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Innovative Romanian Food Biotechnology
2007 by “Dunărea de Jos” University – Galaţi Received July 24, 2007 / Accepted September 26, 2007 RESEARCH ARTICLE
BIOREMEDIATION OF PHENOL USING
MICROBIAL CONSORTIUM IN BIOREACTOR
Dipty SINGH and M. H. FULEKAR1*
1Professor & Head Department of Life Sciences, University of Mumbai, Vidyanagari Campus, Santacruz (E) Mumbai-400 098. Abstract
The aerobic bioremediation of phenol has been carried out in a specially designed bioreactor where activated cow dung slurry was used as a source of microbial consortium containing various concentrations of phenol i.e., 100, 250, 500, 1000 mg/L. A phenol concentration of 1000 mg/L was found inhibitory for cow-dung microbial consortium. Higher concentration (1000 mg/L) of phenol degradation was studied by phenol-acclimated cow dung slurry. The acclimated microbial consortium was found able to degrade higher concentrations of phenol. The present study proved effective in removing phenol of higher concentrations even over a period of 7 days (168 hr). The chemical oxygen demand (COD) has found direct correlation of bioremediation of phenol at concentrations where as decrease in biological oxygen demand (BOD) shows growth and metabolic activity of microorganisms under such conditions the phenol concentration was found decreasing to 100 % at the most in the bioreactor. The technology suggested proved useful for the bioremediation of xenobiotics compounds such as phenol. Key words: Bioremediation, Phenol, Bioreactor, Microorganisms
Introduction
petrochemical plants, coal conversion plants and Phenol, one of the most common environmental phenolic resin industries (Hinteregger et al. 1992). pollutants is a simple organic compound possessing High solubility of phenol in water and the higher a hydroxyl group attached to benzene ring or to content in sewage water testify to a greater another more complex aromatic ring system (Ferhan probability of the phenol phenomenon acting as a water pollutant, and deteriorating the organoleptic et al, 2002). Alongside its derivates, phenol is widely-spread in the environment as a consequence qualities of water (Izmerov, 1984). Phenol is of its common presence in the effluents of many considered to be a toxic compound by the Agency industrial processes such as oil refineries, for Toxic Substances and Disease Registry (Agency for Toxic Substance and Disease Registry 2003) and * Corresponding author : mhfulekar@yahoo.com This paper is available on line at http://www.bioaliment.ugal.ro/ejournal.htm Singh and Fulekar: Bioremediation of phenol Innovative Romanian Food Biotechnology (2007) 1, 31 – 36
using microbial consortium in bioreactor

death with ingestion of phenol ranging from 1 to 32 Bioreactor system and operation
g/L has been reported among adults. Although The bioreactor has been designed for bioremediation
absorbed rapidly through the lungs, the low of the phenol. The bioreactor is made up of Glass volatility of phenol and its affinity with water make and SS having dimensions of 20cm x 20cm x 25cm. oral consumption of contaminated water the greatest The provision for aeration by pump has been risk to humans. Phenol vapors are dangerous in provided to maintain the aerobic condition. The concentrations above 0.001 mg/dm3. The lungs condenser is attached on top of the bioreactor to retain almost 90% of inhaled phenols. The condense the vaporized organic compounds. The maximum permissible concentrations (MPCs) of sampling port has been provided on the top of the lid phenols range from 0.1 to 0.001 mg/dm3. Phenol has connected to the bottom of the bioreactor. The traditionally been removed from industrial effluents bioreactor was operated at 250 rpm, 25 oC, 12 mg/L by costly physico-chemical methods, but dissolved oxygen (DO). biodegradation has been studied recently as an
alternative (Fulekar, 2005a, Fulekar, 2005b), on
account of its lower cost associated with this as well Bioremediation studies
as the possibility of complete mineralization of the xenobiotics. All the bioremediation experiments were carried out
in an especially-designed bioreactor. In a lab setup
The present bioremediation technology using the 100, 250, 500, 1000 mg/L concentrations of phenol microbial consortium in aqueous environment under were taken in activated cow-dung slurry. In another controlled environmental conditions in a bioreactor experimental set up 1000 mg/L phenol was taken in will be useful to treat the hazardous waste acclimated cow dung slurry. Two control sets of containing phenol. In the present study the experiments, one of zero phenol concentration and bioremediation of phenol is carried out in a another set without biomass only phenol in sterile specifically-designed bioreactor using microbial water was set up. The bioremediation conditions consortium of various phenol concentrations. This were monitored in the bioreactor. Samples were technology is also useful higher phenol withdrawn initially from 0 hr up to 6 hr then after Material and Method
Analytical procedure
Microbial biomass preparation and
Samples were centrifuged (5 min, 10000 rpm) to acclimatization
separate cell mass and the supernatant was injected The cow dung has been taken as a source of in a HPLC system using UV-VIS Detector and C – biomass (Satsangee et al. 1990) which is diluted 18 Column. The samples were analyzed using the with water in the ratio 1:25 and filtered through following programme: mobile phase methanol-water sieve (20µm) to remove suspended particles. The 50: 50, wavelength 260 nm, flow rate 1 ml / min, prepared cow-dung slurry was aerated and activated isocratic run for 10 min (Pinto et al. 2002). in a glass vessel for a week. The physico-chemical (Jackson, 1973) and microbial characteristics of the
cow dung have been determined after the activation Results and discussion
with 0.1 mg/ml (NH4)2 SO4, 0.2 mg/ml Dextrose, 0.1 The bioremediation of the phenol was carried out in
mg/ml K2HPO4, 0.1 mg/ml KH2PO4 added as a specially designed bioreactor using activated cow- sources of C: N: P for growth and metabolic activity dung consortium. The varying concentrations of stimulation of microbial biomass. The cow-dung phenol were taken in a separate bioreactor for slurry was further acclimated by adding 50 mg/L bioremediation. The physico-chemical phenol under continuous aeration and stirring in a characteristics of cow dung were determined after glass vessel (Satsangee et al, 1996). activation. The values of physico-chemical parameters are presented in Table 1. This paper is available on line at http://www.bioaliment.ugal.ro/ejournal.htm Singh and Fulekar: Bioremediation of phenol Innovative Romanian Food Biotechnology (2007) 1, 31 – 36
using microbial consortium in bioreactor

Table 1. Physico – chemical characterization of activated
parameters, responsible for bioremediation like pH, temperature, dissolved oxygen and nutrient level (C: N: P), were monitored in bioreactor throughout the No. Physico-chemical
characterization
Table 3. Environmental parameters variation observed
Parameters Values
During the bioremediation the variation of parameter like COD and BOD has been carried out, results are presented in Figure 1 and Figure 2. The Figure 1 demonstrates the decrease in COD levels over a The Table shows the presence of inorganic nutrient period of bioremediation which indicates the in cow dung slurry which is used as a source of degradation of phenol by microorganisms present in nutrients by present microorganisms. The microbial cow dung consortium. The decrease in BOD (Figure assessment of cow dung shows the presence of 2) values indicates the growth of microorganisms in bacteria, fungi, and actenomycetes. Table 2 shows the varying concentration of phenol. the total microbiota of cow dung slurry. The data The present bioremediation study was carried out in shows that increase of the temperature indicating a specially-designed and developed bioreactor. In a bioremediation process which tally with findings bioremediation experiment, initial concentrations of that the rate of bioremediation decreases once phenol (100, 250, 500, 1000 mg/L) were taken in The experimental findings indicate that in the case Table 2. Cow – dung slurry microbiota
of 100 mg/L degradation started immediately i.e., there was no lag phase observed in 100 mg/L Bacteria Fungi
Actinomycetes
concentration; 98.59 % degradation of 100 mg/L of phenol was observed over a period of 24 hr. Similarly, 250 mg/L and 500 mg/L were degraded up to 99.4 and 99.6 % within 72 hr and 96 hr, respectively. Concentration of 1000 mg/L phenol was found inhibitory for cow-dung slurry microbiota It is also in agreement with research findings of as it is not degraded up to 168 hr. Anthony I Okoh (2006) who reported highest rate of The present bioremediation of phenol shows that the bioremediation in aqueous environment which un-acclimated cow-dung consortium can degrade up occurred in range of 20-30 oC. The pH variation was to the 500 mg/L concentration of phenol completely found near neutrality as biodegradation rate is within 120 hr. The degradation pattern of phenol highest at a pH near neutrality (Salleh et al. 2003). with time, by un-acclimated biomass, is presented in Dissolved oxygen was also found decreasing Figure 3. indicates the growth and proliferation of microorganisms. The essential environmental This paper is available on line at http://www.bioaliment.ugal.ro/ejournal.htm Singh and Fulekar: Bioremediation of phenol Innovative Romanian Food Biotechnology (2007) 1, 31 – 36
using microbial consortium in bioreactor Time [Hours]
Figure 1. Chemical oxygen demand (COD) variation during phenol bioremediation by cow-dung slurry
The degradation of an inhibitory concentration of acclimated by adding 50 mg/L of phenol under phenol (1000 mg/L) was carried out by acclimated continuous stirring and aeration (10 mg O2/L) in a cow-dung biomass and results were compared to the glass vessel for a period of 96 hr previous experiment. Cow dung slurry was m
D [
15
BO 10
Time [Hours]
Figure 2. Biological oxygen demand (BOD) variation during phenol bioremediation by cow-dung slurry
This paper is available on line at http://www.bioaliment.ugal.ro/ejournal.htm Singh and Fulekar: Bioremediation of phenol Innovative Romanian Food Biotechnology (2007) 1, 31 – 36
using microbial consortium in bioreactor tion 400
tra
n
e
300
Time [Hours]
Figure 3. Time course plot of phenol concentration modification during bioremediation

Next this acclimated cow dung slurry was taken as a phase of 3hr was noticed for 500 mg/L phenol
source of biomass for bioremediation study under (Figure 3). In so doing, the following study reveals
controlled environmental conditions. The above that acclimated cow dung slurry is capable of phenol
bioremediation study shows that degradation of degradation even for higher concentrations. The time
1000 mg/L phenol started within 4hr of experimental course plot of phenol degradation during
set up (Figure 4). Lag phase of only 4 hr was noticed bioremediation by acclimated cow-dung slurry is
in that case, whereas in un-acclimated slurry lag shown in Figure 4.
tion [
tra
n

Time [Hours]
Figure 4. Time course plot of high concentration (1000 mg/L)] phenol modification during bioremediation by
acclimated (*) and un-acclimated (**) cow-dung slurry * In un-acclimated cow-dung slurry; ** In acclimated cow-dung slurry This paper is available on line at http://www.bioaliment.ugal.ro/ejournal.htm Singh and Fulekar: Bioremediation of phenol Innovative Romanian Food Biotechnology (2007) 1, 31 – 36
using microbial consortium in bioreactor

Conclusion
phenolic compounds by Pseudomonas putida EKII. The present study has been carried out to degrade Appl. Microbiol. Biotechnol., 37, 252-259. the phenol in the aqueous environment by use of Izmerov N.F.(1984) Scientific Reviews of Soviet Literature cow dung consortium. The cow dung consortium on toxicity and hazards of chemical, 61, 1-530 (bacteria, fungi and actinomycetes) was found Jackson M.L (1973) Soil Chemical Analysis. Prentice-effective in degrading phenol ranging from 100 to 1000 mg/L. The present finding will be useful to Leahy J.G., Colwell R.R. (1990) Microbial Degradation treat the waste containing phenol to convert the toxicant into nutrient, biomass and CO2 via biodegradation through their intermediates. This Mamma D., Kalogeris .E, Papadopoulos N., technology will be useful to the Petrochemical Hatzinikolaou D.G., Christrakopoulos P., Kekos D.
industry and Chemical industry which generates the (2004) Biodegradation of phenol by acclimatized waste containing compounds such as phenol. The Pseudomonas putida cells using glucose as an added present technology will also be efficient and growth substrate. J. Environ. Sci. Health A Tox. beneficial to treat the waste generated by chemical Hazard Subst. Environ. Eng., 39(8), 2093-2104 Okoh A.I. (2006) Biodegradation alternative in the cleanup of petroleum hydrocarbon pollutants. References
Biotechnology and Molecular Biology Review, 1 (2), 38-50 Ferhan M., Zahoor A., Riazuddin S., Rajoka M. I. and Pinto G., Pollio A., Previtera L., Temussi F. (2002) Khalid A.M. (2002) Estimation and Removal of Biodegradation of phenols by microalgae. Phenol in Pharmaceutical Industrial Effluents from Biotechnology Letters, 24, 2047-2051 Paracetamol and Aspirin Manufacturing Units. Salleh A.B., Ghazali F.M, Zaliha R.N., Rahman A., Basri OnLine Journal of Biological Science, 2 (9), 587-590 M. (2003) Bioremediation of Petroleum Hydrocarbon Fulekar M.H. (2005a) Environmental Biotechnology. Oxford & IBH Publishing House, New Delhi Satsangee R. and Ghosh P. (1990) Anaerobic degradation Fulekar M.H. (2005b) Bioremediation Technologies for of phenol using an acclimated mixed culture. Appl. Environment. IJEP., 25(4), 358 – 364 Microbio. Biotechnol., 34(1), 127-130 Gonzalez G., Herrera M.G., Garcia M.T., Pena M.M. Satsangee R. and Ghosh P. (1996) Continuous anaerobic (2001) Biodegradation of phenol in a continuous phenol degradation using an adapted mixed culture. process: comparative study of stirred tank and World Journal of Microbiology and Biotechnology, fluidized-bed bioreactors. Bioresources Technology, Hinteregger C., Leitner R., Loidl M., Ferschl A., Biodegradation kinetics of 2,4,6-Trichlorophenol by Streichbier F. (1992) Degradation of phenol and an acclimated mixed microbial culture under aerobic conditions. * Note: Innovative Romanian Food Biotechnology is not responsible if on line references cited on manuscripts are not available any more after the date of publication This paper is available on line at http://www.bioaliment.ugal.ro/ejournal.htm

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