Artificial sea salts

Further experiments were performed to the baseline optimized skimmed-milk flocculation samples to explore effects upon conductivity and centrifuge speed adjustments. For conductivity investigations, the 100-mL primary concentrate was amended by adding 3.33 g artificial sea salts (Sigma-Aldrich, St. Louis, MO, USA) prior to spiking. For centrifuge method investigations, samples were centrifuged at 3500, 4000, and 4500×g.

For DNA and RNA extractions, mycelium from liquid seed cultures of Emericellopsis sp. TS7, A. encephaloides and C. marina in 0.2ASME medium (4 g/L malt extract, 40 g/L artificial sea salts (Sigma), MilliQ-water – hereafter MilliQ) were inoculated in 250 mL of the same medium in 1000-mL baffled culture flasks. The media constituents were dissolved in MilliQ. All media were autoclaved at 121 °C for 30 min before inoculation. Incubations were performed at 10–16 °C at 140 rpm (shaking for liquid cultures only). After 13 days the culture was harvested by vacuum filtration through Miracloth (Merck) and the mycelium was subsequently placed in aluminum foil and stored at − 80 °C until processing.

For both bioaugmentation (BAa and BAp) treatments tested in the bioremediation experiment, a combined sample of the respective triplicates was taken, ten-fold diluted in sterile saline solution (0.85%) and spread out onto three different agar media (MA, SW, and BH) to provide different nutritional conditions and to try to recover the bacterial strains used as inocula. Per litre, agar media were prepared with the following: MA with marine agar (55.2 g L⁻¹) (Pronadisa); SW with 10 g soluble starch (Biochem Chemopharma), 4 g yeast extract (Liofilchem), 2 g peptone (Sigma-Aldrich), 33.3 g artificial sea salts (Sigma-Aldrich), 20 g agar (Liofilchem); BH with 3.27 g Bushnell-Haas broth (Difco™), 1 g sodium acetate anhydrous 99% (Alfa Aesar ^®), 17.5 g agar (Liofilchem), 20 g NaCl (EMSURE ^®). After 3–4 days of incubation at 28 °C, morphologically different colonies were described and purified. Isolated strains were then preserved in 21% glycerol at −80 C and biomass of each strain was also collected for DNA extraction. The 16S rRNA gene sequences of the bacterial identified strains were deposited in GenBank (Table 2).