Year 1

Sampling, microorganism isolation, laboratory growth techniques, identification of microbial biodiversity

Samples of both sediments and liquid were taken from salt crystallisation basins in the Sečovlje saltpans. The particular conditions in these ponds: high salt concentrations, exposure to intense light, and regular desiccation, are ideal for well-adapted microorganisms. We employed a core sampler to collect the first centimetres of sediment.

After sampling we prepared axenic cultures of microalgae and/or cyanobacteria and grew them under laboratory conditions. These included controlled nutrients and salt concentrations, light exposure and stable temperature. The purity of the cultures was regularly assessed using microscopy and molecular biology approaches, such as DNA fingerprinting and barcoding. After identification of the predominant species in laboratory cultures, we  tested different growth conditions (temperature, medium) in order to further enrich/purify microalgae cultures.

Our studies aimed to isolate microalgae of the genus Dunaliella, as well as the cyanobacteria Coleofasciculus chthonoplastes and Spirulina. In addition, we tested the identification of other important organisms such as Chlorella and Nannochloropsis. All of these are known to be amenable to large-scale cultivation and to produce valuable bioactive compounds. Some of these, we purified with the goal of developing the most efficient and environmentally friendly protocols.

Year 2

Large-scale pilot production of microorganisms, developing and testing of cosmetics line prototypes

Sampling continued in the second year to further expand the collection of microorganisms available for cultivation. We started to grow them at larger scales to discern which growth conditions lead to higher yields. At the same time, we characterised the general biodiversity of microorganisms living in the salt pans by means of DNA analysis from samples collected on Year 1. We made a small scale test, comparing the efficiency and purity of DNA isolation methods. Once the best method was identified, DNA from sediments and brine were subjected to shotgun metagenomics library preparation and Illumina sequencing. Reads were taxonomically classified using a variety of bioinformatics approaches. We continued with the purification of bioactive compounds assessing their yield and purity. We also checked the bioactivity of microorganism extracts in batteries of tests.

Year 3

Large-scale pilot production of microorganisms, developing and testing of cosmetics line prototypes.

The identified organisms with the best growth characteristic and valuable bioactive compounds were cultivated in pilot facilities. We refined the growth, harvesting and processing techniques. Furthermore, the extracted compounds were evaluated for their safety and incorporated into prototype cosmetics formulations.

20-L photobioreactor with Dunaliella spp.