International Biodeterioration & Biodegradation 30 (1992) 9-16

Bacterial Util ization 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 isolated from 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

The cumene method of industrial phenol product ion (Kruzhalov & Golovanenko, 1963) has been widely used in many countries from the 1950s to the present day. It is based on the decomposit ion of cumene hydroperoxide to produce phenol and acetone. As a result of direct and

*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 inter- actions, i.e. one compound influences the process of bacterial utilization of another while the latter exercises its own influence on the degradation of the first compound during their microbial decomposition. Meyer et al. (1984) have shown that degradation of the components is difficult to achieve in two compound mixtures.

Many bacterial strains capable of degrading acetophenone, a-methyl- styrene 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 known (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 AND METHODS

Soil samples from the production plants making phenol were treated with a 0.9% solution of sodium chloride. Then 0-1 ml was added to a solid mineral salt medium M9 (Miller, 1972) or to a solidified medium (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 medium of the same composition in order to obtain pure cultures of the bacterial strain. These strains were subcultured on the solid medium M9 containing increasing amounts of the same compound as the sole carbon source.

The ability of the selected strains to utilize various aromatic compounds was determined using the fluid medium M9, which had the same composition and concentration of the substrate as in the solid medium 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. Cumene hydroperoxide concentration in the culture medium 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 AND DISCUSSION

Twenty-four strains of bacteria were isolated from the single compound experiments, 12 from the phenol medium, 7 from the 2-phenyl-2-propanol, 3 from the acetophenone and 2 from a-methylstyrene containing medium. The ability of the selected bacterial strains to utilize these compounds was verified during culture on the fluid medium of the same composition (Table 1).

Of the 24 strains 18 could utilize only the compound 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 CH3 CH3 CH3 I I I I

OH CH3- -C- -OH CH3- -C - -O- -OH C~--~CH2 C~--~O I f i i I

C6H5 On the other hand, the fermentative systems in some strains that can utilize a number 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 compound is cumene hydroperoxide; less inhibitory are

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Bacterial utilization of phenolic wastewater components 13

TABLE 2 Interactions of the Wastewater Components during Their Microbial

Degradation

NN Substrate Inhibitor of the (500 mg l -l) (name of compound) strain

Concentration of inhibitor in medium a

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

1 Phenol Cumene hydroperoxide 15.0 7-5 1.9 3 Phenol Cumene hydroperoxide 8-0 40 1.0

12 Phenol Cumene hydroperoxide 30-0 ! 5.0 3.8 3 Phenol 2-Phenyl-2-propanol 275.0 200-0 100-0 4 Phenol 2-Phenyl-2-propanol 1 000.0 750.0 250. 0

1, 3 Phenol Acetophenone 1 000.0 500-0 I00.0 4 Phenol Aeetophenone 150-0 100.0 25.0

19 2-Phenyl-2-propanol Cumene hydroperoxide 30-0 15.0 3.8 19 2-Phenyl-2-propanol Acetophenone 500-0 250-0 100-0 13 2-Phenyl-2-propanol Phenol 20~0 50-0 15-0 19 2-Phenyl-2-propanol Phenol 200-0 100-0 b 50.0 b 12 Acetophenone Cumene hydroperoxide 15.0 7.5 2.5 12 Acetophenone Phenol 175.0 125.0 b 25.0 b 12 Acetophenone 2-Phenyl-2-propanol 750-0 500.0 125.0

alnhibitory - - concentration of the compound that inhibited substrate degradation completely. Doublelong - - concentration of the compound that increased the period of complete substrate degradation twice. Non-effective - - concentration of the compound 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 co- metabolic 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

14 A. Yu. Fedorov et al.

these wastewaters. However, two strains were investigated further, N12, which utilized a large number of aromatic components of the waste- water, 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-2- propanol 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 1 000 96 Acetophenone 1 250 96 a-Methylstyrene 500 28 Mesityl oxide 1 000 96 Catechol 200 20 Benzoic acid 500 96 2-Aminobenzoic acid 750 36 4-Chlorobenzoic acid 750 48 4-Hydroxybenzoic acid 500 36 2,5-Dihydroxybenzoic acid 500 48 3,4-Dihydroxybenzoic acid 500 20 Bensamide 500 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 waste- water 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 hydro- peroxide was shown by N13, which can utilize this compound (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 cumene hydroperoxide decomposition by N13 began with deactivation of the hydroperoxide group. However, after storage on the solidified mineral medium 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 and the correlation in the chemical structure of both substrates:

CH3 I

Cumene hydroperoxide C6Hs--C--CH 3 and I

O--OH

CH3 I

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

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 compounds in the culture medium decreased by as much as 95% of the initial concentration. The results of thin-layer chromatography suggested that the intermediate compound was 2-

16 A. Yu. Fedorov et al.

phenyl-2-propanol. It should be noted that during culture for 168 h no chemical interaction was observed between the agar gel particles without entrapped bacterial cells and cumene hydroperoxide.

REFERENCES

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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.

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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.

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