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Bioresource Technology 98 (2007) 11151119

Production of vanillin from waste residue of rice bran oil


by Aspergillus niger and Pycnoporus cinnabarinus
Lirong Zheng a, Pu. Zheng
a

a,*

, Zhihao Sun a, Yanbing Bai b, Jun Wang b, Xinfu Guo

The Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi, 214036 Jiangsu, China
b
Zhejiang Hangzhou Xinfu Pharmaceutical Co., Ltd., Hangzhou, 311300 Zhejiang, China
Received 16 November 2005; received in revised form 7 March 2006; accepted 8 March 2006
Available online 16 June 2006

Abstract
A new technology of transforming ferulic acid, which was from waste residue of rice bran oil, into vanillin was developed by a combination of fungal strains Aspergillus niger CGMCC0774 and Pycnoporus cinnabarinus CGMCC1115. Various concentrations of ferulic
acid were compared, and the highest yield reached 2.2 g l 1 of vanillic acid by A. niger CGMCC0774 in a 25 l fermenter when concentration of ferulic acid was 4 g l 1. The ltrate of A. niger CGMCC0774 culture was concentrated and vanillic acid in the ltrate was bioconverted into vanillin by P. cinnabarinus CGMCC1115. The yield of vanillin reached 2.8 g l 1 when 5 g l 1 of glucose and 25 g of
HZ802 resin were supplemented in the bioconversion medium. The 13C isotope analysis indicated that d13CPDB of vanillin prepared
was much dierent from chemically synthesized vanillin.
 2006 Elsevier Ltd. All rights reserved.
Keywords: Waste residue of rice bran oil; Vanillin; Aspergillus niger; Pycnoporus cinnabarinus

1. Introduction
Vanillin (3-methoxy-4-hydroxybenzaldehyde) is one of
the most universally used avors in foods, fragrances,
beverages and pharmaceuticals (Priefert et al., 2001). It
was rstly isolated from vanilla beans in 1816 and its
structure was determined in 1874. Today, the world consumption of vanillin is estimated to be 12,000 t per year.
Approximately 50 t is in the form of natural vanillin
extracted from vanilla pods, with chemically synthetic
vanillin providing the remainder (Li and Rosazza, 2000).
However, under current US and European legislation,
the chemically synthesized avor chemicals could not be
used for natural avors (Muheim and Lerch, 1999).
Therefore, the increasing consumer request for natural
products created many biotechnological processes to produce natural vanillin (Lesage-Meessen et al., 1999).

Corresponding author. Tel.: +86 510 5865133; fax: +86 510 5808498.
E-mail address: zhengpu@sytu.edu.cn (Pu. Zheng).

0960-8524/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.03.028

Microbial transformation from natural substrates, including phenolic stibenes (Yoshimoto et al., 1990), lignin
(Guiraud et al., 1999), isoeugenol (Li et al., 2005), eugenol
(Walton et al., 2000), ferulic acid (Lesage-Meessen et al.,
1996; Sun, 2002), vanillic acid (Stentelaire et al., 2000),
aromatic amino acid (Diaz et al., 2001), sugar beet pulp
(Bonnin et al., 2000, 2001), wheat straw (Klinke et al.,
2002), biomass slurry fuel (Kartal et al., 2004) were investigated, and ferulic acid from raw material was considered
as a suitable precursor for vanillin.
Ferulic acid is an extremely abundant hydroxycinnamic
acid in plant cell wall, which exists in the plant in its free
form, covalently linked to biopolymers, such as polysaccharide compounds (Ishii and Tadashi, 1997), triterpene
alcohols and plant sterols (Rosazza et al., 1995). It occurs
in common agricultural waste residues such as cereal bran,
sugar beet pulp, and was therefore chosen as raw material
for transformation to vanillin by fungi. The biotechnological process to produce vanillin from agro by-products had
been investigated. 105 mg l 1 and 767 mg l 1 of vanillin
were obtained in the two-step bioconversion combining

1116

L. Zheng et al. / Bioresource Technology 98 (2007) 11151119

A. niger I-1472 and P. cinnabarinus MUCL39533 using


sugar beet pulp and maize bran as raw materials respectively (Lesage-Meessen et al., 1999, 2002).
Other agro by-product such as rice bran, which was produced more than 10,000,000 t per year in the rice rening
industry in China, also contained abundant ferulic acid.
Besides esteried to arabinofuranosyl residue of heteroxylans in the cell walls of cereal grains (Bartolome et al.,
1997), ferulic acid was also found in waste residue of rice
bran oil (crude oryzanol) as a mixture of esteried ferulate
of cycloartanol, 24-methylene cycloartanol campesterol, bsitosterol, and cycloartenol (Krishna et al., 2001). Therefore, waste residue of rice bran oil could also be considered
as source of ferulic acid. The aim of this work was to convert ferulic acid prepared from waste residue of rice bran
oil to vanillin by A. niger CGMCC0774 and P. cinnabarinus CGMCC1115.
2. Methods
2.1. Microorganisms
Two strains, A. niger CGMCC0774 and P. cinnabarinus
CGMCC1115 (China General Microbiological Culture
Collection Center) were used in our experiment. The strains
were kept at 4 C on potato agar slants.
2.2. Chemicals
Ferulic acid, vanillic acid, vanillin, vanillyl alcohol and
methoxyhydroquinone were purchased from Sigma Chemical Co. (St., USA). Waste residue of rice bran oil was
provided by Zhejiang Yinhe Pharmaceutical Co., Ltd
(Zhejiang, China). All other chemicals were from local
commercial sources and of analytical grade.
2.3. Preparation of ferulic acid from waste residue of rice
bran oil
Waste residue of rice bran oil was treated with water
ethanol isolation was summarized as following (Fig. 1):
50 g waste residue of rice bran oil was dissolved in ethanol
solution. The solution was adjusted to pH 8.0 with
0.4 mol l 1 NaOH, heated up from 90 to 135 C, and then
hydrolyzed for 5 h, then cooled down to 90 C. Plant sterol
(such as b-Sitosterol, 24-methylene cycloartanol, campesterol, etc.) was precipitated and sodium ferulate enriched
solution (ferulic acid enriched fraction) was obtained. Then
the solution obtained was regulated to pH 7.0, the ferulic
acid enriched fraction was sterilized by ltration for the
next bioconversion.
2.4. Growth and bioconversion
The basal medium for growth of A. niger CGMCC0774
and P. cinnabarinus CGMCC1115 contained (g l 1): maltose 20, ammonium tartrate 1.8, yeast extract 0.5, MgSO4

Waste residue of rice bran oil

Dissolved in ethanol solution


(Adjusted pH with NaOH)

Heated treatment
(135 oC, 5 h)

Cool

The aqueous phase

Ferulic acid enriched fraction

Precipitated
(-Sitosterol ect.)

medicine materials

Fig. 1. Preparation of ferulic acid from waste residue of rice bran oil.

7H2O 0.5, K2HPO4 0.2, and CaCl2 1.3 mg l 1 and VB2


2.5 mg l 1. Either A. niger CGMCC0774 or P. cinnabarinus
CGMCC1115 was inoculated in 250 ml ask containing
100 ml basal medium. Incubation was carried out at
30 C and 150 rpm for 48 h.
Ten milliliters of above culture of A. niger CGMCC0774
was inoculated into 250 ml ask containing 120 ml basal
medium (yeast extract up to 3 g l 1) and incubated as
above. After 48 h, ferulic acid from waste residue of rice
bran oil was added for bioconversion to vanillic acid by
A. niger CGMCC0774. Then the ltered culture broth of
A. niger CGMCC0774, the solution containing vanillic
acid was used as precursor source in the subsequent
bioconversion.
Ten milliliters of growth cultures of P. cinnabarinus
CGMCC1115 was inoculaterd into 500 ml ask containing
200 ml basal medium with (g l 1) glucose 20, beef extract 8,
MgSO4 7H2O 0.5, K2HPO4 0.2, CaCl2 1.3 mg l 1 and VB2
1 mg l 1. The pH of the media was adjusted to 5.0. The cultivation was carried out at 30 C for 48 h. P. cinnabarinus
CGMCC1115 cells was harvested by ltration. Bioconversion with P. cinnabarinus CGMCC1115 was carried out at
35 C and 120 rpm. Filtered P. cinnabarinus cells were
suspended in vanillic acid enriched broth of A. niger
CGMCC0774.
2.5. Analytical methods
Ferulic acid, vanillic acid, vanillin, vanillyl alcohol and
methoxyhydroquinone were determined by a HPLC (Hewlett-Packard Co., USA) equipped with a C18 column
(Lichrospher 100 RP-18, 5 lm, 125 4 mm) and a UV
detector, with methanol and 70% aqueous solution (with
0.01% acetic acid included) as the mobile phase (ow rate
1 ml min 1). The column was maintained at 30 C, and
elutes were detected at 280 nm. The quantication was
performed using external standards (Li et al., 2004).

C isotope analysis of vanillin

The 13C isotope analysis of various vanillin was carried


out by DeltaPlusXL (Bristol, the UK).
3. Results
3.1. Ferulic acid release from waste residue of rice bran oil
Waste residue of rice bran oil contained about 70% esteried ferulate, in which ferulic acid content ranged from 25%
to 30%. Ferulic acid was prepared with the method shown in
Fig. 1. In ferulic acid enriched fraction, 6.8 g ferulic acid was
released from 50 g waste residue of rice bran oil by hydrolysis. Higher concentration of ferulic acid (more than 34 g l 1)
in ferulic acid enriched fraction was detected. It has been
reported that A. niger CGMCC0774 produced some
enzymes to degrade the esteried ferulate in waste residue
of rice bran oil. When waste residue of rice bran oil
was directly added to the growing broth of A. niger
CGMCC0774 directly, ferulic acid and vanillic acid formed
in the fermentation medium (Sun et al., 2005).
3.2. Eect of ferulic acid concentration on the bioconversion
The eect of dierent concentrations of ferulic acid from
ferulic acid enriched fraction on bioconversion was studied.
Initial ferulic acid concentration of 4 g l 1 resulted 2.0 g l 1
of vanillic acid together with a molar yield of 57.7%. With
the increase of initial ferulic acid, the molar yield declined
correspondingly. When using ferulic acid with the initial
concentration of 6 g l 1, the molar yield was only 42%
and 1.2 g l 1 of ferulic acid remained unused. If the initial
ferulic acid concentration exceeded 8 g l 1, the substrate
inhibition occurred. For 72 h bioconversion, the best initial
ferulic acid concentration was considered around the level
of 4 g l 1. On the other hand, 1.1 g l 1 of vanillic acid was
produced from 2.0 g l 1 ferulic acid at 72 h indicating a
higher conversion yield at lower ferulic acid concentration.
During the whole bioconversion period, vanillic acid formation increased with incubation time and then declined
at the point where ferulic acid was completely consumed.
As a result, the best incubation time was at 48 h and a
molar yield of 70.8% was obtained correspondingly
(Fig. 2). This also implied that higher yield of vanillic acid
might be achieved by batch feeding substrate.
3.3. Bioconversion by A. niger CGMCC0774 in a 25 l
fermenter

1117

-1

13

2.0
1.5
1.0
0.5
0.0
0

12

24
36
Time (h)
Vanillic acid

60

72

Ferulic acid

degrading ability, and then ferulic acid from waste residue


of rice bran oil was fed into the culture broth twice for bioconversion of vanillic acid. As shown in Fig. 3, by feeding
2.0 g l 1 of ferulic acid solution from waste residue of rice
bran oil at 36 h, the nal vanillic acid concentration of
2.2 g l 1 was achieved with a molar yield of 63.5% at
72 h and ferulic acid was almost completely used out (residual concentration of ferulic acid <0.2 g l 1).
3.4. Bioconversion of vanillic acid enriched cultures to
vanillin by P. cinnabarinus CGMCC1115
A signicant amount of vanillic acid was produced with a
minimum quantity of ferulic acid remaining in the broth of
A. niger CGMCC0774. By combining the two bioconversion

2.5
2.0
1.5
1.0
0.5
0.0
0

20

40

Ferulic acid

Based on the above results, further experiment was conducted in order to obtain a mass of vanillic acid enriched
broth of A. niger CGMCC0774. The fermentation of A.
niger and bioconversion process converting ferulic acid to
vanillic acid was scaled-up to 25 l level. Under of the optimum fermentation conditions, A. niger CGMCC0774 was
grown for 42 h to produce the enzyme with ferulic acid

48

Fig. 2. The conversion time course when the initial ferulic acid concentration was 2.0 g l 1. The bioconversion was carried out at 37 C, with
shaking speed of 150 rpm.

Ferulic acid/vanillic acid/vanillin


-1
concentration (g l )

2.6. The

Vanillic acid /ferulic acid concentration (g l )

L. Zheng et al. / Bioresource Technology 98 (2007) 11151119

60
80
100
Time (h)
Vanillic acid

120

140

Vanillin

Fig. 3. The bioconversion course of vanillin production combining by A.


niger CGMCC0774 and P. cinnabarinus CGMCC1115. Between 0 h and
72 h, ferulic acid was converted to vanillic acid by A. niger in a 25 l
fementer under 0.1 MPa pressure, 1 vvm aeration rate, 37 C, and
200 rpm. From 72 h to 132 h, vanillic acid in above medium with
treatment was further converted to vanillin by P. cinnabarinus. The
bioconversion was performed at pH 5.0, 35 C and 120 rpm, supplementing with 5 g l 1 of glucose and 10% HZ802 resin.

1118

L. Zheng et al. / Bioresource Technology 98 (2007) 11151119

processes of ferulic acid to vanillic acid and vanillic acid to


vanillin, the ltrate of A. niger CGMCC0774 culture medium could be used as the medium of bioconversion with
P. cinnabarinus CGMCC1115 cells. Glucose and HZ802
resin were added to this medium of conversion. As shown
in Fig. 3, the concentration of vanillin reached 1.1 g l 1 with
a molar yield of 60.7% at 54 h of conversion time.
During the bioconversion of vanillic acid into vanillin
by P. cinnabarinus MUCL39533, vanillic acid was both
oxidatively decarboxylated into methoxyhydroquinone
and reduced to vanillin (Falconnier et al., 1994). In our
study, P. cinnabarinus CGMCC1115 preferentially metabolized vanillic acid to vanillin using a reduction pathway,
subsequently further transformed into vanillyl alcohol with
extension of the conversion time, no methoxyhydroquinone was determined by HPLC. As vanillin accumulated
was not only toxic to microorganisms but also benecial
to vanillyl alcohol emergence (Stentelaire et al., 2000),
adsorbents were used to adsorb vanillin, and HZ802 resin
was considered as the best one (Sun et al., 2005). The quantitative eect of HZ802 resin on vanillin yield and on
the production of by-products was showed in Table 1.
The concentration of vanillin was raised to 1.09 g l 1 and
vanillyl alcohol was controlled within 0.12 g l 1, while
0.63 g l 1 of vanillin and 0.32 g l 1 of vanillyl alcohol were
determined without adding HZ802 resin. The adsorbed
vanillin was increased and the residual in the liquid
decreased with the increasing of resin until added 10 g of
HZ802 resin. The results exhibited that supply of HZ802
resin was necessary and the optimal ratio of vanillic acid
to HZ802 resin was 1:5.
3.5. Eect of concentration of the vanillic acid enriched
cultures on vanillin production
Vanillin production was then tested under dierent concentrations of vanillic acid. Vanillic acid enriched culture
was ltrated and concentrated to dierent concentrations
before it was used as the medium of bioconversion by P.
cinnabarinus cells. The results are shown in Table 2.
It was found that with the increase of both initial vanillic
Table 1
Eect of HZ802 resin on vanillin yield and vanillyl alcohol
HZ802 resin (g)

0
4
6
8
10
12

Residual in
conversion medium
(g l 1)

Adsorbed in HZ802 resin


(g l 1)

Vanillin

Vanillyl
alcohol

Vanillin

Vanillyl
alcohol

0.63
0.29
0.20
0.10
0.04
0.03

0.32
0.15
0.12
0.12
0.12
0.12

0
0.79
0.86
0.92
1.05
1.06

0
<0.01
<0.01
<0.01

The bioconversion was performed on 100 ml medium at 35 C, pH 5.0,


with shaking speed of 120 rpm. HZ802 resin was added after 12 h and
conversion continued 54 h.

Table 2
Eect of vanillic acid concentration on vanillin production
Vanillic acid
(g l 1)

HZ802 resin
(g)

Vanillin
(g l 1)

Molar conversion
yield (%)

2
3
4
5

10
15
20
25

1.1
1.7
2.2
2.5

60.8
62.6
60.7
55.2

The bioconversion was carried out at 35 C, pH 5.0, with shaking speed of
120 rpm. HZ802 resin was added after12 h and conversion continued 54 h.
Table 3
The 13C isotope characteristic of various vanillin
d13CPDB (&)

Vanillin
Chemically synthesized vanillin from guaiacol
Rhovanil natural from ferulic acid
Vanillin from residue of rice bran oil

26.88
36.67
36.11

The determination was carried out with DeltaPlusXL (Bristol, the UK).

acid concentration and resin amount, vanillin formation


increased obviously. Although the maximum concentration
of vanillin was 2.5 g l 1 with bioconversion medium containing 5 g l 1 of initial vanillic acid and 25 g of HZ802
resin, there was 1.6 g l 1 of vanillic acid still unused. The
production of vanillin was further increased to 2.8 g l 1
with a molar conversion yield of 61.9% by extension of
the conversion time to 72 h.
3.6. The

13

C isotope analysis of vanillin

In order to obtain the natural label, the vanillin product was authenticated by the 13C isotope analysis. Isotopic
mass spectrometry reected vanillin from dierent sources
(agricultural or petrochemicals). The 13C isotope of the
crystallized vanillin produced from waste residue of rice
bran oil was identical with Rhovanil Natural (Rhodia
Co. France), and dierent from chemically synthetic vanillin. The result was shown in Table 3.
4. Discussion
Considering the increasingly requirement for natural avors in the food industry, this work was focused on production of vanillin from a natural raw materials by use
of microorganisms. Waste residue of rice bran oil is a very
cheap and abundant by-product of the rice rening industry in China. In this study, we attempt to use waste residue
of rice bran oil as ferulic acid source for vanillin production
at the rst time. With preliminary treatment, free ferulic
acid in ferulic acid enriched fraction released from waste
residue of rice bran oil was converted to vanillic acid by
A. niger CGMCC0774, and then the vanillic acid enriched
broth was further converted to vanillin by P. cinnabarinus
CGMCC1115 cells. Under the optimum condition of
ferulic acid consecutive bioconversion by A. niger and
P. cinnabarinus, the maximum concentration of vanillin
reached 2.8 g l 1 with a molar yield of 61.9% at 72 h. Based

L. Zheng et al. / Bioresource Technology 98 (2007) 11151119

on these results, a technology for the production of vanillin


from waste residue of rice bran oil has been developed.
Furthermore, crystal vanillin extracted from HZ802 resin
was authenticated by 13C isotopic analysis. The results veried that vanillin produced by our technology is a natural product from natural raw materials agricultural
sources, which means it is safe for the consumers.
Majority of agricultural by-products contain ferulic acid
ranged within 0.143.0% (w/w). As a cheap raw material,
many investigations were carried out about the bioconversion of ferulic acid into vanillin in order to decreases the
production cost of vanillin recently. Bonnin et al. (2001)
used sugar beet pulp to produce vanillin. When feruloylated
oligosaccharide enriched fraction prepared from sugar beet
pulp or maize bran was used as a carbon source, a number
of polysaccharide-degrading enzymes, including commercial enzymes (SP 584 or Novozym 342) and feruloyl esterase
excreted from A. niger I-1472, were used to release the ferulic acid from feruloylated oligosaccharide-rich fractions,
and then was further converted to vanillic acid for the production of natural vanillin. The autoclaved pre-treatments
of maize bran was shown as the best source of ferulic acid,
and vanillin concentration reached 767 mg l 1 after 14 d
bioconversion with a molar yield of 71% with adding
XAD-2 resin (Lesage-Meessen et al., 2002). This paper
was tested another agricultural by-product, waste residue
of rice bran oil. About 1/3 of ferulic acid and 2/3 of plant
sterol (sitosterol) were released from waste residue of rice
bran oil after hydrolysis. The ferulic acid enriched fraction
was used as the natural precursor of vanillic acid in A. niger
CGMCC0774 culture, and the by-products (b-sitosterol ect)
could also be used as high value-added raw medicine material. The advantages of this source are that ferulic acid content is high in waste residue of rice bran oil and ferulic acid
is easy to be extracted. This is benecial to obtain higher
yield of vanillin and shorten the whole bioconversion time.
If our bioconversion technology is scaled-up for industrial
production, high value-added vanillin could be produced
with the cheap and abundant raw material, waste residue
of rice bran oil.
Acknowledgements
This research was supported by the State Key Basic
Research and Development Plan of China (No.
2003CB716008) and the Ministry of Science and Technology (No. 2004BA713B05-082).
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