Induction of Auxins Synthesis by Rhodococcus erythropolis IMV Ac-5017 with the Addition of Tryptophan to the Cultivation Medium

Abstract

The ability of surfactant producers to synthesize phytohormones expands the scope of their practical application and provides prospects for the development of microbial preparations with growth-stimulating properties. The possibility to intensify the phytohormone-stimulants synthesis by bacterial strains increases the efficiency of such preparations. Aim. The aim is to research the possibility of extracellular auxin synthesis induction in the presence of tryptophan in the cultivation medium of surfactant producer Rhodococcus erythropolis IMV Ac-5017 and establish the optimal concentration of tryptophan and time of introduction into the medium to ensure maximum synthesis of auxins. Methods. Biochemical, microbiological, biotechnological. Cultivation was performed in the liquid mineral medium using ethanol and waste sunflower oil as substrates. Tryptophan was added to the medium as a 1% solution in an amount of 200 or 300 mg/l at the beginning of the cultivation process or at the end of the exponential growth phase. Phytohormones were isolated by triple extraction with organic solvents from the culture broth supernatant after surfactant extraction. Preliminary purification and concentration of phytohormones was performed by thin layer chromatography. Qualitative and quantitative determination of auxins was performed using high performance liquid chromatography. Results. It was found that regardless of the concentration and time of tryptophan introduction to the culture medium of R. erythropolis IMV Ac-5017 with both substrates, a significant increase (by two to three orders of magnitude) was observed in the amount of synthesized auxins compared to tryptophan-free medium. The highest concentration of auxins (5552–5634 μg/l) was achieved by adding 300 mg/l of tryptophan into the culture medium of R. erythropolis IMV Ac-5017 with ethanol, while without the precursor their amount was only 143 μg/l. In contrast to the cultivation of the strain on culture medium with ethanol, where the synthesis of auxins did not depend on the time of tryptophan introduction, R. erythropolis IMV Ac-5017 formed the maximum amount of auxins when 300 mg/l tryptophan was added to the culture medium with waste oil at the end of the exponential growth phase (2398 μg/l compared to 9.8 μg/l on the medium without tryptophan). As auxin compounds were identified: indole-3-acetic acid, indole-3-carboxylic acid and indole-3-butyric acid. However, the highest amount of indole-3-acetic acid was synthesized, the precursor of which is tryptophan. The synthesis of this auxin (the most common plant auxin) in the presence of 300 mg/l of tryptophan increased more than 40 times on ethanol medium and more than 700 times on medium with waste oil. Induction of auxin synthesis by strain R. erythropolis IMV Ac-5017 correlated with the activity of tryptophan transaminase: when cultured on ethanol without tryptophan, it was 138 nmol·min-1·mg-1 of protein, while cultured in the presence of precursor it was increased by 5.2 times (up to 714 nmol·min-1·mg-1 of protein). The obtained results suggest that indole-3-acetic acid biosynthesis by the strain IMV Ac-5017 occurs due to the formation of indole-3-pyruvate. Conclusions. Thus, it was established the possibility of increasing by two or three orders the amount of synthesized auxins in the case of low concentrations of tryptophan introducing to the culture medium of R. erythropolis IMV Ac-5017 not only with ethanol but also with industrial waste (waste oil). The obtained results can be considered as promising for use of exometabolites of R. erythropolis IMV Ac-5017 with growth-stimulating properties in crop production.

Effect of Tryptophane on Synthesis of Certain Exometabolites by Bacteria of Genus Acinetobacter, Nocardia, and Rhodococcus and Their Properties

“…Later [7,8], we found the possibility of increasing the number of synthesized auxins by two or three orders of magnitude in the case of low concentration of tryptophan (precursor of their biosynthesis) introduced in the culture medium of strains R. erythropolis IMV Ac-5017 and A. calcoaceticus IMV B-7241 not only with ethanol but also with industrial waste (biodiesel production waste or waste oil). Th e obtained results are the basis for increasing the effi ciency of the use of complex preparations with growth-stimulating and antimicrobial properties against phytopathogenic bacteria in crop production.…”

“…Extracellular phytohormones auxins were isolated by the method of redistribution of phytohormones in two phases of solvents that do not mix with each other, as described earlier [7,8]. Ethyl acetate, pH 3.0, was used as an organic solvent.…”

“…Th e qualitative and quantitative compositions of auxins were analyzed by high-performance liquid chromatography (HPLC) using an Agilent 1200 liquid chromatograph (Agilent Technologies, USA) and a mass spectrometry (MS) detector Agilent G1956B. HPLC/MS analysis of auxin extracts of N. vaccinii IMV B-7405 was performed at the Center for Collective Use at the ZIMV of NAS of Ukraine as described in previous works [7,8].…”