# The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria
## 3 The VBNC State in Beneficial Bacteria
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\begin{tabular}{p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline \hline
**Group of Bacteria** & **Species** & **Conditions that Induce VBNC State** & **References** \\ \hline \multirow{8}{*}{_Alphaproteobacteria_} & \multirow{2}{*}{_Acetobacter aceti_} & Treatment with SO\({}_{2}\) at a concentration of 30 and 50 mg/L & [46] \\ \cline{2-4}  & & _Acetobacter pasteurimus_ & High acid stress during fermentation & [47] \\ \cline{2-4}  & & _Methyllosinus sporium_ & & \\ \cline{2-4}  & & _Methyllosinus_ & & \\ \cline{2-4}  & & _trichosporium_ & Freeze drying and & & \\ \cline{2-4}  & & _Methyllocyystis hirsuta_ & cryopreservation (liquid nitrogen) & [25; 30] \\ \cline{2-4}  & & _Methyllocyystis parvus_ & & \\ \cline{2-4}  & & _Methyllocella lundrae_ & & \\ \cline{2-4}  & & _Rhizobium_ & Cupric sulfate to a concentration & [48] \\ \cline{2-4}  & & _leguminosarum_ & of 60 ppm & [49] \\ \cline{2-4}  & & & Incubation at 25 \({}^{\circ}\)C in tap water & & \\ \cline{2-4}  & & & (microcosm-water) & & \\ \cline{2-4}  & & _Sinorhizobium mellioti_ & Incubation under anoxic conditions in liquid microcosms & [16] \\ \cline{2-4}  & & & Incubation in nitrocellulose filters at relative humidity of 22\% for three days at 20 \({}^{\circ}\)C in the dark & & \\ \cline{2-4}  & & _Petaproteobacteriaa_ & _Cupriavidus_ & Incubation in artificial soil at 30 \({}^{\circ}\)C for metallidurans_ & [49] \\ \cline{2-4}  & & _Methyllomonas methanolic_ & & \\ \cline{2-4}  & & _Methyllosarcina fibrata_ & & \\ \cline{2-4}  & & _Methyllocaldum gracile_ & Lyophilization and cryopreservation (liquid nitrogen) & [25; 30] \\ \cline{2-4}  & & _Methyllomicrobium_ & & \\ \cline{2-4}  & & _alcaliphium_ & & \\ \cline{2-4}  & & _Methyllococcus capsulatus_ & & \\ \cline{2-4}  & & _Microbulbifer aggregans_ & Incubation in modified artificial seawater (ASW) for 4 h at 30 \({}^{\circ}\)C & [50] \\ \cline{2-4}  & & _Pseudomonas fluorescens_ & Incubation in saline solution (NaCl & \\ \cline{2-4}  & & 0.9\% \(w/v\)) at 37 \({}^{\circ}\)C & [51] \\ \cline{2-4}  & & Exposure to benzalkonium chloride (BAC) & & \\ \cline{2-4}  & & _Pseudomonas putida_ & Desiccation at 30 \({}^{\circ}\)C and & [4] \\ \cline{2-4}  & & _KT2440_ & 50\% relative humidity & & \\ \hline \multirow{4}{*}{_Vibrio fischeri_} & \multirow{2}{*}{Incubation at 22 \({}^{\circ}\)C in nutrient-limited artificial seawater (ASW)} & \multirow{2}{*}{[52]} & \multirow{2}{*}{[52]} & \multirow{2}{*}{[53]} \\ \cline{2-4}  & & & & \\ \cline{1-1} \cline{3-4}  & & & & \\ \cline{1-1} \cline{3-4}  & & & & \\ \hline \hline \end{tabular}
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Table 1: Main species of beneficial bacteria that enter the viable but non-culturable state. The phylogenetic relationships between different beneficial bacterial species reported to enter the VBNC state were analyzed (Figure 2). The sequences were compared with the data available in the National Center for Biotechnology Information (NCBI) database. To evaluate viable but unculturable strains, phylogenetic trees were constructed by the neighbor-joining method [58] using the Clustal X 2.1, BioEdit 7.7, and Mega 4 (D1993-2011 software. A bootstrap confidence analysis was applied on 1000 replicates to determine the reliability of the topology obtained [59]. The phylogenetic tree showed that these bacteria are very diverse. The furthest phylogenetic group corresponds to _Methylosinus_, _Methylocytosis_, and _Methyllocella_. The genera _Rhizobium_, _Sinorhizobium_, and _Acetobacter_ are groups that are phylogenetically closer to each other. _Methylococcus_, _Methylolandum_, _Methylomicrobium_, _Methyloloxarcina_, and _Methylomonas_ are phylogenetically close to _Vibrio_, _Microbullbifer_, and _Pseudomonas_. Another group that can be identified is formed by _Arthrobacter_, _Bifidobacterium_, _Bacillus_, _Lactobacillus_, and _Oenococcus_, with some strains being phylogenetically closely related and others being very distant species.