A Novel Method Using a Potential-Controlled Electrode for Screening Difficult-to-Cultivate Microorganisms

We developed an electrical retrieval method for living environmental microorganisms using a weak electrical potential applied to an optically transparent electrode. Living microorganisms resuspended in non-nutritive solutions such as calcium- and magnesium-free phosphate-buffered saline [PBS(-)] and artificial seawater were attracted by and selectively attached to an indium tin oxide (ITO) electrode surface to which a negative potential between −0.2 and −0.4 V vs. Ag/AgCl was applied. The electrically attached microorganisms could be cultured on the agar medium after detachment from the ITO electrode by application of a ±20 mV vs. Ag/AgCl, 9 MHz sine wave potential. When a ±0.2 V vs. Ag/AgCl, 12 MHz sine wave potential was applied to the attached microorganisms on the electrode with an applied negative potential, actinomycetes were selectively cultured and were expanded 60- to 4000-fold in terms of colony-forming units (CFUs) as compared with application of a ±20 mV vs. Ag/AgCl, 9 MHz sine wave potential. Using the electrical retrieval method, we identified and isolated Nocardiopsis sp., Dietzia sp., Pseudonocardia sp., Brachybacterium sp., Nesterenkonia sp., Microcella sp., Microbacterium sp., and Streptomyces sp. from deep-sea sediment core samples in Suruga Bay, Japan. Phylogenetic analyses based on 16S ribosomal RNA gene sequences indicated that 59% of the obtained strains are novel species or are highly likely to be novel species. The electrical retrieval method for living microorganisms holds promise as a novel screening strategy for hardly cultivable microorganisms.

Keywords: Actinomycetes, Deep-sea microorganisms, Electrical retrieval, Marine sponge, Potential-controlled electrode.