Chlorophenol degradation by Phanerochaete chrysosporium

ELSEVIER P I I : S 0 9 6 0 - 8 5 2 4 ( 9 7 ) 0 0 0 2 2 - 9

Bioresource Technology 60 (1997) 207-213 © 1997 Elsevier Science Limited

All rights reserved. Printed in Great Britain 0960-8524/97 $17.00

CHLOROPHENOL DEGRADATION BY PHANEROCHAETE CHRYSOSPORIUM

R. Rubio P6rez, G. Gonzfilez Benito* & M. Pefia Miranda

Dpto. de Ingenieria Quimica, Facultad de Ciencias, Universidad de Valladolid, 47011 Valladolid, Spain

(Received 3 December 1996; revised version received 20 January 1997; accepted 31 January 1997)

Abstract Degradation of chlorophenols by P. chrysosporium in static cultures has been studied. The influences o f mycelium acclimation, co-substrate concentration and nitrogen source on phenol degradation were analyzed. With non-acclimated mycelium the maximal concentrations degraded were 150ppm of o-chorophenol and lOOppm of the isomers m- and p-chlorophenol. The substituted ortho-position on the aromatic ring was the preferred attack position. Meta- and para-positions were less reactive and resulted in a slower degradation rate than the ortho position. Nevertheless, with acclimated mycelium, an increase in the ability to degrade chlorophenol and a higher reactivity in meta- and para-positions were observed (degraded chlorophenol increased by up to 70% for the o-isomer and 50% for the m- and p-isomers with respect to non-acclimated mycelium). A decrease in glucose concentration caused a decrease in chlorophenol degradation rate. Twelve days were needed for complete degradation of o-chlorophenol with lO g/l of glucose and 22 days when glucose concentration was decreased to 2.5g/l. The reduction of ammonium tartrate caused a greater lag time, but not a decrease in chlorophenol degradation rate. Replacement of ammonium tartrate by ammonium chloride caused a decrease in chlorophenol degradation rate. © 1997 Elsevier Science Ltd.

Key words: Phanerochaete chrysosporium, chlorophenol isomers, degradation.

INTRODUCTION

Organic chlorinated compounds are environmentally widespread. The main sources of these xenobiotic compounds are pulp chlorine-bleaching industries, textile dyes industries, and biocides and herbicides used in agriculture. The excessive use of pesticides and herbicides in agriculture is an important cause of monochlorophenol appearance in surface- and ground-water due to the breakdown of compounds such as 2-4D and pentachlorophenols (U.S. EPA,

*Author to whom correspondence should be addressed. 207

1993; Virtanen and Hattula, 1982). Chlorophenols are also intermediates in the aerobic metabolism of chlorinated compounds such as chlorobenzenes and chlorobenzoates (Reineke and Knackmuss, 1984). They may also be formed in the chlorination of surface waters and wastewater. They are, then, common estuarine pollutants.

These chlorinated compounds are toxic and inhibitory for conventional anaerobic treatment (Beltrame et al., 1988; Batersby and Wilson, 1989; Madsen and Aamand, 1992) and for denitrification processes (Owen et al., 1996). Mono- and di-chlorophenols are the end products of the anaerobic reductive dehalogenation of organic compounds (Sheldon et al., 1995) and are relatively recalcitrant to reductive dechlorination (Bryant et al., 1991; Woods et al., 1989; Mikesell and Boyd, 1986). The development of new and alternative ways for degrading such compounds is an important subject to be studied. An alternative for these compounds is the use of white-rot fungi (Lamar and Dietrick, 1990; Bumpus et al., 1985; Eaton, 1985). These have non-specific enzymatic systems which are expressed under nutrient-limiting conditions (Jeffries et al., 1981; Kirk et al., 1978).

The aim of this work was the study of the degradation, in a culture medium, of ortho-, meta- and para-chlorophenol isomers by Phanerochaete chrysosporium. Mycelium acclimation, glucose concentration and ammonium nitrogen concentration and ammonium nitrogen source were the parameters studied.

METHODS

Fungus Phanerochaete chrysosporium (ATCC 24725) supplied by the Chemical Engineering Department of the Universidad Aut6noma de Barcelona (Barce- lona, Spain) was used. It was maintained on malt extract (ME) agar slants at 30°C. ME medium con- tained 3% malt extract, 2% glucose, 0.1% casein

208 R. Rubio P&ez, (Yr. GonzMez Benito, M. Peha Miranda

peptone (pH4.5) [Pronadisa (Hispananlab S.A.), Alcobendas, Madrid].

The acclimated fungus was obtained after consec- utive growths of the microorganism with increasing concentrations of each chlorophenol-isomer. Inocu- lum from ME was grown with 50ppm of o-chlorophenol, then an inoculum from this was grown in a culture with 100 ppm, and so on up to 300 ppm of o-chlorophenol at which mycelium did not grow. The highest o-chlorophenol concentration at which growth was observed was 250 ppm. Myce- lium from the last culture of this series was the acclimated mycelium. This mycelium was kept and periodically subcultured on ME. The activity of this acclimated mycelium was also periodically tested by growing it in a culture with 250 ppm of o-chlorophenol and showing that this amount was completely degraded. The same sequence was carried out for meta- and para-isomers.

Chemicals o-Chlorophenol (98% pure), m-chlorophenol (98% pure) and p-chlorophenol (98% pure) were obtained from Merck.

Inoculum and media Cultures were inoculated with two mycelial discs of agar of about 1 cm diameter cut from the zone of active growth of ME Petri plates about five-seven days old and in the stationary phase of growth.

To determine chlorophenol disappearance, cultures of P chrysosporium were incubated at 33°C and pH 4.5 in nitrogen-limited culture medium containing KH2PO 4 2 g/l, MgSO47H20 0.5 g/l, CaCI2 0.1 g/l, glucose 10 g/l, ammonium tartrate 0.2 g/l, thiamine i mg/l and trace elements (Mg +2, Mn +2, Na +, Fe +2, C0+2, Zn+2, Cu+2, Al+3, K+). Except for thiamine solution, the culture medium was autoclaved at 120°C for 20 min. Thiamine solution was sterilized separately by filtration (22 nm HP Millipore filter).

All experiments were carried out in static cultures (surface culture), using 500 ml Erlenmeyer flasks, containing 150ml of the culture medium. The experiments were carried out in triplicate and the values shown are mean values with a standard devia- tion of 10%.

Analytical methods The disappearance of o-, m- or p-chlorophenol was monitored by following the concentrations of the isomers in the culture by HPLC, with a Nucleosil packed column (120 C-18 5 # 25*0.46 cm) at 25°C. The mobile phase was a mixture of acetonitrile/ water in a ratio 1:1 at a flow rate of 0.5 ml/min. (600-700psi of pressure). A spectrophotometer (Waters LC, Lamb model 481) set at a wavelength of 254 nm was used for the detection. The samples were filtered through a GP 45 nm Millipore filter before HPLC measurements.

The pH was measured with a Crison pH/mv-506 pH meter.

RESULTS AND DISCUSSION

Non-acclimated and acclimated mycelium Figure 1 shows o-chlorophenol degradation using non-acclimated mycelium. The highest concentration degraded under these conditions was about 150 ppm, 14-16 days of incubation being needed for its complete disappearance. Growth of mycelium was not observed at o-chlorophenol concentrations of 200 ppm and over.

Figure 2 shows that m- and p-chlorophenol had similar behaviours, both in the maximum concentration degraded and changes with time. The highest concentration degraded was 100 ppm for both isomers and 15 days was needed for complete degradation.

Comparing the results obtained with the three isomers showed different degradation rates for the

400

350

~, 300

250 "5 e-,

200 o.

150 ? o 100

50

~ W e ~ ~ ~ V

0 10 20 time (d)

--x- 50 ppm ~ 100 ppm ~ 150 ppm -v-- 200 ppm --=- 300 ppm

Fig. 1. Changes in concentration of o-chlorophenol in static flask cultures with non-acclimated mycelium of P chrysosporium for different initial o-chlorophenol concentrations.

Chlorophenol degradation by P. chrysosporium 209

200

~150 J

== ~1oo

~ 50 I

0

0 5 10 time (d)

= t I - - ~

15 20 25

-~ - 50 ppm m- -v- 50 ppm p- --=- 100 ppm m-

-E~ 100 ppm p- ~ 150 ppm m- -~ - 150 ppm p-

Fig. 2. Changes in concentration of m- and p-chlorophenol isomers in static flask cultures with non-acclimated mycelium of P. chrysospodum for different initial concentrations.

o-isomer, and meta- and para-isomers. Through all experiments the degradation rates of m- and p- chlorophenols were less than that of the o-isomer, this effect becoming more pronounced with increasing isomer concentration. With an initial concentration of 50 ppm, after three days of cultivation, about 60-70% of each isomer was degraded. When the initial concentration was 100ppm and after the same time of cultivation, 44% of the ortho isomer was degraded, but only 31 and 23% of m- and p-chlorophenols were degraded. Therefore, the ability of P chrysosporium to degrade monochlorophenol depended on the position of the substitute chlorine. A different degradation rate depending on the substituted position was also observed by Rogers et al. (1990) working with fresh sewage, and by Hale et al. (1990) working with two microbial communities from different pond sediments. They found that the dechlorination rates of the monochlorophenol isomers were in the order ortho > meta > para. The transformation of trichlorophenols to dichlorophenols and monochlorophenols by a methanogenic enrichment culture from a sewage

sludge also showed a similar preference in dechlorination (Boyd and Shelton, 1984; Madsen and Aamand, 1992). Zeddel et al. (1993), with low- chlorinated biphenyls, also observed a different dehalogenation rate depending on the substituted position, although they found that para was the preferred attack position.

When acclimated mycelium was used, an increase in the ability to degrade chlorophenols was observed (Fig. 3). The acclimated mycelium tolerated and degraded larger amounts of chlorophenols than did non-acclimated mycelium. The amount of o-chlorophenol degraded increased by 70%, and for the other isomers by 50%. Fifteen-17 days were needed for a complete degradation of these concentrations.

After three days of cultivation with acclimated inoculum in an initial concentration of 100 ppm, the o-chlorophenol isomer was degraded about 70%, and m- and p-chlorophenol isomers about 58%. These results contrast with the values obtained with non-acclimated mycelium in which a large difference between ortho- and para-position was observed in the amount degraded. Therefore, the acclimated

300 - -

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c)

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iiiiiii!iiiiiiiii - iiiiiiiiiiiiiiiiiiii

!iiiiii si !

- iii~i~

o-chlorc ~henol m--chlorophenol p-chlorophenol

D Non acclimated [ ] Acclimated

Fig. 3. H i g h e s t c o n c e n t r a t i o n of c h l o r o p h e n o l i s om er s d e g r a d e d in s ta t ic f lask cu l tu res of n o n - a c c l i m a t e d a n d a c c l i m a t e d m y c e l i u m of P. chrysosporium.

210 R. Rubio P&ez, G. GonzMez Ben#o, M. Peha Miranda

mycelium produced an increase in the ability to degrade chlorophenols, the degradation rate was increased, and the preference for the ortho attack- position was reduced, the degradation rates becoming increasingly similar for the three isomers.

Influence of co-substrate concentrat ion Glucose in the range of 2-10 g/1 was the co-substrate used to determine the influence of carbon source concentration on degradation of chlorophenol isomers. These cultures were inoculated with acclimated mycelium of P chrysosporium.

Figures 4-6 show the degradation time for each chlorophenol isomer. The degradation rate decreased for all isomers when glucose concentration decreased; nevertheless, behaviour was different depending on whether ortho isomer or m-/p-isomers were present.

No lag period for the ortho-chlorophenol isomer was observed in the range of glucose concentration between 10 and 2.5 g/1. After eight days of cultiva-

tion, the removal percentage was about 52%-96% for this range of glucose concentration. However, for a glucose concentration of 2 g/l, mycelium did not grow and therefore o-chlorophenol degradation did not happen. The degradation rate decreased when the glucose was decreased. For meta- and para- chlorophenol isomers, glucose concentrations less than 10 g/1 caused a period of no appreciable chlorophenol degradation. This maximum period seemed to be five days for the para isomer. After this period a rapid isomer disappearance took place. The percentage of chlorophenol removal increased from

.26% (seventh day) up to 90% ( l l th day) for the m- isomer and from 20% up to 76% for p-chlorophenol isomer.

The time courses of degradation of m- and p- chlorophenol isomers were practically coincident. The minimum glucose concentration needed for their degradation was 3.5 g/1. However, for the ortho-isomer 2.5 g/1 was the minimum glucose concentration needed for its complete degradation. For

150

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v

0 t -

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o ¢ -

? 0

100

50

0 25

)< ~ >< x ~ < --____,1<

5 10 15 20 time (d)

glucose 4 - 10 g/L -~- 5 g/L - * - 2,5 g/L -~- 2 g/L

Fig. 4. o-Chlorophenol degradation with different glucose concentrations in static cultures of acclimated mycelium of P. chrysosporium. The initial o-chlorophenol concentration was 100 ppm.

150

E ¢ n

~100 0 ¢,-

t.'- 0 .

e o 50 r -

? E

x

0 0 5 10 15 20 25

time (d)

=lucos= -=- 10 g/L ~ - 5 g/L ~ 3,5 g/L --'- 2,5 g/L

Fig. 5. m-Chlorophenol degradation with different glucose concentrations in static cultures of acclimated mycelium of P chrysosporium. The initial m-chlorophenol concentration was 100 ppm.


150

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100 0 C

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2 o 50 ¢ -

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~lucose

5 10 time (d)

-,,- 10 glk -~- 5 g/L

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15 20 25

~ - 3,5 g/L - ~ 2,5 g/L

Fig. 6. p-Chlorophenol degradation with different glucose concentrations in static cultures of acclimated mycelium of P chrysosporium. The initial p-chlorophenol concentration was 100 ppm.

glucose concentrations below this value mycelium growth did not occur and therefore chlorophenol degradation was not observed.

Influence of nitrogen concentration In order to determine the influence of ammonium nitrogen on chlorophenol degradation, experiments with two different ammonium-nitrogen concentrations were carried out. The ammonium-nitrogen source was ammonium tartrate (0.2 and 0.1 g/l). The cultures were inoculated with acclimated mycelium, and the glucose concentration was 10 g/l.

Figure 7 shows the results obtained with the three monochlorophenols. In all experiments a complete degradation of each isomer occurred. When ammonium tartrate concentration was 0.2 g/1 there was no lag period, chlorophenol degradation was observed from the start of the culture. But, when ammonium tartrate was reduced to 0.1g/1 a period of about three days with no appreciable degradation

appeared. After this period the degradation rate was similar to the rate obtained with the higher ammonium tartrate concentration, so that the time of the degradation was the same. It could be concluded that ammonium tartrate did not exert any influence on the degradation rate of chlorophenols, only on the time that the fungus needed to adapt to the lower nitrogen concentration.

Influence of ammonium nitrogen source Experiments with ammonium chloride instead of ammonium tartrate and for each glucose concentration were carried out in order to determine the influence on chlorophenol degradation of ammonium-nitrogen source. Ammonium chloride is a cheaper nitrogen source and for applications on a large scale cost is an important factor to be con- sidered. The ammonium-nitrogen concentration used was 0.12g/l, corresponding to 0.2g/l of ammonium tartrate and 0.356g/1 of ammonium

0 5 10 15 20 25 time (d)

0.2alL tartrate -m- o-chlorophenol =e- m-chlorophenol -v- p-chlorophenol 0 , 1 a l L tar t rate - 0 - o-chlorophenol ~ m-chlorophenol -~ - p-chlorophenol

Fig. 7. Influence of ammonium tartrate concentration. Changes in chlorophenol isomer concentrations in static cultures of acclimated P. chrysosporium. The concentrations of ammonium tartrate added to the culture medium were 0.2 g/l and

0.1 g/1.

212 R. Rubio P~rez, G. Gonzdlez Benito, M. Peha Miranda

chloride. Figures 8-10 show the results obtained in these experiments. In all of them the chlorophenol degradation rate was lowest when ammonium chloride was used. The effect of glucose concentration was the same for both nitrogen sources; chlorophenol removal rate decreased when glucose concentration decreased. For both ammonium-nitrogen sources the highest disappearance rates occurred with highest glucose concentrations.

In this study, the ability of P. chrysosporium to remove chlorophenol has been shown. As a result of these experiments, it can be noted that the amount degraded increased by up to 70% for the o-isomer and by 50% for m- and p-isomers when acclimated mycelium was used instead of non-acclimated mycelium. The highest amount degraded, 250ppm of

o-chlorophenol after 15-17 days of cultivation, required 10 g/l of glucose and 0.2 g/l of ammonium tartrate for its complete removal, and the ortho position was the preferred attack position. Neverthe- less, more studies are needed in order to obtain better information on the effects of ammonium- nitrogen source and carbon source, as well as the feasibility of use of this fungus in a submerged pro- cess for a further application on a large scale.

R E F E R E N C E S

Battersby, N. S. & Wilson, V. (1989). Survey of the anaerobic biodegradation potential of organic chemicals in digesting sludge. Applied Environ. MicrobioL, 55(2), 433 -439.

120

100

ao o c ® 60 e-

-~ 40

20

0

0 5 10 15 20 25 time (d)

ammonium tartrate - l - lOg/L glucose ~ 5g/L glucose -,,-- 2,5g/L glucose ammonium chlodde -~- lOg/L glucose -~- 5g/L. glucose + 2,5g/L glucose

Fig. 8. Influence of ammonium nitrogen source and glucose concentration, o-Chlorophenol degradation by acclimated mycelium of P chrysosporium in static cultures. Ammonium tartrate (0.2 g/I) and ammonium chloride (0.356 g/l) were the

nitrogen sources used.

1 0 0 ~

i g~ ~ 5o

0 5 10 15 20 25 Time (d)

Ammonium tartrate .4- 10g/L glucose ~ 5g/L glucose -.0- 3,5g/L glucose Ammonium chloride -El-- 10g/L glucose ~ 5g/L glucose -<:3- 3,5g/L glucose

Fig. 9. Influence of ammonium nitrogen source and glucose concentration, m-Chlorophenol degradation by acclimated mycelium of P. chrysosporium in static cultures. Ammonium tartrate (0.2 g/l) and ammonium chloride (0.356 g/l) were the



100 E

0 t '-

e -

e 50 o t"-

? Q .

0

0

ammonium tartrate

ammonium c~hlodde

5 10 15 20 25 time (d)

-m= lOg/L glucose -v- 5 g/L glucose -e- 3,5g/L glucose - ~ lOg/L glucose ~ 5g/L glucose -~- 3.5g/L glucose

Fig. 10. Influence of ammonium nitrogen source and glucose concentration, p-Chlorophenol degradation by acclimated mycelium of P chrysosporium in static cultures. Ammonium tartrate (0.2 g/l) and ammonium chloride (0.356 g/l) were the


Beltrame, P., Beltrame, P. L., Carniti, P., Guardioni, D. & Lanzetta, C. (1988). Inhibiting action of chlorophenols on biodegradation of phenol and its correlation with structural properties of inhibitors Biotechnol. Bioengng, 31, 821-828.

Boyd, S. A. & Shelton, D. R. (1984). Anaerobic biodegradation of chlorophenols in fresh and acclimated sludge. Applied Environ. Microbiol., 47, 272-277.

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Bumpus, J. A., Wright, T. D. & Aust, S. D. (1985). Oxida- tion of persistent environmental pollutants by a white rot fungus. Science, 225(4706), 1434-1436.

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Jeffries, T. W., Choi, S. & Kirk, T. K. (1981). Nutritional regulation of lignin degradation by Phanerochaete chrysosporium. Applied Environ. Microbiol., 42, 290-296.

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enrichment culture. AppL Environ. Microbiol., 58(2), 557-561.

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Chlorophenol degradation by Phanerochaete chrysosporium