Long-term storage of aerobic granules in liquid media: viable but non-culturable status

Long-term storage and successful reactivation after storage are essential for practical applications of aerobic granules on wastewater treatment. This study cultivated aerobic granules (SI) in sequencing batch reactors and then stored the granules at 4 °C in five liquid media (DI water (SW), acetone (SA), acetone/isoamyl acetate mix (SAA), saline water (SS), and formaldehyde (SF)) for over 1 year. The first four granules were then successfully reactivated in 24h cultivation. The specific oxygen uptake rates (SOUR) of the granules followed SI>SS>SA>SAA>SW>SF; and the corresponding granular strengths (10 min ultrasound) followed SI>SA=SS>SAA>SW>>SF. During storage the granular cells secreted excess quantities of cyclic-diguanylate (c-di-GMP) and pentaphosphate (ppGpp) as responses to the stringent challenges. We proposed that to force cells in granules (Alphaproteobacteria, Flavobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Sphingobacteria, and Clostridia) entering viable but non-culturable (VBNC) status is the key of success for extended period storage of granules.