Introduction

The environment, as a consequence of industrial and agricultural revolutions, has been hardened with potentially carcinogenic and mutagenic halogen-substituted aromatic compounds^1-3. Among which phenol is a listed priority pollutant by the U.S. Environmental Protection Agency⁴ and is consider to be a toxic compound by the Agency for Toxic substances and Disease Registry⁵. Phenol and its derivatives which represent major organic pollutant in the effluents, from many industrial activities such as oil refineries, chemical, petrochemical, pharmaceutical, metallurgical, pesticide product, paint and varnish industries, textile and also in the polymer industries, like phenolic resins, bisphenol A, alkylphenols, caprolactums and adipic acid⁶. Phenol is a toxic and hazardous substance even at low concentrations^{7, 8}. The concentration of phenols in wastewaters varies from 10 to 300 mg/l ⁶. The adverse effects of phenol on health are well documented⁹ and death among adults has been reported with ingestion of phenol ranging from 1 to 32 g¹⁰. The low volatility of phenol and its affinity for water make oral consumption of contaminated water, the greatest risk to humans¹⁰.

Efficient treatment methods are necessary to reduce phenol concentration in wastewater to acceptable level, which is 5 ppm (USEPA). Several methods available for treatment of phenol in which bioremediation is receiving the most attention due to its environmentally friendly, its ability to completely mineralize toxic organic compounds without producing innocuous end products and minimum secondary waste generation¹¹ and of low-cost^{12, 10} . Microbial degradation of phenol has been actively studied and these studies have shown that phenol can be aerobically degraded by wide variety of fungi and bacterial cultures such as Acinetobacter species W-17 ^13,14 , Bacillus brevis^{15, 16}; Microbacterium phyllospaerae¹⁷; Pseudomonas cepacia¹⁸; Pseudomonas putida CCRC 14365¹⁹; Pseudomonas aeruginosa²⁰; Pseudomonas fluorescence²⁰; Pseudomonas desmolyticum²¹ ; Fusarium^22,23 ; Candida tropicalis^24-26  and Ankistrodesmus braunii²⁷.

It has been demonstrated that treatment of small volumes of toxic compounds at the point of emission using specific microbial strains and better bioreactors allows a higher control over the process and higher removal efficiencies²⁸ .Thus optimization of process variables is recognized to be an essential aspect of successful fermentation²⁹.

In order to find a strain, able to degrade phenol in a changeable environment, we studied the influence of inoculum size, effect of pH, and effect of temperature on phenol degradation by P. fluorescence .Phenol degradation was also studied with different level of phenol concentrations (100-750 mg/l) at these optimized conditions.

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. fluorescence was obtained from culture collection (NCL) Pune, India and it was maintained 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 30⁰C for further use.

Growth determination

To study the extent of degradation, the cells were grown in a Minimal Salt (MS) medium with the following composition: Phenol 0.100 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 flask containing and inoculated with P. fluorescence. The experimental studies were carried out in shake flasks with agitation at a rate of 120 rpm, temperature at 30⁰C. Bacterial growth was determined in terms of cell mass by measuring optical density at a wavelength of 500nm.

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 30⁰C in MS medium supplemented with 100 mg/l phenol at pH 7 for P. fluorescence in 250 ml Erlenmeyer flasks. At different times, growth and phenol degradation were measured.

Effect of pH of the medium on phenol degradation

fluorescence with an optimum inoculum size, (5%v/v) was grown in MS medium with 100 mg/l of phenol at different pH values 5 to 9.This mixture was contained in 250 ml Erlenmeyer flasks. The cultures were incubated in an orbital shaker at 30⁰C with agitation speed 120 rpm. At different times, growth and phenol degradation were measured.

Effect of temperature of the medium on phenol degradation

fluorescence was grown in MS medium with 100 mg/l of phenol at different temperatures (27°C, 28⁰C, 29⁰C, 30⁰C and 32⁰C), inoculum size 5% v/v and pH 7 in 250 ml Erlenmeyer flasks. The cultures were placed on a shaker (120 rpm) at the above mentioned temperatures. 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 analysis³⁰.

Growth determination

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

Results and Discussion

 Effect of amount of inocula on phenol degradation

Effect of various amounts of inocula 1- 10 %v/v was studied on phenol degradation with a level of phenol concentration, 100 mg/l. Hill and Robinson,1975 concluded the size of inoculum might affect the duration of the lag phase. The data in the Fig.-1 (all the data not shown for clarity) show that phenol was almost completely reduced after incubation for 120 hrs in the presence of different concentrations of incoula in the medium. Whereas the concentration of phenol declined quickly when more cells were inoculated. Phenol degradation which was influenced by the amount of inocula, 5%v/v of biomass concentration provided the best degradation in short duration.

Figure 1: Effect of inoculum size on phenol degradation by by P.fluorescence.   Click here to View figure

Effect of pH of the medium on phenol degradation

The hydrogen ion concentration in the culture medium greatly influences the bacterial growth since p H limits the activity of enzymes. Variations in the p H of the medium resulted in changes in the ionic form of the active site and changes in the activity of the enzymes and hence the biodegradation rate. Changes in p H may also alter the three dimensional shape of the enzymes in microorganisms. For these reasons enzymes in microorganisms are only active over a certain p H range and different microorganisms have different p H optima. The experiments were carried out to optimize the p H. Degradation of phenol at various p H, 5 to 9, which plays a vital role in the growth, degradation and lysis of bacteria is shown in Fig.-2. The most favorable p H for the strain to achieve the maximum rate of phenol degradation was 7 whereas the phenol degradation at other p H values was slower.

Figure 2: Effect of pH on phenol degradation by P.fluorescence.   Click here to View figure

Effect of temperature of the medium on phenol degradation

The high dependence of enzymatic activity and cellular maintenance requirements on temperature makes it an important quantity. Temperature exerts an important regulatory influence on the rate of metabolism. From the Fig. -3, the effect of temperature on phenol degradation by P fluorescence was assessed. The degradation rate increased with increase of temperature form 27 to 30°C. Above the optimal temperature, thermal death occurred so the specific growth and phenol removal rates decreased. The optimum temperature was 30°C at which the degradative enzyme reached the highest activity.

Figure 3: Effect of Temperature on phenol degradation by by P.fluorescence.   Click here to View figure

In order to determine the effect of phenol concentration on microbial growth and phenol removal rates a series of batch experiments were conducted in shake flasks by P. fluorescence at optimum conditions (inoculum size 5% v/v, p H 7, temperature 30°C). Experiments were conducted at various initial phenol concentrations ranging from 100-750 mg/l as shown in the Fig.-4 (All the data not shown for clarity). Results of these studies show that higher the concentrations of phenol the more time it takes to be degraded completely. At lower initial phenol concentrations P. fluorescence degrades phenol immediately with no lag phase indeed at phenol concentrations greater than 480mg/l there occurs a lag phase. At each of the phenol concentrations there was a period of exponential growth period which is further confirmed by substrate being consumed at faster rate.

Figure 4: Effect of phenol concentration.   Click here to View figure

In summary, these results shows P. fluorescence can be tolerated up to 480 mg/l at these optimum values.

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