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International Biodeterioration & Biodegradation 30 (1992) 9-16

Bacterial Utilization of Phenolic Wastewater


Components*

A. Yu. Fedorov, at V. I. Korzhenevich, A. D. Mironov,


V. Yu. Krestyaninov & A. P. Gumenyuk
VNII Genetics and Selection of Industrial Microorganisms, Saratov Branch,
Saratov, Russia
(Received 1 February 1991; revised version received 11 July 1991;
accepted 7 October 1991)

ABSTRACT
The cumene method is widely used to produce industrial phenol, Wastewaters
from this process include phenol, 2-phenyl-2-propanol, cumene hydroperoxide,
acetophenone, mesityl oxide, a-methylstyrene and other compounds. The
inhibitory action of the components of this wastewater during microbial
decomposition has been investigated.
The results of extensive screening experiments with bacterial strains
isolatedfrom soil showed that they were able to degrade the main components
and were consequently used in this study. The ability of some strains to utilize
cumene hydroperoxide has also been demonstrated. The bacterial strains
employed can be recommended for the treatment of phenolic wastewater.

INTRODUCTION
T h e c u m e n e m e t h o d o f industrial p h e n o l p r o d u c t i o n ( K r u z h a l o v &
G o l o v a n e n k o , 1963) has b e e n w i d e l y u s e d in m a n y countries from the
1950s to the p r e s e n t day. It is b a s e d o n the d e c o m p o s i t i o n o f c u m e n e
h y d r o p e r o x i d e to p r o d u c e p h e n o l a n d acetone. As a result o f direct a n d
*This paper was presented at Biodeterioration 8.
~Present address: Lenin av. 134/146-298, Saratov, 410600, USSIL
9
International Biodeterioration & Biodegradation 0964-8305/92/$05.00 1992 Elsevier
Science Publishers Ltd, England. Printed in Great Britain.

10

A. Yu. Fedorov et al.

secondary reactions, during the process, phenol, 2-phenyl-2-propanol,


acetophenone, a-methylstyrene and mesityl oxide are formed (Kruzhalov
& Golovanenko, 1963). Subsequently they are discharged in wastewater
and have been shown to be significant environmental pollutants.
It is considered that these compounds have mutual inhibitory interactions, i.e. one c o m p o u n d influences the process of bacterial utilization
of another while the latter exercises its own influence on the degradation
of the first c o m p o u n d during their microbial decomposition. Meyer et al.
(1984) have shown that degradation of the components is difficult to
achieve in two c o m p o u n d mixtures.
M a n y bacterial strains capable of degrading acetophenone, a-methylstyrene and phenol are known (Cripps, 1975; Cripps et al., 1978; Pilon et
al., 1976; Bestetti et al., 1981; Alieva etal., 1983; Pillis & Davis, 1984;
Korgzenevitch & Shenderov, 1985). However, these strains are unable to
utilize other aromatic compounds.
Bacterial strains able to use a wide variety of aromatic compounds are
k n o w n (Haribabu et al., 1984), but some of the substrates are not found in
the wastewaters of industrial phenol production. Data on microbiological
degradation of2-phenyl-2-propanol, cumene hydroperoxide and mesityl
oxide are unknown.
Bacterial strains that can be used for biological wastewater treatment
must have the ability to resist the inhibitory action of the wastewater
compounds. This can be determined by their resistance to these
compounds and/or their capacity to use them as a source of carbon.

MATERIALS A N D M E T H O D S
Soil samples from the production plants making phenol were treated
with a 0.9% solution of sodium chloride. T h e n 0-1 ml was added to a solid
mineral salt m e d i u m M9 (Miller, 1972) or to a solidified m e d i u m
(Ralston & Vela, 1974), containing one of the wastewater components
(phenol, 2-phenyl-2-propanol, acetophenone, or a-methylstyrene) at a
concentration of 500 mg 1-1. Single colonies that produced the most
intensive growth, on these media, were transferred to a m e d i u m of the
same composition in order to obtain pure cultures of the bacterial strain.
These strains were subcultured on the solid m e d i u m M9 containing
increasing amounts of the same c o m p o u n d as the sole carbon source.
The ability of the selected strains to utilize various aromatic
compounds was determined using the fluid m e d i u m M9, which h a d the
same composition and concentration of the substrate as in the solid
m e d i u m used for strain selection. Measurement of the substrate concen-

Bacterial utilization of phenolic wastewater components

11

tration in the media was carried out spectrophotometrically on a Specord


M-40(DDR) at 200-500 nm. C u m e n e hydroperoxide concentration in
the culture m e d i u m was determined by iodometric titration (Kruzhalov
& Golovanenko, 1963). The mixtures containing the compounds or
wastewater samples were analysed by thin-layer and gas chromatography.
Selected strains of bacteria were immobilized using a modified version
of the method of Nilsson et al. (1983).

RESULTS A N D D I S C U S S I O N
Twenty-four strains of bacteria were isolated from the single c o m p o u n d
experiments, 12 from the phenol medium, 7 from the 2-phenyl-2-propanol,
3 from the acetophenone a n d 2 from a-methylstyrene containing
medium. The ability of the selected bacterial strains to utilize these
compounds was verified during culture on the fluid m e d i u m of the same
composition (Table 1).
Of the 24 strains 18 could utilize only the c o m p o u n d on which they
were selected. Two strains, N 19 isolated on 2-phenyl-2-propanol and N22
isolated on acetophenone, were able to degrade phenol (500 mg/1). The
strain N12 isolated on phenol also utilized acetophenone and mesityl
oxide (500 mg 1-1). Strains N11, 12, 20, 21 and 22 were also able to degrade
mesityl oxide, a non-aromatic compound, but a significant component
of phenolic wastewater.
Thus the majority of the bacterial strains have specific enzymatic
systems which can only deal with certain functional groups. These are
hydroxy, alcohol, hydroperoxy, alken and keto groups whose structural
formulae are as follows:

CH3

I
OH

CH3--C--OH

CH3

I
CH3--C--O--OH

C6H5

CH3

CH3

C~--~CH2 C~--~O

On the other hand, the fermentative systems in some strains that can
utilize a n u m b e r of substrates may be less specific or they may have
several fermentative systems as a result of mutation in their own genomes
or of exchange of genetic material within soil bacterial populations
before isolation.
At the same time, possible inhibitory interactions of wastewater
components on the bacterial strains employed were investigated (Table 2).
The most toxic c o m p o u n d is c u m e n e hydroperoxide; less inhibitory are

Phenol
Phenol
Phenol
2-Phenyl-2-propanol
2-Phenyl-2-propanol
Acetophenone
Acetophenone
a-Methylstyrene

Substrate for the isolation


of the strains

TABLE 1

by Selected Strains

+
+
+
-t
-

+
+
-

+
+
+
th
+
-

Acetophenone

2-Phenyl2-propanol

Phenol

+
+

a-Methylstyrene

Utilized substrate (500 mg I-)

of Various Aromatic Compounds

ONumeration of strains remains the same during all articles.


bPhenol concentration in the medium was 350 mg 1-r.

l-10
II
12
13-18
19
20,21
22
23,24

straina

Mv of the

Degradation

+
+
+
+
-

Mesityl oxide

a
8.
ad
9
P,
r

Bacterial utilization of phenolic wastewater components


TABLE 2
Inhibitory Interactions of the Wastewater Components
Degradation

NN
of the
strain

1
3
12
3
4
1, 3
4
19
19
13
19
12
12
12

Substrate
(500 mg l -l)

Inhibitor
(name of compound)

13

during Their Microbial

Concentration of inhibitor in medium a


Inhibitory Doublelong Non-effective
(rag 1-1)
(mg l - 9
(rag l - 9

Phenol
Phenol
Phenol
Phenol
Phenol
Phenol
Phenol
2-Phenyl-2-propanol
2-Phenyl-2-propanol
2-Phenyl-2-propanol
2-Phenyl-2-propanol
Acetophenone
Acetophenone
Acetophenone

C u m e n e hydroperoxide
15.0
Cumene hydroperoxide
8-0
Cumene hydroperoxide
30-0
2-Phenyl-2-propanol
275.0
2-Phenyl-2-propanol
1 000.0
Acetophenone
1 000.0
Aeetophenone
150-0
Cumene hydroperoxide
30-0
Acetophenone
500-0
Phenol
20~0
Phenol
200-0
Cumene hydroperoxide
15.0
Phenol
175.0
2-Phenyl-2-propanol
750-0

7-5
40
! 5.0
200-0
750.0
500-0
100.0
15.0
250-0
50-0
100-0b
7.5
125.0b
500.0

1.9
1.0
3.8
100-0
250. 0
I00.0
25.0
3.8
100-0
15-0
50.0 b
2.5
25.0 b
125.0

alnhibitory - - concentration of the c o m p o u n d that inhibited substrate degradation


completely.
Doublelong - - concentration of the c o m p o u n d that increased the period of complete
substrate degradation twice.
Non-effective - - concentration of the c o m p o u n d that had no influence on the complete
substrate degradation.
bUtilization of the substrate and inhibitor (phenol) occurred simultaneously.

2-phenyl-2-propanol and acetophenone. Cultures of the strain N19 in


2-phenyl-2-propanol containing medium with added phenol (100 mg 1-~
or less) and cultures of the strain N12 in acetophenone containing
medium supplemented by phenol (125 mg1-1 or less) exhibited cometabolic processes. However, in spite of this inhibitory interactions did
occur.
Cumene hydroperoxide, acetophenone, a-methylstyrene, mesityl
oxide, 2-phenyl-2-propanol simultaneously added to the medium
containing phenol (500 mg l-l), in non-effective amounts (see Table 2),
prolonged the period of complete phenol utilization or fully inhibited
the process. These results lead to the conclusion that the inhibitory
interactions of the individual wastewater components increase according
to the number of compounds.
The majority of the isolated bacterial strains were able to degrade only
one of the compounds and were sensitive to one or two components of

A. Yu. Fedorov et al.

14

these wastewaters. However, two strains were investigated further, N12,


which utilized a large number of aromatic components of the wastewater, and N 19, which co-metabolized phenol and 2-phenyl-2-propanol.
As the result of selection N12 aquired the ability to degrade all the
compounds investigated in higher concentrations and another aromatic
compound previously not degraded by this strain (see Table 3). N12
cultured in a sample of wastewater containing phenol (480 mgl-~),
cumene hydroperoxide (17 mgl-1), a-methylstyrene (10mgl -l) and
traces of acetophenone decreased the amount of phenol by 95% in 96 h.
In other samples of wastewater containing 200 mg 1-~ acetophenone,
52 mg 1-I mesityl oxide, 10 mg 1-j 2-phenyl-2-propanol, after 48 h, N12
reduced these compounds to trace amounts.
Bacterial strain N 19 was shown to utilize 98% of 750 mg 1-~ 2-phenyl-2propanol in 96 hours and other aromatic compounds such as catechol
(100 mg 1-1), benzoic acid (500 mg 1-~) and 3,4-dihydroxybenzoic acid
(500 mg 1-l) in 24 h. On the rotary shaker, strain N19 utilized only 52% of
the phenol (350 mg 1-~) in 96 h. Complete degradation did not occur,
probably due to the inhibitory effect of the unidentified intermediates
(optical density at 310-340 nm). In order to eliminate this inhibitory
effect, cells of N19 were used entrapped in agar. It is known from the
literature that entrapped bacterial cells can utilize phenol with great
efficiency (Anselmo et al., 1985). In the present study entrapped cells of
N19 were able to utilize phenol (350 mg 1-j) completely in 48 h under the
same conditions of culture as free cells, thus confirming the protective
TABLE 3

Degradative Activity of the Strain N12 after Selection

Substrate

Maximum substrate The period for the


concentration (rag l-l) complete utilization

(h)
Phenol
Acetophenone
a-Methylstyrene
Mesityl oxide
Catechol
Benzoic acid
2-Aminobenzoic acid
4-Chlorobenzoic acid
4-Hydroxybenzoic acid
2,5-Dihydroxybenzoic acid
3,4-Dihydroxybenzoic acid
Bensamide

1 000
1 250
500
1 000
200
500
750
750
500
500
500
500

96
96
28
96
20
96
36
48
36
48
20
96

Bacterial utilization of phenolic wastewater components

15

effect of immobilization on the process of bacterial degradation of


xenobiotics.
Strain N19 was also able, under laboratory conditions (culture on the
rotary shaker at 30C), to completely refine from xenobiotics the wastewater sample containing 2-phenyl-2-propanol (520mgl-~), phenol
(32 mg 1-1), acetophenone and methylstyrene as trace compounds.
The principal ability of microbial degradation of cumene hydroperoxide was shown by N13, which can utilize this c o m p o u n d
(300 mg 1-~) and 2-phenyl-2-propanol (750 mg 1-~) completely in 72-96 h
when cultured at 30 . The results of further investigations make it
possible to propose that c u m e n e hydroperoxide decomposition by N13
began with deactivation of the hydroperoxide group. However, after
storage on the solidified mineral m e d i u m M9 containing 300 mg 1-2
cumene hydroperoxide, N13 lost the capacity to utilize this compound,
but was still able to degrade 2-phenyl-2-propanol under the same
conditions as before and at the same concentration. It is therefore
concluded that agar protects bacterial cells from the hydroperoxide
activity of this substrate and leads to a decrease in activity of the first
enzyme or to a lowering of the strain's resistance to the hydroperoxide
group inhibitory effect.
This hypothesis is supported by the ability of strain N13 to utilize
2-phenyl-2-propanol a n d the correlation in the chemical structure of
both substrates:

CH3

C u m e n e hydroperoxide C6Hs--C--CH 3 and

I
O--OH

CH3

2-phenyl-2-propanol C6Hs---C~CH 3

OH
To verify this supposition entrapped cells in agar gel were used for the
degradation of the cumene hydroperoxide (300 mg 1-~). In the first 24 h
the hydroperoxide group was completely deactivated and after a further
48 h all the aromatic c o m p o u n d s in the culture m e d i u m decreased by as
m u c h as 95% of the initial concentration. The results of thin-layer
chromatography suggested that the intermediate c o m p o u n d was 2-

16

A. Yu. Fedorov et al.

phenyl-2-propanol. It s h o u l d be n o t e d that d u r i n g culture for 168 h no


c h e m i c a l interaction was observed between the agar gel particles without
e n t r a p p e d bacterial cells a n d c u m e n e hydroperoxide.

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