Académique Documents
Professionnel Documents
Culture Documents
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
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-
11
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
TABLE 1
by Selected Strains
+
+
+
-t
-
+
+
-
+
+
+
th
+
-
Acetophenone
2-Phenyl2-propanol
Phenol
+
+
a-Methylstyrene
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
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
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
14
Substrate
(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
15
CH3
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
REFERENCES
Alieva, R. M., Iljaletdinov, A. N. & Dgusupova, D. B. (1983). Strain Bacillus cereus
N3, used for the refining of industrial wastewaters from a-methylstyrene.
Author's Certificate N1033542 USSR, C 12 N 15/00//C 02 F 3/34, Publ.
07.08.83, Bul.N29.
Anselmo, A. M., Mateus, M., Cabral, J. M. S. & Novais, J. M. (1985). Degradation
of phenol by immobilized cells of Fusarium flocciferum. Biotechnology
Letters, 7, 889-94.
Bestetti, G., Barbieri, P. & Galli, E. (1981). Evidence for catabolic plasmids in
fluorescent Pseudomonas degrading styrene and ethylbenzene. Annals of
Microbiology, 31, 35-42.
Cripps, R. E. (1975). The microbial metabolism ofacetophenone. Metabolism of
acetophenone by an Arthrobacter species. Biochem. J., 152, 233-41.
Cripps, R. E., Trudgill, D. W. & Whatelly, J. C. (1978). The metabolism of
1-phenyl-ethanol and acetophenane by Nocardia TS and an Arthrobacter
species. Eur. J. Biochem., 86, 175-86.
Haribabu, B., Kamath, A. V. & Vaidyanathan, C. S. (1984). Microbial degradation
of substituted benzoic acids. Journal of Indian Institute of Science, 65(C),
69-104.
Korzhenevich, V. I. & Shenderov, B. A. (1985). Strain of the bacteriumAlcaligenes
faecalis KSV21, capable of degrading phenol. Author's Certificate N 1198118
USSR, C 12 N 15/00, Publ. 15.12.85, Bul.N32.
Kruzhalov, B. D. & Golovanenko, B. I. (1963). Combined Reception of the Phenol
anti the Acetone. Goschimizdat, Moscow.
Meyer, J. S., Marcus, M. D. & Bergman, H. L. (1984). Inhibitory interactions of
aromatic organics during microbial degradation. Environmental Toxicology
and Chemistry, 3, 583-7.
Miller, J. H. (1972). Experiments in Molecular Genetics, Cold Spring Harbor
Laboratory.
Nilsson, K., et al. (1983). A general method for the immobilization of cells with
preserved viability. European Journal of Applied Microbiology, 17, 319-26.
Pillis, L. J. & Davis, L. T. (1984). Microorganism capable of degrading phenolics.
US Patent N4447539, C 12 N 1/20//C 12 R 1/40, Publ. 08.05.84.
Pilon, R., Pons, B. J., Sechet, N. & Duc, N. G. C. (1976). Procede de degradation
biologique de solutions contenant des phenols. French Patent N2311759,
C 02 C 5/10, Publ. 17.12.76.
Ralston, L. R. & Vela, G. R. (1974). A medium for detecting phenol-degrading
bacteria. Journal of Applied Bacteriology, 37, 347-51.