protocol

Methods for isolation of marine-derived endophytic

fungi and their bioactive secondary products
Julia Kjer, Abdessamad Debbab, Amal H Aly & Peter Proksch
Institut fr Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universitt Dsseldorf, Dsseldorf, Germany. Correspondence should be addressed to
P.P. (proksch@uni-duesseldorf.de).
Published online XX XX 2009; doi:10.1038/nprot.2009.233

Marine-derived fungi have been shown in recent years to produce a plethora of new bioactive secondary metabolites, some
of them featuring new carbon frameworks hitherto unprecedented in nature. These compounds are of interest as new lead
structures for medicine as well as for plant protection. The aim of this protocol is to give a detailed description of methods
useful for the isolation and cultivation of fungi associated with various marine organisms (sponges, algae and mangrove
plants) for the extraction, characterization and structure elucidation of biologically active secondary metabolites produced
by these marine-derived endophytic fungi, and for the preliminary evaluation of their pharmacological properties based on
rapid in house screening systems. Some results exemplifying the positive outcomes of the protocol are given at the end.
From sampling in marine environment to completion of the structure elucidation and bioactivity screening, a period of at
least 3 months has to be scheduled.

INTRODUCTION
Terrestrial fungi have for long been known as a rich source of
biologically active secondary metabolites. Since the discovery
of penicillin by Sir Alexander Fleming in 1928, which resulted
in a breakthrough in the treatment of bacterial infections, fungi
have become an important source of drugs for the treatment of a
variety of diseases. Beside other well-known antimicrobial agents
of fungal origin like fusidic acid and griseofulvin1, new semi
synthetic antifungal drugs like anidulafungin (Eraxis), caspafungin (Cancidas) and retapamulin (Altabax) are likewise derived
from fungal metabolites2,3. With the discovery of cyclosporine
isolated from Tolypocladium inflatum in 1971 an important step in
immunopharmacology was taken and improvements in the field
of organ transplantation and treatment of autoimmune diseases
are still in progress, like the introduction of substances such as
the fungal-derived mycophenolic acid (Myfortic) to the market
in 2004 (ref. 2).
Further pharmacologically important fungal metabolites include
antilipidemic drugs collectively known as statins with their parent
compounds mevastatin and lovastatin isolated from Penicillium
citrinum and Aspergillus terreus, respectively. Statins reduce blood
cholesterol levels and are used for the treatment of coronary
diseases2,4.
Fungal metabolites are, however, not only indispensable for
medicine but are also important for plant protection, as demonstrated by the discovery of the strobilurines that were first isolated
from Strobilurus sp. and served as lead compounds for synthetic
fungicidals such as trifloxystrobin (Flint)5.
However, the rediscovery of high numbers of previously
described metabolites has to some extent precluded the study of
traditional terrestrial sources of fungi6 and recently, interest of
natural product chemists and pharmacologists alike has turned
to so far less investigated habitats and ecological niches such as
the oceans. The oceans cover nearly three-quarters of the earths
surface and can be considered a soup of essentially all imaginable
types of microbes6,7. Ecological niches, e.g. deep-sea hydrothermal
vents, mangrove forests, algae and sponges provide distinct
habitats for the isolation of specific micro-organisms 8. Marine

micro-organisms including bacteria, cyanobacteria, microalgae

and fungi have become an important source of new pharmacologically active metabolites. Recent reviews demonstrate the
importance of these organisms as potential sources of pharmaceutical leads625. Especially, marine fungi have shown promising potential as sources of new and bioactive natural products as
suggested by the chemical diversity of their secondary meta
bolites6 even though sample collection and preparation as the
first step might be more difficult for marine-derived fungi than
for terrestrial material because of difficulties inherent to the
collection in marine environment such as the need for scuba
diving. Although the most famous group of bioactive compounds
obtained from marine-derived fungi are still the cephalosporines
with cephalosporine C, first isolated by G. Brotzu in 1945 from
a marine strain of Acremonium chrysogenum26, there are also
more recent promising examples. These include halovir and
several naturally occurring analogs, which are potent inhibitors
of Herpes simplex viruses 1 and 2. Synthetic studies that were
carried out for these substances allowed insight into structure
activity relationships22,27. The new alkaloid sorbicillactone A
isolated from the sponge-derived fungus Penicillium chrysogenum showed promising activity against leukemia cells without
exhibiting notable cytotoxicity. Large-scale fermentation of
the fungus yielded sufficient amounts of the compound for
preclinical investigations22,28. Overall, research on marinederived fungi has led to the discovery of more than 270 new
natural products mainly from the early 1990s to mid 2002
(with more than two-thirds of these compounds isolated from
sponge- and plant-derived fungi), whereas more than 330 new
metabolites were reported only in the 5 years period between 2002
and 2006 (refs. 6,12,22,29) including numerous compounds with
new carbon skeletons. This sharp rise in numbers indicates
a growing interest in marine-derived fungi as sources of new
bioactive metabolites.
The aim of this paper is to provide a detailed protocol on
the purification and cultivation of fungi from various selected
marine macro-organisms (sponges, algae and mangrove plants),
nature protocols | VOL.4 NO.12 | 2009 | 

protocol
Host organisms

15: 1 week

Small-scale fermentation
in liquid medium
+EtOAc
UltraTurrax

15: Isolation of fungi

3 Weeks

Homogenization and
extraction

Several min

Cell suspension
6: Purification
on malt agar

6: 2 weeks

7: 2 d

Pure culture

Several min

Filter
7: Taxonomic identification
(by PCR and gene sequencing,
followed by BLAST search)

Filtrate
Several hours
Water phase

8: Each 3
4 weeks

8: Small-scale
fermentation on rice or
in liquid medium for
screening purpose

Based on chemical
screening/bioactivity results in largescale fermentation of promising strains

EtOAc
Evaporate
~1 h per 1 EtOAc

Residue
90% MeOH
+ n-Hexane

911: 13 d

911: Extraction with EtOAc

~1 h

8: Large-scale
fermentation

90% MeOH
1214: Fractionation and
isolation of secondary products

n-Hexane

Figure 2 | Estimation of the time required for fermentation of fungi and

workup of extracts from liquid medium on small-scale basis.

16: Bioassays
(cytotoxicity and antimicrobial activity)
1216: At least
24 weeks
15: Structure elucidation

Figure 1 | Schematic overview on important steps involved in the isolation

of fungi from marine sources and in the isolation and identification of their
secondary metabolites.

as well as on the isolation, characterization and structure eluci

dation of biologically active secondary metabolites produced
by these fungi when grown in liquid or on solid media. An
overview including an approximate time schedule is shown
in Figure 1.
In this protocol we describe the isolation of endophytic marinederived fungi from inner parts of living tissues of marine invertebrates, algae and mangrove plants. Endophytic fungi are referred
to as fungi that spend, at least, part of their life inside healthy
tissues of host organisms regardless of the fact whether the host
is a plant or an animal30. For the isolation of marine-derived
fungi, especially for the isolation of fungi from sponges, alternative methods compared with those described in this protocol are
available. For example, instead of cutting pieces from surfacesterilized samples, it is also possible to drop squeeze water from
the sponge on an agar slant or to excise an inner piece of the tissue after thoroughly rinsing it with sterilized sea water without
previous surface sterilization3133. Likewise, several other media
for successful isolation and fermentation of marine fungi like
potato dextrose agar, barley spelt solid substrate or solid malt
extract medium17,31 as well as addition of specific nutrients or
of sub-lethal doses of fungicides to restrict fast-growing fungi34,
have been described in the literature. Numerous fermentation conditions such as shaking versus static cultures, application of different temperature regimes or employment of larger
fermenters that can provide varying aeration17,31,35, have likewise
been reported. However, all conditions employed in cultivating marine-derived endophytic fungi suffer from limitations
as different cultivation techniques usually select only a small

 | VOL.4 NO.12 | 2009 | nature protocols

fraction of the fungal community present, thereby providing a

limited and selective window of the actual diversity that is present
in a given sample36. Furthermore, it must be remembered that
different culture conditions might also result in different chemical
patterns of metabolites formed, as already stated, in the OSMAC
(one strain, many compounds) concept that makes deliberate
use of the chemical plasticity of microorganisms as a response
to varying conditions of culture37. However, after conducting
numerous fermentation experiments with endophytic fungi
using different culture conditions, we discovered the methods
described in this protocol as being most suitable for our purposes, as they generally gave reproducible results regarding the
isolation of endophytic fungi and regarding the patterns of
secondary metabolites that are produced by these fungi32,3840.
In our hands we have obtained optimum results when culturing fungi on different solid or in liquid media at room temperature (2025 C), followed by extraction of mycelia and
growth media with organic solvents. Flow charts briefly describing the workup procedures of the respective fungal cultures
and the time needed for each step are shown in Figures 24.
The crude extracts are then separated using various chromatographic
techniques and the isolated bioactive compounds are analyzed
by HPLCDAD for their purity followed by structure elucidation
by MS and NMR using state of the art techniques. Chiral compounds
may be derivatized to determine their absolute configuration.
Chromatographic separation of fungal extracts usually proceeds
guided by bioassays, thereby directing the isolation strategy
towards the discovery of bioactive constituents. After the extraction
of cultures, the obtained extracts are treated in a similar manner
as described earlier for extracts from marine macro-organisms.
As the chromatographic separation, structure elucidation and
bioactivity screening for extracts derived from marine macroorganisms have been described in detail in a previous protocol41,
only those steps that differ from the described procedure are
depicted in this protocol.

protocol
Large-scale
fermentation

Mycelium
Several hours

3 Weeks

Fermentation
on rice medium
+EtOAc
Filter

Medium
EtOAc

Several hours

Evaporate

Residue
Several hours

Residue

90% MeOH
+ n-Hexane

90% MeOH

Water phase EtOAc

Evaporate

Evaporate

~1 h
per 1 EtOAc

Residue
90% MeOH
+ n-Hexane

n-Hexane

~1 h per 1 EtOAc

90% MeOH

small scale, ~ 1 h
large scale, several hours

90% MeOH
+ n-Hexane

Filter
MeOH extract

~1 h
per 1 MeOH

Overnight + 30 min

EtOAc extract

+MeOH
UltraTurrax
Cell suspension

~1 h

4 6 Weeks

Several hours

n-Hexane

Figure 3 | Estimation of the time required for fermentation of fungi and

workup of extracts from liquid medium on large-scale basis.

Two examples taken from our group that focus on polyketides

of fungal origin will be used to highlight the structural variability
of natural products obtained from marine-derived fungi. Other
results can be found in ref.33,38,39,4244.
Experimental design
Sampling and general consideration before isolation of fungi.
Transport the collected material in suitable containers (for
sponges, algae or the like containing sea water). For animal and
algal tissues with high water contents, isolation of fungi must be
carried out within the next hours to avoid growth of ambient
bacteria. Isolation of fungi from plant material like mangrove
leaves can take place within 12 d as long as the samples are
kept at low temperature (510 C) and excess condensation
is prohibited. Otherwise there is the risk that phylloplane fungi
will colonize the plant material leading to false results during the
isolation of endophytes.
Identification of fungi. As we restrict ourselves to cultivation
of non-pathogenic fungi, any fungal cultures with documented
pathogenicity45,46 towards humans are discarded immediately after
taxonomic identification and excluded from further investigation.

90% MeOH

n-Hexane

Figure 4 | Estimation of the time required for fermentation and workup of

extracts from rice medium.

Thus, identification of the isolates is carried out at an early stage

during workup. Taxonomic identification of fungal strains is
achieved by DNA amplification and sequencing of the fungal ITS
region39 as described in Box 1. However, fungi can also be identified by morphological characteristics using e.g., a commercially
available service such as Centraalbureau voor Schimmelcultures
(CBS), Utrecht, The Netherlands.
Biological assays. Biological activities of extracts, fractions
and pure compounds are tested in a number of rapid in house
bioassays that can be used for bioactivity-guided isolations.
Those used in our laboratory are briefly described in Box 2.
Controls. Negative controls during the isolation of endophytic
fungi from marine organisms are necessary to detect contamination from external parts of the slices, from phylloplane or
from airborne micro-organisms. Therefore, surface-sterilized marine samples are pressed onto a petri dish containing
isolation medium before aseptic cutting and inoculation on
a second agar plate. If fungal growth can be observed on the
negative control dishes, the positive plates are not used for
further purification procedures that aim at the isolation of
true endophytic fungi.
For all bioassays, negative and positive controls to compare the
effectiveness of the tested fractions and pure compounds have to
be used as described.
Q1

Box

1 | IDENTIFICATION OF FUNGI

Q10

Taxonomic identification of the fungal strains is achieved by DNA amplification and sequencing of the fungal ITS region39.
For this purpose, a piece of fungal mycelium (0.5 cm2) is sampled from an agar plate, lyophilized in a freeze dryer and powdered in a
mixer mill after adding a tungsten carbide bead.
DNA isolation is carried out using the DNeasy Plant Mini Kit according to the manufacturers protocol. The procedure includes cell
lysis, digestion of RNA by RNAse A, removing of precipitates and cell debris, DNA shearing, DNA precipitation and purification.
This is followed by DNA amplification using Hot StarTaq Master Mix Kit and the primer pair ITS1 and ITS4 in an iCycler thermocycler.
The PCR product is loaded onto agarose gel (2% agarose in 100 ml TBE buffer, 10 l SybrSafe). After electrophoresis at 70 V for
60 min, the band corresponding to the desired PCR product (~size 550 bp) is removed from the gel slice using the PerfectPrep Gel
Cleanup Kit following the manufacturers protocol.
Pure PCR products are submitted for sequencing together with the primer ITS1 to a commercial service (e.g., SeqLab GmbH,
Goettingen and BMBF, Dsseldorf, Germany) and the base sequence is compared with publicly available databases (GenBank) with
the help of Blast-Algorithmus.

nature protocols | VOL.4 NO.12 | 2009 | 

protocol
Box

2 | BIOACTIVITY SCREENING TESTS

Agar diffusion assay
According to the BauerKirby test (DIN 59040), susceptibility disks are impregnated with 250 or 500 g of crude extracts and 50 or
100 g of pure compounds, respectively, and placed on agar plates that have been inoculated with either the standard bacterial strains
Bacillus subtilis DSM 22109 (=ATCC 11774), Escherichia coli DSM 10290 (=ATCC 15766), the yeast Saccharomyces cerevisiae DSM 1333
(=ATCC 9763) or the fungi Cladosporium herbarum DSM 63422 or Cladosporium curcumerinum DSM 62122.
The plates are checked for inhibition zones after 24 h of incubation (37 C for bacteria, 27 C for yeast) or after 7 d (22 C for
fungi), respectively.
Negative controls are only treated with the respective solvents, positive controls are treated with penicillin G, streptomycin and
gentamycin (for bacteria) or nystatin (for fungi)33,47.
CYTOTOXICITY TESTS
Brine shrimp assay
Eggs of Artemia salina are hatched in artificial seawater at room temperature and under daylight conditions. After 48 h, 20 nauplii
are transferred into each test vial using a binocular and seawater is added to 5 ml. Pure compounds are dissolved in 40 l of DMSO
to give final concentrations of compounds of 10 or 100 p.p.m. The experiment is kept under illumination and mortality is determined
after 24 h by counting the survivors with the aid of a 3 magnifying glass. Vials containing 40 l of DMSO (negative controls) are
also prepared48.
The microculture tetrazolium (MTT) assay is carried out as previously described for marine macro-organisms41.

MATERIALS
REAGENTS
Bacto agar (BD, cat. no. 214010)
Malt extract (Merck, cat. no. 105391)
Artificial sea salt (Sera, cat. no. 05420)
Chloramphenicol (Sigma, cat. no. C0378)
Sodium hydroxide (NaOH, Sigma, cat. no. S5881)
Hydrochloric acid (HCl, Sigma, cat. no. 258148)
Yeast extract (Fluka, cat. no. 70161)
Peptone (Merck, cat. no. 111931)
Glucose (Caelo, cat. no. 5247)
Rice (parboiled, any supermarket brand)
Glycerin (Roth, cat. no. 4043)
Penicillin G (Sigma, cat. no. P3032)
Streptomycin (Sigma, cat. no. S6501)
Gentamycin (Serva, cat. no. 22185)
Nystatin (Sigma, cat. no. N4014)
Antimicrobial detergent Melsept SF (B. Braun, cat. no. 18907)
Tungsten carbide bead (Qiagen, cat. no. 69997)
Agarose (Sigma, cat. no. A9414)
TBE buffer (Merck, cat. no. 106177)
DNeasy Plant Mini Kit (Qiagen, cat. no. 69104)
Hot StarTaq Master Mix Kit (Qiagen, cat. no. 203443)
RNAse A (Invitrogen, cat. no. 12091-039)
Primers: ITS1 and ITS4 (Invitrogen; individual order)
SYBR Safe DNA gel stain (Invitrogen, cat. no. S33102)
DNA loading buffer (Eppendorf, cat. no. 0032 006.850)
PerfectPrep Gel Cleanup Kit (Eppendorf, cat. no. 0032 007.740)
Solvents:
Solvents for extraction and chromatographic separation (see REAGENT
SETUP)
Solvents for high performance liquid chromatography, HPLC, for
measuring optical rotation, for NMR spectroscopy are used as described
in a previous protocol41
Spray reagents are used as described in a previous protocol41
N-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfeys reagent, Fluka,
cat. no. 42095)
Standard amino acids (ICN, Sigma)
EQUIPMENT
Sterile plastic petri dishes, diameter 9.4 cm (Greiner, cat. no. 633161)
Parafilm M (Marienfeld, cat. no. 74 038 10)
Sterile tubes, 15 ml, 120 mm 17 mm (Sarstedt, cat. no. 62553)
Susceptibility paper disks (5 mm diameter, Oxoid)
All micro-organisms are acquired from DSMZDeutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH
Artemia salina (Dohse Aquaristik, cat. no. 21350)
 | VOL.4 NO.12 | 2009 | nature protocols

Chromatographic stationary phases: Different materials from different

suppliers are possible, in our laboratory material is used as described in
a previous protocol41
Laminar air-flow (Herasafe HS15, Heraeus)
pH meter (420 Aplus, Orion)
Autoclave (Varioklav, H&P)
Microcentrifuge (Biofuge pico, Heraeus)
Freeze dryer (Lyovac GT2, pump Trivac D10E, Savant)
PCR machine (iCycler, Bio-Rad)
UV transluminator (GVM 20,Syngene)
Mixer mill (MixerMill MM30)
Power supply for electrophoresis (PowerPac 300, Bio-Rad)
Ultra Turrax (T18 basic, IKA)
Vacuum pump (type 4EKF 56CX-4, Greiffenberger Antriebstechnik)
Nitrogen generator (UHPN 3001, Nitrox)
Deep freezer (80 C, 86-Freezer, Forma Scientific)
Shaker SM 30 A (Edmund Bhler, cat. no. 6101 000)
Balance (BL1500, Sartorius)
HPLC, MS and NMR: the equipment as described in a previous protocol is used41
REAGENT SETUP
Organic solvents for separation and purification Many organic solvents
of varying polarities may be used for chromatographic separation and
purification procedures, including acetone (! CAUTION highly flammable
and irritant), acetonitrile (ACN) (! CAUTION highly flammable and toxic),
dichloromethane (DCM) (! CAUTION harmful), ethanol (EtOH) (! CAUTION
highly flammable), ethyl acetate (EtOAc) (! CAUTION highly flammable
and irritant), n-hexane (! CAUTION highly flammable, irritant, harmful,
dangerous for the environment and toxic for reproduction), n-butanol
(n-BuOH) (! CAUTION flammable, harmful and irritant) and methanol
(MeOH) (! CAUTION highly flammable and toxic). They all are of analytical
grade. ! CAUTION All organic solvents must be handled carefully.
Wear protective clothing, safety glasses and gloves. They should be handled
under a fume hood and stored in a ventilated solvent cabinet.
Medium A for isolation of fungal strains from marine macro-organisms
and mangrove plants
Bacto agar

15 g

Malt extract

15 g

Artificial sea salt

24.4 g (for isolation from mangroves 10 g)

Chloramphenicol

0.2 g

pH

7.47.8 (adjusted with NaOH/HCl)

Dem. water

ad 1,000 g

Q2

Q3

protocol
The contents are combined in a flask big enough so that it is filled not
more than two-thirds, covered and autoclaved at 121 C for 20 min. After
cooling down to ~60 C, the medium is poured in sterile plastic petri dishes,
~25 ml medium per plate, under aseptic conditions. The prepared agar
plates can be kept under sterile conditions for upto 1 week before inoculation. ! CAUTION Flasks must not be closed tightly when being autoclaved.
The temperature of the autoclave has to be below 80 C before it can be
opened to prevent hot steam from emerging. Heat protective gloves and
safety glasses should be worn.
Medium B for purification and short term storage of fungal strains
Bacto agar

15 g

Malt extract

15 g

Artificial sea salt

24.4 g (for mangrove-derived fungi 10 g)

pH

7.47.8 (adjusted with NaOH/HCl)

Dem. water

ad 1,000 g

The contents are combined in a flask big enough so that it is filled not
more than two-thirds, covered and autoclaved at 121 C for 20 min. After
cooling down to ~60 C, the medium is poured in sterile plastic petri dishes,
~25 ml medium per plate, under aseptic conditions. The prepared agar plates
can be kept under sterile conditions for upto 1 week before inoculation.
! CAUTION Flasks must not be closed tightly when being autoclaved. The
temperature of the autoclave has to be below 80 C before it can be opened
to prevent hot steam from emerging. Heat protective gloves and safety glasses
have to be worn.
Liquid Wickerhams medium
Yeast extract

3g

Malt extract

3g

Peptone

5g

Glucose

10 g

Artificial sea salt

24.4 g (for mangrove-derived fungi 10 g)

pH

7.27.4 (adjusted with NaOH/HCl)

Dem. water

ad 1,000 g

All contents are mixed thoroughly. In all, 300 ml of the media is poured
in 1,000 ml Erlenmeyer flasks. The flasks are covered with a tissue lid and
autoclaved at 121 C for 20 min. ! CAUTION Tissue lids must be covered
with aluminum foil to prevent soaking during autoclaving. The temperature
of the autoclave has to be below 80 C before it can be opened to prevent hot
steam from emerging. Heat protective gloves and safety glasses have to be
worn. The medium can be stored for 12 d before inoculation.
Solid rice medium
Rice

100 g

Dem. water

110 g

The 1,000 ml Erlenmeyer flasks with the contents are covered with a tissue
lid and kept standing overnight to allow swelling of the rice kernels before
autoclaving at 121 C for 20 min. ! CAUTION Tissue lids must be covered with
aluminium foil to prevent soaking during autoclaving. The temperature of
the autoclave has to be below 80 C before it can be opened to prevent hot
steam from emerging. Heat protective gloves and safety glasses have to be
worn. The medium can be stored for 12 d before inoculation.
MexA medium for long-term storage
Malt extract

20 g

Yeast extract

0.1 g

Glycerin

50 g

Artificial sea salt

24.4 g (for mangrove derived fungi 10.0 g)

Bacto Agar

13 g

Dem. water

ad 1,000 g

The contents are combined in a flask big enough so that it is filled not more
than two-thirds, covered and autoclaved at 121 C for 20 min. ! CAUTION
During autoclaving the flasks must not be tightly closed. The temperature of the
autoclave has to be below 80 C before it can be opened to prevent hot steam
from emerging. Heat protective gloves and safety glasses have to be worn.
After cooling down to ~60 C the medium is poured in sterile tubes, each
with ~6 ml under aseptic conditions. The prepared flasks can be kept under
sterile conditions for upto 1 week before inoculation. CRITICAL All the
autoclaved media have to be stored under aseptic conditions to prevent
contamination with ubiquitous microbes from the environment.

PROCEDURE
Isolation and cultivation of fungi from marine organisms TIMING 69 weeks
1| Cut the samples (pieces of sponge tissue, algal biomass or mangrove leaves or other organs) into small segments
of approximately 1 cm 1 cm and rinse three times with sterile sea water to eliminate adherent surface debris.
CRITICAL STEP It is crucial to maintain aseptic conditions by working under a laminar air-flow for the isolation,
purification, transfer and growth of the fungal cultures to prevent contamination by ubiquitous micro-organisms.
2| Immerse a piece of the sample in EtOH 70% (vol/vol) for 60120 s for surface sterilization.
CRITICAL STEP If the treatment with EtOH is too short, the sterilization of the outer part is not complete; if the
sterilization time is too long, EtOH kills fungi in the inner parts of the tissue.
3| Dry the piece of tissue with sterile cotton cloth (or rinse with sterile artificial sea water) to stop the sterilization
with EtOH.
4| Streak the piece carefully over the surface of a first petri dish containing isolation medium A with sterilized tweezers,
then put it back onto sterile cotton cloth and cut it into smaller segments with a sterile razor blade (negative control).
5| Place the small pieces on a second petri dish containing isolation medium A so that the freshly cut edges are in direct
contact with the agar surface. Seal the agar dish with parafilm, label and store it at 2025 C.
Cultures are kept between 20 C and 25 C under daylight. Fungal growth from the cut segments usually begins after
23 d until 14 d after the onset of experiment.
? TROUBLESHOOTING
nature protocols | VOL.4 NO.12 | 2009 | 

protocol
6| Usually different fungal strains will develop from one sample. Isolate the individual strains by transferring hyphal tips
growing out of the cut tissue pieces with a sterile loop onto a fresh petri dish containing medium B.
CRITICAL STEP For purification of the fungal strains this step might be repeated several times until the colony is deemed
pure following macroscopic and microscopic analysis.
Depending on the culture condition and different fungal strains, growth can be observed after 23 d. Pure strains must
grow for ~12 weeks before further workup.
7| Submit pure fungal strains for taxonomic identification as described in Box 1.
8| For small-scale and large-scale fermentation, inoculate a pure fungal strain in a 1,000 ml Erlenmeyer flask containing
either 300 ml of liquid Wickerhams medium or 210 g of solid rice medium. For this purpose, cut a strain that covers the
surface of the inoculated petri dish into small pieces of ~1.5 cm 1.5 cm and transfer these pieces with a sterile loop into
an Erlenmeyer flask containing the sterilized medium.
Cultivation is carried out at room temperature under static conditions and daylight. Depending on the fungal growth,
cultures on liquid medium are incubated for 34 weeks, while on rice for 46 weeks.
9| Bring the fermentation to an end by adding 250 ml EtOAc to the culture flask and leave the flask closed for at least
24 h. EtOAc will increase the wettability of the spores and decrease the number of spores, which will get airborne once
the flask is opened.
! CAUTION To prevent the distribution of fungal fragments or spores, flasks should only be opened under a laminar air-flow.
10| For long-term storage, transfer the pure fungal strains to 10 ml BD Falcon tubes containing ~5 ml MexA medium.
When growth can be observed (after ~3 d, depending on the fungal strain), freeze the fungal strain stepwise: first place
it in a fridge at 4 C for 2 h, then in a freezer at 20 C for 2 h and finally place it in a deep freezer at 80 C.
PAUSE POINT Frozen pure fungal strains can be stored at 80 C.
For recovery from 80 C, thaw the frozen culture quickly in a water bath at 37 C and transfer a small piece with a sterile
loop to a petri dish containing medium B.
! CAUTION To prevent contamination of the environment with viable material, equipment that has been in contact with
fungal material must be autoclaved at 134 C for 15 min before reuse or disposal.

Q4

Extraction of small scale cultures TIMING ~2 d

11| Extract the cultured material by following the steps in options (A) small-scale liquid medium (Fig. 2); (B) small-scale
rice medium (Fig. 4); (C) large-scale liquid medium (Fig. 3); or (D) large-scale rice medium (Fig. 4).
(A) Small-scale liquid medium (Fig. 2)
(i) Mix the content of the culture flask including the added 250 ml of EtOAc thoroughly in an Ultraturrax at 4,000 min1
for cell destruction and extraction for 10 min.
(ii) Filter the mixture under vacuum using a Buchner funnel and discard the mycelium residue.
(iii) Transfer the culture filtrate into a separation funnel. Separate the EtOAc and H2O phases and extract the aqueous phase
twice with 300 ml EtOAc each. Wash the combined EtOAc phases with 100 ml dem. water to eliminate any remaining
sea salt.
CRITICAL STEP Each step of the extraction should be carried out with care under a fume hood. Complete separation
of the two immiscible liquid phases should be achieved before continuing.
For optimal extraction of the fungal biomass, usually three cycles are sufficient. However, if the EtOAc phase is still
colored after the third extraction, further exhaustive extraction with EtOAc should follow until the color fades.
! CAUTION To prevent contamination of the environment with viable material, an antimicrobial detergent (Melsept SF)
has to be added to the medium and to the disrupted fungal cells and kept in it for at least 1 h before disposal. Autoclaving is not possible at this stage because of the remaining traces of EtOAc that may cause explosion in the autoclave.
(B) Small-scale rice medium (Fig. 4)
(i) Cut the culture medium containing the mycelium into small pieces to allow exhaustive extraction with EtOAc.
For optimum extraction the mycelium with added extraction solvent should be kept on a shaker for 1 d.
(ii) Filter the contents under vacuum using a Buchner funnel and repeat the extraction with EtOAc until exhaustion.
(iii) Wash the combined EtOAc phases with 300 ml water to eliminate remaining sugar and starch.
CRITICAL STEP The aqueous and EtOAc phases should be left in a separation funnel until complete separation of
the two immiscible liquid phases is achieved.
For optimal extraction of the fungal biomass, usually three cycles are sufficient. However, if the EtOAc phase is still
colored after the third extraction, further exhaustive extraction with EtOAc should follow until the color fades.

 | VOL.4 NO.12 | 2009 | nature protocols

protocol
! CAUTION To prevent contamination of the environment with viable material, an antimicrobial detergent (Melsept SF) has to
be added to the medium and to the disrupted fungal cells and kept in it for at least 1 h before disposal. Autoclaving is not
possible at this stage because of the remaining traces of EtOAc that may cause explosion in the autoclave.
(C) Large-scale liquid medium (Fig. 3)
(i) Separate fungal mycelia from the culture media and cover the mycelia with ~5 liters of MeOH.
! CAUTION To prevent the distribution of fungal fragments or spores, flasks are only opened under a laminar air-flow.
The mycelia are left in MeOH overnight.
(ii) Disrupt and extract the cells for 10 min at 4000 min1 using an Ultraturrax.
(iii) Filter the mixture under vacuum using a Buchner funnel.
(iv) Repeat extraction in the same manner until exhaustion (23 times).
(v) Extract the culture media in the same manner as described for the extraction of small-scale cultures to obtain the
EtOAc extract.
! CAUTION To prevent contamination of the environment with viable material, antimicrobial detergent has to be added
to the extracted mycelia and growth medium and kept in it for at least 1 h before disposal. Autoclaving of the residues
is not possible because of the remaining solvent.
(D) Large-scale rice medium (Fig. 4)
(i) The extraction of the large-scale rice media is carried out in the same manner as described for the small-scale cultures
using ~10 liters of EtOAc.
? TROUBLESHOOTING
Workup of obtained crude extracts TIMING several daysweeks
12| Workup of the crude extracts following the steps in option (A) for smallscale extracts and option (B) for large-scale
extracts, respectively.
(A) Small-scale extracts
(i) Dry the EtOAc extract (either from rice or liquid culture) under vacuum (~200 mbar) using a rotary evaporator at
40 C to give a solid or oily residue.
Depending on the amount of EtOAc used and on the size of the round-bottom flask this step takes about 40 min liter1
of EtOAc.
PAUSE POINT The dried extract can be stored in the deep freezer until further workup.
(ii) Partition the residue between n-hexane and 90% (vol/vol) aqueous MeOH in a ratio of 1:1 (vol/vol) (~150 ml each).
CRITICAL STEP The solvent fractionation should be carried out with care under a fume hood until complete
separation of the two immiscible liquid phases is achieved.
(iii) Dry each fraction under vacuum (~200 mbar) using a rotary evaporator at 40 C to give an oily or solid residue.
(iv) Submit all fractions to TLC, analytical HPLC, LCMS and also to bioactivity assays (see Box 3, ref. 41).
(v) Further investigation depends on the results of the chemical and biological screening and on the taxonomic identity
of the fungus. If the fungus is non-pathogenic and if the extract displays promising activity in bioactivity screening,
cultivate the fungal strain on a large-scale basis either in 20 liters of liquid Wickerhams medium (67 Erlenmeyer flasks)
or on 4.2 kg solid rice medium (20 flasks). From these large-scale fermentations, usually an amount of crude extract
sufficient for the subsequent isolation and structural identification of pure secondary metabolites can be obtained.
(B) Large-scale extracts
(i) Dry the EtOAc or the MeOH extracts (either from rice or liquid culture) under vacuum (~200 mbar) using a rotary
evaporator at 40 C to give solid or oily residues.
Depending on the volume of EtOAc used and on the size of the round flask this step takes about 40 min liter1
of the solvent.
PAUSE POINT The dried extract can be stored in the deep freezer until further workup.
(ii) Partition each residue between n-hexane and 90% (vol/vol) MeOH in a ratio of 1:1 (vol/vol), using as little solvent
as possible.
CRITICAL STEP The solvent fractionations should be carried out with care under a fume hood. Complete separation
of the two immiscible liquid phases should be achieved before continuing.
(iii) Submit all fractions to TLC, analytical HPLC, LCMS and also bioactivity assays such as the agar-diffusion assay or
brine shrimp assay described in Box 2 or the MTT assay described in reference 41.
CRITICAL STEP Based on the obtained results, the success of the solvent fractionation can be monitored easily by
differences in both bioactivities and HPLC/TLC profiles of the different fractions.
13| In accordance with the diverse properties of the components of the fractions, two different procedures for purification
can be applied:
nature protocols | VOL.4 NO.12 | 2009 | 

protocol
Box

3 | DERIVATIZATION METHODS

Q11

Q5
Q6

Determination of absolute stereochemistry by Mosher reaction has been described in detail in a protocol on isolation and
structural elucidation of metabolites from marine invertebrates38,41.
Determination of the absolute configuration of amino acids by Marfeys analysis
Marfeys reagent (FDAA=N-(2,4-dinitro-5-fluorophenyl)-L-alaninamide) is used as a reagent for derivatization of D- and L-amino acids
that are obtained after hydrolysis of cyclic or linear peptides to determine their absolute configuration. The obtained diastereoisomers
can be easily differentiated and identified by their retention times following HPLC analysis on RP columns and comparison with
commercially available D- and L-amino acids that have been treated in the same way47,49.
1. Mix 50 l of 50 mM of each commercially available standard amino acid (D- or L-form) that is of interest in H2O, 20 l 1 M NaHCO3
and 100 l of 1% Marfeys reagent in acetone and heat at 40 C for 1 h.
2. Stop the reaction by addition of 10 l of 2 M HCl.
3. Freeze the derivatized product to 80 C, dry it in a freeze dryer, redissolve it in MeOH and analyze it by HPLC or by LCMS.
4. Hydrolyze your isolated peptide (0.51 mg) in 12 ml 6 N-HCl at 110 C for 24 h under N2 atmosphere until complete hydrolysis and
liberation of the amino acids.
5. Cool the hydrolysate containing the mixture of free amino acids, dry it and redissolve it in water to achieve a final concentration of
~50 M. Proceed in the same way as applied to standard amino acids (see 13).
6. Compare the retention times (HPLC or LCMS) of the derivatized standard amino acids and of the derivatized amino acids obtained
following hydrolysis of the peptide to distinguish D- and L-amino acids.

For low- or medium-polar compounds refer to option (A), for polar compounds refer to option (B).
(A) Low- or medium-polarity fractions
(i) Fractions containing low- or medium-polar compounds are further fractionated and purified using MPLC methods like
VLC. Then, further purification proceeds by column chromatography (CC) using either normal or reversed stationary
phase and a suitable mobile phase to elute the components.
Refer to reference 41 for advice on the choice of stationary phase and how to setup the experiment.
(B) Polar fractions
(i) Highly polar fractions contain water-soluble organic compounds. In our experience, a good procedure is to use
reversed phase CC (e.g., RP-18) or ion-exchange resin beds such as HP-20 eluted gradually from water to MeOH, to
eliminate the remaining mineral salts and sugars present in these fractions. Refer to REAGENT SETUP for further advice.
Chromatographic methods are carried out as described in a previous protocol dealing with workup of extracts derived
from marine macro-organisms41.
? TROUBLESHOOTING

14| Continue the purification procedures until compounds of sufficient purity is obtained to allow structural elucidation.

Q7

15| Structure elucidation is carried out using various spectroscopic methods, mainly MS and NMR (1 d and 2 d). For the
elucidation of the absolute configuration of new chiral natural products, derivatization methods such as the preparation
of Mosher esters (see reference C) or by using Marfeys method (see Box 3) are sometimes necessary (alternatively, the
absolute configuration may be elucidated by X-ray crystallography or by CD spectroscopy followed by quantum chemical
calculations32).

16| Once the individual components are isolated in pure form and structurally identified, they should be subjected to
bioactivity testing.
17| Study the structureactivity relationships to identify optimized compounds which may serve as drug leads from natural
sources.
Q8

TIMING
Steps 15: 1530 min per isolate (growth ~12 weeks)
Step 6: 5 min per strain
Step 7: see Box 1
Step 8: ~5 min per strain (growth ~35 weeks)
Steps 9 and 10: each ~5 min
Step 11 A(iiii): 4560 min
Step 11 B/D(i): 5/30 min (plus 1 d for extraction)

 | VOL.4 NO.12 | 2009 | nature protocols

protocol
Step 11 B/D(ii): ~10 min ( plus 1 d for extraction) (twice)
Step 11 B/D(iii): ~10 min
Step 11 C(i): ~5 min (plus extraction overnight)
Step 11 C(iiv): 2030 min (23 times)
Step 11 C(v): 23 h
Steps 1317: The timing of these steps is dependent on chosen columns, complexity of the extract and the kind of isolated
compounds.
See also Figures 14.
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
Table 1 | Troubleshooting table.
Step

Problem

Possible reason

Solution

Fungal growth on
negative controls

Insufficient surface sterilization

If you are not sure how delicate the surface of your source
organism is, then try different times for surface sterilization,
e.g. 30, 60 and 120 s. The plates with growth on the respective negative controls or those without any growth can be
discarded

No growth at all

Time of surface sterilization toolong,

fungi residing in the tissue are killed

11

Insufficient separation
of the two phases

Formation of an emulsion because of

extracted compounds

Leave standing overnight in the separation funnel; centrifugation also helps

13

No or insufficient
separation

Elution of the column is too fast

Reduce the speed of the mobile phase (for Sephadex material

~10 drops min1, silica <20 drops min1, depending on the
size of the column)
Always test on TLC to check stationary and mobile phase
before column chromatography
Combine all fractions again and find a better separation
system

Inappropriate stationary or mobile

phases
Column too small
13, 14

Loss of substance

Adsorption on stationary phases

Q20

Always keep some of your fraction to be able to repeat

separation steps

ANTICIPATED RESULTS
Examples taken from our group that illustrate the application of the described procedures for the isolation of spongeand mangrove-derived fungi and the subsequent isolation
and structure determination of bioactive compounds will be
described.

O
O

O
H

HO

OH

OH

OH

Anthraquinones and betaenones (Fig. 5)

Two new betaenone derivatives (1,2) and three new

1,3,6,8-tetrahydroxy anthraquinone congeners (35) were
obtained from the fungus Microsphaeropsis sp. isolated from
the inner parts of the marine sponge Aplysina aerophoba32.
The EtOAc extract of the fungus that had been grown in
liquid medium (including 2.4% (wt/vol) of artificial sea salt)
under static conditions at room temperature for 41 d,
was evaporated under reduced pressure. The residue was
partitioned between EtOAc (4 400 ml) and H2O (400 ml)
to eliminate the remaining salt. The organic phase was
taken to dryness and subjected to VLC on silica gel using
step gradient employing CH2Cl2 and MeOH (100:0, 98:2,
95:5, 90:10, 80:20 and 30:70 vol/vol) as solvent systems.

HO
O

OH

HO

OH

OH

OH

OH

OH

OH

O
HO
OH

OH

OH
O

HO
HO

OH
O

Figure 5 | New betaenones and anthaquinones from the sponge-derived

fungus Microsphaeropsis sp.

nature protocols | VOL.4 NO.12 | 2009 | 

protocol
Fractions 3 and 4 from the obtained ten fractions were pooled and chromatographed over a Sephadex LH-20 column
with MeOH as eluent. From the obtained 17 fractions, fractions 6 and 7 contained the betaenone congeners, whereas the
anthraquinone derivatives were obtained from the yellow colored fractions 11, 12 and 13.
Purification of the betaenones was accomplished by chromatography on an RP-18 column. 10-Hydroxy-18-methoxybetaenone
(1) was obtained from fraction 7 with MeOH:H2O:TFA (95:5:0.1 vol/vol) as eluent and 10-hydroxy-18-N-2-naphthyl-N-phenyl
aminobetaenone (2) from fraction 6 with MeOH:H2O:TFA (90:10:0.1 vol/vol) as eluent.
The anthraquinone congeners were purified by semi-preparative HPLC on a C18 column using a gradient of H2O and
MeOH as follows: 0 min, 80% MeOH (vol/vol); 20 min, 90% MeOH (vol/vol); 25 min, 90% MeOH (vol/vol); and 30 min,
100% MeOH (vol/vol).
All compounds were readily identified by their spectroscopic data. Through-bond homonuclear and heteronuclear correlations were used to establish assignments and atom connectivities. The relative stereochemistry of 10-hydroxy-18-methoxy
betaenone (1) was determined from a ROESY experiment.
As similar metabolites had previously been described as inhibitors of protein kinases, which are of particular interest as
targets for the development of novel anticancer drugs, the isolated compounds were tested for inhibition of protein kinases
in vitro.
For a first assessment of the kinase inhibitory potential, an isoenzyme of the protein kinase family C, the cyclin-dependent
kinase 4 in complex with its activator cyclin D1 and the tyrosine kinase domain of the epidermal growth factor receptor
were selected.
Of the two obtained betaenone derivatives only 10-hydroxy-18-methoxybetaenone (1) inhibited all three kinases
(with ED50 values of 36.0, 11.5 and 10.5 M, respectively), whereas 10-hydroxy-18-N-2-naphthyl-N-phenylaminobetaenone
(2) showed no inhibitory effect, thus suggesting the enone side chain as a promising position for further optimization of
such compounds towards higher potency and better selectivity.
Alternaria metabolites (Fig. 6)

From the endophyte Alternaria sp. isolated from leaves of the Chinese mangrove plant Sonneratia alba, several natural
products, some of them structurally related to alternariol (611), perylene quinones and new compounds (1217) were
isolated40.
The crude EtOAc extract obtained after fermentation of the fungus on solid rice medium was partitioned between n-hexane
and 90% (vol/vol) aqueous MeOH. After VLC of the MeOH extract using silica gel as stationary phase and a step gradient
employing CH2Cl2 and MeOH as solvent
systems, fraction 3 (80% (vol/vol)
O
O
HO
OH
HOOC
OH
O
OH
O
CH2Cl2) was chromatographed over
HO
silica gel using CH2Cl2 and MeOH (90:10
HO
HO
vol/vol) as eluent mixture. Fractions 3
O
HO
O
HO
and 4 were combined and re-chromato
graphed by normal-phase VLC using a
O
step gradient of CH2Cl2 and MeOH as
O
O
O
OH
O
OH
eluent. Fraction 5 was further purified
HO
HO
using a RP-18 column and H2O:MeOH
HO
O
OH
(7:3 vol/vol) as eluent. Fraction 3
O
yielded altenusin (6), fraction 5 gave
O
OH
altenuene (7), 4-epialtenuene (8)
and 2,5-dimethyl-7-hydroxychromone
COOH
OH
OH
(9) after semi-preparative HPLC using
OH
HO
an RP-18 column with MeOH:H2O (25:75
O
vol/vol) as eluent. Alternariol (10) and
O
O
OH
altertoxin I (13) were obtained from
fraction 7 and final purification was
carried out by semi-preparative HPLC
OH
OH
OH
OH
O
O
COOH
using a RP-18 column with MeOH:H2O
COOH
O
O
OH
(35:65 vol/vol) as eluent.
OH
OH
OH
The new metabolite alternarienonic
H
H
OH
HO
acid (12) was obtained from the combined VLC fractions 5 and 6 following
OH
O
OH
O
O
OH
O
OH
purification by Sephadex LH-20 CC
|
Figure 6 Natural products from the mangrove-derived fungus Alternaria sp.
with MeOH as eluent.
10 | VOL.4 NO.12 | 2009 | nature protocols

protocol
Fraction 5 of the VLC separation yielded the new natural products xanalteric acid I (14) and II (15), which were purified
over silica gel using CH2Cl2 and MeOH as solvent system and subsequent semi-preparative HPLC with MeOH and H2O as eluent.
Upon fermentation for 3 weeks in liquid Wickerhams medium, compounds 68, 10, 13, 14 and 15 were likewise detected
as constituents of the crude extract using HPLC, whereas 9 and 12 were missing. In addition, the known compounds
alternariol-5-O-methylether (11) and the perylene derivatives stemphyperylenol (16) and alterperylenol (17) were also
obtained after fractionation of the extract by VLC using silica gel as stationary phase followed by semi-preparative HPLC,
thus confirming the OSMAC concept that a given microbial strain can yield different metabolites upon varying fermentation
conditions.
Both crude EtOAc extracts (obtained following fermentation on solid medium or in liquid medium) exhibited promising
antiproliferative activities as detected by the MTT assay. Alternariol (10) and altenusin (6) exhibited strong antiproliferative activities against the murine L5178Y cell line with EC50 values of 1.7 g ml1 and 6.8 g ml1, respectively, whereas the
new metabolites epialtenuene (8), alternarienonic acid (12), xanalteric acid I (14) and II (15), as well as altenuene (7),
altertoxin I (13) and 2,5-dimethyl-7-hydroxychromone (9) were inactive in this bioassay38.

Acknowledgments We are indebted to numerous previous coworkers and to

several colleagues who were indispensable for our studies on marine-derived
fungi. Continued support by BMBF to P.P. is gratefully acknowledged.

Q9 AUTHOR CONTRIBUTIONS

Published online at http://www.natureprotocols.com/.

Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.

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Please provide the page range in reference 19.

AQ15

Please provide the page range in reference 23.

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nature protocols | VOL.4 NO.12 | 2009 | 13

QUERY FORM
Nature Protocol
Manuscript ID
Author
Editor
Publisher
AUTHOR:

The following queries have arisen during the editing of your manuscript. Please answer queries by making the requisite corrections directly on the galley proof. It is also imperative that you include a typewritten list of all corrections and comments, as
handwritten corrections sometimes cannot be read or are easily missed. Please verify receipt of proofs via e-mail

Query No.

Nature of Query

AQ16

Please provide the page range in reference 25.

AQ17

Please provide the place of the publisher in reference 34.

AQ18

Please provide the updates in reference 40.

AQ19

Please provide the updates in reference 46.

AQ20

Solution for the problem?

AQ21

Please provide the title of chapter/article, location as a single city.

AQ22

Please provide title of the chapter/article and page range.

AQ23

Please provide the country details for Refs. 22, 23 and 24.

AQ24

Please provide the single city, country of publisher.

14 | VOL.4 NO.12 | 2009 | nature protocols

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