Monoaromatic Compounds Biodegradation by Hydrocarbon-Oxidizing Actinobacteria

Abstract

Monoaromatic substances belong to the most widespread and dangerous environmental pollutants. They are components of wastewater from oil refineries and the coal and chemical industries and are often found in soil, groundwater, and surface water. The use of actinobacteria for bioremediation of natural and industrial objects contaminated by these substances is an environmentally and economically advantageous alternative to physicochemical cleaning methods. Therefore, it is actual to search for new actinobacteria strains capable to assimilate of monoaromatic compounds for their further use in biotechnologies of purification of the environment polluted by these substances. Aim. To determine the ability of hydrocarbon-oxidizing strains of actinobacteria of the Ukrainian Collection of Microorganisms to assimilate monoaromatic compounds − derivatives of benzene and phenol. Methods. The strain cultivation was carried out in a liquid mineral medium at an initial concentration of monoaromatic compounds of 500 mg/L as the only source of carbon and energy. The expression activity of the catA gene encoding catechol 1,2-dioxygenase, the key enzyme at the initial stage of the monoaromatic compounds biodegradation, was assessed under culture growth conditions with phenol and glucose. Relative expression of the catA gene was evaluated using RT-qPCR. Fatty acid methyl esters were obtained by hydrolysis of cells in a 5 % solution of acetyl chloride in methanol, followed by extraction with an ether-hexane mixture. Methyl esters were identified using an Agilent 6800N/5973 inert GC/MS system (Agilent Technologies, US). Fatty acid content was determined by AgilentChemStation software. Results. It was established that the studied strains of actinobacteria belonging to the species Dietzia maris, Gordonia rubripertincta, Rhodococcus aetherivorans, and Rhodococcus erythropolis differ in their ability to assimilate monocyclic aromatic compounds − benzene and phenol derivatives. Most strains grew better in media with benzene and its derivatives. All strains well grew on a mixture of ethylbenzene, ortho-, meta-, and para-xylene (EX), most of them grew with different intensities on a mixture of benzene, toluene, and ortho-xylene (BTX), and on monosubstrates – benzene, benzotriazole, and benzoate. Toluene was used by 50 % of the studied strains, and only one of them (R. aetherivorans UCM Ac-602) grew on ortho-xylene. A smaller amount of strains grew in media with phenol derivatives. They did not assimilate ortho-cresol and hydroquinone but grew with different intensities on phenol, paranitrophenol, resorcinol, and catechol. Only a few strains grew in the presence of meta- and para-cresols. The widest range of monoaromatic substances was used by R. aetherivorans UCM Ac-602. It was shown that in the process of phenol assimilation by strain R. aetherivorans UCM Ac-602, the level of transcriptional activity of the catA gene encoding the key enzyme of an ortho-pathway of aromatic ring cleavage, catechol 1,2-dioxygenase, increased almost 2-fold after 48 h compared to 24 h of strain growth on this substrate and 3-fold compared to glucose. The obtained data indicate the ability of R. aetherivorans UCM Ac-602 strains to assimilate monoaromatic substances through theortho-cleavage way. During growth on benzene, toluene, BTX and EX substances, the fatty acid pool of cells of this strain was dominated by hexadecanoic C16:0 (32.73-39.81 %), hexadecenoic C16:1 cis-9 (5.66-15.11 %), and octadecenoic C18:1 cis-9 (7.09-11.83 %) acids, as well as 10-methyloctadecanoic 10-Me-C18:0 (29.88-34.76 %) acid. The composition of fatty acids of cells grown on aromatic compounds, unlike those grown on glucose, contained 2.4-4.0 times less unsaturated C18:1 cis-9 acid and 2.8-3.2 times more methyl-branched acid 10Me-C18:0. Conclusions. Assimilation of monoaromatic compounds − derivatives of benzene and phenol - is a strain-specific characteristic of the actinobacteria species. Using strain R. aetherivorans UCM Ac-602 as an example, it was shown that the assimilation of monoaromatic substances in studied actinobacteria occurs through the ortho-cleavage pathway. One of the adaptation mechanisms of strain R. aetherivorans UCM Ac-602 to the assimilation of these substances is a significant decrease in unsaturated fatty acids and an increase in methyl branched acids, which contributes to an increase in membrane rigidity and cell resistance to the toxic effect of these substrates. The data obtained indicate a high level of adaptation of actinobacteria to the assimilation of monoaromatic compounds and the prospects for their further study to be used in biotechnologies for environmental purification from these pollutants.