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Biodegradation of phenol and nitrobenzene in the interactional

inhibitory system through the selective bioaugmentation

Xuewei HU1, 2 Ping Ning 1* Nguyễn Đình Trung1

1 Department of Environmental Science & Engineering Kunming University of science & technology,

Kunming 650093, P.R. China

Aimin LI2 * 2 State Key Laboratory of Pollution Control and Resources Reuse School of the Environment Nanjing University, Nanjing 210008, P.R. China

Abstract:

This work studies the feasibility of combining selective

adsorption and bioaugmentation to treat the interactional

inhibitory wastewater containing nitrobenzene and phenol.

Through adjusting the pH value of mixed wastewater and

the adsorption flow rate, nitrobenzene could be separated

from the mixed wastewater. Nitrobenzene adsorbed into

resin could be desorbed by the driving force of

concentration difference and be biodegraded through

bioaugmentation. At the same time the adsorption capacity

of resin HU-03 was recovered partly. Without the toxic

inhibition of nitrobenzene, the residual phenol in

adsorption effluent could be degraded effectively through

bioaugmentaiton. After the recycle experiments of 60 days,

the adsorption selectivity of the resin HU-03 to

nitrobenzene decreased to some extent and then kept at a

constant level. This method not only reduces the

interactional toxic inhibition of two compounds, but also

realizes the respective biodegradation to different

pollutants.

Key words: Adsorption ； Selective bioaugmentation； Interactional inhibition system； Nitrobenzene； Phenol; Wastewater treatment

I. INTRODUCTION

The objective of this study is to explore an effective

method to deal with mixed wastewater and realized the

pertinence biodegradation on different pollutants.

Nitrobenzene and aniline were chosen as target

recalcitrant substances in a binary mutual inhibitory

wastewater system. First, nitrobenzene was separated

effectively from wastewater through selective adsorption.

Without the toxic inhibition of nitrobenzene and the

biological competition of different constrains, the

residual phenol in adsorption effluent could be degraded

effectively through bioaugmentation. The nitrobenzene

adsorbed onto resin was desorbed and degraded through

bioaugmentation. At the same time the sorption capacity

of resin HU-03 has been recovered. The work described

here is of practical significance to explore an effective,

environmental friendly and economical method to treat

multicomponent wastewater. II. Material and methods

2.1 Material

Resin HU-03 was obtained from Research Center

for Organic Toxicant Control and Resource Reuse of

Jiangsu province, China. All other chemicals and

solvents used were analytical grade or better (Beijing

Chemical Reagent Co.).The nitrobenzene-degrading

bacterium and the phenol-degrading bacterium were

original screened respectively from the sewage sludge in

the chemical plant.

2.2 Preparation of adsorbents and bacteria Prior to their initial use, HU-03 were firstly washed

with 50 ml 5% hydrochloric acid, 50 ml tap water and 50

ml 5% sodium hydroxide at 10 ml h-1 at least three times,

then extracted by acetone for 8 h and dried for 24 h

under vacuum at 70 ℃. Two bacteria were inoculated

into its respective inorganic nutrient culture and then

shaken at 120 r min-1 and 25○C for 36h. The pre-grown

mixed cultures were centrifuged at 5000 r min-1 for 5 min

and the upside liquid was discarded. The centrifugal

products were diluted with its respective inorganic

nutrient solution until their OD600 were adjusted to 0.15

and then they would be used in the subsequent

experiments. 2.3 The effect of pH value on the adsorption selectivity

Briefly, 0.1 g adsorbents were added to a 250 ml

978-1-4244-5089-3/11/$26.00 ©2011 IEEE

conical flasks containing 100 ml synthetic wastewater

and the initial pH value of suspension were adjusted

from 7 to 12 with 1 mol L–1 NaOH. Preliminary kinetic

experiments demonstrated that equilibrium time 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 rpm at 25 ℃ for

24 h. After the equilibrium being reached, the

concentrations of nitrobenzene and phenol were

determined. 2.4 The effect of flow rate on the adsorption selectivity

The dynamic adsorption was performed to identify

the effect of flow rate on the adsorption selectivity. The

dynamic adsorption was conducted using a 100 mm ╳

4.6 mm I.D. glass column packed with 10 ml (about 7.2

g dry weight) resin HU-03 and connected with a 6672

reciprocating pump (Beijing Analytical Instrument Plant)

at 30 ℃. Under the optimized pH value condition, the

synthetic wastewater with nitrobenzene (72 mg l-1) and

phenol (251 mg l-1) was employed for the dynamic

adsorption tests at different flow rate (30 ml h-1, 70 ml h-1,

110 ml h-1, 150 ml h-1). Previous experiments

demonstrated that the phenol-degrading bacterium could

degrade phenol effectively when the nitrobenzene

concentration is less than 3.5 mg l-1. So when the

nitrobenzene concentration in dynamic adsorption

effluent is more than 3 mg l-1, the dynamic adsorption is

finished.

III. Results and discussion

3.1 Effect of pH value on the adsorption selectivity to

nitrobenzene and phenol

Figure 1 The effect of pH on adsorption selectivity.

The impact of pH on the adsorptive selectivity to

nitrobenzene and phenol was investigated. As shown in

figure 1, phenol equilibrium concentration in synthetic

wastewater increased when pH rose, which meant resin

HU-03 lost its sorption capacity to phenol gradually.

When the pH was elevated to 11, the phenol

concentration approached a maximum. However, the

nitrobenzene concentration did not have significant

change with pH variation. These observations can be

explained through the adsorb capability of organic ions

on the hydrophobic adsorbents. Organic ions with small

molecular weights does not adsorb on the hydrophobic

adsorbent in contradistinction to undissociational

molecules of the same matter. Therefore the adsorption

capacity of resin HU-03 to phenol depends on its

molecules dissociation extent. When the solution pH

exceed to its pKa (9.48), phenol as organic ion could not

be adsorbed by resin HU-03. Their difference of

chemical properties under different pH is the base of

selective adsorption. From this result it could be

concluded that nitrobenzene and phenol can be separated

effectively through adjusting the aqueous pH.

Considering the operation cost and the performance, the

optimized pH was chosen to 10.5. 3.2 Effect of the adsorption flow rate on the adsorption

selectivity

Figure 2 The effect of adsorption flow rate on the adsorption

selectivity.

The adsorption flow rate is a very important factor,

so their impact on the adsorptive selectivity was

investigated. As shown in figure 2, during the dynamic

adsorption process, the phenol concentration in effluent

approached its initial concentration, which is not affected

by the variation of flow rates in the experimental range.

When the adsorption flow rate is below 110 ml h-1, the

nitrobenzene concentration in adsorption effluent is not

over 3 mg L-1, which is important to the following

phenol biodegradation. When the flow rate reached to

150 ml h-1, the nitrobenzene concentration began to

beyond 3 mg L-1, which concentration might inhibit the

degradation activity of phenol-degrading bacterium and

caused the uptake of the following biological treatment.

When the nitrobenzene concentration in effluent

exceeded 3 mg l-1, the dynamic adsorption has been

finished. Through the pretreatment about 540 mg

nitrobenzene has been adsorbed into resin HU-03, about

74 mg nitrobenzene had been adsorbed by per gram resin

HU-03. 3.3 Adsorption isotherm of nitrobenzene onto resin

HU-03

The adsorption reversibility is the basic of

nitrobenzene continuous biodegradation and resin

bio-regeneration. To investigate whether the adsorption

of nitrobenzene is reversible or not, the accurate

adsorption isotherms were determined. Adsorption

isotherms obtained at 25 ℃ and the respective

experimental data were represented in figure 3. The

adsorption experimental data were analyzed using

Freundlich equation

log qe = log KF + 1/n log Ce,

where KF and n are characteristic constants.

20 40 60 80 100 120 140 160200

225

250

275

300

phenol nitrobenzene

adsorption flow rate, (ml h-1)

Cn

itor

ben

zen

e (

mg

l-1)

0

1

2

3

4

5

Cp

hen

ol (m

g l -1)

Figure 3 nitrobenzene sorption equilibrium values obtained by

adsorption and desorption processes, at 25 ℃.

From the fitting curve we obtained of the

nitrobenzene adsorption isotherm values in mineral

medium was q=87.51 × C0.33. Three experimental

desorption values are shown in Figure 3. Desorption

experiments confirmed the reversibility of the adsorption

process. Comparing seven experimental data about

adsorption and desorption, desorption experiments

confirmed the reversibility of the adsorption process.

Table 2 Effect of different abiotic parameters on the adsorption of

nitrobenzene onto resin HU-03

Parameters q (mg/ g ) Parameters q (mg/ g )

pH =4 159 pH =8 173

pH =6 163 pH =10 162

T=288 K 174 T=308 K 159

Comparing the values of the adsorbed concentration

for nitrobenzene solutions at the four pH values (5~9)

and three temperatures (10~30 ℃), it was observed that

5% is the maximum relative difference obtained (Table

2). In this way, it is possible to conclude that

nitrobenzene adsorption is independent of environmental

factors in the range tested. In summary, the variation of

environmental factors tested did not affect the adsorption

process which suggested that resin HU-03 can be

successfully used to separate nitrobenzene from mixed

wastewater, under most actual conditions. 3.4 Biodegradation of nitrobenzene and

bio-regeneration of nitrobenzene-laden adsorbents

through bioaugmentation

Figure 4 Growth and free molinate depletion by mixed

culture, respectively, corresponding to 74 mg nitrobenzene

per g of dry resin HU-03

Bio-regeneration was performed using bacterium

suspension, and was able to degrade nitrobenzene to

aniline below 1.2 mg l-1. For aniline could be adsorbed

by HU-03, there has no accumulation of other

degradation products. The capability of bacterium

suspension to degrade nitrobenzene and to regenerate

resin HU-03 has been assessed. As shown in Figure 4,

for the continuous metabolism of bacterium, the

nitrobenzene concentration in solution decreased

gradually with increasing incubation time. As mentioned

above that the adsorption to nitrobenzene is reversible,

the dynamic adsorption equilibrium exists between the

aqueous and the adsorbed nitrobenzene. Degradation of

nitrobenzene in the aqueous solution broke up the

equilibrium. The lower concentration of nitrobenzene in

the aqueous phase serves as a driving force to desorb

nitrobenzene from resin continuously. The bacterium

utilized nitrobenzene adsorbed by resin HU-03 as the

sole source of energy and carbon. During this process

their biomass (OD600) increased from 0.32 to 1.21.

The complete desorption from adsorbents with high

volume of micropores is not usual. When the

nitrobenzene concentration did not further declined,

nitrobenzene remaining in resin HU-03 was extracted

and analyzed. The analysis revealed that most

nitrobenzene remained adsorbed into the resin (about 55

mg nitrobenzene adsorbed onto per g dry resin) and not

be consumed completely. Since the extent of

nitrobenzene degradation was limited by the

bioavailability. Using the concentration values of free

nitrobenzene predicate by the isotherm equation, the

limit of nitrobenzene bioavailability was about 1.2 mg l-1,

under this condition the adsorption capacity of resin

HU-03 is 51 mg nitrobenzene per g of dry resin.

Comparing with previous adsorption amount, about 20

mg nitrobenzene per g of dry resin has been degraded

through bioaugmentation in each bio-regeneration

recycle. 3.5 Phenol biodegradation through bioaugmentation

0 12 24 36 48 60 720

50

100

150

200

250

Cph

enol

(m

g/L

)

time (h)

without pretreatment with pretreatment

Figure 5 Phenol biodegradation as a function of incubation time

with/without selective adsorption pretreatment, corresponding to

mixed wastewater of nitrobenzene (71 mg l-1) and phenol (251 mg

l-1)

As shown in Figure 5, without the selective

adsorption pretreatment, at first the phenol concentration

decreased to some extent and then did not further decline.

That is because 72 mg l-1 nitrobenzene is high enough to

complete inhibit the degrading activity of

phenol-degrading bacterium. The first decrease of phenol

concentration might caused by the bio-adsorption.

Through the selective adsorption pretreatment most

nitrobenzene had been removed and its concentration in

adsorption effluent is less than 3 mg l-1, which is below

the bearing limit of phenol-degrading bacterium. Without

nitrobenzene toxic inhibition, the residual phenol in

adsorption effluent could be degraded effectively and

below to 1 mg l-1 within 60 h.

3.6 Assessment of resin HU-03 adsorption selectivity

through bio-regeneration

0 1 2 3 4 5 6 7 8 90.4

0.6

0.8

1.0

1.2

1.4

1.6

ni t r obenzene

Cn

itro

ben

zen

e(m

g l-1

)

number of repeated sorption

Figure 6 The effect of bio-regeneration on the adsorption

performance of resin HU-03

The use of high amounts of substrate and the

conversion to biomass, needed to evaluate the

deterioration of the resin, namely due to the effect of

metabolites and/or cellular debris on adsorption capacity.

During the 60 days of recycle experiment, the

nitrobenzene concentration variation in the adsorption

effluent can be shown in Figure 6. The results showed

that the resin can be re-utilized, although a progressive

deterioration will occur, although due to metabolic

activity. During the regeneration recycles, adsorption

capacity of resin HU-03 to nitrobenzene has decrease to

some extent at first and then their performance is kept at

a constant level. After the first bio-regeneration, the

nitrobenzene concentration in effluent has increased from

0.5 mg l-1 to 1.4 mg l-1. It is because that the extent of

resin bio-regeneration was limited by the bio-availability

during the first recycle process. Loss of adsorbent

capacity during bio-regeneration has been attributed to

decay products of microbial cells (Ha et al., 2001;

Vinitnantharat et al., 2001). Although the nitrobenzene

concentration approached 2 mg l-1, it is still below the

bearing limit of phenol-degrading bacterium. After 60

days of recycle experiment, the adsorption selectivity of

resin HU-03 is stable. 4. Conclusion

Through adjusting the pH value to 10 and the

adsorption flow rate to 110 ml h-1, nitrobenzene in the

mixed wastewater of nitrobenzene and phenol could be

separated. Most of nitrobenzene had been removed

through the selective adsorption and its concentration in

adsorption effluent did not significantly change. For

the adsorption to nitrobenzene is reversible, nitrobenzene

that adsorbed by resin HU-03 could be desorbed and

degraded through biodegradation. The regeneration

ability of resin HU-03 was limited by the nitrobenzene

bioavailability. About 20 mg nitrobenzene per g of dry

resin HU-03 has been degraded through bioaugmentation.

Without the toxic inhibition of nitrobenzene, phenol

remained in adsorption effluent could also be degraded

effectively through bioaugmentation. Through sixty days

for repeated experiment, the separate performance of

resin did not decline obviously, which ensure it could be

used in a long term. This method realizes the respective

biodegradation to different pollutants in mixed

wastewater and provides a promising method to treat

wastewater containing complex component.

Acknowledgments This work was jointly supported by the National

Natural Science Foundation of China （ 50578073,

50908109），the Natural Science Foundation of Yunnan

（2008E035M）.

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