Journal of Biotechnology 50 (1996) 107- 113

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus
Laurence Lesage-Meesse@*, Michel Delattrea, Mireille Haona, Jean-Franqois Thibaultb, Benoit Colonna Ceccaldi, Pascal Bruneriec, Marcel Asthera
Laboratoire de Biotechnologie des Champignons Filamenteux, INRA, Centre dEnseignement Superieur en Biotechnologie, ESIL, Fact&t des Sciences de Luminy, 163 Avenue de Luminy, Case Postale 925, 13288 Marseille cedex 09, France bLaboratoire de Biochimie et Technologie des Glucides, INRA, rue de la Geraudiere, BP1627, 44316 Nantes cedex 03, France Pernod-Ricard, Centre de Recherche, I20 Avenue du Marechal Foch, 94015 Creteil, France

Received 4 March 1996; revised 14 May 1996; accepted 17 May 1996

Abstract
A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg l- with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg 1- with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.
Aspergillus Keywords:

Vanillin; Ferulic acid; Aspergillus

niger;

Pycnoporus

cinnabarinus

1. Introduction Vanillin (3-methoxy-4-hydroxybenzaldehyde) is the major component responsible for the charac* Corresponding author. Tel: + 33-91828600; fax: 91828601; e-mail: Laurence.Lesage@esil.univ-mrs.fr
+ 33

teristic

aroma

of vanilla

bean extracts.

Because

of

the high cost of vanilla flavour, vanillin is mainly

0168-1656/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved

PII SOl68-1656(96)01552-0

Ferulic acid Polymerisation

I I i I I

Ferulic acid Metabolism

Pathway 1 : Fcrulic acid reduction

laccast:

unsolublepolymers

N--------

I I
E-Ferulic acid E-Coniferyl aldehyde E-Coniferyl alcohol

Ill

Pathway 2 : Ferulic acid propenoic chain degradation

Vanillic acid n

Vanillin

Vanillyl alcohol

co2

-4
w

Pathway 4 : Vanillic acid dccarboxylation

Methoxyhydroquinone

of synthetic origin (Clark, 1990). In recent years, the consumer demand for natural products has stimulated the search for alternative means of natural flavour production. An advantageous way to obtain natural flavours is the use of biotechnological processes involving microorganisms, since the EEC legislation incorporates under the term natural products those produced from biological sources by living cells or their components, including enzymes. Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and occurs mainly in cell walls covalently linked to lignin and other polymers (Harris and Hartley, 1980; Hartley and Harris, 1981; Higuchi, 1990: Graf, 1992). A number of industrial and food applications were reported for ferulic acid, especially based on its microbial degradation to vanillin (Gross et al.. 1991) and its antioxidant properties (Graf, 1992). The high level of ferulic acid hydrolysis from plant cell wall materials rich in this acid such as 1995; cereal brans (Faulds and Williamson,

Faulds et al., 1995) or sugar beet pulp (Micard et al., 1994) would provide a sufficient natural source of ferulic acid. Ferulic acid degradation by filamentous fungi was frequently studied. Non-oxidative decarboxylation of ferulic acid to vanillin via 4-vinylguaiaco1 was described for Fusarium soluni (Nazareth and Mavinkurve, 1986) and Paeci1om)xe.y variotii (Rahouti et al., 1989). A route involving a preliminary demethylation to caffeic acid, followed by side-chain shortening to yield protocatechuic acid was described for ferulic acid metabolism by Peni1990). cillium rubrum (Tillett and Walker, TramettJs sp. reduced ferulic acid to coniferyl alcohol which was further degraded to vanillic acid, methoxyhydroquinone alcohol and vanillyl (Nishida and Fukuzumi, 1978). Gupta et al. (I 98 1) studied ferulic acid metabolism in Sporetrichum pulverulentum and reported high levels of reduced products such as coniferyl alcohol and small amounts of vanillin, vanillic acid and

L. Lesage-Meessen

et al. /Journal

of Biotechnology 50 (1996) 107-113

109

methoxyhydroquinone. Ferulic acid metabolism by Pycnoporus cinnabarinus (Fig. 1) occurred essentially via the propenoic chain degradation to vanillic acid which was subsequently reduced either to vanillin and vanillyl alcohol, or decarboxylated into methoxyhydroquinone (Falconnier et al., 1994). From these results, a process of vanillin production (64 mg l- ) was patented (Gross et al., 1991). In order to improve level of vanillin production from ferulic acid, a new strategy was elaborated. A two-step bioconversion process combining two filamentous fungi was examined. In the first step, a micromycete Aspergillus niger transformed ferulic acid to vanillic acid, and in the second step vanillic acid was transformed to vanillin by the basidiomycete Pycnoporus cinnabarinus. This work was recently patented (Lesage-Meessen et al., 1994).

tose (20 g l- ) as carbon source and diammonium tartrate (1.82 g l- ) as nitrogen source. Cultures were inoculated with either 2 x lo5 A. niger conidiospores per ml or with mycelium fragments of P. cinnabarinus as described by Falconnier et al. (1994). Incubations were carried out at 30C in 250-ml baffled flasks, containing 100 ml medium and shaken at 120 rpm. After 3 days of incubation, a single or a sequential addition of ferulic acid or vanillic acid was performed. Phenolic precursors were used as filter-sterilized sodium salt solutions. In one case, vanillic-acid-enriched culture medium of A. niger was used as precursor source in the P. cinnabarinus 3-day-old cultures. Each experiment was run in triplicate and repeated at least twice. The standard deviation of analyses was less than 5%. 2.4. Growth measurements Growth was measured in terms of dry weight of mycelium after filtration on glass-fiber filters (GF/ D, Whatman, Maidstone, UK) and drying overnight at 105C. 2.5. Maltose and glucose determination An aliquot of culture medium (1 ml) was removed daily and filtered through 0.2-pm nylon filters (Gelman Sciences, Acrodisc 13, Ann Arbor, MI, USA). Isocratic HPLC analysis was immediately carried out (25 ~1 injected) on an ion-exchange column maintained at 80C (Bio-Rad, Richmond, CA, USA; Aminex ion-exclusion HPX-87P, 300 x 7.8 mm) using a HPLC model 1050 (Hewlett-Packard, Rockville, MD, USA), equipped with a refractive index detector and a 34-position autosampler-autoinjector. The eluant, at a flow rate of 0.4 ml min -I, was water. The chromatograph was connected to the HP 3365 Chem Station for chromatographic data handling and the quantification was performed using external standards. 2.6. Ammonium determination Ammonium was measured quantitatively according to the Spectroquant 14752 Ammonium

2. Material and methods

2.1. Fungal strains

Two strains were used in this study: Aspergillus Nationale de Culture de Microorganismes, Institut Pasteur, Paris, France) and Pycnoporus cinnabarinus MUCL39532, a monokaryotic lactase-deficient strain belonging to the Mycothbque de 1UniversitC Catholique de Louvain (Louvain-La-Neuve, Belgique). The strains were maintained on malt agar slants.
niger I-1472 (Collection

2.2. Chemicals The phenolic compounds used were obtained from Prolabo (Paris, France) for vanillin, Fluka (Saint-Quentin Fallavier, France) for ferulic acid, acid, and vanillic 2-methoxyhydroquinone Aldrich (Saint-Quentin Fallavier, France) for vanillyl alcohol. The solvents were of HPLC grade. 2.3. Medium and culture conditions Cultures were grown in a basal medium previously described by Gross et al. (1990) with mal-

method (Merck, Darmstadt, Germany). In alkaline medium, ammonium reacted with hypochloride and thymol to form blue indophenol. The reaction was monitored at 690 nm; ammonium chloride was used as standard. 2.7. HPLC metabolites quantitative unal_)Jses ($ phmolic

Filtrates were immediately analyzed by HPLC (25 ~1 injected). Separation was achieved on a Merck (Darmstadt, Germany) Cl8 column maintained at 30C (Lichrospher 100 RP-18, 5 pm, 125 x 4 mm) using a HPLC model 1050 (HewlettPackard, Rockville, MD, USA), equipped with a variable UVjVIS detector set at 280 nm and a 34-position autosampler-autoinjector. The mobile phase, at a flow rate of 1 ml min , comprised a mixture of two solvents: A, water with 0.01% acetic acid, and B, methanol. The following elution profile was used: solvent B started at 20%. holding 4 min, then increased to 40% at 24 min and 100% at 27 min, holding 2 min. Solvent B was returned to 20% at 30 min and the column was re-equilibrated for at least 5 min, before the next injection. The quantification was performed using external standards.

to a lesser extent, methoxyhyacid and, droquinone. Vanillic-acid-enriched culture media obtained on day 7 of A. niger cultures were recovered and used as vanillic acid source for 3-day-old P. cinnabarinus cultures (Fig. 2B). As defined in Falconnier et al. ( 1994), supplementation of vanillic acid was performed with a final concentration of 300 . At the optimum of metabolite producmg 1 was totally consumed. vanillic acid tion, Methoxyhydroquinone was the major product. but 80 mg 1 of vanillin and 5 mg 1~ of vanillyl alcohol were obtained. The high level of methoxy-

3. Results 3.1. A new two-step vanillin production bioconversion process

,fijt

In order to improve vanillin production by fungal bioconversion of ferulic acid, a two-step examined which consisted of process was combining two filamentous fungi exhibiting complementary capabilities of bioconversion. In the first step, ferulic acid was metabolized to vanillic acid by a micromycete, A. niger and in the second step vanillic acid was metabolized to vanillin by a basidiomycete, P. cinnabarinus. Ferulic acid metabolism by A. niger cultures supplemented on day 3 with 1 g 1 feruhc acid is reported in Fig. 2A. At the optimum of metabolite production, 98% of ferulic acid was consumed. The major products formed were vanillic

Fig. 2. Aromatic compounds at the optimum of vanillic acid production (on day 7) in Asper~ilh niger 3-day-old cultures grown in the presence of I g 1 ferulic acid (A) and at the optimum of vanillin production (on day 5) in Pycno~porus (.jn~?u~f~~iff~s J-day-old cultures grown in the presence of vaniltic-acid-enriched culture medium from A. niger containing 300 mg I vanillic acid (B). FA, ferulic acid; Va AC, vanillic acid; Va, vanillin; MHQ, methoxyhydroquinone; Va Ale, vanillyl alcohol.

L. Lesage-Meessen

et al. / Journal of Biotechnology 50 (1996) 107- I13

111
, 25

25 r

TIME OF INCUBATION (DAYS) Fig. 3. Formation and disappearance of aromatic compounds in cultures of Aspergillus niger supplemented daily from days 3 to 6 with 300 mg 1- ferulic acid in relation to mycelial biomass, carbon and nitrogen consumption. (A) ferulic acid, W; vanillic acid, 0; methoxyhydroquinone, A. (B) maltose, l; glucose, +; nitrogen, A; biomass, W.

hydroquinone was due to an active oxidative system of vanillic acid in P. cinnabarinus but also to vanillic acid source containing methoxyhydroquinone produced by the A. niger metabolism. 3.2. Increased vanillic acid production from ferulic
acid by Aspergillus niger

completely consumed (Fig. 3B). Nitrogen level decreased from 20 mM to 5 mM over the experimental period. 3.3. Increased vanillin production from vanillic acid by Pycnoporus cinnabarinus As previously realized with ferulic acid, cultures were daily supplemented with 300 mg 1- vanillic acid from days 3 to 6. Metabolites produced in relation to carbon and nitrogen consumption and mycelial biomass are shown in Fig. 4A and Fig. 4B. The rate of vanillic acid disappearance was constant, each addition of 300 mg lP being consumed totally. Under these conditions, vanillin production reached an optimum on day 7 with a concentration of 237 mg 1- (22% of molar yield) and then decreased while the vanillyl alcohol concentration increased. In addition, more than 750 mg 1- of methoxyhydroquinone were obtained. Growth rate and mycelial biomass of P. cinnabarinus were significantly lower than those of A. niger. The carbon and nitrogen sources were very slowly consumed; at the end of the experiment, 10 g 1- maltose and 15 mM nitrogen were detected in the culture medium. Vanillic acid bioconversion

In order to improve vanillic acid production, A. niger cultures were supplemented daily with 300 mg 1- ferulic acid from days 3 to 6. Metabolites produced in relation to carbon and nitrogen consumption and mycelial biomass were followed. As shown in Fig. 3A, each daily addition of ferulic acid was consumed and transformed to vanillic acid. Under these conditions, high concentrations of vanillic acid were reached with an optimum of 920 mg 1- on day 7 corresponding to a molar yield of 88%. Only 95 mg 1- of methoxyhydroquinone was detected at this time. No significative variation of vanillic acid concentration was observed between days 7 and 8, which indicated that vanillic acid was accumulated or slowly metabolized by the fungus. On day 7, at the optimum of vanillic acid production, maltose and glucose, liberated from maltose metabolism, were

TIME OF INCUBATION

(DAYS) of P~~~uporu,s c*~~crhrrrinu.s supplemented daily from days carbon and nitrogen consumption. (A) vanillic acid, l : l ; nitrogen, A; biomass, H.

Fig. 4. Formation and disappearance of aromatic compounds in cultures 3 to 6 with 300 mg I- vanillic acid in relation to mycelial biomass, methoxyhydroquinone. A: vanillin, +: vanillyl alcohol. jA. (3) maltose.

therefore occurred starvation.

without

carbon

and

nitrogen

4. Discussion A process involving the Basidiomycete strain P. I-937 in the bioconversion of ferulic acid, a phenolic compound present in plant cell walls, to vanillin was previously reported (Gross et al., 1991). Vanillin production reached 64 mg I -I, which is not an industrially realistic concentration. In this context, a new strategy was elaborated to improve vanillin production from ferulic acid. A two-step process combining two filamentous fungi exhibiting complementary capabilities of bioconversion was examined. In the first step, ferulic acid was metabolized to vanillic acid by a micromycete, A. niger and in the second step vanillic acid was metabolized to vanillin by a basidiomycete, P. c*innabarinus. When A. niger was grown in the presence of ferulic acid, the culture medium was shown to contain mainly a high level of vanillic acid. No reduced products such as coniferyl alcohol or vanillin and vanillyl alcohol were observed from

chabarinus

ferulic acid as described for Sporotrichum puluerukmtum (Gupta et al., 1981) and P. cinnabarinus (Falconnier et al., 1994). Methoxyhydroquinone was, however, recovered by subsequent oxidative decarboxylation of vanillic acid. The enzyme responsible for this conversion, the vanillate hydroxylase, has already been described in the white-rot fungi (Buswell et al., 1981). The supplementation of a vanillic-acid-enriched culture medium of A. niger as vanillic acid source in P. cinnabarims culture resulted successfully in vanillin production. As indicated by Falconnier et al. (1994), P. cinnabarinus metabolized vanillic acid through a reductive pathway leading to vanillin and vanillyl alcohol and through an oxidative pathway conducting to methoxyhydroquinone via the vanillate hydroxylase activity. Little is known about the reduction of vanillic acid to vanillin and vanillyl alcohol. The reduction steps in the fungal metabolism of aromatic compounds would be a part of a detoxification system to maintain the level of inhibitory compounds under a threshold concentration (Gupta et al., 1981). In order to increase the yields of vanillic acid and vanillin production by 4. niger and P. cinnubut-inus, respectively, and considering the high

L. Lesage-Meessen

et al. / Journal oj Biotechnology 50 (1996) 107-l 13

113

toxicity of phenolic compounds (Ander et al., 1980), sequential additions of precursors were performed. Under these conditions, vanillic acid production from ferulic acid by A. niger and vanillin production from vanillic acid by P. cinnabarinus attained very high levels of 920 and 237 mg 1~ , respectively. Although vanillin concentration has been increased 4-fold as compared to the process of Gross et al. (1991), the oxidative pathway producing methoxyhydroquinone was predominant. Standing cultures of S. pulverulentum with less oxygen available to the fungal pellet surface were shown to exhibit a lower decarboxylating pathway compared to agitated cultures (Ander et al., 1980). Further investigations will be necessary to decrease vanillate oxidative decarboxylation at the extent of the reduction pathway leading to vanillin. As a result of these experiments, a new two-step bioconversion process involving two filamentous fungi for the production of vanillin from ferulic acid can be proposed. This process was recently patented (Lesage-Meessen et al., 1994). The use of a cheap source of ferulic acid associated with a better control of the different fungal metabolic pathways involved should lead to the industrial feasibility of the process.

Acknowledgements

This work was supported by the Commission of the European Communities, Directorate-General XII for Research, Technological Development and Demonstration in the Field of Agriculture and Agro-Industry and the Conseil Regional Provence-Alpes-Cote dAzur (France).

References
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