To verify the hypothesis of cryptic growth and viable but nonculturable (VBNC) state, survival responses of Escherichiacoli cells were examined under oligotrophic microcosm conditions for an extended period. In the case of filtered distilled water at 4°C, E. coli cells definitely entered the VBNC state within 56 days. However, culturability and viability increased while the total number of cells declined after 110 days. This phenomenon can be explained by considering three possible states. The first is the existence of the VBNC state, the second is cryptic growth, and the third is the death of E. coli cells. In the case of artificial seawater at 4°C, VBNC E. coli cells confirmed the existence of two log units of elongated VBNC cells. Moreover, elongated VBNC cells showed the most significant change among all the other transformed cells. Also, E. coli cells in microcosms at 28°C indicated the entrance to the classical starvation survival state. In resuscitation tests, 1% diluted Luria-Bertani agar medium showed the highest level of resuscitation among amended agar media. To evaluate the survival ability of E. coli cells in the activated sludge samples, we used an E. colistrain XL-1 blue containing plasmids pQ2 including GFPcDNA (XL/GFP). In supernatant of activated sludge (SUP) at 28°C, XL/GFP cells entered the VBNC state after 10 days, whereas existence of VBNC cells was not detectable in resuspended activated sludge (ACT) at 28°C.
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This research was financially supported by a grant from the Japanese Ministry of Education, Environmental Protection Board, Science, and Technology.
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Na, S.H., Miyanaga, K., Unno, H. et al. The survival response of Escherichia coli K12 in a natural environment. Appl Microbiol Biotechnol 72, 386–392 (2006). https://doi.org/10.1007/s00253-005-0268-3
- Green Fluorescent Protein
- Activate Sludge
- Green Fluorescent Protein Fluorescence
- Dormant Cell
- VBNC State