Studies Of Secondary Metabolites In Two Marin Bacteria Cultivated Under Different Conditions
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This work has focused on the identification of secondary metabolites possessing antibacterial activity in two isolates collected from the Trondheim fjord. The isolate MP 113-05 was collected from a sponge at 60 meters depth. Trough 16S rDNA analysis it was found to be a completely novel actinobacterium belonging to the family Streptomycetaceae. MP 131-18, collected from sediment sample at 450 meters depth, was found to belong to the genus Streptomyces. Gene clusters for secondary metabolite biosynthesis had been revealed through draft genome sequencing for both strains. Isolates were provided from the Trondheim fjord Actinomycetes collection at SINTEF/NTNU,Though genome sequencing, performed at NTNU, had revealed several interesting gene clusters, production of secondary metabolites by these two strains had not yet been studied. Secondary metabolism of both strains was attempted stimulated through various cultivation conditions in deep-well plates. The different conditions were: five different media, three dilutions (undiluted, 3X and 6X) of medium, medium with or without resin, two oxygenation levels (450 l and 700 l medium volume), two temperatures (20oC and 25oC) and four different incubation periods. Extracts from freeze-dried total culture were made with DMSO and analyzed for antibacterial and antifungal properties against the test organisms Micrococcus luteus, Enterococcus faecium (vanA positive) and Candida albicans. Production of secondary metabolites from MP 113-05 was best under the conditions 6X diluted medium10, without resin, 700 l medium volume, 25oC, 19 days of incubation, and undiluted medium 2, without resin, 700 l medium volume, at 25oC, 23 days. Extracts showed activity against M. luteus. MP 131-18 showed good production under several conditions, many exhibited the same amount of activity on both M. luteus and E. faecium. Upscale of bioactive compound, from micro-wells to 500 ml shake flasks added 5 g/flask glass beads was successful for MP 131-18 cultivated in the conditions 125 ml undiluted medium 4 with resin and 10 with resin, 14 days, 25oC. For MP 113-05, bioactivity in up scaled 500 ml shake flask added 3 g/flask glass beads, was reproduced with cultivation conditions: 200 ml medium 10 added 1 g/l TSB, 3 g/flask glass beads, 14 days, 25oC.Freeze dried material from total culture in shake flasks was tried extracted in water, ethanol and methanol additionally to DMSO. This together with precipitation analysis of ethanol, methanol and DMSO extract with ion free water would indicate some of the polar properties of the antibacterial compound. MP 113-05 was only soluble in DMSO and did not precipitate when added ion free water. The antibacterial compound had thus both hydrophobic and hydrophilic properties. MP 131-18 was soluble in ethanol, methanol and DMSO and both methanol and DMSO extract precipitated when added ion free water. Antibacterial compounds of MP 131-18 had Fractionation and LC-TOF of MP 131-18 extracts, showed presence of two active compounds in the sample. A list of potential masses possessing the antibacterial activity identified from fractionation was generated based on TOF results. Search in three databases gave a suggestion based on UV-profile and ppm error that the active compound in sample could be either be Leptofuranin B or Lagunapyrone B, but further work need to be done to either confirm or e these.Regarding the antibacterial in MP 113-05 sample, these initial investigation of masses from LC-TOF analysis did not identify any already known compounds.