Production of halogenated compounds by - Springer Link

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Jan 4, 1994 - 1986; Harper and Hamilton 1988). Both systems are active with chloride, bromide and iodide ions but not with fluoride ions. In this work we also ...
Appl Microbiol Biotechnol (1994) 42:212-221

© Springer-Verlag 1994

H.-E. Spinnler • E. de Jong • G. Mauvais E. Semon • J.-L. le Quere

Production of halogenated compoundsby Bjerkandera adusta

Received: 4 January 1994/Received revision: 21 March 1994/Accepted: 18 April 1994

Abstract The white-rot fungus Bjerkandera adusta produces volatile chlorinated phenyl compounds. The main compounds identified were 3-chloro-4-methoxybenzaldehyde (3-chloro-p-anisaldehyde), 3-chloro4-methoxybenzyl alcohol (3-chloro-p-anisyl alcohol), 3,5-dichloro-4-methoxybenzaldehyde (3,5-dichloro-panisaldehyde), and 3,5-dichloro, 4-methoxybenzyl alcohol (3,5-dichloro-p-anisyl alcohol), p-Anisaldehyde, veratraldehyde and the corresponding alcohols, p-anisyl alcohol and veratryl alcohol were produced simultaneously. Even with a very low concentration of chloride in the medium (< 10-SM), chlorinated aromatic compounds were still observed. Addition of bromide to the culture medium led to the production of brominated compounds: 3-bromo-4-methoxybenzaldehyde, 3-bromo4-methoxybenzyl alcohol, 3,5-dibromo-4-methoxybenzaldehyde and 3-bromo-5-chloro-4-methoxybenzaldehyde. These brominated compounds have not previously been reported as natural products. Although iodo-aromatic compounds were not produced by supplementation of the medium with iodide, isovanillin was found in the culture broth under these conditions. This compound may be formed by substitution of the iodine intermediate by a hydroxyl group on the third carbon of the ring. Diiodomethane or chloroiodomethane were also found. It is the first time that the production of halomethane has been

H.-E. Spinnler1 • G. Mauvais • E. Semon - J.-L. le Quere INRA, Laboratoire de Recherches sur les Aromes, 17 rue Sully, 21034 Dijon, Cedex, France E. de Jong Division of Industrial Microbiology, Department of Food Science, Agricultural University of Wageningen, P.O. Box 8129, 6700 EV Wageningen, The Netherlands Present address: 1 IFR-Reading laboratory, Earley Gate, Whiteknights Road, Reading RG6 2EF, UK

related to the production of halogenated aromatic compounds. All the strains tested have these capabilities.

Introduction Numerous micro-organisms, especially fungi, are known to produce volatile odorous compounds. At the moment, biotechnological production of aromas still remains marginal. However, many ligninolytic fungi (basidiomycetes) produce several flavour compounds containing an aromatic skeleton (Gallois et al. 1990). Bjerkandera adusta, a ligninolytic basidiomycete, produces several aromatic volatiles in high quantities and was addressed as a promising micro-organism for the production of valuable flavour compounds (Berger et al. 1987). During the screening of basidiomycetes capable of producing flavour compounds (Gallois et al. 1990), we detected, surprisingly, many volatiles with the characteristic mass spectra of chlorinated compounds in extracts of B. adusta media. They were among the most important volatile compounds produced. Part of these results were recently confirmed for another strain, Bjerkandera sp. strain BOS55, by de Jong et al. (1992, 1994a). They reported the production of several chlorinated compounds, including 3-chloro-4-methoxybenzaldehyde (3-chloro-p-anisaldehyde) and 3-chloro-4methoxybenzyl alcohol (3-chloro-p-anisyl alcohol) by this strain. Halogenated compounds are not only of anthropogenic origin, since at present many micro-organisms are known to biosynthesize over 1500 different organohalogen metabolites (Gribble 1992). It has been anticipated that they are usually produced by chloroor bromoperoxidases, enzymes of biological importance in cellular differentiation (Morris et al. 1987), plant hormone production (Engvild 1986) and mammalian defence (Albrich et al. 1981). However, in

213 microbiological systems the concentrations of the c h l o r i n a t e d c o m p o u n d s d e t e c t e d a r e u s u a l l y low. P r e viously, only the significant production of chlorom e t h a n e b y s a p r o p h y t i c fungi h a s b e e n r e p o r t e d . I t h a s b e e n i m p l i e d t h a t this b i o l o g i c a l p r o d u c t i o n is a m a j o r c o n t r i b u t o r to t h e g l o b a l c h l o r o m e t h a n e p o o l (Edw a r d s et al. 1982; H a r p e r 1985). I n t h e p r e s e n t w o r k , t h e a m o u n t a n d s p e c t r u m of both halogenated and non-halogenated volatile comp o u n d s p r o d u c e d b y s e v e r a l different B. adusta s t r a i n s is r e p o r t e d . T h e t w o m a i n c h l o r i n a t e d m e t a b o l i t i e s a r e identified by several spectroscopic techniques: mass spectrometry (MS) Fourier-transform infrared spectroscopy, (FTIR) and nuclear magnetic resonance (NMR). In addition, the mass spectra of two other halogenated metabolities are presented. In an attempt to alter the r a t i o b e t w e e n h a l o g e n a t e d a n d n o n - h a l o g e n a t e d volatiles t h e effect o f r e d u c e d c h l o r i d e c o n c e n t r a t i o n s a n d t h e a d d i t i o n of p r e c u r s o r s o n t h e m e t a b o l i t e s p e c t r u m h a s b e e n i n v e s t i g a t e d . T y r o s i n e a p p e a r s to b e a n efficient p r e c u r s o r o f all v o l a t i l e a r o m a t i c c o m p o u n d s , a n d u n d e r all c u l t u r e c o n d i t i o n s h a l o g e n a t e d c o m p o u n d s w e r e d e t e c t a b l e . T h e h a l o g e n a t i o n s y s t e m o f Bjerkandera spp. c a n b e aspecific for h a l i d e s , so t h e a d d i t i o n o f o t h e r h a l i d e s to t h e c u l t u r e m e d i u m h a s b e e n i n v e s t i g a t e d . T h i s r e s u l t e d in t h e p r o d u c t i o n o f s e v e r a l o t h e r halogenated compounds, including brominated anisyl aldehydes and halomethanes.

follows: riboflavin, 50 mg; pyridoxine, 50 mg; calcium pantothenate, 500 mg; p-aminobenzoic acid, 50 mg; H20 up to 1 1. This solution was sterilized on a 0.2-/~m filter (Sartorius, Palaiseau, France). As in the Vogel medium, phenylalanine (50 mg/1), threonine (36 mg/1) and tryptophane (20 mg/l) were also added to this medium. The high ammonium tartrate medium (LCM-HAT) was used to test the effect of 1 g/1 of tyrosine and/or 41 mM calcium chloride addition. Halides were added as their potassium salts at a concentration of 20 mM except for KF (10mM) to the SM-HAT medium. All media were adjusted to pH 5.5 with 0.5 M H2SO4. Media (100 ml) were dispensed in 500-ml erlenmeyer flasks. They were sterilized for 20 rain at 110°C, inoculated with i x 106 spores/ml and routinely incubated under agitated conditions (200 rpm, 3.6 cm diameter) at 25°C. Chloride content was verified using a chloride measurement apparatus made up of a pH meter (Minisis 800, Tacussel, Lyon, France), a reference electrode (TR200, Tacussel) and a chloride measurement electrode (X5210, Tacussel). The standardization was made with KC1 10 -s M to 1 M. The concentration in the LCM was lower than 10 -5 M. The SM contained 17 mM of chlorides provided mainly by the casamino acids (16 mM at 0.1% concentration) and only 1.6 mM by yeast extract (0.1%).

Extraction and analysis of volatile compounds Each culture was filtered through cheese cloth. The mycelium retained was dried in an oven (70°C) until constant weight to measure growth. 1,3-Dimethoxy benzene (64 ~tg/100 ml) was added to the culture filtrate before extraction and used as a standard for quantification. The filtrates were extracted with bidistilled CH2C12 (1:0.3 v/v and 1:0.1 v/v) (bidistilled diethyl ether was used in the assay with low chlorinated medium). Solvent extracts were concentrated to 100 gl as described previously (Gallois et al. 1990). All chemicals used in analytical procedures were the purest found on the market.

Materials and methods Organisms

Gas chromatography (GC)

The strains used in this study were B. adusta [(Wild. ex Fr.) Karst] CBS 595.78, DSM 3375, or INRA MIC 64. The basidiomycetes used in this study were obtained as described before (Gallois et al. 1990). The strain INRA MIC 64 was kindly provided by Dr. M. Asther (LMCF, INRA Marseilles, F). When the strain number is not specified the strain used was B. adusta CBS 595.78. The strains were maintained at 4°C on potato-dextrose agar slants. Spore inocula were prepared in Roux flasks in the same medium and harvested after 3 weeks with 150 ml Tween 80 solution (0.5%, v/v) containing ten glass balls (3 mm diameter).

Volatile compounds were analysed on a DB 1701 bonded fused silica capillary column (J&W Scientific, 30 m, 0.32 i.d., 1 gm). The injection (1 pl) was splitless/split(30 s) the temperature of the injector was 230°C and that of the flame ionization detector was 240°C. The temperature raised from 60°C to 240°C at 3°C/rain. The Hz carrier gas flow rate was 50 cm/s. Kovats indices were calculated using a personal-computer based, multichannel chromatography workstation "Coconut" (R. Almanza and P. Mielle, INRA, Dijon, France). Quantification was made as internal standard equivalents by the following calculation: Qx = (Ax/Ae)*Qe

Liquid media and culture conditions A standard medium with a high ammonium tartrate level was usually used. The composition of this medium (SM-HAT) was: glucose, 10 g; KH2PO4, 0.2 g; MgSO4-7HzO, 0.5 g; casamino acids, 1 g; yeast extract, 1 g; ammonium tartrate, 0.368g; distilled water, 1 1. A low ammonium tartrate medium (SM-LAT) was used as control with the culture made on a medium without chloride; the concentration of ammonium tartrate was in this case (0.0184 g/l). A chemically defined medium was used in attempt to reduce the concentrations of chloride. This medium was a low ammonium tartrate medium (LCM-LAT) (0.0184 g/l). A concentrated vitamin solution (1 ml) replaced the casamino acids and yeast extract. This vitamin solution, first defined by Vogel (1964) was modified as

Qe and Qx being the respective concentrations of the internal standard and of the compound x, and Ae and Ax being the area of the corresponding GC peaks.

Mass spectrometry Mass spectra were obtained with a Nermag R10-10 coupled with a Girdel 31 GC. The column and the GC conditions were as above. Electron impact was recorded with an ion source energy of 70 eV. The scanning rate was 0.8 s from 25 to 300 atom mass unit. Infrared spectra were measured in the gas phase on a Bruker 1FS85 FTIR spectrometer coupled to a Carlo Erba HRGC 5160.

214 1H-NMR spectra

These were recorded on a Bruker WM400 spectrometer at 400.13 MHz. The residual proton signal of the deuterated solvent (CDC13) was used as the internal reference (d = 7.24 ppm).

HPLC analysis

Products were purified using a preparative Lichrosorb diol column (250 x 10 mm) in two steps: (1) CH2Clz (10%)/pentane (90%) to 100% CH2C12 for 40 min; (2) 20% CH2Clz/pentane 80% to 50% CH2C12 for 40 rain. In both cases the flow rate was 3 ml/min. UV detection was at 214 nm.

Results Profile of volatile compounds produced by B. adusta The c o m p o u n d s quantified here were less volatile than the dichloromethane or ether used as solvent for extraction. The quantification of very volatile c o m p o u n d s such as chloromethane, b r o m o m e t h a n e , i o d o m e t h a n e would need to use headspace techniques, which we did not do. The main volatiles o b t a i n e d in a 21-day-old culture of B. adusta on the S M - H A T m e d i u m are shown in Fig. 1. In this profile, the main c o m p o u n d s are the chlorinated c o m p o u n d B (35.1 _+ 2.2 mg/1), benzyl alco-

Fig. 1. Profile of the volatile SIGNAL compounds produced by 1000 Bjerkandera adusta from a 21day-old culture on glucose medium: 1 N,Ndimethylacetamide, 2 benzaldehyde, 3 benzyl alcohol, 4 internal standard (1,3-dimethoxybenzene), 5 anisaldehyde, 6 anisyl alcohol, 7 compound A (3,5-dichloro4-methoxybenzaldehyde), 8 compound B (3-chloro-4methoxybenzaldehyde), 9 veratraldehyde, 10 veratryl alcohol, 11 3-chloro-4methoxybenzyl alcohol, 12 ?undecanolide, 13 V500 dodecanolide, 14 unknown chlorinated compound, 15 3,5dichloro-4-methoxybenzyl alcohol

2

hol (7.5 + 0.7 mg/1), p-anisaldehyde (9.5 __+0.6 mg/1), veratryl alcohol (2.05 + 1.5mg/1), p-anisyl alcohol (1.3 + 0.4 mg/1) and c o m p o u n d A (0.42 + 0.05 mg/1). As we were not able to identify directly by G C - M S the chlorinated c o m p o u n d s produced, N M R spectra of the pure c o m p o u n d s were obtained after H P L C isolation. These c o m p o u n d s were found in the culture b r o t h of the three strains of B. adusta tested.

Identification of compounds A and B From 1.5 1 of culture broth, 1.1 mg of thin white crystals of compound A and 37.8 mg of compound B were obtained after two purification steps. The mass spectrum of A displayed the major ions in decreasing abundance at m/z 207(14.9), 206(44.8), 205(69), 204(75), 203(100), 133(13.4). The isotopic distribution revealed a dichlorinated molecule with a molecular mass of 204 (35C1) and suggested a formula of C8H602C12 . The importance of the fragment ions doublet at m/z 203 and 205 (major peaks), attributed to (M - 1) ÷, suggested an aldehyde. This was confirmed with the G C - F T I R spectrum, which displayed characteristic features of an aromatic aldehyde (n C = O , 1726 cm; 2826 cm, aldehydic n C - H ; 2716 cm, aldehydic 2 d C - H (Nyquist 1984). The gas phase infrared spectrum revealed an ether functionality (1273cm, n C - O - C ) . The 1 H - N M R

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215 spectrum of A revealed an aldehydic proton at d 9.85, two equivalent aromatic protons at d 7.81 and a methoxy singlet at d 3.97. All these spectroscopic features indicated the structure 3,5-dichloro-4methoxybenzaldehyde (3,5-dichloro-p-anisaldehyde) for compound A. The isotopic distribution around the molecular ion in the mass spectrum of B revealed a monochlorinated aldehyde of molecular mass 170 (3sC1), with the major peak at m/z 169 attributed to [M - 1] +, suggesting a molecular formula C8H702C1. The aldehydic function was confirmed with the GC-FTIR spectrum, which again displayed characteristic features of an aromatic aldehyde (1720 cm, n C = O; 2810 cm, aldehydic n C-H; 2719 cm, aldehydic 2 d C-H). An absorption band at 1269 cm was attributed to the C - O - C stretching of an ether function. The 1H-NMR spectrum of B revealed an aldehydic proton at d 9.84 and a methoxy singlet at d 3.98. Three aromatic protons were found at d 7.90, 7.76 and 7.03 ppm, respectively. The former revealed a meta coupling (J = 2.0 Hz) and the latter an ortho coupling (J = 8.48 Hz). The proton at d 7.76 displayed both ortho (J = 8.48 Hz) and meta (J = 2.0 Hz) couplings. These spectroscopic features supported the formula 3-chloro-4-methoxybenzaldehyde (3-chloro-p-anisaldehyde). The corresponding alcohols 3,5-dichloro-panisyl alcohol and 3-chloro-p-anisyl alcohol could also be detected in the profile (Fig. 1). The identifications were based on comparing the mass spectra with reported data (Pfefferle et al. 1990; de Jong et al. 1992). However, the concentrations were too low for further purification. An interesting fact was the detection of two more oxidized chlorinated compounds in old stationary cultures. Based on the recorded mass spectra (Fig. 2) and those reported in the literature (Valli and Gold 1991; Joshi and Gold 1993), the compounds are tentatively identified as 2-chloro-l,4-dimethoxybenzene and 2,6dichloro-l,4-dimethoxybenzene; in agitated cultures we were not able to detect those two degradation products. From these data and the mass spectra of brominated compounds presented in Fig. 3, we identified also brominated compounds when bromide was added in the culture medium. Effect of chloride concentration on organohalogen production For flavour production chlorinated compounds are undesirable. We therefore tried to prevent their production by drastically reducing the concentration of chloride ions. The low chloride concentration of LCM-LAT medium (< 10-SM) did not completely stop the synthesis of chlorinated compounds. In LCM-LAT medium, the biomass obtained was much lower than in SM-LAT medium (Table 1). The average production of

chlorinated compounds was much lower on LCMLAT medium than on SM-LAT medium. However, the difference was not significant because of the high standard deviation. After 10 days the concentrations measured were always below 3.5 I~M inLCM-LAT medium, in accordance with the initial concentration of chloride present. This testifies to the high yield of chloride conversion, which was roughly estimated to be 55%. The chlorinated/flavours ratio was significantly decreased in LCM-LAT medium as compared with SM-LAT medium (P < 0.1%) but the production of chlorinated compounds per gram of biomass was quite constant on both media and equal to 3.2 gmol/g dry weight. Addition of calcium chloride and tyrosine in SM-HAT medium As chloro-p-anisaldehyde derivatives were the more concentrated, we looked for the effect of tyrosine addition with high or low chloride concentration. For this assay we chose LCM-HAT medium. The results were analysed with a model of variance taking into account the changes due to calcium chloride, tyrosine, and their interaction. The addition of chloride in the medium (41 mu) significantly improved the production of aromatic alcohols (benzyl alcohol, anisyl alcohol, veratryl alcohol) and significantly decreased the production of the aromatic aldehydes (benzaldehyde and p-anisaldehyde) (Table 2). However both the total production and the relative distribution of the chlorinated aromatic compounds were not significantly influenced by the addition of extra chloride ions to the medium. The addition of tyrosine to this medium resulted in a strong increase in the total amount of volatiles produced. Especially, the amount of 3-chloro-p-anisaldehyde produced was strongly raised, but also the amounts of p-anisyl alcohol and p-anisaldehyde were elevated. The production of benzyl alcohol and benzaldehyde, compounds not containing a substitution on the para position, were decreased. Effect of the addition of other halides to SM-HAT medium The addition of bromide to the medium brought about the production of brominated phenyl compounds (Table 3). Three of these compounds were identified by their mass spectra (Fig. 3), the breakdown of these molecules by the electron flux resulted in the same pattern as observed with the chlorinated compounds. The product spectrum with bromide ions was similar to that with chloride ions since the main compound produced was 3-bromo-p-anisaldehyde (14 mg/1). This is the bromo analogue of the main chlorinated compound produced. Although chloride ions and bromide ions were initially available at about the same concentration,

216 Fig. 2A, B Mass spectra of 2-chloro-l,4-dimethoxybenzene (A) and 2,6-dichloro1,4-dimethoxybenzene(B)

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m/e the total a m o u n t of b r o m i n a t e d aromatic c o m p o u n d s exceeded the total a m o u n t of chlorinated aromatic c o m p o u n d s by m o r e than eight times. The p r o d u c t i o n of a b r o m o - c h l o r o a r o m a t i c c o m p o u n d was also ob-

served in the culture broth. It is anticipated that 3-bromo-5-chloro-p- anisaldehyde was formed but the respective position of the two halogens on the ring have not yet been determined. 3-Methoxybenzaldehyde was

217 Fig. 3 Mass spectra of 3-bromo-panisyl alcohol (A), 3-bromo-panisaldehyde (B), 3,5-dibromop-anisaldehyde (C), 3-bromo-5-chlorop-anisaldehyde (D) (continued on next page)

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m/e found only in the extract with extra bromide ions and not with the addition of chloride ions. However, as the Kovats indice of this c o m p o u n d was close to that of the 3-chloroanisyl alcohol, it could be hidden by this compound. The addition of iodide ions to the culture broth resulted in a strong reduction in the total a m o u n t of chlorinated aromatic c o m p o u n d s produced. This reduction was comparable to the reduction seen with bromide ions (Table 3). The only new aromatic metab-

olite detected with iodide ions addition was isovanillin (3-hydroxy-p-anisaldehyde). In the extracts its concentration was 276 _+ 85 gg/1. N o t e w o r t h y was the detection of di- and trihalogenated methanes, chloroiodomethane, diiodomethane and dichloroiodomethane, in the culture broth. All the c o m p o u n d s quantified here were less volatile than the dichloromethane or ether used as solvents for extraction. It is possible also that very volatile c o m p o u n d s such as chloromethane, b r o m o m e t h a n e and i o d o m e t h a n e are

218 Fig. 3 (continued)

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m/e produced. However, quantification of these kinds of compounds would require a different experimental set-up and headspace analysis.

Discussion Basidiomycetous fungi are a potential source for the biotechnological production of flavours (Gallois et al.

1990). One of the promising strains is B. adusta (Berger et al. 1986). However, several strains of B. adusta consistently produced the same spectrum of chlorinated anisyl metabolites on several different media concurrently with several interesting flavour compounds containing an aromatic skeleton. The main chlorinated compound produced was 3-chloro-p-anisaldehyde. Minor concentrations of 3,5-dichloro-p-anisaldehyde and the corresponding alcohols were also found. These results confirm and extend earlier reports on the

219

production of 3-chloroanisyl alcohol and aldehyde by Bjerkandera sp. strain BOS55 (de Jong et al. 1992) and of 3,5-dichloroanisyl alcohol and aldehyde by several basidiomycetes, including Hypholoma, Stropharia, and Bjerkandera spp. (Pfefferle et al. 1990; de Jong et al. 1994a). For the production of flavours, chorinated aromatic metabolities are unwanted side products. Therefore, we examined the influence of the chloride ion concentration on the chlorinated/non-chlorinated aromatics ratio and on the total amount of flavours produced. We

did not succeed in the preparation of a medium absolutely free of chloride ions. When B. adusta was grown in LCM medium, containing less then 10-5 M of CI-, the total amount of the chlorinated metabolites produced was strongly reduced. These results show that B. adusta can very efficiently convert chloride ion to organohalogen at very low chloride ion concentrations. The LCM was very suitable for strongly decreasing the chlorinated/non-chlorinated aromatics ratio while at the same time the amount of flavours (of dry biomass/ gram) remains high. The addition of extra chloride ions to the medium did not result in higher amounts of chlorinated aromatic compounds. It has been proposed that Phanerochaete chrysosporium biosynthesizes veratryl alcohol via phenylalanine, 3,4-dimethoxycinnamyl alcohol and veratryl glycerol (Shimada et al. 1981), but it was recently demonstrated that phenylalanine as well as benzaldehyde are veratryl alcohol precursors (Jensen et al. 1994). In this work, it has been shown that the addition of tyrosine results in elevated levels of p-anisyl alcohol, p-anisaldehyde and 3-chloro-p-anisaldehyde. These results indicate that tyrosine is an efficient precursor of those metabolites. In fact, tyrosine may even be an intermediate in the biosynthesis route for these (chlorinated-)anisyl metabolites. At present it is not known what kind of enzyme system is able to chlorinate the aromatic ring. However, we did not succeed in producing chlorinated monochlorodimedone, normally used to measure haloperoxidase activity, with crude culture supernatant (unpublished results). Lignin peroxidase, a ligninolytic enzyme, from P. chrysosporium, exhibits only brominating but no chlorinating activity with monochlorodimedone (Renganathan et al. 1987).

Table 2 Effect of the addition of tyrosine (5.65 mM) and/or chloride (41 raM) on the synthesis of volatiles (in equivalent mg/1 of 1,3-dimethoxybenzene) by Bjerkandera adusta on SM-HAT

medium (see Materials and methods) after 21 days of culture (means of four repetitions). The pH was the same in all media (3.4-3.86)

Table 1 Change in concentrations between cultures grown on SMLAT medium and cultures grown on LCM-LAT medium after 10 days; for media, see Materials and methods. Flavour compound means (benzaldehyde, benzyl alcohol, p-anisaldehyde, p-anisyl alcohol, veratraldehyde, veratryl alcohol) and chlorinated compound means (3-chloro-p-anisaldehyde, 3-chloro-p-anisyl alcohol and 3,5dichloro-p-anisaldehyde) are given. All concentrations were estimated in equivalents of the internal standard (x mean, SD standard deviation)

Parameters Biomass (g/l) Flavour compounds (~tM) Chlorinated compounds (~tM) Ratio (chlorinated/ flavours) Specific production of chlorinated compounds (gmol/g dry biomass)

Standard medium

Medium without chloride

x

x

SD 2.92

11.6

0.59 8.4

SD

0.59 32.9

0.01 21.5

9.13

7.5

1.94

1.56

0.79

0.17

0.06

0.01

3.13

3.29

Without tyrosine

Parameters

With tyrosine

With chloride

Without chloride

With chloride

Without chloride

Mean

Mean

SD

Mean

Mean

0.86 2.67 2.31 0.15 0.83 0.27

0.31 1.25 0.75 0.06 0.13 0.16

0.31 3.13 4.35 0.82 0.66 1.22

Benzaldehyde Beuzyl alcohol p-Anisaldehyde p-Anisyl alcohol Veratraldehyde Veratryl alcohol

0,45 3.9 2.39 0.18 1.66 1.3

Total non-chlorinated (flavours)

9.88

3-Chloro-p-anisaldehyde 3-Chloro-p-anisyl alcohol 3,5-Dichloro-p-anisaldehyde

26.3 0.50 0.90

Total chlorinated Ratio (chlorinated/flavours) Dry biomass (g/l)

27.7 2.8 0.81

SD 0.026 0.26 0.14 0.045 1.94 0.49

7.09 1.26 0.06 0.10

27.9 0.72 0.98

0.04

29.6 4.17 0.84

SD 0.020 0.56 0.23 0.16 0.02 1.32

10.49 4.85 0.53 0.13

39.8 0.34 1.06

0.05

41.2 3.9 0.74

0.3 0.94 6.92 0.17 0.74 0.0

SD 0.005 0.17 0.37 0.026 0.03 /

9.07 0.68 0.03 0.08

46.0 0.29 0.79

3.10 0.06 0.11

0.12

47.1 5.19 0.97

0.14

220 Table 3 Variations in the concentrations of volatiles of B. adusta cultures when different halides, (20 mM) except for F - (10 mM), were added to SM-HAT medium (in equivalent mg/1 of 1,3-

dimethoxybenzene). Cultures were stopped after 10 days of growth, (CV coefficient of variation, / not detected)

Halogen F-

C1-

Br-

I-

Compounds

Kovats Indices

Mean

CV(%)

Mean

CV (%) Mean

CV(%)

Mean

c v (%)

3-Chloro-4-methoxybenzaldehyde 3,5-Dichloro-4-methoxybenzaldehyde 3-Chloro-4-methoxybenzyl alcohol Chloroiodomethane Diiodomethane Dichloroiodomethane 3-Bromo-4-methoxybenzaldehyde 3-Bromo-4-methoxybenzyl alcohol 3-Bromo-5-chloro-4-methoxybenzaldehyde 3,5-Dibromo-4-methoxybenzaldehyde Benzaldehyde Benzyl alcohol Anisaldehyde Anisyl alcohol Veratraldehyde Veratryl alcohol Isovanillin

1658 1624 1517 810 1036 943 1820 1876 1759 1845 1090 1217 1443 1503 1691 1749 1744

16.1 0.59 0.003 / / / / / / / 2.7 2.07 2.76 0.02 1.87 0.06 0

46 61 119 / / / / / / / 31 49 29 58 176 26 /

35.1 0.42 1.83 / / / / / / / 1.16 6.38 3.36 1.34 1.53 2.64

6.2 13 28 / / / / / / / 33 19 88 23 19 30

1.14 0.02 0 / / / 8.8 0.026 0.016 0.054 1.9 2.04 2.28 0 0.37 0

22 75 / / / / 37 44 20 50 75 85 34 / 5 /

1.78 0.02 0.009 0.176 0.013 0.002 / / / / 0.89 0.31 5.35 0 0.48 0.18

20 11 27 57 70 45 / / / / 58 54 21 / 26 32

/

0

/

0.28

31

Although it has been shown that the chlorinated anisyl metabolites are quite resistant against their own extracellular ligninolytic enzymes (de Jong et al. 1994b), the discovery of trace amounts of 2-chloro- and 2,6dichloro- 1,4-dimethoxybenzene points to limited metabolism of the chlorinated compounds. Both phenol oxidases and vanillate hydroxylase can convert vanillate to methoxy(hydro) quinone (Eriksson et al. 1990). It is anticipated that a hydroxyl group on the aromatic ring is a prerequisit e for further degradation of the chlorinated anisyl metabolites. However, at present it is unclear how the chlorinated anisyl metabolites. are demethylated. Recently, the metabolism of 2,4-dichlorophenol and 2,4,5-trichlorophenol by whole cultures and by ligninolytic enzymes of P, chrysosporium has been elucidated (Valli and Gold 1991; Joshi and Gold 1993). In culture fluids of P. chrysosporium supplemented with chlorophenols the relatively stable sideproducts 2-chloro- and 2,5-dichloro-l,4-dimethoxybenzene were detected. Both chloroperoxidases and the chloromethane-producing system in hymenochaetous fungi are not very specific for the halogen used (Neidelman and Geigert 1986; Harper and Hamilton 1988). Both systems are active with chloride, bromide and iodide ions but not with fluoride ions. In this work we also showed that the addition of fluoride ions to the medium did not give rise to the production of fluorinated compounds, whereas the addition of the other three halogens resulted in the production of organohalogens. The brominated compounds are very common in marine environments (Neidelman and Geigert 1986; Gribble 1992). Brominated compounds have also been reported in sedi-

0

ments (Watanabe et al. 1985) and in treated water (Sithole and Williams 1986) and bromophenols were detected as a side reaction in chloroperoxidase assays (Wannstedt et al. 1990). The occurrence of the halogenation with bromide as well as with chloride is a new element for Bjerkandera spp. and supports the assumption that a comparable system as described above is active. When iodide ions were added to the medium, surprisingly no iodinated aromatic compounds were detected. However, the occurrence of isovanillin in the culture supernatants confirms the specific attack of carbon 3 of the ring. As the carbon-iodine bond is reported as a low energy bond (Weast et al. 1989), iodine may be easily substituted by a hydroxyl group (Neidelman and Geigert 1986). An interesting aspect of the addition of iodide ions was the occurrence of iodide-containing methane. This is the first report of halomethane production by B. adusta. Chloromethane production is a well-known fact of hymenochaetous fungi, such as Phellinus pomaceus and many other Phellinus spp. (Cowan et al. 1973; Harper et al. 1988; McNally and Harper 1991). Unfortunately, the methodology used prevented the detection of highly volatile halomethanes such as chloromethane, dichloromethane, bromomethane, dibromomethane or iodomethane. Chloromethane has a primary role in the methylation of aromatic compounds such as acids or phenols (Harper et al. 1989). Also, fungi belonging to other families such as P. chrysosporium, which does not emit detectable amounts of chloromethane, use this compound as a methylating agent in the biosynthesis of veratryl alcohol (Harper et al. 1990). The detection of

221

di- and trihalogenated methanes makes it likely that also B. adusta strains possess the chloromethanemethylating sytem. One can speculate if the chlorination mechanism of the aromatic ring has something in common with the methylation of hydroxyl groups with chloromethane. Acknowledgements We wish to thank Dr. Patrick Etirvant, Dr. Jim Field, and Pr. Jan de Bont for helpful discussions, Dr. Marcel Asther for providing the strain Mic 64 and Michel Delattre for chloride measurements.

References Albrich JM, Mc Carthy CA, Hurst JK (1981) Biological reactivity of hypochlorous acid:implications for microbicidal mechanisms of leucocyte myeloperoxidase. Proc Natl Acad Sci USA 75:210-214 Berger RG, Neuhauser K, Drawert F (1986) Characterization of the odor principles of some basidiomycetes: Bjerkandera adusta, Poria aurea, Tyromyces sambuceus. Flavour Fragrance J 1:181-185 Berger RG, Drawert F, H/idrich S (1987) Microbial sources of flavour compounds. In: Schreier P, (ed) Bioflavour'87, de Gruyter, Berlin, pp 415-434 Cowan MI, Glen AT, Hutchinson SA, Mc Cartney ME, Mackintosh JM, Moss AM (1973) Production of volatile metabolites by species of Fomes. Trans Br Mycol Soc 60:347-360 Edwards. PR, Campbell I, Milne GS (1982) The impact of chloromethanes on the environment. Chem Ind 574-578 Engvild KC (1986) Chlorine-containing natural compounds in higher plants. Phytochemistry 25:781-791 Eriksson KE, Blanchette RA, Ander P (1990) Microbial and enzymatic degradation of wood and wood components. Springer series in wood science, Springer, Berlin Heidelberg New York Gallois A, Gross B, Langlois D, Spinnler HE, Brunerie P (1990) Influence of culture conditions on production of flavour compounds by 29 ligninolytic basidiomycetes. Mycol Res 94:494-504 Gribble GW (1992) Naturally occurring organohalogen compounds--a survey. J Nat Prod (Lloydia) 55:1353-1395 Harper DB (1985) Halomethane from halide ion--a highly efficient fungal conversion of environmental significance. Nature 315:55-57 Harper DB, Hamilton JTG (1988) Biosynthesis of chloromethane in Phellinus pomaceus. J Gen Microbiol 134:2831-2839 Harper DB, Kennedy JT, Hamilton JTG (1988) Chloromethane biosynthesis in poroid fungi. Phytochemistry 27:3147-3153 Harper DB, Hamilton JTG, Kennedy JT, McNally KJ (1989) Chloromethane, a novel methyl donor for biosynthesis of esters and anisoles in Phellinus pomaceus. Appl Environ Microbiol 55:1981-1989 Harper DB, Buswell JA, Kennedy JT, Hamilton JTG (1990) Chloromethane, methyl donor in veratryl alcohol biosynthesis in Phanerochaete ehrysosporium and other lignin-degrading fungi. Appl Environ Microbiol 56:3450-3457

Jensen KA, Evans KMC, Kirk TK, Hammel KA (1994) Biosynthetic pathway for veratryl alcohol in the ligninolytic fungus Phanerochaete chrysosporium. Appl Environ Microbiol 60:709-714 Jong E de, Field JA, Dings JAFM, Wijnberg, JBPA, Bont JAM de (1992) De novo biosynthesis of chlorinated aromatics by the white rot fungus Bjerkandera sp. BOS55. FEBS lett 305:220-224 Jong E de, Field JA, Spinnler HE, Wijnberg JBPA, Bont JAM de (1994a) Significant biogenesis of chlorinated aromatics by fungi in natural environments. Appl Environ Microbiol 60:264-270 Jong E de, Cazemier AE, Field JA, Bont JAM de (1994b) Physiological role of chlorinated aromatics biosynthesized de novo by Bjerkandera sp. BOS55. Appl Environ Microbiol 60:271-277 Joshi DK, Gold MH (1993) Degradation of 2,4,5-trichlorophenol by the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl Environ Microbiol 59:1779-1785 McNally KJ, Harper DB (1991) Methylation of phenol by chloromethane in the fungus Phellinus pomaceus. J Gen Microbiol 137:1029-1032 Morris HR, Taylor GW, Masento MS, Jermyn KA, Kay RR (1987) Chemical structure of the morphogen differentiation inducing factor from Dictyostelium discoideum. Nature 328:811-814 Neidleman SL, Geigert J (1986) Biohalogenation: principles, basic roles and applications. Ellis Horwood (Wiley, New York), p. 39 Nyquist RA (1984) The interpretation of vapor phase infared spectra, vol. 1, Sadtler Research Laboratories, Philadelphia, p. 93 Pfefferle W, Anke H, Bross M, Steglich W (1990) Inhibition of solubilized chitin synthase by chlorinated aromatic compounds isolated from mushroom cultures. Agric Biol Chem 54:1381-1384 Renganathan, Miki K, Gold MH (1987) Haloperoxidase reactions catalyzed by lignin peroxidase an extracellular enzyme from the basidiomycete Phanerochaete chrysosporium. Biochemistry 26:5127-5132 Shimada M, Nakatsubo F, Kirk TK, Higushi T (1981) Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete chrysosporium. Arch Microbiol 129:321-324 Sithole B, Williams DT (1986) Halogenated phenols in water at forty Canadian potable water treatment facilities. J Assoc Off Anal Chem 69: 807-810 Valli K, Gold MH (1991) Degradation of 2,4-dichlorophenol by the lignin-degrading fungus Phanerochaete chrysosporium. J Bacteriol 173:345-352 Vogel HJ (1964) Distribution of lysine pathways among fungi: evolutionary implications. Am Naturalist 98:435446 Wannstedt C, Rotella D, Sinda JF (1990) Chloroperoxidase mediated halogenation of phenols. Bull Environ Contam Toxicol 44:282-287 Watanabe 1, Kashimoto T, Tatsukawa R (1985) Brominated phenol and anisoles in river and marine sediment in Japan. Bull Environ Contam Toxicol 35:272-278 Weast RC, Astle M J, Beyer WH (1989) CRC handbook of chemistry and physics. CRC Press, Boca Raton, Fla., pF-185