Electrical Retrieval of Living Microorganisms from Cryopreserved Marine Sponges Using a Potential-Controlled Electrode

Abstract

The purpose of this study was to develop a novel electrical retrieval method (ER method) for living sponge-associated microorganisms from marine sponges frozen at −80 °C. A −0.3-V vs. Ag/AgCl constant potential applied for 2 h at 9 °C induced the attachment of the sponge-associated microorganisms to an indium tin oxide/glass (ITO) or a gallium-doped zinc oxide/glass (GZO) working electrode. The electrically attached microorganisms from homogenized Spirastrella insignis tissues had intact cell membranes and showed intracellular dehydrogenase activity. Dead microorganisms were not attracted to the electrode when the homogenized tissues were autoclaved for 15 min at 121 °C before use. The electrically attached microorganisms included cultivable microorganisms retrieved after detachment from the electrode by application of a 9-MHz sine-wave potential. Using the ER method, we obtained 32 phyla and 72 classes of bacteria and 3 archaea of Crenarchaeota thermoprotei, Marine Group I, and Thaumarchaeota incertae sedis from marine sponges S. insignis and Callyspongia confoederata. Employment of the ER method for extraction and purification of the living microorganisms holds potential of single-cell cultivation for genome, transcriptome, proteome, and metabolome analyses of bioactive compounds producing sponge-associated microorganisms.

Electrical Retrieval of Living Streptomycete Spores Using a Potential-Controlled ITO Electrode

“…1A). [31][32][33] A large electrode chamber device [31][32][33] was used for collecting microorganisms from the deep-sea sediment core samples. The microelectrode array 35 was formed by the plane ITO electrode fabricated with SiO 2 and water-repellent coating with fluorine compound (Geomatec Co., Ltd.; the contact angle of water droplets is about 115°).…”

“…3,24,30 In our previous study, we found that numerous actinomycetes including the genus Streptomyces could be electrically retrieved from deep-sea sediments 31 and cryopreserved marine sponges. 32 Living microorganisms suspended in nonnutritive solution such as PBS(¹) and artificial seawater (ASW) were selectively attached to a negative potential-applied optically transparent electrode against gravitational force. 31, 32 Microorganisms suspended in Luria-Bertani medium and glucose solution were not attracted to the electrode.…”

“…32 Living microorganisms suspended in nonnutritive solution such as PBS(¹) and artificial seawater (ASW) were selectively attached to a negative potential-applied optically transparent electrode against gravitational force. 31, 32 Microorganisms suspended in Luria-Bertani medium and glucose solution were not attracted to the electrode. 31 Dead microorganisms were also not attracted to the electrode.…”

“…[31][32][33] The mechanisms by which the high-frequency wave potential induced electrical detachment involve both oscillation of the negative zeta potential-charged cells and insertion of water molecules between the electrode surface and attached cells. [31][32][33][34] High-frequency wave potential-induced cell detachment is practically noncytotoxic, [31][32][33][34] since 91% of the residual soil microorganisms attached to the ITO electrode remained alive after 60 min of a «10 mV vs. Ag/AgCl, 9 MHz triangular-wave potential application in PBS(¹). 31 The electrically retrieved sponge-associated microorganisms could be cultured on agar medium after detachment from the ITO electrode by a «20 mV vs. Ag/AgCl, 9 MHz sine-wave potential application for 20 min in ASW.…”

“…31 The electrically retrieved sponge-associated microorganisms could be cultured on agar medium after detachment from the ITO electrode by a «20 mV vs. Ag/AgCl, 9 MHz sine-wave potential application for 20 min in ASW. 32 Using the electrical retrieval method, we obtained a wide range of microorganisms including 19 phyla of deep-sea sediment bacteria, 31 32 phyla of sponge-associated bacteria, 32 three phyla of spongeassociated archaea, 32 budding yeast, 35 and symbiotic bacteria from the deep-sea bivalve Calyptogena okutanii. 33 Microorganisms such as Escherichia coli recognized small regions of the negative appliedpotential microelectrode and selectively attached to the 5 µmT circular microelectrode array.…”