Removal of Phenol in Batch Culture by Pseudomonas putida AP 11 , AP 9 , AP 6 and AP 7 Isolated from the Aromatic Hydrocarbon Contaminated Soils

Phenol widely used in industries, are of growing concern owing to their toxicity and wide distribution in industrial wastes. The aims of the study were to characterize some of the locally isolated bacteria and to develop suitable methods for the degradation of phenol using them. Locally isolated AP11, AP9, AP6 and AP7 were identified as Pseudomonas putida using the classical methods. Pseudomonas putida AP11 and AP9 were able to remove 600 ppm phenol completely, but Pseudomonas putida AP6 and AP7 were able to remove 400 ppm phenol completely. The maximum degradation rates for freely suspended culture of Pseudomonas putida AP11, AP9, AP6, AP7 were 10.83, 10.42, 8.33, and 8.33 (ppm/h) respectively. The isolates AP11, AP9, AP6 and AP7 can be used to wastewater containing phenol in effluent treatment systems.

treatments often produce other toxic end products, requiring further processing steps [6][7][8].On the other hand, biodegradation has been studied as an alternative approach due to the low costs associated with this option, as well as the possibility of complete mineralization of the xenobiotic [9].Phenol biodegradation has been studied detail using pure and mixed cultures of suspended bacteria [10][11].Phenol and other phenolic compounds are common constituents of many industrial effluents and suitable microorganism based process need to be developed for the effective degradation of phenol and then the effluents could be disposed safely [12].
The aim of this study was to identify and characterize the potential phenol degrading bacteria, isolated from the contaminated sites and their ability to degrade various concentrations of phenol when supplied as the sole carbon source was investigated.

Test organisms and identification
Isolates were obtained from Microbiology and Industrial Irradiation Division, Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Savar, Dhaka, Bangladesh.The organisms were previously isolated from soils of aromatic compound contaminated sites.The isolates were identified using cultural, morphological characteristics and biochemical tests according to methods described in Bergey's Manual of Systematic Bacteriology [13].The bacteria were maintained on phenol agar medium and stored at 4 0 C for around 1 month and then sub-cultured.

Phenol agar
Bacteriological agar at a concentration of 1.5% (w/v) was added to the minimal medium.Following sterilization by autoclaving, the medium was allowed to cool.Immediately prior to pouring phenol was added to the medium to give the appropriate concentrations.Phenol broth was used for biodegradation studies, the composition of which was exactly similar to the phenol agar except that no agar was added to it.

Cultural conditions for biodegradation studies
Isolates were grown in nutrient broth for 24 hours, centrifuged at 5000 rpm for 10 minutes and washed twice with potassium phosphate buffer.Five ml of bacterial suspension (10 7 cells/ml) was used to inoculate 95 ml sterile minimal medium [14] containing phenol in 250 ml conical flasks.After inoculation, flasks were incubated in an orbital shaker at 120 rpm at 37°C.Control flasks were run in parallel.Samples were aseptically removed at regular intervals and analyzed for growth and substrate removal.The study period for phenol was 0-96 h.

Measurement of the growth of bacterial cells
Growth of the bacterial cells was monitored turbidimetrically by measuring the optical density (OD) at 660 nm using a UV visible spectrometer.

Chemical Analyses
Concentrations of phenol were determined by colorimetric method using 4aminoantipyrene based on the procedure detailed in standard methods for the examination of water and wastewater [15].

Identification and characterization of bacterial isolates
Cultural and morphological characteristics of the isolates are shown in Table 1.The cell characteristics were noted following cultivation on nutrient agar at 30°C.All the isolates were gram negative, motile, non-spore forming and rod shaped.None of them showed acid fast staining.A variety of biochemical tests were performed on all the strains to Table 1.Colony characteristics and microscopic observation of the bacteria isolated from aromatic hydrocarbon contaminated soils.
A wide range of sugar utilization was also observed by all of the isolates (Table 3).Isolates AP11 and AP9 were able to grow on most of the sugars except lactose.The AP6 grew in the presence of four sugars out of ten while the AP7 utilized six sugars.Comparing the cultural, morphological and biochemical characteristics of isolates with the properties listed in the Bargey,s Manual for systematic Bacteriology [13], it was found that all of the isolates belong to Pseudomonas putida.Pseudomonas putida are regarded as one of the most common species of phenol degrading bacteria isolated from contaminated sites [16][17][18][19].The ability of this species particularly to utilize aromatic hydrocarbons has been widely documented [20][21].It was also able to use a wide diversity of carbon energy substrates and this together with their ability to compete effectively with other bacteria, is undoubtedly responsible for their dominance [22].Pseudomonas putida EKII, Pseudomonas cepacia G4, Pseudomonas putida Q5, Pseudomonas putida MTCC were investigated as phenol degrader as well as all of them were non pathogenic [16,[23][24][25][26].

Growth of pseudomonas putida isolates and their degradation capacity in liquid culture media containing phenol in different concentrations
All the organisms were grown in the liquid culture containing 100 ml minimal medium with various concentrations of phenol (400-800 ppm).The results of phenol degradation and their corresponding removal rates and removal efficiency are presented in Fig. 1 and Table 4. AP11 was found to be the most effective bacteria comparing with the others isolates.Complete removal of 400 and 600 ppm of phenol was observed by the organism.Removal efficiency was higher with the progressing growth.Highest bacterial growth (OD 0.507 at 660 nm) was observed at 72 h.However, complete removal of 800 ppm was not observed.It was possible to remove about 41.25% within the time 96 h and the bacterial growth was increasing slowly with removal of phenol (Table 4).Almost similar patterns of growth, phenol degradation, removal rate and removal efficiency were observed for the isolate AP9.
Pseudomonas putida AP6 degraded 400 ppm of phenol completely within 72 h.Complete degradation of 600 ppm phenol was not achieved within 96 h.It was possible to degrade only 66%.The bacterial growth was highest at 72 h for 400 ppm phenol.The increasing growth was not in similar fashion at 600 ppm compared to 400 ppm.Maximum growth (OD 0.370) was found at 96 h with 600 ppm phenol.Maximum removal rate was 8.33 (Table 4).The isolates AP7 also showed almost similar patterns of growth, phenol degradation, removal rate and removal efficiency as observed for AP6.It was reported that Pseudomonas putida CP1 is capable to remove 600-800 ppm of phenol, Pseudomonas putida A(a) also degraded 600 ppm phenol within 24 h and 800 ppm phenol within 48 h [18].The bacterial growth varied with time and concentration of phenol.The Pseudomonas sp.A4CP2, exhibited the highest growth (OD = 0.55) growing on 800 ppm phenol [19].A number of bacteria have been evaluated for their usefulness in controlling phenol, a hazardous pollutant, which is produced in oil refineries, petrochemical plants, pharmaceutical industries etc. [27][28][29].The isolates AP11, AP9, AP6 and AP7 were capable to degrade phenol at a concentration very much similar to other reported strains, therefore, the isolates can be used to wastewater containing phenol in effluent treatment systems.

Conclusions
Four isolates, namely AP11, AP9, AP6 and AP7 obtained from aromatic hydrocarbon contaminated soils were identified as Pseudomonas putida.Complete degradation of 600 ppm phenol was found for isolates AP11 and AP9, but complete degradation of 400 ppm phenol was found for isolates AP6 and AP7.The maximum degradation rates for suspended culture of Pseudomonas putida AP11, AP9, AP6, AP7 were 10.83, 10.42, 8.33, and 8.33 (ppm/h), respectively.

Fig. 1 .
Fig.1.The removal of various concentrations of (400 -800 ppm) phenol by Pseudomonas species when supplied as the sole sources of carbon and energy (solid line represents degradation and corresponding broken line represents growth).

Table 2 .
their identification and the results are shown in Table2.In the biochemical test, all of the isolates were found as oxidase and catalase positive.Production of H 2 S and urease were not observed for any of the isolates.In Voges proskauer and methyl red test, different response was observed.Biochemical tests of four bacterial isolates. enable

Table 3 .
Carbohydrate utilization by four isolates.

Table 4 .
Maximum concentration of phenol degradation, degradation time, and growth and removal efficiency of different isolates of Pseudomonas putida AP11, AP9, AP7 and AP6.