Biodegradation of Phenolic Industrial

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Biodegradation of phenolic industrial wastewater in a ¯uidized bedbioreactor with immobilized cells of Pseudomonas putida

G. Gonzalez *, G. Herrera, Ma.T. Garcõa, M. Pe~na

Department of Chemical Engineering, University of Valladolid, Paseo Prado de la Magdalena s/n, Valladolid 47011, Spain

Received 21 August 2000; received in revised form 3 April 2001; accepted 9 April 2001

Abstract

The paper presents the main results obtained from the study of the biodegradation of phenolic industrial wastewaters by a pure

culture of immobilized cells of Pseudomonas putida ATCC 17484. The experiments were carried out in batch and continuous mode.The maximum degradation capacity and the in¯uence of the adaptation of the microorganism to the substrate were studied in batch

mode. Industrial wastewater with a phenol concentration of 1000 mg/l was degraded when the microorganism was adapted to the

toxic chemical. The presence in the wastewater of compounds other than phenol was noted and it was found that Pseudomonas putida

was able to degrade these compounds. In continuous mode, a ¯uidized-bed bioreactor was operated and the in¯uence of the organic

loading rate on the removal eciency of phenol was studied. The bioreactor showed phenol degradation eciencies higher than 90%,

even for a phenol loading rate of 0.5 g phenol/l d (corresponding to 0.54 g TOC/l d). Ó 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Phenol degradation; Pseudomonas putida; Immobilized cells; Fluidized bed

1. Introduction

Some industrial wastewaters, especially those comingfrom the production processes of phenolic resins, contain

high concentrations (>10 g/l) of phenolic compounds

(Patterson, 1985). Several physico-chemical and biolog-

ical treatments have been suggested in the last 20 years to

remove eciently these compounds: adsorption with

bone char or zeolites, stripping with air or steam (Zilli

et al., 1993), wet air oxidation (Lin and Chuang, 1994) or

biological treatments with pure or mixed cultures of mi-

croorganisms (Lakhwala et al., 1992; Buitron et al., 1998;

Kapoor et al., 1998; Loh et al., 2000) have been used.

Several works appeared in the literature concerning

the biodegradation of phenol especially from model

solutions, by Pseudomonas putida (Zilli et al., 1993;Hannaford and Kuek, 1999; Mordocco et al., 1999),

with high removal eciencies.

On the other hand, the biodegradation of industrial

wastewaters can be improved if the microorganism is

previously adapted to the toxic chemical (Zilli et al.,

1993), especially when high phenol concentrations are

present. Moreover, other compounds dierent from

phenol, also present in the industrial wastewater, can

aect the biodegradation process.

The biodegradation of phenol from model solutionshas been studied by the authors and reported in previous

papers (Gonzalez and Herrera, 1995, 2001). These ex-

periments were carried out with free and immobilized

cells of P. putida in batch and continuous mode and best

results were obtained when a continuous ¯uidized bed

bioreactor with immobilized cells was operated.

This work presents the results obtained for the bio-

degradation of high phenol concentrations from indus-

trial wastewaters, by cells of P. putida immobilized in

calcium-alginate gel beads. Batch experiments were

made in order to obtain the maximum phenol degra-

dation capacity, analyzing the in¯uence of the adapta-

tion of the microorganism to the medium. Then,continuous experiments in a ¯uidized-bed bioreactor, in

order to determine the maximum phenol loading rate to

be treated, were carried out.

2. Methods

2.1. Raw wastewater

The raw wastewater came from the industrial pro-

duction of phenolic resins. Some of the characteristics of

Bioresource Technology 80 (2001) 137±142

* Corresponding author. Tel.: +1-349-83-423-170; fax: +1-349-83-

423-616.

E-mail address: [email protected] (G. Gonzalez).

0960-8524/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 9 6 0 - 8 5 2 4 ( 0 1 ) 0 0 0 7 6 - 1



this wastewater were pH, 4.5; phenol, 42,000 mg/l; total

organic carbon (TOC), 45,000 mg C/l and chemical

oxygen demand (COD), 124,000 mg O2=l.

The theoretical TOC and COD corresponding to the

analytical phenol concentration were calculated. Results

obtained were 32,000 mg C/l and 100,100 mg O2=l for

TOCphenol and CODphenol, respectively. The dierence

between experimental and calculated values indicated

the presence of other compounds, dierent from phenol,

that contributed to the organic load of the industrial

wastewater. It was intended to identify these other

compounds by HPLC. While some peaks appeared in

the analysis, it was not possible to identify any com-

pound and in the paper these are named ``non-phenolic

compounds''.

Due to the high phenol concentration, it was neces-

sary to dilute the raw wastewater before each experi-

ment.

2.2. Culture and growth medium

A strain of P. putida ATCC 17484, biotype B, from

the Laboratory of Microbiology Voor (Gante, Belgium)

was used as pure culture. The maintenance medium was

reported in a previous work (Gonzalez and Herrera,

1995). The cultures were grown aerobically in 250 ml

¯asks ®lled to 150 cm3 with growth medium and stirred

in a orbital shaker at 250 rpm at a constant temperature

of 30°C and pH 6.6. The growth medium was also

reported in the previous work with the only dierence in

the substrate used for this work: a mixture of the in-

dustrial wastewater and commercial phenol was em-

ployed, with a ®nal phenol concentration of 75 mg/l.

2.3. Immobilization protocol

The microorganisms were immobilized by entrap-

ment in calcium-alginate gel beads hardened with Al3.

Bioparticles were formed by mixing a suspension of

P. putida with a solution of sodium alginate (3% w/w;

Protonal LF 10/60), according to Bravo and Gonzalez

(1991). These bioparticles (spherical gel beads) obtained

had an average diameter of 1±2 mm. The concentration

in the bioparticles was 3 Â 1010 mg microorganism per

litre of alginate.

2.4. Batch experiments

Volumes of 250 ml of the wastewater, with bioparti-

cles and a varying phenol concentration in the range

200±1000 mg phenol/l, were disposed in a 500 ml ¯asks.

Initially the pH was adjusted to 6.6 with sodium hy-

droxide in order to attain the pH value necessary to

carry out the biodegradation process. The ¯ask was

continuously stirred in an orbital shaker at 250 rpm and

the temperature was maintained at 30°C. Periodical

samples were taken in order to analyze the operating

parameters pH, phenol, COD and TOC concentration.

2.5. Fluidized-bed bioreactor

The continuous biodegradation of phenol was carried

out with a ¯uidized-bed bioreactor (FBB). The reactor

body consisted of a jacketed cylinder, made of methyl

methacrylate (420 mm height and 140 mm internal di-

ameter). An enlargement at the top (170 mm height and

250 mm internal diameter) was provided, to ensure the

degassi®cation of the liquid and to avoid the loss of

bioparticles. The working volume was 3 l. The reactor

was thermostated.

Sterile air was supplied from the bottom of the col-

umn at a ¯ow rate of 85 l/h (43 vvm) through a porous

glass distributor (average pore diameter 20±40 lm). The

bioreactor worked with immobilized cells of the micro-

organism and bioparticles were suspended in the column

by air up¯ow.Several openings at the top of the bioreactor allowed

for the insertion of dierent probes (pH, DO), the ad-

dition of chemicals (nutrients, acid/base and antifoam

agents), and liquid sampling. The reactor was fed using

peristaltic pumps of variable ¯ow rates.

A software developed in a previous work (Vallejo,

1994) was used for the implementation of control closed

loops in the bioreactor. The structure of the control can

be considered as a feed-forward system with a feedback

loop cascade. This software allowed for the acquisition

and the registration of the main control variables (dis-

solved oxygen, pH and temperature).The following experimental conditions were main-

tained in the bioreactor: pH 6.6, temperature 30°C and

air¯ow, 43 vvm. In order to attain the initial pH nec-

essary to carry out the biodegradation, the addition of

sodium hydroxide was necessary.

Initially the reactor was operated in batch mode: the

bioreactor was initially ®lled up with a solution con-

taining the bioparticles necessaries to attain a concen-

tration level of 5 mg cells/l solution, plus 250 mg/l of

phenol from diluted industrial wastewater. No nutrients

were added during the biodegradation process and air

was supplied to provide the dissolved oxygen necessary

for the bacteria (2±4.5 mg/l).When almost complete phenol degradation was at-

tained, an amount of the industrial wastewater neces-

sary to obtain again a phenol concentration of 250 mg/l

in the bioreactor was added, in order to obtain a better

adaptation of the bioparticles. When no phenol was

detected in the euent, the reactor was switched to

continuous ¯ow conditions, feeding the reactor with a

diluted solution of the industrial wastewater (250 mg

phenol/l). Initially, a HRT of 4 days was ®xed in the

bioreactor. When a steady-state was reached (no phenol

or a very low phenol concentration in the euent was

138 G. Gonzalez et al. / Bioresource Technology 80 (2001) 137±142



observed), the in¯uent loading rate was increased, rais-

ing the phenol concentration in the feed from 250 to 500

mg phenol/l, until the steady-state was again obtained.

Then, phenol in the in¯uent was still increased and the

phenol concentration range studied was 250±2500 mg/l.

Periodical samples of the mixed liquor were taken

throughout all the experiments in order to measure the

concentrations of phenol, COD and TOC.

2.6. Analytical methods

Phenol was determined by HPLC, using a Nucleosil

120 C-18 column (250 Â 46 mm inner diameter; 5 lm

particle size) in combination with a Waters LC-Spec-

trophotometer. The mobile phase was acetonitrile: water

(65:35 v/v) pumped at 0.6 ml/min. COD was measured

according to standard methods (APHA, 1993). TOC

was determined by a Shimadzu Analyzer, TOC-5050

model. A selective electrode (Ingold U 457-S7) con-

nected to a pH-meter (Aqua Lytic pH 21) was used forpH determinations.

3. Results and discussion

3.1. Batch experiments: in¯uence of adaptation on the

degradation capacity

Several experiments were carried out varying the

initial phenol concentration of the wastewater in the

range 200±1000 mg phenol/l, to obtain the maximum

concentration of phenol that could be degraded byP. putida. The results showing a degradation capacity of

500 mg phenol/l and 25 h gave the maximum degrada-

tion.

Then new batch experiments were programmed in

order to increase both the capacity and rate of phenol

degradation. The culture was subjected to successive

adaptation tests (Zilli et al., 1993), from low to high

concentrations of phenol, using as inoculum the euent

from the preceding run. The method was described in a

previous work (Gonzalez and Herrera, 1995).

The results showed an increase in the phenol degra-

dation capacity from 500 to 1000 mg/l when acclimated

bioparticles were employed and the greater the numberof adaptations the lesser was the time necessary to carry

out the biodegradation. When a phenol concentration of

1000 mg/l was used, the degradation time decreased

from 340 h (one adaptation) to 260 h for the second

adaptation and also the lag phase decreased, as shown in

Fig. 1. Similar results were obtained when phenol con-

centrations of 200 and 500 mg/l were tested.

P. putida was able to degrade the compounds, other

than phenol, present in the industrial wastewater. The

change in TOC and phenol concentrations along with

time was followed for every experiment and the contri-

bution of phenol to the TOC and the TOC corre-

sponding to the other compounds determined. The

results obtained for TOCtotalTOCphenol and

TOCnon-phenol when immobilized cells of P. putida and

an initial phenol concentration of 1000 mg/l were used,

are shown in Fig. 2.

It was observed that an increase in the number of

adaptations led to an appreciable decrease in the lag

phase during the degradation of phenol, but the degra-

dation of the other compounds did not start until a

percentage (33% approximately) of the phenol had been

removed.The phenol degradation was complete in every ex-

periment. However, only the 75% of the non-phenolic

compounds present in the industrial wastewater had

been removed.

The maximum phenol concentration degraded, 1000

mg phenol/l was lower than that obtained with P. putida

Fig. 1. In¯uence of adaptation on the phenol degradation capacity

when immobilized cells and an initial phenol concentration of 1000

mg/l were used for the biodegradation process. (}) without any ad-

aptation; (w) after one adaptation; (Ã) after two adaptations.

Fig. 2. Experimental values for TOCtotalx), TOCphenolr) and

TOCnon-phenolicj and Paris predicted ( ± ), when an initial phenol

concentration of 1000 mg/l, and adapted immobilized cells were used.

G. Gonzalez et al. / Bioresource Technology 80 (2001) 137±142 139



and model solutions of phenol (Gonzalez et al., 2001):

2000 mg phenol/l. This result could indicate the negative

in¯uence of the non-phenolic compounds on the bio-

degradation process.

3.2. Kinetic model

The Haldane equation has been frequently used to

describe the phenol biodegradation process by pure or

mixed cultures Allsop et al. (1993), Buitron et al. (1998),

and Gonzalez and Herrera (1995). Usually, the param-

eters from the Haldane equation have been obtained

from batch experiments. Some authors (Hutchinson and

Robinson, 1988; Wang et al., 1996; Magbanua et al.,

1994) have introduced in the model equation the eect

of other substrates, dierent from phenol.

In addition, some authors (Wang et al., 1996) have

proposed a competitive cross-inhibitory kinetics while

others (Hutchinson and Robinson, 1988) proposed a

dual-substrate growth model, including the inhibition of both phenol and non-phenolic compounds in the kinetic

model. However, when acclimated microorganisms were

used, the Monod model appeared to be suitable to de-

scribe the biodegradation process, since the inhibition

due to the substrate could be neglected. Nevertheless,

this model could not take into account the inhibition

due to the presence of refractory compounds in the

wastewater. On the other hand, when relatively small

concentrations of phenol are tested, the model of Paris is

frequently used (Paris et al., 1982; Gonzalez and Her-

rera, 1995).

The Paris and Monod models were selected to ®t theexperimental data obtained in our batch experiments

with immobilized cells of P. putida, due to the simplicity

of both models.

The Paris model considers a second-order kinetics

(order one for both substrate and microorganisms) and

it was applied successfully in a wide range of phenol

concentrations (200±1000 mg/l). Fig. 2 shows data ob-

tained when an industrial wastewater with 1000 mg

phenol/l TOCtotal1000mgC=l;TOCphenol750mgC=l;

TOCnon-phenolic 250 mgC=l was tested. As can be ob-

served, there is a good agreement between experimental

and ®tted values. The kinetic parameters were

k x2:3 Â 10À4 l=mgh and Y xs (0.15 mg microorg/mgphenol).

The Monod model was also applied when experi-

ments with high phenol concentration and adapted mi-

croorganisms were made. Results with the industrial

wastewater, containing 1000 mg phenol/l. TOCtotal 1000 mgC=l; TOCphenol 750 mgC=l; TOCnon-phenolic 250 mgC=l are shown in Fig. 3 and the kinetic

parameters were lmax0:03 hÀ1; K s280 mg=l and Y xs

(0.15 mg/mg).

The comparison of these results with those obtained

when data from batch experiments with model solutions

were tested (Gonzalez and Herrera, 1995) shows the

lower values of kinetic parameters when the industrialwastewater was used in the biodegradation process. This

fact indicates less degradation capacity and a slower

kinetic, probably due to the non-phenolic compounds

present in the industrial wastewater.

3.3. Continuous biodegradation process

Continuous essays were planned to study the bio-

degradation of phenolic compounds from industrial

wastewaters. The methodology used in the tests was the

same as described when model solutions were treated

(Gonzalez et al., 2001).With the aim of attaining a good adaptation of the

bioparticles, the bioreactor was started in the batch

mode, introducing a solution of the industrial waste-

water containing 250 mg/l of phenol and the amount of

bioparticles required to attain a ®nal concentration of

5 mg microorg/l solution. The initial pH value was

®xed at 6.6 and no reagents were needed during the

biodegradation process to maintain this pH value.

Complete phenol degradation was observed after 6

days and the bioreactor was again operated in batch

mode to attain a better adaptation of the bioparticles,

introducing the amount of industrial wastewater to

attain initially 250 mg/l. After 10 days, the biodegra-dation was completed.

The operation was then switched to the continuous

mode and the reactor was fed with a diluted industrial

wastewater containing a concentration of 250 mg phe-

nol/l, and a pH value of 4.5. In continuous mode, the

reactor was operated with 4 days HRT (phenol loading

rate of 62.5 mg/l d) and no phenol was detected in the

euent. Then, the phenol concentration in the in¯uent

was increased progressively from 250 to 2500 mg/l of

phenol (phenol loading rates from 62.5 to 625 mg/l d),

working with a constant HRT of 4 d.

Fig. 3. Experimental values for (TOCtotalx; TOCphenolr and

TOCnon-phenolicj and Monod predicted ( ± ), when an initial phenol

concentration of 1000 mg/l, and adapted immobilized cells were used.




Fig. 4 shows the behavior of the system operated with

dierent in¯uent phenol concentrations, in terms of

in¯uent and euent concentration values of phenol and

TOC. Within the range of concentrations between 250

and 2000 mg/l, the system operation was very stable and

the phenol and TOC in the euent were always lower

than 1 mg phenol/l and 15 mg C/l, respectively. Similar

results were obtained when the COD content was fol-

lowed: euent lower than 120 mg O2=l and removal

eciencies higher than 95%. The removal of non-

phenolic compounds, evaluated as TOCnon-phenolic , was

always higher than 95%.At higher concentrations (2500 mg phenol/l), the

operation in the bioreactor became unstable and the

phenol and TOC concentration in the euent increased

progressively. After this time, the bioreactor was fed

with industrial wastewater containing 2000 mg phenol/l,

and an euent free of phenol was obtained after a 10-

days operation. With these conditions, the bioreactor

showed the same performance for more than 60 days

(data not shown).

The phenol loading rate susceptible to degradation

(0.5 g phenol/l d) was lower than that obtained in a

previous paper (Gonzalez et al., 2001) when model so-

lutions were treated (4 g phenol/l d). However, the

biodegradation eciencies are comparable (>98% for

phenol, >95% for COD and TOC) and the phenol

concentration in the euent was lower than 1 mg/l.

4. Conclusions

The experimental results showed that it was possible

to treat industrial euents containing high phenolconcentrations. When it was operated in batch mode,

phenol concentrations up to 1000 mg/l were degraded

and high removal eciencies (>90%) were attained for

both phenol and non-phenolic compounds. The opera-

tion of an FBB bioreactor, with a phenol loading rate of

500 mg/l d (COD: 1500 mg/l d; TOC: 525 mg/l d), was

proven to be ecient and euent phenol concentrations

were lower than the highest discharge limit permitted for

the Spanish legislation (1 mg/l). The COD and TOC

contents in the euent were always lower than 120 and

40 mg/l, respectively.

Fig. 4. In¯uent and euent concentrations of phenol and TOC in the FBB operated under dierent phenol concentrations and a HRT of 4 d: phenol

concentration in the in¯uent () and euent and (); TOC in the in¯uent (w) and euent x.

G. Gonzalez et al. / Bioresource Technology 80 (2001) 137±142 141



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Biodegradation of Phenolic Industrial