You are on page 1of 10

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 ABSTRACT This paper reports the tolerance and biodegradation of phenol
Ainon Hamzah, 1 by a heavy metal–adapted environmental bacterial consortium, known as
and Salmijah Surif 1 consortium culture (CC). At the highest tolerable phenol concentration of
1
School of Environmental and 1200 mg/L, CC displayed specific growth rate of 0.04 h¡1, phenol degradation
Natural Resource Sciences,
rate of 6.11 mg L¡1 h¡1 and biomass of 8.45 § 0.35 (log10 colony-forming
Faculty of Science and
Technology, Universiti units [CFU]/ml) at the end of incubation. Phenol was degraded via the
Kebangsaan Malaysia, Selangor, ortho-cleavage pathway catalyzed by cathechol-1,2-dioxygenase with specific
Malaysia activity of 0.083 (mmol min¡1 mg¡1 protein). The different constituent
2
School of Biotechnology, Dublin bacterial isolates of CC preferentially grow on benzene, toluene, xylene,
City University, Dublin, Republic ethylbenzene, cresol, and catechol, suggesting a synergistic mechanism
of Ireland
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-
Address correspondence to Kok Kee ronment and industrial effluents by physicochemical methods such as ozona-
Wong, School of Environmental and
Natural Resource Sciences, Faculty of tion, activated carbon adsorption, chemical oxidation, and Fenton’s reaction or
Science and Technology, Universiti using conventional biological methods of activated sludges (Gogate and Pandit
Kebangsaan Malaysia, 43600, UKM
Bangi, Selangor, Malaysia. E-mail:
2004). However, these processes are usually complex, ineffective, expensive,
anson_gulf@yahoo.co.uk need enormous amounts of chemicals, leading to secondary pollution and
104
produce hazardous end products (Contreras et al. was then rinsed twice and resuspended using 0.85%
2008). For these reasons, the interest in the use of bio- NaCl to give an OD600nm of 0.5. The consortium cul-
logical methods is increasing. ture was constructed by mixing each of the single iso-
In recent years, phenol degradation focused on lates in equal portion and top up with 0.85% NaCl to
microorganisms widely reported to have evolved cata- give a final reading of OD600nm 0.5, corresponding to
bolic pathways to degrade aromatic compounds (Prad- approximately 1 £ 107 colony-forming units (CFU)/
han and Ingle 2007). However, biocatalytic activities ml as determined by spread plate method (Fellie et al.
via enzymatic reaction in microorganisms can be inhib- 2012). This was used as the starting culture in all subse-
ited by the presence of heavy metals (Mathew, Thanga, quent experiments.
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
Phenol Degradation by Consortium
cadmium (Cd) are among the most toxic metals pre-
dominantly found in wastewater of various industries,
Culture
including hydrocarbon processing, iron/steel All phenol biodegradation studies were performed
manufacturing, electronics manufacturing, and smelt- using freshly prepared Difco (Strasbourgh, France)
ing (The World Bank Group 1999). In this scenario, Bushnell-Haas (BH) medium that contained (g/L)
the use of a mixed culture acclimatized to heavy metals MgSO4, 0.2; CaCl2, 0.02; KH2PO4, 1.0; (NH4)2HPO4,
can have the distinct advantage to overcome the addi- 1.0; KNO3, 1.0; and FeCl2, 0.05. The pH of the
tional stress exerted by metals. medium was adjusted to 7.0 § 0.2 and autoclaved at
In the present research, the use of a consortium cul- 121 C for 15 min.
ture (CC) isolated from heavy metal–contaminated sites A series of Bushnell-Haas (BH) medium containing
with good Cr(VI), Cu(II), and Pb(II) metal removal phenol at 200, 400, 600, 800, 1000, 1200, and
capability (Sannasi et al. 2006) to degrade phenol was 1400 mg/L were inoculated with 1.0 ml (1% v/v) of the
studied. The objective of this study was to investigate CC. The cultures were incubated in an orbital shaker at
the ability of CC to degrade phenol, the effects of Cd 150 rpm (InforsHT Multitron, Einsbach, Germany), at
and Pb towards the biodegradation, and the simulta- 30 C for 28 days. At every 4 days intervals, 1.0 ml of
neous removal of the two metals from the mixed pollu- samples were removed and analyzed for the remaining
tants. The mechanism of which metals ions were phenol residues according to the 4-aminoantipyrine col-
removed from aqueous solutions by CC is discussed. orimetric method based on the Standard Methods for
the Examination of Water and Wastewater (APHA-
AWWA-WPCF 1995). Briefly, bacterial cells from the
MATERIALS AND METHOD culture were removed by centrifuge at 3000 rpm for
10 min. The resulting supernatant was diluted when
Source of Culture necessary using distilled water to fit into the range of
The metal-adapted consortium culture (CC) consist- phenol concentration in the standard curve. To each
ing of six gram-negative (Agrobacterium sp., Chryseomo- test tube 250.0 ml NH4OH (0.5 N) was added and the
nas sp., Flavobacterium sp., Pseudomonas sp., Serratia sp., pH was immediately adjusted to 7.9 § 0.1 using phos-
Xanthomonas sp.) and three gram-positive (Arthobacter phate buffer (104.5 g K2HPO4; 72.3 g KH2PO4 in 1 L).
sp., Bacillus sp., Micrococcus sp.) bacteria was developed To this, 100 ml of 4-aminoantipyrine solution (2% w/v)
from environmental isolates obtained from 10 waste- was added followed by 100 ml of K3Fe(CN)6 (8% w/v),
water metal-based industrial premises in Malaysia. resulting in the formation of red color. The tubes were
They were isolated, selected, characterized, and main- mixed well and allowed to stand for 20 min at room
tained in 10 ppm lead (Pb) and have been shown to temperature. The absorbance of the mixture was read at
successfully remove heavy metals (Sannasi et al. 2006). OD500nm, and the phenol residues were calculated from
Each of these isolates was grown on nutrient broth the standard curve prepared using phenol solutions in
(18 h), and the bacterial cells pelleted by centrifugation the concentration range of 0–0.01 g/L.
(4000 rpm for 15 min at 4 C; Eppendorf Centrifuge The growth of bacterial cells was determined spec-
5804R, New Brunswick, New Jersey, USA). The pellet trophotometrically using 1.0 ml of samples read at

105 Mixed Bacterial Consortium for Phenol Biodegradation


OD600nm using ultraviolet-visible (UV-vis) spectropho- The bacterial cell pellet obtained was resuspended in
tometer (Hitachi U-1900; Tokyo, Japan) as well as by 0.05 M Tris-HCl buffer (pH 9) at 4 C. The bacterial
performing cell counts on Oxoid plate count agar cells were then broken by sonication (15 min, 4 C;
(Fischer Scientific, Leicestershire, UK) using the spread Vibra-Cell; Connecticut, USA), and cell debris were
plate method (Fellie et al. 2012) removed by centrifugation (10,000 rpm, 20 min, 4 C).
The phenol biodegradation rate Qs (mg L¡1 h¡1) was The supernatant was used as the crude enzyme source
calculated from the steepest slope obtained from the for all assays.
plot of phenol residue (mg/L) versus time of incuba- The assay of enzyme catechol-1,2-dioxygenase (E.C.
tion (h), as suggested by Pirt (1975). 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
Growth Analysis reagents were added to a quartz cuvette: 2.0 ml of
Specific growth rate, m (h¡1) of bacterial consortium 50 mM Tris-HCl (pH 8), 0.7 ml of distilled water,
culture was calculated from the maximum slope 0.1 ml of 100 mM 2-mercaptoethanol, and 0.1 ml of
obtained from the exponential growth phase in the supernatant. The contents of the cuvette were mixed
plot of cell density (log10) versus incubation time by inversion several times; finally, 0.1 ml of catechol
(Chen and Taylor 1995). (1 mM) was then added and the contents mixed by
pipetting. Cis,cis-muconic acid formation, the product
Specific growth; m D log10 ðX ¡ X0 Þ=ðt ¡ t0 Þ of ortho-cleavage, was followed by an increase in the
absorbance at 260 nm over a period of 5 min.
where m is specific growth rate (h¡1); X is number of Catechol-2,3-dioxygenase (E.C. 1.13.11.2) activity was
cells (cell/ml) at the end of exponential phase; X0 is followed by measuring the formation of 2-hydroxymu-
number of cells (cell/ml) at the beginning of exponen- conic semialdehyde, the meta-cleavage product of cate-
tial phase; t is time point (h) for [X] and t0 is time point chol. The following reagents were added into a quartz
(h) for [X0]. cuvette in the following order: 2.0 ml of 50 mM Tris-
The yield coefficient YX/S (mg cell mg¡1 phenol) of HCl buffer (pH 7.5), 0.6 ml of distilled water, and 0.2 ml
phenol was calculated using the initial phenol concen- of supernatant. The content in the cuvette was mixed
trations in the culture (Kumar, Kumar, and Kumar using a pipette. Finally, 0.2 ml of catechol (100 mM) as
2005). The value of dry biomass achieved by the CC at substrate to start the reaction was added into the cuvette
the end of exponential phase (from the plot of and mixed again (Farrell and Quilty 2002). Increase in
OD600nm versus time) was used to calculate the bio- absorbance at 375 nm due to the formation of 2-hydroxy-
mass produced as a result of phenol consumption. muconic semialdehyde was recorded over a period of
For dry biomass measurement, overnight culture was 5 min (Feist and Hegeman 1969).
rinsed twice and diluted with NaCl (0.85%) to get a series Specific activity for catechol-1,2-dioxygenase was
of OD600nm reading (0.1–1.2). A 1-ml sample from each calculated by using the extinction coefficient of cate-
OD600nm reading was then pippetted into a preweighted chol of 16,000 M¡1 cm¡1 at 260 nm and for catechol-
Eppendorf tube and spin at 10,000 rpm for 15 min to 2,3-dioxygenase, at absorbance 375 nm equaled to
remove the supernatant. The sample was then transferred 36,000 M¡1cm¡1 (Farrell and Quilty 2002). One unit
into an infrared oven (AND AD4712; Scientech, Brad- of enzyme activity was taken as the amount that cata-
ford, Massachusetts, USA) to dry at 100–105 C for lyzed the formation of 1 mmole of product/min. Spe-
15 min then cooled and weighted. The step was repeated cific activity was calculated as product/min/mg of
until a constant reading was achieved. The dried biomass protein. The amount of protein in the culture was
versus OD600nm reading was then plotted. determined using Bradford (1976) assay.

Catechol Dioxygenase Enzyme Assay


About 20 ml of the CC incubated with 1200 mg/L
Ecotoxicity Test Using Microtox Assay
phenol was harvested (5000 rpm, 10 min, 4 C) after For toxicity testing, supernatant from culture of days
more than half of the phenol was degraded (day 16). 0, 8, 16, and 28 were collected and evaluated using

K. K. Wong et al. 106


Microtox Analyzer 500 (Azur Environmental 1998; Measurement of residual phenol in culture was done
Cambridge, UK). The experimental procedure was according to the 4-aminoantipyrene colorimetric
adopted from the official standards of France (standard method of the Standard Methods for the Examination
AFNOR T90-320-1991). In this, the light emitted by of Water and Wastewater (APHA-AWWA-WPCF
the Vibrio fisheri is reduced upon exposure to toxic sample 1995). Growth of bacterial CC was determined using
and this reduction is directly related to the relative toxicity spread plate method. Analyses of the Cd and Pb resi-
of the sample. The effective concentration for 50% inhibi- dues in the culture medium were performed according
tion of luminescence (EC50) after 5-min incubation was to Sannasi et al. (2006) by filtering the culture (0.45
calculated using a supporting computer software with a mM nitrocellulose membrane), and the heavy metal
standard log-linear model. A color correction was done contents in the filtrate were analyzed using inductively
according to the Microtox instructions. To obtain 50% coupled plasma mass spectrometry (ICP-MS; Perki-
inhibition, the supernatants were diluted wherever neces- nElmer ELAN 9000, Massachusetts, USA).
sary with purified water containing 2% NaCl. This diluent
is also used as nontoxic control.
Statistical Analysis
Data were analyzed by means of one-way analysis of
Growth of Consortium Culture variance (ANOVA) with 95% confidence level using
on Other Aromatic Hydrocarbons SPSS PASW Statistic 17 (SPSS, Chicago, IL, USA) soft-
ware. Results are reported as the mean § standard devi-
Growth of the individual bacterial isolates of CC on
ation (n D 3).
various aromatic hydrocarbons including benzene, tol-
uene, ethylbenzene, xylene, cresol, catechol, and phe-
nol was tested using BH medium containing the given RESULTS
hydrocarbon at 0.5% (v/v; w/v). The BH medium was
inoculated with 5% (v/v) of CC and then incubated at Effects of Phenol on Growth of
30 C on a shaker incubator set at 150 rpm for 7 days. Consortium Culture
When no difference was observed in the turbidity com- Figure 1 and Table 1 present the effects of phenol at
pared with day 0, it was taken as no growth (¡). Signifi- varying initial concentrations (200, 400, 600, 800, 1000,
cant increase in turbidity was taken as good growth (C) 1200, 1400 mg/L) on CC in terms of time needed to
and luxuriant (CC) when the turbidity was >4-fold fully degrade phenol, initial lag phase, specific growth
compared with day 0. rate (m h¡1), and maximum cell biomass achieved (log10
CFU/ml). The measurement of phenol concentration in

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 FIGURE 1 Effects of initial phenol concentration on biodegra-
to reflect conditions of contamination. dation of phenol by the consortium culture.

107 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 § 0.21a


400 — 0.03 4.40 § 0.18b
600 4 0.05 5.96 § 0.23c
800 4 0.06 7.60 § 0.14d
1000 4 0.06 9.44 § 0.31e
1200 8 0.04 8.45 § 0.35e
*Different letters represent significant differences (p < .05). FIGURE 2 Biodegradation of phenol and the growth curve of
the consortium culture at the maximum tolerable concentration
of phenol.

the culture was followed until all the initial phenol pres- growth. After the initial lag phase, rapid degradation
ent was completely degraded. Results as shown in Fig- was observed, with increasing cell growth.
ure 1 indicated that when the initial concentration of The degradation rate, Qs (mg L¡1 h¡1) of CC, as cal-
phenol was increased, more time was needed for com- culated from the highest slope determined from the
plete degradation to be achieved by CC. The toxicity of graph of residual phenol, was 6.11, whereas the specific
different phenol concentration on the growth of CC growth rate (m) and yield coefficient (YX/S) were of
can be evaluated by comparing their respective specific 0.07 h¡1 and 0.04 mg cell mg¡1 phenol, respectively.
growth rate, m (h¡1), and by the colonies formed (log10 Although Pradhan and Ingle (2007) reported on Serratia
CFU/ml) on nutrient agar plates (Table 1). When the plymuthica preadapted to 100 mg/L phenol with similar
phenol concentration was increased from 200 to degradation rate of 6.83 mg L¡1 h¡1 and specific
1000 mg/L, the specific growth rate tripled, i.e., from growth rate (m) of 0.074 h¡1, the phenol concentration
0.02 to 0.06 (h¡1). However, at the highest concentra- used was much lower at 50 mg/L. This study shows
tion of 1200 mg/L, the specific growth rate dropped to that CC is more efficient to degrade phenol at increas-
0.04 h¡1. Similarly, the maximum biomass achieved ing toxicity to support growth even at phenol concentra-
increased accordingly to 4-fold when the phenol con- tions 200-fold higher compared with the phenol-
centration was increased from 200 to 1000 mg/L. At the adapted Serratia plymuthica (Pradhan and Ingle 2007).
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.
Catechol Dioxygenase Activities
However, once the phenol concentration reached The ability of CC to degrade phenol was further
1200 mg/L, it started to be toxic to CC, as can be seen investigated by assaying the activities of catechol-1,2-
by the decrease in specific growth rate (0.04 h¡1) and dioxygenase and catechol-2,3-dioxygenase sampled
biomass (8.45 log10 CFU/ml), a longer lag phase of from day 16 when more than half of the initial phenol
8 days, and the longer duration needed to degrade phe- concentration had been degraded (Table 2). The crude
nol (28 days). This fact is supported by the observation cell extract of CC showed catechol-1,2-dioxygenase
that at the next tested concentrations of 1400 mg/L, activity, but none was detected for catechol-2,3-dioxy-
CC failed to adapt and died. genase. From the chemistry perspective, the presence of
To elucidate the effect of the highest tolerable phe- catechol-1,2-dioxygenase activity demonstrated that
nol concentration on CC, the phenol consumption CC was able to metabolize the benzene-ring structure
curve (mg/L) was plotted against the corresponding cell of phenol via the ortho-cleavage pathway under aerobic
growth (OD600nm) (Figure 2). The degradation curve condition (Wang et al. 2007).
showed little loss of phenol during the initial period of In Table 3, it is shown that different single isolates
day 0 to day 8, which corresponded to the lag phase in within the CC preferentially utilized benzene, toluene,

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

analyzed for Pb and Cd contents at the end of 72 h of


incubation. Figure 4 shows that CC was able to remove metal exposure, more metals were available to be
Pb and Cd at both tested concentrations after 72 h. removed by CC.
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-
DISCUSSION
tration of Pb from 0.1 to 1.0 mg/L did not significantly This study demonstrated the capability of CC con-
inhibit the percentage of Pb removal from the medium. sisting of mixed gram-positive and gram-negative iso-
However, increasing the Cd from 0.01 to 0.1 mg/L sig- lates to live and biodegrade phenol. As many
nificantly reduced (p < .05) the CC ability to remove biodegradation studies focused in using defined single
Cd by 23.6%. But when viewed from the amount cultures, the present research using mixed bacterial cul-
(mg/L) of metals removed, CC was able to remove ture offers more advantages to solving issues pertaining
0.02 (mg/L) Cd from the initial 0.1 mg/L Cd, whereas to mixed pollutants in wastewater. The CC, which con-
only 0.005 mg/L Cd was removed from the initial sists of Agrobacterium sp., Chryseomonas sp., Flavobacte-
0.01 mg/L Cd spiked into the culture medium. In the rium sp., Pseudomonas sp., Serratia sp., Xanthomonas sp.,
case of Pb, 0.45 mg/L was removed from the 1.0 mg/L Arthobacter sp., Bacillus sp., and Micrococcus sp., is
Pb and 0.03 mg/L from the initial 0.1 mg/L Pb. From shown here to degrade much higher concentration of
the results, when the Pb and Cd concentrations were phenol at 1200 mg/L compared with other studies that
increased 10-fold, the metals removed by CC increased uses only single isolates. Kumar, Kumar, and Kumar
by 15- and 4-fold, respectively. This is because at higher (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
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 an intermediate metabolite, catechol. The ring struc-
of incubation. ture of catechol was then broken down by catechol-1,

K. K. Wong et al. 110


2-dioxygenase to produce cis,cis-muconic acid. This In nature, most polluted sites are not confined to a
metabolite could then be further metabolized through single pollutant. Hydrocarbon-related industries such as
a series of catalytic enzymes to produce acetyl-coen- oil refineries and petrol stations are frequently contami-
zyme A (acetyl-CoA) and enters the tricarboxylic acid nated by heavy metals as well. It is known that heavy
cycle (TCA cycle) to produce energy, with CO2 as non- metals, particularly Pb and Cd, have an inhibitory effect
toxic end product (Ellis, Roe, and Wackett 2006). on microbial growth and activity, consequently reducing
Many other hydrocarbons with aromatic ring struc- the degradative ability of microbes (Ke et al. 2010).
ture are reported to be metabolized by catechol dioxye- Exposing CC to 1.0 mg/L Pb or 0.1 mg/L Cd did not
nases. Naphthalene (2 rings), anthracene (3 rings), and impair its ability to degrade phenol, which is a great
phenanthrene (3 rings) were found to be degraded by a advantage with respect to bioremediation, since the con-
Pseudomonas putida G7 by first converting these polyaro- centrations of Pb and Cd used were 10 times higher
matic hydrocarbon (PAH) compounds into cis-dihydro- than the permissible levels allowed by the Environmen-
diol by dioxygenase and then oxidized into catechol tal Quality Act 1974 (Sewage and Industrial Effluents),
(Haritash and Kaushik 2009). The catechol can be reflecting levels of Pb and Cd contamination scenario.
completely degraded via the ortho-pathways catalyzed by Combination of metals and hydrocarbon com-
catechol-1,2-dioxygenase that goes into TCA cycle with- pounds has been reported to cause structural and func-
out generating toxic dead-end metabolites. Thus, the tional damages to bacteria. The combined effects of
advantage of CC displaying catechol-1,2-dioxygenase fluoranthrene and copper resulted in ultrastructural
activity suggests that it can also be used for more effi- impairments, including cytoplamic vacuolization,
cient and rapid biodegradation of various hydrocarbons organelle changes, and anomaly on the multilayered
with similar aromatic structures, such as benzene, tolu- cell walls of marine diatom Phaeodactylum tricornutum
ene, xylene, and ethylbenzene (Fellie et al. 2012). (Wang and Zheng 2008). Amor, Kennes, and Veiga
Although the population dynamics of the CC was (2001) further reported on growth inhibition in Bacillus
not studied during phenol degradation, but based on sp. on alkylbenzene and cadmium (0.33 mM). The
the results on Table 3, the ability of Chryseomonas sp. presence of metal caused additional stress through the
to grow on phenol and not on catechol suggested that induction of reactive oxygen species (ROS), which
the bacteria most probably secreted phenol hydroxy- damage cells membrane, chloroplasts, and mitochon-
lase, but not catechol-1,2-dioxygenase, to metabolize dria, resulting in the inhibition of growth, physiologi-
phenol into intermediate metabolite catechol, to fur- cal activities, and metabolism (Mittler 2002). The
ther cleave the catechol to complete the degradation. ability of CC to tolerate both Pb and Cd can be attrib-
The implication of this is that Chryseomonas sp. is vital uted to the mixed bacterial culture displaying various
in initiating the first step of metabolizing phenol into binding sites such as carboxylate (–COO¡), phospho-
catechol. This probably resulted in a reservoir of cate- ryl/phosphodiester (–PO43¡), and hydroxyl (–OH¡)
chol to be further metabolized by Pseudomonas sp., Ser- within the cell wall that allowed unspecific bindings
ratia sp., Bacillus sp., Arthobacter sp., and Flavobacterium towards metal cations, as previously reported by our
sp., which were shown able to grow on catechol as sole group (Sannasi et al. 2009). In our previous study, CC
carbon source. This pointed to the synergistic mecha- culture exposed to Pb and Cd observed under transmis-
nism occurring within a consortium that consists of dif- sion electron microscope (TEM) showed a dense area
ferent species of bacteria working closely together to around the bacteria compared with control. The dense
complete the degradation of phenol. This is important area showed metal adsorption on the surface of bacte-
because when catechol dioxygenases are inhibited, the rial cells, possibly coated with biosurfactant (exopoly-
key metabolite, catechol, will accumulate and is toxic saccharide), suggesting this to be the mechanism
to microbes (Feist and Hageman 1969; Kumar, Kumar, employed by CC to remove metals from the culture
and Kumar 2005). The Microtox assay further sup- solution to reduce metals toxicity (Wong, Quilty, and
ported this whereby the supernatant showed no inhibi- Surif 2013). This postulation was subsequently demon-
tion of luminescence, demonstrating that complete strated by our ensuing studies in the laboratory show-
phenol degradation has been achieved by CC without ing that CC culture secreted exopolysaccharide (EPS)
generating toxic dead-end metabolites, a distinct advan- under natural circumstance (Mohammad et al. 2014),
tage in bioremediation strategies. and under saline condition (Wong et al. 2014), which

111 Mixed Bacterial Consortium for Phenol Biodegradation


again suggests that CC could bind metals ions on the Contreras, E. M., M. E. Albertario, N. C. Bertola, and N. E. Zaritzky. 2008.
Modelling phenol biodegradation by activated sludges evaluated
cell surface coated by EPS and remove them from the through respirometric techniques. J. Hazard. Mater. 258:366–374.
culture medium. The present study demonstrated that Ellis, L. B. E., D. Roe, and L. P. Wackett. 2006. The university of Minnesota
biocatalysis/biodegradation database: The first decade. Nucleic
CC was adaptable and robust, with tolerance towards
Acids Res. 34:517–521.
toxicity posed by phenol and heavy metals (Pb and Farrell, A., and B. Quilty. 2002. Substrate-dependent autoaggregation of
Cd). In addition, CC’s ability to degrade phenol and Pseudomonas putida CP1 during the degradation of mono-chloro-
phenols and phenol. J. Ind. Microbiol. Biotechnol.28:316–324.
simultaneously remove Pb or Cd is most beneficial for Fellie, E. A., P. Sannasi, K. K. Wong, S. Salmijah, and J. Kader. 2012. Tol-
the purpose of bioremediation in cases of mixed pollu- erance and Biodegradation of benzene, toluene, ethylbenzene and
xylenes (BTEX) by a metal acclimatized bacterial consortium culture.
tants, which are increasingly common due to highly
Res. J. Biotechol. 7:52–58.
developed industrialized area. Feist, C. F., and G. D. Hegeman. 1969. Phenol and benzoate metabolism
by Pseudomonas putida: Regulation of tangential pathways. J. Bac-
teriol. 100:869–877.
Gogate, P. R., and A. Pandit. 2004. A review of imperative technologies
CONCLUSION for wastewater treatment I: Oxidation technologies at ambient
conditions. Adv. Environ. Res. 8:501–551.
Results confirm that CC is able to grow in phenol Haritash, A. K., and C. P. Kaushik. 2009. Biodegradation aspects of polycyclic
aromatic hydrocarbons (PAHs): A review. J. Hazard. Mater. 169:1–15.
and withstand the toxicity to a relatively high concentra- Ke, L., L. J. Luo, P. Wang, T. G. Luan, and N. F. Y. Tam. 2010. Effects of
tion of 1200 mg/L. Complete degradation of phenol metals on biosorption and biodegradation of mixed polycyclic aro-
matic hydrocarbons by a freshwater green alga Selenastrum capri-
via ortho-pathway catalyzed by catechol-1,2-dioxygenase
cornutum. Bioresour. Technol. 101:6950–6961.
was achieved without generating toxic metabolites. CC Kumar, A., S. Kumar, and S. Kumar. 2005. Biodegradation kinetics of
ability to degrade phenol was not compromised in the phenol and catechol using Pseudomonas putida MTCC 1194. Bio-
chem. Eng. J. 22:151–159.
presence of Pb and Cd. Furthermore, CC was able to Lovell, C. R., N. T. Eriksen, A. J. Lewitus, and Y. P. Chen. 2002. Resistance
remove metals during the phenol degradation process. of the marine diatom Thalassiosira sp. to toxicity of phenolic com-
The present findings managed to demonstrate the suc- pounds. Mar. Ecol. Prog. Ser. 229:11–18.
Mathew, A., V. S. G. Thanga, and J. K. Reshma. 2010. Simultaneous phe-
cessful use of CC targeted upon multicomponent pollu- nol degradation and chromium (VI) reduction by bacterial isolates.
tants, which represent a model closer to the real Res. J. Biotechnol. 5:46–49.
Mittler, R. 2002. Oxidative stress, antioxidants and stress tolerance.
situations found in the natural environment. Trends Plant Sci. 7:405–410.
Mohammad, H., A. M. Sahilah, S. A. Palsan, K. K. Wong, and S. Surif.
2014. Enhanced crude oil hydrocarbon degradation by self-immo-
bilized bacterial consortium culture on sawdust and oil palm empty
FUNDING fruit bunch. Ann Microbiol. doi: 10.1007/s13213-014-0821-3.
Pirt, S. J. 1975. Principles of cell cultivation.London: Blackwell Scientific.
The work described in this paper was supported by Pradhan, N., and A. O. Ingle. 2007. Mineralization of phenol by Serratia
plymuthic strain GC isolated from sludge sample. Int. Biodeterior.
Exxon-Mobil STGL-008-2006 and the Malaysian Min-
Biodegrad. 60:103–108.
istry of Science, Technology and Innovation (MOSTI), Saha, N. C., F. Bhunia, and A. Kaviraj. 1999. Toxicity of phenol to fish and
escience fund 06-01-02-SF0469. aquatic ecosystem. Bull. Environ. Contam. Toxicol. 63:195–202.
Sannasi, P., J. Kader, B. S. Ismail, and S. Salmijah. 2006. Sorption of Cr
(VI), Cu (II) and Pb (II) by growing and non-growing cells of bacterial
consortium. Bioresour. Technol. 97:740–747.
REFERENCES Sannasi, P., S. Salmijah, J. Kader, and O. Othman. 2009. Physical growth
and biomass characterization of bacterial cells exposed to Cd(II), Cr
AFNOR T90-320. 1991. Determination de l’inhibition de la luminescence (VI), Cu(II), Ni(II), and Pb(II). J. Environ. Res. Dev. 4:8–18.
de P. phosphoreum. The World Bank Group. 1999. Pollution and abatement handbook: Toward
Amor, L., C. Kennes, and M. C. Veiga. 2001. Kinetics of inhibition in the cleaner production.Washington, DC: The World Bank Group.
biodegradation of monoaromatic hydrocarbons in presence of Wang, T., Y. Tian, B. Han, H. B. Zhao, J. N. Bi, and B. L. Cai. 2007. Bio-
heavy metals. Bioresour. Technol. 78:181–185. degradation of phenol by free and immobilized Acinetobacter sp.
APHA-AWWA-WPCF (American Public Health Association, the American strain PD12. J. Environ. Sci. 19:222–225.
Water Works Association, Water Pollution Control Federation). Wang, L., and B. Zheng. 2008. Toxic effects of fluoranthe and copper on
1989. Standard Methods for the Examination of Water and Waste- marine diatom Phaeodactylum tricornutum. J. Environ. Sci.
water.Washington, DC: American Public Health Association. 20:1363–1372.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation Wong K. K., B. Quilty, and S. Surif. 2013. Degradation of crude oil in the
of microgram quantities of protein utilizing the principle of protein- presence of lead (Pb) and cadmium (Cd) by a metal-adapted con-
dye binding. Anal. Biochem. 72:248–254. sortium culture. Adv. Environ. Biol. 7:577–585.
Chen, C. I., and R. T. Taylor. 1995. Thermophilic biodegradation of BTEX Wong, K. K., H. Mohammad, A. M. Sahilah, S. A. Palsan, and S. Surif.
by two thermos species. Biotechnol. Bioeng. 48:614–624. 2014. Self-immobilised bacterial consortium culture as ready-to-
Coleman, R. N., and A.A. Qureshi. 1985. Microtox! and Spirillum volu- use seed for crude oil bioremediation under various saline condi-
tans tests for assessing toxicity of environmental samples. Bull. Envi- tions and seawater. Int. J. Environ. Sci. Technol. doi: 10.1007/
ron. Contam. Toxicol. 35:443–451. s13762-014-0619-7.

K. K. Wong et al. 112


Copyright of Bioremediation Journal is the property of Taylor & Francis Ltd and its content
may not be copied or emailed to multiple sites or posted to a listserv without the copyright
holder's express written permission. However, users may print, download, or email articles for
individual use.