Department of Chemical Engineering (Biotechnology), College of Engineering, Andhra University, Visakhapatnam 530 003, Andhra Pradesh, India
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Phenolic compounds are hazardous pollutants that are toxic relatively at low concentrations. Accumulation of phenol creates toxicity both for flora and fauna. Because of its toxicity, there is a need to decontaminate the phenol-laden soils. Hence, bioremediation is a very useful alternative to conventional clean-up methods. The aim of this work was to study the effect of two variables – temperature (300C, 320C, 330C, 340C) and carbon (0.5, 1, 2, 3, 4 gm/L) to identify the significant effects and interactions in the batch studies. It was found that the degrading potential of Pseudomonas aeruginosa (NCIM 2074) was strongly affected by the variations in carbon and temperature. Optimum conditions of the variables for the growth of P. aeruginosa (NCIM 2074) and for maximum biodegradation of phenol are temperature (320C) and carbon (0.5gm/L). These results are useful to understand the physiological and biochemical properties of P. aeruginosa (NCIM 2074) before its optimum use in environmental application and these data will assist in choosing the right phenol degrader for a changeable environment.
Biodegradation, Temperature, Carbon, Phenol, Pseudomonas aeruginosa.
The massive increase in the synthesis of organic chemicals by man has led to the production of wide variety of compounds, some of which are xenobiotic. Their xenobiotic character means that their structures are not easily recognized by existing degradative enzymes and as a result they accumulate in the environment (Singleton, 1994). As they persist in the environment, they are capable of long-range transportation, bioaccumulation in human and animal tissue and biomagnifications in food chain. Phenol and its higher homology are aromatic molecules containing hydroxyl group attached to the benzene ring structure. 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). The presence of phenol in water imparts carbolic odor to receiving water bodies and can cause toxic effects on aquatic flora and fauna (Ghadhi and Sangodkar, 1995). It is lethal to fish even at relatively low concentrations of 5-25 mg/L (Nuhoglu and Yakin, 2005). Phenols are toxic to human beings and effects several biochemical functions (Saha et al. 1999).The concentration of phenols in waste waters varies from 10 to 300 mg/L. Phenol is also a priority pollutant and is included in the list of EPA (1979) (Indu Nair et al. 2008). As a result, phenol - containing effluents have to be properly treated prior to discharge (Keith, 1976; Jungclaus et al. 1978; Parkhurst et al. 1979; Pfeffer, 1979; Delfino and Dube, 1976). Efficient treatment methods are necessary to reduce phenol concentration in waste water to acceptable level, which is 5 ppm (USEPA).
Conventional methods of treatment for phenolic wastes have been largely chemical or physical methods like chlorination, advanced oxidation process (Santiago Esplugas et al. 2002), adsorption, solvent extraction, coagulation, flocculation, reverse osmosis, ozonation, photo catalysis, and electrolytic oxidation (Arutchelvan et al. 2006), but these processes have led to secondary effluent problems. Biological treatment for the bulk removal of these pollutants is therefore generally preferred. Biological degradation of phenol has been extensively studied using pure and mixed cultures (Kang and Park, 1997; Hugues and Cooper, 1996; Wang et al. 1996; Ha et al. 2000; Chirwa and Wang, 2002. Several studies have been carried out with the bacterium P. aeruginosa in pure cultures (Agarry et al. 2008) in which phenol is degraded via the meta-pathway (Sala-Trepat et al.1972). The success of bioremediation may depend on the availability of microbial strains that can mineralize high levels of phenol and withstand adverse conditions to compete under in situ conditions. An effective bacterial inoculum should be able to tolerate high levels of phenol while maintaining a high level of activity to provide efficient mineralization (Shaw et al. 1997). Understanding the physiological and biochemical properties of phenol degrading bacteria is required before optimum use of bacteria in environmental applications.
The biodegradation of phenol by P. aeruginosa (NCIM 2074), a potential biodegradent of phenol has been investigated for its degrading potential under different operating conditions. Chandana and Sridevi (2009) identified the optimum conditions on phenol degradation by P. aeruginosa (NCIM 2074): pH 7, inoculum size 6% v/v. This is a continuation of previous studies. Two variables of temperature, glucose (as carbon source) were used to identify the significant effects and interactions in the batch studied.
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) was obtained from culture collection (NCL) Pune, India. The microorganism was maintained on a medium containing Beef extract: 1.0 gm/L, Yeast extract: 2.0 gm/L, Peptone: 5.0 gm/L, NaCl: 5.0 gm/L and Agar: 20 gm/L The pH of the medium was adjusted to 7.0 by adding 1N NaOH. It was stored at 320C for further use.
To study the extent of degradation, the cells were grown in a Minimal Salts (MS) medium with the following composition: Phenol 0.500 gm/L; K2HPO4, 1.5 gm/L; KH2PO4, 0.5 gm/L; (NH4)2SO4, 0.5 gm/L; NaCl, 0.5 gm/L; Na2SO4, 3.0 gm/L; Yeast extract, 2.0 gm/L; Ferrous sulfate, 0.002 gm/L; CaCl2,0.002 gm/L in conical flasks containing and inoculated with P. aeruginosa (NCIM 2074). 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 temperature of the medium on phenol degradation
Pseudomonas cells were grown in MS medium with 500 mg/L of phenol at different temperature values (300C, 320C, 330C, 340C) at pH 7 and inoculum size 5%v/v (Chandana and Sridevi, 2009). This mixture was contained in 250 mL Erlenmeyer flasks. The cultures were placed on a shaker (120rpm) at the above temperatures. At different times, growth and phenol degradation were measured.
Effect of glucose on phenol degradation
The effect of glucose (0.5, 1, 2, 3, 4gm/L), on phenol degradation was tested. Cells were grown as shake cultures at 320C in MS medium supplemented with 500 mg/L phenol at pH 7 and inoculum size 5%v/v 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 APHA, (1989).
Bacterial growth was determined in terms of cell mass by measuring optical density at a wavelength of 500nm.
Biological treatment using P. aeruginosa (NCIM 2074) was the most effective method for removal of phenol. It is also a time saving method compared to other conventional methods.
Influence of temperature of the medium on phenol degradation
Temperature exerts an important regulatory influence on the rate of metabolism. However, little work has been done on the microbiological activity of the organisms present in the water treatment plants operating at lower temperatures. But conventional biological waste treatment processes can operate at low temperature provided sufficient time is allowed for these organisms to degrade in organic wastes. Microbiological degradation of phenol in industrial waste water is affected by temperature in an unexpected manner. The efficiency of treatment by microbiological activity on phenol and other contaminants were significantly good. Four temperature values from (300C, 320C, 330C, 340C) were investigated in Fig.1. Phenol was degraded rapidly at 320C. At this temperature value, phenol degradation was high compared to other values. However, the phenol degradation at temperature 300C, 330C, 340C was slower. These results showed that P. aeruginosa (NCIM 2074) degraded more phenol per day at 320C than at any other temperature value.
Effect of carbon on phenol degradation
Phenol was degraded by P. aeruginosa (NCIM 2074), at different concentrations of glucose (0.5, 1, 2, 3, 4gm/L) as shown in Fig. 2. The presence of glucose in the culture medium increased the tolerance of the organisms to high phenol concentrations by providing a good source readily metabolisable carbon to support cell growth. Hence, it was concluded that glucose on MS medium supported phenol degradation. In summary, these results show that P. aeruginosa (NCIM 2074) is able to tolerate higher levels of phenol when supplemented with glucose as additional sources. The optimum level of glucose was 0.5gm/L. This value shows that higher concentration of glucose however had no effect on phenol degradation. In addition, the rate of phenol degradation was tested.