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Bioresource Technology 99 (2008) 5703–5709

Investigation of the biotransformation of pentachlorophenol

and pulp paper mill effluent decolorisation by the bacterial strains
in a mixed culture
Shail Singh a, R. Chandra a, D.K. Patel b, M.M.K. Reddy b, Vibhuti Rai c,*

a
Environmental Microbiology Section, Industrial Toxicology Research Centre, Post Box No. 80, M.G. Marg, Lucknow, UP 226 001, India
b
Analytical Chemistry Section, Industrial Toxicology Research Centre, Post Box No. 80, M.G. Marg, Lucknow, UP 226 001, India
c
School of Life Sciences, Pandit Ravi Shankar Shukla University, Raipur, CG 492 010, India

Received 25 March 2007; received in revised form 10 October 2007; accepted 11 October 2007
Available online 26 November 2007

Abstract

Mixed culture of two bacterial strains Bacillus sp. and Serratia marcescens showed potential pentachlorophenol (PCP) degradation
and decolorisation of pulp paper mill effluent. The physico-chemical quality of pulp paper mill effluent has been analyzed after 168 h
incubation period degraded by mixed culture. The study revealed that it has decreased high load of BOD, COD, TS, TDS, TSS, sul-
phate, phosphate, total nitrogen, total phenols, metals and different salts (i.e. chloride, sodium, nitrate, potassium) at 168 h incubation
period. PCP degradation in pulp paper mill effluent was confirmed by HPLC analysis. Mixed culture was found to degrade PCP up to
(94%) present in pulp paper mill effluent with 1% glucose and 0.5% peptone (w/v) at 30 ± 1 C, pH 8.0 ± 0.2 at 120 rpm in 168 h
incubation period. The simultaneous release of chloride ion up to 1200 mg/l at 168 h emphasized the bacterial dechlorination in
the medium. The pulp paper mill effluent degradation was also supported by decline in pH, AOX (absorbable organic halides), color,
D.O., BOD, COD and PCP. The analysis of pulp paper mill effluent degradation products by GC–MS analysis revealed the formation
of low molecular weight compound like 2-chlorophenol (RT = 3.8 min) and tetrachlorohydroquinone (RT = 11.86 min) from PCP
extracted degraded sample. Further, mixed culture may be used for bioremediation of PCP containing pulp paper mill waste in
the environment.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Dechlorination; GC–MS; Mixed culture; Pentachlorophenol; Pulp paper mill effluent

1. Introduction 47,000–80,000 gallons of wastewater containing lignin

There are 308 paper mills in India, producing a million
tonne of paper using variety of wood materials (Pandey
and Carney, 1998). Out of these there are 34 large mills,
which contribute more than 51% of the total paper produc-
tion and rest 274 small paper mill produces 49% of the total
paper produce in country. It is estimated that about 273–
455 M3 (60,000–1,00,000 gallons) of water is required per
tonne of paper production and discharges more than
*
Corresponding author. Tel.: +91 (0)771 2262631; fax: +91 (0)771
2262636.
E-mail address: vr_raipur@yahoo.co.in (V. Rai).
and chlorophenols (CPs), which causes soil as well as aqua-
tic pollution where lignin is the polymer of phenolic com-
pounds such as P. coumaryl alcohol, coniferyl alcohol,
sinapyl alcohol and are main component of vascular
plants, providing the plants strength and rigidity (Burnow,
2001). Lignin is heterogenous polymer containing various
biologically stable carbon-to-carbon linkages are inter-
spersed with hemicelluloses. The color of effluent is mainly
due to the presence of lignin and its derivatives. Color not
only is aesthetically unacceptable but also leads to chain of
adverse effect on the aquatic ecosystem. Similarly, penta-
chlorophenol (PCP) from category of CPs, one of the most
hazardous classes of environmental pollutants, has been

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.10.022
5704 S. Singh et al. / Bioresource Technology 99 (2008) 5703–5709

produced in thousand of tons annually by the pulp and Table 1

paper and agrochemical industries. The combined waste- Physico-chemical analysis of pulp paper mill effluent after its biodegra-
dation as compared to control from M/s. Centuary pulp paper mill
water coming out in each process is toxic to all trophic level effluent
in the aquatic ecosystem, affecting flora and fauna, both in
Parameters Control Pulp paper mill effluent after
water column and sediment (Sodergren, 1993). Seasonal biodegradation at 168 h
bacteriological analysis and physico-chemical characteris-
pHa 8.0 ± 0.1 7.0 ± 0.1
tics of Gola river water contaminated with century pulp BOD 4500 ± 5 81.25 ± 10
paper mill effluent was reported by Chandra et al. (2006b). COD 12800 ± 10 128 ± 9
PCP is expected to be recalcitrant to aerobic biodegra- TS 10370 ± 2.08 1350 ± 2
dation because it is highly chlorinated and, in general, aro- TDS 7760 ± 1 1330 ± 4
matic compounds with higher amounts of chlorine are TSS 2610 ± 2 20 ± 1.5
Sulphate 22090 ± 4 9950 ± 5
more resistant to biodegradation (Anandarajah et al., Total phenol 1440 ± 0.01 72.96 ± 1
2000). Bleached chemical pulp mill effluents have been Total nitrogen 14 ± 0.5 Nil
identified as toxic according to the Canadian Environmen- Phosphate ðPO34 Þ 18150 ± 5 6250 ± 16
tal Protection Act due to the presence of high quantity of Nitrate ðNO3Þ 32.88 ± 0.02 16.8 ± 0.08
CPs (Schnell et al., 2000). Potassium (K+) 34.45 ± 0.05 18 ± 0.45
Sodium (Na+) 285 ± 2 59 ± 6
To treat pulp paper mill effluent, biological treatment is Carbon dioxide (CO2) 56.2 ± 0.1 70.01 ± 0.3
superior to the physicochemical methods, because it has
Heavy metals
low treatment costs and possibilities of causing a secondary
Cadmium (Cd) 0.135 ± 0.003 Nil
pollution. Biological treatment of PCP attracts more atten- Chromium (Cr) <0.001 Nil
tion than physical and chemical methods, because a variety Copper (Cu) 0.216 ± 0.004 Nil
of microorganisms are known to utilize PCP as their sole Iron (Fe) 0.182 ± 0.003 Nil
carbon or energy source (Haggblom et al., 1992). Manganese (Mn) 0.04 ± 0.01 Nil
Nickel (Ni) 0.122 ± 0.003 Nil
We have previously described about the Serratia marces-
Lead (Pb) <0.025 ± 0.001 Nil
cens (DQ002385) degrading 300 ppm of PCP in 168 h incu- Zinc (Zn) 0.062 ± 0.001 Nil
bation period by Chandra et al. (2006a) simultaneously a
All values are in mg/l except pH.
Chandra et al. (2007) also reported about the Bacillus sp.
(AY952465) could degrade and decolorize kraft lignin up
to 500 ppm in 120 h. However, in this study mixed culture 2.2. Culture condition
of these two bacterial strains was prepared and attempted
to degradation of PCP and pulp paper mill effluent The mixed culture was used in this study was identified
decolorisation. as Bacillus sp. (AY952465) and S. marcescens (DQ002385)
based on biochemical and 16S rRNA gene sequencing
2. Methods (Chandra et al., 2006a, 2007). One percentage of inoculum
was taken from each bacterial strains Bacillus sp. potential
2.1. Sampling sites and sample collection for kraft lignin degradation and decolorisation designated
as ITRC S8 and S. marcescens potential for PCP degrada-
Pulp paper mill effluent samples were collected from tion designated as ITRC S9 grown in 250 ml Erlenmeyer
M/s. Centuary pulp paper mill, Lalkuan, Nainital, Uttar- flask containing 98 ml of mineral salt media (MSM) of fol-
anchal, India which is located (7910 0 E longitude and lowing composition (mg/l): K2HPO4, 85; KH2PO4, 17;
293 0 N latitude) at foot hills of Himalayas from outlet final MgSO4, 30; FeSO4 Æ 7H2O, 30; CaSO4, 30; MnSO4 Æ H2O,
treated effluent having pH 8.0 containing significant 30; (NH4)2SO4, 17, trace element solution, 1 ml/l contain-
amount of PCP (50.31 mg/l) and lignin (413.8 mg/l) (Raj ing PCP (300 mg/l) and amended with 1% glucose (w/v)
and Chandra, 2004) near wastewater discharged drain site as additional carbon source at pH 7.0 ± 0.2, incubated at
of pulp paper mill. The samples were taken into pre-steril- 30 ± 1 C at 120 rpm (Innova 4230, NJ, USA). Each was
ized containers, test tubes and immediately preserved at capable of growth in MSM containing PCP as sole carbon
4 C. Further, the physico-chemical analysis of pulp paper source. The stability of mixed culture was maintained in
mill effluent as such as a control sample were analyzed for same media and further their mixed culture used for decol-
total solid (TS), total dissolved solid (TDS), total sus- orisation and degradation of pulp paper mill effluent.
pended solid (TSS), biochemical oxygen demand (BOD),
chemical oxygen demand (COD), carbon dioxide (CO2),
total phenols, total nitrogen, sulphate and phosphate as 2.3. Pulp paper mill effluent degradation
per method (APHA, 1998). Metals and different salts (i.e.
chloride, sodium, nitrate and potassium) were analyzed The degradation studies were performed by inoculat-
by atomic absorption spectrophotometer and ion meter ing 1% inoculum of pure and mixed culture in 99 ml
(Orion Model 960) using selective ion electrode at 168 as of filter-sterilized pulp paper mill effluent sample in
shown in Table 1. 250 ml Erlenmeyer flask amended with 1% glucose and
 S. Singh et al. / Bioresource Technology 99 (2008) 5703–5709
0.5% peptone (w/v) as additional carbon and nitrogen
source incubated at 30 ± 1 C in a refrigerated incubator
shaker (Innova 4230, NJ, USA) at 120 rpm up to 168 h.
The culture sample was removed under aseptic condi-
tions. The growth of the mixed culture was determined
by measuring optical density at 620 nm at every 24 h
interval up to 168 h incubation period. Pulp paper mill
effluent (without inoculum) was used as control during
the experimental period. Simultaneously, the PCP dechlo-
rination in pulp paper mill effluent was determined by
estimation of chloride ion released at every 24 h interval
up to 168 h by Orion ion analyzer model 960 (Boston,
USA) using calibrated selective chloride ion electrode.
pH were determined at every 24 h interval up to 168 h
by Orion ion analyzer model 960 (Boston, USA) using
calibrated selective electrode (9172 BN). D.O. (dissolved
oxygen) was measured as partial oxygen pressures using
a Clark-type polarographic DO probe model-835A,
USA of (083010F) electrode, Thermo Orion according
to the manufacture’s instructions (detection level,
0.1 mg/l). AOX (adsorbable organic halide) analyzer
measured AOX. Color reduction during study was mea-
sured according to Bajpai et al. (1993). One milliliter
sample was diluted with 3 ml phosphate buffer (pH
8.0 ± 0.2) to maintain equal pH and to dissolve KL that
adsorb on cells. The samples were centrifuged at 8000g
for 30 min and absorbance was measured at optical den-
sity at 465 nm by using UV–vis spectrophotometer (GBC
Cintra-40, Australia) at every 24 h interval up to 168 h.
The uninoculated media were used as control throughout
the degradation period. At 168 h incubation period over-
all physico-chemical parameters were studied. All experi-
ments were carried out in triplicates. The values were
presented as mean ± SD (n = 3).
2.4. HPLC analysis of PCP in pulp paper mill effluent
In order to estimate the PCP concentration, degraded
pulp paper mill effluent sample were centrifuged at 5000g
for 20 min (Remi C-24) and the supernatant was collected.
For HPLC analysis, the supernatant was initially acidified
to pH 2.0 using 1 N HCl and subsequently extracted three
times using ethyl acetate (99.5%) in 1:1 ratio in a separating
funnel by intermittent shaking. The extracted upper
organic layer containing residual PCP was filtered through
sodium sulphate to absorb excess water. Filtered the sam-
ple with Millipore (0.22 lm filter). Filtered samples were
evaporated to dryness at room temperature, subsequently
resuspended in 5.0 ml acetonitrile (HPLC grade) and ana-
lyzed using Waters 515 model equipped with UV–vis
(Waters-2487, Milford, USA) detector operating at
320 nm. Separation was carried out with a reverse phase
watersmake 5 lm C-18 column (250 · 4.6) mm and the iso-
cratic mobile phase was acetonitrile and water (70:30, v/v)
with a flow rate of 1 ml/min. PCP standard was analyzed
under the same conditions and the utilization of PCP was
estimated by measuring the peak area of the compound.
5705
2.5. GC–MS analysis
The same extracted sample given in above section were
used for GC–MS analysis for qualitative estimation of
PCP and its metabolites degraded by mixed culture at
168 h incubation period along with experimental control.
For GC–MS analysis, 2 ll of control and degraded sample
by mixed culture were injected in GC–MS equipped with a
split/splitless injector and a PE Auto system XL gas chro-
matograph interfaced with a Turbomass spectrometric
mass spectrometric mass selective detector system were
used. The MS was operated in the EI mode (70 eV).
Helium was employed as carrier gas and its flow rate was
adjusted to 1 ml/min. The analytical column connected to
the system was a PE-5MS capillary column (length 20 m,
Diameter i.d. (180 lm), 80 lm film thickness). The GC col-
umn temperature was programmed at 250 C. A solvent
delay of 3 min was selected. The injector temperature was
set at 250 C, and all injections were carried out on the
splitless mode. The GC–MS interface was maintained at
250 C. The oven programme was 55 C hold 2 min/@
15 C/min to 160 C hold 0 min @ 5 C/min to 250 C hold
3 min. The MS was operated in the total ion current (TIC)
mode, scanning from m/z 30 to 400. In the full scan mode,
electron ionization (E1) mass spectra in the range of 30–
400 (m/z) were recorded at electron energy of 70 eV. The
metabolic intermediates were derived from pulp paper mill
effluent degradation identified by comparing their retention
time (RT in min) and mass spectra with that of the
National Institute of Standard and Technology (NIST)
library available RT and mass spectra in the software or
by comparing the RT with those of standard compounds
available.
3. Results and discussion
3.1. Physico-chemical analysis
The physico-chemical analysis of pulp paper mill efflu-
ent (control) and after it degradation at 168 h by mixed cul-
ture as showed in (Table 1). The pulp paper effluent was
found slightly alkaline, as the pH was recorded 8.0 that
decreased up to 7.0 with decrease level of TS (1350),
TDS (1330), TSS (20), COD (128), BOD (81.25), sulphate
(9950), phosphate (6250), total nitrogen (Nil), CO2
(70.01) and phenol (72.96 mg/l), respectively. Heavy metals
such as Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn were decreased
up to zero level and effluent was also characterized by
decrease concentration of ions such as NO 3 (16.8), K
+
+
(18) and Na (59 mg/l) at 168 h incubation period, respec-
tively. The HPLC analysis of effluent revealed that the
effluent was found to contain significant amount of lignin
(413.8 mg/l), which decrease to zero level and PCP
(50.31 mg/l), which decreased up to 3 mg/l. However, the
pulp paper mill effluent did not comply with the permissible
Minimum National Standards (MINAS) limits for other
characteristics such as BOD and COD (CPCB, 1993).
5706 S. Singh et al. / Bioresource Technology 99 (2008) 5703–5709
The presence of oxidisable organic material in pulp paper
mill effluent is associated with increase of BOD, which
causes anoxic condition as evident in this study. High TS,
TDS and TSS of the pulp paper mill waste are directly
associated with raw material used for paper manufacturing
e.g. sugarcane bagasse.
It is well recognized that microorganisms can remove
both soluble and particulate forms of inorganic and
organic chemicals including heavy metals from industrial
effluents by a variety of mechanisms (Gadd, 1988; Silver,
1991; Lovely et al., 1991, 1993; Niu et al., 1993; Erasmus
et al., 2000; Chandra, 2001). The uptake of essential and
non-essential chemicals by biomass can take place by an
active mode (dependent on the metabolic activity) known
as bioaccumulation or by a passive mode (sorption and/
or complexation) termed as biosorption. Shumate and
Strandberg (1985) defined biosorption as ‘‘a non-directed
physico-chemical interaction that may occur between toxic
chemicals and the cellular compounds of biological
species’’.
Microbes accumulate metals such as Cu, Zn, U, Ni, Cd,
Sn, Hg, Mn and non-metals like phosphate, sulphate,
nitrate, sodium, potassium in amounts higher than the
nutritional requirement (Gadd, 1988; Erasmus et al.,
2000; Chandra, 2001). The cell wall of the fungi is the first
to come into contact with metal ions in solution, where the
metals can be deposited on the surface or within the cell
wall structure before interacting with the cytoplasmic
material or the other cellular parts. In extreme cases, for
the living cells, intracellular uptake may take place due to
the increased permeability as a result of cell wall rupture
and subsequent exposure of the metal-binding sites (Gadd,
1990). Biosorption of metal and non-metal ions primarily
occurs by surface binding, including ion-exchange reac-
tions and complexation with the functional groups present
on the cell surface. Various functional groups believed to
be involved in metal binding include carboxyl, amine,
hydroxyl, phosphate and sulfhydryl groups (Strandberg
et al., 1981).
3.2. Pulp paper mill effluent degradation
When the degradation assay was carried out in pulp
paper mill effluent by mixed culture in batch shake flasks
at 30 C, 120 rpm, a marked increase in optical density
growth at 620 nm showed that the small lag phase up to
24 h and then reached prolonged log phase up to 96 h
and thereafter decline phase as shown in Fig. 1. The release
of chloride ion (1200 mg/l) maximum at 96 h and then
decreases up to (134 mg/l) has given strong evidence for
the bacterial degradation of PCP as shown in Fig. 1.
Further, the pure bacterial strains (Bacillus sp. and S.
marcescens) showed the stationary phase after 144 h incu-
bation period as showed in Fig. 1. Mixture of two bacterial
strains showed that it supported the growth of each other
and simultaneously degraded pulp paper mill effluent fast
as compared to alone (data not shown). They grow equally
2.4
Mixed culture
1400
Bacillus sp. 1200
2
Serratia
Absorbance at 620 nm
marcescens 1000
Chloride (mg l-1)
1.6 Chloride ion
release
800
1.2
600
0.8
400
0.4
200
0 0
0 24 48 72 96 120 144 168
Time (h)
Fig. 1. Growth curve of pure [Bacillus sp. (AY952465) and Serratia
marcescens (DQ002385)] and mixed culture and chloride ion release of
mixed culture during pulp paper mill effluent degradation.
well in the medium and are compatible with each other, the
proportion of each strains were estimated at the end of
treatment. The combinations of three isolates manifested
a similar growth pattern to that observed with the pure cul-
ture previously reported by Yu and Ward (1996).
During pulp paper mill effluent degradation, BOD and
COD is simultaneously decreases. Mixed culture concomi-
tantly utilized glucose and PCP for its growth which was
shown by change in color of pulp paper mill effluent from
dark brownish black color to yellowish due to the acidic
pH resulting from release of Cl ion as well as H+ from
PCP aromatic ring during dechlorination in the medium
(Martins et al., 1997) concomitantly low molecular weight
compounds formation due to PCP degradation. This phe-
nomenon indicated that PCP degradation was occurring.
This finding was similar as reported earlier by Premalatha
and Rajkumar (1994) for PCP degradation by Pseudomo-
nas aeruginosa.
The present study supported by Singh and Thakur
(2006), which reported the sequential anaerobic and aero-
bic treatment in two steps bioreactor was performed for
removal of color in the pulp and paper mill effluent. The
anaerobically treated effluent was separately applied in bio-
reactor in presence of fungal strain, Paecilomyces sp. and
bacterial strain, Microbrevis luteum. Data of study indi-
cated reduction in color (95%), AOX (67%), lignin (86%),
COD (88%) and phenol (63%) by Paecilomyces sp. where
as M. luteum showed removal in color (76%), lignin
(69%), COD (75%) AOX (82%) and phenol (93%) by day
third when 7 days anaerobically treated effluent was further
treated by aerobic microorganisms. Change in pH of the
effluent and increase in biomass of microorganisms sub-
stantiated results of the study, which was concomitant to
the treatment method.
The PCP utilized by a mixed culture in pulp paper mill
effluent as a sole source of carbon and energy. Vicuna
(1988) previously reported that a glucose and nitrogen
 S. Singh et al. / Bioresource Technology 99 (2008) 5703–5709 5707

source were essential for degradation of PCP present in

1400 Color Control
pulp paper mill effluent. The simultaneous release of chlo-
ride ion also corroborated the previous findings of dechlo-
1200
rination during PCP degradation as described by Mohn
and Kennedy (1992). Murialdo et al. (2003) and Puhakka
1000
et al. (1995) also reported the degradation of PCP by mixed
culture.

Color (Co-Pt)
800
The PCP degradation by mixed culture leads to a
decrease in from pH 8.0 to 4 at 24 h and then pH value
600
increased up to seven concomitantly D.O. of the media
were changes from 4.5 to 0.1 mg/l at 24 h and then
400
increase up to 2 mg/l as shown in Fig. 2. Mixed culture
decreases AOX from 9.4 to 0.2 mg/l at 168 h as shown
200
in Fig. 2. The treatment resulted in the reduction of color
up to 80% (Fig. 3) was removed from pulp paper mill
0
effluent by mixed culture as compared to control in 0 24 48 72 96 120 144 168
168 h incubation period. A major part of reductions in
Time (h)
these parameters occurred within first 96 h of the treat-
ment, which was also characterized by a steep decline in Fig. 3. Color (platinum–cobalt units) removal from pulp paper mill
the pH of the effluent. effluent by mixed culture.
The degradation of PCP was further monitored by
HPLC chromatography. HPLC showed PCP peak (con- and consumption of oxygen during the biodegradation of
trol) at a retention time of 2.470 min. Biodegraded phenol PCP under aerobic conditions. Further, HPLC analysis
samples showed a very small peak of PCP after 168 as com- confirmed the degradation of PCP (94%) by showing disap-
pared to control at 2.470 min by mixed culture with addi- pearance of the peak of PCP as compared to control.
tional peaks at 1.297, 4.293, 7.550, 11.050, 11.419, 12.761, In the standard degradation experiment described
20.429 min. This observation correlated well with the trans- above, the incubation mixtures were in cotton-plugged
formation of PCP. shake flasks. Under these conditions, atmospheric oxygen
In addition, there was additional peak suggesting that is admitted to the system. However, in present investiga-
there was metabolite formation; which was further con- tion the D.O. content at 48 h was changed for mixed cul-
firmed by GC–MS revealed the biotransformation rather ture as compared to control (Fig. 2). As degradation was
than complete degradation in the low concentration of oxy- not apparently affected by low O2 concentration, inhibition
gen. The data obtained from mixed culture treatment is more likely to be a metabolism dependent event. The
revealed that the presence of the each bacterial strain in decrease of D.O. may be attributed to glucose fermenta-
culture medium showed cumulative enhancing effect for tion, contribute to decrease its concentration in the med-
growth and PCP degradation rather than inhibition. This ium. However, the degradation at low oxygen
observation correlated well with the increase in growth concentrations can be considered to indicate the facultative
microaerophilic nature of the bacteria.

12 4.5

pH AOX DO
3.3. GC–MS analysis
4
10
3.5 GC–MS analysis of PCP degraded sample by mixed cul-
ture and its control in ethyl acetate-extractable products
8 3
showed that in control sample, only PCP (RT = 11.5 min)
pH and AOX

D.O. (mg l-1)

2.5 is identified but in degraded sample the possibility that the

6
2
intermediate was identified using the NIST mass spectral
database in several new peaks were showed, such as 2-chlo-
4 1.5 rophenol (RT = 3.94 min) and tetrachlorohydroquinone
1 (RT = 11.86 min) at 168 h. At 168 h about 94% of the
2 PCP had been degrade showed small peak and only very
0.5 less PCP remained in media, indicating the formation of
0 0 small peak of tetrachlorohydroquinone and large peak of
0 24 48 72 96 120 144 168 2-chlorophenol. It assures from the results that the PCP
Time (h) degradation is at its end of destination.
Fig. 2. pH, AOX and D.O. of mixed culture during pulp paper mill The identification of 2-chlorophenol and tetrachlorohy-
effluent degradation. droquinone would suggest that their products are produced
5708 S. Singh et al. / Bioresource Technology 99 (2008) 5703–5709
by a reductive dechlorination were intermediates prior to
ring cleavage in PCP degradation. GC–MS analysis
revealed the formation of low molecular weight com-
pounds release from PCP degradation was previously
reported by Gerlach and Emon (1997). This result showed
a considerable qualitative difference in the pattern of com-
pounds obtained by PCP degradation by mixed culture in
comparison to that of control sample. Controls did not
show the formation of metabolites from PCP. Metabolites
formation mechanisms based on bond cleavage. The C–Cl
bonds are more fragile in PCP although the O–H bond is
not very much stronger. They originate due to weakening
of the C–Cl bonds of PCP due to decrease in bond
strength. The results verify that the PCP dechlorination
pathway can be tracked from the GC–MS analysis. Similar
to the results of previously reported by Piccinini et al.
(1998), Hong et al. (2000) and Suegara et al. (2005). Spe-
cific metabolites were identified based only on the interpre-
tation of the mass spectra and RT with those of standards
identified compared with those present in the mass analyzer
NIST library. Therefore, this mixed culture may be used
for bioremediation of pulp paper mill effluent.
4. Conclusion
In present work, we describe a potential mixed culture
degrading pulp paper mill effluent with permissible limit
of PCP. It consequently has potential applications in the
biotreatment of high-strength PCP and lignin contami-
nated pulp paper mill effluent.
Acknowledgements
The authors are thankful to Dr. Jai Raj Behari, Head,
Analytical Chemistry Section, Industrial Toxicology Re-
search Centre, Lucknow, India for his suggestions in lignin
and PCP degradation products by GC–MS.
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