Introduction

Microorganisms are exposed to a wide variety of environmental stresses such as nutrient deficiency, temperature, osmotic stress, suboptimal pH, high oxygen concentration, heavy metals, inorganic salts, and antibiotics (Matilla 2018; Jia et al. 2020; Pazos-Rojas et al. 2023). When exposed to these stresses or adapted to unfavorable conditions, they enter a state known as Viable But Non-Culturable (VBNC) to survive (Sachidanandham et al. 2009; Yamamoto 2000). This state means that microorganisms lose their ability to reproduce and cannot be produced by conventional culture methods. Cells in this state have an intact membrane, undamaged genetic material, and cytoplasmic membrane. Therefore, these microorganisms are still alive and continue to perform various molecular, physiological, and metabolic activities (Kim et al. 2018; Ayrapetyan et al. 2018; Dong et al. 2020; Yoon and Lee 2020; İzgördü et al. 2022). In addition, there are various changes in cells in the VBNC state compared with culturable cells. Previous studies demonstrated that cell wall structure (Signoretto et al. 2000) and cell morphology (Takeda 2011) change in VBNC cells, and a decrease in cell size occurs (Ye et al. 2020). It was determined that the rod-shaped cells change to a coke and similar form and the spiral-shaped cells change to a spherical form when they switch to the VBNC state. These morphological changes are closely related to changes in cell wall components such as fatty acid composition, peptidoglycan cross-links, lipoprotein, and glycan chains in cytoplasmic membranes (Ding et al. 2006; Nocker and Camper 2009; Gin and Goh 2013; Zhang et al. 2015; Ding et al. 2012; Traag et al. 2013; Setlow and Christie 2020). The same applies to biofilm formation, where microorganisms come together to protect themselves from environmental stresses. During biofilm formation, microorganisms adapt to environmental conditions by altering gene expression and metabolic activities. In particular, there is an increase in RNA synthesis and expression by upregulating the expression of various genes to ensure communication and coordination between cells within the biofilm (Ghaz-Jahanian et al. 2013; Martínez and Vadyvaloo 2014; Mitra and Mukhopadhyay 2023; Condinho et al. 2023). Consequently, during the adaptation of bacteria to environmental stresses, the increase in the amount of RNA may be part of their strategy to preserve genetic material and potentially preserve their ability to reproduce. This may be an important adaptation mechanism for bacteria to survive for long periods and adapt to stress conditions while in the VBNC state.

Conclusion

The findings of the study suggest a complex interplay of biomolecular changes during the induction of the VBNC state, with specific alterations in RNA, protein, and nucleic acid concentrations. The consistency of the findings across different stress conditions, as validated by ROC analysis, reinforces the reliability of the proposed RNA band at 995 cm− 1 as a robust biomarker for E. coli W3110 in VBNC status. The observed reduction in protein and nucleic acid amounts under certain stress conditions hints at the dynamic nature of cellular responses to environmental stressors, further emphasizing the need for a comprehensive understanding of VBNC-associated modulations. Overall, the combination of chemometric analysis, band integration quantification, and ROC validation strengthens the credibility of the identified spectrochemical biomarker and provides a nuanced perspective on the quantitative changes in biomolecular constituents during the VBNC state induced by different stress factors in E. coli. These findings contribute to the advancement of our understanding of bacterial physiological responses and may have practical implications in the rapid detection of VBNC states in laboratory settings. The ability to detect and understand VBNC cells has also serious implications for public health, food safety, environmental monitoring, and microbial control. In conclusion, the application of IR spectroscopy coupled with chemometric analysis proves to be a powerful tool for characterizing and differentiating VBNC states in E. coli W3110 under diverse stress conditions. The identified spectral discriminators offer a molecular-level understanding of the biomolecular modulations associated with the VBNC state, contributing to our broader comprehension of bacterial responses to environmental stressors.