# The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria
## 5 What Happens during the VBNC State?  
There are different characteristics shown by cells in the VBNC state. Several bacterial species decrease their size, producing several metabolic changes, including the depletion of energy reserves, altered gene expression, and DNA replication [3]. Biosynthesis is a process that does not stop during this state, with cells forming new proteins related to starvation and cold shock [108; 109]. ATP levels rapidly decrease in dead cells; however, in VBNC cells, these levels are high [110]. [PERSON] et al. (2019) detected the expression of the _opr_H, _mut_S, and _mut_L genes and 16S RNA in _P. putida_ KT2440 VBNC cells under desiccation stress [4]. These studies suggest that some genes may be effective monitors for viability, but whether they are involved in the entrance or exit from the VBNC state is still unknown.  
At the structural level, several characteristics have been observed in non-culturable cells [111], including thickened cell envelopes that potentially provide enhanced resistance to various stressors, contributing to their sustained viability. Changes in the cell membrane composition aid in maintaining ionic homeostasis and, consequently, the intracellular water content. A granular cytoplasmic appearance may indicate reduced ribosome numbers or protein aggregation. Nucleoid compaction likely plays a role in preserving the genome more effectively. Recent proposals suggest that alterations such as cellular structure reorganization, global protein aggregation, and ribosome dimerization may facilitate the transition of bacteria into the VBNC state when exposed to different types of stress [112].  
At the DNA level, three types of stable condensation can form: nanocrystalline structures, liquid crystalline structures, and a structure similar to a folded nucleosome. This type of rearrangement may be the result of complex interactions and associations of proteins to protect the integrity of the genome [113]. [PERSON] et al. (2017) [114] reported that the crystallization of nucleoids in complexes with DNA-binding protein of starvation (Dps) or small acid-soluble proteins (SASP) can offer protection against damaging factors, representing a necessary form of structural organization for DNA in VBNC microorganisms. These discoveries could elucidate the long-term survival mechanisms in conditions inhibiting growth, thereby facilitating species preservation.  
In the cytoplasmic membrane, it has been described that VBNC cells may suffer damage and modifications in the composition of fatty acids, strongly suggesting that changes may be essential for entrance into this state [4; 115]. Cells may undergo biochemical changes in cell walls, and genes involved in peptidoglycan biosynthesis could be a necessary characteristic of VBNC cells to create a more rigid wall than in actively dividing cells [116].  
Bacteria can detect stressors through histidine kinases bound to the membrane, mediating the cellular response through the differential expression of the target genes [117]. Therefore, early signaling could be a determining factor to induce the VBNC state in bacteria. As mentioned above, cellular energy is one of the key characteristics of the VBNC state, triggering the induction of genes codifying subunits of proton pumps such as NADH, or ubiquinone oxidoreductase, a protein that is essential for processes requiring energy in VBNC cells as well as under normal conditions [118]. This suggests that when bacterial cells enter the VBNC state in response to an environmental change, the activity of complex I of the respiratory chain and the NADPH-generating systems are critical for the maintenance of cell viability [118]. In addition, selective permeability to nutrients and metabolites provided by ABC transporters may be a prerequisite for the VBNC state [119]. Although this understanding has been developed within pathogenic bacteria, it is possible to consider that this behavior is widely distributed among beneficial bacteria.