Isolation and Identification of Phenol-Degrading Bacteria from Oil-Contaminated Sites

This work is aimed at isolating and identifying phenol-degrading bacteria from oilcontaminated sites. Five soil samples from three auto-mechanic workshops within Katsina metropolis were collected. The samples were analyzed by selective enrichment technique, which resulted in the isolation of four bacterial species. The species were further subjected to the Vitek 2 compact microbiological system analysis. Cupriavidus pauculus, Pontoea spp, Proteus mirabilis 1 and Proteus mirabilis 2 were identified. Result from the present study showed that the bacteria could utilize phenol as their carbon source. Proteus mirabilis 1 and Proteus mirabilis 2 showed lower phenol degradation potential, under similar conditions. Cupriavidus pauculus and Pontoea sp. showed significant increases (p<0.05) in their optical densities. The optical density increment is strongly correlated with increase in colony forming units of the bacteria. This study further showed that the isolates could tolerate high phenol concentrations and may serve as strong putative isolates in bioremediation of phenol-contaminated sites.


Introduction
The exploration of the environment by man in the quest to survive has generated some negative impact on the ecosystem (Ojuederie and Babalola, 2017). Pollution is one of the major impacts of man's activity which has caused threat to life. Phenol is one of the major pollutants found in oil contaminated sites (Gami et al, 2014). Phenol and its compounds like chlorophenols and nitrophenols, among others, are injurious to humans and other flora and fauna at oil-contaminated sites even at low concentrations. Symptoms including irritation of the skin and eyes, diarrhea and vomiting have been associated with hydrocarbon pollutants. Other significant morbidities like cancers, coronary heart diseases, kidney, and liver failure have been reported in individuals exposed to phenol and other aromatic pollutants (Abubakar and Shukor, 2017).
Physicochemical and biological methods can be applied in order to remediate the toxic effect of phenols (Riser-Roberts, 2020). Biological methods have been shown to be cost-effective in bioremediation of phenols. This study is aimed at identifying, isolating phenol degrading bacteria from oil contaminated sites and to determine the phenol degrading ability of such bacteria at oil contaminated sites within Katsina Metropolis.

Sample Collection
Samples were collected in clean polythene bags from contaminated sites. The samples were homogeneously mixed together to form a bulk (Ayandiran and Dahunsi, 2017).

Isolation and identification of Hydrocarbon Utilizing Bacteria
The total population of the hydrocarbon-utilizing bacteria was obtained by pour plate method on minimal salt medium (MSM) using phenol as the sole source of carbon. The morphological characteristics of the bacterial isolates were identified by Gram staining and biochemical reactions (Udeani et al., 2009). All the isolates were screened for phenol utilization capabilities in mineral salt broth medium (Okpokwasili and Nweke 2006). Vitek 2 compact system (A microbiological analyzer) was used to identify the bacteria (Garcia-Garrote et al, 2000).

Determination of phenol biodegradation of selected bacterial isolates
Isolates that showed good utilization potentials of phenol during the screening test were selected for biodegradation studies. The ability of the bacterial isolates to degrade phenol was confirmed by inoculating each isolate into 250 mL Erlenmeyer flask containing 100 mL mineral salt medium. Phenol (0.6%) was used to serve as carbon source, while the strains were tested for growth by turbidity formation as described by Mills et al. (1978) with slight modifications.
The concentration of phenol was increased to 1% and the growths of individual organisms were also monitored over 15 days. The pH of the culture broth was taken at intervals.

Results
Isolates were screened for phenol utilization capabilities in mineral salt broth medium with phenol (0.6%) as carbon source. Four bacterial isolates (Cupriavidus pauculus, Proteus mirabilis 1, Proteus mirabilis 2 and Pantoea Spp) were identified by Gram staining and biochemical reactions ( Table 1). The strains were bacillus specie, among which only Pantoea spp tested positive to oxidase reaction. Pantoea spp did not produce hydrogen sulphide and could not ferment glucose as compared to the other three bacteria isolates (Cupriavidus pauculus, Proteus mirabilis 1 and 2) (2)  (1) had the least OD and pH (0.393 and 6.71 respectively). The optimum growth of the organisms was observed after 9 th day. Tolerance of Cupriavidus pauculus in phenol (1%) and minimal salt medium was observed for 15 days at 30 ºC. The optical density (OD) at the optimum pH was 0.26. There is an exponential growth of Cupriavidus pauculus corresponding to the rate of degradation from the 1 st day to the 9 th day. The pH of the culture media was continuously measured during the growth. The pH at the optimum growth was 6.89 (Figure 1). There was a decrease in pH of the culture when Cupriavidus pauculus was grown in 1% phenol. A significant decrease in optical density (0.26) was observed when Cupriavidus pauculus was grown in 1% phenol concentration as compared to 0.6%. The decrease in OD might be due to the increased concentration of phenol.  degradation from the 1 st day to the 9 th day. The pH of the culture media was continuously measured during the growth. The pH at the optimum growth (0.26) was 8.66 (Figure 2). Proteus mirabilis (2) was also cultured in 1% of phenol with minimal salt medium for 15 days to observe its tolerance in phenol. There optimum growth was obtained at 0.24 OD, at pH of 7.41. An exponential growth was also seen of between the 1 st and the 9 th days (Figure 3).

Both proteus species have an optimum growth
in alkaline media at 1% phenol concentration. The slight increase in pH and a decrease in OD signiy that increase in pH might have affected the growth of the microbes.  Tolerance of Pantoea sp. in phenol (1%) and minimal salt medium was observed for 15 days at 30ºC. The optical density OD at optimum pH was 0.36, this showed an exponential growth of Pantoea sp. corresponding to the rate of degradation from the 1 st day to the 9 th day. The pH of the culture media was continuously measured during the growth. The pH at the optimum growth was 7.93 (Figure 4) and this is a clear indication that Pantoea sp. requires an alkaline pH for the degradation of phenol. There was no significant difference in the pH when Pantoea sp. was grown in 1% and 0.6% phenol concentrations. Figure 4: Tolerance of phenol with Pantoea sp. on minimal salt medium at 30ºC for 15 days

Discussion
This study was aimed at isolating and identifying phenol-degrading bacteria from oil-contaminated sites in Katsina Metropolis. Four isolates showed biodegrading ability towards phenol. The isolates were identified using biochemical reaction tests (Table 1). The isolates were further identified using an automated microbiological system (Vitek 2) as Pantoea spp., Proteus mirabilis (1) and (2)  Screening showed the good ability of the bacteria to tolerate phenol as the sole source of carbon and the isolates were able to grow well on phenol when screened for hydrocarbon utilization (Figures 3 to 6). The pH values of each medium containing the isolates showed a significant increment (p<0.05) with increasing number of days of incubation. This signified that the isolated bacteria increased the pH of the medium to slightly alkaline and that biodegradation of phenol may be best achieved at slightly alkaline pH. The result obtained in this work is similar with the studies conducted by Mbachu et al. (2014)  corresponding to the rate of degradation clearly indicates that the microbe was able to tolerate such high concentration of phenol and also able to utilize it as its carbon and energy source. The maximum growths for all the tested bacterial isolates were obtained in day 12. Cupriavidus pauculus showed the highest utilizing potential of phenol having the highest aerobic mesophilic bacterial count of 62.4x10 8 cfu/g, which is an evidence of utilization of phenol. The least in terms of aerobic mesophilic bacterial count among all the isolates is Proteus mirabilis (1) having the aerobic mesophilic bacterial count of 8.7 x 10 8 cfu/g. According to Gomez et al (2013), bacteria isolated from phenol rich contaminated sites could tolerate phenol considering their better proliferation when incubated in phenol rich media. The optical density increment correlates to an increase in cell number. This showed that the bacterial isolates can grow effectively in phenol-rich substrate. The present study demonstrates the phenol biodegradation potentials of these isolates.