Year 1

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

Samples of both sediments and liquid are 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 employ a core sampler to collect the first centimetres of sediment.

After sampling we will try to prepare axenic cultures of microalgae and/or cyanobacteria and grow them in laboratory conditions. Nutrients, salt concentration, light exposure and temperature are all controlled parameters. Culture purity will be regularly assessed using optical microscopy and molecular biology approaches, such as DNA electrophoresis and barcoding. After identification of the predominant species in laboratory cultures, we will test different growth conditions (temperature, medium) in order to further enrich/purify microalgae cultures.

Our studies aim to isolate microalgae of the genus Dunaliella, as well as the cyanobacteria Coleofasciculus chthonoplastes and Spirulina. In addition, we envision the possibility of identifying 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 will try to purify with the goal of developing the most efficient and environmentally friendly protocols.

Year 2

Sampling, small- and pilot-scale growth of microorganisms, DNA analysis of biodiversity, and identification of active biocompounds.

Sampling will continue in the second year to further expand the microorganisms available for research. We will also start growing them at larger scales to discern which growth conditions lead to higher yields. At the same time, we will characterise the general biodiversity of microorganisms living in the salt pans by means of DNA analysis from samples collected on Year 1. We will make a small scale test, comparing the efficiency and purity of DNA isolation methods. Once the best is identified, DNA from sediments and brine will be subjected to shotgun metagenomics library preparation and Illumina sequencing. Reads will be taxonomically classified using a variety of bioinformatics approaches. We will continue the purification of bioactive compounds assessing their yield and purity. We will also check 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 will be cultivated in large-scale pilot facilities. We will refine the growth, harvesting and processing techniques. Furthermore, the extracted compounds will be evaluated for their  safety and incorporated into prototype cosmetics formulations.