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VANILLIN PRODUCTION BY PHANEROCHAETE

CHRYSOSPORIUM GROWN ON GREEN COCONUT AGRO-
INDUSTRIAL HUSK IN SOLID STATE FERMENTATION
Elisabete dos Santos Barbosa,a* Daniel Perrone,a Ana Lúcia do Amaral Vendramini, b and
Selma Gomes Ferreira Leiteb

Agro-industrial residues have become an important source for the

production of chemical compounds using biological pathways,
contributing to preservation of the environment and making the overall
process economically supportable. Vanillin is a very important aromatic
compound for the food, beverage, and pharmaceutical industries. The
aim of the present study was to evaluate the vanillin production by solid-
state fermentation on green coconut residue using the basidiomycete
Phanerochaete chrysosporium. Solid-state fermentation was carried on a
support of green coconut husk treated in two different ways: sun-dried
and mechanical-pressed. A Plackett-Burman experimental design was
used to screen the compounds of liquid medium culture of the vanillin
production. Nineteen variables were studied to optimize the culture
conditions, and eleven of them were significant. The screening improved
the production of vanillin from 44.4 μg/g of support to 52.5 μg/g of
support in 24 hours of fermentation. Sun-dried coconut husk was found
to be superior to mechanical-pressed coconut husk for production of
vanillin. HPLC was used for the quantification of vanillin aroma.

Keywords: Vanillin; Solid-state fermentation; Phanerochaete chrysosporium

Contact information a: Technology Center; Department of Biochemistry, Federal University of Rio de

Janeiro, NC 21949-900, Rio de Janeiro, Brazil; b: Technology Center, Department of Biochemistry
Engineering, Federal University of Rio de Janeiro, NC 21949-900, Rio de Janeiro, Brazil.
*Corresponding author: [email protected]

INTRODUCTION

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the major component of natural

Vanilla planifolia, which is one of the most widely used flavouring agents in a large
range of foods, and also used in some fragrances (Walton et al. 2000; Walton et al. 2003).
For a long time, the most important source of vanillin was eugenol, the main component
of cloves. Compared with the cost of synthetic vanillin (~US$15Kg-1), natural vanilla
extract is relatively expensive, with a current cost of ~US$4000Kg-1, depending on
quality. With an increasing interest in natural products, alternative process are being
developed that use biotechnological methods involving fungi to produce vanillin (Lesage-
Meessen et al. 1996).
Lignin represents one of the main sources of natural aromatics. This insoluble
polymer is known to be attacked by microorganisms, particularly by white-rot
basidiomycetes, which release aromatic aldehydes such as vanillin when growing on

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lignin. Vanillic and ferulic acids are also well-known products of the degradation of
lignin related substances by white-rot fungi (Walton et al. 2000; Pandey 2003). 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; Graf 1992). A number of industrial and food applications have
been 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 agro-industrial
residues would provide a sufficient natural source of ferulic acid for biotechnological
processes (Soccol and Vandenberghe 2003).
Green coconut water is becoming a very attractive product to the Brazilian
market, growing 20% in a year. Brazil is the world leader of green coconut production
with a planted area of 90,000 ha. The increasing of green coconut consumption generates
6.7 million tons of husk in a year (Embrapa Agroindústria Tropical, 2005).
However, this great production generates a large amount of residues that causes
serious environmental problems (Pandev and Soccol 1998; Soccol and Vandenberghe
2003).
The application of agro-industrial residues in bioprocesses provides alternative
substrates, and it also helps to solve pollution problems. Biotechnological processes,
especially the solid-state fermentation (SSF) technique, have contributed enormously to
such utilization (Soares et al. 2000; Soccol and Vandenberghe 2003).
Traditionally, SSF methods have been characterized by the development of
microorganisms in a low water environment on a non-soluble material that acts both as
physical support and source of nutrients; however it is not necessary to combine the role
of support and substrate, but rather to reproduce the conditions of low water activity and
high oxygen transference by using a nutritionally-inert material soaked with a nutrient
solution (Pandey 2003). Improvement in productivity of metabolic microbial by the
organisms is done by manipulating the nutritional parameters, physical parameters, and
strain improvement so that it can better be evaluated through experimental design. The
Placket-Burman statistical method offers a design where initial screening of the
ingredients is done to understand the significance of their effect on the products
formation, and then the most suitable ingredients for the increasing of the production of
the studied compound are selected for further optimizations (Carvalho et al. 1997;
Naveena et al. 2005).
The aim of the present study was to evaluate vanillin production by solid-
state fermentation on green coconut agro-industrial husk using the basidiomycete
Phanerochaete chrysosporium.

EXPERIMENTAL

Materials
Fungal strains and growth media
Phanerochaete chrysosporium CCT-1999 (ATCC 24725) was selected as a
suitable organism for bio-processing of SSF due to its ability to degrade lignin effectively

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(Kirk and Farrel 1987). The cultures were grown at 28°C ±1°C during five days on
Potato Dextrose Agar (PDA) medium, and it was stored at 4°C.

Obtaining the inoculum

The spores were produced during five days on Potato Dextrose Agar (PDA)
medium at 28°C ± 1°C, and they were suspended in NaCl 0.9% p/v solution. The spores
were quantified in a Neubauer chamber. In solid-state fermentation 1 mL of spore
suspension containing 2.7 x 106 spores/mL was added (Vane 2003).

Solid support
The solid support consisted of green coconut agro-industrial husk provided by
Empresa Brasileira de Pesquisa Agropecuária, Agroindústria Tropical, Brazil. The green
coconut husk was treated in two ways: sun-dried and mechanical-pressed. The solid
support was submitted to granulometric classification (20 mesh Tyler screen), autoclaved
at 121°C for 20 minutes, and stored at room temperature until its use.

Methods
SSF technique
Experiments were conducted in 250 mL Erlenmeyer flasks containing 2g of
autoclaved support (sun-dried green coconut husk or mechanical-pressed green coconut
husk). It was impregnated to 550% moisture content, with 10 mL of the liquid nutrient
medium, and 1 mL of spore suspension, but it didn’t develop a submerged fermentation.
The liquid nutrient medium contained: 1g glucose; 0.2g KH2PO4; 0.05g MgSO4.7H2O;
0.022g ammonium tartrate; 0.1mg thiamine; 0.02g yeast extract; 0.01g CaCl2.H2O; 6.7mg
veratryl alcohol; and 0.1mL trace solution element in 100 mL of pH 4.5 buffer-solution
(Contarini 1992; Vane 2003). The liquid nutrient medium was autoclaved at 121°C for 15
minutes, with the exception of the thiamine and veratryl alcohol. These components had
been filtered through cellulose acetate membrane (pore size: 0.25μm; Millipore) and
inoculated at the moment of the fermentation.
The Erlenmeyer flasks were incubated at 28°C ±1°C, in the absence of shaking.
The samples extractions were done with the mixture of two solvents (60% methanol, 40%
water) in each 24 hours until the end of 96 hours of culture. The mixture then was filtered
with a cellulose acetate membrane (pore size: 0.25μm, Millipore). All the experiments
were done in triplicate.

Screening of liquid medium components using Plackett-Burman design

The Plackett-Burman design was used for screening in order to improve vanillin
production. Among the components of the liquid medium culture, sucrose and
Polyoxyethylenesorbitan Monooleate (POE-SM) surfactant, were tested for their
significance in vanillin production. The effects of 19 variables were considered in 20
experiments with 5 central points and three levels (-1, concentration 0g/L; 0, half of the
maximum concentration; +1, maximum concentration used in the liquid medium) (Tables
1 and 2). The Plackett-Burman design was applied in accordance with optimum result of
the first stage of the fermentation. The results were analyzed using the Statistica 6.0
(Statsoft) software. Values for vanillin production were expressed in μg/g of the support.

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Table 1: Assigned Concentrations of Variables at Different Levels in Plackett-
Burman Design for Vanillin Production in SSF
S. no. Variables with designation (g/L) -1 0 +1
1 X1 Glucose 0.0 5.0 10.0
2 X2 Sucrose 0.0 5.0 10.0
3 X3 KH2PO4 0.0 1.0 2.0
4 X4 MgSO4.7H2O 0.0 0.125 0.250
5 X5 Ammonium tartrate 0.0 0.11 0.22
6 X6 Thiamine 0.0 0.0005 0.0010
7 X7 Yeast extract 0.0 0.1 0.2
8 X8 Veratryl alcohol 0.0 0.0335 0.0670
9 X9 POE-MS surfactant 0.0 0.05 1.0
10 X10 CaCl2.H2O 0.0 0.5 1.0
11 X11 NaCl.H2O 0.0 0.0005 0.0010
12 X12 Nitrilotriacetic acid 0.0 0.00075 0.00150
13 X13 MnSO4.H2O 0.0 0.00025 0.00050
14 X14 CoSO4.7H2O 0.0 0.00009 0.00018
15 X15 CuSO4.5H2O 0.0 0.00009 0.00018
16 X16 FeSO4.7H2O 0.0 0.00005 0.00010
17 X17 ZnSO4.7H2O 0.0 0.00009 0.00018
18 X18 NaMoO4 0.0 0.000005 0.000010
19 X19 H3BO3 0.0 0.000005 0.000010

Table 2: Plackett-Burman Design for 19 Variables with Coded Values along with
Observed Results for Vanillin Production in SSF
S. Vanillin
no. X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 production
1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 27,498
2 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 32,936
3 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 31,343
4 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 25,110
5 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 27,195
6 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 30,851
7 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 30,108
8 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 28,821
9 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 29,317
10 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 27,911
11 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 31,928
12 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 32,694
13 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 30,351
14 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 26,648
15 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 23,319
16 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 25,229
17 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 27,988
18 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 26,343
19 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 27,858
20 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 24,123
21 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28,251
22 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 27,956
23 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28,196
24 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28,350
25 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28,500

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Analytical procedures
Filtrates were analyzed by HPLC, and the quantification was performed using a
vanillin standard. Separation was achieved with a Spherisorb® S5 ODS2 column (150 ×
2 mm, Waters Corp., USA) maintained at 40°C using HPLC model Shimadzu, Kyoto,
Japan. The mobile phase used a mixture of two solvents with 75% of aqueous formic acid
at 0.3% and 25% of methanol, using an isocratic elution with 0.2 mL/min of flow rate.
The data were obtained by LCMSsolution (Shimadzu Corp., version 2.00, 2000)
software. The vanillin concentration produced (μg/g of support) was determined by:

Vanillin = (quantification of vanillin for HPLC . volume of the sample obtained)

g of support used at the process

RESULTS AND DISCUSSION

Kinetic Profile of Vanillin Production by Phanerochaete chrysosporium on

Solid Support
The present study demonstrated the ability of Phanerochaete chrysosporium to
release compounds from the cell walls of green coconut husk and then to convert it to
vanillin, according with the parameters established by solid-state fermentation. The
highest production achieved with the mechanical-pressed support happened in 48 hours
of fermentation, while just 24 hours was required to obtain the highest production with
the sun-dried support, being 28.95 μg/g of the support and 44.4 μg/g of the support,
respectively (Fig. 1). It was possible to observe the influence of the type of treatment of
the support (sun-dried and mechanical-pressed) on the production, with the highest
production rates (p<0.05) achieved with the sun-dried support (Fig. 1). Therefore, the
solid-state fermentation on sun-dried support was more efficient to produce the
compound vanillin in comparison with the mechanical-pressed support.

Fig. 1. Kinetic profile of vanillin production by Phanerochaete chrysosporium on two solid

supports

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Several studies have been recently focused on the production of specialty
chemicals such as flavor compounds from residues of the agro-industry as a possible way
of both disposing them and producing an item of value. Green coconut agro-industrial
husk, one of the most widely produced agricultural residues in Brazil (Pandey and Soccol
1998; Carrijo et al. 2002; Soccol and Vandenberghe 2003), can be of interest in vanillin
production as it is a potential source of ferulic acid, from which vanillin can be obtained
via microbial conversion. Ferulic acid is linked to cell wall polysaccharides through ester
bonds, which have to be hydrolyzed to release ferulic acid (Walton et al. 2000; Pandey
2003; Mathew et al. 2006). Hydrolysis can be done through the action of specific
enzymes, especially lignolytic enzymes. The white rot fungus Phanerochaete
chrysosporium, under nitrogen or carbon limitation, produces both extracellular
peroxidase lignin (LiP) and manganese peroxidase (MnP) (Podgornik et al. 2001).

Plackett-Burman design
The Plackett-Burman design was implemented just in the case of the sun-dried
green coconut husk support to optimize the liquid medium compounds in 24 hours of the
solid-state fermentation. In the present study Phanerochaete chrysosporium produced a
high yield of vanillin in combinations 2 and 12 (Table 2), and the experimental analysis is
shown in Table 3. Fourteen variables out of nineteen, namely glucose, sucrose, KH2PO4,
ammonium tartrate, thiamine, veratryl alcohol, POE-SM surfactant, nitrilotriacetic acid,
MnSO4.H2O, CoSO4.7H2O, CuSO4.5H2O, FeSO4.7H2O, ZnSO4.7H2O, and NaMoO4, all
had values of p<0.05 (Table 3), implying that these variables influenced the
fermentation process significantly, being part of the experimental model equation.
However, three of these variables (Table 3) namely POE-SM surfactant, ZnSO4.7H2O,
and NaMoO4 had negative coefficients over vanillin production, and they must be used
on the low level (Table 1) established by the design. The other five variables,
MgSO4.7H2O, yeast extract, CaCl2.H2O, NaCl.H2O, and H3BO3, were found to be
insignificant with value p>0.05 (Table 3).
The negative coefficients of POE-SM surfactant in this experimental model did
not coincide with the data presented for Giese et al. (2004), in which the presence of the
surfactant in the liquid medium culture stimulated the activity of lignolytic enzymes in
basidiomycetes. However, it can be suggested that in this study, the surfactant did not
serve as a stimulant of the activity of lignolytic enzymes in the basidiomycete
Phanerochaete chrysosporium for release of the ferulic acid of wall cells of solid support
for further conversion in vanillin.
Phanerochaete chrysosporium used both carbon sources present in the liquid
medium, the glucose and sucrose. The veratryl alcohol also appeared to be an important
compound of the liquid medium. This can be explained due to the inductive effect of
veratryl alcohol on the microorganism production of lignolytic enzymes, which probably
contributed to release the ferulic acid from support cell walls (Leisola et al. 1984; Leisola
et al. 1985; Schoemaker and Leisola 1990).

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Table 3: Coefficient of each Variable, Confidence interval (CI) at 95%

Confidence Level and p Value for Production of Vanillin in a 19-Variables
Plackett-Burman Design
S. Variables Coefficient Coefficient + or – (CI) Value p Significance
no.
1 Glucose 1.71176 1.46206 1.96145 0.000045 significant
2 Sucrose 3.21402 2.96432 3.46371 0.000004 significant
3 KH2PO4 0.70418 0.45448 0.95388 0.001437 significant
4 MgSO4.7H2O 0.12008 -0.12961 0.36978 0.252718 *
5 Ammonium tartrate 2.45332 2.20363 2.70302 0.000011 significant
6 Thiamine 1.56345 1.31375 1.81315 0.000064 significant
7 Yeast extract 0.12833 -0.12136 0.37803 0.226754 *
8 Veratryl alcohol 0.25119 0.00149 0.50089 0.049160 significant
9 POE-SM surfactant -1.24219 -1.49189 -0.99250 0.000159 *
10 CaCl2.H2O -0.20772 -0.45742 0.04197 0.082057 *
11 NaCl.H2O 0.13889 -0.11081 0.38858 0.197392 *
12 Nitrilotriacetic acid 0.39021 0.14051 0.63990 0.012264 significant
13 MnSO4.H2O 0.25539 0.00569 0.50509 0.046880 significant
14 CoSO4.7H2O 0.27996 0.03026 0.52966 0.035770 significant
15 CuSO4.5H2O 0.94921 0.69951 1.19890 0.000456 significant
16 FeSO4.7H2O 0.59772 0.34803 0.84742 0.002661 significant
17 ZnSO4.7H2O -0.69562 -0.94532 -0.44593 0.001505 *
18 NaMoO4 -1.96101 -2.21071 -1.71132 0.000026 *
19 H3BO3 -0.14062 -0.39032 0.10908 0.192951 *
*Insignificant.

In accordance with the Plackett-Burman design, confirmatory experiments were

done in triplicate in the same conditions imposed in this process of solid-state
fermentation on the solid support sun-dried green coconut. The optimization of liquid
compounds in the medium increased the vanillin production by 20%, from 44.4 μg/g of
support to 52.5 μg/g of support in 24 hours of fermentation. For this study, the Plackett-
Burman design not only helped to reduce the number of compounds in the liquid
medium, selecting the more significant contributions, to 11 compounds (Table 3) namely
glucose, sucrose, KH2PO4, ammonium tartrate, thiamine, veratryl alcohol, nitrilotriacetic
acid, MnSO4.H2O, CoSO4.7H2O, CuSO4.5H2O and FeSO4.7H2O4 being used on the high
level (Table 1) established by the design, but also ensured a better vanillin production,
making the process interesting for industrial production.

CONCLUSIONS

Phanerochaete chrysosporium can release compounds from the cell walls of

green coconut husk. The sun-dried green coconut husk was a better support solid for the
vanillin production by solid-state fermentation. The Plackett-Burman design made it
possible to reduce the number of compounds in the liquid medium, removing 8 of them,
and it increased the vanillin production by 20%.

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ACKNOWLEDGMENTS

The authors are grateful for the support of the CNPq, CAPES, FAPERJ and
Embrapa Agroindústria Tropical.

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Article submitted: August 23, 2007; Peer review completed: Sept. 23, 2007; Revised
version received: August 25, 2008; Revised version accepted: August 26, 2008;
Published: August 28, 2008

Barbosa et al. (2008). “Vanillin from coconut husk,” BioResources 3(4), 1042-1050. 1050