Selective Isolation of Actinobacteria from Marine Invertebrates: A Bio-Prospective Search for New Producer Strains

Abstract

Actinobacteria are spore-forming Gram-positive bacteria, with high G-C content in their genome. Their ability to metabolize complex compounds helps them survive different environmental conditions and form diverse associations with different organisms. These characteristics make Actinobacteria good producers of natural products with useful bioactivity. However, Actinobacteria are slow growers and could be outcompeted by fast-dividing microbes with simple metabolic systems in a given sample. Therefore, optimizing the selective isolation of actinobacteria to increase their yield is essential in bioprospecting. This work is aimed at optimizing the selective isolation method used in Schneider et al., 2022, to isolate actinobacteria from marine invertebrates, using a selective media M1 supplemented with antibiotics (1.0ml of Nalidixic acid 30mg/ml in 0.3M NaOH) and fungicide (5.0ml of cycloheximide 10mg/ml in EtOH) and 10mins heat pre-treatment at 550C, 650C, and 750C respectively, as well as a chemical pre-treatment with phenol alone (3%phenol) and a combination of 3% phenol and 650C heat treatment before culturing. At the end of this work, there was a yield of 43 bacteria isolates, 1 from the 650C treatment group and 42 from the control group with no treatment. Of the 43 isolates, 3 were from the class Gammaproteobacteria (7%), 5 were of the class Bacilli (12%), 7 were Alphaproteobacteria (16%), 13 were Flavobacteriia (30%), and 15 were Actinomycetes (35%). Though there were relatively high isolates of Actinomycetes, the curiosity to investigate the high inhibition in the treatment group led to the next phase of this research. Firstly, to test the heat tolerance of the isolated Actinomycetes, 7 tentatively identified Actinomycetes genus were subjected to the heat treatment and there was a total inhibition at 750C except for the sample A010 (tentatively identified as Microbacterium Sp.) however, there was growth on the 550C and 650C in all 7 samples. This was the second phase of this work, but to further investigate the inhibition effect of the heat and phenol pre-treatment and prolonged sample storage at -800C in 20% glycerol, the third phase started with collecting fresh samples of breadcrumb sponge at the shorelines of Uteng, Tromsø. This is because the five animal stock samples were not enough to continue the research studies. The bread crumb samples were homogenized and separated into three conditions, unfrozen, 1 day frozen, and 15 days frozen. To aliquots from these condition groups were given the same heat treatments for 10 mins except the unfrozen sample which received three different phenol treatments (0.1%, 0.5%, and 1%) in addition to the heat treatment received by all. There was a total inhibition on the 1% phenol and 550C, 650C, and 750C of all the conditions except the 650C of the unfrozen condition which showed growth. There was also growth in the 0.5% and 0.1% phenol, and the control groups of all conditions. At the end of this project, we obtained a 35% yield of actinobacteria from the control group with no treatment. We observed that, though heat pre-treatment has proven to be good for the selective isolation of Actinobacteria in marine samples like soil samples and sediments as in Schneider et al., 2022, it may not be suited for the selective isolation of Actinobacteria in marine animal samples, as most treatment groups showed high inhibition, but growth was observed in the control groups with no treatment. Also, Phenol treatment at 1% and 3% concentrations were observed to be bactericidal both in this research and the previous study by Scheider et al., 2022, but can be used at 0.5% and 0.1% concentrations for selective isolation. Finally, we recommend the use of a sample-specific selection method like the “selective filter membrane method” described by Savitha, et al., 2022 to increase the percentage yield in the isolation of Actinobacteria from marine animal samples.