ARTICLE IN PRESS

Microbiological Research 161 (2006) 232—237

www.elsevier.de/micres

Experimental studies on the growth and usnic acid

production in ‘‘lichen’’ Usnea ghattensis in vitro
B.C. Behera, Neeraj Verma, Anjali Sonone, Urmila Makhija

Plant Science Division, Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India

Accepted 11 August 2005

KEYWORDS Summary
Lichen;
The study was aimed to optimize the culture conditions for the production of usnic
Culture condition;
acid in the cultured cell aggregates composed of symbionts in lichen Usnea ghattensis
Usnic acid
in vitro. The cultured lichen tissue composed of symbionts appeared after about 2–3
weeks of inoculation in water–agar and malt–yeast extract (MYE) media and shown
the production of usnic acid after 2–3 months of inoculation. However, the growth of
symbionts was strongly affected by different culture conditions. The addition of
excess carbon and nitrogen sources in the media has significantly enhanced the growth
as well as usnic acid content. The cultured symbionts in MYE medium having 4%
sucrose, 4% polyethyl glycol (PEG) gave 7.63 g dry biomass with 3.9 mg usnic acid/g dry
biomass. In water–agar medium having 4% sucrose and 4% PEG gave 3.08 g dry biomass
with 1.11 mg usnic acid/g dry biomass. The positive effects of medium on the growth of
symbionts and the production of usnic acid are seemed to be due to nutritional
factors.
& 2005 Elsevier GmbH. All rights reserved.

Introduction 2001; Ingolfsdottir, 2002; Behera et al., 2005).

Lichens are symbiotic organisms composed of a
fungus and an alga. They produce characteristic
secondary metabolites ‘‘lichen substances’’ that
seldom occur in other organisms. Lichens and their
metabolites have many biological activities such as
antimicrobial, antiviral, antiprotozoal, antiproli-
ferative, antioxidant, antiinflammatory and an-
algesic activity (Yamamoto et al., 1998; Muller,
Inspite of the wide spectrum of biological activities
shown by the lichens, they have long been
neglected by mycologists and overlooked by phar-
maceutical industry because of its slow growth in
nature and difficulties in the artificial cultivation of
the organisms (Crittenden and Porter, 1991). Hence
the large-scale industrial production of the lichen
metabolites have never been accomplished. How-
ever, in general, lichen cultures grow much faster

Corresponding author.
E-mail address: bcbehera2002@yahoo.co.in (B.C. Behera).

0944-5013/$ - see front matter & 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.micres.2005.08.006
 ARTICLE IN PRESS
Experimental studies of ‘‘lichen’’ in vitro 233

than the natural thalli, but more slowly than many

other microorganisms (Yamamoto et al., 1993).
Growth rates must be improved if the lichen tissue
cultures are to be used for industrial production of
lichen substances.
Considering the well-known potential biological
activities of usnic acid, the production of usnic acid
by the cell aggregates derived from their natural
thallus of Usnea ghattensis could be useful in
screening experiments directed towards future
pharmaceutical applications. With this background,
the aim of the present study was to optimize the
culture conditions to improve the growth rate and
the production of lichen metabolites in the lichen
U. ghattensis in vitro.

Materials and methods

Lichen material

The natural lichen thallus of U. ghattensis

Awasthi. was collected from Silver Oak trees in
Mahabaleswar (Satara-District, Maharashtra state,
India) in the month of July 2003. The colonies of
the symbionts and mycobiont from the fragment of
natural thalli of U. ghattensis were obtained using
the following techniques.

Lichen culture

Lichen cultures were started within 7 days after

the collection using methods described earlier
(Yamamoto et al., 1985, 1987). Lichen thalli were
cut into pieces 1 cm long and were washed with tap
water for overnight and homogenized with 5 ml of
distilled water under sterile conditions. The sus-
pensions were passed through a sterilized stainless
filter with a 500 mm mesh. Small segments from the
second filteration were picked up with stainless
loop and were planted on slant media in Petri
dishes of 9 cm diameter and cultured at 18 1C in
light (400 lux) with daily cycle (8 h light/16 h dark).
The following culture media were used: malt–yeast
extract (MYE) medium; malt extract (20 g/l) and
yeast extract (2 g/l) solidified with agar (20 g/l).
After 3 months, the cultured symbionts and the
natural thallus were extracted with acetone. The
chemical data have been obtained by the standar-
dized method of TLC (Culberson, 1972) using
standard solvent systems BDA: benzene:dioxane:a-
cetic acid, 180:45:5, 230 ml and HEF: hexane:ethyl
ether:formic acid, 130:80:20, 230 ml. Lichen sub-
stances were identified by comparison with stan-
dard lichen substances and samples of several
Figure 1. (A) Tissue obtained from the lichen thallus of
U. ghattensis grown in malt–yeast media having 4%
sucrose and 4% polyethyl glycol (PEG). (B) Cell aggregates
composed of colorless fungal hyphae and green algal cells
in groups (micro-preparations from the same culture).
species containing atranorin, norstictic acid and
usnic acid and the corresponding natural thalli
(Figs. 1 and 2).
After confirmation of the symbionts in culture
derived from the natural lichen thallus fragments
of U. ghattensis, the symbionts were further
subcultured under various culture conditions in
order to optimize the conditions to enhance the
growth rate and production of usnic acid.
The cultured symbionts were further gently
crushed in a sterile mortar and pestle. They were
further subcultured in the Petri plates containing
solid or semi-solid water–agar and MYE media with
the addition of different amount of carbon and
nitrogen sources and was kept under 18 1C in light
(400 lux) with daily cycle (8 h light/16 h dark) for 6
 ARTICLE IN PRESS
234
Figure 2. TLC chromatogram of lichen substances in
natural thallus of U. ghattensis and its derived cultured
tissue in laboratory in solvent system BDA (benzene:diox-
ane:acetic acid, 180:45:5, 230 ml, v/v). Lane 1: Natural
thallus of U. ghattensis, Lane 2: derived cultured tissue
of U. ghattensis, Lane 3: atranorin and Lane 4: norstictic
acid.
months. In the first set of experiments, carbon
sources (glucose, sucrose or polyethyl glycol
(PEG)), amino acids (L- or D-asparagine, glutamine,
alanine, glycine) in concentrations of 2%, 4%, 8%,
16% or 32% (w/v) and vitamins (thiamin (B1),
riboflavin (B2), ascorbic acid (C) or biotin (H) in
concentrations of 1, 10, 100 or 1000 ppb were
individually added in the MYE media. In the second
set of experiments, symbionts were subcultured
in MYE media prepared with the addition of extra
4% sucrose, 4% PEG and 2% glycine (w/v). In all
experiments, the pH of the media were adjusted
to 5.8–6.2. After every 2 months and finally
after 6 months, cultured tissues were taken
out from the Petri dishes and dried at 40 1C for
72 h. The biomass of the cultured symbionts
obtained under various conditions mentioned above
were weighed and then proceeded for the estima-
tion of the production of usnic acid content in the
tissue.
Estimation of usnic acid content
The estimation of usnic acid produced by the
cultured symbionts was carried out spectrophoto-
B.C. Behera et al.
metrically following the procedure (Jayasankar and
Towers, 1968). After identification of the spot of
usnic acid in TLC chromatogram of acetone extract,
they were removed carefully with a scalpel,
transferred to a centrifuge tube, and further
extracted with 3 ml of glacial acetic acid. To a
1 ml aliquot was added 2 ml of p-dimethylamino-
benzaldehyde (PDAB) reagent and the mixture was
heated on a boiling water bath for 5 min. After
cooling, 5 ml of ethanol was added and the optical
density of the colored product measured against a
reagent blank at 617 nm. The amount of usnic acid
in dry biomass of the symbionts was calculated
using a standard curve obtained with the usnic acid
(sigma).
In all cases, three independent experiments,
each with five measurements, were performed.
The results are the means of these measurements.
Results and discussion
The influence of various culture conditions on the
growth of symbionts and the production of usnic
acid (lichen substance) of lichen U. ghattensis
under laboratory conditions have been studied. The
growth of symbionts and the production of usnic
acid content were found to be varied in the
water–agar and MYE media. In case of water–agar
medium, the growth of symbionts was found to
be very slow (0.68–1.77 g dry biomass) and less
amount of usnic acid (0.08–0.17 mg/g dry biomass)
was produced. Although the lower concentration
(5%) of agar in media initially promoted the growth
of symbiont but that did not produce usnic acid
even after 6 months of inoculation (Fig. 5).
However, when the agar concentration was in-
creased from 10% to 20% in water–agar medium the
growth of symbionts and the production of usnic
acid increased. The lichen symbionts grown in the
MYE medium having 20% agar yielded 2.44 g dry
biomass weight with 0.41 mg usnic acid/g dry
biomass after 6 months of inoculation which was
approximately double than that in the water–agar
medium after 6 months of inoculation. It has been
reported that lichen substances in culture have
been induced by the increased agar concentration
in the nutrient media (Kinoshita et al., 1993;
Yasuhiro et al., 2001).
Many lichen species also have been grown in MYE
and other media supplemented with excess carbon
or nitrogen for the enhancement of the growth of
mycobiont alone or the symbionts and the produc-
tion of lichen substances (Hamada, 1993; Hamada
et al., 1996; Behera and Makhija, 2001). With this
 ARTICLE IN PRESS
Experimental studies of ‘‘lichen’’ in vitro 235

in mind we have also conducted experiments by tion of usnic acid was very slow than the carbon
adding excess carbon and nitrogen sources in the sources added in the MYE media (Fig. 3).
water–agar and MYE media to improve the growth The cultures grown in media having asparagine
rate of the symbionts and the production of usnic and alanine did not promote usnic acid production
acid of lichen U. ghattensis in culture. The carbon (data not shown). The MYE media having 2% glycine
sources, i.e. sucrose or PEG concentrations ranging although maintained the symbionts growth rate but
from 2% to 32% in MYE medium showed linear it started production of usnic acid in 3–4 months
increment in growth rate of symbionts and the after inoculation and finally produced 0.16 mg/g dry
production of usnic acid (Fig. 4). The PEG and biomass (Fig. 4). As far as the addition of vitamin as
sucrose in water–agar and MYE media had approxi- nitrogen sources in the media is concerned, initially
mately equivalent effects on growth rate and the growth of mycobiont was observed after 3
production of usnic acid. However, irrespective of weeks of inoculation but throughout the culture
the concentrations of glucose added in the MYE period neither we could observe the growth of
medium, it promoted only the growth of mycobiont photobiont nor they did produce usnic acid. When
for 3 months and the symbionts could be visible we further tried with the addition of 4% sucrose or
only after four months of inoculation but it could 4% PEG with 2% glycine in the the MYE media that
not promote the production of usnic acid in culture yielded 5.62 g dry biomass of the symbionts with
even after 6 months of inoculation. The combina- 1.53 mg usnic acid/g dry biomass (Fig. 6). These
tion of 4% sucrose and 4% PEG in MYE medium results suggests that the addition of amino acid as
produced 7.63 g dry biomass with 3.9 mg usnic acid/ nitrogen sources in the media only promoted the
g dry biomass compared to 2.4 g dry biomass and growth of the symbionts but less effective than the
0.41 mg usnic acid/g dry biomass in MYE without sucrose and PEG as far as production of usnic acid in
having excess carbon sources. The addition of culture is concerned.
sucrose or PEG individually or combined in water–- Our results are in agreement with those that
agar medium, the growth of symbionts and the reported suppression of usnic acid production by
production of usnic acid were found to be lower amino acids (Yasuhiro et al., 2001). However, in
(Fig. 6). general, increased in the carbon concentration has
As far as nitrogen sources such as amino acids: increased the growth of biomass of the symbionts
glycine, asparagine, alanine or vitamins: thiamin and also the production of usnic acid in this lichen
(B1), riboflavin (B2), ascorbic acid (C) or biotin (H) species. This indicates that the excess sucrose or
on the growth of symbionts and production of usnic PEG enhance the growth and activate the pathways
acid are concerned, glycine did promote the growth producing lichen substances. Thus at least some
of symbionts but their rate of growth and produc- parts of physiological activation of mycobionts

6 3.5 3.0 5
Usnic acid content (µg / g dry biomass)

Usnic acid content (µg / g dry biomass)

3.0 2.5 4
5
Symbiont dry biomass (g)
Symbiont dry biomass (g)

2.5
4 2.0 3
2.0

3 1.5 1.5 2

1.0 1.0 1
2
0.5
1 0.5 0
0.0

0 0.0
60 120 180 4 8 12 16 20 24 28 32
Days Concentration (g / l)

Figure 3. The symbiont growth and the production of Figure 4. Influences of various carbon and nitrogen
usnic acid content of lichen U. ghattensis at various days concentrations in the malt–yeast extract (MYE) media
under various culture conditions: on the growth of symbionts and the production of usnic
acid in lichen U. ghattensis in vitro.
Symbiont biomass Usnic acid content
Sucrose Sucrose Symbiont biomass Usnic acid content
PEG PEG Sucrose Sucrose
Glycine Glycine PEG PEG
Sucrose+PEG+glycine Sucrose+PEG+glycine glycine glycine
 ARTICLE IN PRESS
236 B.C. Behera et al.

3.0 0.45 Many lichenologists have also studied the effects

Symbiont dry biomass 0.40 on cultures of various carbon and nitrogen sources.
2.5 Usnic acid content

Usnic acid (µg / g dry biomass)

In a more recent study a small amounts of salazinic
Symbiont dry biomass (g)

0.35
2.0 and usnic acids in axenic cultures of Usnea
0.30
orientalis grown on a malt–yeast medium was
1.5 0.25 detected and the production of both compounds
0.20 increased when it was grown together with its
1.0
0.15
natural photobiont (Kon et al., 1997).
0.5 In our study sucrose was supplied for an alter-
0.10
native reason. The U. ghattensis is an epiphytic
0.0 0.05 lichen which grows on the bark of trees, and is
ar ar ar ar ar
Ag Ag Ag Ag Ag attached by holdfast. Sucrose is the transport sugar
5% 10
%
20
% 0 % 0 %
E-1 E-2
MY MY in higher plants and since most epiphytic lichens
Culture conditions growing on trees have holdfast which tightly
anchors to the tree bark, it seems reasonable to
Figure 5. Symbiont growth and production of usnic acid
of lichen U. ghattensis in different concentrations of agar suggest that they might absorb sucrose from the
in water and malt–yeast extract (MYE) after 6 months of tree bark. Such carbon transfer has yet to be
inoculation. demonstrated. However, the relatively high growth
rates of U. ghattensis cultures on the sucrose
10 5 containing medium suggests that the mycobiont
Symbiont dry biomass might have a strong preference for sucrose con-
4
sistent with the utilization of this sugar as a carbon
Usnic acid (µg / g dry biomass)

8 Usnic acid content

Symbiont dry biomass (g)

3
source in nature.
6 In conclusion, our results suggest that the
2 production of the usnic acid by the symbionts in
4 culture may be due to the combined effects of the
1 high osmotic pressure of the medium as well as the
2 nutritional conditions. Lichenization seems to be
0
important not only for the carbon-source, but also
0
for giving constant environmental (culture) condi-
su c
ros
e
%
G
PE glycin
e
%
G e
PE glycin ucro
s
se
%
PE
G
gly
cin
e
%
G
PE glycin
e
tions to the symbionts.
4% r +4 2% s e +4 2% +4% r + 4 + 2% e + 4 + 2%
ar
+
Ag 20%
a
Ag Agar
+
%
suc
ro
%
PE
G +
Ag
ar
0%
Ag
a
Ag
ar
su c
ro s
PE
G The present culture methods afforded a rela-
20% 20% ar +4 +4 20% E + 2 + 20% r +4% e + 4%
Ag
20% 4% s
u crose
M Y E+ MY
M Y E A ga
ucro s tively high yield (7.63 g dry biomass) that could be
20% 4% s
ar
+ E+ ar + easily scaled up to provide significant quantities of
Ag MY Ag
20% 20%

MY
E+ usnic acid. This could encourage future investiga-
Culture conditions
tors to screen this and related metabolites for their
bioactivity and possible applications in pharmaceu-
Figure 6. Comparision of symbiont growth and produc- tical research.
tion of usnic acid of lichen U. ghattensis in water–agar
and malt–yeast extract (MYE) media supplemented with
carbon and nitrogen sources.
Acknowledgements
substituted by sucrose and PEG. Over all increasing We are very grateful to the Department of
of agar concentration in the media increased the Biotechnology, Government of India, New Delhi,
usnic acid production by the symbionts in culture for the financial support (Grant No. BT/PR 3133/
probably due to the osmotic conditions. Further- BCE/08/237/2002 dated 21.02.2003).
more, our results are in agreement with those that
reported the periods of desiccation and higher agar
concentration required for the production of larger
amount of usnic acid in lichen culture in vitro
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