Environmental Context User Case Study #3
Dalibor Stys
Standard Reporting Requirements for Biological Samples in Metabolomics
Experiments: Environmental Context Case Study
Bioprospection of 120 cyanobacterial strains of Nostoc genus
1) Participants
Dalibor Stys, Jiri Masojidek, Pavel Hrouzek, Petr Kohout, Jan Urban and Jan Vanek, Institute of Physical Biology, University of South Bohemia in Nove Hrady, Czech
Republic
Jiri Kopecky, Petr Zelík, Petra Drápalová, and Pavel Soucek – Institute of Microbiology,
Academy of Sciences of the Czech Republic in Trebon, Czech Republic
Jiri Soukup – Code Farms Inc. Richmond, Ontario, Canada
Barbara Entler, Rudolf Lucas, Christoph Wiesner, Wolfgang Schütt and Christoph Kleber
– Polytechnical University Krems a.D., Austria
2) Background
This work was undertaken as a part of INTERREG III twin-projects “Partnering for
Science” awarded to Institute of Microbiology, Academy of Science of the Czech
Republic in Trebon, Czech Republic and “Cell Biotechnology” awarded to IMC
University of Applied Sciences Krems a.D., Austria. The long-term aim of the project is
to screen microalgae and cyanobacteria for potential pharmaceuticals. Originally, the
scope of screening was broader; currently it focuses almost solely to the most promising
genus Nostoc. The panel of tests is being extended as new methods are introduced.
Samples are tested for allelopathic (algicidal), antiinflamatory, fungicidal and
antibacterial activity, acetylcholinesterase inhibition, cytotoxicity (antitumor activity),
and protease inhibition activity. Samples are characterised for their content of water
soluble (mainly secondary) metabolites.
Microalgal strains are obtained from international collections, regional laboratories as
well as original samples collected in nature. The samples from international collections
are generally well characterised, for other types of samples the characterisation is being
done in our laboratories in collaboration with colleagues collecting samples.
The work involves cultivation of each organism in bioreactors of various types and sizes.
For the primary testing, the Erlenmayer flasks or 300-ml glass cylinders (bubble-column
type) illuminated by fluorescent lamps are used; for structural characterisation and more
detailed studies large-volume bioreactors are used to obtain enough biomass. New types
of bioreactors are being developed for biotechnological applications.
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3) Application of Standard Reporting Requirements for Biological Samples in
Metabolomics Experiments: Environmental Context
S1 – Description of the biological sample involved in the study1:
See also the requirements in P1 – P6
 Taxonomic classification of organism(s) – Nostoc genus i.e. N. calciola, N.lickia
etc. but majority of strains have not been characterised previously and are
denominated by numbers in the particular collection
o Also, include where possible:
 Common name(s) (vernacular) – not existent
 Genotype(s) – under characterisation
 Ecotype(s) – soil and freshwater cyanobacteria
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Sample composition. Please, provide details of the organism(s) that constitutes the
sample. Amounts may be described in ‘absolute’ (number of individuals) or
relative terms (50% Organism X; 25% Organism Y; 25% Unknown) – The strains
are being isolated by test cultivation on Petri dishes as long as there are no
observable contaminating strains. The cultivation experiments are being
performed in 3 replicas, chemical experiments are generally repeated five-tmes.
Despite of the strain uniqueness, however, the metabolic composition varies
significantly. The reason of this variability has not been discovered yet. Most
probably, it lies in the systemic behaviour of the culture which is currently under
complex evaluation.
Sample Type. Please give details of the type of sample (For example;
Community, Population, Whole Organism, Organ, Biofluid, etc). Refer to an
ontology, such as the Ontology for Biomedical Investigations (OBI) http://obi.sourceforge.net/ - culture in water soluble media
Condition of specimen(s). Please give details of general observations on health,
etc. - The cultures were grown in the medium of Allen and Arnon (1955) in 350ml glass cylinders bubbled by the mixture of air + 2% carbon dioxide. The
optimization of light and temperature regime was carried out in the temperature
range between 25°C and 35°C and irradiance between 140 and 560 µmol photon
m-2 s-1 (banks of cool-white fluorescent tubes, Osram Dulux L, 55W/12-950). The
growth rate was calculated from nitrate consumption determined by ion
chromatography (Dionex, IC90, USA).
Phenotypic characteristic(s) – Assessed for each sample individually, key
characteristics represent:
Weight of specimen(s) – individual for each strain and experiment type
Age(s) of specimen(s) – not applicable
Sex(es) of specimen(s) – not applicable
Stage(s) of development – not applicable
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Dalibor Stys
Image data. Please, provide copies of any photographs of samples taken in the
field during collection (or URLs to such images) - Not taken.
Requirement Group – Environment (E)
E2 – Description of ANY laboratory environment:
See also the requirements in S1 and P1 – P6
Please, refer also to the MSI – In vivo context requirements and the MSI Plant context
requirements (Both at: http://msi-workgroups.sourceforge.net/bio-metadata/reporting/)
(Griffin et al., Nikolau et al., this issue).
 Laboratory address and contact details
Experiments are performed:
1) at the Institute of Microbiology, Academy of Sciences of the Czech Republic,
Opatovicky mlyn, CZ-37981 Trebon, Czech Republic, e-mail: kopecky@alga.cz,
web: www.alga.cz
2) in the Technology Incubator of the Institute of Physical Biology, University of
South Bohemia, Academic and University Center, Zamek 136, 373 33 Nove
Hrady, Czech Republic e-mail: stys@jcu.cz, web
http://www.greentech.cz/cbt/en/index.html
3) in laboratories of the IMC University of Applied Sciences Krems,
Campus Krems, A-3500 Krems, Austria, e-mail: barbara.entler@fh-krems.ac.at,
www: http://www.imc-krems.ac.at/IMC/Uni/Content/studiengaenge/bio/info/
E3.1 – Description of terrestrial environments:
See also the requirements in E1, E2.
 Inclination and aspect – described for each sample, however, not always available
in detail from distant collections
 Substrate type – water soluble medium, usually Allen and Arnon (1955)
 Substrate temperature – controlled in the range between 25-35oC, depends on the
optimum for each strain. Nevertheless, Antarctic strains with the optimum below
15oC and thermophilic strains with the growth optimum above 45 oC were also
tested.
 All other measured parameters. For example:
o Substrate pH - 7.1.
o Substrate organic content – solely inorganic substrate.
o Some kind of measure of water availability (suggestions) – n.a.
o Cation exchange capacity - Not measured.
Requirement Group – Process (P)
Any description of a process must be accompanied by information identifying who
performed the action, and the time-point (relative or absolute) or interval over which
it occurred. Relative time points can be expressed in terms of a specific interval, since or
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until, an identifiable event, i.e. 4 hours since dose of environmental toxicant, or 20
minutes after sunrise.
P1 – Description of harvesting/sampling of sample or organism(s):
 Description of harvesting/sampling procedure. The culture was harvested after 2week cultivation by centrifugation at 800 x g for 15 min.
 Means of harvesting/sampling. For example: Netted, electrically stunned,
anaesthetised, razor cut etc. Refer to an ontology, such as the OBI http://obi.sourceforge.net/ - see above
 Purpose for collection - The supernatant was directly tested for the presence of
bioactive compounds and the biomass was freeze-dried (Lyovac GT3, LeyboldHeraeus).
 Other collection parameters - N/A.
P2 – Description of storage/preservation conditions of sample(s):
 Description of storage/preservation procedure - Freezing.
 Means of storage/preservation. For example: Liquid nitrogen, dry ice,
formaldehyde addition, etc. Refer to ontology, such as the OBI http://obi.sourceforge.net/ - Freeze-dried biomass was disintegrated by abrasion
with sea-sand and extracted by 70% methanol (6 ml per 200 mg of dry biomass).
The extract was centrifuged at 800 x g for 5 min. and the supernatant was
evaporated under vacuum and used directly for enzymatic assay. The residue was
dissolved in 100 % acetone and partitioned in the water/hexane mixture for
pigment removal. Samples were frozen in liquid nitrogen and stored in separate
vials prior to analysis. The acetone extract containing the active compounds was
applied to a C8 column of preparative HPLC chromatograph.
 Purpose for storage/preservation - To prevent degradation before processing and
analysis.
 Other storage/preservation parameters. For example:
o Temperature - All samples were stored at -80 oC.
P3 – Description of maintenance of organism(s):
Please also refer to the MSI – In vivo context requirements and the MSI Plant context
requirements (Both at: http://msi-workgroups.sourceforge.net/bio-metadata/reporting/)
(Griffin et al., Nikolau et al., this issue).
 Description of maintenance procedure – cultures are grown on agar plates and reinoculated every two months,
 Means of maintenance. For example: Cage, aquaria, continuous culture, seed bag
or plant pot etc. Refer to ontology, such as the Ontology Biomedical
Investigations (OBI) - http://obi.sourceforge.net/ - see above.
 Purpose for maintenance – necessity to repeat experiment under modified
conditions
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Other maintenance parameters. For example:
o Any relevant experimental design parameters – Generally an agar plate
contains the growth medium by Allen and Arnon (1955), the exact
conditions of illumination, temperature etc. are individual for each strain,
o Feeding regime – n.a.
o Cage dimensions – n.a.
P4 – Description of transportation of samples or organism(s):
Transportation involves storage and/or maintenance; see also the requirements in either
P2 or P3 as appropriate.
 Description of transportation procedure – Organisms are delivered on agar plates.
The extracts are transported in freeze-dried form at ambient temperature.
 Means of transportation. For example: Refrigerated container. Refer to ontology,
such as the OBI - http://obi.sourceforge.net/ - Samples were transported by car.
 Purpose for transportation - Biological activity tests of the extract.
 Other transportation parameters – None.
P5 – Description of acclimation of organism(s):
Acclimation involves maintenance; see also the requirements in P3 as appropriate.
 Description of acclimation procedure:
 Means of acclimation. For example: Cage, aquaria, continuous culture, plant pot
or plant growth room etc. Refer to ontology, such as the OBI http://obi.sourceforge.net/ see above
 Purpose for acclimation – the primary biotechnological goal of the project
requires stable metabolite composition as much as possible
 Other acclimation parameters - None.
P6 – Description of general manipulation of sample or organism(s):
Record details of any controlled treatment as part of the study
 Manipulation type. For example: Perturbation such as an exposure to toxicants,
dissection, sacrifice etc. not applicable
 Description of manipulation procedure. For example: Perturbation of specific
environmental parameter; dissection of specific tissue – not applicable
 Reason for manipulation – not applicable
 Other manipulation parameters - None.
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