title: Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria.
abstract: Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium.
authors.affiliation: Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Gent, Belgium.
authors.forename: Sven
authors.initials: S
authors.lastname: Hoefman
authors.forename: Koenraad
authors.initials: K
authors.lastname: Van Hoorde
authors.forename: Nico
authors.initials: N
authors.lastname: Boon
authors.forename: Peter
authors.initials: P
authors.lastname: Vandamme
authors.forename: Paul
authors.initials: P
authors.lastname: De Vos
authors.forename: Kim
authors.initials: K
authors.lastname: Heylen
mesh_terms: D015925:Cryopreservation; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005612:Freeze Drying; D008772:Methylococcaceae / Q000166:cytology* / Q000502:physiology; D014199:Trehalose / Q000737:chemistry
chemical_list: D014199:Trehalose; D004121:Dimethyl Sulfoxide