Phenol Biodegradation by some Bacterial Species Isolated from Oil Contaminated Environments

Phenols are poisonous chemical molecules that affect human and other mammals as well as the aquatic environment, particularly higher species in fresh water environments. useing microorganisms through bioremediation is an efficient technique for reducing dangerous chemicals and detoxification of soil contaminated with hydrocarbons. The aim of this study is to investigate the efficiency of some local bacterial species to degrade phenol. Bacterial species were isolated from soils contaminated with hydrocarbons from Al-Diwaniyah and Al-Basrah governorates. The remaining concentration of phenol was examined usingthe 4-aminoantipyrine assay. The results showed that the rate of decomposition of Enterobacter cloacae complex was 100% after an incubation period of 120h, pantoea spp was 100% after an incubation period of 120h, Acinetobacter lwoffii was 100% after an incubation period of 144h, Aeromonas salmonicida was 100% after an incubation period 168h, Escherichia hermannii was 100% after an incubation period of 168h, Sphingomonas paucimobilis was 100% after an incubation period of 168h, Streptococcus thoraltensis was 100% after an incubation period of 168h, Bacillus licheniformis was 97.98%after an incubation period of 168h, Bacillus subtilis was 95.91% after an incubation period of 168h, and Bacillus firmus was 70.91% after an incubation period of 168h. The tested bacterial strains can offer a potential basis for bioremediation of phenol polluted locations.


Introduction
The environmental protection agency has listed phenol as a priority pollutant [1,2].Phenol is an atomic organic compound that is dangerous to humans and mammals and disturbs the environment, especially higher freshwater organisms as a result of improper treatment by various industrial waste.In the production of resin, oil, petrochemicals, medicines, and leather processing [3], phenols are used.Before being released into the environment, phenol-containing soil from these companies may not have received the required treatment.Ground water, surface water, and soil are all very concentrated [4].Adsorption, extraction, electrodialysis, and ozonation are few techniques that have been used for phenol removal from soils.However, biological approaches have obtained more consideration than physical and chemical means as numerous diverse bacteria are acknowledged to consume phenolic composites as the single basis of carbon and vitality [5].The decomposition of 1259 (2023) 012038 IOP Publishing doi:10.1088/1755-1315/1259/1/012038 2 phenol by bacteria has been investigated.Many bacterial species have been reported to be able to degrade phenols such as Acinetobacter baumanni [6], and Klebsiella oxytoca [7].In bacterial aerobic catabolism of phenolic compounds, single hydroxyl group is added to the aromatic ring of the phenol to create catechol by phenol hydroxylase.The catechol then is degraded either by catechol 1,2dioxygenase or catechol 2,3dioxygenase [8,9].Bioremediation methods that use indigenous microorganisms reflected a major ecofriendly alternative way to the removal of toxic compounds such as phenols [10,11].Therefore, the aim of the current study is is to examine the efficacy of certain bacterial strains isolated from locally contaminated environments to degrade phenol.

Phenol Degradation Assay
In 100ml of liquid mineral salts medium with phenol added at various concentrations of 100-500-1000 mg/L , phenol removal studies were conducted in flasks with a capacity of 250ml.Isolated colonies were grown in LB medium for 24h at 30c in a shaker incubator.A volume of 50μL of the culture was taken and added to the medium of liquid mineral salts, to which phenol was added at various concentrations.The mixture was then incubated for 168h at 30c and a PH of 7 using the same conditions.4-aminoantipyrine assay was used to test the ability of the bacterial isolates to degrade phenol.In order to calculate the amount of dissolved phenol present in the liquid medium, samples were obtained from 0 hours to 168h .OD460 manometers of wavelength were used to evaluate absorbance .The of phenol degradation percentage was obtained based on the following equation [12].% Degradation = [(Absorbance at 0 h-Absorbance after incubation)/ (Absorbance at 0h) ]*100

Amplification of Phenol Hydroxylalase Gene
PCR reactions were done using the primers listed in table (1).The DNA fragments of phenol hydroxylalase gene were produced using DNA Polymerase of Go Taq ® G2 Green Master Mix kit, 2X.PCR was carried out in a reaction mixture that contains 25 μL of Go Taq ® G2 Green Master Mix kit, 2X, 0.5 μM for each of the primers, 100 ng/ 50 μL of DNA.The final volume of reactions was designated to be 50 μL.The thermal cycler run for PCR was set as: denaturation for 5 minute at 94 °C, then for 1 minute (30 rounds), annealing at 58 for 1 minute, elongation at 72 °C for 1 minute, and last extension at 72 °C for 10 min.The resulting bands were detected by 1.5% of agarose gels.The gels were run using electrophoresis and visualized using a Bio-Rad ChemiDoc MP after soaking with ethidium bromide stain.
Table 1.The sequences of the primers that used to detect phenol hydroxylalase gene [13].

Phenol Degradation Ability by the Tested Bacterial Strains
The results showed the ability of Aeromonas salmonicida to completely degrade phenol, with a degradation rate of (100%) for a concentration of 100 mg/L after an incubation period of 7 days, with an increase in the bacterial growth rate.For a concentration of 500 mg/L, the degradation rate for phenol was 19.25% as shown in figure 1.As for Bacillus subtilis, the results of the decomposition of this bacteria at a concentration of 100 mg/L were 95.91% during the incubation period of 7 days.The ability of Bacillus subtilis to degrade phenol decreased to be 21.71% at a concentration of 500 mg/L as shown in figure 2. For the results of Enterobacter cloacae complex, the decomposition of phenol by this strain was (100%) at a concentration of 100 mg/L for an incubation period of 5 days while for a concentration of 1000 mg/L, the degradation rate was 24.69% as shown in figure 3. The results of the degradation of phenol by Bacillus Firmus were also shown in figure 4, where the degradation rate was 70.91% at a concentration of 100 mg/L, and the degradation rate of phenol at a concentration of 500 mg/L was 21.23% after an incubation period for both concentrations of 7 day.The results of phenol degradation by Escherichia hermannii showed that the degradation rate was 100% at a concentration of 100 mg/L after an incubation period of 7 days, while for a concentration of 1000 mg/L the degradation rate was 27.1% (figure5).Figure 6 shows the results of phenol degradation of Bacillus licheniformis at a concentration of 100 mg/l proved to be 97.98% for a period of 7 days incubation.As for a concentration of 500 mg/l, the phenol degradation rate was 18.86% .The results of the degradation of phenol by the Sphingonas paucimobilis are shown in figure7.At a concentration of 100 mg/L, it was 100% after an incubation period of 7 days, and at a concentration of 1000 mg/L, it was 18.86%.For Acinetobacter lwoffii, the degradation rate of phenol was 100% at a concentration of 100 mg/L after an incubation period of 6 days while it was 24.68 at a concentration of 1000 mg / liter as shown in figure 8.The results of phenol degradation by Pantoea spp at a concentration of 100 mg/L are 100% during the incubation period of 5 days and at a concentration of 1000 is 24.69% (figure9).The results of the decomposition of phenol by Streptococcus thoraltensis are shown in figure 10.It was100% at a concentration of 100 mg/L after an incubation period of 7 days, and 8.94% at a concentration of 1000 mg/L (figure 10).PCR fragments for phenol hydroxylalase gene (444bp) were detected in all of the tested strains as shown in figure 11.In this study, reliance was made on bacterial isolates that are efficient in growth and decomposition of hydrocarbon compounds, and a depiction of the efficiency of each isolate was given.The results of the current study are similar to previous studies [14][15][16][17][18][19].

Figure 3 .
Figure 3.The percentage of phenol degradation by Enterobacter cloacae complex.

Figure 6 .
Figure 6.The percentage of phenol degradation by bacteria Bacillus licheniformis.

Figure 9 .
Figure 9.The percentage of phenol degradation by bacteria Pantoea spp.