Synthesis of hydroxytyrosol, 2-hydroxyphenylacetic acid, and 3-hydroxyphenylacetic acid by differential conversion of tyrosol isomers using Serratia marcescens strain

Article abstract of DOI:10.1021/jf050972w

We investigated to develop an effective procedure to produce the potentially high-added-value phenolic compounds through bioconversion of tyrosol isomers. A soil bacterium, designated Serratia marcescens strain, was isolated on the basis of its ability to grow on p-tyrosol (4-hydroxyphenylethanol) as a sole source of carbon and energy. During growth on p-tyrosol, Ser. marcescens strain was capable of promoting the formation of hydroxytyrosol. To achieve maximal hydroxytyrosol yield, the growth state of the culture utilized for p-tyrosol conversion as well as the amount of p-tyrosol that was treated were optimized. The optimal yield of hydroxytyrosol (80%) was obtained by Ser. marcescens growing cells after a 7-h incubation using 2 g/L of p-tyrosol added at the end of the exponential phase to a culture pregrown on 1 g/L of p-tyrosol. Furthermore, the substrate specificity of the developed biosynthesis was investigated using m-tyrosol (3-hydroxyphenylethanol) and o-tyrosol (2-hydroxyphenylethanol) as substrates. Ser. marcescens strain transformed completely m-tyrosol and o-tyrosol into 3-hydroxyphenylacetic acid and 2-hydroxyphenylacetic acid, respectively, via the oxidation of the side chain carbon of the treated substrates. This proposed procedure is an alternative approach to obtain hydroxytyrosol, 2-hydroxyphenylacetic acid, and 3-hydroxyphenylacetic acid in an environmentally friendly way which could encourage their use as alternatives in the search for replacement of synthetic food additives.

Full text of DOI:10.1021/jf050972w

J. Agric. Food Chem. 2005, 53, 6525−6530 6525
Synthesis of Hydroxytyrosol, 2-Hydroxyphenylacetic Acid, and
3-Hydroxyphenylacetic Acid by Differential Conversion of
Tyrosol Isomers Using Serratia marcescens Strain
NOUREDDINE ALLOUCHE AND SAMI SAYADI*
Laboratoire des Bio-proce´de´s, Centre de Biotechnologie de Sfax: B.P “K”. 3038, Sfax, Tunisia
We investigated to develop an effective procedure to produce the potentially high-added-value phenolic
compounds through bioconversion of tyrosol isomers. A soil bacterium, designated Serratia
marcescens strain, was isolated on the basis of its ability to grow on p-tyrosol (4-hydroxyphenylethanol)
as a sole source of carbon and energy. During growth on p-tyrosol, Ser. marcescens strain was
capable of promoting the formation of hydroxytyrosol. To achieve maximal hydroxytyrosol yield, the
growth state of the culture utilized for p-tyrosol conversion as well as the amount of p-tyrosol that
was treated were optimized. The optimal yield of hydroxytyrosol (80%) was obtained by Ser.
marcescens growing cells after a 7-h incubation using 2 g/L of p-tyrosol added at the end of the
exponential phase to a culture pregrown on 1 g/L of p-tyrosol. Furthermore, the substrate specificity
of the developed biosynthesis was investigated using m-tyrosol (3-hydroxyphenylethanol) and o-tyrosol
(2-hydroxyphenylethanol) as substrates. Ser. marcescens strain transformed completely m-tyrosol
and o-tyrosol into 3-hydroxyphenylacetic acid and 2-hydroxyphenylacetic acid, respectively, via the
oxidation of the side chain carbon of the treated substrates. This proposed procedure is an alternative
approach to obtain hydroxytyrosol, 2-hydroxyphenylacetic acid, and 3-hydroxyphenylacetic acid in
an environmentally friendly way which could encourage their use as alternatives in the search for
replacement of synthetic food additives.
KEYWORDS: Tyrosol, bioconversion, hydroxytyrosol, antioxidant, 3-hydroxyphenylacetic acid, 2-hy-
droxyphenylacetic acid, Serratia marcescens
INTRODUCTION
allow its potential application in food, as well as in the cosmetic
and pharmaceutical industries (13). Recent works demonstrated
that the bioavailability of hydroxytyrosol makes it a beneficial
addition to the diet (14, 15). Several investigations developed
various methods to produce hydroxytyrosol by means of
chemical synthesis (16), through conversion of oleuropein (17,
18), by enzymatic synthesis using tyrosinase as biocatalyst (19),
and by bench-scale purification from olive mill wastewaters (20,
21). Recently, we developed a continuous procedure for the
extraction and the purification of hydroxytyrosol from olive mill
wastewaters (22) and a microbial conversion procedure of
p-tyrosol into hydroxytyrosol using Pseudomonas aeruginosa
(23).
Hydroxylation of aromatic rings is a fundamental reaction
used for the preparation of several active compounds. Compared
with chemical production, hydroxylation of aromatic compounds
by microorganisms is an interesting and promising method to
synthesize the desired products in a single-step reaction, with a
high regioselectivity and a stereoselectivity and under mild
conditions (1). Furthermore, the products of such bioconversions
are considered natural, since the European Community legisla-
tion includes products which occur in nature and that are
produced by living cells or enzymes using starting materials
from a natural source under the term “natural products” (2).
Hydroxylation of aromatic rings is a fundamental reaction
used for the synthesis of several high-added-value compounds,
including ortho-diphenols. Hydroxytyrosol (3,4-dihydroxyphen-
ylethanol) is the most abundant ortho-diphenol compound
occurring in olive oil (3) as well as in olive mill wastewaters
(4, 5). Several investigations reported that hydroxytyrosol is
characterized by its highly antioxidant property and presents
several interesting aspects for human health (6-12) that would
Besides hydroxytyrosol, 2-hydroxyphenylacetic acid and
3-hydroxyphenylacetic acid stand out as aromatic compounds
of a high-added-value. These two products are frequently used
in the pharmaceutical industry where they are used as intermedi-
ates in the preparation of some biologically active products such
as antihypertensive agents (24). In a recent investigation, it was
demonstrated that 3-hydroxyphenylacetic acid would be a
starting material for the synthesis of three drugs which have
relatively high affinity at gamma-hydroxybutyric acid (GHB)
sites. (GHB) is a therapeutic and a neurotransmitter with a
* Author to whom correspondence should be addressed. Tel/fax: +216-
74-440 452; e-mail: sami.sayadi@cbs.rnrt.tn.
10.1021/jf050972w CCC: $30.25
© 2005 American Chemical Society
Published on Web 07/14/2005

6526 J. Agric. Food Chem., Vol. 53, No. 16, 2005
Allouche and Sayadi
(34). A 50-µL reaction mixture contained 50 mg of genomic DNA, 1
µL of each primer, 5 µL of 10× buffer, 200 µL of deoxynucleoside
triphosphate, 3.5 mM of MgCl2, and 2.5 U of Taq polymerase
(Promega). PCR was carried out by an initial denaturation at 94 °C for
1 min followed by cycles of annealing at 55 °C for 30 s, extension at
72 °C for 45 s (depending on the lengths of the amplified fragments),
and denaturation at 94 °C for 30 s, followed by cycles of annealing at
55 °C for 30 s, extension at 72 °C for 45 s, and finally an extension
cycle of 72 °C for 10 min.
HPLC and TLC Analyses. Aromatic compound identification and
quantification were carried out by HPLC analysis as described by
Akasbi et al. (35). It was performed on a Shimadzu C-R6A liquid
chromatograph. The separation was carried out in a C18 column (length,
250 mm; internal diameter, 4.6 mm; Waters chromatography). The
compounds were eluted with a gradient, acetonitrile (70%)-H3PO4
(0.1%), in which the concentration of acetonitrile varied as follows: 0
min, 10%; 0-20 min, increase to 50%; 20-25 min, 50%; 25-30 min,
decrease to 10%. The column temperature was maintained at 40 °C
and the flow rate was 0.5 mL/min. Sample detection was achieved at
280 nm with a Shimadzu SPD-6AUV detector connected to a Shimadzu
C-R6A integrator. The injection volume was 20 µL.
Samples (1 mL) were withdrawn periodically from the culture
medium and centrifuged at 7000g (10 min). The supernatant was
analyzed directly by HPLC for substrate and intermediary-metabolite
quantification. The compounds were identified and quantified by
comparison of retention times and peak areas with those of authentic
samples. The molar yields of produced compounds were calculated as
follows: molar yield of produced compound ) molar concentration
of produced compound (mol/L)/initial molar concentration of tyrosol
(mol/L) × 100.
complex mechanism of action in vivo (25). 2-Hydroxyphenyl-
acetic acid and 3-hydroxyphenylacetic acid were chemically
synthesized and commercially available products. 2-Hydroxy-
phenylacetic acid is a natural phenolic product found in the
genus Astilbe and it derives from the shikimic acid pathway
via phenylpyruvic acid (26). 3-Hydroxyphenylacetic acid is a
major phenolic acid degradation product of proanthocyanidin
metabolism in humans (27). These two phenolic compounds
are synthesized by cultivating a fungus in the presence of
phenylacetic acid (24, 28).
The present work deals with the development of simple and
highly convenient microbial transformations for the synthesis
of hydroxytyrosol, 2-hydroxyphenylacetic acid, and 3-hydroxy-
phenylacetic acid using, respectively, p-tyrosol (4-hydroxyphen-
ylethanol), m-tyrosol (3-hydroxyphenylethanol), and o-tyrosol
(2-hydroxyphenylethanol) as precursors. These substrates natu-
rally occur in olive mill wastewaters (29). They can also be
obtained from olive cake (30), olive stones (31), white wine
(32), and beer (33). The developed biosynthesis methods could
prove useful for laboratory applications as well as for a possible
industrial exploitation.
MATERIALS AND METHODS
Chemicals. p-Tyrosol (4-hydroxyphenylethanol) and its isomers
m-tyrosol (3-hydroxyphenylethanol) and o-tyrosol (2-hydroxyphenyl-
ethanol) were purchased by Fluka (France). Hydroxytyrosol, used as
standard, was purfied from olive mill wastewaters as described by
Allouche et al. (22).
The thin-layer chromatography (TLC) was monitored using silica
gel sheets (Kieselgel 60 F254; 0.2 mm layers; Merck) eluted with toluene-
ethyl acetate-acetic acid (7:2:1, V/V/V). The constituents were visual-
ized by exposure to UV light and were revealed by resublimed iodine
fumes.
GC-MS Analysis. GC-MS was performed with a Hewlett-Packard
model 5872A chromatograph apparatus, equipped with a capillary
HP5MS column (length, 30 m; internal diameter, 0.32 mm; film
thickness, 0.32 µm). The carrier gas was He, used at a 1.7 mL/min
flow rate. The oven temperature program was as follows: 1 min at
100 °C, from 100 to 260 °C at 4 °C/min, and 10 min at 260 °C. A
sample from the culture medium (1 mL) was extracted with ethyl
acetate, and 100 µL of bis-(trimethylsilyl)-acetamide was added to 100
µL of the organic extract. The obtained solution was incubated 30 min
at 60 °C. Ethyl acetate and bis-(trimethylsilyl)-acetamide were evapo-
rated under a N2 current, and the residue was redissolved in ethyl acetate
(1 mL) and was analyzed by GC-MS.
Bacterial Strain Isolation. For bacterial isolation, an enrichment
culture method was used. In this experimentation, soil (1 g), regularly
irrigated with olive mill wastewaters (100 m3/ha) for 5 years, was mixed
with 100 mL minimal medium containing 0.1 g/L of p-tyrosol and 1
g/L of glucose both as carbon sources. The obtained culture was
incubated overnight in an orbital shaker at 180 rpm and 30 °C. Each
day, an aliquot (5 mL) from the incubated culture was used to inoculate
a fresh medium of 100 mL in which the concentration of p-tyrosol
was increased (0.1 g/L was added) while decreasing the glucose
concentration. This procedure was repeated daily until the p-tyrosol
concentration reached 1 g/L and the glucose was completely removed
from the culture medium. Once p-tyrosol was completely metabolized,
an aliquot (5 mL) from the last incubated culture was used to inoculate
another fresh medium containing 1 g/L of p-tyrosol. This step was
repeated four times. The disappearance of p-tyrosol was confirmed by
thin-layer chromatography (TLC) analysis. Aliquots (0.1 mL) of 10-2
to 10-8 dilutions from the last acclimated culture were plated onto
p-tyrosol (1 g/L) minimal agar medium and were incubated overnight
at 30 °C. Single colonies were picked and used for screening.
Culture Conditions. Bacteria were grown in a minimal medium
containing (g/L): Na2HPO4, 2.44; KH2PO4, 1.52; (NH4)2SO4, 1.5;
MgSO4‚7H2O, 0.2; and CaCl2‚H2O, 0.05 and 10 mL of trace-element
solution which contains (% W/V): EDTA, 5; ZnSO4‚7H2O, 2.2; CaCl2,
0.55; MnCl2‚5H2O, 0.5; (NH4)6Mo7O24‚4H2O, 0.11; CuSO4‚5H2O, 0.16;
CoCl2‚6H2O, 0.16. The pH of the medium was adjusted to 7.2. The
carbon substrates were filter sterilized. Solid media were prepared by
the addition of 1.5% agar. Cultures (25 mL) were inoculated with 107
cells pregrown in Luria-Bertani broth and were incubated in an orbital
shaker at 180 rpm and 30 °C. The growth rate was followed by
measuring the changes in turbidity at 600 nm using a Shimadzu UV-
visible-light spectrophotometer (UV-160A). All cultures used for
studying the bioconversion of tyrosol isomers by the selected strain
were undertaken in triplicate. Three samples were withdrawn at the
same time from each culture for analysis, and standard deviation (SD)
is indicated in each figure.
RESULTS
Isolation of p-Tyrosol Degrading Strain. To isolate different
p-tyrosol-tolerant microorganisms, an enrichment culture method
was conducted according to the protocol described in the
Materials and Methods section. This enrichment culture was
designed to select strains able to grow on p-tyrosol as sole
carbon source and energy. The substrate degradation was
followed by TLC and HPLC analysis. Among several p-tyrosol
tolerant strains, one microorganism was isolated for further
studies on the basis of its capacity to produce hydroxytyrosol
during p-tyrosol degradation. This strain was able to grow on
high concentration of p-tyrosol (up to 4 g/L). However, it was
unable to metabolize either m-tyrosol or o-tyrosol (data not
shown). By means of the 16S-rDNA sequence analysis, the
isolated strain was identified as Serratia marcescens.
Optimization of Conditions for Hydroxytyrosol Produc-
tion by Ser. marcescens Growing Cells. To achieve maximal
yield of hydroxytyrosol production by growing cells of Ser.
marcescens strain, two parameters were optimized. For this
purpose, the relationship between hydroxytyrosol production and
DNA Extraction and PCR Amplification. Total DNA was isolated
from the isolated strain by using the alkaline-lysis method with certain
modifications. The universal primers Fd1 and Rd1 were used to obtain
a PCR product of approximately 1.5 Kb corresponding to base positions
8-1542 on the basis of Escherichia coli numbering of the 16S-DNA

Differential Conversion of Tyrosol Isomers
J. Agric. Food Chem., Vol. 53, No. 16, 2005 6527
Figure 2. Time course of Ser. marcescens strain growth in minimal
medium containing 2 g/L p-tyrosol. Optical density at 600 nm (
4), p-tyrosol
Figure 1. Time course of hydroxytyrosol formation by growing cells of
Ser. marcescens strain with increasing concentrations of p-tyrosol. 1 g/L
([), 1.5 g/L (0), 2 g/L (2), 2.5 g/L (4), 3 g/L (9), 4 g/L (]). Each point
represents the mean of three determinations and three independent
experiments (SD < 15% of the mean).
(
9
), hydroxytyrosol ( ), aromatic compounds ( ). Each point represents
0
2
the mean of three determinations and three independent experiments (SD
< 12% of the mean).
Table 1. Abbreviated Mass Spectra of the Bioconversion Products of
p-Tyrosol, o-Tyrosol, and m-Tyrosol by Ser. marcescens Strain
(i) the growth state of the used culture and (ii) the amount of
treated p-tyrosol was examined.
mass spectra
TMS derivatives of
hydroxytyrosol
(m/z and % of the base peak)
Three Ser. marcescens cultures were prepared in minimal
medium on p-tyrosol (1 g/L). These batch cultures were then
supplemented with p-tyrosol (1 g/L) at different growth states:
at the middle of the exponential phase (cell concentration )
0.6 g/L) for the first culture, at the end of the exponential phase
(cell concentration ) 1.14 g/L) for the second culture, and at
the stationary phase (cell concentration ) 1.24 g/L) for the third
culture. These cultures were then incubated aerobically, in an
orbital shaker at 180 rpm and 30 °C.
+
370 (M , 39); 267 (90); 193 (25);
179 (12); 73 (100)
+
2-hydroxyphenylacetic acid
3-hydroxyphenylacetic acid
296 (M , 18); 281 (12); 253 (25);
164 (20); 147 (40);73 (100)
+
296 (M , 22); 281 (20); 253 (10);
164 (35);147 (25);73 (100)
scens strain was assessed by the addition of p-tyrosol (2 g/L)
to a culture previously grown on p-tyrosol (1 g/L) until the end
of the exponential phase. The bioconversion was monitored by
HPLC analyzing samples withdrawn from the culture medium
at different times. Figure 2 shows the time courses of p-tyrosol
depletion and transient accumulation of hydroxytyrosol in the
medium during the growth of Ser. marcescens strain. During
the first 4 h of incubation, OD600nm remained constant (Figure
2). This lag-phase period could be explained by the toxicity of
p-tyrosol toward the isolated strain. During this period, Ser.
marcescens transformed 60% of p-tyrosol into hydroxytyrosol
without aromatic compound consumption. After this lag phase,
hydroxytyrosol concentration increased in the medium, but
degradation of aromatic compounds started and consequently
an increase of the OD600nm was observed. After 7 h, hydroxy-
tyrosol degradation started, and it was completely removed from
the culture after 15 h. Maximal hydroxytyrosol concentration
(1.78 g/L), resulting in a molar yield of 80%, was achieved at
7 h. The identification of hydroxytyrosol in the culture medium
was achieved according to the comparison of the obtained
GC-MS spectra with GC-MS apparatus library (Table 1).
Moreover, the obtained mass fragments agreed with those
described by Capasso (36).
Conversion of m-Tyrosol and o-Tyrosol by Ser. marce-
scens. To study the substrate specificity of the hydroxylation
reaction by Ser. marcescens strain, the bioconversions of
m-tyrosol (3-hydroxyphenylethanol) and o-tyrosol (2-hydroxy-
phenylethanol) were investigated. For this aim, 1 g/L of each
compound was added to the corresponding culture pregrown
on p-tyrosol (1 g/L). It should be stressed that Ser. marcescens
strain did not grow on m-tyrosol and on o-tyrosol (data not
shown). Using GC-MS analysis (Table 1), it was deduced that
m-tyrosol and o-tyrosol were converted by Ser. marcescens into
The depletion of p-tyrosol and the increase of hydroxytyrosol
concentration in each medium were monitored by frequent
HPLC sample analysis. The obtained results showed that the
maximum level of hydroxytyrosol (0.5 g/L) was accumulated
in the second culture where p-tyrosol was added at the end of
the exponential phase of growth. This corresponded to a
bioconversion molar yield of 44.8%. However, for the first and
the third cultures, molar yields of 18% and 25% were obtained,
respectively.
The effect of p-tyrosol concentration on the bioconversion
rate was then examined. Six batch cultures pregrown on
p-tyrosol (1 g/L) until the end of the exponential phase were
supplemented, respectively, with different concentrations of
p-tyrosol (1, 1.5, 2, 2.5, 3, 4 g/L). The optimal p-tyrosol
concentration should be considered to be that which allows
higher yields of hydroxytyrosol to be obtained, and afterwards,
a delayed decrease in the metabolite. Figure 1 shows the
variation in the level of hydroxytyrosol produced with the
treated p-tyrosol concentration. The maximum yield of the
produced hydroxytyrosol increased with the amount of p-tyrosol
used for concentrations up to 2 g/L. An excess of p-tyrosol
concentration (over 2 g/L) was detrimental to the increase in
the quantity of hydroxytyrosol produced. Furthermore, Figure
1 shows that for tyrosol concentrations higher than 2.5 g/L, the
bioconversion reaction is stopped after reaching its maximal
product level. The consumption of the hydroxytyrosol produced
did not start after more than 24 h. This may be explained by
the toxicity of the p-tyrosol. Thus, the optimal p-tyrosol
concentration would be 2 g/L which resulted in a bioconversion
yield of 80%.
Kinetic Formation of Hydroxytyrosol by Ser. marcescens
Grown Cells. The formation of hydroxytyrosol by Ser. marce-

6528 J. Agric. Food Chem., Vol. 53, No. 16, 2005
Allouche and Sayadi
DISCUSSION
In the present study, we investigated the possibility of
producing high-added-value products by microbial transforma-
tions using isomers of synthetic tyrosol as precursors. During
the screening of microorganisms able to utilize p-tyrosol as a
sole carbon and energy source, a strain, further characterized
as Ser. marcescens, was isolated. This strain was capable of
producing hydroxytyrosol using a high concentration (up to 2
g/L) of p-tyrosol. This result is in agreement with previous
investigations demonstrating that Ser. marcescens strain tolerates
high vanillin concentrations to be converted into vanillic acid
(37). Furthermore, Ser. marcescens strain was capable of
transiently accumulating a significant amount of hydroxytyrosol
which resulted from an ortho-hydroxylation of p-tyrosol. Using
optimal conditions, 1.6 g of hydroxytyrosol was obtained after
a 7-h incubation of Ser. marcescens in 1 L of minimal medium
in the presence of 2 g of p-tyrosol. This yield would be improved
using the resting cell technique as was recently performed using
Pseudomonas aeruginosa (23).
The produced hydroxytyrosol by the isolated strain Ser.
marcescens resulted from an ortho-hydroxylation of p-tyrosol.
In a previous investigation, Mason (38) noted that monooxy-
genases are commonly involved in the aromatic ring-hydroxy-
lation of a wide range of aromatic compounds, and the most
frequently encountered aromatic monooxygenases are of the
p-hydroxybenzoate hydroxylase type. These enzymes add a
single hydroxyl group to an already hydroxylated substrate to
generate a product with vicinal hydroxyl groups.
The substrate-specificity of the performed hydroxylation
reaction was examined. The obtained result suggested that the
enzymatic system of the isolated strain Ser. marcescens was
affected by the position of the hydroxyl group of the aromatic
substrate. Ser. marcescens hydroxylated the tyrosol containing
a hydroxyl group in the para position, but it oxidized the side
chain carbon when the hydroxyl group was in the meta or ortho
position. On the other hand, Ser. marcescens strain transformed
completely m-tyrosol and o-tyrosol into, respectively, 3-hy-
droxyphenylacetic acid and 2-hydroxyphenylacetic acid by
oxidizing the side chain carbon of the corresponding precursors.
This result confirms the findings by De la Fuente et al. (39)
who reported that grown and resting cells of Ser. marcescens
oxidize a variety of aromatic aldehydes. The productions of
3-hydroxyphenylacetic acid and 2-hydroxyphenylacetic acid
developed in the present study were achieved after 4 and 30 h
of incubation time, respectively. Staudenmaier et al. (24, 28)
patented the preparation protocols of 3-hydroxyphenylacetic acid
and 2-hydroxyphenylacetic acid via microbial hydroxylation of
phenylacetic acid in the meta and the ortho positions, respec-
tively. In these investigations, researchers used a fungus to
convert phenylacetic acid entirely. However, compared with our
procedure, the used protocols are very slow. Indeed, the
performed reactions were accomplished after 7 days of incuba-
tion time with a molar yield of 100%. The produced 3-hydroxy-
phenylacetic acid and 2-hydroxyphenylacetic by the isolated Ser.
marcescens strain would be used in the pharmaceutical industry
as alternatives to the synthetic intermediates which are involved
in the production of various medicinal products as was suggested
in previous works (24, 28).
Figure 3. Transformation time courses of m-tyrosol and o-tyrosol into
3-hydroxyphenylacetic acid (A) and 2-hydroxyphenylacetic acid (B),
respectively, using Ser. marcescens strain. m-Tyrosol or o-tyrosol (
3-hydroxyphenylacetic acid or 2-hydroxyphenylacetic acid ( ), optical
density at 600 nm ). Each point represents the mean of three
]),
9
(
2
determinations and three independent experiments (SD < 15% of the
mean).
3-hydroxyphenylacetic acid and 2-hydroxyphenylacetic acid,
respectively. This result showed that Ser. marcescens hydroxy-
lated the aromatic ring only when the existing hydroxyl group
is in the para position (p-tyrosol). However, with the two other
isomers (m-tyrosol and o-tyrosol), the side chain carbon was
oxidized into acetic acid. Hence, the hydroxylation of p-tyrosol
by Ser. marcescens strain is a substrate-specific reaction.
The conversion of m-tyrosol (1 g/L) and o-tyrosol (1 g/L)
using Ser. marcescens strain pregrown on p-tyrosol was
monitored by HPLC analyzing samples withdrawn from the
corresponding cultures at different times. Figure 3 shows the
time courses of the m-tyrosol (Figure 3A) and the o-tyrosol
(Figure 3B) depletion and the accumulation of the 3-hydroxy-
phenylacetic acid and the 2-hydroxyphenylacetic acid, respec-
tively. During the incubation period, OD600nm remained constant
in the two-culture media. Ser. marcescens strain transformed
entirely m-tyrosol and o-tyrosol to 3-hydroxyphenylacetic acid
and 2-hydroxyphenylacetic acid, respectively, without further
aromatic consumption. Maximal 3-hydroxyphenylacetic acid and
2-hydroxyphenylacetic acid concentrations (1.1 g/L), resulting
in a molar yield of 100%, were achieved after 4 and 30 h,
respectively. In addition, the bioconversion reactions started after
0 h in m-tyrosol and after 4 h in o-tyrosol. This could be
explained by the hydrogen bond effect. This latter is more
important in the chemical structure of 2-hydroxyphenylacetic
acid than it is in the chemical structure of 3-hydroxyphenylacetic
acid.
In comparison with our previous study dealing with hydroxy-
tyrosol production using P. aeruginosa (23), Ser. marcescens
is a more promising strain for several reasons. Indeed, Ser.
marcescens transformed entierly m-tyrosol and o-tyrosol into,
respectively, 3-hydroxyphenylacetic acid and 2-hydroxyphenyl-
acetic acid with molar yields of 100% while P. aeruginosa was

Differential Conversion of Tyrosol Isomers
J. Agric. Food Chem., Vol. 53, No. 16, 2005 6529
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ACKNOWLEDGMENT
Authors wish to thank Mr. He`di Aouissaoui and Mr. Mohamed
Hammami for their help in HPLC and GC-MS analysis.
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Received for review April 27, 2005. Revised manuscript received June
10, 2005. Accepted June 13, 2005. This research was supported by
“Contrats Programmes MRSTDC”, Tunisia.
JF050972W