Monitoring and screening of bioactive compounds and toxins produced by Antarctic cyanobacterial strains.

The photosynthetic microorganisms from the sea, soil and lakes represent a profitable resource for the production of biologically active compounds. The harsh environment of Antarctic with low level of nutrients available for the growth and survival of phototrophic microorganisms (algae and cyanobacteria) often induce them, under extreme low temperature conditions, to produce novel biochemical compounds.

The main objective of this research is to investigate bioactive nature of different Antarctic cyanobacterial strains, both filamentous and unicellular strains, giving particular attention to heterocystous group classified by Rippka (1979). The research studies utilized different selective screening methods to isolate as many strains as possible from different Antarctic regions, aiming to create a collection center of Antarctic cyanobacteria at national level. Studies were carried out to understand their growth physiology and production of bio-metabolites. The collected strains are valuable inheritance of this national program of Antarctic research and could contribute to supply the strains for future basic research of various scientific institution.

Evaluation and selection of the principle active fractions for pharmacological applications, has importance in the research on bioactive metabolites hunting. Majority of the terrestrial groups of cyanobacteria studied have demonstrated to possess interesting compounds with antimicrobial activity. Through selective screening bioassays, namely antimicrobial activity, cytotoxic assay, antitumour activity assays and enzyme inhibitors studies were conducted to find potential microorganisms capable of producing compounds useful in pharmacology and in medicine. A defined protocol was adopted to guarantee uniform extraction procedures of the cyanobacterial biomass. The different extracts (crude, partially purified and that very pure) were examined using the above bioassays reported. Fractionating of biomass extracts were carried out to have active components and guided isolation were carried out through the modern chromatographic techniques (column chromatography, HPLC). In parallel the level of each single constituent was evaluated with the help of GC-MS and LC-MS and NMR, in order to obtain a wide view of more common metabolites and to define eventual variations in their level. The integration of acute cytotoxicity assays gave rapid indication of possible anti fungal and anti-tumour nature of the compound A discovery of new compound with new molecular structure could help to formulate and register new patents of national and international value.

Cyanobacterial isolation
Enrichment of the samples collected were carried out by transferring, a small aliquot of each sample into 50 ml Erlenmeyer flask, containing about 30 ml of BG11, BG 110, Cyano and ASW media. They were incubated at room temperatures ranging from 15 °C to 30°C, under continuous illumination with cool white fluorescent lamps at a low light intensity. Periodical observations of both the natural samples and the isolated strains of cyanobacteria were carried out using a Nikon Eclipse E600 microscope and the photomicrography was done using Nomarski differential interference contrast.

The enrichment cultures were cleaned off the contaminants, mainly bacteria and fungi, by repeated separation and plating of trichomes and hormogones until to obtain clones of each strain free of contaminants. The characterisation of the species up to genus level was performed on morphological basis through consultation of literature (Bourrelley 1966, 68, 70, Germain (1981) and Desikachary (1959), Anagnostidis and Komàrek (1988), Komàrek (1999), Castenholz et al (2001) Broady (1979, 84) and Rippka 1990, Stanier 1978).

The following procedures were adopted for the isolation of cyanobacteria:
a)- A suspension of the sample was fragmented in a sterile potter and was diluted several times with sterile physiological solution, a few drops of which were streaked over the media BG11, BG110 (Rippka et al 1979) and Cyano (Juttner et al 1983), containing 1.2% w/v agar. The plated samples were incubated at different temperatures under continuous illumination at low light intensity. The developing colonies were isolated and conserved in fresh nutrient media.
b)- The above fragmented samples were serially diluted and plated in agar media in Petri plates and were incubated at 30°C under continuous illumination at a light intensity of 5 E m-2 s-1. Single colonies of different cyanobacterial strains that appeared after 10-20 days were picked out and transferred to liquid and solid media.
c)- The fragmented samples were serially diluted in sterile nutrient medium and filtered aseptically through 8µ membrane and placed on selective nutrient media. After the incubation under low light intensity, at temperatures from 15 to 30°C, the single cyanobacterial colonies were picked out and transferred to agar slants or to liquid nutrient media.
d)- Small portion of cyanobacterial clusters developed in the enriched culture were transferred to sterile tubes containing nutrient media, and fragmented by shaking vigorously with sterile glass beads diluted serially with sterile medium and plated on nutrient media. Incubation was done as described above. The individual colonies were picked out and transferred to agar slants and liquid media.
e)- direct isolation from the soil samples was also tired following the method of Broady and Weinstein (1998), by spreading over nutrient agar media few mg of the sample and the agar plates were incubated at low temperature varying from 10-25°C under low light intensity. The cultures were examined as and when the colonies made their appearance and transferred to nutrient media.

Extraction and bioassays
A defined protocol will be adopted to guarantee an uniform extraction procedure of the cyanobacterial biomass. The different extracts (crude, partially purified and that very pure) will be examined using the following bioassays:-Antibacterial assay against few human pathogens
This assay is important to reveal that those cyanobacteria that are unable to synthesise antitumoral agents, can have high activity against human eubacterial cells. The antimicrobial assays of the crude and purified extracts will be tested against Gram+ve and Gram-ve bacteria (Staphylococcus aureus, Bcillus subtilis, E. Coli and Pseudomonas aeruginosa).
-Brine shrimp assay This method is very rapid and provides a simple and inexpensive screening for cytotoxic compounds. Artemia salina , a rotifer, will be used as a test organism. A serial dilution of the crude methanol extract will be made and portions will be added to the brine shrimp in sea water to determine the LC50 of the extracts.
-Anti tumoural bioassay
1) A protocol adopted by the NCNPDDG (National Co-operative Natural Product Drug Discovery Group), will be used to assay the extracts to demonstrate the anti tumour activities.
2) MTT cytotoxicity assay with cell lines (YAC-1 and WEH1). Cell viability assays are important tools in oncological research and clinical practice to assess the tumour cell sensitivity.
The fractionation of the biomass will start at the beginning of the extraction. Successfully the active component's guided-isolation will be carried out through the modern chromatographic techniques (column chromatography, HPLC, techniques at medium pressure etc.). In parallel, the level of each single constituent will be evaluated with the help of GC-MS in order to obtain a wide view of the more common metabolites and to define eventual variations at their levels. Finally the characterization of the specific structures will be followed through the spectroscopic techniques (IR, UV, NMR, MS).