# The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria
## 3 The VBNC State in Beneficial Bacteria
### Alphaproteobacteria and the VBNC State  
Within the group of Alphaproteobacteria that enter the VBNC state are organisms belonging to the genera _Acetobacter_, _Methylosinus_, _Methylocitis_, _Methyllocella_, _Rhizobium_, and _Sinorhizobium_ (Table 1). _Acetobacter aceti_ and _Acetobacter pasteurianus_ have a great relevance in vinegar production, since they can transform ethanol into acetic acid through oxidative fermentation [60]. The VBNC state in _A. aceti_ has been documented in wine production [46], and _A. pasteurianus_ enters the VBNC state under high acid stress generated during the fermentation process [47]. Studies are still lacking that support the idea that _A. aceti_ and _A. pasteurianus_, under a non-culturable state, can follow the fermentation process, which would help to increase production. The ability of these species to persist under adverse conditions rep \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Group of Bacteria** & **Species** & **Conditions that Induce VBNC State** & **References** \\ \hline \multirow{8}{*}{_Actinobacteria_} & _Bifidobacterium animalis_ & 1. & Storage in fermented foods & \\  & subsp. _lactis_ & 2. & Refrigerated storage of butter for & \\  & subsp. _lactis_ & 3. & Microcapsules with full-fat goat & \\  & & & milk and inulin-type fructans & \\ \cline{2-4}  & _Bifidobacterium longum_ & Storage in fermented foods & [54] \\ \cline{2-4}  & _Arthrobacter albidus_, & Absence of resuscitation promoting & \\  & reclassified as & factor (Rpf) protein in the & [55; 56] \\  & _Sinomonas albida_ & culture medium & \\ \hline \multirow{8}{*}{_Firmicutes_} & _Bacillus coagulans_ & Incubation at pH 2 for 24 h and & \\  & _Bacillus coagulans_ & subsequent incubation at 140 \({}^{\circ}\)C for & [57] \\  & & 5 min & \\ \cline{1-1} \cline{2-4}  & _Lattiplantibacillus_ & Treatment for 30 min at 100 \({}^{\circ}\)C or & \\ \cline{1-1}  & _Dattarum_ & with 1 mol/L HCI & [20] \\ \cline{1-1} \cline{2-4}  & _Oenococcus oeni_ & Incubation in beer at 0 \({}^{\circ}\)C temperature & \\ \cline{1-1} \cline{2-4}  & _Oenococcus oeni_ & Sulfur dioxide and histidine & \\ \cline{1-1}  & & decarboxylase activity in wines & [46] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._resents a challenge in developing novel ways to improve industrial yields, enhance wine quality, and drive vinegar production on a larger scale. The methylotrophic bacteria _Methylosinus gorium_, _Methylosinus trichosporium_, _Methylocystis hirsuta_, _Methylocystis parvus_, and _Methylocella tundrae_ are methanotrophic microorganisms, also called methane-oxidizing bacteria (MOB), capable of generating energy through the oxidation of methane gas [61]. MOB have different biotechnological applications, mainly the biological mitigation of the methane greenhouse gas, the production of high-value products from methane, and the bioremediation of pollutants [62; 63]. Because of the ecological importance of these microorganisms, it is important to understand how different conditions can affect methane consumption. One of the conditions that induces the VBNC state in MOB is freeze-drying and cryopreservation (Table 1). This represents a challenge  
Figure 2: Phylogenetic tree of beneficial bacterial species entering the VBNC state. Phylogenetic trees were constructed by the neighbor-joining method [58] using Clustal X 2.1, BioEdit 7.7, and Mega 4 ©1993–2011 software. A bootstrap confidence analysis was applied on 1000 replicates to determine the reliability of the topology obtained. to researchers, because culturable cells are needed for beneficial application. Most studies focus on how to avoid the loss of cultivability, but it is likely that MOB can return to a culturable state when in an environment with methane gas. This has been observed with different pathogen species of bacteria that, when found in favorable conditions, leave the non-culturable state [27]. Research with this group of bacteria in the VBNC state is scarce, representing a challenge in understanding this survival strategy.