Available online at www.sciencedirect.com
Bioresource Technology 99 (2008) 4529–4533
Short Communication
Biotreatment of p-nitrophenol and nitrobenzene in mixed
wastewater through selective bioaugmentation
Xuewei Hu, Aimin Li *, Jun Fan, Conglin Deng, Quanxing Zhang
State Key Laboratory of Pollution Control and Resources Reuse, School of the Environment, Nanjing University, Nanjing 210093, PR China
Received 16 May 2007; received in revised form 19 August 2007; accepted 21 August 2007
Available online 22 October 2007
Abstract
This work combined selective adsorption and bioaugmentation to treat mixed wastewater of nitrobenzene and p-nitrophenol. The
mixed wastewater of nitrobenzene (217 mg/L) and p-nitrophenol (500 mg/L) was adjusted its pH to 8 and then passed through the
adsorption column at 100 mL/h. In effluent the nitrobenzene concentration was less than 4 mg/L. Without the toxic inhibition of nitrobenzene, p-nitrophenol in effluent could be degraded within 60 h through bioaugmentation. About 23 mg/g of nitrobenzene adsorbed the
dry resin HU-05 could be desorbed and degraded through bioaugmentation. During this process the adsorption capacity of the resin
HU-05 was recovered partly. The recovered extent was limited by nitrobenzene bioavailability. The performance of the resin HU-05 kept
stably in the recycle experiments of 60 days.
2007 Elsevier Ltd. All rights reserved.
Keywords: Selective bioaugmentation; Competitive adsorption; Mixed wastewater; Nitrobenzene; p-Nitrophenol
1. Introduction
During the past years, bioaugmentation has been recognized as a potential technology for its biodegradation to
the persistent or toxic organic pollutants (Chen et al.,
2005; Gentry et al., 2004). Under the single pollutant condition, the objective pollutant can be degraded effectively
by the introduced bacteria (Singer et al., 2005). However,
an actual wastewater often contains different pollutants,
even some toxic or inert pollutants. Under these conditions, on the one hand, for either inhibited by the toxic pollutants or prior to metabolize the easy degradable
pollutants, bacteria lose their degradation ability to the
objective pollutants. Quan demonstrated that the introduced 2,4-dichloroPNP-degrading bacterium was prior to
metabolize phenol and lost its degrade ability to 2,4-dichloroPNP when phenol existed in synthetic wastewater
(Quan et al., 2003). On the other hand, the introduced
strains may fail to the competition with the indigenous
*
Corresponding author. Tel.: +86 25 86087698; fax: +86 25 85722627.
E-mail address: [email protected] (A. Li).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.08.039
microorganism and hardly keep their population dominance. In one well-documented instance, bioaugmentation
with the aerobic denitrifying bacterium not only did not
improve nitrification but it resulted in the rapid disappearance of the exogenous bacterium owing to their extensive
predation by protozoa that gained a competitive advantage
(Bouchez et al., 2000). As a result, bioaugmentation is still
experimental although it has been practiced in agriculture
and in wastewater treatment for years (Agathos and El
Fantroussi, 2005). Thus, it is needed to explore one effective technology to avoid these problems above mentioned.
Adsorption is an appropriate technique to separate
objective pollutants from mixed wastewater (Xu et al.,
2003). In recent years some researchers explored to combine adsorption and bioaugmentation for environmental
remediation and obtained some constructive results. The
biological regeneration of HDTMA-modified montmorillonite was realized through bioaugmentation. Nunes
explored the feasibility of through the adsorption and biological regeneration procedure of polymeric adsorbent
XAD-4 for the treatment of effluents contaminated with
molinate (Nunes et al., 2004). However, in their researches
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X. Hu et al. / Bioresource Technology 99 (2008) 4529–4533
they still focused their attention on the single component
wastewater treatment. There are few researches about the
treatment of multicomponent wastewater although it
widely existed in actual environment.
Nitrobenzene and p-nitrophenol coexisted in pesticides
wastewater (Bielska and Szymanowski, 2004). In this
wastewater, the nitrobenzene concentration varied from
20 to 230 mg/L and the p-nitrophenol concentration varied
from 100 to 500 mg/L. For their biological toxicity to
microorganism, the traditional biotreatment could not
treat them economically and effectively. The aim of this
work is to explore a novel method to realize the respective
biodegradation to different pollutants in mixed wastewater.
ter was confected with nitrobenzene (70 mg/L) and p-nitrophenol (470 mg/L). Briefly, 0.1 g (dry weight) adsorbents
were added to a 250 mL conical flask containing 100 mL
synthetic wastewater. The initial pH of the suspension
was adjusted in the range of 4–11 with 1 mol/L NaOH.
Preliminary kinetic experiments demonstrated that the
equilibrium was reached within 24 h. The conical flasks
were then completely sealed with Teflon liner and placed
in a model G25 incubator shaker with 120 r/min at 25 C
for 24 h. After the equilibrium was reached, the aqueous
concentrations of nitrobenzene and p-nitrophenol were
determined.
2.4. The effect of flow rate on adsorption selectivity
2. Methods
2.1. Materials
Polymeric adsorbent HU-05, a synthetic resin constituted by a crosslinked polymer (polystyrene reticulated with
vinylbenzene) which derived its adsorptive properties from
its patented macroreticular structure (large internal surface,
726 m2/g), was obtained from Research Center for Organic
Toxicant Control and Resource Reuse of Jiangsu Province,
China. Nitrobenzene and p-nitrophenol were obtained from
Shanghai Reagent Company. Nitrobenzene and p-nitrophenol which were technical pure had not further purified, other reagents (A.R. grade) used for analysis. The
formula of Nitrobenzene is C6H5NO2, which COD is
about 1.95 Cnitrobenzene, and the formula of p-nitrophenol
is HOC6H4NO2, which COD is about 1.82 Cp-nitrophenol,
where C is the respective aqueous concentration.
Two bacteria used in experiments were previously isolated from the sewage sludge of chemical plants. Bacillus
subtilis, a nitrobenzene-degrading bacterium, can degrade
nitrobenzene when its concentration is less than 140 mg/
L. The p-nitrophenol-degrading bacterium is able to
metabolize p-nitrophenol as sole carbon and energy source
at aqueous p-nitrophenol concentration as high as 800 mg/
L. However, the p-nitrophenol degradation can be inhibited by nitrobenzene toxicity when the nitrobenzene concentration is more than 4 mg/L in mixed wastewater.
2.2. Preparation of the polymeric resin HU-05
Prior to their initial use, the resin HU-05 was firstly
washed with 5 BV 5% hydrochloric acid, where BV was
volume of resin bed, 5 BV tap water and 5 BV 5% sodium
hydroxide at 1 BV/h for three times, where BV was the
total volume of adsorbent bed, then extracted by acetone
for 8 h and dried for 24 h under vacuum at 70 C.
2.3. The effect of pH on adsorption selectivity to
nitrobenzene and p-nitrophenol
Batch experiments were performed to identify the effect
of pH on adsorption selectivity. Synthetic mixed wastewa-
Dynamic adsorption was conducted using a
43 cm · 6.6 mm I.D. glass column packed with 10 mL
(about 7.2 g dry weight) resin HU-05 and connected with
a 6672 reciprocating pump at 25 C. Synthetic wastewater
containing nitrobenzene (217 mg/L) and p-nitrophenol
(500 mg/L) was adjusted pH to 9 and employed for the
dynamic adsorption tests. Under the identical operation
this wastewater passed through the adsorption column at
different flow rate (50, 100, and 150 mL/h). When the nitrobenzene concentration in effluent was over 4 mg/L, the
dynamic adsorption was over.
2.5. The adsorption reversibility of the resin HU-05 to
nitrobenzene
Equilibrium adsorption of nitrobenzene was performed
at 25 C. Briefly, 0.10 g resin was introduced directly into
a 250 mL conical flask, and 100 mL aqueous solution of
nitrobenzene was added into each flask. The initial concentrations (C0) of the solutions were 61, 145, 297, and
492 mg/L, respectively. The flasks were completely sealed
and placed in a G25 model incubator shaker at 25 C
and were shaken under 120 r/min for 24 h. The nitrobenzene equilibrium concentrations (Ce) were determined.
Thus qe (mmol/g), the adsorption capacity, was calculated
according to
qe ¼ V 1 ðC 0 C e Þ=MW ;
where V1 is the volume of solution (L), W is the weight of
dry resin (g), and M is the molecular weight of nitrobenzene. The nitrobenzene-laden resin was taken out and
mixed with inorganic solution. Under the identical condition the desorption process was monitored.
2.6. Nitrobenzene degradation and resin HU-05 regeneration
After the dynamic adsorption was over, 10 mL nitrobenzene-laden resin HU-05 sample was added to a 250 mL
conical flask containing 40 mL inorganic nutrient solution.
After the suspensions were adjusted to neutral, 50 mL
nitrobenzene-degrading bacterium suspension in exponential phase was added to the conical flasks. The mixture
X. Hu et al. / Bioresource Technology 99 (2008) 4529–4533
18
Cp-nitrophenol ( mg L-1)
2.7. The degradation of p-nitrophenol after selective
adsorption
20
500
400
16
300
14
200
12
10
Cnitrobenzene (mgL-1)
was shaken with 120 r/min at 25 C for designed incubation period. The aqueous concentrations of nitrobenzene
were determined regularly. When the nitrobenzene concentration did not further decline, the nitrobenzene remaining
in the resin HU-05 was extracted with ethanol and was
determined. The regenerated resin through bioaugmentation could be then used directly in its wet form in the next
cycles following the same procedures.
4531
100
8
0
To evaluate the effect of pretreatment on p-nitrophenol
degradation, the batch experiments were carried out. The
adsorption effluent was adjusted to neutral and mixed with
the p-nitrophenol-degrading bacterium suspension. After
added essential nutrimental material, the suspension was
introduced to bioreactor for p-nitrophenol degradation.
As comparison, under the identical condition wastewater
without pretreatment was also introduced to another bioreactor directly. The p-nitrophenol degradation process was
monitored regularly.
2.8. Analysis method
Samples were filtered on syringe nylon membrane filters
(0.45 lm pore-size) in order to remove the microorganism.
p-nitrophenol and nitrobenzene were quantified by highperformance liquid chromatograph (HPLC) (JASCO,
PU-1580, Japan) equipped with UV detector (UV-1575)
and C18 reverse-phase column (250 · 4.6 mm, 5 lm
ODS, Kromasil, China). The ratio of methanol and water
in buffer solution was 70:30.
3. Results and discussion
2
10
pH
Fig. 1. The effect of pH on adsorption selectivity. The initial concentration of p-nitrophenol (j) and nitrobenzene(.) were 470 mg/L and 70 mg/
L at 25 C, respectively.
that nitrobenzene and p-nitrophenol can be separated effectively only through adjusting the aqueous pH.
3.2. Effect of the flow rate on the adsorption selectivity
As shown in Fig. 2, during the dynamic adsorption process the p-nitrophenol concentration in adsorption effluent
increased quickly and then approached its initial concentration. With the flow rate rose from 50 mL/h to 150 mL/
h, the nitrobenzene concentration in effluent also increased
from 1.2 mg/L to 3.5 mg/L. When the flow rate reached to
150 mL/h, the nitrobenzene concentration began to over
4 mg/L, at which concentration nitrobenzene might inhibit
the degrading activity of p-nitrophenol-degrading bacterium and cause uptake of following bioaugmentation treatment. So the optimized flow rate was 100 mL/h. When the
3.1. Effect of pH on adsorption selectivity
700
4.0
3.5
Cnitrobenzene ( mgL-1)
600
3.0
500
2.5
2.0
400
1.5
300
Cp-nitrophenol ( mg L-1)
As shown in Fig. 1, the aqueous concentration of pnitrophenol increased when pH rose, which meant the resin
HU-05 lost its sorption capability to p-nitrophenol gradually. When the pH was elevated to 8, the p-nitrophenol concentration approached a maximum. However, the aqueous
nitrobenzene concentration did not have significant change
with this pH variation. These observations can be
explained through the adsorb capability of organic ions
on the hydrophobic adsorbents. Organic ions with small
molecular weights do not adsorb on the hydrophobic
adsorbent in contradistinction to undissociational molecules of the same matter. Therefore the adsorption capacity
of p-nitrophenol depends on the dissociation extent of its
molecules. When the solution pH rose to 8 and exceeded
to its pKa (7.14), p-nitrophenol as organic ion could not
be adsorbed by polymeric resin HU-05. Their difference
of chemical properties under different pH is the base of
selective adsorption. From this result it can be concluded
8
6
4
1.0
200
0.5
0
5
10
15
20
25
adsorption amount (100mL)
Fig. 2. Effect of the flow rate on adsorption selectivity. The nitrobenzene
concentration in the effluent at flow rate of 50 mL/h (j), 100 mL/h (.)
and 150 mL/h (m), respectively. The p-nitrophenol concentration in the
effluent at flow rate of 50 mL/h (h), 100 mL/h (5) and 150 mL/h (4),
respectively. The initial pH was adjusted to 8 in mixed wastewater
containing nitrobenzene (217 mg/L) and p-nitrophenol (500 mg/L).
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X. Hu et al. / Bioresource Technology 99 (2008) 4529–4533
dynamic adsorption was over, about 520 mg nitrobenzene
had been adsorbed in 7.2 g (dry weight) resin HU-05.
3.3. Biodegradation of nitrobenzene and bio-regeneration of
adsorbents
The adsorption and desorption experimental data were
analyzed using Freundlich equation
log qe ¼ log K F þ 1=n log C e ;
where KF and n are characteristic constants.
From the fitting curve we obtained of the nitrobenzene
adsorption isotherm values in mineral medium was
q = 87.51 · C0.33. Desorption experiments confirmed the
reversibility of the adsorption process. So the dynamic
equilibrium of nitrobenzene existed between the aqueous
and the adsorbed nitrobenzene. Degradation of nitrobenzene in wastewater broke up the adsorption equilibrium.
The lower concentration of nitrobenzene in the aqueous
phase served as a driving force to continuously adsorbed
nitrobenzene from the resin. As a result, nitrobenzene
adsorbed in the resin had been desorbed and degraded until
the concentration in aqueous solution was too low to support the growth of nitrobenzene-degrading bacterium.
During this process the adsorption capacity of the resin
HU-05 had been recovered to some extent.
Nitrobenzene remaining in the resin had been extracted
with ethanol and then analyzed. It is well known that the
total desorption from adsorbents with high volume of
micropores is not usual. The results of the extract experiment showed that some nitrobenzene (approximately
360 mg) still remained in the resin HU-05 and become
unavailable for biodegradation. The extent of nitrobenzene
degradation was limited by nitrobenzene bioavailability.
When the aqueous equilibrium concentration of nitrobenzene was below 1.4 mg/L, the nitrobenzene in aqueous
solution was too low to support the growth of nitrobenzene-degrading bacterium. For the initial 520 mg nitrobenzene adsorbed in the resin HU-05, in each bioregeneration
recycle about 160 mg nitrobenzene could be degraded
through bioaugmentation.
3.4. p-Nitrophenol biodegradation through bioaugmentation
To investigate the effect of pretreatment on the p-nitrophenol degradation, the comparing experiments were carried out. Without the selective adsorption pretreatment,
the p-nitrophenol concentration did not declined obviously
during the incubation process. The reason was that 72 mg/
L of the nitrobenzene concentration was so high that the
metabolic activity of p-nitrophenol-degrading bacterium
had been absolute inhibited. The first decrease of the pnitrophenol concentration might be caused by the biosorption. Through the pretreatment most of nitrobenzene had
been removed and its concentration in effluent was less
than 3 mg/L, which was below the bearing limit of p-nitrophenol-degrading bacterium. Without its toxic inhibition,
the p-nitrophenol-degrading bacterium could degrade pnitrophenol in effluent effectively, and the p-nitrophenol
concentration decreased from 400 mg/L to 0 mg/L within
60 h.
3.5. Assessment of resin HU-05 adsorption selectivity during
bioaugmentation
The use of high amounts of substratum and the
respective conversion to biomass, permitted to evaluate
the deterioration of the resin, namely due to the effect
of metabolites and/or debris on adsorption capacity.
After the recycle experiments of 60 days, the adsorption
selectivity of the resin HU-05 to nitrobenzene decreased
to some extent and then kept at a constant level. After
the first bioregeneration cycle, the nitrobenzene concentration in effluent increased from 0.5 mg/L to 1.2 mg/L.
because some nitrobenzene remained in the resin and
formed the new dynamic equilibrium. Thereafter the
nitrobenzene concentration in effluent increased continuously until it reached 1.6 mg/L. Degeneracy of adsorbent
selectivity has been attributed to decay products of
microbial cells (Ha et al., 2001; Vinitnantharat et al.,
2001).
4. Conclusion
Through combining selective adsorption and bioaugmentation, our work realized the respective biodegradation
of nitrobenzene and p-nitrophenol in mixed wastewater
and provided a promising novel method to treat multicomponent wastewater containing different inhibiting pollutants for bioaugmentaion.
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