Keywords

1 Introduction

Many pollutants that are very hazardous for the nature are discharged from chemical, pharmaceuticals and oil refinery industries including many aliphatic and aromatic hydrocarbons (Taghreed and El-Naas 2014). Phenol is one of them, which has the capacity to damage the gastrointestinal tract, irritation of respiratory tracts, muscle tremors. Damage of liver, kidney, and nervous system are the adverse effects of phenol. Also phenol is very much hazardous to the aquatic ecosystems (Szczyrba et al. 2016). Therefore, it is mandatory to treat the phenol and phenolic wastes properly before disposal into the nature (Szczyrba et al. 2016; Kumar et al. 2005). The maximum permissible level of phenol in land water is 1 ppm according to the Central Pollution Control Board (CPCB) and IS:2490-1974 (Cheela et al. 2014; Lathasree et al. 2004; Saravanan et al. 2009). As per World Health Organization (WHO), the safety limit of phenol in drinking water should not exceed 1 mg/L (Bakhshi et al. 2011; Saravanan et al. 2008; Wang et al. 2010).

Several treatment methods are there to treat phenol such as adsorption, chlorination, ozonation as well as many physicochemical methods (Szczyrba et al. 2016; Tamer et al. 2010). Due to high cost effect of these methods and production of toxic intermediate compounds, involvement of biological processes is necessary in the treatment of phenol (Szczyrba et al. 2016). These biological treatments may be biosorption, biodegradation, bioaccumulation, etc., involving bacteria, algae, fungi, etc. Many such studies have been done previously as Mohanty and Jena (2017) did his experiment on biodegradation of phenol using Pseudomonus sp. NBM11 that was able to degrade up to 1000 ppm phenol completely in the temperature ranging between 30 and 32 ℃ and pH 6.8–7.2. Another study was done by Parvathy and Prabhakumari (2017) involving Pseudomonas aeruginosa, isolated from industrial soil to remove catechol.

The current study has been carried out aiming to isolate the most potent bacterial strain to degrade phenol and the identification of the strain.

2 Materials and Methods

2.1 Soil

Soil was collected from the local hospital area.

2.2 Reagents

Phenol-(analytical grade), 4-amino antipyrene, potassium ferricyanide, ammonium chloride and ammonium hydroxide solution. Our targeted concentration of phenol was prepared by mixing the properly weighed phenol to distilled water.

2.3 Bacterial Media

The nutrient agar medium with following composition was used for cultivation of bacteria.

Peptone—0.5%, beef extract—0.3%, agar—3.0%.

2.4 Isolation and Screening of Most Potent Bacterial Strain

The bacterial strain capable of removing phenol was isolated by soil enrichment and serial dilution plate count method. The soil was enriched with 500 ppm phenol for 10 days. After serial dilution and plate count isolated colonies were transferred in individual slant. A total of 29 isolated colonies were transferred to slant.

Screening was done to select most potent strain. Each of the isolated colonies was transferred to liquid medium and incubated 37 ℃ for 24 h in the presence of 500 ppm phenol. After fermentation, the fermented broth was centrifuged and the clear supernatant was used for spectrophotometric estimation of residual phenol content.

2.5 Estimation of Residual Phenol Content

Residual phenol concentration was measured in spectrophotometer at 510 nm wavelength followed by APHA method. Residual phenol content was calculated from the standard curve made with the known concentrations of phenol.

2.6 Morphological, Biochemical, and Phylogenetic Characterization of the Isolated Bacterial Strain

Morphological and biochemical characterization of the isolated bacterial strain was done as per Bergey’s Manual of Determinative Bacteriology (Holt et al. 1993). Phylogenetic assay was made by 16s rDNA method.

3 Results and Discussion

3.1 Isolation and Screening of Most Potent Bacterial Strain

After serial dilution of the enriched soil sample and transfer of the diluted soil sample into nutrient agar medium, 29 isolated colonies were obtained. Colony characteristics of the isolated colonies are shown in Table 1.

Table 1 Screening of the most potent bacterial strain
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After screening, it was found that the strain marked as P25 showed maximum phenol removal capability (~99.44%) (Table 1), so it was selected for further study.

3.2 Morphological, Biochemical, and Phylogenetic Characterization of the Isolated Bacterial Strain

Morphological characterization of the isolated bacterial strain P25 is shown in Tables 2 and 3. Gram characteristics and spore characteristics are shown in Figs. 1, 2, and 3. Physicochemical characteristics are shown in Table 4. The phylogenetic tree is shown in Fig. 4.

Table 2 Colony characterization of the isolated bacterial strain
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Table 3 Growth characterization on slant
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Fig. 1
figure 1

Simple staining

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Fig. 2
figure 2

Gram staining

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Fig. 3
figure 3

Spore staining

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Fig. 4
figure 4

Phylogenetic tree of the strain P25

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Table 4 Physicochemical characteristics of the isolated bacterial strain P25
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4 Conclusion

The isolated most potent bacterial strain was identified as Brevibacillus formosus strain NRRL NRS-863, which was able to reduce almost 99.44% of phenol. However, the mechanism of removal of phenol by the isolated strain i.e. whether the strain degrades it or accumulates it is not known till now. Our further study will reveal it.