Appl Microbiol Biotechnol (2007) 74:783–790
DOI 10.1007/s00253-006-0735-5
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Enhanced vanillin production from ferulic acid using
adsorbent resin
Dongliang Hua & Cuiqing Ma & Lifu Song & Shan Lin &
Zhaobin Zhang & Zixin Deng & Ping Xu
Received: 26 July 2006 / Revised: 23 October 2006 / Accepted: 25 October 2006 / Published online: 24 November 2006
# Springer-Verlag 2006
Abstract High vanillin productivity was achieved in the
batch biotransformation of ferulic acid by Streptomyces sp.
strain V-1. Due to the toxicity of vanillin and the product
inhibition, fed-batch biotransformation with high concen-
tration of ferulic acid was unsuccessful. To solve this
problem and improve the vanillin yield, a biotransformation
strategy using adsorbent resin was investigated. Several
macroporous adsorbent resins were chosen to adsorb
vanillin in situ during the bioconversion. Resin DM11
was found to be the best, which adsorbed the most vanillin
and the least ferulic acid. When 8% resin DM11 (wet w/v)
was added to the biotransformation system, 45 g l−1 ferulic
acid could be added continually and 19.2 g l−1 vanillin was
obtained within 55 h, which was the highest vanillin yield
by bioconversion until now. This yield was remarkable for
exceeding the crystallization concentration of vanillin and
therefore had far-reaching consequence in its downstream
processing.
Keywords Vanillin . Ferulic acid . Streptomyces sp .
Biotransformation . Adsorbent resin
D. Hua : C. Ma (*) : L. Song : S. Lin : Z. Zhang : P. Xu (*)
State Key Laboratory of Microbial Technology,
Shandong University,
Jinan 250100, People’s Republic of China
e-mail: macq@sdu.edu.cn
e-mail: pingxu@sdu.edu.cn
Z. Deng : P. Xu
Key Laboratory of Microbial Metabolism, Ministry of Education,
College of Life Science and Biotechnology,
Shanghai Jiao Tong University,
Shanghai 200240, People’s Republic of China
Introduction
Vanillin is one of the most important aromatic compounds
used in foods, beverages, perfumes, and pharmaceuticals. It
is mainly produced from lignin and petrochemicals through
chemical synthesis on a scale of 10,000 tons per year (Clark
1990; Dignum et al. 2001). Nowadays, the increasing
customer-led demands for natural flavors, which are
consistence with the attributes “natural”, “healthy”, and
the serving of “bio” marketing claims, has led to a growing
interest of flavor industry to produce natural vanillin from
raw materials by biotechnology (Priefert et al. 2001; Rao
and Ravishankar 2000).
Most biotechnological production processes of vanillin
are based on bioconversions of ferulic acid, phenolic
stilbenes, isoeugenol, or eugenol and on de novo biosyn-
thesis, applying bacteria, fungi, plant cells, or genetically
engineered microorganisms (Priefert et al. 2001; Rao and
Ravishankar 2000). Because the vanillin yields from most
processes are very low, it is considered that the unique
commercial biocatalytic process is a route based on the
biotransformation of ferulic acid (Schrader et al. 2004).
Ferulic acid is an abundant naturally occurring phenolic
compound in common agricultural residues such as cereal
bran and sugar-beet pulp (Rosazza et al. 1995; Saulnier and
Thibault 1999). It was found to be linked to different
positions on the arabinose residues in the arabinoxylans
through ester linkages. Free ferulic acid can be released by
a combination of physical and enzymatic processing, which
provides a sufficient natural source of ferulic acid (Faulds
and Williamson 1995; Mathew and Abraham 2004).
As ferulic acid is a suitable precursor for vanillin
production, the biotransformation of ferulic acid to vanillin
was studied and optimized (Civolani et al. 2000; Lesage-
Meessen et al. 1997; Muheim and Lerch 1999; Oddou et al.
784 Appl Microbiol Biotechnol (2007) 74:783–790

1999; Stentelaire et al. 1998). Most of the researchers paid
much attention to prevent further degradation of vanillin by
optimizing bioconversion condition, metabolic engineering,
and enzyme inhibition involved in these reactions (Berry
1996; Civolani et al. 2000; Oddou et al. 1999; Overhage et
al. 2000). However, the vanillin yields were merely
improved. Attempts have been made to reduce the further
transformation of vanillin to vanillyl alcohol or vanillic acid
by cellobiose and adsorbent resins such as Amberlite XAD-
2 and Diaion HP20, but failed (Bonnin et al. 1999;
Stentelaire et al. 1998). The most important factors limited
the vanillin production were high toxicity of vanillin and
product inhibition in biotransformation. So, high concen-
tration of ferulic acid in fed-batch bioconversions did not
lead to high vanillin yield. Therefore, vanillin yields still
remain at a low level (Lopez-Malo et al. 1997; Priefert et al.
2001).
In this paper, we elucidated a successful biotransforma-
tion strategy using adsorbent resin. Suitable macroporous
adsorbent resin was chosen and its application in the fed-
batch biotransformation of ferulic acid was studied, which
led to a great enhancement of vanillin yield.
Materials and methods
Chemicals
Ferulic acid and vanillin were purchased from Sigma
Chemical (St. Louis, MO, USA). The hydrophobic cross-
linked polystyrene resin HZ803 and HZ816 were purchased
from the Huachang Polymer (Shanghai, China). Other
macroporous adsorbent resins used were purchased from
Shandong Lukang Pharmaceutical Group (Shandong, China).
Detailed properties of these resins were shown in Table 1. All
other chemicals were of analytical grade and were commer-
cially available.
Strain, medium, culture condition, and biotransformation
The strain V-1 used for the biotransformation of ferulic acid
was isolated from soil samples taken from greenhouse,
according to the screening method described by Karmakar
et al. (2000). It was identified as Streptomyces sp. from
physiological, biochemical properties according to Bergey’s
manual of determinative bacteriology and the 16S rRNA
gene sequence (1–500 nt) comparisons with those of several
Streptomyces strains (98–99%) held in the GenBank
database (Holt et al. 1994; Hopwood et al. 1985). Strain
V-1 was deposited at the China Center for Type Culture
Collection (CCTCC M 206065).
Cultures were first incubated in seed medium (200 rpm,
30°C and 30 ml in 300-ml flask), which contained 10 g l−1
glucose, 5 g l−1 yeast extract, 10 g l−1 peptone, 5 g l−1 malt
extract, and 10 g l−1 NaCl (pH 7.2). The midlogarithmic-
stage preculture was then inoculated (6%, v/v) into the
biotransformation medium. The biotransformation medium
was composed of 15 g l−1 glucose, 8 g l−1 yeast extract,
0.8 g l−1 MgSO4, and 2 g l−1 NaCl (pH 7.2). After
incubation for 24 h (200 rpm, 30°C and 50 ml in 500-ml
flask), the cultures for the biotransformation were prepared.
Ferulic acid was dissolved in 1 M NaOH solution (1:10,
w/v) and the pH value was adjusted to 8.5 with 6 M HCl. In
the batch biotransformation, ferulic acid solution was
directly added to the mature cultures described above. The
biotransformation was conducted at 200 rpm, 30°C and
50 ml medium in 500-ml flask. At regular time intervals,
samples were removed from flasks to determine the
concentrations of ferulic acid and vanillin, pH and OD620nm
of the culture.
In the fed-batch biotransformation, adding ferulic acid
was timely monitored by thin layer chromatography (TLC).
When the concentration of ferulic acid was below 2 g l−1,
additional 5 g l−1 ferulic acid was added. Adsorbent resin
was added to the biotransformation system when additional
precursor was supplied for the first time. Samples were
taken and analyzed at average time intervals.

Selection of adsorbent resins

Table 1 Properties of macroporous adsorbent resins Six agents known to be highly adsorbent for aromatic
compounds including CAD40, CD180, DM11, DM130,
Resins Water Granularity Specific Bulk density HZ803, and HZ816 were used. Before use, the resins were
content (mm) surface area (g ml−1)
soaked in methanol to remove impurities and then washed
(%) (m2 g−1)
with distilled water sufficiently to remove the solvent. The
CAD40 60–70 0.32–1.25 550–600 0.65–0.75 affinity of aromatic compounds for these adsorbents was
CD180 70–80 0.16–0.42 40–90 0.70–0.80 measured as follows: 50 ml sterilized biotransformation
DM11 60–70 0.32–1.25 ≥500 0.70–0.80 medium containing 10 g l−1 ferulic acid and 10 g l−1
DM130 65–75 0.32–1.25 500–550 0.65–0.75 vanillin was transferred into a 500-ml flask. The pH value
HZ803 57–64 0.2–1.0 450–550 0.65–0.67
of the solution was adjusted to 8.5, which was similar with
HZ816 45–53 0.3–1.2 500–550 0.77–0.87
that of the fermentation broth. The resin was added into the
Appl Microbiol Biotechnol (2007) 74:783–790
flask and shaken at 200 rpm for 2 h at 30°C. Triplicate
experiments were performed under the same condition.
Concentrations of ferulic acid and vanillin in the aqueous
phase were analyzed by gas chromatography (GC).
Adsorption of vanillin by DM11
Suitable amount of DM11 used in the fed-batch biotrans-
formation was tested. The concentrations of ferulic acid and
vanillin were both 10 g l−1, which was dissolved in the
same sterilized biotransformation medium. Five groups of
experiments were performed, in which DM11 was added at
the concentration of 5, 8, 10, 12, and 15%, respectively
(wet w/v). Adsorption was carried out at 200 rpm for 2 h at
30°C. Triplicate experiments were performed under the
same condition. Concentrations of ferulic acid and vanillin
in the aqueous phase were analyzed by GC.
The capacity of DM11 to adsorb high concentration of
vanillin in the absence or presence of ferulic acid was also
investigated. Total vanillin added were 0.8, 1, 1.2, 1.5, and
2% (w/v), respectively. The concentration of ferulic acid
was 5 g l−1 if added. Adsorption condition was as described
above and samples were analyzed.
Desorption of vanillin from the resin
The resin in which vanillin was adsorbed was collected and
eluted with butyl acetate (1:2, w/v). Desorption was
performed shaking at 200 rpm for 2 h at 30°C. From this
way, 100% of the adsorbed vanillin could be released.
Analytical methods
Cell growth was measured spectrophotometrically at a
wavelength of 620 nm (OD620nm). The pH value of the
culture was determined by Orion828 pH meter (Orion
Research).
TLC analysis was carried out on 0.25-mm silica gel
(GF254, JYD, Qingdao, China). About 0.5 μl sample after
centrifugation (8,000×g, 10 min) were spotted and devel-
oped with the solvent system of hexane–chloroform–ether–
acetic acid (4:3:2:0.1, v/v/v/v). Plates were dried at room
temperature and visualized under a universal UV lamp at
254 nm.
Before GC analysis, the pH value of the sample was
adjusted to 2 with 6 M HCl and extracted with butyl acetate
(1:2, v/v). Concentrations of ferulic acid and vanillin were
analyzed by a VARIAN-3380 gas chromatography,
equipped with a 30-m SPB-5 capillary column (30 m×
0.25 mm×0.25 μm, Supelco, USA). The temperatures of
the injector and flame ionization detector were both 280°C.
The column oven temperature was maintained at 140°C
for 2 min, then raised to 275°C at a rate of 20°C min−1,
785
and maintained at this level for 2 min. Quantitative
data were obtained by comparing the peak areas of the
query compounds with those of standards of known
concentrations.
In the fed-batch biotransformation with the addition of
adsorbent resin, the total vanillin concentration was
calculated as described by Yang et al. (1994). It was made
up of the vanillin retained in the aqueous phase and that
adsorbed onto the resin.
Results
Batch biotransformation of ferulic acid to vanillin
Batch biotransformation without ferulic acid supplement
was first carried out. The initial ferulic acid concentration in
the culture was 9.0 g l−1. From Fig. 1, we could see the
changes of ferulic acid and vanillin during the biotransfor-
mation. And guaiacol was also produced as a byproduct. As
shown in Fig. 2, the optical density of the broth at OD620nm
increased a little and then maintained at the highest level,
which led to a high bioconversion activity. During the first
5 h, the production of vanillin was slow. In the next 10 h,
the consumption of ferulic acid and the production of
vanillin were very quick, and within 20 h nearly no ferulic
acid remained. After reaching the highest yield, vanillin
was further transferred and therefore its concentration
decreased accordingly. During the biotransformation, the
pH value of the bioconversion broth first decreased from
9.3 to 8.2, and then it increased a little. The slightly
decrease of the pH value might be due to the production of
the acidic byproduct vanillic acid (Muheim and Lerch
1999). The highest concentration of vanillin was achieved
at 18 h, which was 5.24 g l−1, with a molar yield 74.6%.
The pH value was 8.6 when the batch biotransformation
finished.
Selection of adsorbent resins
The affinities of the selected adsorbents for ferulic acid and
vanillin were shown in Fig. 3. The choice of the most
suitable adsorbent was based on two criteria: a strong
ability to adsorb vanillin and a high specificity to avoid
ferulic acid fixation. After a 2-h incubation, all the resins
showed the ability to adsorb vanillin. CD180, CAD40,
DM130, and HZ803 had a relative poor adsorption
capacity. No more than 25% of the added vanillin was
adsorbed by the resins CAD40, DM130, and HZ803.
Adsorption capacity of CD180 was the poorest, in which
only 10% vanillin was adsorbed. DM11 and HZ816
showed better adsorption ability for vanillin, in which more
than 30% of the added vanillin was adsorbed, but for the
786 Appl Microbiol Biotechnol (2007) 74:783–790

Fig. 1 Gas chromatography

spectra of samples before bio-
transformation and after bio-
transformation. The
concentration of ferulic acid
added was 9 g l−1. Samples were
extracted with butyl acetate (1:2,
v/v) and analyzed by GC. The
retention times of guaiacol,
ferulic acid, and vanillin were
2.45, 3.80, and 4.63 min,
respectively

later near 10% of the added ferulic acid was also adsorbed.
DM11 was the most suitable, with which about 40% of
added vanillin was adsorbed, while almost all the ferulic
acid was retained in the aqueous phase. Therefore, in the
next experiments, DM11 was used.
that 8 and 10% DM11 added almost have the same
adsorption ability, in which the most vanillin was adsorbed
(more than 50%) without the loss of ferulic acid (less than
3%). On the basis of economy, we concluded the optimal
concentration of resin DM11 was 8%.

Suitable amount of adsorbent resin used Adsorption capacity for high concentration of vanillin

Suitable amount of adsorbent resin used in the fed-batch
biotransformation was tested. It was performed with ferulic
acid and vanillin solution dissolved in the sterilized
biotransformation medium. As shown in Fig. 4, nearly no
ferulic acid was adsorbed when the resin added was under
10%, and exceeding this range more than 20% of the added
ferulic acid was also adsorbed. This was not preferable in
the bioconversion, though with the increasing of DM11
used, a little more vanillin was also adsorbed. It was clear
The capacity of resin DM11 to absorb high concentration of
vanillin in the absence or presence of ferulic acid was
investigated. The concentrations of vanillin were between
0.8 and 2% (w/v) and the concentration of ferulic acid was
5 g l−1, which were similar with their concentrations during
the fed-batch biotransformation. As shown in Fig. 5a,b, the
presence of ferulic acid did not affect the adsorption ability
of DM11 for vanillin. In both experiments, more than 50%
of the vanillin could be absorbed until vanillin concentra-
Appl Microbiol Biotechnol (2007) 74:783–790
Fig. 2 Batch biotransformation of ferulic acid to vanillin. The initial
ferulic acid concentration was 9 g l−1. Biotransformation was carried
out on a rotary shaker with 200 rpm at 30°C. Concentrations of ferulic
acid and vanillin were analyzed by GC. Triplicate assays were
performed and the error bars indicated standard deviations. Filled
square pH; empty circle OD620nm; multiplication symbol ferulic acid;
filled triangle vanillin
tion was higher to 20 g l−1. This would greatly decrease the
toxicity of vanillin and the product repression in the fed-
batch biotransformation.
Fed-batch biotransformation with adsorbent resin DM11
Fed-batch biotransformation with adsorbent resin DM11
was performed. The initial ferulic acid concentration was
10 g l−1. Resin DM11 (8%, wet w/v) was added to the
biotransformation system at 12 h, at which the concentra-
Fig. 3 The adsorption capacity of different resins. Six adsorbent resins
including CAD40, CAD180, DM11, DM130, HZ803, and HZ816 were
tested. The resins used were 5% (wet w/v). Initial concentrations of
ferulic acid and vanillin were both 10 g l−1. The pH value of the
adsorption mixture was adjusted to 8.5. Adsorption was carried out on
a rotary shaker with 200 rpm at 30°C. After a 2-h adsorption, the
reaction was stopped and the concentrations of ferulic acid and
vanillin in the aqueous phase were analyzed by GC. The values were
means of three replicates, and the error bars indicated standard
deviations
787
Fig. 4 The adsorption capacity of DM11 at different additional
concentrations. DM11 was added at the concentration of 5, 8, 10, 12,
and 15% (wet w/v). The initial concentrations of ferulic acid and
vanillin were both 10 g l−1. Adsorption was also carried out on a
rotary shaker with 200 rpm at 30°C. The control experiment was
carried out without the adding of resin. After a 2-h adsorption,
concentrations of ferulic acid and vanillin in the aqueous phase were
analyzed by GC. The values were means of three replicates, and the
error bars indicated standard deviations
tion of ferulic acid was below 2 g l−1. At the same time,
continually precursor supplements began. Along with the
addition of ferulic acid, the strain still had high biotrans-
formation ability, though which was a little lower than that
in the batch biotransformation. As shown in Fig. 6, seven
times of precursor addition (5 g l−1 ferulic acid every time)
were carried out continually and the total ferulic acid
concentration reached 45 g l−1. More ferulic acid added
would lead to the entirely loss of the biotransformation
ability and no more vanillin was produced (data not
shown). When the biotransformation was finished, 19.2 g
l−1 vanillin was achieved in 55 h, with a high molar yield of
54.5%. To our knowledge, this is by far the highest vanillin
production from ferulic acid by biotransformation.
Discussion
The high chemical activity and the toxicity of vanillin have
been considered to be the reason for low vanillin yield from
ferulic acid (Dignum et al. 2001; Lopez-Malo et al. 1997;
Priefert et al. 2001; Rao and Ravishankar 2000). In many
biotransformation processes, it is usually found that little
vanillin was accumulated due to the higher degrading rate
of vanillin than that of ferulic acid. And vanillin of high
concentration also causes great production repression in the
fermentation, which results in the decrease of biotransfor-
mation efficiency. Moreover, oxygen tension in the reaction
system is found to play an important role in the production
of vanillin.
788 Appl Microbiol Biotechnol (2007) 74:783–790

Fig. 5 The capacity of DM11 to

adsorb high concentration of
vanillin in the absence or pres-
ence of ferulic acid. Adsorption
condition was simulated with
that of the fed-batch biotransfor-
mation. Total vanillin added
were 0.8, 1, 1.2, 1.5, and 2%
(w/v), respectively. The concen-
tration of ferulic acid added was
5 g l−1. Adsorption was carried
out on a rotary shaker with
200 rpm at 30°C.
After a 2-h adsorption, concen-
trations of vanillin in the aque-
ous phase were analyzed by GC.
The values were means of three
replicates, and the error bars
indicated standard deviations.
a Adsorption in the absence of
ferulic acid; b adsorption in the
presence of 5 g l−1 ferulic acid

In our preliminary study, the role of oxygen tension in
this biotransformation had been investigated, which was
carried out with different agitation speed and medium
volume in the flask. And the agitation speed of 200 rpm
and the medium volume of 50-ml medium in 500-ml flask
were found to be the best. Lower (low agitation speed,
more medium volume) or higher oxygen tension (high
agitation speed, less medium volume) were both disadvan-
tageous to the vanillin production. When the oxygen
Fig. 6 Fed-batch biotransformation of ferulic acid to vanillin with the
adding of DM11. Biotransformation was carried out on a rotary shaker
with 200 rpm at 30°C. Consequential adding of ferulic acid (5 g l−1) was
carried out when the concentration of ferulic acid was below 2 g l−1.
Adsorbent resin DM11 was added to the biotransformation system
when additional precursor was supplied for the first time. Concen-
trations of ferulic acid and vanillin were analyzed by GC. The values
were means of three replicates, and the error bars indicated standard
deviations. Solid line ferulic acid; filled triangle vanillin
tension was low, the production of vanillin was very slow
and the biotransformation time was much longer. But when
the oxygen tension was higher, the biotransformation speed
was too quick and the byproduct such as guaiacol and
vanillic acid produced.
In the batch biotransformation described in this paper,
5.24 g l−1 vanillin was achieved from 9 g l−1 ferulic acid
(molar yield 74.6%), which showed that strain V-1 was a
good vanillin producer. In the next experiment, fed-batch
biotransformation was attempted. However, when more
ferulic acid was added to the system, the bioconversion rate
and the molar yield were much lower (data not shown).
Therefore, high concentration of ferulic acid could not be
added and vanillin production was only improved a little.
The potential benefits introduced by the application of in
situ product removal (ISPR) techniques to a biotechnolog-
ical process lie in: avoid or reduce inhibition or toxicity;
product stabilization and facilitate further downstream
processing of the product. Among ISPR techniques, in situ
product adsorption (ISPA) using adsorbent resin is widely
used in the biotransformation productions of natural flavors
and antibiotics, which are toxic to the producing organisms
(Freeman et al. 1993). In the fermentation of kirromycin
and rubradirin, adsorbent resins have been successfully
applied to decrease the toxicity and repression of the
fermentation products (Gastaldo et al. 1996; Marshall et al.
1990). Solid adsorbents also have been found useful in the
fermentation of benzaldehyde, which shifted the biotrans-
formation pathways of L-phenylalanine into benzaldehyde
and greatly enhanced benzaldehyde production (Lomascolo
et al. 1999, 2001). However, there are few reports on the
Appl Microbiol Biotechnol (2007) 74:783–790
adsorbent resins used in the vanillin production (Stentelaire
et al. 1998) and vanillin yields in these processes were very
low.
The use of macroporous adsorbent resins DM11 well
solved these problems. In the fed-batch biotransformation,
quite a lot of the produced vanillin was fixed onto DM11,
which largely reduced its chemical toxicity and the product
repression. Therefore, ferulic acid (up to 45 g l−1) could be
continually added and vanillin was produced at a relatively
high rate, which led to a high vanillin yield.
The previous best results of vanillin production were
performed by the strains Streptomyces setonii
ATCC39116 and Amycolatopsis sp. HR167. With these
two strains, the highest vanillin concentrations were about
12 g l−1, in which the total molar yields were lower than
30% (Müller et al. 1998; Rabenhorst and Hopp 1997).
Most other vanillin productions reported were below
10 g l−1 (Priefert et al. 2001). In this study, with the
addition of adsorbent resin DM11 vanillin production was
greatly enhanced and 19.2 g l−1 vanillin was obtained by
fed-batch biotransformation (with a total molar yield
54.5%), which was much higher than those biotransformed
by above two strains.
It should be noted that vanillin has a crystallization
concentration of 10 g l−1 at 20°C (Clark 1990) and a
vanillin concentration above 10 g l−1 is considered to
provide great convenience for the product recovery.
Therefore, high vanillin concentration near to 20 g l−1
reported in this paper will undoubtedly make the down-
stream processing more feasible and easy to be carried out
in the industrial application. At the same time, much of the
vanillin adsorbed onto the resins in situ also greatly
lessened the fermentation broth pretreatment and facilitated
the extraction process (Freeman et al. 1993; Perez 2001;
Priefert et al. 2001).
Acknowledgments The authors gratefully acknowledged the finan-
cial support of this work by Shanghai Apple Flavor & Fragrance
(China). The work was supported in part by the financial support of
the State Major Basic Research Development Program (China) (grant
2007CB707800).
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