*2.3. Bacterial Growth*

To evaluate the production of the anthraquinones by the isolated bacteria, we selected the actinobacterial representatives for growth experiments that were most closely related to known producers of the genera *Streptomyces*, *Micromonospora* and *Verrucosispora*. In addition, we also grew the *Dietzia* representative due to the lack of comprehensive information about its secondary metabolite production. On the other hand, *Rhodococcus*, *Arthrobacter*, and *Cellulosimicrobium* were omitted here because of their known poor production of secondary metabolites.

The growth yield of the selected Actinobacteria was in the range of 20 to 100 mg crude extract. The chromatograms of HPLC analyses of the crude extracts were compared in order to facilitate the metabolic comparison between grown bacteria, the sea anemone and the pure substances (Figure 4).

**Figure 4.** HPLC chromatograms of the crude extracts of the sea anemone *Gyractis sesere*, its respective actinobacterial isolates, and the purified anthraquinones, Lupinacidin A (**1**) and Galvaquinone B (**2**). Approximate retention times of Lupinacidin A (**1**) and Galvaquinone B (**2**) are highlighted by boxes.

By comparison of chromatograms and retention times, we observed that Lupinacidin A (**1**) and Galvaquinone B (**2**) were only present in the crude extract of the sea anemone *Gyractis sesere* and *Verrucosispora sp*. SN26\_14.1, and not in the other actinobacterial representatives. Clearly, *Verrucosispora* must be the producer of the anthraquinones. Further, it is obvious that the metabolites of *Verrucosispora sp.* strain SN26\_14.1 are dominant in the marine invertebrate. The chromatograms of *Verrucosispora* and sea anemone extracts are nearly identical and differ only slightly in the region of retention time 20–23 min. Notably the chromatogram of the sea anemone extract also does not show any peaks that sugges<sup>t</sup> the presence of metabolites of any other of the cultivated bacteria. Together, this strongly suggests *Verrucosispora* appeared to be the most abundant microbe in the sea anemone biomass during the collection.

The subtle difference in the metabolite profiles between *Verrucosispora* and sea anemone extract in the retention time region 20–23 min appears to be to metabolites produced by the sea anemone itself. Overall, the amount appears to be surprisingly small. This may however be caused by the isolation methodology (chloroform extraction), that prioritizes lipophilic substances and selects against the isolation of polar compounds such as peptides.
