Metabolism differences of biofilm and planktonic Pseudomonas aeruginosa in viable but nonculturable state induced by chlorine stress

More than 95% of the bacteria in environment are viable but nonculturable (VBNC). However, it is difficult to elucidate directly the metabolic characteristics of these VBNC bacteria and the differences between biofilm-VBNC bacteria and planktonic-VBNC bacteria. In this study, VBNC P. aeruginosa induced by chlorine was used to clarify the metabolism characteristics and mechanism of differential metabolism between biofilm-VBNC bacteria and planktonic-VBNC bacteria. Results showed that P. aeruginosa in biofilm state was more likely to enter VBNC state. The mechanisms of differential metabolism were involved in the difference of reactive oxygen species production owing to the protection of extracellular polymers. 15N and 2H labeled single-cell Raman spectra directly proved that VBNC state bacteria still maintained low material and energy metabolism, and the metabolic activity of biofilm-VBNC P. aeruginosa was lower than that of planktonic-VBNC P. aeruginosa. GC-MS/MS analysis showed 51 metabolites with significant differences. KEGG analysis showed that the types and contents of extracellular metabolites from P. aeruginosa in VBNC states were significantly lower than those in the culturable state (p < 0.05), mainly involving in citrate cycle, glutathione metabolism, phenylalanine metabolism, tyrosine metabolism and fatty acid degradation. Also, the contents of most extracellular metabolites from P. aeruginosa in biofilm-VBNC state were lower than those in VBNC planktonic state. The significant differences (p < 0.05) were mainly involved in alanine, aspartate and glutamate metabolism, glycolysis/gluconeogenesis, D-Alanine metabolism and glycerophospholipid metabolism. The result of this research was favorable to the accurate identification of VBNC bacteria, the health risk assessment and scientific control of harmful VBNC bacteria.