MOL () at 25°C and 400 rpm. TSB Data represents the mean ± SE of three independent replicates. Asterisks (*) symbols within each sampling period denote significant differences between TSB and optimized media according to Tukey HSD test (P < 0.05).

Fig. 5

– Bacterial growth curve (log CFU mL⁻¹) () and pH () evolution of growth curve in a 2-L bioreactor of the bacterium P. oryzihabitans PGP01 in the optimized-wastes-medium based on frozen peels and pulps (FPP*: 300 g L⁻¹ FPP, 10 g L^− 1 TRP, 10 g L⁻¹ MOL and 5 g L⁻¹ NaCl) at 25°C, 400 rpm and 0.75 vvm of oxygen flux. Initial pH of the culture medium was adjusted to 7 by the addition of K₂HPO₄ 0.2 M. Each value of the log (CFU mL⁻¹) of the curve represents the mean ± SE of at least three replicates.

Fig. 6

- Effect of the bacterium P. oryzihabitans PGP01 in the number of roots (A1), root/shoot fresh weight (FW) (A2) and root/plant FW ratio (A3) as well as in root morphology (B) of in vitro rooted Rootpac® 20 plantlets when growing in the absence of microorganism (control) (B1), or in the presence of P. oryzihabitans PGP01 growing in the commercial medium tryptone soy broth (TSB) (B2) or in the optimized potato wastes-based-medium FPP* (300 g L⁻¹ FPP + 10 g L⁻¹ TRP + 10 g L⁻¹ MOL + 5 g L⁻¹ NaCl + 2.5 g L^− 1 K₂HPO₄) (B3). Data represents the mean ± SE of at least ten independent replicates. Different letters indicate significant differences amongst control, commercial and optimized medium according to Tukey HSD test (P < 0.05).