Introduction

Environmental pollution has been considered as a side effect of industrial society. Soil, lakes, rivers and seas are highly contaminated with different toxic compounds (Alexander, 1981). An example of such compound is phenol. Phenol is released into the environment from industrial discharges (Keith, 1976, Jungclaus et al, 1978, Parkhurst et al, 1979, Pfeffer, 1979) and spills (Delfino and Dube, 1976). According to Prasad and Ellis (1978), phenols and its derivatives are among the most frequently found pollutants in rivers, industrial effluents and landfill run-off waters. Hence, populations residing near waste disposal sites, landfill sites or phenol spills may be at risk for higher exposure to phenol than other populations. The origin of phenol in the environment is both natural and industrial. Natural sources of phenol include forest fire, natural run off from urban area where asphalt is used as the binding material and natural decay of lignocellulosic material. Industrial sources such as oil refineries, chemical, petrochemical, pharmaceutical, metallurgical, pesticide products, paint and varnish industries, textile and also in the polymer industries like phenolic resins, bisphenol A, alkylphenols, caprolactums and adipic acid (Paula  et al, 1998). Hence removal of phenol from industrial aqueous effluents is an important practical problem. Their presence in sewage and river water has also been confirmed (Thomas, 1973). Because of their high reliability and water solubility, phenols impart taste and odour problems to drinking water supplies even at parts per billion levels (Gosselin et al, 1984). Phenols are protoplasmic poisons, hence damage all kinds of cells. They are alleged to have caused an astonishing number of poisonings since they have come into general use. Fatal doses of phenols can be absorbed by the skin.

The removal of phenol from industrial effluents has attracted researchers from different fields (Yang and Humphrey, 1975, Shingler, 1996). The increasing awareness on the environment in both developed and developing countries has initiated more studies of possible solutions for treating phenol.

Environmental biotechnology relies on the pollutant – degrading capacities of naturally occurring microorganisms (Liu and Suflita, 1993). It has been reported to be advantageous over physical and chemical treatments due to its relatively low cost and has less ecological impact to the environment (Head, 1998, Edington, 1994). Researchers are studying pollutant-degrading microorganisms which inhabit polluted environments (Kumaran, 1980, Kapoor et al, 1998, Yap et al, 1999, Heinaru et al, 2000, Komarkova et al, 2003, Santos and Linardi, 2004, Margesin et al, 2005) as well as uncontaminated environment (Bastos et al, 2000a, Kounty et al, 2003). Harnessing the potential of microbes ( Ahmed, 1995, Fulthorpe and Allen, 1995, Bastos et al, 2000b, Ruiz-Ordaz et al, 2001, Vojta et al, 2002, Paca Jr. et al, 2003) to degrade phenol has been an area of considerable study to develop bioremediation approaches which has been considered as a “green-option “ ( Singleton, 1994) for treatment of environmental contaminants.

Many researchers support the biological treatment of phenols. A number of studies with prokaryotic microorganisms have been carried out for the purpose to improve the technological processes of biodegradation. Some examples are Pseudomonas sp., have demonstrated the ability to mineralize phenol ( Ehrhardt and Rehm, 1989, Hinteregger et al, 1992, Ahmed, 1995, Chitra et al,1995, Dapaah and Hill, 1992, Fulthorpe and Allen, 1995, Fava et al, 1995, Loh and Wang, 1998).The biodegradation of phenol by P. aeruginosa and P. desmolyticum, potential degradents of phenol have been investigated for its degrading potential under different operating conditions and were compared. Two variables of pH and inoculum size were used to identify the significant effects and interactions in the batch studied. Phenol degradation was studied by P. aeruginosa and P. desmolyticum with different phenol concentrations (500 mg/l, 700 mg/l, 900 mg/l and 1000 mg/l) at their optimized conditions. The maximum tolerance limit of phenol for P. aeruginosa and P. desmolyticum were also tested.

Materials and Methods

Chemicals

Phenol (99% pure, chemical grade) 4-amino antipyrine and all other chemicals used were from Merck.

Source of Organism

The microorganism P. aeruginosa (NCIM 2074) and P. desmolyticum (NCIM 2028) were obtained from culture collection (NCL) Pune, India. The microorganisms were maintained separately on a medium containing Beef extract: 1.0 g/l, Yeast extract: 2.0 g/l, Peptone: 5.0 g/l, NaCl: 5.0 g/l and Agar: 20 g/l. The pH of the medium was adjusted to 7.0 by adding 1N NaOH. It was stored at 32⁰C for further use.

Growth determination

To study the extent of degradation, the cells were grown in a Minimal Salts (MS) medium with the following composition: Phenol 0.500 g/l; K₂HPO₄, 1.5 g/l; KH₂PO₄, 0.5 g/l; (NH₄)₂SO₄, 0.5 g/l; NaCl, 0.5 g/l; Na₂SO₄, 3.0 g/l; Yeast extract, 2.0 g/l; Ferrous sulfate, 0.002 g/l; CaCl₂, 0.002 g/l in conical flasks containing and inoculated with  P. aeruginosa(NCIM 2074) and P.desmolyticum(NCIM 2028) individually. The experimental studies were carried out in shake flasks with agitation at a rate of 120 rpm, temperature at 32⁰C. Bacterial growth was determined in terms of cell mass by measuring optical density at a wavelength of 500nm.

Influence of pH of the medium on phenol degradation

Pseudomonas Cells were grown in MS medium with 500 mg/l of phenol at different pH 5 to 9. This mixture was contained in 250 ml Erlenmeyer flasks. The cultures were placed on a shaker (120rpm) at 32⁰C. At different times, growth and phenol degradation were measured.

Effect of inoculum size on phenol degradation

The effect of inoculum size (1 – 10% v/v) on phenol degradation was tested. Cells were grown as shake cultures at 32⁰C in MS medium supplemented with 500mg/l phenol at pH 7 in case of P. aeruginosa and pH 6 for P. desmolyticum in 250 ml Erlenmeyer flask. At different times, growth and phenol degradation were measured.

Estimation of phenol

Phenol was determined quantitatively by the Spectrophotometric method (DR/ 4000 V, Hach) using 4-amino antipyrine as the color reagent (λ _max: 500nm) according to standard methods of analysis (APHA, 1989).

Growth determination

Bacterial growth was determined in terms of cell mass by measuring optical density at a wavelength of 500nm.

Results and Discussions

aeruginosa (All the data is not plotted for clarity)

Influence of pH of the medium on phenol degradation

pH values from 5 to 9 were investigated (Fig. – 1). Phenol was degraded rapidly at pH 7. At this pH value, phenol degradation was high compared to other pH values. However, the phenol degradation at pH 5, 6, 8, and 9 was slower and phenol concentration decreased rapidly after 24 hrs inoculation. These results showed that P.  aeruginosa degraded more phenol per day at pH 7 than at any other pH value.

Figure 1: Effect of  pH on phenol degradation.   Click here to View figure

Effect of inoculum size on phenol degradation

Phenol was degraded by P. aeruginosa during all the inoculum sizes (1-10% v/v) tested (Fig. – 2). At 5% v/v the phenol concentration began to decrease rapidly after 5 hrs and reached 5mg/l after approximately 70 hrs. Cultures inoculated with 5 % v/v inoculum size showed the highest rate of phenol degradation, while the cultures inoculated with the other inoculum sizes showed a decrease in phenol consumption.

Figure 2: Effect of  inoculum size on phenol degradation.   Click here to View figure

desmolyticum (All the data is not plotted for clarity)

Influence of pH of the medium on phenol degradation

pH values from 5 to 9 were examined (Fig. – 3). At pH 6 phenol concentration started decreasing after 24 hrs inoculation when compared to other pH values. These results showed that P.desmolyticum degraded more phenol per day at pH 6 than at any other pH values.

Figure 3: Influence of  pH on phenol degradation.   Click here to View figure

Effect of inoculum size on phenol degradation

Phenol degradation by P. desmolyticum at inoculum sizes (1-10% v/v) was tested (Fig. – 4). The phenol degradation was optimized at 4% v/v and phenol concentration began to decrease rapidly after 5 hrs and reached 50 mg/l after approximately 70 hrs.

Figure 4: Influence of  inoculum size on phenol degradation.   Click here to View figure

At these optimized values of P. aeruginosa (pH 7, 5%v/v) and P.desmolyticum (pH 6, 4% v/v), the phenol degradation was tested and compared. Fig. – 5, show the experimental variation of the phenol concentration with time. As can be seen, P. aeruginosa at its optimum conditions degraded 1000mg/l of phenol concentration and P. desmolyticum at its optimum conditions could not tolerate 1000 mg/l of phenol concentration and could degrade only upto 700 mg/l. Hence, maximum removal efficiency of phenol was achieved at the optimum process conditions by P. aeruginosa.

Figure 5: Experimental values of phenol concentration during fermentations by  P.aeruginosa and P.desmolyticum.   Click here to View figure

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