Biotransformation of eugenol to vanillin by a novel strain Bacillus safensis SMS1003

Article abstract of DOI:10.1080/10242422.2018.1544245

Due to the extensive applications of vanillin as flavored compound and increasing consumers concern for its natural and environment friendly mode of production, present work was focused on the selection of bacterial isolate capable of producing vanillin using eugenol biotransformation. Bacterial strain SMS1003 is evidenced as the potential strain for vanillin production and identified as Bacillus safensis (GeneBank accession no. MG561863) using biochemical tests and molecular phylogenic analysis of its 16S rDNA gene sequence. Molar yield of vanillin reached up to 10.7% (0.055 g/L) at 96 h of biotransformation using growing culture of B. safensis SMS1003 in following culture conditions: eugenol concentration 500 mg/L; temperature 37 °C; initial pH 7.0; inoculum volume 4%; volume of culture media 10%; and shaking speed 180 rpm. Vanillin was detected as the single metabolite with a molar yield of 26% (0.12 g/L) at 96 h using resting cells of B. safensis SMS1003. Product confirmation was based on spectral scan using photodiode array detector, Fourier-transform infrared spectroscopy, high-performance liquid chromatography, and mass spectroscopy.

Full text of DOI:10.1080/10242422.2018.1544245

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Biotransformation of eugenol to vanillin by a novel
strain Bacillus safensis SMS1003
Archana Singh, Kunal Mukhopadhyay & Shashwati Ghosh Sachan
To cite this article: Archana Singh, Kunal Mukhopadhyay & Shashwati Ghosh Sachan (2018):
Biotransformation of eugenol to vanillin by a novel strain Bacillusꢀsafensis SMS1003, Biocatalysis
and Biotransformation, DOI: 10.1080/10242422.2018.1544245
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2018.1544245
RESEARCH ARTICLE
Biotransformation of eugenol to vanillin by a novel strain Bacillus
safensis SMS1003
Archana Singh, Kunal Mukhopadhyay
and Shashwati Ghosh Sachan
Department of Bio-Engineering, Birla Institute of Technology, Ranchi, Jharkhand, India
ABSTRACT
ARTICLE HISTORY
Received 26 July 2018
Accepted 28 October 2018
Due to the extensive applications of vanillin as flavored compound and increasing consumers
concern for its natural and environment friendly mode of production, present work was focused
on the selection of bacterial isolate capable of producing vanillin using eugenol biotransform-
ation. Bacterial strain SMS1003 is evidenced as the potential strain for vanillin production and
identified as Bacillus safensis (GeneBank accession no. MG561863) using biochemical tests and
molecular phylogenic analysis of its 16S rDNA gene sequence. Molar yield of vanillin reached up
to 10.7% (0.055g/L) at 96 h of biotransformation using growing culture of B. safensis SMS1003
in following culture conditions: eugenol concentration 500mg/L; temperature 37 ꢀC; initial pH
7.0; inoculum volume 4%; volume of culture media 10%; and shaking speed 180rpm. Vanillin
was detected as the single metabolite with a molar yield of 26% (0.12 g/L) at 96 h using resting
cells of B. safensis SMS1003. Product confirmation was based on spectral scan using photodiode
array detector, Fourier-transform infrared spectroscopy, high-performance liquid chromatog-
raphy, and mass spectroscopy.
KEYWORDS
Biotransformation; eugenol;
vanillin; PDA; molar yield;
Bacillus safensis
1. Introduction
appropriate precursor compounds act as a substitute
in the biosynthesis of these aromatic compounds.
Microbial whole cells, immobilized cells as well as their
enzymatic preparations are considered as efficient and
environment friendly bioconversion tools (Ashengroph
et al. 2012).
During the past few years, worldwide increasing mar-
ket demand for healthy food additives as well as com-
pounds of various cosmetic and pharmaceutical uses,
primes the development of safe and ecofriendly indus-
trial processes for their production. Generally, various
aromatic compounds impart a significant role in food
and chemical industries. Vanillin (4-hydroxy-3-methox-
ybenzaldehyde) is one such widely used aromatic
compound used as a flavor in food, confectionary,
beverage production and also has certain applications
in medical fields and pharmaceuticals (Priefert et al.
2001). Natural vanillin production was based on the
fermentation of cured pods obtained from two differ-
ent species of vanilla orchids: Vanilla planifolia and
Vanilla tahitensis. Traditionally, a chemical method of
synthesis was used for their production leading to the
formation of undesirable racemic mixtures, lacking
substrate selectivity and reduce process efficiency by
increasing the downstream processing cost and cause
environmental pollution (Longo and Sanroman 2006;
Xu et al. 2007). Microbial transformation that is fer-
mentation (= de novo) and biotransformation of
Plant derived phenylpropanoids which are a diverse
family of the organic compounds synthesized by the
plants can act as the potential precursor for microbial
biotransformation (Giedraityte and Kalediene 2014).
Various plant-derived phenylpropanoids (eugenol, iso-
eugenol, and ferulic acid) can be used as the natural
renewable and environment friendly sources for the
production of fine chemicals (Ashengroph et al. 2011).
Out of all potential sources, use of ferulic acid has
been widely investigated for biotransformation
(Giedraityte and Kalediene 2014). However, due to the
limited availability of natural ferulic acid and its high
price, eugenol was explored as an alternative precur-
sor for enzymatic oxidative transformation to other
valuable eugenol derivatives like ferulic acid and vanil-
lin (Rabenhorst 1996).
Eugenol (4-allyl-2-methoxyphenol) is the major aro-
matic constituent present in the clove oil extracted
CONTACT Shashwati Ghosh Sachan
ssachan@bitmesra.ac.in
Department of Bio-Engineering, Birla Institute of Technology, Mesra, Ranchi,
Jharkhand 835215, India
Supplemental data for this article can be accessed here.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
A. SINGH ET AL.
from the clove plant, Syzygium aromaticum and has
been explored as a potential precursor for the produc-
tion of various methoxyphenol derivatives. It is an
inexpensive substrate with the market price of around
US$50/kg and also commercially available. According
to the US and European legislation production of fla-
vored compounds using a raw material through bio-
technological route is “natural’’ (Hua et al. 2007). Use
of eugenol as a food additive and potential precursor
for industrial vanillin production by microorganisms is
amount of vanillin (0.12 g/L) was obtained as a sin-
gle metabolite.
2. Materials and methods
2.1. Chemicals
Standards such as eugenol (99%), coniferyl aldehyde
(98%), ferulic acid (99%), vanillin (99%), vanillic acid
(98%), and protocatechuic acid (98%) were purchased
from Sigma-Aldrich (Bangalore, India). Analytical grade
methanol and ethyl acetate as well as high-perform-
ance liquid chromatography (HPLC)-grade solvents
such as methanol, and Trifluoroacetic acid were pro-
cured from Himedia (Mumbai, India). All other chem-
ical used as media components were of analytical
grade and purchased from Hi-Media Laboratory India.
generally
recognized as safe (Gallage and
Moller 2015).
Vanillin as well as its associated metabolites formed
during eugenol biotransformation have widespread
applications and are of great economic importance
(Ashengroph et al. 2011). Coniferyl aldehyde exhibits
antibacterial, pesticidal and chemoattractant proper-
ties. Similarly, various biomedical, pharmaceutical and
industrial applications have been associated with the
use of ferulic acid. It acts as a potential antioxidant,
anti-diabetic and anti-ageing agent and is an effective
component of various Chinese medicinal herbs (Graf
1992; Mishra et al. 2013).
Rabenhorst developed a method of eugenol bio-
transformation to vanillin using Pseudomonas sp. strain
(Rabenhorst 1996). Various other microorganisms,
including Pseudomonas fluorescens, P. putida,
Corynebacterium sp., Fusarium solani, Byssochlamys
fulva, and Penicillium simplicissimum, were also
reported on the basis of their ability to degrade
eugenol (Tadasa and Kayahara 1983; Furukawa and
Nagasawa 1998; Priefert et al. 2001; Giedraityle and
Kalediene 2014). Biodegradation of eugenol to vanillin
was also reported using Bacillus sp (Kadakol and
Kamanavali 2010). Although, various microorganisms
have been explored so far for vanillin production
using eugenol (Tadasa and Kayahara 1983; Rabenhorst
1996; Furukawa and Nagasawa 1998; Priefert et al.
2001; Kadakol and Kamanavali 2010; Giedraityle and
Kalediene 2014). Extensive screening of other potential
microorganisms needs to be done with the aim to
improvise the yield of vanillin as a single metabolite.
This study is focused on the isolation and selection of
a bacterial strain with the potential to transform
eugenol into value added methoxyphenol derivatives
like vanillin.
2.2. Isolation of bacterial cultures
Eugenol metabolizing bacteria was isolated from soil
samples of Ocimum and Petunia sp. cultivated field of
BIT Mesra medicinal garden. Soil suspension was pre-
pared using 10 g of the wet soil sample in 100 ml of
sterile saline water, and appropriate dilutions of these
suspensions were prepared. From the above prepared
dilutions, 100 ml were plated into the sterilized bio-
transformation (BT) medium (ingredients: gram per
liter of distilled water: (Na2)HPO4, 4; KH2PO4, 1; yeast
extract, 1; NaCl, 0.2; MgSO4.7H2O, 0.2 and anhydrous
CaCl2.2H2O, 0.05; agar, 15; pH 7.0 7.2 supplemented
with 100 mg/L of eugenol as sole carbon and energy
source to obtain strains capable of utilizing eugenol at
37 ꢀC for 24
h
(Muheim and Lerch 1999).
Morphologically different microorganisms were
selected from master plates and transferred on min-
imal agar media with eugenol (100 mg/L) by quadrant
streak plate technique to obtain the cultures which
showed the capacity to utilize eugenol as a sole car-
bon source for growth and energy in pure form.
Isolates were further sub cultured and maintained for
future use in minimal medium slants at
with eugenol.
4
ꢀC
2.3. Screening of transforming strain
The most effective one step screening method was
adopted. Cells were cultured in a 100-ml flask contain-
ing 25 ml of sterile (BT) medium. Starter cultures of
selected bacterial isolates were prepared ꢀindividually
from stock cultures and incubated at 37 C for 24 h
followed by the transfer of 1 ml of inoculum from
selected starter cultures individually to sterilized BT
In the current study, a novel strain of Bacillus safen-
sis, labelled as SMS1003, capable of transforming
eugenol into derivatives like vanillin and coniferyl
aldehyde was identified. Further, in response to opti-
mization of various culture conditions, a significant

BIOCATALYSIS AND BIOTRANSFORMATION
3
medium supplemented with filter-sterilized eugenol
(100 mg/L) as a sole carbon source. These were incu-
bated upto 144 h at 37 C initially under static condi-
sample of 25 ml culture broth obtained after opti-
mized days of incubation was centrifuged (1000 rpm,
10 min, 4 C) and filtered using 0.22 mm syringe filters.
ꢀ
ꢀ
tion. After the incubation, each isolate inoculated was
processed individually. The supernatant was acidified
with conc. HCl to pH 2 and extracted using ethyl acet-
ate in a ratio of 1:1. Phase separation was followed by
the collection of upper organic phase which was then
concentrated in Rotary Vacuum Evaporator to dryness
under vaccum at 30 ꢀC. Dried samples were resus-
pended in 1 ml of aqueous methanol (50% v/v). Both
qualitative and quantitative analysis of the processed
sample was performed.
Samples were injected on a Kinetex C18 reverse phase
HPLC column (250 ꢁ 4.6 mm, 5 lm particle size;
Phenomenex, Torrance, CA, USA) keeping injection
volume 20 ml and a constant flow rate of 1 ml/min.
Separation of samples and standard solutions was per-
formed at room temperature. Sample elutions were
performed using mobile phases consisting of 1 mM
trifluoroacetic acid in water (Solvent A) and methanol
(Solvent B). The elution gradient optimized was: t =
0–20 min: 30% B; t = 25 min: 70% B; t = 40–45 min:
100% B followed by the equilibration of the column
for 10–20 min. Total run time was 45 min. Ambient
column temperature followed by two detection wave-
length of 254 and 310 nm was used. Detection wave-
length of 310 nm was used for vanillin, coniferyl
aldehyde, ferulic acid and that of 254 nm was used for
eugenol, vanillic acid, and protocatechuic acid.
Comparative analysis of the analytes present in the
samples and their respective reference standards were
performed based on their retention times and
PDA spectra.
2.4. Phenotypic and genotypic characterization of
transforming strains
Based on the HPLC analysis, the strain showing signifi-
cant yield (mg/L) of desired metabolites was selected
and characterized at morphological, biochemical and
molecular levels. Genus identification was based on
Bergey’s Manual of Determinative Bacteriology (Holt
et al. 1994). Morphological characterization involved
Gram staining, endospore staining, motility, colony
shape, size, and color as well as growth on nutrient
agar and MacConkey agar plate. Biochemical tests per-
formed for gelatinase, amylase, catalase, and oxidase
activities. Other biochemical tests included indole pro-
duction, Voges-Proskauer, carbohydrate fermentation,
methyl red, citrate utilization, hydrogen sulfide pro-
duction, nitrate reduction, Tween 20 hydrolysis, and
Tween 80 hydrolysis. Species identification was done
using 16S ribosomal RNA (rRNA) gene sequencing
(Xcelris Labs Ltd. Ahmedabad, India). BLASTN (http://
www.ncbi.nlm.nih.gov/Blast) program was used for the
homology searches of the resulting consensus
sequence. Molecular evolutionary genetics analysis
(MEGA) software version 6.0 was used for alignment,
phylogenetic and molecular evolutionary analyses by
using the neighbor-joining method. Reliability of the
phylogenetic tree was tested using bootstrap analysis
based on 1000 replicates (Saitou and Nei 1987).
2.6. Identification of methoxyphenol derivatives
Metabolites produced by B. safensis strain SMS1003
using eugenol were confirmed through a PDA scan,
Fourier-transform infrared spectroscopy (FTIR), and
mass spectroscopy. Waters HPLC coupled with a
photodiode array detector was used for the spectral
scan in the region of 200–400 nm. Identification of the
compounds was based on the comparison of max-
imum adsorption of the identified compound with
that of its purified standard compounds. FTIR spectra
were recorded using a Shimadzu Corporation FT/IR-
Prestige 21, Japan in the region 4000–400 cmꢂ1 with
KBr pellet technique. Mass spectra were recorded
using Thermo Scientific LTQ XL ion trap mass spec-
trometer. Spectra were generated in positive ion
mode at 30 eV. Continuum mode across the range
from 100 to 1000 m/z was used to record the mass
spectra of metabolites obtained during biotransform-
ation and compared with those of its authentic stand-
ard compounds as references.
2.5. Quantitative analysis using HPLC
Quantification was based on the HPLC profile of
methoxyphenol derivatives obtained during eugenol
metabolism. A Waters binary HPLC pump (Waters
1525) equipped with Waters photodiode array
detector (PDA Waters 2996) was used, Waters 717 plus
Autosampler and Waters Empower login software
were used for instrument control and data handling. A
2.7. Optimization of culture conditions for
effective eugenol biotransformation
Lab scale experiments were carried out in Erlenmeyer
flasks and setup was designed for the optimization of

4
A. SINGH ET AL.
Figure 1. Overlay profile of HPLC indicating authentic standards of protocatechuic acid (Peak 1) at 310 nm vanillic acid (Peak 2)
at 254 nm, vanillin (Peak 3) at 310 nm, ferulic acid (Peak 4) at 310nm, coniferyl aldehyde (Peak 5) at 310 nm, and eugenol (Peak
6) at 254 nm. HPLC, high-performance liquid chromatography.
various process parameters (Environmental and nutri-
tional) using the classical approach of one factor at a
time. Different environmental parameters investigated
were effect of substrate concentration (100–1000 mg/
L), temperature (28, 30, 37, and 45 ꢀC), initial pH
(5.0–9.0), inoculum volume (4%–16%), volume of the
culture medium (10%–30%), and shaking speed (0–200
rpm). Similarly, the effect of various carbon and nitro-
gen sources were investigated as the nutritional
parameters on the biotransformation of eugenol by B.
safensis SMS1003 (Tilay et al. 2010). To study the effect
of various nutritional parameters, BT media was sup-
plemented with various additional carbon sources like
glucose, sucrose, and glycerol at the concentration of
1% and organic nitrogen sources like beef extract and
peptone as well as inorganic nitrogen sources like
ammonium sulphate and ammonium chloride at the
concentration of 0.05%.
2.9. Biotransformation by cell free extract
Cells were allꢀowed to grow in the presence of eugenol
for 24 h (37 C, 180 rpm, in MBT medium). Harvesting
and washing of cells were performed according to the
previously reported method (Giedraityle and Kalediene
2014). Washed cells were then disrupted by ultrasonic
treatment at 22 kHz in short bursts of 30 s (5 min,
titanium probe with tip diameter 13 mm, 35% cycle, 4
ꢀC (VCX 750 Sonics and Materials Inc., Newton, CT,
USA). The soluble fraction of the cell free extract was
obtained by centrifugation (15,000 rpm, 20 min, and 4
ꢀC). The Resulting supernatant was further concen-
trated using Amicon Ultra-0.5 CFU (Merck Millipore,
Burlington, MA, USA) membrane and used as cell free
crude extract for in vitro enzyme assays.
In vitro eugenol bioconversion were examined
ꢀ
using cell free extract preparation at 37 C. A reaction
mixture of 1.5 ml was prepared to check the in vitro
conversion of eugenol containing 200 ml of cell
extract, 1.8 mM eugenol, and 1.3 mM nicotinamide
adenine dinucleotide (NADþ), 100 mM sodium phos-
phate buffer pH 7.0. A Similar analysis was carried out
to check the in vitro conversion of coniferyl aldehyde
by supplementing the reaction mixture with 1.3 mM
NADþ and replacing eugenol with the coniferyl alde-
hyde. Ferulic acid bioconversion was also tested using
an in vitro condition reported earlier (Ghosh et al.
2007). All the reaction mixtures were incubated for 12
h and stopped using an equal volume of acetic acid:
methanol prepared in the ratio of 1:4.
2.8. Preparation of resting cells
To enhance the molar yield of vanillin further using
eugenol, the resting cell reaction assay was performed.
Cells of B. safensis SMS1003 were cultured in modified
biotransformation (MBT) medium and were centri-
ꢀ
fuged (10,000 rpm, 10 min, 4 C) at the late-exponen-
tial phase of growth. Harvested cells were then
washed twice with cold sodium phosphate buffer (100
mM, pH 7.0) and then pelleted cells were resuspended
in the same buffer at a concentration of about 3 g
(dry cell weight)/L and used as resting cell culture for
all experiments. The biotransformation was conducted
in 250 ml flasks with 25 ml of resting cell preparation
in the presence of a sterile eugenol solution (500 mg/
3. Results
3.1. Isolation and screening of eugenol
transforming strain
ꢀ
L) and incubated at 37 C and 180 rpm for 24–96 h. A
Similar experiment was carried out for the control con-
taining 25 ml phosphate buffer without resting cells
under the same testing conditions. Experiments were
performed in triplicate.
In the current study total sixteen morphologically dif-
ferent bacterial colonies were isolated using the soil
samples collected from an Ocimum sp and Petunia sp
cultivated field, as both the species contains about

BIOCATALYSIS AND BIOTRANSFORMATION
5
Figure 2. Stack plot of HPLC at 310 nm and 254 nm showing eugenol biotransformation in processed culture filtrate of Bacillus
safensis SMS1003. Chromatogram a and b represents standard vanillin (1) and coniferyl aldehyde (2) at 310 nm. Chromatogram c
represents control containing standard eugenol (3) at 254nm. Chromatogram d (3) represents eugenol utilization in a filtrate proc-
essed at 96 h of eugenol biotransformation at 254 nm. Chromatogram e represents the formation of vanillin (1) and coniferyl alde-
hyde (2) in a filtrate processed at 96 h of eugenol biotransformation at 310 nm. HPLC, high-performance liquid chromatography.
75% phenylpropanoids including eugenol and isoeu-
genol (Kasana et al. 2007). The isolates which were
found capable of utilizing eugenol at the concentra-
tion 100 mg/L as a sole carbon and energy source
were further examined for their ability to transform
eugenol into other valuable methoxyphenol deriva-
tives using a developed method of RP-HPLC. The
retention times of authentic standards obtained in the
present developed method were as follows: protocate-
chuic acid, 4.6 0.1 min; vanillic acid, 8.1 0.1 min;
vanillin, 9.9 0.5 min; ferulic acid, 17.3 0.7 min; con-
sequence of strain SMS1003. The sequence showed
99% similarity with B. safensis strain NBRC 100820
(Figure 5). Strain SMS1003 was identified as B. safensis
based on its phenotypic and genotypic characteristics.
The 16S rRNA sequence of SMS1003 was submitted to
GenBank under accession number, MG561863.
3.3. Biotransformation using growing culture of
B. safensis
Growing culture of selected bacterial strain SMS1003
was examined to biotransform eugenol into valuable
methoxyphenol derivatives initially at the concentra-
tion of 100 mg/L. As analyzed by HPLC, coniferyl alde-
hyde was initially detected in the processed culture
supernatant after 24 h of incubation. Vanillin was
formed after 48 h of incubation. Both coniferyl alde-
hyde and vanillin were accumulated in the BT medium
at the concentration of 0.07 mg/L (Molar yield 0.06%)
and 0.08 mg/L (Molar yield 0.09%) respectively, at 144
h of incubation under un-optimized culture conditions.
Eugenol transformation was not observed in control
(un-inoculated media) which was incubated under the
same conditions.
iferyl aldehyde, 20.1
0.4 min; eugenol, 31.8
0.1
min; (Figure 1). Various methoxyphenol derivatives
produced during eugenol biotransformation were then
calibrated with the external standards for quantifica-
tion. Each experiment was performed in a set of three
and results were expressed as the mean
standard
deviation. Methoxyphenol derivatives like coniferyl
aldehyde, ferulic acid, vanillin, and protocatechuic acid
were detected as the biotransformed products using
HPLC (Supporting Information Table S1). Under the
given conditions, among the eugenol degrading
microorganisms isolated from soil, a bacterial stain
designated as SMS1003 produced a significant amount
of both coniferyl aldehyde and vanillin after 144 h of
incubation (Figure 2).
3.4.1. Effect of substrate concentration
The effects of different concentration of eugenol were
investigated on the biotransformation of eugenol as
shown in Figure 6. The selected bacterial strain of B.
safensis SMS1003 was inoculated in 25 ml of nutrient
broth to prepare its starter culture and allowed to
grow for 24 h. Experimental runs were then carried
out by supplementing the BT medium with eugenol at
the concentrations of 100, 300, 500, 700, and 1000
mg/L, inoculated with 4% v/v of the starter culture fol-
lowed by incubation at 37 ꢀC for 24–144 h. It was
found that increased eugenol concentration in the
range of 100–500 mg/L proximately increased the con-
centration of both vanillin and coniferyl aldehyde in
Products were confirmed using PDA spectral analysis
(Figure 3) and mass spectral analysis (Figure 4). In com-
parison to other isolates, strain SMS1003 was chosen
for further optimization studies keeping in view rela-
tively the low productivity of vanillin using eugenol
due to its potential oxidation into vanillic acid as well
as the toxicity of eugenol (Overhage et al. 1999).
3.2. Characterization of the strain SMS1003
Microbial characteristics of strain SMS1003 as studied
are enlisted in Table 1. Phylogenetic analysis was per-
formed for the sequence similarity search of 16S rRNA

6
A. SINGH ET AL.
Figure 3. PDA spectral scan in the range of 200–400 nm of a processed culture filtrate obtained using growing culture of Bacillus
safensis SMS1003 (a) A control containing eugenol at 0 h of incubation (b) Shift in the scan due to eugenol biotransformation into
vanillin and coniferyl aldehyde using growing culture of B. safensis at 96 h of incubation. PDA, photodiode array detector.
Figure 4. Mass spectra of authentic standards and sample generated in ESI positive ion mode for the detection of eugenol
metabolites. (a) Standard eugenol (b) Standard coniferyl aldehyde (c) Standard vanillin (d) Processed sample. CALD, coniferyl alde-
hyde; ESI, electrospray ionization; EU, eugenol; VAN, vanillin.
the culture medium. However, a further increase in
the concentration of eugenol decreased the concen-
tration of both the derivatives, which may be due to
the substrate toxicity at the higher concentration or
due to the poor solubility of eugenol in water as it is
found to be lypophilic in nature (Ashengroph et al.
2011). Moreover, vanillin toxicity was also reported to
organisms at high concentrations (Chen et al. 2016).
Due to the negative effect of both eugenol and
vanillin at higher concentrations on the growth and
metabolism of microorganism, eugenol concentration
of 500 mg/L was chosen as the optimum substrate
concentration to promote growth and biotransform-
ation efficiency of B. safensis SMS1003. Under the
optimum substrate concentration of 500 mg/L,
vanillin and coniferyl aldehyde were accumulated with
the molar yield of 0.13% and 0.09%, respectively at
96 h.

BIOCATALYSIS AND BIOTRANSFORMATION
7
Table 1. Morphological and biochemical characteristics of strain SMS1003.
Tests
Observation
Tests
Observation
Colony morphology
Configuration
Margin
Elevation
Surface
Density
Gram reaction
Shape
Biochemical tests
Growth on MacConkey agar
Indole
Round
ꢂ
ꢂ
ꢂ
Irregular margin
Undulate
Smooth
Non-luminescent
Positive
Rods
Nitrate test
Voges proskauer test
Citrate utilization
Starch hydrolysis
Casein hydrolysis
Lipid hydrolysis
Tween 20 hydrolysis
Tween 80 hydrolysis
H2S production
Oxidase
ꢂ
þ
þ
þ
ꢂ
ꢂ
ꢂ
þ
þ
O
Size
Arrangement
Motility
Moderate
Single
þ
Endospore formation
þ
Growth at the temperature (ꢀC)
4
10
20
30
37
45
55
þ
þ
þ
þ
þ
þ
–
Catalase
Oxidation/fermentation
Gelatin liquefaction
Acid production from
D-Glucose
Ribose
Galactose
Fructose
Maltose
Mannitol
Sucrose
ꢂ
þ
þ
þ
þ
þ
þ
þ
þ
Growth at pH
4
5
6
7
8
9
–
þ
þ
þ
þ
þ
Glycerol
Growth on NaCl (%)
2.5
5
7.5
9
10
15
þ
þ
þ
þ
þ
þ
Figure 5. Phylogenetic tree indicating the estimated relationship of strain SMS1003 to other Bacillus sp. based on 16S rRNA gene
sequence similarity. Bootstrap values (expressed as percentages of 1000 replications) greater than 50% are shown at the branch
points. Scale bar, 0.005 substitutions per nucleotide position.
biotransformation processes (Zhao et al. 2005), the
effect of varying pH conditions were studied on the
eugenol biotransformation by B. safensis and repre-
sented in Figure 7(b). A significant increase in the
concentration of both vanillin and coniferyl alde-
hyde was observed when the initial pH of the
medium was 7. pH 7 was selected as the optimum
pH for the growth and eugenol metabolism by B.
safensis SMS1003.
3.4.2. Effect of incubation temperature
Figure 7(a) depicts the effect of four dꢀifferent incuba-
tion temperatures (25, 30, 37, and 45 C) on eugenol
biotransformation by B. safenꢀsis SMS1003. At incuba-
tion temperatures 30 and 37 C, the amount of vanil-
lin and coniferyl aldehyde obtained was 0.59 mg/L
and 0.47 mg/L respectively. Whereas the concentration
of both the metabolites decreased when the tempera-
ꢀ
ꢀ
ture was below 30 C and beyond 45 C.
3.4.3. Effect of pH
3.4.4. Effect of inoculum size
Since pH has a profound effect on microbial growth
as well as regulation of enzymes involved in
The effect of different inoculum sizes was investigated
on the biotransformation of eugenol by B. safensis

8
A. SINGH ET AL.
vanillin up to 55 mg/L (Molar yield 10.7%). Therefore,
the following optimized parameters were found to
show a significant improvement in vanillin production:
temperature, 37 ꢀC; initial pH, 7.0; inoculum volume,
4%; eugenol concentration, 0.5 g/L; the volume of cul-
ture medium, 10%; and shaking speed, 180 rpm.
3.5. Biotransformation using resting cells of B.
Safensis SMS1003
Biotransformation of eugenol was also studied using
the resting cells of B. safensis strain SMS1003 in an
optimized conversion condition. In the present study,
use of resting cells resulted in a higher production of
vanillin and was detected as the single major metabol-
ite in the culture filtrate obtained during eugenol bio-
transformation shown in Figure 8. Product
confirmation was achieved using PDA spectral analysis
(Figure 9; Supporting Information Figure S1) and FTIR
spectroscopy (Supporting Information Figure S2). No
biotransformation was observed in the control culture
supplemented with eugenol at the concentration of
500 mg/L, which was incubated under the same test-
ing condition. It was also reported in the previous
studies that biomass under non proliferating condi-
tions (resting cells) can be used in a recycled form for
at least four times without the loss of bioconversion
ability (Torre et al. 2004). The initial eugenol concen-
tration was decreased with an increase in the vanillin
concentration and biomass of B. safensis SMS1003 as
shown in Figure 10. Vanillin concentration reached up
to 120 mg/L (Molar yield 26%) at 96 h of incubation.
Vanillin was the single metabolite obtained and was
found to be stable in the buffer for at least 10 h.
Figure 6. Biotransformation of eugenol by Bacillus safensis
SMS1003 using different substrate concentration. Results are
expressed as means of at least three experiments and the
error bars indicate standard deviations.
SMS1003 as shown in Figure 7(c). Inoculum sizes were
varied from 4% to 16% and 4% was chosen as the
optimum for further optimization studies.
3.4.5. Effect of carbon and nitrogen sources
BT media supplementation with additional carbon
sources increased the microbial growth but did not
show any biotransformation (Tilay et al. 2010). It can
be inferred from the results that the selected strain
was not able to utilize eugenol once exposed to differ-
ent carbon sources in the BT media. As eugenol plays
a vital role as an inducer of the enzymatic machinery
involved in microbial transformation, no biotransform-
ation was observed in media supplemented with glu-
cose. Presence of beef extract and ammonium
sulphate (data not shown) as the additional nitrogen
sources in the media increased the vanillin and coni-
feryl aldehyde concentration up to 5.5 and 4 mg/L,
respectively.
3.4.6. Effect of media volume and agitation
3.6. Metabolic pathway of eugenol in SMS 1003
One of the most important factors is oxygen concen-
tration which not only affects the growth of the
microorganisms but also influences the biotransform-
ation efficiency (Chen et al. 2016). Since oxygen con-
centration is influenced by the media volume and
agitation condition, the effect of varied culture media
volume (%) and various shaking speeds were studied
on the biotransformation of eugenol by SMS1003 and
graphically represented in Figure 7(d) and Figure 7(e).
Results imply that vanillin was accumulated at a max-
imum concentration of 36 mg/L by using 10% volume
of culture medium. Further increase in the percentage
volume decreased the biotransformation efficiency. It
was also observed that incubation of the 10% volume
of the inoculated culture media at a shaking speed of
180 rpm significantly increased the concentration of
To confirm the metabolic pathway of eugenol biotrans-
formation by the selected strain of B. safensis SMS1003
possible pathway intermediates such as coniferyl alco-
hol, coniferyl aldehyde, and ferulic acid were added
into the BT media at the concentration of 100 mg/L.
Bacterial strain was able to grow in the presence of all
the mentioned intermediates. No change was observed
in the concentration of intermediates prepared as a
control. Analyses of the degradation products formed
using various intermediates were also carried out after
48–96 h of incubation. Formation of vanillin was
observed in a processed culture media containing
ferulic acid at 48 h of incubation. Coniferyl aldehyde
was found to get biotransformed into ferulic acid after
48 h of incubation. Vanillin degradation product(s) such

BIOCATALYSIS AND BIOTRANSFORMATION
9
Figure 7. Effect of different culture parameters on biotransformation of eugenol into coniferyl aldehyde and vanillin (mg/L) at
96 h of incubation by Bacillus safensis SMS1003. (a) Effect of different incubation temperature on the concentration of coniferyl
aldehyde and vanillin (mg/L). (b) Effect of pH on the concentration of coniferyl aldehyde and vanillin (mg/L). (c) Effect of inoculum
volume on the biotransformation of eugenol by B. safensis SMS1003. (d) Effect of volume of the culture medium on the biotrans-
formation of eugenol by B. safensis SMS1003. (e) Effect of shaking speed on the biotransformation of eugenol by B. safensis
SMS1003. Triplicate assays were performed and the vertical error bars indicate standard deviations.
as vanillic acid, vanillyl alcohol, protocatechuic acid
were not detected. These observations suggested vanil-
lin as the end product of eugenol metabolic pathway
followed by B.safensis SMS1003 via intermediates like
coniferyl aldehyde, ferulic acid, and vanillin (Supporting
Information Figure S3).
catalysis of biotransformation process in B. safensis
SMS1003. Since methoxyphenol derivatives like coni-
feryl aldehyde and vanillin were detected as the
major metabolites, hence, assays were performed for
previously reported enzymes of eugenol metabolic
pathway such as eugenol dehydrogenase, coniferyl
aldehyde dehydrogenase, feruloyl CoA synthetase.
Growing cells of B. safensis was found to transform
eugenol into vanillin via coniferyl aldehyde. Ferulic
acid was not detected as the intermediate in the
3.7. Biotransformation by cell free extract
Soluble cell free extract preparations were checked
for the presence of major enzymes involved in the
reaction
mixture
processed
after
eugenol

10
A. SINGH ET AL.
Figure 8. HPLC chromatograms at 254 nm and 310 nm obtained during eugenol (500 mg/L) biotransformation by resting cells of
Bacillus safensis strain SMS1003. (a) HPLC profile of control at 254 nm. (b) Overlay of HPLC profile at 310 and 254 nm obtained
using sample culture filtrate processed at 96 h of incubation. HPLC, high-performance liquid chromatography.
Figure 9. PDA spectral scan in the range of 200–400 nm of a processed culture filtrate obtained using resting cells of Bacillus
safensis SMS1003. (a) A control containing eugenol at 0 h of incubation. (b) Change in the absorbance of eugenol due to vanillin
formation at 96 h of incubation using resting cells of B. safensis. PDA, photodiode array detector.
biotransformation using growing culture of B. safen-
sis. But cell free extract incubated with eugenol has
shown both the consumption of eugenol and its bio-
transformation into coniferyl alcohol and ferulic acid
after 12–14 h. Again when the extract was incubated
with coniferyl aldehyde, its biotransformation into
vanillin was observed. Results indicated the active
presence of both eugenol dehydrogenase as well as
coniferyl aldehyde dehydrogenase in the cell free
extract. When vanillin was used as the substrate, its
major degradation product(s) such as vanillic acid,
protocatechuic acid was not detected, only change
in the absorbance of vanillin was observed as com-
pared to control. From the above results, it is evident
that enzymatic catalysis was present in the obtained
cell free extract containing a mixture of crude
enzymes involved in the pathway followed by B.
safensis SMS1003. In the present work, the whole
microbial cell was preferred for biotransformation as
it is considered as an important and efficient tool for
microbial biotransformation for large scale synthesis
as compared to enzymatic catalysis (Ruiz-Teran et al.
2001; Serra et al. 2005; Xu et al. 2007).
4. Discussion
Due to the vital role of microorganisms in the produc-
tion of natural substituted methoxyphenols like

BIOCATALYSIS AND BIOTRANSFORMATION
11
vanillin, sixteen bacterial isolates were isolated and
screened on the basis of their ability to utilize and bio-
transform eugenol as a sole carbon source. The isolate
SMS1003 which showed reasonable vanillin production
was selected for further optimization studies.
Production of several substituted methoxyphenols
from eugenol using microbial transformation has been
reported and is summarized in Table 2. In many of the
above biotransformation processes, vanillin was
detected in trace amounts and its accumulation was
followed by other intermediate byproducts. It was also
reported that bioconversion based vanillin production
is unstable, as it is utilized as a sole carbon and
energy source by some microorganisms. Due to the
toxicity of vanillin at higher concentration, soon after
accumulation, it gets oxidized to vanillic acid by the
microorganisms (Di et al. 2011). So taking into consid-
eration the ineffective conversion of eugenol into van-
illin as well as its instability and reactivity, B. safensis
SMS1003 was screened as an efficient vanillin produ-
cer from eugenol at the initial concentration of 100
mg/L. On the basis of previous reports, selected envir-
onmental and nutritional parameters were optimized
to improvise the yield (Satomi et al. 2006; Tilay et al.
2010). Eugenol concentration of 500 mg/L was found
to be the optimum substrate concentration for the
selected bacterial strain. B. safensis is a mesophilic
organism ꢀthat can grow over the temperature range
of 20–40 C and pH range of 5–9 (Satomi et al. 2006).
In the present study, an incubation temperature of 37
ꢀC and pH 7 was found optimum both for its growth
and biotransformation ability. For the optimization of
nutritional parameters, earlier studies have revealed
that supplementation of the media with co-substrate
as additional carbon and nitrogen sources helped in
augmenting the product yield (Rabenhorst 1996).
Importance of nitrogen sources like yeast extract, tryp-
tone, casein hydrolysate, beef extract, and peptone
were demonstrated so far to enhance vanillin produc-
tion (Torre et al. 2004; Tilay et al. 2010). In the present
study, only beef extract and ammonium sulphate as
additional nitrogen sources enhanced the concentra-
tion of products in the media. Further, media volume
of 10% and agitation condition of 180 rpm raised the
vanillin concentration rapidly up to 96 mg/L at 96 h of
incubation. A Similar effect was highlighted earlier on
the bioconversion ability of Escherichia coli JM109
(pBB1; Torre et al. 2004). Optimized culture conditions
Figure 10. Biotransformation kinetics of eugenol by Bacillus
safensis resting cells. Experiments were performed in 250-ml
flasks containing 25 ml of sodium phosphate buffer (pH 7.0) at
37 ꢀC for 96 h with shaking at 180 rpm. Results expressed
as mean SD.
Table 2. Biotransformation of eugenol to various methoxyphenol derivatives by microorganisms.
Microorganisms
Metabolites detected
Vanillin concentration (mg/L)
Trace amount
References
Corynebacterium sp.
Ferulic acid, vanillin, vanillic acid, pro-
tocatechuic acid, and keto-
adipic acid
Tadasa and Kayahara (1983)
Pseudomonas sp.
Pseudomonas sp.
P. putida
Coniferyl alcohol, ferulic acid, vanillin,
and vanillic acid
Coniferyl alcohol, ferulic acid, and
vanillic acid
Ferulic acid, vanillin, and
280
Washisu et al. (1993)
Rabenhorst (1996)
Trace amount
Trace amount
440
Muheim and Lerch (1999)
Priefert et al. (1999)
Svanillyl alcohol
Pseudomonas sp. HR199 (genetic-
ally modified)
Ferulic acid, coniferyl alcohol, coni-
feryl aldehyde, ferulic acid, vanillin,
and vanillic acid
Escherichia coli (genetically modified)
P. nitroreducens Jin1
Coniferyl alcohol, coniferyl aldehyde,
ferulic acid, and vanillin
Coniferyl alcohol, ferulic acid, vanillin,
and vanillic acid
4-vinyl guaiacol, vanillin, vanillic acid,
protocatechuic acid, and keto-
adipic acid
300
Overhage et al. (2003)
Unno et al. (2007)
Trace amounts
Not detected
Bacillus cereus PN24
Kadakol and Kamanavali (2010)
P. resinovorans SPR1
Bacillus sp.
Ferulic acid, coniferyl alcohol, coni-
feryl aldehyde, ferulic acid, vanillin,
and vanillic acid
240
Ashengroph et al. (2011)
Sindhwani et al. (2012)
Vanillin
0.32

12
A. SINGH ET AL.
of eugenol metabolism by the growing cultures of B.
safensis SMS1003 increased vanillin concentration but
also displayed residual amounts of coniferyl aldehyde.
Hence to achieve vanillin as the exclusive metabolite,
resting cells of B. safensis SMS1003 were prepared in
optimized culture conditions and evaluated for vanillin
production. Since the age of inoculum is considered
as a decisive factor for biotransformation, cells in the
late exponential growth phase were selected as inocu-
lum. Current work also evidenced the role of prior
substrate induction to the microbial culture enhanced
the product yield (Rivas Torres et al. 2009). Similar
experiments were performed using resting cells of
Pseudomonas resinovorans SPR1 (Ashengroph et al.
2011) and Bacillus sp. (Sindhwani et al. 2012) for
enhanced vanillin production from eugenol. The pre-
sent study proposed the efficient and stable vanillin
production of 120 mg/L (Molar yield 26%) as a single
metabolite at 96 h using the resting cells of nature
isolated B. safensis SMS1003 in optimized cul-
ture conditions.
Acknowledgment
The authors highly acknowledge Birla Institute of
Technology for the infrastructure facilities.
Disclosure statement
The authors declare that they have no conflicts of interest.
Ethical approval
This article does not contain any studies with human partici-
pants or animals performed by any of the authors.
Funding
This study was funded by the Ministry of Science and
Technology of India through its funding agency DST SERB
(Science and Engineering Research Board grant no SB/YS/LS-
308/2013).
ORCID
Kunal Mukhopadhyay
2213-8134
Shashwati Ghosh Sachan
2129-0062
http://orcid.org/0000-0003-
http://orcid.org/0000-0003-
5. Conclusion
The proposed work aimed at the isolation, screening,
and characterization of potential bacterial strain cap-
able of showing the transformation of eugenol into
valuable methoxyphenol derivative like vanillin.
Attempts were made to enhance the yield of vanillin
using controlled and optimized culture conditions.
Due to emerging market demand of biotechnological
based vanillin production using the natural raw mater-
ial, eugenol was used as a potential precursor for bio-
transformation. The prime goal of the current work
was to optimize the yield of vanillin using a selected
bacterial strain of SMS1003. The strain was identified
as B. safensis by phenotypic characterization and 16S
rRNA analysis. Our results showed that an appreciable
amount of vanillin was achieved using a nature iso-
lated novel strain of B. safensis SMS1003. In contrast
to other microbial species reported so far (as dis-
cussed above), vanillin accumulated as the major end
product without any other intermediates in the media.
By far, current biotransformation study for vanillin pro-
duction is evidenced for the first time using B. safensis
SMS1003. An attempt is being made to optimize the
feasibility for large-scale production of vanillin using B.
safensis SMS1003. Identification and characterization of
the genes through the proposed metabolic pathway
would help in the metabolic engineering of this indus-
trially important bacterium.
References
Ashengroph M, Nahvi I, Esfahani HZ, Momenbeik F. 2011.
Pseudomonas resinovorans SPR1, a newly isolated strain
with potential of transforming eugenol to vanillin and
vanillic acid. New Biotechnol.28:656–664.
Ashengroph M, Nahvi I, Zarkesh-Esfahani H, Momenbeik F.
2012. Conversion of isoeugenol to vanillin by
Psychrobacter sp. strain CSW4. Appl Biochem Biotechnol.
166:1–12.
Chen P, Yan L, Wu Z, Li S, Bai Z, Yan X, Wang N, Liang N, Li
H. 2016. A microbial transformation using Bacillus subtilis
B7-S to produce natural vanillin from ferulic acid. Sci Rep.
6:20400
Di D, Luziatelli F, Negroni A, Grazia A, Fava F, Ruzzi M. 2011.
Metabolic engineering of Pseudomonas fluorescens for the
production of vanillin from ferulic acid. J Biotechnol. 156:
309–316.
Furukawa H, Nagasawa T. 1998. Novel double bond transfer-
ring hydroxylation reactions in microbial metabolism of
eugenol. Annals NY Acad Sci. 864:81–86.
Gallage NJ, Moller BL. 2015. Vanillin-bioconversion and bio-
engineering of the most popular plant flavor and its de
novo biosynthesis in the vanilla orchid. Mol Plant. 8:
40–57.
Ghosh S, Sachan A, Sen SK, Mitra A. 2007. Microbial trans-
formation of ferulic acid to vanillic acid by Streptomyces
sannanensis MTCC 6637. J Ind Microbiol Biotechnol. 34:
131–138.

BIOCATALYSIS AND BIOTRANSFORMATION
13
Giedraityte G, Kalediene L. 2014. Biotransformation of
eugenol via protocatechuic acid by thermophilic
Geobacillus sp. AY 946034 strain. J Microbiol Biotechnol.
24:475–482.
Graf E. 1992. Antioxidant potential of ferulic acid. Free Radic
Biol Med. 13:435–448.
with a new Pseudomonas sp. Appl Microbiol Biotechnol.
46:470–474.
ꢀ
Rivas Torres B, Aliakbarian N, Torre P, Perego P, Domınguez
JM, Zilli M, Converti A. 2009. Vanillin bioproduction from
alkaline hydrolyzate of corn cob by Escherichia coli JM109/
pBB1. Enzyme Microb Tech. 44:154–158.
Ruiz-Teran F, Perez-Amador I, Lopez-Munguia A. 2001.
Enzymatic extraction and transformation of glucovanillin
to vanillin from vanilla green pods. J Agric Food Chem.
49:5207–5209.
Saitou N, Nei M. 1987. The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol
Evol. 4:406–425.
Satomi M, Myron T, Duc L, Venkateswaran K. 2006. Bacillus
safensis sp. nov., isolated from spacecraft and assembly-
facility surfaces. Int J Syst Evol Microbiol. 56:1735–1740.
Serra S, Fuganti C, Brenna E. 2005. Biocatalytic preparation
of natural flavours and fragrances. Trends Biotechnol. 23:
193–198.
Sindhwani G, Uk I, Aeri V. 2012. Microbial transformation of
eugenol to vanillin. J Microbiol Biotechnol Res. 2:313–318.
Tadasa K, Kayahara H. 1983. Initial steps of eugenol degrad-
ation pathway of a microorganism. Agric Biol Chem. 47:
2639–2640.
Tilay A, Bule M, Annapure U. 2010. Production of biovanillin
by one-step biotransformation using fungus Pycnoporous
cinnabarinus. J Agric Food Chem. 58:4401–4405.
Torre P, De Faveri D, Perego P, Ruzzi M, Barghini P, Gandolfi
R, Converti A. 2004. Bioconversion of ferulate into vanillin
by Escherichia coli strain JM109/pBB1 in an immobilized-
cell reactor. Ann Microbiol. 54:517–527.
Torre P, De Faveri D, Perego P, Converti A, Barghini P, Ruzzi
M, Faria FP. 2004. Selection of co-substrate and aeration
conditions for vanillin production by E. coli JM109/pBB1.
Food Technol Biotechnol. 42:193–196.
Holt JG, Krieg NR, Sneath PHA, Staley JT, William ST. 1994.
Bergey’s manual of determinative bacteriology. Baltimore,
MD: Williams and Wilkins.
Hua D, Ma C, Lin S, Song L, Deng Z, Maomy Z, Zhaobin Z,
Yu B, Xu P. 2007. Biotransformation of isoeugenol to vanil-
lin by a newly isolated Bacillus pumilus strain: identifica-
tion of major metabolites. J Biotechnol. 130:463–470.
Kadakol JC, Kamanavalli CM. 2010. Biodegradation of
eugenol by Bacillus cereus strain PN24. E-J Chem. 7:74–80.
Kasana RC, Sharma UK, Sharma N, Sinha AK. 2007. Isolation
and identification of a novel strain of Pseudomonas chlor-
oraphis capable of transforming isoeugenol to vanillin.
Curr Microbiol. 54:457–461.
ꢀ
Longo MA, Sanroman MA. 2006. Production of food aroma
compounds: microbial and enzymatic methodologies.
Food Technol Biotechnol. 44:335–353.
Mishra S, Sachan A, Sachan SG. 2013. Production of natural
value-added compounds: an insight into the eugenol bio-
transformation pathway. J Ind Microbiol Biotechnol. 40:
545–550.
Muheim A, Lerch K. 1999. Towards a high-yield bioconver-
sion of ferulic acid to vanillin. Appl Microbiol Biotechnol.
51:456–461.
Overhage J, Priefert H, Rabenhorst J, Steinbuchel A. 1999.
Biotransformation of eugenol to vanillin by a mutant of
Pseudomonas sp. strain HR199 constructed by disruption
of the vanillin dehydrogenase (vdh) gene. Appl Microbiol
Biotechnol. 52:820–828.
Overhage J, Steinbuchel A, Priefert H. 2003. Highly efficient
biotransformation of eugenol to ferulic acid and further
conversion to vanillin in recombinant strains of
Escherichia coli. Appl Environ Microbiol. 69:6569–6576.
Priefert H, Overhage J, Steinbuchel A. 1999. Identification
and molecular characterization of the eugenol hydroxy-
lase genes (ehyA/ehyB) of Pseudomonas sp. strain HR199.
Arch Microbiol. 172:354–363.
Priefert H, Rabenhorst J, Steinbuchel A. 2001.
Biotechnological production of vanillin. Appl Microbiol
Biotechnol. 56:296–314.
Rabenhorst J. 1996. Production of methoxyphenol-type nat-
ural aroma chemicals by biotransformation of eugenol
Unno T, Kim SJ, Kanaly RA, Ahn JH, Kang SI, Hur HG. 2007.
Metabolic characterization of newly isolated Pseudomonas
nitroreducens Jin1 growing on eugenol and isoeugenol. J
Agric Food Chem. 55:8556–8561.
Washisu Y, Tetsushi A, Hashimoto N, Kanisawa T. 1993.
Manufacture of vanillin and related compounds with
Pseudomonas. Japan patent 5,227,980.
Xu P, Hua D, Ma C. 2007. Microbial transformation of prope-
nylbenzenes for natural flavour production. Trends
Biotechnol. 25:571–576.
Zhao LQ, Sun ZH, Zheng P, Zhu LL. 2005. Biotransformation
of isoeugenol to vanillin by a novel strain of Bacillus fusi-
formis. Biotechnol Lett. 27:1505–1509.