Bioremediation Journal, 19(2):104–112, 2015
Copyright Ó 2015 Taylor and Francis Group, LLC
ISSN: 1088-9868 print / 1547-6529 online
DOI: 10.1080/10889868.2014.995368
Phenol Biodegradation and Metal Removal
by a Mixed Bacterial Consortium
Kok Kee Wong,1 Brid Quilty,2
Ainon Hamzah, 1
and Salmijah Surif 1
1
School of Environmental and
Natural Resource Sciences,
Faculty of Science and
Technology, Universiti
Kebangsaan Malaysia, Selangor,
Malaysia
2
School of Biotechnology, Dublin
City University, Dublin, Republic
of Ireland
Address correspondence to Kok Kee
Wong, School of Environmental and
Natural Resource Sciences, Faculty of
Science and Technology, Universiti
Kebangsaan Malaysia, 43600, UKM
Bangi, Selangor, Malaysia. E-mail:
anson_gulf@yahoo.co.uk
ABSTRACT This paper reports the tolerance and biodegradation of phenol
by a heavy metal–adapted environmental bacterial consortium, known as
consortium culture (CC). At the highest tolerable phenol concentration of
1200 mg/L, CC displayed specific growth rate of 0.04 h¡1, phenol degradation
rate of 6.11 mg L¡1 h¡1 and biomass of 8.45 § 0.35 (log10 colony-forming
units [CFU]/ml) at the end of incubation. Phenol was degraded via the
ortho-cleavage pathway catalyzed by cathechol-1,2-dioxygenase with specific
activity of 0.083 (mmol min¡1 mg¡1 protein). The different constituent
bacterial isolates of CC preferentially grow on benzene, toluene, xylene,
ethylbenzene, cresol, and catechol, suggesting a synergistic mechanism
involved in the degradation process. Microtox assay showed that phenol
degradation was achieved without producing toxic dead-end metabolites.
Moreover, lead (Pb) and cadmium (Cd) at the highest tested concentration of
1.0 and 0.1 mg/L, respectively, did not inhibit phenol degradation by CC.
Simultaneous metal removal during phenol degradation was achieved using
CC. These findings confirmed the dual function of CC to degrade phenol and
to remove heavy metals from a mixed-pollutant medium.
KEYWORDS biodegradation, cadmium, consortium culture, lead, phenol
INTRODUCTION
Phenol and their derivatives are prevalent in wastewaters of many industries,
including oil refineries and chemical plants (Pradhan and Ingle 2007), and their
traces have been found in both freshwater and marine waters (Saha, Bhunia,
and Kaviraj 1999; Dimou et al. 2006). Pollution by phenols could modify the
biota of environment because most of these recalcitrant compounds exhibit a
high degree of toxicity and they can accumulate in sediments or at different lev-
els in the trophic chain (Lovell et al. 2002); thus, the removal of phenols from
contaminated waters is much desired. Phenols can be removed from the envi-
ronment and industrial effluents by physicochemical methods such as ozona-
tion, activated carbon adsorption, chemical oxidation, and Fenton’s reaction or
using conventional biological methods of activated sludges (Gogate and Pandit
2004). However, these processes are usually complex, ineffective, expensive,
need enormous amounts of chemicals, leading to secondary pollution and
104
produce hazardous end products (Contreras et al.
2008). For these reasons, the interest in the use of bio-
logical methods is increasing.
In recent years, phenol degradation focused on
microorganisms widely reported to have evolved cata-
bolic pathways to degrade aromatic compounds (Prad-
han and Ingle 2007). However, biocatalytic activities
via enzymatic reaction in microorganisms can be inhib-
ited by the presence of heavy metals (Mathew, Thanga,
and Reshma 2010). In most cases, industrial wastes are
mixed wastes, containing organic contaminants such
as phenols as well as heavy metals. Lead (Pb) and
cadmium (Cd) are among the most toxic metals pre-
dominantly found in wastewater of various industries,
including hydrocarbon processing, iron/steel
manufacturing, electronics manufacturing, and smelt-
ing (The World Bank Group 1999). In this scenario,
the use of a mixed culture acclimatized to heavy metals
can have the distinct advantage to overcome the addi-
tional stress exerted by metals.
In the present research, the use of a consortium cul-
ture (CC) isolated from heavy metal–contaminated sites
with good Cr(VI), Cu(II), and Pb(II) metal removal
capability (Sannasi et al. 2006) to degrade phenol was
studied. The objective of this study was to investigate
the ability of CC to degrade phenol, the effects of Cd
and Pb towards the biodegradation, and the simulta-
neous removal of the two metals from the mixed pollu-
tants. The mechanism of which metals ions were
removed from aqueous solutions by CC is discussed.
MATERIALS AND METHOD
Source of Culture
The metal-adapted consortium culture (CC) consist-
ing of six gram-negative (Agrobacterium sp., Chryseomo-
nas sp., Flavobacterium sp., Pseudomonas sp., Serratia sp.,
Xanthomonas sp.) and three gram-positive (Arthobacter
sp., Bacillus sp., Micrococcus sp.) bacteria was developed
from environmental isolates obtained from 10 waste-
water metal-based industrial premises in Malaysia.
They were isolated, selected, characterized, and main-
tained in 10 ppm lead (Pb) and have been shown to
successfully remove heavy metals (Sannasi et al. 2006).
Each of these isolates was grown on nutrient broth
(18 h), and the bacterial cells pelleted by centrifugation
(4000 rpm for 15 min at 4
C; Eppendorf Centrifuge
5804R, New Brunswick, New Jersey, USA). The pellet
105
was then rinsed twice and resuspended using 0.85%
NaCl to give an OD600nm of 0.5. The consortium cul-
ture was constructed by mixing each of the single iso-
lates in equal portion and top up with 0.85% NaCl to
give a final reading of OD600nm 0.5, corresponding to
approximately 1 £ 107 colony-forming units (CFU)/
ml as determined by spread plate method (Fellie et al.
2012). This was used as the starting culture in all subse-
quent experiments.
Phenol Degradation by Consortium
Culture
All phenol biodegradation studies were performed
using freshly prepared Difco (Strasbourgh, France)
Bushnell-Haas (BH) medium that contained (g/L)
MgSO4, 0.2; CaCl2, 0.02; KH2PO4, 1.0; (NH4)2HPO4,
1.0; KNO3, 1.0; and FeCl2, 0.05. The pH of the
medium was adjusted to 7.0 § 0.2 and autoclaved at
121
C for 15 min.
A series of Bushnell-Haas (BH) medium containing
phenol at 200, 400, 600, 800, 1000, 1200, and
1400 mg/L were inoculated with 1.0 ml (1% v/v) of the
CC. The cultures were incubated in an orbital shaker at
150 rpm (InforsHT Multitron, Einsbach, Germany), at
30
C for 28 days. At every 4 days intervals, 1.0 ml of
samples were removed and analyzed for the remaining
phenol residues according to the 4-aminoantipyrine col-
orimetric method based on the Standard Methods for
the Examination of Water and Wastewater (APHA-
AWWA-WPCF 1995). Briefly, bacterial cells from the
culture were removed by centrifuge at 3000 rpm for
10 min. The resulting supernatant was diluted when
necessary using distilled water to fit into the range of
phenol concentration in the standard curve. To each
test tube 250.0 ml NH4OH (0.5 N) was added and the
pH was immediately adjusted to 7.9 § 0.1 using phos-
phate buffer (104.5 g K2HPO4; 72.3 g KH2PO4 in 1 L).
To this, 100 ml of 4-aminoantipyrine solution (2% w/v)
was added followed by 100 ml of K3Fe(CN)6 (8% w/v),
resulting in the formation of red color. The tubes were
mixed well and allowed to stand for 20 min at room
temperature. The absorbance of the mixture was read at
OD500nm, and the phenol residues were calculated from
the standard curve prepared using phenol solutions in
the concentration range of 0–0.01 g/L.
The growth of bacterial cells was determined spec-
trophotometrically using 1.0 ml of samples read at
Mixed Bacterial Consortium for Phenol Biodegradation
OD600nm using ultraviolet-visible (UV-vis) spectropho-
tometer (Hitachi U-1900; Tokyo, Japan) as well as by
performing cell counts on Oxoid plate count agar
(Fischer Scientific, Leicestershire, UK) using the spread
plate method (Fellie et al. 2012)
The phenol biodegradation rate Qs (mg L¡1 h¡1) was
calculated from the steepest slope obtained from the
plot of phenol residue (mg/L) versus time of incuba-
tion (h), as suggested by Pirt (1975).
Growth Analysis
Specific growth rate, m (h¡1) of bacterial consortium
culture was calculated from the maximum slope
obtained from the exponential growth phase in the
plot of cell density (log10) versus incubation time
(Chen and Taylor 1995).
Specific growth; m D log10 ðX ¡ X0 Þ=ðt ¡ t0 Þ
where m is specific growth rate (h¡1); X is number of
cells (cell/ml) at the end of exponential phase; X0 is
number of cells (cell/ml) at the beginning of exponen-
tial phase; t is time point (h) for [X] and t0 is time point
(h) for [X0].
The yield coefficient YX/S (mg cell mg¡1 phenol) of
phenol was calculated using the initial phenol concen-
trations in the culture (Kumar, Kumar, and Kumar
2005). The value of dry biomass achieved by the CC at
the end of exponential phase (from the plot of
OD600nm versus time) was used to calculate the bio-
mass produced as a result of phenol consumption.
For dry biomass measurement, overnight culture was
rinsed twice and diluted with NaCl (0.85%) to get a series
of OD600nm reading (0.1–1.2). A 1-ml sample from each
OD600nm reading was then pippetted into a preweighted
Eppendorf tube and spin at 10,000 rpm for 15 min to
remove the supernatant. The sample was then transferred
into an infrared oven (AND AD4712; Scientech, Brad-
ford, Massachusetts, USA) to dry at 100–105
C for
15 min then cooled and weighted. The step was repeated
until a constant reading was achieved. The dried biomass
versus OD600nm reading was then plotted.
Catechol Dioxygenase Enzyme Assay
About 20 ml of the CC incubated with 1200 mg/L
phenol was harvested (5000 rpm, 10 min, 4
C) after
more than half of the phenol was degraded (day 16).
K. K. Wong et al.
The bacterial cell pellet obtained was resuspended in
0.05 M Tris-HCl buffer (pH 9) at 4
C. The bacterial
cells were then broken by sonication (15 min, 4
C;
Vibra-Cell; Connecticut, USA), and cell debris were
removed by centrifugation (10,000 rpm, 20 min, 4
C).
The supernatant was used as the crude enzyme source
for all assays.
The assay of enzyme catechol-1,2-dioxygenase (E.C.
1.13.11.1) was measured spectrophotometrically by fol-
lowing the formation of cis,cis-muconic acid at absor-
bance 260 nm (Farrell and Quilty 2002). The following
reagents were added to a quartz cuvette: 2.0 ml of
50 mM Tris-HCl (pH 8), 0.7 ml of distilled water,
0.1 ml of 100 mM 2-mercaptoethanol, and 0.1 ml of
supernatant. The contents of the cuvette were mixed
by inversion several times; finally, 0.1 ml of catechol
(1 mM) was then added and the contents mixed by
pipetting. Cis,cis-muconic acid formation, the product
of ortho-cleavage, was followed by an increase in the
absorbance at 260 nm over a period of 5 min.
Catechol-2,3-dioxygenase (E.C. 1.13.11.2) activity was
followed by measuring the formation of 2-hydroxymu-
conic semialdehyde, the meta-cleavage product of cate-
chol. The following reagents were added into a quartz
cuvette in the following order: 2.0 ml of 50 mM Tris-
HCl buffer (pH 7.5), 0.6 ml of distilled water, and 0.2 ml
of supernatant. The content in the cuvette was mixed
using a pipette. Finally, 0.2 ml of catechol (100 mM) as
substrate to start the reaction was added into the cuvette
and mixed again (Farrell and Quilty 2002). Increase in
absorbance at 375 nm due to the formation of 2-hydroxy-
muconic semialdehyde was recorded over a period of
5 min (Feist and Hegeman 1969).
Specific activity for catechol-1,2-dioxygenase was
calculated by using the extinction coefficient of cate-
chol of 16,000 M¡1 cm¡1 at 260 nm and for catechol-
2,3-dioxygenase, at absorbance 375 nm equaled to
36,000 M¡1cm¡1 (Farrell and Quilty 2002). One unit
of enzyme activity was taken as the amount that cata-
lyzed the formation of 1 mmole of product/min. Spe-
cific activity was calculated as product/min/mg of
protein. The amount of protein in the culture was
determined using Bradford (1976) assay.
Ecotoxicity Test Using Microtox Assay
For toxicity testing, supernatant from culture of days
0, 8, 16, and 28 were collected and evaluated using
106
Microtox Analyzer 500 (Azur Environmental 1998;
Cambridge, UK). The experimental procedure was
adopted from the official standards of France (standard
AFNOR T90-320-1991). In this, the light emitted by
the Vibrio fisheri is reduced upon exposure to toxic sample
and this reduction is directly related to the relative toxicity
of the sample. The effective concentration for 50% inhibi-
tion of luminescence (EC50) after 5-min incubation was
calculated using a supporting computer software with a
standard log-linear model. A color correction was done
according to the Microtox instructions. To obtain 50%
inhibition, the supernatants were diluted wherever neces-
sary with purified water containing 2% NaCl. This diluent
is also used as nontoxic control.
Growth of Consortium Culture
on Other Aromatic Hydrocarbons
Growth of the individual bacterial isolates of CC on
various aromatic hydrocarbons including benzene, tol-
uene, ethylbenzene, xylene, cresol, catechol, and phe-
nol was tested using BH medium containing the given
hydrocarbon at 0.5% (v/v; w/v). The BH medium was
inoculated with 5% (v/v) of CC and then incubated at
30
C on a shaker incubator set at 150 rpm for 7 days.
When no difference was observed in the turbidity com-
pared with day 0, it was taken as no growth (¡). Signifi-
cant increase in turbidity was taken as good growth (C)
and luxuriant (CC) when the turbidity was >4-fold
compared with day 0.
Effects of Lead and Cadmium on
Phenol Degradation
A total of four sets of flasks each containing 120 mg/
L phenol in 100 ml of Bushnell-Haas (BH) medium
and lead at 0.1 or 1.0 mg/L and cadmium at 0.01 or
0.1 mg/L, respectively, were inoculated with 5% (v/v)
of consortium culture (CC). The cultures were incu-
bated in an orbital shaker at 150 rpm (InforsHT Multi-
tron), at 30
C for 72 h. At 24 and 72 h, samples were
drawn out and the phenol residue, bacterial growth,
and heavy metal contents were analyzed. The lead (Pb)
and cadmium (Cd) concentrations used in this present
study were chosen based on the permissible level sug-
gested by Environmental Quality Act 1974 Malaysia
(Sewage and Industrial Effluents), and 10 times higher
to reflect conditions of contamination.
107
Measurement of residual phenol in culture was done
according to the 4-aminoantipyrene colorimetric
method of the Standard Methods for the Examination
of Water and Wastewater (APHA-AWWA-WPCF
1995). Growth of bacterial CC was determined using
spread plate method. Analyses of the Cd and Pb resi-
dues in the culture medium were performed according
to Sannasi et al. (2006) by filtering the culture (0.45
mM nitrocellulose membrane), and the heavy metal
contents in the filtrate were analyzed using inductively
coupled plasma mass spectrometry (ICP-MS; Perki-
nElmer ELAN 9000, Massachusetts, USA).
Statistical Analysis
Data were analyzed by means of one-way analysis of
variance (ANOVA) with 95% confidence level using
SPSS PASW Statistic 17 (SPSS, Chicago, IL, USA) soft-
ware. Results are reported as the mean § standard devi-
ation (n D 3).
RESULTS
Effects of Phenol on Growth of
Consortium Culture
Figure 1 and Table 1 present the effects of phenol at
varying initial concentrations (200, 400, 600, 800, 1000,
1200, 1400 mg/L) on CC in terms of time needed to
fully degrade phenol, initial lag phase, specific growth
rate (m h¡1), and maximum cell biomass achieved (log10
CFU/ml). The measurement of phenol concentration in
FIGURE 1 Effects of initial phenol concentration on biodegra-
dation of phenol by the consortium culture.
Mixed Bacterial Consortium for Phenol Biodegradation
TABLE 1 Specific Growth Rate, Maximum Biomass, and the
Observed Lag Phase of CC Grown under 200 to 1200 mg/L Phenol
Phenol Specific Maximum
concentration Lag phase growth m biomass*
(mg/L) (days) (h¡1) (log10 CFU/ml)
200 — 0.02 2.60
400 — 0.03 4.40
600 4 0.05 5.96
800 4 0.06 7.60
1000 4 0.06 9.44
1200 8 0.04 8.45
*Different letters represent significant differences (p < .05).
the culture was followed until all the initial phenol pres-
ent was completely degraded. Results as shown in Fig-
ure 1 indicated that when the initial concentration of
phenol was increased, more time was needed for com-
plete degradation to be achieved by CC. The toxicity of
different phenol concentration on the growth of CC
can be evaluated by comparing their respective specific
growth rate, m (h¡1), and by the colonies formed (log10
CFU/ml) on nutrient agar plates (Table 1). When the
phenol concentration was increased from 200 to
1000 mg/L, the specific growth rate tripled, i.e., from
0.02 to 0.06 (h¡1). However, at the highest concentra-
tion of 1200 mg/L, the specific growth rate dropped to
0.04 h¡1. Similarly, the maximum biomass achieved
increased accordingly to 4-fold when the phenol con-
centration was increased from 200 to 1000 mg/L. At the
highest concentrations of 1200 mg/L, the biomass
(log10 CFU/ml) slightly declined to 8.45 compared with
9.44 of 1000 mg/L. This shows that increasing the phe-
nol from 200 to 1000 mg/L has no toxic effect on CC.
However, once the phenol concentration reached
1200 mg/L, it started to be toxic to CC, as can be seen
by the decrease in specific growth rate (0.04 h¡1) and
biomass (8.45 log10 CFU/ml), a longer lag phase of
8 days, and the longer duration needed to degrade phe-
nol (28 days). This fact is supported by the observation
that at the next tested concentrations of 1400 mg/L,
CC failed to adapt and died.
To elucidate the effect of the highest tolerable phe-
nol concentration on CC, the phenol consumption
curve (mg/L) was plotted against the corresponding cell
growth (OD600nm) (Figure 2). The degradation curve
showed little loss of phenol during the initial period of
day 0 to day 8, which corresponded to the lag phase in
K. K. Wong et al.
§ 0.21a
§ 0.18b
§ 0.23c
§ 0.14d
§ 0.31e
§ 0.35e
FIGURE 2 Biodegradation of phenol and the growth curve of
the consortium culture at the maximum tolerable concentration
of phenol.
growth. After the initial lag phase, rapid degradation
was observed, with increasing cell growth.
The degradation rate, Qs (mg L¡1 h¡1) of CC, as cal-
culated from the highest slope determined from the
graph of residual phenol, was 6.11, whereas the specific
growth rate (m) and yield coefficient (YX/S) were of
0.07 h¡1 and 0.04 mg cell mg¡1 phenol, respectively.
Although Pradhan and Ingle (2007) reported on Serratia
plymuthica preadapted to 100 mg/L phenol with similar
degradation rate of 6.83 mg L¡1 h¡1 and specific
growth rate (m) of 0.074 h¡1, the phenol concentration
used was much lower at 50 mg/L. This study shows
that CC is more efficient to degrade phenol at increas-
ing toxicity to support growth even at phenol concentra-
tions 200-fold higher compared with the phenol-
adapted Serratia plymuthica (Pradhan and Ingle 2007).
Catechol Dioxygenase Activities
The ability of CC to degrade phenol was further
investigated by assaying the activities of catechol-1,2-
dioxygenase and catechol-2,3-dioxygenase sampled
from day 16 when more than half of the initial phenol
concentration had been degraded (Table 2). The crude
cell extract of CC showed catechol-1,2-dioxygenase
activity, but none was detected for catechol-2,3-dioxy-
genase. From the chemistry perspective, the presence of
catechol-1,2-dioxygenase activity demonstrated that
CC was able to metabolize the benzene-ring structure
of phenol via the ortho-cleavage pathway under aerobic
condition (Wang et al. 2007).
In Table 3, it is shown that different single isolates
within the CC preferentially utilized benzene, toluene,
108
TABLE 2 Activities of Catechol Dioxygenases of the Consortium Culture
Specific activity (mmol min¡1 mg¡1 protein)

Total protein (mg/mL) Catechol-1,2-dioxygenase Catechol-2,3-dioxygenase

0.52 0.083 Not detected

xylene, ethylbenzene, cresol, catechol, and phenol. All
the species within CC can grow on phenol, showing
degradation to support growth. Pseudomonas sp., Serra-
tia sp., and Bacillus sp. were able to adapt and grow
well in all the other hydrocarbons tested. Arthobacter
sp. and Flavobacterium sp. can grow on phenol, cate-
chol, and in the case of the latter, cresol as well. Chrys-
eomonas sp. could not grow on cresol and catechol, the
latter being the pivotal and most frequently encoun-
tered intermediate metabolite before ring cleavage dur-
ing phenol degradation.
Ecotoxicity Test Using Microtox Assay
The Microtox test showed inhibition of lumines-
cence from culture samples of days 0, 8, and 16 but
no inhibition for culture obtained at day 28
(Table 4). The different toxicities observed were
reflected by the results of phenol assay whereby phe-
nol degradation at days 8, 16, and 28 were 5.2%,
28.6%, and 100%, respectively (Figure 2). As the ini-
tial phenol concentration decreased due to degrada-
tion, the inhibition of luminescence also decreased.
Results of Microtox assay suggest that complete phe-
nol degradation was achieved at day 28 by CC with-
out producing toxic metabolites that can cause
inhibition of luminescence.
Effects of Lead and Cadmium on
Phenol Degradation
Figure 3 shows that CC culture was able to degrade
phenol in the presence of lead (Pb) or cadmium (Cd). It
can be seen that from 24 h to the end of 72 h, the percent-
age of phenol degraded by CC was significantly increased
despite that the culture was exposed to Pb and Cd at
increasing concentrations. When comparing the two con-
centrations of Pb tested, the percentage of phenol
degraded by CC was not significant different except at
time point 48 h. However, increasing the Cd from 0.01 to
0.1 mg/L significantly decreased the percentage of phenol
degraded by 8.1%, 19.1%, and 2.2% at 24, 48, and 72 h,
respectively. At the end of the 72-h incubation, only CC
exposed to 0.1 mg/L Cd displayed significantly lower
(22.1–23.2%) percentages of phenol degraded when com-
pared with Pb and Cd (0.1 mg/L). These results showed
Cd at higher concentration of 0.1 mg/L increased the
inhibition on phenol degradation. Increasing the Pb from
0.1 to 1.0 mg/L did not inhibit degradation of phenol by
CC. The results also suggest that the efficiency of degrada-
tion by CC was both metal and dose dependent.
Lead and Cadmium Removal
To investigate the ability of CC to degrade phenol
and remove metals simultaneously, the filtrate was

TABLE 3 Growth of Different Bacterial Isolates on Different Hydrocarbons

Hydrocarbon

Isolate Benzene Toluene Xylene Ethyl benzene Cresol Catechol Phenol

Agrobacterium sp. C CC CC CC C CC CC
Chryseomonas sp. C C C CC ¡ ¡ C
Flavobacterium sp. ¡ ¡ ¡ ¡ C C C
Pseudomonas sp. CC CC CC CC CC CC CC
Serratia sp. CC CC CC CC CC CC CC
Xanthomonas sp. C C C CC CC CC CC
Arthobacter sp. ¡ ¡ ¡ ¡ ¡ C C
Bacillus sp. CC CC CC CC CC CC CC
Micrococcus sp. C CC C CC C C CC
Note. Semiquantitative level of growth of different isolates tested on different hydrocarbons: CC (luxuriant); C (good); ¡ (none).

109 Mixed Bacterial Consortium for Phenol Biodegradation

TABLE 4 Toxicity Results of CC Incubated with 1200 mg/L
Phenol at 30
C for 28 Days
Day Microtox (Vibrio fisheri)
0 5 (highly toxic)
8 18 (highly toxic)
16 62 (toxic)
28 >100 (nontoxic)
Note. Coleman and Qureshi (1985) suggested following rating of
toxicity on the basis of % inhibition of luminescence towards
Microtox for assessing toxicity of environmental samples: EC50 (%)
<25 (highly toxic); 25–50 (moderately toxic); 51–75 (toxic); >75
(slightly toxic); >100 (nontoxic).
analyzed for Pb and Cd contents at the end of 72 h of
incubation. Figure 4 shows that CC was able to remove
Pb and Cd at both tested concentrations after 72 h.
After 72 h, highest metal removal in terms of per-
centage was found in 0.1 mg/L Pb (44.9%), followed
by 0.01 mg/L Cd (45.3%), 0.1 mg/L Pb (30.0%), and
lowest in 0.1 mg/L Cd (21.7%). Increasing the concen-
tration of Pb from 0.1 to 1.0 mg/L did not significantly
inhibit the percentage of Pb removal from the medium.
However, increasing the Cd from 0.01 to 0.1 mg/L sig-
nificantly reduced (p < .05) the CC ability to remove
Cd by 23.6%. But when viewed from the amount
(mg/L) of metals removed, CC was able to remove
0.02 (mg/L) Cd from the initial 0.1 mg/L Cd, whereas
only 0.005 mg/L Cd was removed from the initial
0.01 mg/L Cd spiked into the culture medium. In the
case of Pb, 0.45 mg/L was removed from the 1.0 mg/L
Pb and 0.03 mg/L from the initial 0.1 mg/L Pb. From
the results, when the Pb and Cd concentrations were
increased 10-fold, the metals removed by CC increased
by 15- and 4-fold, respectively. This is because at higher
FIGURE 3 Percentages of phenol degraded in the presence of
Pb (0.1 and 1.0 mg/L) and Cd (0.01 and 0.1 mg/L) by CC after 72 h
of incubation.
K. K. Wong et al.
FIGURE 4 Pb and Cd removal by CC exposed to phenol at the
end of 72 h of incubation.
metal exposure, more metals were available to be
removed by CC.
DISCUSSION
This study demonstrated the capability of CC con-
sisting of mixed gram-positive and gram-negative iso-
lates to live and biodegrade phenol. As many
biodegradation studies focused in using defined single
cultures, the present research using mixed bacterial cul-
ture offers more advantages to solving issues pertaining
to mixed pollutants in wastewater. The CC, which con-
sists of Agrobacterium sp., Chryseomonas sp., Flavobacte-
rium sp., Pseudomonas sp., Serratia sp., Xanthomonas sp.,
Arthobacter sp., Bacillus sp., and Micrococcus sp., is
shown here to degrade much higher concentration of
phenol at 1200 mg/L compared with other studies that
uses only single isolates. Kumar, Kumar, and Kumar
(2005) reported on Pseudomonas putida MTCC 1194
that degraded maximum 1000 mg/L phenol. Similarly,
Pradhan and Ingle (2007) reported on Serratia plymuth-
ica degrading up to 1050 mg/L of phenol. When a
mixed culture is used, more enzymes can be harnessed
to facilitate and enhance the degradation process. Of
the numerous enzymes involved in the phenol meta-
bolic pathway, key enzymes such as catechol dioxyge-
nase or phenol dehydrogenase are responsible for
cleaving the benzene ring of the phenol structure (Feist
and Hegeman 1969). Catechol-1,2-dioxygenase activi-
ties indicated that the recalcitrant aromatic ring is bro-
ken via ortho-cleavage pathway by CC, whereby the
benzene ring of phenol was first hydroxylated to form
an intermediate metabolite, catechol. The ring struc-
ture of catechol was then broken down by catechol-1,
110
2-dioxygenase to produce cis,cis-muconic acid. This
metabolite could then be further metabolized through
a series of catalytic enzymes to produce acetyl-coen-
zyme A (acetyl-CoA) and enters the tricarboxylic acid
cycle (TCA cycle) to produce energy, with CO2 as non-
toxic end product (Ellis, Roe, and Wackett 2006).
Many other hydrocarbons with aromatic ring struc-
ture are reported to be metabolized by catechol dioxye-
nases. Naphthalene (2 rings), anthracene (3 rings), and
phenanthrene (3 rings) were found to be degraded by a
Pseudomonas putida G7 by first converting these polyaro-
matic hydrocarbon (PAH) compounds into cis-dihydro-
diol by dioxygenase and then oxidized into catechol
(Haritash and Kaushik 2009). The catechol can be
completely degraded via the ortho-pathways catalyzed by
catechol-1,2-dioxygenase that goes into TCA cycle with-
out generating toxic dead-end metabolites. Thus, the
advantage of CC displaying catechol-1,2-dioxygenase
activity suggests that it can also be used for more effi-
cient and rapid biodegradation of various hydrocarbons
with similar aromatic structures, such as benzene, tolu-
ene, xylene, and ethylbenzene (Fellie et al. 2012).
Although the population dynamics of the CC was
not studied during phenol degradation, but based on
the results on Table 3, the ability of Chryseomonas sp.
to grow on phenol and not on catechol suggested that
the bacteria most probably secreted phenol hydroxy-
lase, but not catechol-1,2-dioxygenase, to metabolize
phenol into intermediate metabolite catechol, to fur-
ther cleave the catechol to complete the degradation.
The implication of this is that Chryseomonas sp. is vital
in initiating the first step of metabolizing phenol into
catechol. This probably resulted in a reservoir of cate-
chol to be further metabolized by Pseudomonas sp., Ser-
ratia sp., Bacillus sp., Arthobacter sp., and Flavobacterium
sp., which were shown able to grow on catechol as sole
carbon source. This pointed to the synergistic mecha-
nism occurring within a consortium that consists of dif-
ferent species of bacteria working closely together to
complete the degradation of phenol. This is important
because when catechol dioxygenases are inhibited, the
key metabolite, catechol, will accumulate and is toxic
to microbes (Feist and Hageman 1969; Kumar, Kumar,
and Kumar 2005). The Microtox assay further sup-
ported this whereby the supernatant showed no inhibi-
tion of luminescence, demonstrating that complete
phenol degradation has been achieved by CC without
generating toxic dead-end metabolites, a distinct advan-
tage in bioremediation strategies.
111
In nature, most polluted sites are not confined to a
single pollutant. Hydrocarbon-related industries such as
oil refineries and petrol stations are frequently contami-
nated by heavy metals as well. It is known that heavy
metals, particularly Pb and Cd, have an inhibitory effect
on microbial growth and activity, consequently reducing
the degradative ability of microbes (Ke et al. 2010).
Exposing CC to 1.0 mg/L Pb or 0.1 mg/L Cd did not
impair its ability to degrade phenol, which is a great
advantage with respect to bioremediation, since the con-
centrations of Pb and Cd used were 10 times higher
than the permissible levels allowed by the Environmen-
tal Quality Act 1974 (Sewage and Industrial Effluents),
reflecting levels of Pb and Cd contamination scenario.
Combination of metals and hydrocarbon com-
pounds has been reported to cause structural and func-
tional damages to bacteria. The combined effects of
fluoranthrene and copper resulted in ultrastructural
impairments, including cytoplamic vacuolization,
organelle changes, and anomaly on the multilayered
cell walls of marine diatom Phaeodactylum tricornutum
(Wang and Zheng 2008). Amor, Kennes, and Veiga
(2001) further reported on growth inhibition in Bacillus
sp. on alkylbenzene and cadmium (0.33 mM). The
presence of metal caused additional stress through the
induction of reactive oxygen species (ROS), which
damage cells membrane, chloroplasts, and mitochon-
dria, resulting in the inhibition of growth, physiologi-
cal activities, and metabolism (Mittler 2002). The
ability of CC to tolerate both Pb and Cd can be attrib-
uted to the mixed bacterial culture displaying various
binding sites such as carboxylate (–COO¡), phospho-
ryl/phosphodiester (–PO43¡), and hydroxyl (–OH¡)
within the cell wall that allowed unspecific bindings
towards metal cations, as previously reported by our
group (Sannasi et al. 2009). In our previous study, CC
culture exposed to Pb and Cd observed under transmis-
sion electron microscope (TEM) showed a dense area
around the bacteria compared with control. The dense
area showed metal adsorption on the surface of bacte-
rial cells, possibly coated with biosurfactant (exopoly-
saccharide), suggesting this to be the mechanism
employed by CC to remove metals from the culture
solution to reduce metals toxicity (Wong, Quilty, and
Surif 2013). This postulation was subsequently demon-
strated by our ensuing studies in the laboratory show-
ing that CC culture secreted exopolysaccharide (EPS)
under natural circumstance (Mohammad et al. 2014),
and under saline condition (Wong et al. 2014), which
Mixed Bacterial Consortium for Phenol Biodegradation
again suggests that CC could bind metals ions on the
cell surface coated by EPS and remove them from the
culture medium. The present study demonstrated that
CC was adaptable and robust, with tolerance towards
toxicity posed by phenol and heavy metals (Pb and
Cd). In addition, CC’s ability to degrade phenol and
simultaneously remove Pb or Cd is most beneficial for
the purpose of bioremediation in cases of mixed pollu-
tants, which are increasingly common due to highly
developed industrialized area.
CONCLUSION
Results confirm that CC is able to grow in phenol
and withstand the toxicity to a relatively high concentra-
tion of 1200 mg/L. Complete degradation of phenol
via ortho-pathway catalyzed by catechol-1,2-dioxygenase
was achieved without generating toxic metabolites. CC
ability to degrade phenol was not compromised in the
presence of Pb and Cd. Furthermore, CC was able to
remove metals during the phenol degradation process.
The present findings managed to demonstrate the suc-
cessful use of CC targeted upon multicomponent pollu-
tants, which represent a model closer to the real
situations found in the natural environment.
FUNDING
The work described in this paper was supported by
Exxon-Mobil STGL-008-2006 and the Malaysian Min-
istry of Science, Technology and Innovation (MOSTI),
escience fund 06-01-02-SF0469.
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