Formation of polymeric pigments from syringaldehyde in the presence of bacteria. Comparison with chemical oxidative coupling

Eur. Polym.J. Vol. 27, No. 6, pp. 527-536, 1991 Printed in Great Britain.All rightsreserved

0014-3057/91 $3.00+0.00 Copyright © 1991PergamonPress plc

FORMATION OF POLYMERIC PIGMENTS FROM SYRINGALDEHYDE IN THE PRESENCE OF BACTERIA. COMPARISON WITH CHEMICAL OXIDATIVE COUPLING T. ATARHOUCH,A. DARO and C. DAVID* Universit6 Libre de Bruxelles, Facult6 des Sciences, Campus Plaine, CP 206/1, Boulevard du Triomphe, 1050 Brussels, Belgium (Received 19 July 1990) Abstract--The formation of polymeric pigments from hydroxy-substituted benzoic acid in the presence of some strains of Pseudomonas putida was investigated as a possible route to new aromatic polymers. The rate of disappearance of aromatic compounds such as syringaldehyde and its degradation products syringic acid and 3-O-methylgallic acid have been measured. The structure of the pigment has been investigated by GPC, u.v. and FTIR spectroscopy, NMR and elemental analysis. It has been compared with that of the polymeric pigments obtained by chemical oxidative coupling in the absence of bacteria. The bacterial pigments were found to be of badly defined structure different from that of the chemical pigments, and probably similar to humic acids; indeed amino acids are incorporated in the polymer chain. The role of the bacteria at the various steps of the metabolization of the initial aromatic compounds and of pigment formation is discussed.

INTRODUCTION This work is a part of a more general research concerned with the valorization of ligno-cellulosic materials [1-3]. A comprehensive study of pretreatments of wood, straw and bagasse was first performed in order to increase the yield of the enzymatic hydrolysis of cellulose into glucose. Although very high yields could be obtained using HCIO-NaCIO pretreatments, valorization of the lignin fraction of these materials is required to develop economically viable processes. Therefore, our attention was turned to the degradation of lignin model compounds by Pseudomonas. Vanillin and vanillic acid are known to be metabolized by most Pseudomonas strains when they are used as sole carbon source. The problem of syringaldehyde, syringic acid and their demethylated products is more complicated. The presence of a third ---OH or - - O C H 3 group on benzoic acid makes their degradation much more difficult. Most bacteria are not able to grow on these compounds. Many studies have been concerned with their degradation by various strains of Pseudomonas putida [4-10]. The most probable degradation path is:

Syringic acid is first demethylated with the formation of formaldehyde which could be metabolized by the bacteria. Methanol resulting from the second mOCHa group is most probably not used. Oxaloacetate and pyruvate are also formed after ring-opening at the meta position. An exploratory study of the growth of two strains of Pseudomonas (SR and SN) on syringaldehyde, a model compound of lignin, as sole carbon source, indicated a qualitative difference between them. The first one induced a moderate growth revealed by turbidity; the aromatic compound was shown to disappear rapidly. The second one was characterized by negligible growth, the slow disappearance of the aromatics and the formation of a coloured pigment. A detailed investigation was then undertaken in order to elucidate the behaviour of these two strains. The present work has a double justification: - - A strain which is able to degrade syringic type aromatic compounds could be valuable to degrade lignin type aromatic compounds. - - T h e pigment formed by the other strain could be a high value aromatic polymer of well defined structure. CHpH

COOH

COOH

COOH

+

o

II t,

NADH CH 3

OCH 3 OH

02

~ H

OH

OCH 3 OH

COOCH 3 COOH

HOOC

- C - CH 2 - COOH +

o

II

HOOC -C -CH3

Syringicacid

3 - O - methylgallicacid

*To whom all correspondence should be addressed. 527

T. ATARHOUCHet al.

528

It was thus interesting to isolate it and to compare its structure with those obtained by chemical oxidative coupling in the absence of bacteria. Oxidative coupling in the presence of metal salts is currently used to synthetize poly-p-phenylene oxide from phenol and substituted phenols [11]. Oxidative coupling by electrochemical methods has also been reported for the synthesis of high value polymers from such substituted phenols [12]. EXPERIMENTAL PROCEDURES

Micro -organisms Pseudomonasputida spp (SR and SN) were isolated from soil.

Suspension cultures Micro-organisms were inoculated into preculture flasks containing NH4C1 5 g/l, KH2PO4 1.5g/l, K2HPO4 3 g/l, MgSO4 0.2 g/l, FeSO4' (NH4)2 ' SO4' 6H20 10 mg/1, yeast extract 0.5 g/1 and tryptone 1 g/1. They were then transferred into culture medium containing the same salts and carbon substrates: syringaldehyde (0.5 g/l) and vanillin (0.1 g/l) or syringaldehyde (0.5 g/l) and lactate (2 g/l). The aromatic compounds were Aldrich products except 3-O-methylgallic acid which was synthesized in the laboratory according to the method proposed by Scheline [13].

Chemical oxidative couplings At pH 11.5, 3-O-methylgallicacid was stirred for 15 days in NaOH solution; it was readjusted each day at this pH. At pH 7.8, 3-O-methylgallic acid was stirrred in the (KH2PO4/KzHPO4) buffer in the culture conditions.

--Incubation of P. putida SR in the presence of syringaldehyde as the sole carbon source. --Incubation of P. putida SN in the presence of syringaldehyde as the sole carbon source. --Incubation of P. putida SN in the presence of syringaldehyde and vanillin. --Incubation of P. putida SN in the presence of syringaldehyde and lactate. This was a macroculture (1 1. solution containing 500 mg syringaldehyde and 2 g lactate) performed in order to isolate enough pigment to characterize it by the usual methods (C, H, O, N analysis, IH- and 13C-NMR, i.r. and u.v. absorption spectroscopy). Various methods were used to follow the changes of the cultures. The bacterial density was determined by the method of viable count as a function of the incubation time. The disappearance of syringaldehyde and the formation and identification of the catabolites were monitored by HPLC. The disappearance of syringaldehyde and the formation of the pigment were also followed by u.v. absorption spectroscopy and by gel permeation chromatography.

Incubation of P. putida SR in the presence of syringaldehyde. The disappearance of syringaldehyde as a function of time is shown in Fig. 1. The growth of bacteria is quite good, 2. 108bact/ml being obtained at the plateau attained after two days. The substrate is completely consumed after 4 days. No intermediate product could be detected in amount larger than 5 mg/1. The medium becomes light yellow.

Incubation of P. putida S N in the presence of syringaldehyde. The rate of disappearance of

u.v. ,Spectral analyses u.v. Spectra of culture supernatants were recorded as a function of time with a Perkin-Elmer lambda 3A spectrophotometer.

i.r. Spectroscopy FTIR spectra of the acidified pigment (extracted with ethylacetate and isolated by lyophilization) were recorded with a Bruker IFS45 spectrophotometer in KBr discs.

Liquid chromatography After sterile filtration, the aliquots were analysed using a Perkin Elmer chromatograph, model 601, equipped with a high performance Waters Novapak C 18 column. The eluant was a ternary mixture of water/acetonitrile/acetic acid in a ratio 88/10/2, with a flow rate of 0.5 ml/min. Detection was made at 275 nm with a Perkin-Elmer LC55 u.v. spectrophotometer; calibration was performed with Aldrich standards and with 3-O-methylgallic acid synthesized according to Scheline [13].

Gel permeation chromatography The column was a Waters Ultrahydrogel 250 for high performance GPC of water soluble polymers. The eluant was a phosphate buffer at pH 7 (KH2PO4 25 mM/K2HPO4 25 mM) with a flow rate of 0,8 ml/min. Detection was made at 265 nm with a u.v. Perkin-Elmer LC55 spectrophotometer.

syringaldehyde is slower than in the preceding case (Fig. 2); it is almost quantitatively transformed into syringic acid which accumulates in the medium and disappears after 10 days. The pigment appears as a brown colouration at the same time. The growth and decay of small quantities of 3-O-methylgallic acid was also observed. Low molecular weight compounds were identified by injection of standard samples and comparison of retention time. The number of bacteria grows from 105 at the seeding time to 107bact/ml at the plateau. This is almost insignificant since the same bacterial density were observed on sterile distilled water seeded under the same conditions [15].

0.4

A

~e

03

Q2

0.1

I

RESULTS

Metabolism of syringaldehyde in the presence of bacteria Four types of experiments have been performed:

0

J 2

4

I 6

I

I 8

t (days)

Fig. I. Change of the culture medium composition as a function of time during growth of P. putida SR in phosphate buffer plus syringaldehyde (0.5g/l). O, Syringaldehyde.

A~ O.4

Formation of polymeric pigments from syringaldehyde

529

0.5

0,5

0.3 0,4 O2 0.1

o

2

4

6

,o

e

12

~4

,6

,e

~o

t (days)

Fig. 2. Change of the culture medium composition as a function of time during growth of P. putida SN in phosphate buffer plus syringaldehyde (0.5 g/l). 0 , Syringaldehyde;/X, Syringic acid; *, 3-O-methylgallic acid. The formation of the pigment has also been followed by GPC (Fig. 3). This pigment starts to form after 10 days with a retention time tR of 10-11 min, but the yield is very low. Assuming an e value of 6.000 1 • mol- ~• cm- ' as determined for the pigment obtained from the macroculture, only 2-3% of the initial syringaldehyde transforms into pigments. Incubation o f P. putida S N in the presence o f syringaldehyde and vanillin. Figure 4 shows the very fast decay of syringaldehyde and the rapid formation of syringic acid and 3-O-methylgallic acid. Other cultures have shown that syringic acid forms first and transforms into 3-O-methyigallic acid. The number of bacteria tends to 2.108 per ml at the plateau. The appearance of a brown colour after 3 days indicates that pigment is formed as confirmed by the GPC diagram given in Fig. 5. The sum of syringic acid and 3-O-methylgallic which are not separated in these conditions, eluates with a tR of 17.3 min. A broad but small pigment peak eluates at 10-11 min. It is accompanied by several small peaks characterized by longer t R. The quantity of polymer formed is larger than in the preceding case by a factor of about three. Macroculture. After 2 days, the concentration of syringaldehyde which was initially 0.5 g/l falls to 0 g/1 and is replaced by syringic acid (0.35 g/l). This last product reaches a plateau of 0.25 g/1 after 10 days

0

5

10

15

t (days)

Fig. 4. Change of the culture medium composition as a function of time during growth of P. putida SN in phosphate buffer plus syringaldehyde (0.5 g/l) and vanillin (0.1 g/l). O, Syringaldehyde; A, syringic acid; *, 3-O-methylgallic acid. when the culture is stopped. At this point the residual concentration of syringaldehyde, syringic acid and 3-O-methylgallic acid are respectively 0, 0.25 and 0 g/1. The density of bacteria is 10s per ml. After sterile filtration, the medium is acidified up to pH 2 and concentrated under vacua up to 0.3 1, from 1 1. During this operation, a suspension was separated from the solution and was isolated by filtration. It will be called fraction 2. The aqueous solution is then extracted with ethyl acetate. The pigment (fraction 1) is isolated by evaporation of the ethyl acetate in vacua. A total of 35 wt% of the initial syringaldehyde has been transformed into pigments, the first and second fractions corresponding respectively to 30 and 5%. The polymeric pigment corresponding to fraction 1 was then purified by dialysis and isolated by lyophilization. The obtained sample is the 15% highest molecular weight part of fraction 1. It was used for elemental analysis, i.r., u.v. and N M R spectra.

7

I

I

g ~o

22 days

i

~

13 days

18 days 16 days

- 8d0ys

14 days

5d0ys

10 d0ys D

tR

Fig. 3. GPC diagrams of the culture medium (dil. 2 × ) as a function of time during growth of P. putida SN in phosphate buffer plus syringaldehyde (0.5 g/l).

D tR

Fig. 5. GPC diagrams of the culture medium (dil. 2 x ) as a function of time during growth of P. putida SN in phosphate buffer plus syringaldehyde (0.5 g/l) and vanillin (0.1 g/l).

530

T. ATARHOUCHet

al.

o

O

~

--

OO ~m

O

o

o

~o ~ O

N~D

/ I

I

I

I

4000

3500

3000

2500

I

I

2000

1800

I

I

1600

Wovenumbers

1400

I

I

I

I

I

1200

1000

800

600

400

[ c m -~)

Fig. 6. i.r. Spectrum of fraction I of the bacterial pigment formed from syringaldehyde. The elemental analysis gives: C(5.44%), H(4.06%), O(37.87%), N(2.52%). The incorporation of nitrogen probably results, as in humic substances [16], from reaction of the condensation products with amino-acids excreted by bacteria. The i.r. spectra of the first and second fraction are given in Figs 6 and 7. They are quite similar between 1515 and 500 cm-L The minor fraction is characterized by a shoulder at 1715 cm-l and by a large peak at 1590cm-L No strong C------O absorption is observed near 1700 c m - ' for either fraction. Vibrations corresponding to OH deformation and C. O stretching are responsible for the absorption at 1310, 1210 and l l l 0 c m - L The complex absorption between 1715 and 1660 cm -] is probably due to the superposition of different C-------Ogroups in acids, ketones and mainly structures resulting from the incorporation of nitrogen from amino-acids.

The u.v. spectrum is given in Fig. 8. The absorption bands characterized by their 2ma~at 210, 260 and 280nm are most probably due to the presence of the aromatic groups although conjugated aliphatic hydrocarbons, aldehydes and ketones could also absorb there. The broad absorption near 420 nm could not be identified. Its e value is of the order of 26001. cm-1 per mol of monomeric unit assumed to be 3-O-methylgallic acid. Substituted semi-aldehydes of the muconic type resulting from ring-opening of substituted aromatic compounds have been reported to absorb at 410--420nm but should have a much lower e value. The ]H-NMR of fraction l, although very badly resolved, reveals the presence of methyl protons, aromatic protons and unsaturated and saturated aliphatic protons resulting probably from ring-opening and from amino-acids. o

~ O

o

~o~

0

o ~;

m

o~

I 4000

I

I

3000

2000 Wovenumbers

I 1500

I 1000

I 500

( c m -I)

Fig. 7. i.r. Spectrum of fraction 2 of the bacterial pigment formed from syringaldehyde.

Formation of polymeric pigments from syringaldehyde E_ o ea

b

E

= O

E ¢

O

E o

I 200

I 300

I 500

I

400

X (nrn)

Fig. 8. u.v. Spectrum of the bacterial pigment.

Oxidative coupling of syringic and 3-O-methylgallic acids in the absence of bacteria at pH 11.5 and 7.8 The pigment formed in the presence of bacteria was

531

first suspected to be formed by oxidative coupling of syringic acid without any participation of the bacteria. Therefore, the oxidative coupling of the acid in the absence of bacteria has been investigated separately in order to compare the structure of the formed pigments. Two pH conditions were chosen, one strongly alkaline (pH 11.5) generally used for oxidative coupling and another not far from neutrality (pH 7.8), identical to that of the bacterial culture medium. Syringic acid failed to react under any of these conditions. Since o- and p-diphenols are known to be more reactive than monophenols such as syringic acid, the coupling of 3-O-methylgallic acid formed by demethylation of syringic acid was then investigated. Pigments are formed at pH 11.5 and 7.8; the disappearance of 3-O-methylgallic acid is much more rapid at pH 11.5 as shown by comparison of GPC diagrams evolution. Figure 9 shows that pigment is already detectable by GPC after 0.5 hr at pH 11.5 and that 98.5% of the initial aromatic acid has disappeared after 3 days. At pH 7.8 the polymer is detectable after 3 days; 8.8 and 3.3% of the initial

~D 0

r, 0

o ¢.__ 0.5 hr

2 0 hr

3 doys

8

d0ys

Fig. 9. Change of the GPC diagrams as a function of time of the chemical pigment obtained by oxidative coupling of 3-O-methylgallic acid at p H I 1.3 (I g/l). 0.5 hr (dil. 10 x ), 20 hr (dil. 2 × ), 3 days (dil. 2 × ), 8 days (dil. 2 x ).

i.... 0

a 0

0.5 hr

3

days

6 days

9

days

tR

Fig. 10. Change of the GPC diagrams as a function of time of the chemical pigment obtained by oxidative coupling of 3-O-methylgallic acid at pH 7.8 (0.5 g/l). 0.5 hr (dil. 10 x ), 3 days (dil. 2 x ), 6 days (dil. 2 x ), 9 days (dil. 2 × ).

T. ATARHOUCH et al.

532

aromatic compound are unreacted after respectively 6 and 9 days (Fig. 10). The u.v. spectrum of the pigment formed at pH 7.8 [Pch(7.8)] is given in Fig. 11. It is continuous and shows two bands with maxima at 210 and 258 nm with ~ value of 4000 and 2000. The broad absorption with Amax at 410nm is absent at pH 7.8 and at pH 11.5. The i.r. spectra of both pigments isolated by acidification, dialysis and lyophilization are given in Figs 12 and 13. They are very different from each other and also very different from the bacterial pigment Pb=ct. The broad badly resolved absorptions are difficult to assign. The thin absorption peaks characteristic of the aromatic structure at 1600, 1500 and 900-600 cm -~ are absent in the case of Pch (11.5) while present for Pch (7.8). The C----O absorption is present at 1720cm -~ in both cases. Both spectra are dominated by the - - - C - - O vibration near 1400 cm -~. ~H-NMR suggests a partly aromatic

o

¢M I

E

I

200

I

300

400

I

500

X (nm)

Fig. 11. u.v. Spectrum of the chemical pigment obtained by oxidative coupling at pH 7,8. o

4000

I

I

3000

2000

L 1500

Wavenumbers

I

I

1000

500

( crn -1)

Fig. 12. i.r. Spectrum of the chemical pigment formed by oxidative coupling of 3-O-methylgallic acid at

pH 11.3. o

0

0

g

I 4000

3000

I 2000 Wavenurnbers

I 1500

I 1000

I 500

( crn -1)

Fig. 13. i.r. Spectrum of the chemical pigment formed by oxidative coupling of 3-O-methylgallic acid at pH 7.8.

Formation of polymeric pigments from syringaldehyde and partly unsaturated aliphatic structure for Pch (1 1.5), the only saturated alkanes identified belonging to the - - O C H 3 groups; the structure of Pch (7.8) is mainly aromatic.

DISCUSSION All the methods used to determine the structure of the pigments indicate that Pbact, Pch (11.5) and Pch (7.8) are polymers of ill-defined structure which arc very different from each other although they have similar molecular weights. Pbact contains nitrogen most probably provided by reaction of the aromatic intermediates with amino-acids excreted by the bacteria in the reaction medium. The structure is mainly aromatic and few carboxylic acid groups seem to be present. Pch (11.5) is mainly an unsaturated aliphatic compound of ill-defined structure as revealed by the broad badly resolved i.r. and N M R spectra. Pch (7.8) is mainly aromatic. The results are summarized in Scheme 1 (A, B, C and D). These structures will now be related: - - t o the metabolism of syringaldehyde in the presence of Pseudomonas SN and SR and to probable condensation reactions of the formed metabolitcs. - - t o the oxidative coupling of 3-O-mcthylgallic acid at pH 11.5 and 7.8 in the absence of bacteria. Pseudomonas SR metabolizes syringaldehyde and can use it as sole carbon source. Although intermediates do not accumulate, the classical mechanism of degradation can be proposed. Syringaldehyde is oxidized into syringic acid; demethylation and ringopening then occur according to equation (1) and Scheme I(A). Pseudomonas SN is not able to open the aromatic ring probably because the enzymes responsible for the demethylation ofsyringic acid and/or the ring-opening are deficient. If syringaldehyde is the only carbon source, the number of bacteria is low but sufficient to oxidize slowly the aldehyde into acid. Indeed, syringic acid is not formed by air oxidation when syringalde-

@.

.e OH

hyde is stirred for 15 days in the incubation medium and conditions in the absence of bacteria. In the absence of other carbon source, syringic acid accumulates in the medium. The stationary concentration of 3-O-methylgallic acid resulting from its demethylation is very low. Syringic acid and 3-O-methylgallic acid finally disappear with the formation of a small quantity of pigment. Other unidentified pathways are also probably operative. They could involve ringopening and growth or cometabolization. These different steps are summarized in Scheme I(B). In the presence of vanillin as cosubstrate, the bacterial growth is more important since vanillin has been shown to support the growth of most Pseudomonas and particularly of Pseudomonas SN [14]. Vanillin induces the formation of the enzymes responsible for the demethylation as shown by the larger amount of 3-O-methylgallic acid formed in the presence of vanillin. Syringic acid and 3-O-methylgallic acid finally disappear with the formation of a quantity of pigment which is larger than the preceding case. Pigment is most probably formed from 3-O-methylgallic acid. Indeed the yield of pigment increases with the stationary concentration of this compound which contains two free OH substituents and is thus more reactive than its monohydroxylated equivalent syringic acid. Other competitive unidcntiffed pathways are also probably followed as in the preceding case. The successive steps are thus qualitatively similar to those of Scheme I(B) but the quantity of pigment formed and the stationary concentration of bacteria and 3-O-methylgallic acid are higher. Lactate also induces the enzyme responsible for the demethylation. It promotes the formation of pigment which is isolated in much higher yield in the slightly more alkaline conditions which prevail when it is present (pH 8.5 at the end of the reaction). The polymerization reactions most probably involve coupling. The oxidant is oxygen in the absence of bacteria. It is either oxygen or H202 excreted by the bacteria when the pigment is formed in the presence of bacteria. Oxidative coupling can be schematized by electron-abstraction from mono-ionized diphenols followed by cross recombination of the mesomeric forms of the formed radicals [17, 18]:

OH

O-

OH 0

0

I

II

III

"

~

OH

0

OH

0~

IV

o

while

II+Ill

OH

O°

- - C - - O - - and - - C - - C - - bonding can thus occur. Typical examples of coupling are:

I+II

533

D o= ~ i OH

OH

<~

T. ATAI~-IOUCH et al.

534

E

E

o

O

~J

r~

b~ E

E

o

o

8

O

O

.=.

Z

E

E

e~

E Z

= o O

5

o

E .E

.E

o

cq

Formation of polymeric pigments from syringaldehyde

535

IV and V respectively rearrange into: OH

HO

OH

HO

OH

Ito VI

VII

Vl and VII are also diphenols which can be further oxidized and undergo coupling with other radicals. Coupling of I + I, II + II, HI + HI, II + HI can be similarly considered. In the case of 3-O-methylgallic acid, HI would be COOH

CH

30/[~ OH O

Coupling of such radicals has often been shown to involve decarboxylation. Incorporation of amino-acids can be proposed to occur according to [16]:

COOH O ===<

COOH

>=== O + H 2 N-CH

i,

O ===(k

/~===

N-CH

l

l

R

R

These few examples demonstrate the complexity of the random oxidative coupling which would occur if the reaction is not controlled by enzymes or chemical catalysts. The structure of the pigment is thus far from the regular structure of poly-p-phenylene oxide R

-(O ~ ~ - -

),

R

obtained in the presence of copper catalysts [11]. In strongly alkaline medium, oxidative ring-opening of aromatic cycle of tri- and tetrasubstituted aromatics of the guaiacyl and syringyl type could occur according to [19]:

% "~"-"~ R

OCH3 O(')

OCH 3 R/

~ (') O

=

OCH3 R

l[ O

This reaction which is used in the bleaching of paperpulp, most probably modifies the polymer obtained by oxidative coupling and justifies the unsaturated, complex aliphatic structure of Pch (11.5). CONCLUSION The synthesis of the bacterial pigment Pbact from syringaldehyde in the presence of P. putida SN involves the participation of the bacteria at different levels - - i n the oxidation of the syringaldehyde and demethylation of the syringic acid

OO()

lm

OCH3~ R

/ x O (')O O')

OCH3 R

// x O O O(')

- - i n the synthesis of the amino acids or other nitrogen containing molecules which are incorporated in the pigment - - p r o b a b l y in the condensation steps since the main structure of the polymer is to be different from that of Pch (7.8): it contains combined nitrogen and has different u.v. and i.r. absorption spectra. The synthesis of the chemical pigment Pch (7.8) involves random oxidative coupling; that of the chemical pigment Pch (11.5) involves random oxidative coupling followed by ring-opening.

536

T. ATARHOUCHet al.

Although none of the formed pigments exhibit the expected regular structure, interesting information has been obtained concerning the metabolization of syringaldehyde by bacteria and the chemical oxidative coupling of 3-O-methylgallic acid. Pseudomonas putida SR which is able to open aromatic rings o f the syringic acid type could degrade softwood lignin and lignin model compounds. Acknowledgements--We thank Professor J. Dony (University of Brussels), Dr M. Mergeay (SCK-CEN) and Ir Merenyi (UCL) for their collaboration and helpful discussions. We are grateful to the EEC for financial support to the laboratory. REFERENCES

i. C. David, R. Fornasier and P. Thiry. Eur. Polym. J. 22, 515 (1986). 2. C. David and R. Fornasier. Macromolecules 19, 552 (1986). 3. C. David, R. Fornasier, W. Lejong and N. Vanlautem. J. appl. Polym. Sci. 36, 29 (1988). 4. S. Dagley. Microbiological metabolism of aromatic

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

compounds. In Comprehensive Microbiology. The Principles, Applications and Regulations of Biotechnology in Agriculture and Medicine (edited by Murray Moo Young), Chap 25, p. 483 (1983). B. T. Tack, P. J. Chapman and S. Dagley. J'. biol. Chem. 247, 6438 (1972). V. L. Sparnins and S. Dagley. J. Bact. 124, 1374 (1975). Y. L. T. Lee, V. L. Sparnings and S. Dagley. Appl. Environ. Microbiol. 35, 817 (1978). M. I. Donnelly and S. Dagley. J. Bact. 142, 916 (1980). M. I. Donnelly and S. Dagley. J. Bact. 147, 471 (1981). B. F. Taylor. Appl. Envir. Microbiol. 46, 1286 (1983). A. S. Hay. J. Polym. Sci. 58, 581 (1962). G. Mengali. Adv. Polym. Sci. 33, 1 (1979). R. Scheline. Acta Chem. Scand. 20, 1182 (1966). C. David et al. (to be published). L. A. Carson, M. S. Favero, W. W. Bond and N. J. Petersen. Appl. Microbiol. 25, 476 (1973). F. J. Stevenson. Humus Chemistry. Wiley-Interscience, New York (1980). A. E. Scott. Quart. Rev. 67, 1 (1967). W. A. Waters. J. chem. Soc. B 4302 (1971). J. Gierer and F. Imsgard. Svensk Paperstid, 80, 510 (1977) Ref. cited in D. Fengel and G. Wengener Wood. Chemistry, Ultra-structure and Reactions, p. 310. de Gruyter, New York (1984).

Resum~-La formation de pigments polymrriques par certaines souches de Pseudomonas putida en prrsence d'acides benzoiques bydroxy-substiturs, a 6t6 6tudire en tant que voie de synthrse possible pour de nouveaux polymrres aromatiques. La vitesse de disparition de composrs aromatiques tels que la syringaldehyde et ses produits de drgradation (acides syringique et 3-O-methylgallique) a 6t6 mesurre. La structure du pigment a 6t6 analysre par GPC, spectroscopie u.v., i.r., NMR ainsi que par analyse 616mentaire. Elle a 6t6 comparre ~i celle des pigments polymrriques obtenus par couplage oxydatif en l'absence de bactrries. Le pigment form6 en prrsence de bactrries a une structure mal drfinie diffrrente de celle des pigments chimiques et probablement voisine de celle des acides humiques; en effet des acides aminrs sont incorporrs dans la cha~ne. Le r61e des bactrries dans les diffrrentes 6tapes de m&abolisation du compos6 initial et de formation du pigment est discutr.