Phenol-oxidizing activity and fatty acid Profile of Brevibacillus centrosporus f14 strain

Biotechnological treatment of enterprises wastewater, which contains toxic phenolic compounds, is the most acceptable method today. therefore, the search for new non-pathogenic biochemically active micro organisms - destructors of phenol and their identification is relevant. the aim of this study was to determine phenol-oxidizing activity and the fatty acid profile of Brevibacillus centrosporus F14 – destructor of phenol, isolated from pharmaceutical plant wastewater. Fatty acids analysis of the microorganism culture was car ried out using automatic system of microorganisms identification Midi sherlock based on gas chromato graph agilent 7890. the phenol-oxidizing activity of bacteria was determined by photometric method according to the degree of phenol extraction from the water. it was experimentally confirmed that Brevibacillus sp. F14 strain had a phenol-destructive activity. When phenol-containing water was treated with Brevibacillus sp. F14 cells, phenol concentration in the water decreased from 200.0 ± 12.0 mg/l to 6.8 ± 0.8 mg/l during 15 days of exposure. chromatographic analysis showed that saturated fatty acids i-14:0 (14.9%), i-15:0 (14.8%), a-15:0 (34.9%), i-16:0 (11.10%) dominated in the strain’s fatty acids profile. the total content of the branched saturated fatty acids i-14:0,

G ram-positive bacteria of the genus Brevibacillus are widespread in various habitats [1]. They are characterized by a high growth rate, better transformation efficiency using electroporation, the presence of transport vectors, the production of a small amount of extracellular protease and the constitutive expression of heterologous proteins [1]. Representatives of this genus are of great biotechnological interest -they are a source of various enzymes, antitumor peptides, and are capable of biodegrading polyethylene [1,2]. The authors of [3] indicate that the strain Brevibacillus borstelensis AK1 has the genes for the decom-lected from Textile Industry, Bhaluka, Bangladesh) have the abilities to degrade Reactive Violet 5R. The decolourization rate was different for the different concentrations of the same dye. Brevibacillus centrosporus displayed a decolorization rate of 94.55%, 90.79%, 91.17% when inoculated and incubated in an SM broth containing the azo dye reactive violet 5R at 1% (v/v), 3% (v/v) and 5% (v/v) concentrations respectively for a consecutive 5 days [4].
Fatty acid composition of total cellular lipids as important species and intraspecific chemotaxono mic characteristics correlates with the results of molecular genetics indicators' identification [5,6]. For microorganisms, it is used as the chemotaxonomic feature that allows identification of microorganisms using fatty acid profiles' libraries [7].
It is known that the FAME data have diagnostic value for a few Brevibacillus species [8]. Some differences in the fatty acid profile of six species of the bacteria of the genus Brevibacillus: Brevibacillus agri, Brevibacillus borstelensis, Brevibacillus lateosporus, Brevibacillus parabrevis, Brevibacillus choshinensis and Brevibacillus invocatus, are presented in [8]. For the last two species, the mean of the ratio a-15:0 versus i-15:0 was above 3.0, while for the other four species it was, at most, 1.0. Furthermore, Brevibacillus borstelensis and Brevibacillus parabrevis could be discriminated from these other species by having much lower amounts of i-14:0 (below 4%). Finally, Brevibacillus laterosporus showed less than 1% of 16:1 w7c alcohol while all others had at least 2% or more of this component [8].
Thus, сlosely related microorganisms are clearly distinguished by the presence and percentage of fatty acids in the composition of their cellular lipids. Particular attention is paid to the search for new nonpathogenic microorganisms intended for the purification of wastewater from the production of pharmaceuticals, medical institutions with a predominant content of toxic phenolic and other cyclic aromatic compounds in them [9].
The aim of this study was to determine the phenoloxidizing activity and the fatty acid profile of Brevibacillus centrosporus F14 -destructor of phenol, isolated from wastewater pharmaceutical plant.

Materials and Methods
The object of the study was the bacterial strain Brevibacillus sp. F14 isolated in 2017 (the southern region of Ukraine) from the wastewater of a pharmaceutical plant.
Bacteria were cultured on Tryptic soy agar (Merck, Germany) at 28 ± 1 °C for 24 h. Cellular fatty acids (FAs) were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification system (version 6.21; MIDI) and FAs methyl esters profile was determined by gas chromatography by referencing the STBA6 Microbial Identification System (MIS) standard library [10].
To analyze the composition of cellular lipids, one complete loop of wet biomass was placed in glass vials for further chemical lysis of cells and saponification of the lipids of the test organism. Saponification was performed by adding 50% methanol and 3.7 M NaOH.
The prepared sample was kept for 30 min at 95-100 °C. Fatty acid methylation was carried out by heating the reaction mixture at 80 ºC for 10 min after adding a solution of acidic methanol. Extracted methyl esters of fatty acids were neutralized with a 0.3 M solution of NaOH [11].
Samples of 2 ml volume were transferred into the evaporator in the mode split with a coefficient 40:1, evaporator's temperature -250 °C. The separation was carried out in temperature programming mode -initial temperature of 170 °C with a gradient of 5 °C/min to 270 °C.
Chromatographic separation of methyl FA esters was conducted at Agilent 7890 gas chromatograph (Agilent Technologies, USA) with a capillary column ULTRA 2 and flame ionization detector. Fatty acid content was expressed as a percentage to the total sum of peaks' squares.
Evaluation of the phenol-oxidizing activity of free bacterial cells of Brevibacillus sp. F14 was carried out according to the degree of extraction of phenol from water (α,%), which was calculated by the equation: where C 0 and С are concentrations of phenol in water before (200 mg/l) and after treatment.
The concentration of phenol in the control and experimental samples before and after treatment was determined by a photocolorimetric method based on the formation of colored phenol compounds with 4-aminoantipyrine in the presence of potassium hexacyanoferrate (III) at pH 10.0 ± 0.2 [12].
The significance of differences between the mean values of the residual concentration of phenol in water was determined by the Student's criterion at a significance level of at least 95% (p ≤ 0.05). Five series of tests (n = 5) were conducted.
Data processing was carried out using the program Microsoft Office Excel (2003).

results and discussion
Preliminary studies have shown that, on the basis of the morphological, molecular-biological and biochemical properties of the strain F14, identified as Brevibacillus sp. F14, had destructive activity against N-cetylpyridinium bromide [9,13].
The results of the evaluation of the phenol-oxidizing ability of the test bacterial culture of Brevibacillus sp. F14 are presented in Table 1. As a result of research, it was found that with a single injection of free cells of the strain of Brevibacillus sp. F14 in the amount of 5.5×10 5 CFU/ml in the water contaminated with phenol on the 8 th day of exposure, the concentration of phenol in water decreased from 200.0 ± 12.0 mg/l to 66.0 ± 4.7 mg/l (the degree of phenol extraction from water is 67%). On the 8 th day of exposure, the degree of excretion of phenol from water increased to 86%, reaching a maximum of 97% for 15 days.
To clarify the species of the test strain Brevibacillus sp. F14 was studied the fatty acid composition of the cell wall of bacteria -destructor of phenol.
The fatty acid composition of the studied phenol-oxidizing strain with a high similarity index (Sim Index = 0.645) was identified as Brevibacillus centrosporus F14. The interpretation of the obtained chromatograms for the fattyacid profile of the strain Brevibacillus centrosporus F14 is presented in Table 2. The absence of chemical markers of hydroxy acids in their fatty acid profile indicates the attribution of this strain to gram-positive bacteria. In the spectrum of fatty acids of total cellular lipids of the test strain, no peaks corresponding to the series of 2-and 3-hydroxy acids from the composition of the biomass were found.
As a result of the analysis of the fatty acid profile of the strain Brevibacillus centrosporus F14 was total detected 17 fatty acids with 12 to 18 carbon atoms , mostly branched structure ( Table 2). The total fatty acid content (saturated and unsaturated) of the branched structure was 85% of the total fatty acid pool found on the chromatograms. The 12-methyltetradecanoic acid a-15:0 was dominant, the mole fraction of which was 34.89%. The main fatty acids of the strain Brevibacillus centrosporus F14 include fatty acids i-14:0 (14.85%), a-15:0 (34.89%), i-15:0 (14.81%), i-16:0 (11.10%), characteristic of bacteria of the genus Brevibacillus [14]. The fatty acid profile of Brevibacillus centrosporus F14 was similar to that of other Brevibacillus species, but differed from them, as were the strains of Brevibacillus limnophilus sp. nov. DSM 6472 (12.40%) [15], Brevibacillus invocatus JCM 12215T (7.9%) [16], with a high content of i16:0 fatty acid. For example, in the fatty acid profile of the strain Brevibacillus agri NBRC 15538T, the i-16:0 fatty acid was recorded in an amount up to 2% [16]. It is important to note that the authors of work [17], when identifying a denitrifying bacterium isolated from the soil of a ginseng field in the province of Pocheon in South Korea, showed that the fatty acid profile of the strain Gsoil 3088T mainly inclu ded i-15:0 (36.3%), i-14:0 (15.0%) and a-15:0 (14.8%). These chemotaxonomic data confirm the belonging of the strain Gsoil 3088T to the genus Brevibacillus.
It should be noted that on the chromatograms of the strain of Brevibacillus centrosporus F14 we studied were not recorded even in trace amounts of fatty acid i-15:1 w9c, isomers of saturated fatty acids of branched structure: i-13:0 and a-13:0 anteiso, which are characteristic of pathogenic bacteria Brevibacillus agri NBRC 15538T, Brevibacillus invocatus JCM 12215T, causing waterborne diseases [16].