Isolation and Identification of some Phenol Degrading Bacterial Species from some Contaminated Environments

Phenol and its derivatives are toxic substances that harm a variety of living organisms, including plants, animals, and aquatic life. Therefore, in order to save the environment, these substances must be effectively removed. The purpose of this study is to screen and select potent phenol degrading strains from oil contaminated soils as well as testing their capacity to break down phenols. After growing on the phenol-containing mineral salts medium (MM), the chosen strains were identified using biochemical testing by Vitek 2 compact system in addition to morphological identification. After 168 hours of incubation, the chosen strains were identified as Aeromonas salmonicida, Escherichia hermannii, Acinetobacter lwoffii, Enterobacter cloacae complex, Sphingomonas paucimobilis,, and Pantoea spp. All of the selected strains were grown efficiently at a concentration of 100mg/ml, so they may be putative species for bioremediation of phenol contaminated environments.


Introduction
Soil pollution caused by petroleum hydrocarbons is a major global issue that has aroused social concern in recent decades.Individual actions are important aspects that lead to leakage of hydrocarbons.Although petroleum is one of the main sources of energy that sustains the country's profitability and social progress, petroleum products have proven to be significant types of organic pollutants due to massive leaks from destructive storage tanks and accidents during transportation and release [1].As a result, oil contamination occurrences have spread over the world, posing significant threats to the environment through their effects on food supplies and soil chemistry [2].There is a significant emission of organic chemicals into the atmosphere.Many of these substances end up in landfills after being synthesized chemically.Because it is poisonous to human health and other plants, phenol is both a widespread contaminant and one of the most dangerous in the environment [3].Phenol is a liquid or solid with a low melting point but a high boiling point due to the hydrogen in its structure.It is water soluble because it can form hydrogen bonds with water.Phenol complexes are critical to many manufacturing industries.They can be detected in the wastes of many industrial processes, such as oil refineries, industrial plants for food preparation, pharmaceuticals, and the manufacture of polymers and dyes (7) which effectively contain phenolic compounds [4].Physical and chemical processes can be used to clean the environment of any lingering phenolics.However, these methods are unfeasible because of their hefty price tags and the byproducts they produce.Chlorine, for instance, can change phenol into chlorophenol, another hazardous chemical.[5].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 source of carbon.In order to obtain local potential phenol degrading bacterial species and understand their heterogeneity in the soil community, the objective of the current study was screen and select potent phenol degrading strains from oil contaminated soils.

Sample Collection
40 samples were collected from different locations contaminated by petroleum products.The soil was stored in sterilized glass bottles and transferred to a laboratory for testing [6,7].

Growth Media
The selecting process took place in a mineral salt medium (MM).Components include MgSO 4.7 H 2 O (0.02 g), NH4 (2SO 4 ) (0.1 g), NaCl (0.01 g), CaCl 2 (0.01 g), K 2 HPO 4 (0.45), and FeCl 3 (0.002 g).The components were sterilized for 20 minutes at 121 °C and pH 7 after being dissolved in one litter of distilled water and thoroughly mixed to assure solubility.After reaching the correct concentration, phenol was added to the medium as the only carbon source.

Selection of Phenol Tolerant Bacterial Strains
To a volume of (50 mL) liquid mineral salt medium (MM), 5 grams of each soil sample was added.Phenol was added at a starting concentration of 100 mg/l.The flasks were kept in a 30 degree Celsius incubator for 5 days (7).After incubating the bacterial solution at 30°C for 5 days, 0.1 ml of it was plated onto MM agar medium with the same quantity of phenol.This process was repeated twice more to generate pure colonies and verify that the isolated strain could grow on phenol-based media.

Identification of the Phenol Degrading Bacteria
Phenotypic identification on (MM agar, MacConkey agar, and(or) blood agar) and Vitek 2 compact system biochemical assays were used to determine the identities of the isolated bacterial strains.The Vitek 2 compact system's diagnostic kit includes 64 wells, each of which can be inoculated with a 24hour-old bacterial suspension and then incubated for another 24 hours, after which the device can record the color changes caused by the bacteria's growth [8,9].

Statistical Analysis
The Least Significant Differences Test (LSD) was used.

Screening and Selecting Phenol Degrading Strains
42 bacterial isolates were obtained from the tested 40 oil contaminated samples.29 samples were positive and 11 were negative (no growth).Table 1, shows the number of samples distributed according to isolation areas.All bacterial isolates were cultured on the medium of liquid mineral salts containing (100) mg / liter of phenol and the value of the OD600 was recorded for 7 days.Strains with different colony shape and high OD600 values were selected for identification.6 strains, namely (ZA1-ZA6) were selected for the purpose of diagnosis.Table (2) shows the distribution of bacterial species isolated from soil contaminated with petroleum products from different Locations.The predominance of Gram-negative bacteria is because they contain an outer membrane works to protect them from external influences by reducing the permeability of different types of molecules and because the outer membrane contains fats that help it to obtain the largest possible amount of hydrocarbon compounds from the environment in which it resides [10].

Cultural Identification
Aeromonas salmonicida colonies appeared on the mineral salt medium in the form of round white colonies, while on MaCconky agar, it appeared as pink colonies (figure1).Enterobacter Cloacae complex colonies appeared on the medium of mineral white , ceramic, small to medium in size, while on MaCconky agar, it appears as small and white colonies (figure 2).Escherichia hermannii appeared on the medium of mineral salts in the form of white dotted colonies.On the medium of Maconkey agar, it appeared in the form of semi-transparent white colonies (figure 3).Sphingomonas paucimobilis on the medium of solid mineral salts and MaCconkey agar appeared as white round colonies (figure 4).Acinetobacter lwoffii are white medium size colonies on mineral salts medium and small size on Maconkey agar medium as shown in figure (5).Pantoea spp appeared on the medium of solid mineral salts as white, round, small to medium colonies.While on blood agar, the colonies appeared in a gray color, small to medium size (figure 6).

Identification Using VITEK2 System
This test was conducted using the VITEK compact2 device according to the method [6].

Testing the Ability of Isolated and Identified Bacterial Species to Grow in Different Concentrations of Phenol
Table (3) shows the ability of the isolated and diagnosed bacterial species to grow at phenol concentrations ranging from (100-1000) mg/l.The results showed the ability of isolates of each of Sphingomonas paucimobili, Escherichia hermannii, ,Enterobacter cloacae complex, Acinetobacter lwoffii , Pantoea spp ,and Aeromonas salmonicida to grow on solid mineral salts medium containing phenol at different concentrations (100,200,300,400,500,600,700,800,900,1000) mg/l.

Measuring the Efficiency of Bacterial Isolates to Grow at Different Concentrations of Phenol
The efficiency of the isolated and diagnosed bacterial species on growth was compared with different concentrations of phenol, as the highest concentration was tested in which the bacterial species showed the ability to grow on the solid culture medium according to Table (3) and compared with the initial concentration used for isolation by measuring the optical density values OD600 for 7 days.Table (4) shows a decrease in the growth rate of the bacteria Aeromonas salmonicida at a concentration of 1000 mg/ml, as the value of OD600 was 0.0098 compared to the value of OD600 at a concentration of 100 mg/ml, which was 0.6891.LSD (days) = 0.000212 LSD con.= 0.000106 LSD interferance = 0.000300 The results also showed decrease in the growth rate of Enterobacter cloacae complex at a concentration of 1000 mg/L as the value of OD600 was 0.0091 compared to the value of OD600 at a concentration of 100 mg/L which was 0.3544( table5).Table (6) shows the efficiency of Escherichia hermannii to grow at a concentration of 100 and1000 mg/L.The growth rate of Escherichia hermannii was decreased at a concentration of 1000 mg/L as the value of OD600 was 0.0048 compared to the value of OD600 at a concentration of 100 mg/L which was 0.5833.The efficiency of Sphingomonas paucimobilis to grow at a concentration of 100 and1000 mg/L is shown in table 7. The results showed a decrease in the growth rate of sphingomonas paucimobillis at a concentration of 1000 mg/ml, as the value of OD600 was 0.0075, compared to its value at a concentration of 100 mg/L, which was 0.3543.LSD days = 0.000184 LSD con.= 0.000092 LSD interferance = 0.000261 The results showed a decrease in the growth rate of Acinetobacter lwoffii at a concentration of 1000 mg/ml, as the value of OD600 was 0.0133, compared to its value at a concentration of 100 mg/L, which was 0.3933 (table8).Table 9 shows that OD600 of Pantoea spp was 0.0087 at a concentration of 1000 mg/L compared to its value at a concentration of 100 mg/L, which was 0.2926.LSD days = 0.000300 LSD con.= 0.000150 LSD interferance = 0.000424 In this study, reliance was made on bacterial isolates that are efficient in growth and degradation of hydrocarbon compounds, and a depiction of the efficiency of each isolate was given.The goal of using microorganisms is to remove pollutants from the environment because the conversion of toxic compounds into non-toxic or low-toxic compounds.It is also less expensive than physical and chemical processes.In addition to the ability of these organisms to adapt to the surrounding environmental conditions over time.This adaptation leads to enhancing their ability to decompose and consume toxic hydrocarbons and convert them into non-toxic and low-toxic compounds [12][13][14][15][16][17][18].

Figure 1 .
Figure 1.Phenotypic characteristics of Aeromonas salmonicida grown on mineral salts medium and MaCconky agar medium.

Figure 2 .
Figure 2. Phenotypic characteristics of Enterobacter Cloacae complex growing on mineral salts medium and MacConkey agar medium.

Figure 3 .
Figure 3. Phenotypic characteristics of Escherichia hermannii grown on mineral salts medium and Maconkey agar medium.

Figure 4 .
Figure 4. Phenotypic characteristics of Sphingomonas paucimobilis growing on mineral salts medium and MaCconkey agar medium.

Figure 5 .
Figure 5. Phenotypic characteristics of Acinetobacter lwoffii grown on mineral salts medium and Maconkey agar medium.

Figure 6 .
Figure 6.Phenotypic characteristics of Pantoea spp.growing on mineral salt medium and blood agar medium.

Table 1 .
Number of samples distributed according to isolation areas.

Table 2 .
Bacterial species isolated from soil contaminated with petroleum products from different Locations.

Table 3 .
Testing the efficiency of isolated and identified bacterial species on growth with different concentrations of phenol.

Table 4 .
The efficiency of Aeromonas salmonicida to grow at concentrations (100&1000) mg/l of phenol.

Table 5 .
The efficiency of Enterobacter cloacae complex on growth at a concentration of 100&1000 mg/L.

Table 6 .
The efficiency of Escherichia hermannii bacteria to grow at a concentration of 100&1000 mg/L.

Table 7 .
The efficiency of Sphingomonas paucimobilis bacteria to grow at a concentration of 100&1000 mg/L.

Table 8 .
The efficiency of Acinetobacter lwoffii to grow at a concentration of 100&1000 mg/L.

Table 9 .
The efficiency of Pantoea spp to grow at a concentration of 100&1000 mg/L.