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BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Alternaria sp. MG1, a resveratrol-producing fungus: isolation,
identification, and optimal cultivation conditions for resveratrol
production
Junling Shi &Qin Zeng &Yanlin Liu &Zhongli Pan
Received: 29 October 2011 / Revised: 19 March 2012 /Accepted: 19 March 2012 /Published online: 13 April 2012
#Springer-Verlag 2012
Abstract Due to its potential in preventing or slowing the
occurrence of many diseases, resveratrol (3,5,4′-trihydroxys-
tilbene) has attracted great research interest. The objective of
this study was to identify microorganisms from selected plants
that produce resveratrol and to optimize the conditions for
resveratrol production. Endophytes from Merlot wine grapes
(Vitis vinifera L. cv. Merlot), wild Vitis (Vitis quinquangularis
Rehd.), and Japanese knotweed (Polygonum cuspidatum
Siebold & Zucc.) were isolated, and their abilities to produce
resveratrol were evaluated. A total of 65 isolates were
obtained and 21 produced resveratrol (6–123 μg/L) in liquid
culture. The resveratrol-producing isolates belonged to seven
genera, Botryosphaeria,Penicillium,Cephalosporium,
Aspergillus,Geotrichum,Mucor,andAlternaria.The
resveratrol-producing capability decreased or was completely
lost in most isolates after three rounds of subculture. It was
found that only the strain Alternaria sp. MG1 (isolated from
cob of Merlot using GA1 medium) had stable and high
resveratrol-producing capability in all subcultures. During
liquid cultivation of Alternaria sp. MG1 in potato dextrose
medium, the synthesis of resveratrol began on the first day,
increased to peak levels on day 7, and then decreased sharply
thereafter. Cell growth increased during cultivation and
reached a stable and high level of biomass after 5 days. The
best fermentation conditions for resveratrol production in
liquid cultures of Alternaria sp. MG1 were an inoculum size
of 6 %, a medium volume of 125 mL in a 250-mL flask, a
rotation speed of 101 rpm, and a temperature of 27 °C.
Keywords Resveratrol .Endophyte .Alternaria .Response
surface method
Introduction
Resveratrol (3,5,4′-trihydroxystilbene) is a widely recognized
compound that is known for preventing and slowing the
occurrence of some diseases, including cancer (Jang et al.
1997), cardiovascular disease (Bradamante et al. 2004), and
ischemic injuries (Wang et al. 2002; Sinha et al. 2002). It has
also been shown that resveratrol can enhance stress resistance
(Howitz et al. 2003) and extend the lifespan of various organ-
isms ranging from yeasts to vertebrates (Valenzano et al.
2006). Resveratrol has been found in grapes (Viti s v inif e ra),
a variety of other berries, peanuts, and medicinal plants such
as Japanese knotweed (Polygonum cuspidatum Siebold &
Zucc.) (Baur and Sinclair 2006). The resveratrol found in
grapes can be transferred to wine during winemaking and is
Electronic supplementary material The online version of this article
(doi:10.1007/s00253-012-4045-9) contains supplementary material,
which is available to authorized users.
J. Shi (*):Q. Zeng
College of Food Science and Engineering,
Northwest A & F University,
28 Xinong Road,
Yangling, Shaanxi Province 712100, China
e-mail: sjlshi2004@yahoo.com.cn
Y. Liu
College of Enology, Northwest A & F University,
23 Xinong Road,
Yangling, Shaanxi Province 712100, China
Z. Pan
Processed Foods Research Unit, USDA-ARS-WRRC,
800 Buchanan Street,
Albany, CA 94710, USA
Z. Pan
Department of Biological and Agricultural Engineering,
University of California–Davis,
One Shields Avenue,
Davis, CA 95616, USA
Appl Microbiol Biotechnol (2012) 95:369–379
DOI 10.1007/s00253-012-4045-9
directly related to the resveratrolcontentinwine(Casasetal.
2009). The pretreatment (crushing and pressing) and vinification
process in winemaking could also affect the resveratrol content
found in wine. Wine has attracted much attention because it is
one of the primary sources of resveratrol for humans due to its
high resveratrol content. Therefore, many efforts have been
made to increase the resveratrol content in grape fruits by using
short anoxic treatments or enriched ozone atmosphere in grape
storage (Jiménez et al. 2007; Artés-Hernández et al. 2007), and
in wine by using transgenic yeast for fermentation (Wang et al.
2011), respectively. Currently, resveratrol extracts are mainly
produced in China using an extraction process from Japanese
knotweed (Changsha Nutramax Inc. 2009).
Microbial fermentation has been used to produce various
valuable compounds on a large scale due to its high efficiency,
ease of operation, and low cost (Chemler and Koffas 2008).
Therefore, researchers have explored ways to produce resver-
atrol using recombinant microorganisms (Beekwilder et al.
2006;Shinetal.2011), chemical synthesis, and plant cell
cultures (Fan et al. 2010). Identifying microorganisms able to
produce resveratrol should provide new resources for genes,
or new pathways for producing resveratrol.
At present, most genes involved in the trans-resveratrol
biosynthesis pathway have not been described in microor-
ganisms (Kiselev 2011). Currently, the genes used for pro-
ducing trans-resveratrol in vitro are from plant origin,
including grape, tobacco, and peanut (Beekwilder et al.
2006; Shin et al. 2011). These genes encode 4-coumarate
CoA-ligase (4CL) and stilbene synthase (STS), which are
required for resveratrol biosynthesis when 4-coumaric acid
is added to the culture medium as a precursor. So far, only
one microbial gene for a putative 4CL has been identified in
Aspergillus flavus, and it is available in the National Center
for Biotechnology Information database (NCBI) (Genbank
accessing number: XM002380137). Homology searches
using the encoded protein sequences indicate that 4CL genes
might be abundant in fungi.
Many endophytes exhibit the capability to produce the
same functional compounds as their hosts (Kumaran et al
2010; Suryanarayanan et al. 2009; Strobel and Daisy 2003)
and live asymptomatically within plant tissues (Schulz et al.
1993). Therefore, endophytes have been studied for new
medicine development and plant disease management (Arnold
et al. 2003; Azevedo et al. 2000;Bergetal.2004;Murrayetal.
1992). However, endophytes able to produce resveratrol have
not been reported.
The aim of this study was to identify microorganisms that
can produce resveratrol and optimize the conditions for
producing resveratrol through microbial fermentation. Plant
Merlot wine grape (V. vinifera L. cv. Merlot), wild Vitis
(Vitis quinquangularis Rehd),and Japanese knotweed
(P. cuspidatum Siebold & Zucc) were used as the sources
for isolation of these microorganisms.
Materials and methods
Materials
Three different plants were used to isolate resveratrol-
producing endophytes: grape cultivar Merlot (V. vinifera
L. cv. Merlot), wild Vitis (V. quinquangularis Rehd), and
Japanese knotweed (P. cuspidatum Siebold & Zucc) grown in
Yangling Town, Xianyang City of Shaanxi Province;
Luocheng Town, Hechi City of Guangxi Province; and
Taochuan Town, Taibai County, Baoji City of Shaanxi Province,
China, respectively. The fruits of the plants were picked during
harvest. The stems were also collected together with the fruits
for both Merlot and wild Viti s , and the whole stem tuber was
collected for Japanese knotweed. The fruits were placed in
sterilized bags and kept on ice during transportation. Samples
were analyzed within 24 h of acquisition.
Media
Three media were used for the isolation of endophytes
according to that of Liu et al. (2010): Gause medium G-1
(GA1: soluble starch 20 g, KNO
3
1g,K
2
HPO
4
0.5 g,
MgSO
4
·7H
2
O 0.5 g, NaCl 0.5 g, FeSO
4
·7H
2
O 0.01 g, agar
20 g, and distilled water 1 L; pH 7.4–7.6), beef-protein
medium (BP: beef extract 3 g, peptone 10 g, NaCl 5 g, agar
20 g, and distilled water 1 L; pH 7.4–7.6), and potato
dextrose agar (PDA: potato (peeled and diced) 200 g, dex-
trose 20 g, agar 20 g, and water 1 L). The liquid phases of
these media (without agar) were used to determine if the
obtained isolates could produce resveratrol. The broth of
potato dextrose medium (PDB) was used to study the opti-
mum conditions for Alternaria sp. MG1 to produce
resveratrol.
Czapek yeast extract agar medium (sucrose 30 g, yeast
extract 5 g, NaNO
3
3 g, KCl 0.5 g, MgSO
4
·7H
2
O0.5g,
FeSO
4
·7H
2
O 0.01 g, K
2
HPO
4
1 g, agar 20 g, and water 1 L)
was used to identify strains of the genera Penicillium and
Aspergillus according to the method reported by Pereyra et
al. (2011) and Pitt and Hocking (1997). Potato carrot agar
medium ((PCA: potato (peeled and diced) 20 g, carrot
(peeled and diced) 20 g, agar 20 g, and water 1 L) was used
to identify Alternaria sp. (Sørensen et al. 2009). Other
isolates were observed on PDA plates.
Isolation of endophytes
Endophytes were isolated by cultivating the tissues of
berries, cobs, and stems of Merlot and wild Vitis, and the
root and stem tissues of Japanese knotweed. All samples
were cultivated on GA1, BP, and PDA after the sample
surfaces were sterilized and rinsed with sterile water (Larran
et al. 2002). The rinse water was collected after the last
370 Appl Microbiol Biotechnol (2012) 95:369–379
rinsing and also cultivated to ascertain a complete surface
sterilization (Naik et al 2009). All inoculated plates were
cultivated in darkness at 28± 1 °C. During cultivation, colo-
nies were transferred to a fresh medium from which the
isolates were obtained for purification. All experiments for
each test were conducted in triplicate.
Screening resveratrol-producing endophytes
In order to produce comparable results, spore suspensions of
10
4
/mL (measured using a hemacytometer) from all of the
endophytic fungi were prepared. Samples of 1-mL suspension
were used to inoculate three parallel cultures of 100 mL of a
liquid medium that corresponded to the solid medium used for
isolation. After incubation in darkness at 28± 1 °C with a
rotation speed of 100 rpm for 7 days, the liquid broth and
cells were separated by centrifugation at 3500 × gfor 15 min.
The collected cells were crushed into powder after freezing in
liquid nitrogen and then extracted with 80 % ethanol (15 mL
ethanol per 1 g cell weight) three times (10 h for each extrac-
tion) to obtain the resveratrol inside cells. The liquid broth and
ethanol extracts of the cells were combined and concentrated
to 100 mL at 45 °C using a vacuum evaporator (R-200,
BUCHI, Flawil, Switzerland). The 100-mL sample was parti-
tioned three times with 50 mL ethyl acetate each time. The
three samples of the ethyl acetate phase were combined and
then partitioned with three volumes 20 mL 3 % NaHCO
3
to
remove water. All the obtained ethyl acetate phase was evap-
orated to dryness at 40 °C and then dissolved in 2-mL meth-
anol. Finally, to determine resveratrol content, the methanol
solution was analyzed by high performance liquid chroma-
tography (HPLC) after being filtered through a Millex-HV
filter membrane (0.45-μM, 13-mm diameter; Millipore,
Billerica, MA, USA). The resulting resveratrol content in the
liquid culture (in micrograms per liter) was determined and
reported as the average of the three parallel cultures.
Conditions for HPLC measurement of resveratrol
concentration
Resveratrol concentration in the methanol extracts obtained
above was measured using a Shimadzu Essentia LC-15 C
analytical HPLC system (Shimadzu, Kyoto, Japan)
equipped with LC-15 C pumps, a SIL-10AF automated
sample injector, a SPD-15 C dual-wavelength detector, a
Shimadu Wondasil C18-column (250×4.6 mm), and LC
solution software. The column was operated at a tempera-
ture of 35 °C. The mobile phase was acetonitrile (Sigma, St.
Louis, MO, USA) (eluant A) and double-distilled water
(eluant B). A multistep gradient was used for all tests at a
flow rate of 1 mL/min according to the following steps:
0–28 min: 95 % (v/v)B;28–33 min: 40 % B; 33–40 min:
15 % B; and 40–50 min: 95 % B. The sample injection
volume was 20 μL. Trans-resveratrol (≥99 % ([GC], Sigma)
was used as the standard for measurements.
Identification of resveratrol-producing endophytes
Colony appearance and sporulation of the isolates were observed
on the corresponding identification media for morphological
identification (Xing and Guo 2011). Specimens for light micros-
copy (BA 400; Motic, Richmond, Canada) were mounted in 3 %
KOH or sterile distilled water and observed (Yuan et al. 2011).
The sequence of the internal transcribed spacer (ITS) regions of
ribosomal DNA (rDNA), including ITS1, 5.8S rDNA, and ITS2
(GenBank accession number: JN102357), was determined for
molecular identification of strain Alternaria sp. MG1. For se-
quencing, cultivated mycelium was freeze-dried and ground with
liquid nitrogen (Dey et al. 2011). Genomic DNA was extracted
from the obtained mycelia using a Fungus Genomic DNA
extraction kit (BioFlux, Kyoto, Japan), and the amplification of
the whole ITS region of rDNA was performed by using a Fungi
Identification PCR Kit (TaKaRa, Kyoto, Japan) and primers, 5′-
GAGCGGATAACAATTTCACACAGG-3′and 5′-
CGCCAGGGTTTTCCCAGTCACGAC-3′, according to the
manufacturer’s instructions.
Profiles of cell growth and resveratrol production
of Alternaria sp. MG1
The strain Alternaria sp. MG1 was selected to obtain profiles
of cell growth and resveratrol production in vitro in 100-mL
liquid PDB in a 250-mL flask in an incubator at 28 °C with a
rotation speed of 100 rpm. Dry cell weight was measured
every day after drying the mycelia at 60 °C for 48 h, and the
resveratrol produced was measured using the above described
methods from days 2 to 11 of cultivation. The resveratrol
contents in the cell-free broth and inside the cells were also
measured separately at days 5 and 7 to determine the distri-
bution of resveratrol within the cultures. For separate analysis
of the resveratrol accumulation in liquid broth and inside cells,
the liquid broth and cells were treated separately after centri-
fugation. Similar to the treatment described above, cells were
extracted with ethanol and ethyl acetate, while the liquid broth
was extracted with ethyl acetate directly, dried with 3 %
NaHCO
3
and finally dissolved in 2-mL methanol for HPLC
measurement. The resulting resveratrol content in the liquid
culture (in micrograms per liter) and within the cells (in
micrograms per gram) was determined and reported as the
average of three parallel samples.
Optimum production conditions of resveratrol by Alternaria
sp. MG1
The single-factor design and the Box–Behnken design were
used to optimize the production conditions of resveratrol by
Appl Microbiol Biotechnol (2012) 95:369–379 371
Alternaria sp. MG1. The four parameters of temperature,
rotation speed, inoculum size, and medium volume of
flask were investigated. In the single-factor design, un-
less specified, four parameters were set as follows:
temperature028 °C, rotation speed 0100 rpm, inoculum
size05 %, and medium volume of flask0100 mL in a
250 mL flask. The inoculum size was measured as the
volume ratio of spore suspension (1× 10
4
spore/mL) to
the liquid medium. The levels of each parameter in the single-
factor design were set at 14, 21, 28, 35, and 42 °C for
temperature test; 60, 90, 100, 120, 140, 160, and 180 rpm
for rotation speed test; 1, 5, 10, and 15 % for inoculum size
test; and 100, 125, 150, 175, and 200 mL for medium volume
of flask test.
Based on the results obtained from the single-factor
experiments, a set of 29 experiments were designed and
carried out according to the Box–Behnkendesignwith
four factors (see Table S1in the supplementary materi-
als) to determine the optimum levels for each parameter
under interaction. The results of the Box–Behnken de-
sign are explained by Eq. (1) (Khajeh and Ghanbari
2011):
Y¼b0þX
k
i¼1
biciþX
k
i¼1
biic2
iþX
k
i¼1
X
k
j¼1
bijcicjþ"ð1Þ
The statistical software package Design-Expert (Version
7.0.0; Stat-ease Inc., Minneapolis, MN, USA) was used to
make the response surface methodology analysis and ana-
lyze the experimental results.
Results
Isolated endophytes and resveratrol-producing endophytes
A total of 65 isolates were obtained from the three tested
plants:30 from Merlot, 15 from wild Vitis, and 20 from
Japanese knotweed (Table 1). Only five isolates were iden-
tified as bacteria, and the remaining 60 were fungi. In total,
21 strains, including seven from Merlot, six from wild Vitis,
and six from Japanese knotweed, produced resveratrol in the
range of 6–123 μg/L (Table 2). Figure S1(see supplementary
materials) shows the HPLC chromatogram of standard trans-
resveratrol and the culture of Alternaria sp. MG1. The HG6,
MP1, MP15, YG3, and MG1 strains produced >90 μg/L
resveratrol (Table 2). The resveratrol production capability
of most isolates, however, declined and even disappeared for
some isolates after being subcultured three times. However,
only the MG1 strain (isolated from Merlot using GA1 medi-
um) remained the most stable and retained the highest pro-
duction capability (Table 2).
Identification of resveratrol-producing isolates
All resveratrol-producing isolates were identified and con-
firmed based on their specific morphological and sporulation
characteristics. As shown in Table 2, the resveratrol-producing
fungi belonged to seven genera including Alternaria (nine
strains), Botryosphaeria (one strain), Penicillium (two
strains), Cephalosporium (five strains), Aspergillus (one
strain), Geotrichum (one strain), and Mucor (one strain). All
Table 1 Endophytes isolated from Merlot, wild Vitis, and Japanese knotweed
Plant Strain Types Tissue Medium Number of isolates
Merlot MB1, MB2 Bacterium Cob BP 2
MG1, MG2, MG3, MG4, MG8, MG9, MG10 Fungus Cob GA1 7
MG5, MG6, MG7 Fungus Stem GA1 3
MP1, MP2, MP3, MP7, MP8, MP13, MP14, MP16, MP17 Fungus Stem PDA 9
MP4, MP5, MP9, MP10, MP1, MP12, MP15, MP18 Fungus Skin PDA 8
MP6 Fungus Cob PDA 1
Wild Vitis YN1 Bacterium Cob BP 1
YG3, YG4, YG5 Fungus Stem GA1 3
YG6, YG8 Fungus Cob GA1 2
YP2, YP12, YP15, YP20 Fungus Skin PDA 4
YP3, YP5, YP30 Fungus Stem PDA 3
YP7, YP8 Fungus Cob PDA 2
Japanese knotweed HN1, HN2, HN3, HN4 Bacterium Root BP 4
HG5, HG6, HG8, HG10, HG14, HG7, HG11, HG15 Fungus Stem GA1 8
HG16, HG17 Fungus Root GA1 2
HP4, HP6, HP9, HP7, HP8 Fungus Root PDA 5
GA1 Gause medium G-1, BP beef-protein medium, PDA potato dextrose agar medium
372 Appl Microbiol Biotechnol (2012) 95:369–379
the resveratrol-producing strains isolated from Merlot
belonged to genus Alternaria. The strain MG1 was studied
further to provide information for potential future commercial
production.
The strain MG1 showed felty and brown colony mor-
phology with a dark color in reverse side of the medium
after cultivation on PCA for 5 days (Fig. 1). The colony
growth showed a concentric appearance (Fig. 1a) with the
mycelium displaying smooth subhyaline branches consist-
ing of septate hyphae and 3.5–4μm in width. Conidio-
phores were abundant, subhyaline, erect or ascending from
both submerged and aerial hyphae, simple or branched,
septate, 50–70 μm×3–3.5 μm in length and width, with
three to six uniperforate geniculations (Fig. 1b). The conidia
were obclavate, ovoid, obpyriform, with three to seven
transverse septa (commonly four), zero to three longitudinal
septa, smooth, brown to dark brown, and 4.3–12.5 × 18.2–
40 μm in length and width. Conidial chains appeared after
7 days of cultivation and were multi-branched with sizes of
1–15 conidia (see Fig. 1c). These morphological character-
istics were consistent with that of Alternaria sp. (von Arx
1981).
The DNA sequence of the ITS regions of strain MG1,
together with the 5.8 S rDNA, was 622 bp long and archived
in the GenBank database under the accession number
JN102357. The sequences were subjected to a sequence
similarity search performed through the NCBI database
using the Basic Local Alignment Search Tool (BLAST)
(http://www.ncbi.nih.gov/BLAST/). BLAST results showed
a highest identity value of 91 % between the ITS sequence
data of MG1 and the best hit to a sequence available at
GenBank. The ITS sequence data for strain MG1 and closest
related species available at GenBank were selected for the
construction of a phylogenetic tree using the neighbor-
joining method in the MEGA (version 4.0) program after
sequence alignment with ClustalX (version 1.8) (Thompson
Table 2 Reveratrol
production of the obtained
resveratrol-producing
fungi
a
Standard deviation of three
replicates
Plant Tissue Strain Genus Resveratrol production (μg/L)
First
subculture
Second
subculture
Third
subculture
Merlot Cob MG1 Alternaria 97 ± 4
a
104± 1 101 ± 3
Cob MG2 Alternaria 34 ± 3 28 ±3 26± 4
Cob MG3 Alternaria 23 ± 1 21 ±3 20± 6
Skin MG6 Alternaria 11± 3 0 0
Skin MG7 Alternaria 12±1 0 0
Skin MP11 Alternaria 6±3 0 0
Skin MP15 Alternaria 95±3 90 ± 1 92 ±1
Wild Vitis Stem YG3 Botryosphaeria 94 ±1 88± 4 85 ± 3
Stem YP30 Penicillium 17±4 11±1 0
Cob YG6 Cephalosporium 35 ± 6 0 0
Cob YG8 Aspergillus 38 ±1 25±6 0
Cob YP7 Cephalosporium 20 ± 3 21 ±4 17± 4
Skin YP2 Penicillium 24 ± 3 0 0
Japanese knotweed Root HP7 Geotrichum 43 ± 4 23±3 0
Root HP9 Mucor 21 ±3 17± 4 0
Root HG16 Cephalosporium 67± 1 13 ± 6 0
Stem HG5 Cephalosporium 11 ± 4 0 0
Stem HG6 Alternaria 123 ±6 44± 3 0
Stem HG7 Cephalosporium 56±7 9± 6 0
Fig. 1 Colony (a), conidiophore
(b), and conidial chain (c)of
Alternaria sp. MG1. Pictures
were taken after 5 days (a), after
3days(b), and after 7 days of
incubation (c). The cultivation
was performed on potato carrot
agar medium (PCA)
Appl Microbiol Biotechnol (2012) 95:369–379 373
et al. 1997; Saitou and Nei 1987; Tamura et al. 2007). The
confidence values for individual branches were determined
by bootstrap analyses (1,000 replications) and maximum
parsimony. As shown in Fig. S2(supplementary materials),
the sequences with the highest identity (91 %) to MG1 were
Alternaria sp. Hf3 (GenBank accession number: GU183167.1)
with a bootstrap value of 76. Thus, isolate MG1 was identified
as Alternaria sp. consistent with the morphological identifica-
tion. Isolate MG1 was deposited in the China Center for Type
Culture Collection (Wuhan, China) and coded Alternariasp.
CCTCC M 2011348.
Profiles of cell growth and resveratrol production
by Alternaria sp. MG1 in liquid culture
The peak cell density of strain MG1 in potato dextrose
medium was reached after continuously growing for 5 days
(Fig. 2). A decrease in dry cell weight was observed after
8 days of cultivation. The resveratrol accumulation, both
within cells and in the medium, started from the first day
of cultivation and approached its highest value of 376±
13 μg/L after continuously increasing for 7 days, and resver-
atrol amounts sharply decreased. In the early stage of culti-
vation, the cell growth and total amounts of resveratrol
within cells and in the medium increased almost simulta-
neously (Fig. 2). However, in a parallel analysis of resver-
atrol amounts within cells and in the medium, no resveratrol
was detected in the medium. These results revealed that the
intracellular amounts of resveratrol on days 5 and 7 of
cultivation were 224±25 μg/L medium and 353±24 μg/L
medium, respectively, corresponding to 69±8 μg/g dry cell
weight and 113± 6 μg/g dry cell weight, respectively. There-
fore, resveratrol may be a constitutive product that accumu-
lates within cells of Alternaria sp. MG1.
Optimum conditions for resveratrol production
by Alternaria sp. MG1
The effect of each factor (inoculum size, medium volume of
flask, rotation speed, and temperature) on resveratrol pro-
duction from the single-factor tests is shown in Fig. 3. All
factors had a similar effect on the resveratrol production
showing an initial increase followed by a decrease. Accord-
ing to the highest values of resveratrol production obtained
from the single-factor tests, the middle values for the Box–
Behnken design were set as 5 % for inoculum size, 125 mL
for medium volume of flask, 100 rpm for rotation speed, and
28 °C for temperature. The test results are listed in the
supplementary materials (Table S2).
Based on the obtained data, a statistical model (Eq. 2)
was obtained to determine the yield trends of resveratrol
production.
R1¼413:88X11:80X23:62X385:36X4
40:84X1X2þ93:09X2X3þ44:55X2X4
40:78X2
160:1X2
279:61X2
3105:40X2
4ð2Þ
where R
1
is the amount of resveratrol (in micrograms per
liter), X
1
is the inoculum size (percentage), X
2
is the medium
volume of flask (in milliliters per 250 mL), X
3
is the rotation
speed (in revolutions per minute), and X
4
is temperature (in
degrees Celsius).
Analysis of variance for the model demonstrated that the
model is highly significant based on Fisher Ftest
(F036.2789) and a low probability value (P00.0001). The
high coefficient of determination (R
2
00.9591) and adjusted
coefficient of determination (adjusted R
2
00.9327) indicated
the high significance of the model. The linear coefficients
Fig. 2 Profiles of resveratrol
production (closed square) and
cell growth (open square)in
liquid fermentation of
Alternaria sp. MG1. The
cultivation was carried out in
broth of potato dextrose
medium (PDB) at 28 °C with a
rotation speed of 100 rpm. The
profile of cell growth was
drawn according to the dry cell
weight. The bars show the
standard deviation of the mean
of three replicates
374 Appl Microbiol Biotechnol (2012) 95:369–379
(X
1
,X
4
), four quadratic term coefficient (X
1
2
,X
2
2
,X
3
2
, and
X
4
2
), and cross product coefficients (X
1
X
2
,X
2
X
3
, and X
2
X
4
)
were statistically significant (P< 0.05). The coefficients of
other parameters were not significant (P> 0.05). Tempera-
ture (X
4
) showed the most significant effect on resveratrol
production, followed by inoculum size (X
1
), rotation speed
(X
3
), and medium volume of flask (X
2
). By solving the
model, the optimum conditions for the highest resveratrol
production were obtained as 6 % (v/v) inoculum size,
125 mL/250 mL medium volume of flask, 101 rpm rotation
speed, and 27 °C temperature. Under these conditions
resveratrol production was 451 μg/L by predicted value
and 422±18 μg/L experimentally.
Response surface methodology is usually used to study
the effects of several factors at different levels and their
interactions (Priya and Kanmani 2011). The two-
dimensional contour plots of the parameters show the effect
of two factors on resveratrol production at a time (Fig. 4).
The resveratrol production values in all figures were
obtained along with two variables, while the other two
variables were kept at level zero (middle value of the testing
ranges). Elliptical contours mean that there is a significant
interaction between the independent variables. It can be seen
that the interaction between the medium volume of flask and
rotation speed was the most significant (Fig. 4b), followed
by medium volume of flask and temperature (Fig. 4c), and
then inoculum size and medium volume of flask (Fig. 4a).
Discussion
Fungal endophytes are gaining increased importance because
of their enormous potentials for the production of novel bio-
active compounds for medicine and agriculture (Aly et al.
2011). Among the various secondary metabolite-producing
endophytes, resveratrol-producing fungi were discovered for
the first time from different plant tissues and fruits.
Resveratrol-producing endophytes were isolated from plants
with a high content of resveratrol, which is consistent with the
coevolution theory reported by other research on endophytes
(Amna et al. 2006;Eybergeretal.2006;Kusarietal.2009a,
2009b;Purietal.2005,2006; Stierle et al. 1993). In addition,
isolation of resveratrol-producing isolates was influenced
when different plant tissues and media were used (as shown
Fig. 3 Effect of inoculum size (a), medium volume of flask (b),
rotation speed (c), and temperature (d) on the resveratrol production
of Alternaria sp. MG1 in liquid cultivation. The experiment was
carried out at conditions of (a) 28 °C, rotation speed of 100 rpm, and
medium volume of flask of 100 mL/250 mL, (b) 28 °C, rotation speed
of 100 rpm, and inoculum size of 5 %, (c) 28 °C, inoculum size of 5 %,
and medium volume of flask of 100 mL/250 mL, and (d) inoculum size
of 5 %, medium volume of flask of 100 mL/250 mL, with a rotation
speed of 100 rpm. All the cultivations were carried out in liquid potato
dextrose medium (PDB) and in dark environment by using a sheet of
black paper to cover the incubator. The bars show the standard devi-
ation of the mean of three replicates
Appl Microbiol Biotechnol (2012) 95:369–379 375
in Tables 1and 2). Arnold et al. (2003) and Sun et al. (2011)
have also found that there was a wide diversity of endophytic
fungi in different plants and in different parts of a same plant.
The strain MG1 became the principal focus of this study
since it exhibited a high and stable resveratrol-producing
capability during subculture. For several other isolates, the
resveratrol-producing capability decreased and was even
abolished after subculture. Similar decrease and loss of the
capability for production of other compounds has also been
reported in other metabolite-producing endophytes isolated
from plants (Wang et al. 2010). The instability of resveratrol
production could possibly be caused by the absence of the
host plant in vitro culture of the endophytes, which would
cause unstable expression of the related genes and the trans-
formation of metabolite pathway within cells. Studies on the
biosynthesis pathways and discovery of the key genes
encoding these pathways would provide essential informa-
tion for the mechanism of loss of production.
During the liquid cultivation of the strain MG1, resvera-
trol accumulation was mainly found inside the cells and was
shown to increase with increased biomass. This was differ-
ent from the biosynthesis of secondary products by most
microorganisms, which began at the late stage of exponen-
tial phase of cell growth (Chomcheon et al. 2009; Kornsa-
kulkarn et al. 2011; Papagianni 2003). The profiles of cell
growth and resveratrol accumulation showed that resveratrol
should be a constitutive product in Alternaria sp. MG1.
The discovery of resveratrol-producing fungi in this study
revealed the existence of resveratrol-producing microorgan-
isms in nature. The genus Alternaria is normally reported as a
pathogen for many plants (Akamatsu et al. 1999;Akamatsu
2004). Alternaria spp. have also been widely found in grapes
as an endophyte or a fungus causing rotting or toxin produc-
tion (Gonzalez and Tello 2011; Tournas and Stack 2001;Ostry
2008). Discovery of the resveratrol-producing capability of
Alternaria sp. in this study indicates the possibility of a new
Fig. 4 Contour plots of the effects of interactions between medium
volume of flask and inoculum size (a), between medium volume of
flask and rotation speed (b), and between medium volume of flask and
temperature (c) on resveratrol production of Alternaria sp. MG1 in
liquid cultivation. The cultivation was carried out in liquid potato
dextrose medium (PDB)
376 Appl Microbiol Biotechnol (2012) 95:369–379
pathway or new genes for the biosynthesis of resveratrol in
microorganisms.
The effect of inoculum size, medium volume of flask, and
temperature on resveratrol production in liquid fermentation
apparently relates to the cell growth of Alternaria sp. MG1. In
the batch cultivation method used in this study, low inoculum
sizes resulted in a slow increase of cell growth, while inoculum
sizes that were too large possibly exhibited cell degradation
and thus resulted in low cell weight within a given period. A
low medium volume of flask leads to a high relative inoculum
size, while a high medium volume of flask corresponds to a
low relative inoculum size. Therefore, similar cell growth data
were obtained due to the influence of both inoculum size and
medium volume of flask. The effect of temperature on resver-
atrol production is expected to be related to the temperature
adaptability of the strain. The optimum temperature for resver-
atrol production was the same as that for the cell growth of
Alternaria sp. MG1 (28 °C). The decrease in resveratrol pro-
duction at a high rotation speed could be due to resveratrol
being unstable or produced in lower amounts in the presence of
oxygen, since high rotation speeds resulted in high oxygen
content in the culture (King et al. 2006;Santosetal.2011).
Recombinant microorganisms have been created for resver-
atrol production. For example, Escherichia coli strain JM109
has been transformed with the 4CL gene from Arabidopsis
thaliana and the STS gene from Arachis hypogea, upon which
itwasdemonstratedtobeabletoconvert4-coumaricacidinto
resveratrol with a yield of 100 mg/L (Watts et al. 2006).
However, when E. coli received the 4CL gene from Lithosper-
mum erythrorhizon and the STS gene from A. hypogea,and
coumaroyl-CoAwas used as a substrate, resveratrol production
reached 171 mg/L (Katsuyama et al. 2007). Significant prog-
ress in increasing the resveratrol content in plant cells has been
achieved by transforming grape cells with the rolC gene of
Agrobacterium rhizogenes (Dubrovina et al. 2010) and treating
the cell cultures with methyljasmonate and cyclodextrins that
resulted in a resveratrol content of 1,600 μmol/g (Lijavetzky et
al. 2008). In the current study, we found that the resveratrol
production by Alternaria sp. MG1 was 353 μg/L in total
culture and 113 μg/g within cells after 7 days of cultivation
in PDB. The yield by fermentation of Alternaria sp. MG1 was
much lower than that of plant cells and genetically modified
bacteria in total culture, but comparable when the cells were
collected for extraction. Collectively, these data show potential
for improving the resveratrol production capability may be
achievable given appropriate metabolic controls and the devel-
opment of overproducing mutant strains.
Acknowledgments The authors wish to thank Prof. Bai Xianjin for
providing the materials of wild Vitis, Prof. Xia Xiaodong for revising
professional issues, and Dr. Evan Burkala for English polishing, and to
acknowledge the financial support from the Agriculture Department of
China through Project Numbers of nycytx-30 and 201003021.
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