# The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria
## 7 Resuscitation of Bacteria under VBNC State  
As mentioned in the previous sections, bacteria in the viable but non-culturable state do not grow in routine bacteriological media. However, they are still alive; thus, this state may constitute a survival strategy under stressful conditions. The cells in the non-culturable state must be able to increase their metabolic activity to return to the culturable state [2]. Numerous studies have demonstrated that the return to a cultivable state becomes possible upon removal of the stressor. It has been a topic of discussion and a great challenge to demonstrate that cells can return to the culturable state and that observed cells that grow again on media do not simply correspond to the growth of other surviving bacteria. In _P. putida_ KT2440 cells in the VBNC state induced by desiccation, the colonization of the rhizosphere of maize plants, short rehydration in the presence of root exudates, and prolonged rehydration with only distilled sterile water were shown to allow the return to a culturable state [4]. Interestingly, it has been observed that interaction with higher organisms can function as a biological mediator in the return to the culturable state. For example, in _L. pneumoniae_, which enters the VBNC state under starvation and hypochlorite treatment, it can return to the culturable state in the presence of protozoa _Acanthamoeba polyphalag_ and _Acanthamoeba castellanii_[131; 132]. Regarding beneficial bacteria, a recent study has shown that VBNC cells of _P. putida_ KT2440 can colonize the rhizosphere of maize plants and this interaction allows the return to a culturable state [4]. Furthermore, the root exudates of maize can return these bacteria to the culturable state [4]; thus, it is of interest to study bacterial associations with their hosts, since this influences the survival of the bacteria even in the VBNC state. Other factors that could be decisive for the return to the culturable state are based on the study of extracellular proteins such as the resuscitation promoting factor (Rpf) [133] and the YeaZ protein with protease activity [134; 135]. The promoting effect of YeaZ may be correlated with its protease activity, but the mechanisms that help in the recovery of cultvability need further investigation. Additionally, certain mechanisms have been studied to facilitate the return to the cultivable state. These include the activity of a peptidoglycan hydrolase involved in the digestion of the cell wall and cell division [116; 134; 136] and the \"autoinducer of growth\", which were found to be stable to heat and were secreted by Gram-positive and Gram-negative bacteria growing in culture media with the hormone norepinephrine [137]. It would be interesting to determine whether beneficial bacteria in the VBNC state are also susceptible to resuscitation by these autoinducers. Molecules such as sodium pyruvate have been studied for their role in the return to a culturable state. This molecule is an intermediate key metabolite in glycolysis and a H\({}_{2}\)O\({}_{2}\) degrading compound [138]. It was observed that VBNC cells can return to a culturable state in media supplemented with sodium pyruvate [139]. It was suggested that sodium pyruvate, catalase, and superoxide dismutase, due to their H\({}_{2}\)O\({}_{2}\) or reactive oxygen degrading effects, can induce the return to a cultivable state [140]. It has been proposed that cells in the VBNC state could utilize their remaining ATP to synthesize NAD+, primarily aimed at restoring the energy production machinery and reviving cellular metabolic activity. This mechanism facilitates activity at a transduction level, promoting resuscitation processes, essentially directing their resources towards restarting energy production as a precursor to subsequent resuscitation [141].