Microbial production of 4-vinylguaiacol from ferulic acid by Bacillus cereus SAS-3006

Article abstract of DOI:10.3109/10242422.2014.974573

Ferulic acid is an abundant cinnamic acid derivative found in the plant kingdom. It is a commercially available substrate utilized to produce flavor compounds such as 4-vinylguaiacol (4-VG), vanillin, and vanillic acid. The isolate Bacillus cereus SAS-3006 was screened and selected based on its ability to produce 4-VG upon ferulic acid biotransformation. It was identified based on morphological and physiochemical characteristics and its 16S ribosomal DNA sequence (GenBank accession number: KF699134). A maximum amount (79.4 mg/L) of 4-VG accumulation was observed on the 5th day of incubation when the culture was grown on 2.5 mM ferulic acid as sole carbon source. Further conversion of 4-VG to other intermediates such as vanillin, vanillic acid, protocatechuic acid, acetovanillone, and vanillyl alcohol was not observed. In-vitro conversion of ferulic acid to 4-VG was also studied with cell extracts of B. cereus SAS-3006. The present study provides the first evidence for production of 4-VG as the sole product using B. cereus SAS-3006.

Full text of DOI:10.3109/10242422.2014.974573

Biocatalysis and Biotransformation, 2014; 32: 259–266
ORIGINAL ARTICLE
Microbial production of 4-vinylguaiacol from ferulic acid by
Bacillus cereus SAS-3006
ShaShank MiShra1, aShiSh Sachan1, aMbariSh Sharan Vidyarthi2
& ShaShwati GhoSh Sachan1
1Department of Bio-engineering, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India,
and 2Department of Biotechnology, Birla Institute of Technology, Mesra, Patna Extension center, Patna, Bihar, India,
Abstract
Ferulic acid is an abundant cinnamic acid derivative found in the plant kingdom. it is a commercially available substrate
utilized to produce flavor compounds such as 4-vinylguaiacol (4-VG), vanillin, and vanillic acid.the isolate Bacillus cereus
SaS-3006 was screened and selected based on its ability to produce 4-VG upon ferulic acid biotransformation. it was
identified based on morphological and physiochemical characteristics and its 16S ribosomal dna sequence (Genbank
accession number: kF699134). a maximum amount (79.4 mg/L) of 4-VG accumulation was observed on the 5th day of
incubation when the culture was grown on 2.5 mM ferulic acid as sole carbon source. Further conversion of 4-VG to
other intermediates such as vanillin, vanillic acid, protocatechuic acid, acetovanillone, and vanillyl alcohol was not observed.
In-vitro conversion of ferulic acid to 4-VG was also studied with cell extracts of B. cereus SaS-3006. the present study
provides the first evidence for production of 4-VG as the sole product using B. cereus SaS-3006.
Keywords: Biotransformation, ferulic acid, 4-vinylguaiacol, metabolite
Introduction
major component of lignin and could be an economic
substrate for a biotransformation process to produce
flavor compounds such as 4-vinylguaiacol (4-VG),
vanillin, and vanillic acid. 4-VG is a volatile pheno-
lic compound which has an opaque, spicy, and
clove-like aroma and is used in the food industry
(baqueiro-Peña et al. 2010). it is mainly found in
coffee, beer, wine, chocolate, whiskey, and soy sauce
(Mathew et al. 2007). it has a 40-fold higher eco-
nomic value than ferulic acid and can be further
transformed into various value-added compounds
such as apocynin (acetovanillone), ethylguaiacol,
vanillin, vanillyl alcohol, and vanillic acid (karmakar
et al. 2000; Landete et al. 2010). 4-VG is also used
in the ophthalmic field, as a contact lens solution and
eye-drop formulation (Salamone & Xia 2006).
besides its commercial value as a flavor and fra-
grance substance, 4-VG has medicinal value due to
its antioxidant activity (bortolomeazzi et al. 2007;
terpinc et al. 2011) and possible anti-cancer activity
(Jeong & Jeong 2010).
Flavor and fragrance compounds can be obtained
naturally from plant products or can be produced
chemically. although the majority of flavors and
fragrances used globally are produced by chemical
synthesis (krings & berger 1998; Serra et al. 2005;
Xu et al. 2007) at low cost, consumers prefer natural
compounds because of increasing health and nutri-
tion consciousness (abraham et al. 1994). Phenolic
compounds have antioxidant properties and protect
the gastrointestinal tract against damage by reactive
species present in foods or produced within the
stomach and intestines (halliwell 2007). the USa
and European legislation have stated that “natural fla-
vor substances could be prepared by enzymatic or microbial
process” (Li et al. 2008). hence, microorganisms are
used to produce various value-added compounds
through biotransformation. Ferulic acid is obtainable
from grasses, corn hulls (ishii 1997; oosterveld et al.
2000), wheat, maize, and rice bran (walton et al.
2003; Mariod et al. 2010), where it is present as a
correspondence: Shashwati Ghosh Sachan, department of biotechnology, birla institute of technology, Mesra, ranchi, Jharkhand – 835 215, india.
tel: 91651 2276223. Fax: 91651 2276590. E-mail: ssachan@bitmesra.ac.in
(Received 19 March 2014; revised 1 September 2014; accepted 4 October 2014)
iSSn 1024-2422 print/iSSn 1029-2446 online © 2014 informa Uk, Ltd.
doi: 10.3109/10242422.2014.974573

260 S. Mishra et al.
a wide variety of aromatic compounds can be
produced using ferulic acid metabolism by four major
pathways, namely i) non-oxidative decarboxylation,
ii) side chain reduction, iii) coenzyme-a-dependent
deacetylation, and iV) coenzyme-a-independent
deacetylation. of these, 4-VG is obtained by non-
oxidative decarboxylation of ferulic acid (rosazza
et al. 1995; Lee et al. 1998; donaghy et al. 1999).
the bioconversion of ferulic acid to 4-VG has been
studied using a variety of microorganisms, such as
Bacillus coagulans, Debaryomyces hansenii, white rot
fungus, Aspergillus niger, Lactobacillus farciminis,
Streptomyces setonii, Enterobacter soli, and E. aerogenes
(karmakar et al. 2000; Mathew et al. 2007; Mabinya
et al. 2010; baqueiro-Peña et al. 2010; adamu et al.
2012; Max et al. 2012; hunter et al. 2012). to the
best of our knowledge, there is no report on the
biotransformation of ferulic acid into 4-VG using
B. cereus strains. in this paper, we report the produc-
tion of 4-VG as sole metabolic product in the medium
with no other by-products.
0.20-mm nylon filter (Sartorius, Minisart) before
their addition to minimal medium.
Seed culture preparation and screening of
biotransformation ability
to screen bacterial isolates with the potential for
converting ferulic acid, cells of individual isolates
were inoculated into 25 mL of nutrient broth and
grown for 24 h at 37°c for seed culture preparation.
Growth was measured as optical density at 600 nm.
to check the biotransformation ability of isolate,
1 mL of homogenous seed culture of individual iso-
lates was transferred to 100-mL flask containing
25 mL of sterile minimal medium supplemented
with 1.0 mM ferulic acid as the sole carbon and
energy source. Simultaneously, a control experiment
was carried out by adding 1.0 mM ferulic acid in the
same medium without cultures. Samples were with-
drawn at intervals of 2, 4, and 6 days and were ana-
lyzed by thin-layer chromatography (tLc) and
UV–Vis spectrophotometry (Lambda 25, Perkin
Elmer, USa). the culture with the highest produc-
tion of 4-VG was selected for further study.
Materials and methods
Chemicals and reagents
Analytical procedures
Ferulic acid (99%) and 4-VG (99%) used in this
experiment were purchased from Sigma-aldrich
(Gmbh, Uk). high-performance liquid chromatog-
raphy (hPLc)-grade methanol and formic acid were
purchased from himedia (india). other chemicals
were of analytical grade.
culture supernatant was acidified (ph, 1–2) and
extracted with an equal volume of ethyl acetate.the
aqueous phase was discarded and organic phase
evaporated by rotary vacuum evaporation under
reduced pressure. the extract was resuspended in 1
mL of methanol (50% v/v), and spotted on to atLc
plate.tLc analysis was performed using 2% aqueous
formic acid as solvent system (dey et al. 2003). 4-VG
was directly visualized under a dual-wavelength
Bacterial isolation and culture medium
For isolation of bacteria, the serial dilution method
was used. a soil sample was collected from a har-
vested paddy field at Mesra, ranchi, approximately
5.0 cm below the soil surface. the soil suspension
was plated on minimal medium supplemented with
ferulic acid as the sole carbon source. this was fol-
lowed by 24–72 h of incubation at 37°c and the
colonies obtained were further purified by quadrant
streaking on plates of the same medium. a single
colony thus obtained was maintained on minimal
medium slants supplemented with ferulic acid as the
sole carbon source at 4°c for future work. Minimal
medium was prepared by the addition of basal inor-
ganic salts, ammonium nitrate (3.0 g/L) as a nitrogen
source, sodium chloride (0.2 g/L), hydrated magne-
sium sulfate (0.2 g/L), potassium dihydrogen phos-
phate (1.0 g/L), disodium hydrogen phosphate (1.0
g/L), and calcium chloride (0.05 g/L) (Muheim &
Lerch 1999). the ph was adjusted to 7.0. all the
carbon sources were sterilized and passed through a
table i. Morphological and biochemical characteristics of isolated
strain B. cereus SaS-3006.
Characteristics
Description
Shape
rod
Gram reaction
catalase
oxidase
Endospore forming
Motility
citrate utilization
carbohydrate Fermentation
Glucose
Gram-positive
Positive
Positive
Positive
Positive
negative
Positive/no gas
Positive/no gas
negative
Sucrose
Lactose
h2S production
Gelatin liquefaction
Methyl red test
Voges–Proskauer test
Starch hydrolysis
negative
Positive
negative
Positive
Positive

4-vinylguaiacol from ferulic acid by bacillus cereus SAS-3006 261
Figure 1. Phylogenetic tree of SaS-3006 as constructed using MEGa 5.05 software by the neighbor-joining method. numbers at nodes
indicate levels of bootstrap values (%) based on the neighbor-joining analysis of 1000 replicates; only values50% are given.
(254/310 nm) UV lamp (Uvitec Uk).thetLc chro-
matogramcorrespondingtothestandardwasdetected
on the plate and confirmed by overlay of standards
on hPLc (waters, USa). the hPLc conditions
were set as follows: column, Symmetry® c18
(4.6150 mm, 5 mm, waters); mobile phase, 68%
aqueous trifluoroacetic acid (solvent a) and 32%
methanol (solvent b); flow rate, 1 mL/min; injection
volume, 20 mL; and detection wavelength, 254 nm
(Photodiode array detector-2996, waters). analysis
of the sample was performed using the following gra-
dient: 0–15 min in 68% a and 32% b, and 15–30 min
Figure 2. (a) tLc analysis showing separation of standards and processed culture supernatant of B. cereus SaS-3006. Lane 1: Ferulic
acid; lane 2: 4–VG; lanes 3 and 4: processed culture filtrate of B. cereus SaS-3006 incubated for 3 and 5 days, respectively. (b) Stack plot
of hPLc chromatogram at 254 nm showing ferulic acid degradation by processed culture filtrate of B. cereus SaS-3006. chromatogram
[a] represents 4-VG (1), ferulic acid (2), acetovanillone (3), vanillin (4), vanillic acid (5), and protocatechuic acid (6) as standards.
chromatogram [b] represents conversion of ferulic acid (2) to 4-VG (1) on 3rd day of incubation and [c] represents decrease in the
amount of ferulic acid (2) with an increase in formation of 4-VG (1) as the sole metabolite on 5th day of incubation.

262 S. Mishra et al.
35
glucose). to study the effect of each parameter on
product formation, samples were withdrawn at fixed
intervals and analyzed using tLc, UV–Vis spectro-
photometry, and hPLc. analyses were performed
in triplicate and repeated at least twice.
Supp with FA (1 mM)
Supp with FA (2.5 mM)
30
25
20
15
10
5
Degradation of 4-VG
the metabolite (4-VG) previously detected in the
media of ferulate-grown B. cereus SaS-3006 was inde-
pendently supplied to the medium as the sole carbon
source. analyses were carried out after 3 and 5 days of
incubation for the detection of product using hPLc.
0
2
4
6
Incubation time (days)
Cell extract preparation
Figure 3. time course of 4-VG accumulation in the culture media
of B. cereus SaS-3006 supplemented with 1.0 and 2.5 mM ferulic
acid as the sole carbon source after 2, 4, and 6 days of incubation.
cells grown on ferulic acid as substrate were har-
vested during the mid-exponential phase of growth
by centrifugation at 10,000 rpm for 20 min in a
refrigerated centrifuge (5804 r, Eppendorf, ham-
burg, Germany).the supernatant was removed and
cell pellet washed twice in 50 mM cold sodium
phosphate buffer (ph, 6.0), and then resuspended
in the same buffer containing 5 mM dithiothreitol.
all procedures were carried out at 4°c. the cell
suspension was sonicated in a VcX 750 sonicator
(Sonics and Materials inc., USa) with a titanium
alloy probe having a tip diameter of 13 mm and
operating at an amplitude of 35% with 15000 joule
energy. Sonication was applied in short bursts of
20 s with a total exposure time of 6 min. after
sonication, the cell extract was again centrifuged
and the resultant supernatant was collected and
concentrated using an amicon Ultra-15 centrifu-
gal Filter (Millipore, USa) membrane. this con-
centrated eluate was used as a crude extract for
in-vitro assay of 4-VG.
in 40% a and 60% b, followed by equilibration time
of 10 min using a binary hPLc Pump (waters). com-
pounds were identified by comparison of retention time
and peak with the standards (1 mg/mL). 4-VG was
further quantified by absorbance at 260 nm. Measure-
ments of concentrations were read from the standard
curve in the range of 1–10 mg/mL.
Identification and characterization of selected isolate
the isolate giving the highest 4-VG yield was
selected. Preliminary identification of the isolate was
done by morphological and biochemical character-
izations (breed et al. 1957). Species identification of
the selected bacterial isolate was confirmed on the
basis of 16S ribosomal rna (rrna) gene sequenc-
ing (Xcelris Labs Ltd. ahmedabad, india).the con-
sensus sequence was aligned with the sequence of
the type strain using clustalw (thompson et al.
1997). the homology of the sequences was exam-
ined using bLaSt of national center for biotech-
nology information (ncbi).Molecular Evolutionary
Genetics analysis (MEGa) software version 5 (hall
2013) was used to construct phylogenetic trees using
the neighbor-joining method (Saitou & nei 1987).
495
450
405
360
315
270
225
180
135
90
90
80
70
60
50
40
30
20
10
0
Conc. of 4-VG
Conc. of FA
Optimization of various cultural parameters
for maximum 4-VG production
45
the various cultural parameters considered for
maximum 4-VG production were ferulic acid con-
centration (1.0 and 2.5 mM), incubation tempera-
ture (37 and 28°c), ph (6–8), organic nitrogen
source (0.05% w/v yeast extract), agitation (120
rpm), and additional carbon source (0.1% w/v
0
0
1
2
3
4
5
6
7
8
9
Incubation time (days)
Figure 4. degradation of ferulic acid with formation of 4-VG by
B. cereus SaS-3006 at 37°c, after 1–8 days of incubation and
agitation of 120 rpm.

4-vinylguaiacol from ferulic acid by bacillus cereus SAS-3006 263
Figure 5. Stack plot of hPLc eluates measured at 254 nm showing potential 4-VG degradation products in processed culture filtrate of
B. cereus SaS-3006. chromatogram [a] represents 4-VG (1), ferulic acid (2), acetovanillone (3), vanillin (4), vanillic acid (5), and
protocatechuic acid (6) as standards. chromatogram [b] represents 4-VG (1) used as the sole carbon source on 3rd day of incubation
and [c] shows the decrease in the amount of 4-VG (1) without any product formation by the 5th day of incubation.
In-vitro conversion of ferulic acid
score, sequences were selected and aligned using the
multiple alignment software clustalw. a phyloge-
netic tree was constructed using MEGa 5 software
(Figure 1). analysis of the 16S rdna sequence indi-
cated that isolate SaS-3006 showed the closest sim-
ilarity to B. cereus tJ (JX506728, 99% identity
according to bLaSt, ncbi). the 16S rdna
sequence of SaS-3006 was submitted to dna data
bank of Japan (ddbJ)/European Molecular biology
Laboratory (EMbL)/Genbank under accession
number, kF699134 (http://www.ncbi.nlm.nih.gov/
nuccore/kF699134).
the ability of a cell extract to convert ferulic acid
into 4-VG was examined.the assay mixture (1 mL)
consisted of 50 mM sodium phosphate buffer, ph:
6.0, 5.0 mg/mL of ferulic acid, and 100 mL of cell
extract (degrassi et al. 1995). the sample was then
analyzed for ferulic acid decarboxylase activity using
UV–Vis spectrophotometry for product detection,
by incubating the mixture at 37°c for 120 min.
wavelength ranges were set between 200 and 400
nm to confirm the optimal absorption wavelength.
In-vitro degradation of 4-VG
Transformation of ferulic acid by B. cereus SAS-3006
analyses were carried out in the same way as
described above except that ferulic acid was replaced
with 5.0 mg/mL of 4-VG (degrassi et al. 1995).
in order to examine the ability of B. cereus SaS-3006
to biotransform ferulic acid into 4-VG, a cell suspen-
sion (4% v/v) of B. cereus SaS-3006 was inoculated
into minimal medium containing ferulic acid (1.0
mM) as the sole carbon source.the culture was incu-
bated at 37°c for a maximum period of 6 days. cul-
ture media were processed and analyzed by tLc to
detect any formation of metabolite(s) (Figure 2a).the
presence of 4-VG as the sole product was further con-
firmed by hPLc (Figure 2b). the product obtained
was quantified using UV–Vis spectrophotometry.
Results and discussion
Identification of the bacterial isolate
based on their morphology, 17 different bacterial
isolates were obtained and further checked for their
ability to produce 4-VG. among the cultures
obtained, SaS-3006 was the only one which pro-
duced 4-VG. Morphological and biochemical char-
acterizations were used for preliminary identification
of this microorganism as given in table i (supple-
mentary file). according to these preliminary inves-
tigations, the isolate was identified as B. cereus. For
further confirmation, sequencing of the 16S rrna
gene was executed (Xcelris Labs Ltd. ahmedabad,
india) and the consensus sequence was used for
similarity search using bLaSt algorithm with ncbi
Genbank nrdatabase. based on a maximum identity
Standardization of culture parameters for enhanced
4-VG production
a culture of B. cereus SaS-3006 was inoculated in
minimal medium containing different concentra-
tions of ferulic acid (1.0, 2.5, and 5.0 mM) as the
sole carbon and energy source. Using 2.5 mM ferulic
acid as sole carbon source, a maximum amount of
32.9 mg/L or 0.219 mM 4-VG was observed in the

264 S. Mishra et al.
1.10
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0 min
30 min
60 min
90 min
120 min
0.00
200.0
220
240
260
280
300
320
340
360
380
400.0
Wavelength (nm)
Figure 6. depletion of ferulic acid in the reaction mixture containing crude cell extract of B. cereus SaS-3006 incubated for
0–120 min.
medium on 6th day of incubation (Figure 3). when
5.0 mM ferulic acid was provided, very little utiliza-
tion of the substrate was observed (data not shown).
consumption of ferulic acid (2.5 mM) was also
monitored showing that the substrate was completely
consumed after 4 days of incubation. it was also
observed that a medium ph of 7.0 and incubation
at 37°c favored the formation of 4-VG. Further-
more, yeast extract 0.05% (w/v) was added to the
minimal medium and supplemented with 2.5 mM
ferulic acid at 37°c and agitation of 120 rpm.there
was a significant increase in the amount of 4-VG
(79.4 mg/L) obtained on the 5th day of incubation.
consumption of ferulic acid was also examined and
it was observed that the ferulic acid was fully utilized
after the 4th day of incubation (Figure 4). in order
to obtain a high-density culture of B. cereus SaS-
3006, the microorganism was allowed to grow in
minimal media supplemented with glucose (0.1%
w/v) as the sole carbon source. it has previously been
reported that this approach increased the biomass
which reduced the time period of product formation
(oddou et al. 1999). when almost all the carbon
source was utilized by the microorganism for its
growth, ferulic acid (2.5 mM) was added into the
minimal medium. however, the use of an additional
carbon source did not show a significant increase in
product formation (data not shown).
the medium as the sole carbon source. this experi-
ment was performed according to the strategy
approved by Estrada alvarado et al. (2001). the
metabolite previously detected in the ferulate-grown
B. cereus SaS-3006 served as an independent sub-
strate in the medium. analyses were carried out after
3 and 5 days of incubation for the detection of prod-
uct. in previous research it was observed that 4-VG
accumulated as an intermediate, which was then fur-
ther degraded to intermediates such as vanillin, van-
illic acid, and protocatechuic acid (karmakar et al.
2000); vanillin, vanillic acid, and acetovanillone
(Mabinya et al. 2010); vanillin, vanillyl alcohol, and
vanillic acid (baqueiro-Peña et al. 2010; hunter
et al. 2012); and protocatechuic acid, vanillyl alco-
hol, vanillic acid, and vanillin (Max et al. 2012).
these metabolites resulted from side chain cleavage
of ferulic acid (c2-cleavage) or cleavage of 4-VG
(c1-cleavage) in the culture medium. when 4-VG
(2.5 mM) was used as the sole carbon source with
B. cereus SaS-3006, no degradation products were
detected.this observation suggests that 4-VG is the
sole metabolite formed upon consumption of ferulic
acid in the medium and does not undergo further
degradation to other intermediates (Figure 5). this
is only further utilized as the sole carbon source by
B. cereus SaS-3006 when ferulic acid has been com-
pletely utilized.
Catabolic route of ferulic acid degradation
Biotransformation of ferulic acid by cell extract
in order to trace the catabolic pathway of ferulic acid
degradation, 4-VG was independently supplied to
Ferulic acid decarboxylase was produced by
B. cereus SaS-3006 as demonstrated by the enzymatic

4-vinylguaiacol from ferulic acid by bacillus cereus SAS-3006 265
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shift in the peak maxima. the transformation of fer-
ulic acid (lmax 289, 318 nm) to a product with
absorption maxima at 210, 260 nm could be attrib-
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Acknowledgement
this work is financially supported by University
Grants commission for the Major research Project
[F.no. 37-115/2009 (Sr)]. Shashank Mishra grate-
fully acknowledges the centre of Excellence and
department of biotechnology, birla institute of
technology, Mesra, ranchi, for providing the infra-
structure facilities. the authors acknowledge dr.
Sanjaya Swain, research officer, central instru-
mentation Facility, bit, Mesra for providing ana-
lytical facilities.
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Declaration of interest:the authors report no dec-
larations of interest. the authors alone are responsi-
ble for the content and writing of the paper.
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