April 2011, Volume 2, No.

2
International Journal of Chemical and Environmental Engineering

Phenol Removal from Synthetic Wastewater by

Alcaligenes Faecalis: Online Monitoring
M. Manafi, M. R. Mehrnia*, M. H. Sarrafzadeh
School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran
*Corresponding author: mmehrnia@ut.ac.ir

Abstract
Phenol, a compound regarded as a priority pollutant by the USA environmental protection agency, is a toxic contaminant in
wastewaters and industrial effluents. Biodegradation of phenol have been extensively investigated using pure or mixed cultures in
different kind of bioreactors. In this work, biological removal of phenol by Alcaligenes faecalis has been studied. For petrochemical
industry, even at low concentrations (400 mg/L), phenolic compounds can inhibit microbial growth. Therefore the experiments have
been conducted at different initial concentrations from 400 to 1000 mg/L. In this range the bacterium could utilize phenol in 22 to 100
hours. The Alcaligenes faecalis growth rate and substrate degradation rate were only limited by substrate concentration at fixed
temperature, initial pH and aeration rate. The metabolic pathway for the phenol degradation occurs via catechol derivates, before ortho
oxidation ring cleavage. The ortho oxidation chain contains many stages with different acidity, so pH and DO changes have been
monitored. pH increases to more than 7.16 and then decreases to about 5. There was a logical relation between changes in pH, DO,
cell growth rate and phenol degradation rate. The results of this study can help to control phenol biodegradation more effectively.
Keywords: Phenol biodegradation, Inhibition, Alcaligenes faecalis, Pure culture.

1. Introduction
Phenol is a pollutant in wastewaters and effluent from
Industrial processes such as oil refineries, petrochemical
plants, ceramic plants, steel plants, coal conversion
processes, phenolic resin and pharmaceutical industries
which are containing high concentrations of phenol and
phenolic compounds [1]. Due to the toxic properties,
including permeabilisation of cellular membranes and
cytoplasmic coagulation, phenolic contaminants can
damage sensitive cells and cause profound health and
environmental problems [2]. Therefore the World Health
Organization has limited phenol concentration in drinking
water to 1 mg/L [3]. Toxicity of phenolic compounds
inhibits biological treatment or even eliminates sensitive
micro-organisms from biological wastewater treatment
process and significantly reduces the biodegradation of
the other components [4]. This is why the degradation of
phenol is such a difficult and crucial biological process.
Many aerobic bacteria have been confirmed to use
aromatic compounds as the sole source of carbon and
energy. A typical metabolizing pathway for phenol
degradation is to dihydroxylate the benzene ring to form
catechol and then opening the ring through ortho (Fig. 1)
or meta oxidation pathway [5, 6].
Species of bacteria that are capable of phenol
metabolizing are among Alcaligenes sp. and
Acinetobacter sp., Acromobacter sp., Candida tropicalis,
Rhodococcus sp., Rhodococcus erythropolis, and Candida
maltose, Bacillus thermoleovorans, Pseudomonas putida,
and fungi such as Aspergilius sp., Fusarium sp.,
Penicillum sp., and Graphium sp. [2]. Many bacteria can
degrade phenol at a concentration of about 50–100 mg/L.
due to the increased toxicity of phenol to the microbial
population and in some cases cellular lysis, treatment of
higher concentrations will be difficult [7].
Although phenol can be biodegraded under both
aerobic and anaerobic processes by well acclimated
micro-organisms, aerobic processes are often preferred
[5]. Supplying sufficient oxygen is so important to
guarantee the efficiency of aerobic biodegradation
process. Respirometric measurements can provide
monitoring organic pollutants in wastewater and their
microbial degradability [8]. One such method is the
dissolved oxygen test which can help us to estimate the
efficiency of biological process and microbial growth
through the treatment process.
The follow-up of the medium pH indicates the phenol
degradation progress and can be a significant factor to
demonstrate the success of the biological phenolic
wastewater treatment. Decrease in pH will occur while
the biological degradation of phenol is in progress [9]. As
a result pH monitoring can help to estimate removal
efficiency.
Objective of this study is to monitor the phenol
degradation process with pure culture of Alcaligenes sp.
which has the ability to degrade high concentrations of
phenol with rapid adaptation [10]. It can help us to
determine the variation of parameters through the
biological metabolization. The results of this work can be
 used to indicate phenol biodegradation efficiency more
effectively.
2. Materials and Methods
2.1 Microorganism
The Alcaligenes faecalis PTCC 1624 (ATCC 8750)
was obtained from the Persian Culture Type Collection
(PTCC), Tehran, Iran. The identification of strains
obtained by a range of techniques including
morphological, phenotypic characteristics and DNA/RNA
patterns and sequences. Stork culture of this strain was
maintained by periodic sub transfer on nutrient agar plates
containing 20 g agar per liter medium and then incubated
at 37 °C for about 24 h in an incubator and stored at 4 ⁰C
refrigerator, in sterile conditions. Pure culture of this
microorganism was used in this work.
2.2 Preparation of Microbial Culture
The startup of the experiments was sub transferring
the stork culture on nutrient agar plate which was
incubated at 37°C for about 24 h and then inoculating 10
mL LB medium with microbial strain from nutrient agar
plate.
Figure 1. chemical reaction equations of Phenol degradation
pathway.
After 18 h of incubation at 25 ⁰C, subculture was
obtained by inoculating 0.5 mL of the cell culture to 100
mL fresh LB medium in 250 mL flask at initial pH of 7.2
which was carried out in shaking incubator at 140 rpm
and 25 ⁰C. 7% v/v of subculture medium in the late
exponential growth phase was added to synthetic influent.
The late exponential growth phase was estimated by
optical density (OD600) around 1.2 absorbance units.
2.3 Sterilization
All media and apparatus were autoclaved at 121 ⁰C
for 15 min. Phenol was filter-sterilized through phenol
resistant membranes with pore size of 0.2 µm.
2.4 Analytical Methods
Samples were taken duplicated under positive
pressure. Cell density was determined by measuring the
absorbance of the microorganism at the wavelength of
600 nm (OD600) with a UV spectrophotometer. Then
OD600 results were converted to dry cell weight by a
104
calibration curve, which was obtained by plotting dry
weight of biomass per liter against OD600 [11].
The phenol concentration in supernatant was
evaluated by the photometric method [12]. The method is
based on rapid condensation of phenol with 4-
aminoantipyrene, followed by oxidation with potassium
ferricyanide under alkaline conditions to give a wine red-
colored antipyrine dye. Absorbance of the dye was
measured with a UV spectrophotometer at the wavelength
of 500 nm.
2.5 Experimental system
The bioreactor base unit (W/D/H) was 395x370x550
mm. the vessel was a single wall borosilicate glass with
2.5 L total volume and 1.2 L working volume size.
The bioreactor and probes were autoclavable.
Temperature, stirrer speed, pH, proportion of dissolved
oxygen (pO2) were controllable parameters. Achieving of
data and control was simple. Ports in the head plate were
provided for addition of temperature, pH, and dissolved
oxygen (DO) sensors.
The bioreactor was operated with suspended free cells
of the pure culture microorganism. Filter-sterilized air
through hydrophobic membrane with pore size of 0.2 µm
was supplied at a flow rate of 3 vvm. All experiments
were conducted at fixed stirrer speed of 50 rpm and
temperature of 25 ⁰C.
DO controller output, pO2, is based on the proportion
of dissolved oxygen to the degree of difference between
the actual value and its set point that was 100 for oxygen
saturated medium.
In this study influent contains essential mineral
nutrients with concentrations which are mentioned in
Table 1, in addition to phenol with specific concentration
as will mention in experiment (400, 700 and 1000 mg/L).
3. Results and discussion
In this work batch culture of Alcaligenes faecalis was
studied in media containing just phenol as a sole carbon
and energy source. As a result biodegradation is limited
by phenol concentration only. The most important factor
that can effect negatively on biodegradation progress is
phenol inhibition which is stronger at high phenol
concentration more than 200 mg/L the process. As the
subjective of this study is to evaluate performance of
phenol removal process, the studied phenol
concentrations were 400, 700 and 1000 mg/L while in
1200 mg/L the inhibitory effect of phenol could stop cell
growth and phenol biodegradation. So the inhibitor
concentration of phenol is 1200 mg/L which means the
bacteria could not tolerate substrate toxicity in this case.
therefore studied concentrations is limited to 1000 mg/L
and higher concentrations are not examined.
In order to evaluate the possibility of phenol removal
by aeration or mixing, control experiments were
conducted in the same condition without the bacterium.
The experiments results confirmed that aeration and
mixing do not cause phenol volatilization.
 Fig. 2, shows phenol concentration decrease in
different initial phenol concentrations. Evaluation of
phenol concentration in supernatant demonstrated that the
biodegradation of 400, 700 and 1000 mg/L takes 20, 46
and 94 h and 80% of initial phenol removed in 15, 38 and
72 h. These results proved that the higher is the initial
phenol concentration, the longer it takes the complete
removal. In higher phenol concentration substrate
inhibition was stronger, so increase in initial phenol
concentration to 2.5 times (400 to 1000 mg/L) caused
increasing the degradation time about 5 times (20 to 94
h). In higher substrate concentration inhibition was so
strong that the bacterium failed to degrade phenol.
Table 1. Composition of mineral salt medium in synthetic
wastewater.
Component Concentration (mg/L)
K2HPO4 0.4
KH2PO4 0.2
NaCl 0.1
MgSO4 0.1
MnSO4.H2O 0.01
Fe2(SO4)3.H2O 0.01
Na2MoO4.2H2O 0.01
(NH4)2SO4 0.4
The overall cell concentration at different initial
phenol concentrations is shown in Fig. 3. Results indicate
that in higher phenol concentration, the inhibition was
stronger and therefore the lag phase became longer. In
this case, the lag phase of 400, 700 and 1000 mg/L takes
6, 12 and 26 h. In higher initial phenol concentrations,
before cells got rid of the lag phase, their growth
terminated and death phase occurred.
The stationary phase began while 79% of phenol was
degraded in 400 mg/L experiment (53 mg/L phenol
concentration) which was about 92% and 99% for 700
and 1000 mg/L tests (86 and 12 mg/L phenol
concentration).
Consumption of inhibitory substrate was used mostly
to counteract phenol inhibition not just to synthesis new
cells [10]. This result indicates that in higher phenol
concentration more than 700 mg/L which can cause
higher inhibitory effect, stationary phase of cell growth
was delayed by decrease in phenol concentration through
biodegradation. Due to the removal progress and decrease
in phenol concentration, the inhibition will decrease and
phenol consumption mostly leads to synthesis new cells.
Phenol degradation pathway by Alcaligenes faecalis is
to dihydroxylate the benzene ring to form catechol and
then opening the ring through ortho oxidation to form
cis,cis-muconic acid [10]. Through this pathway that is
shown in Fig. 1, a chain of chemical substances will be
produced besides the 2 enzymes that have different pH
levels. The pH of growth medium at different initial
phenol concentrations can be seen in Fig. 4. pH increases
to more than 7.16 and then decreases to about 5. The pH
increase stage can be a result of catechol formation and its
concentration increase. On the other hand, the decrease in
105
pH can be a sign of cis,cis-muconic acid increase through
ortho oxidation. So pH monitoring in medium can be
used to estimate the stage of degradation pathway.
Fig. 5, shows results of pO2 monitoring at different
initial phenol concentrations. The figure illustrated that,
decrease in pO2 and rate of dissolved oxygen decline was
more significant in low initial phenol concentration.
Results can show clearly that inhibitory effect of high
phenol concentration can cause lower respiration rate at
the start up of biodegradation process.
With progress in phenol removal and so reduction in
substrate inhibition, oxygen consumption increases to its
maximum level. By decrease in phenol concentration
more than 80%, bacterium access to substrate became
limited. The substrate limitation leaded to reduction in
bacterium activity and oxygen consumption.
Fig. 6, shows all discussed parameter for 1000 mg/L
initial phenol concentration experiment simultaneously.
The figure can be split into 5 regions as below:
I. In this region all parameters are changing slowly.
Since cell growth is in lag phase, oxygen
consumption is not noticeable, except at the
beginning of inoculation. While pH is going to
increase, phenol degradation is begun.
II. In the second region, since cell growth is passing the
lag phase, pO2 is decreasing slowly. Phenol removal
is happening faster and pH reaches its maximum.
III. Third region is the main section of the process. In
this region cell growth is in the exponentially phase,
which causes a great decrease in oxygen and phenol
consumption. More than 70% of phenol is degrading
in this period and pH drops to lower than 5.5. At the
end of this phase, the dissolved oxygen reaches its
minimum and cell concentration is maximized.
IV. The forth region is the rapid reduction phase in cell
growth, phenol and oxygen consumption. In this
section pO2 is suddenly increases to a level lower
than its value
Figure 2. Phenol concentration decrease vs. time for different
initial phenol concentrations.
 Figure 3. Cell concentration vs. time for different initial
phenol concentrations.

Figure 4. Variation of pH vs. time for different initial phenol

concentrations.

Figure 6. Phenol concentration, cell concentration, pH and pO2 vs.

time for 1000 mg/L initial phenol concentration.

before the inoculation. It indicates that cells are going

to a situation like the first region, which means cell
growth will stop but this time because of substrate
deficiency.
V. The fifth region is the last period of the experiment in
which all parameters are in stationary phase till
Figure 5. pO2 variation vs. time for different initial phenol
concentrations. complete phenol removal that takes 94 h.

4. Conclusion
In this work batch culture of Alcaligenes faecalis was
studied in media containing just phenol as a sole carbon
source. The studied initial phenol concentrations were
400, 700 and 1000 mg/L. In higher initial phenol
concentrations before cells got rid of the lag phase, death
phase begun. Biodegradation of 400, 700 and 1000 mg/L
phenol takes 20, 46 and 94 h and 80% of initial phenol
removed in 15, 38 and 72 h. results proved that the higher

106
 is the initial phenol concentration, the longer it takes the
complete consumption.
In higher phenol concentration with stronger
inhibition, the lag phase became longer. Due to the
removal progress and decrease in phenol concentration,
the inhibition will decrease and phenol consumption
mostly leads to synthesis new cells.
The ortho degradation pathway chain contains many
stages with different acidity, so pH changes have been
monitored. At first pH increases and then decreases
extremely. The pH increase stage can be a result of
catechol formation through benzene ring dihydroxylation
and on the other hand, the decrease in pH can be a sign of
cis,cis-muconic acid increase through ortho oxidation.
Decrease in pH happened through exponential phase of
cell growth so the pH reduction indicates the phenol
degradation progress and its monitoring can be used to
estimate the stage of degradation pathway.
Results can prove that inhibitory effect can cause
lower respiration rate at the start up of experiments. In the
lag phase of cell growth dissolved oxygen change was not
noticeable which indicates that synthesis of new cells is
the most oxygen demand phase. Through a continuous
process, in the stationary phase of bacterium growth,
oxygen demand can be minimized.
With progress in phenol removal and reduction in
substrate inhibition as a result, oxygen consumption
increases to a maximum level. With decrease in phenol
concentration more than 80%, bacterium access to
substrate is going to be limited which causes decrease in
bacterium activity and oxygen consumption.
107
REFERENCES
[1] V.L. Santos and V.R. Linardi, “Biodegradation of phenol by a
filamentous fungi isolated from industrial effluents_ identification
and degradation potential,” Process Biochem., vol. 39, pp. 1001-
1006, 2004.
[2] G. Tziotzios, M. Teliou, V. Kaltsouni, G. Lyberatos and D.V.
Vayenas, “Biological phenol removal using suspended growth and
packed bed reactors,” Biochem. Eng. J., vol. 26, pp. 65-71, 2005.
[3] J. Yan, W. Jianping, B. Jing and W. Daoquan, “Phenol
biodegradation by the yeast candida tropicalis in the presence of
m-cresol,” Biochem. Eng. J., vol. 29, pp. 227-234, 2006.
[4] W. Gernjak, T. Krutzler, A.G.S. Malato, J. Caceres, R. Bauer, and
A.R. Fernandez-Alba, “Photo-Fenton treatment of water
containing natural phenolic pollutants,” Chemosphere, vol. 50, pp.
71-78, 2003.
[5] J.S. Melo, S. Kholi, A.W. Patwardhan and S.F. D’Souza, “Effect
of oxygen transfer limitations in phenol biodegradation,” Process
Biochem., Vol. 40, pp. 625-628, 2005.
[6] A.B. Martinez, E. Barbot, M.P. Moulin and N. Roche,
“Degradation of synthetic phenol-containing wastewaters by
MBR,” J. Membr. Sci., vol. 281, pp. 288-296, 2006.
[7] Y. Li and Ch. Wang, “Phenol biodegradation in hybrid hollow-
fiber membrane Bioreactors,” World J. Microbiol. Biotechnol.,
vol. 24, pp. 1843-1849, 2008.
[8] E.M. Contreras, M.E. Albertario, N.C. Bertola and N.E. Zaritzky,
“Modelling phenol biodegradation by activated sludges evaluated
through respirometric techniques,” J. Hazard. Mater., vol. 158, pp.
366-374, 2008.
[9] B. Marrot, A. Barrios-Martinez, P. Moulin, and N. Roche,
“Biodegradation of high phenol concentration by activated sludge
in an immersed membrane bioreactor,” Biochem. Eng. J., vol. 30,
pp. 174-183, 2006.
[10] Y. Jiang, J. Wen, J. Bai, X. Jia and Z. Hu, “Biodegradation of
phenol at high initial concentration by Alcaligenes faecalis,” J.
Hazard. Mater., vol. 147, pp. 672-676, 2007.
[11] J. Bai, J.P. Wen, H.M. Li, and Y. Jiang, “Kinetic modeling of
growth and biodegradation of phenol and m-cresol using
Alcaligenes faecalis,” Process Biochem., vol. 42, pp. 510-517,
2007.
[12] L.S. Clesceri, A.E. Greenberg, A.D. Eaton, Standard Methods for
the Examination of Water and Wastewater, 20th ed., American
Public Health Association, American Water Works Association,
Water Pollution Control Federation, Washington, DC., 1998.