Salinity and Phenol‐Induced VBNC State of Quorum‐Quenching Bacterium and Its Resuscitation Strategies for Biofilm Control in Industrial Wastewater Treatment

ABSTRACT

Quorum quenching (QQ) is an effective biological strategy for mitigating membrane biofouling in membrane bioreactors (MBRs), yet the persistence of QQ bacteria under harsh industrial wastewater conditions remains poorly understood. Here, the induction, resuscitation and functional recovery of the viable but nonculturable (VBNC) state in the efficient QQ bacterium Brucella sp. ZJ1 was investigated under salinity and combined phenol‐salinity stress. Combined stress markedly accelerated VBNC formation (36 h vs. 10 days under salinity alone) and caused greater oxidative damage, metabolic suppression, structural deterioration and loss of QQ activity. Salinity‐induced VBNC cells recovered following stress removal, whereas phenol‐salinity‐induced cells required resuscitation‐promoting factor (Rpf) for efficient revival. Rpf‐mediated resuscitation substantially restored both QQ activity and biofilm inhibition capacity. Transcriptomic analysis revealed that VBNC formation was accompanied by coordinated repression of genes involved in central metabolism, DNA replication and protein biosynthesis, together with activation of osmotic adaptation, membrane transport, quorum sensing and oxidative stress response pathways. These findings demonstrate that the VBNC state is an active adaptive strategy that preserves the potential for functional recovery and provides new insights for improving the antifouling performance of QQ‐based MBR systems treating high‐strength industrial wastewater.

Quorum‐quenching bacteria serve as an important approach for mitigating biofouling in MBR for industrial wastewater treatment, yet their survival in stressful conditions remains poorly understood. Here, we characterise the VBNC state formation under combined stress and resuscitation strategies by the Rpf protein, highlighting a novel bioaugmentation strategy for MBR biofouling control.

1. Introduction

Phenol is an important industrial feedstock widely used in coal chemical processing, petroleum refining, textile manufacturing, synthetic fibre production and numerous chemical industries (Sun et al. 2026). Consequently, large volumes of phenol‐containing wastewaters are generated worldwide. In many industrial sectors, phenolic wastewater is simultaneously characterised by elevated salinity, resulting from the extensive use of inorganic salts during production processes. It has been estimated that more than 5% of global industrial wastewater is highly saline (Zhang et al. 2021). Salinity concentrations commonly range from 50 to 150 g/L in wastewaters from petroleum refining, coal chemical industries, pharmaceutical manufacturing and resin production (Lefebvre and Moletta 2006; Yang, Kalam, et al. 2024). The coexistence of phenol and high salinity imposes combined toxic and osmotic stresses on microorganisms, leading to reduced microbial activity and impaired biological treatment efficiency (Su et al. 2019, 2018). Developing robust biological strategies capable of maintaining microbial function under these harsh conditions; therefore, remains an important challenge for industrial wastewater treatment.

Membrane bioreactors (MBRs), which integrate biological degradation with membrane filtration, have been widely adopted for treating phenolic saline wastewater, offering high pollutants' removal efficiency, compact footprint and high‐quality effluent (Luo et al. 2018). Nevertheless, membrane fouling, primarily driven by microbial attachment and biofilm formation, remains one of the major limitations restricting their long‐term operation (Bhatt et al. 2023). It is well established that biofilm development on the membrane surface is largely regulated by bacterial quorum sensing (QS), predominantly mediated by N‐acyl homoserine lactones (AHLs), which coordinate extracellular polymeric substance (EPS) production and surface colonisation (Liu et al. 2019; Mishra et al. 2022). Consequently, quorum quenching (QQ), which disrupts QS signalling through enzymatic degradation of AHLs, has emerged as an effective biological strategy for mitigating membrane biofouling (Lee et al. 2016; Liu et al. 2019). A variety of QQ bacteria, including Rhodococcus sp. BH4, Pseudomonas sp. (KS2, KS10, 1A1), Pseudoalteromonas sp. L11, Altererythrobacter sp. S1‐5, Brucella sp. ZJ1 and Proteus sp. ZJ5, have been successfully applied for biofouling control in MBRs (An et al. 2024; Oh et al. 2017; Shah and Choo 2020; Xu et al. 2024). Many studies have demonstrated that different QQ media can effectively mitigate membrane biofouling by 5‐ to 10‐fold under controlled MBR conditions (Kim et al. 2024; Lee et al. 2023, 2016). Nevertheless, their long‐term performance depends critically on the physiological stability of the QQ bacteria themselves under environmental stress.

One important but largely overlooked challenge is the transition of bacteria into the viable but nonculturable (VBNC) state (Xie et al. 2021; Zhang et al. 2025). The VBNC state is a well‐recognised bacterial survival strategy that enables persistence under unfavourable conditions, including toxic chemicals, osmotic stress, nutrient limitation and other environmental stresses (Yu et al. 2022, 2025; Zhu et al. 2022). Although VBNC cells remain metabolically active and viable, they frequently exhibit reduced physiological activity and impaired ecological functions. Previous studies have demonstrated that functional bacteria involved in pollutant degradation readily enter the VBNC state under industrial wastewater‐related stresses, resulting in diminished biodegradation performance (Shi et al. 2024; Xie et al. 2021; Yang, Zhang, et al. 2024). Similarly, the recent work showed that transition into the VBNC state substantially reduced the QQ activity of Brucella sp. ZJ1 under phenolic stress (Zhang et al. 2025). However, whether the combined stresses imposed by phenol and high salinity further accelerate VBNC formation and compromise QQ function remains unknown.

Resuscitation of VBNC cells provides a potential strategy for restoring microbial function. While some VBNC cells spontaneously regain culturability after stress removal, others require resuscitation‐promoting factors (Rpfs), a family of cytokine‐like proteins originally identified in Micrococcus luteus that can stimulate the recovery of dormant bacteria at picomolar concentrations (Shi et al. 2024; Yu et al. 2025). Rpf supplementation has been shown to enhance degradation of recalcitrant pollutants, promote recovery of microbial communities in saline phenolic wastewater treatment systems and accelerate resuscitation of VBNC bacteria (Su et al. 2023, 2019; Zhou et al. 2023). Nevertheless, it remains unclear whether Rpf can restore not only culturability but also the specific QQ function of bacteria exposed to combined phenol‐salinity stress. Moreover, the molecular mechanisms governing VBNC formation and subsequent functional recovery in QQ bacteria have yet to be elucidated.

Therefore, this study investigated the induction, resuscitation and functional recovery of the VBNC state in a representative QQ bacterium, Brucella sp. ZJ1, under salinity and combined phenol‐salinity stress. The specific objectives were to: (1) evaluate the induction of VBNC state under salinity alone and combined phenol‐salinity stress; (2) assess the resuscitation potential of different strategies, including stress removal and supplementation with Rpf; (3) characterise the changes in QQ activity, morphology and physiological properties among VBNC, resuscitated and normal cells; and (4) elucidate transcriptomic changes associated with VBNC state formation. This study provides new insights into the stress adaptation of QQ bacteria and offers a theoretical basis for improving the long‐term stability of QQ‐based biofouling control in MBR treating complex industrial wastewater.

2. Materials and Methods

2.1. Determination of the Salinity Stress Threshold for QQ Strain ZJ1

The QQ strain of Brucella sp. ZJ1, previously isolated and characterised (An et al. 2024; Wang et al. 2023), was cultured in Luria–Bertani (LB) medium at 30°C for 14 h to the exponential growth phase. The culture was subsequently inoculated into fresh LB medium supplemented with NaCl at final concentrations of 0, 10, 20, 40, 60, 80, 100 and 120 g/L. Cultures were incubated at 30°C with shaking at 160 rpm. Bacterial growth was monitored every 3 h by measuring optical density at 600 nm (OD₆₀₀) and colony‐forming units (CFUs). The salinity threshold inducing significant growth inhibition was determined from the growth responses across the NaCl gradient and subsequently used for VBNC induction experiments.

2.2. Induction of the VBNC State and Physiological Characterisation

Based on the results of Section 2.1, 80 g/L NaCl was identified as the salinity stress level for VBNC induction. Exponential‐phase ZJ1 cells were harvested and resuspended in mineral salt medium (MSM) containing either 250 or 1500 mg/L phenol at an initial density of approximately 10⁸ CFU/mL. Cultures containing 250 mg/L phenol plus 80 g/L NaCl were designated as the salinity‐stress (SS) group, whereas cultures containing 1500 mg/L phenol plus 80 g/L NaCl were designated as the combined phenol‐salinity stress (CS) group.

Culturable cells were quantified periodically by plate counting until culturability was completely lost. Total cell numbers were determined by acridine orange staining followed by fluorescence microscopy, whereas viable cells were quantified using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Devices, USA), as previously described (Yang, Zhang, et al. 2024; Zhang et al. 2025).

During VBNC induction, intracellular Na⁺/K⁺‐ATPase, superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and reactive oxygen species (ROS) were determined using commercial assay kits (Titan Scientific, China) according to the manufacturer's instructions. Enzyme activities were normalised to total cellular protein content determined by the bicinchoninic acid (BCA) assay to minimise the influence of stress‐induced changes in biomass and cell morphology.

2.3. Preparation of Rpf, Resuscitation of VBNC Cells and Evaluation of QQ Activity

Recombinant Rpf was prepared as previously described with minor modifications (Su et al. 2023). Briefly, the rpf gene from M. luteus NCIMB 13267 was cloned into pET‐28a(+), expressed in Escherichia coli BL21(DE3) and purified using a Ni‐NTA‐agarose column. The purified protein was dialysed against 25 mM Tris–HCl buffer, and protein concentration was determined using a modified Bradford assay kit (Sangon Biotech, China). The purified Rpf stock solution (about 0.35 mg/mL) was stored at −20°C until use.

For resuscitation assays, VBNC cells were harvested immediately after complete loss of culturability while retaining detectable viability. Cell pellets were washed twice with sterile 0.9% NaCl solution and resuspended in fresh LB medium to an initial OD₆₀₀ of approximately 1.0. Purified active Rpf was supplemented at 1.5% (v/v) of the stock solution (equivalent to about 5.25 mg protein/L), whereas cultures without Rpf addition served as controls. The resulting suspensions were incubated at 30°C with shaking at 150 rpm for 24 h. To exclude regrowth originating from residual culturable cells, stationary‐phase culturable cells and VBNC cells without Rpf supplementation were included as reference controls. Cell growth was monitored by measuring OD₆₀₀ every 2 h (Xie et al. 2021; Zhang et al. 2025).

The QQ activity of normal, VBNC and Rpf‐resuscitated cells was evaluated using N‐hexanoyl‐DL‐homoserine lactone (C6‐HSL) as a model QS signal molecule (Sun et al. 2026). Briefly, bacterial suspensions containing equal amounts of biomass were incubated with C6‐HSL (1 mg/L) in Tris–HCl buffer at 30°C and 150 rpm. Samples were collected every 30 min for 4 h. Residual C6‐HSL was extracted with an equal volume of ethyl acetate containing 0.1% formic acid, evaporated, reconstituted in methanol and quantified using an Agilent 1290 Infinity LC system coupled to an Agilent 6495 triple quadrupole mass spectrometer equipped with an Agilent Poroshell 120 EC‐C18 column (2.1 × 100 mm, 2.7 μm). Quantification followed the method described by Wang et al. (2025). To account for physiological and morphological changes associated with the VBNC state, biomass was normalised based on total cellular protein content. QQ activity was expressed as the biomass‐normalised degradation efficiency of C6‐HSL relative to its initial concentration, thereby reflecting specific QQ activity independent of variations in cell abundance.

To determine whether recovery of QQ activity translated into restoration of antibiofilm function, biofilm inhibition assays were conducted using Pseudomonas sp. HR1, an AHL‐dependent biofilm‐forming gram‐negative bacterium commonly employed as an indicator strain for QQ evaluation (Shukla and Rao 2013; Zhang et al. 2025). Normal, VBNC and resuscitated ZJ1 cells were co‐cultured with HR1 in sterile 96‐well polystyrene microplates at 30°C for 24 h. Biofilms were stained with 0.1% (w/v) crystal violet, destained with 33% acetic acid and quantified by measuring absorbance at 570 nm (Müsken et al. 2010).

2.4. Morphological and Physiological Analyses

VBNC and resuscitated cells from both SS and CS groups, along with normal cells, were harvested by centrifugation, washed three times with sterile 0.9% NaCl solution and fixed in glutaraldehyde at 4°C. Samples were dehydrated through a graded ethanol series (30%–100%), freeze‐dried, sputter‐coated with gold–palladium and examined using a scanning electron microscope (SEM; Hitachi S‐480, Japan). Changes in EPS were characterised by three‐dimensional excitation‐emission matrix (3D‐EEM) fluorescence spectroscopy using an RF‐6000 spectrofluorometer (Shimadzu, Japan). Excitation and emission wavelengths ranged from 200 to 600 nm with a spectral interval of 5 nm.

Functional‐group compositions were further analysed using Fourier‐transform infrared (FT‐IR) spectroscopy. Washed cell pellets were deposited onto aluminium‐coated glass slides and analysed using a Nicolet Nexus 670 FT‐IR spectrometer (Thermo Nicolet, USA) over the range of 500–4000 cm⁻¹ at a resolution of 4 cm⁻¹. Protein secondary structures were evaluated from the amide I region (1700–1600 cm⁻¹). Second‐derivative spectra were generated using a third‐order Savitzky–Golay algorithm with a 7‐point smoothing window (Zhang et al. 2025). Peak positions identified from the derivative spectra were subjected to Fourier self‐deconvolution using OMNIC software (Thermo Electron Corp.), followed by curve fitting with PeakFit v4.12 (Fevzioglu et al. 2020) to quantify the relative contributions of α‐helix, β‐sheet and other secondary structures.

2.5. Transcriptomic Analysis of VBNC State Formation

Comparative transcriptomic analysis was performed using normal cells and VBNC cells induced under combined phenol‐salinity stress (CS group), as described previously (Yang, Zhang, et al. 2024; Zhang et al. 2025). Total RNA was extracted, and ribosomal RNA was removed using the RiboCop rRNA Depletion Kit for Mixed Bacterial Samples (Lexogen, USA). The resulting mRNA was randomly fragmented into approximately 200 bp segments and reverse transcribed into double‐stranded cDNA. Sequencing libraries were constructed using the Illumina Stranded mRNA Prep, Ligation protocol (San Diego, CA, USA) and sequenced on an Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA) using a paired‐end strategy. Three independent biological replicates were analysed for each condition.

Raw sequencing reads were filtered to remove adaptor sequences, poly‐N reads and low‐quality reads to obtain clean reads. Differential expression analysis was performed using the Majorbio Cloud Platform (www.majorbio.com). Differentially expressed genes (DEGs) between VBNC and normal cells were identified using the DESeq2 algorithm with thresholds of |log₂(fold change)| ≥ 1 and adjusted p < 0.001. Functional enrichment analyses of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were subsequently performed (Xie et al. 2021; Zhang et al. 2025).

To validate the RNA‐seq results, eight representative DEGs associated with VBNC state formation were selected for reverse transcription quantitative PCR (RT‐qPCR). Total RNA from VBNC and normal cells was reverse‐transcribed using SuperScript III reverse transcriptase, and quantitative PCR was performed on a LightCycler 480 system (Roche, Switzerland) using gene‐specific primers (Table S1). Each reaction (10 μL) followed the thermal cycling protocol: 95°C for 10 min, followed by 35 cycles of 95°C for 10 s, 62°C for 30 s and 72°C for 10 s. Relative transcript abundances were quantified using the 2^−ΔΔCT method (Chen et al. 2018; Zhang et al. 2025).

2.6. Statistical Analysis

Unless otherwise stated, all experiments were performed using three independent biological replicates (n = 3), and data are presented as mean ± standard deviation (SD). Temporal variations in enzyme activities were assessed using one‐way analysis of variance (ANOVA), followed by Tukey's and Waller–Duncan multiple‐comparison tests at a significance level of 0.05. Differences in cell growth between resuscitated cells with and without Rpf supplementation were evaluated by one‐way ANOVA followed by the least significant difference (LSD) test. For transcriptomic analyses, enrichment of GO terms and KEGG pathways was assessed using Goatools (v1.4.4) and KOBAS 2.0 based on Fisher's exact test. Resulting p values were adjusted using the Benjamini–Hochberg procedure to control the false discovery rate (FDR), and adjusted p < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA), whereas transcriptomic analyses were conducted using the Majorbio Cloud Platform.

3. Results and Discussion

3.1. Transition of QQ Strain ZJ1 Into the VBNC State Under Salinity and Phenol‐Salinity Stress

Salinity tolerance analysis revealed that the growth of Brucella sp. ZJ1, as determined by both OD₆₀₀ and CFU measurements, was markedly inhibited at 80 g/L NaCl (Figure S1), indicating the onset of severe osmotic stress. Such elevated salinity is expected to increase extracellular osmotic pressure and trigger adaptive physiological responses that ultimately promote entry into the VBNC state, a common bacterial survival strategy under unfavourable environmental conditions (Su et al. 2019). Based on these preliminary results, 80 g/L NaCl was selected to establish a reproducible VBNC induction model. As shown in Figure 1, under salinity stress alone (Figure 1A), culturable cells gradually declined to undetectable levels after 10 days, while viable cell counts remained at 8.07 × 10⁶ CFUs/mL, indicating a transition into the VBNC state. Under combined phenol‐salinity stress (Figure 1B), loss of culturability occurred substantially earlier (36 h), while viable cells stabilised at 7.24 × 10⁶ CFU/mL. Based on the time required to reach complete loss of culturability, VBNC formation occurred approximately 6.67‐fold faster under combined stress than under salinity alone, indicating that phenol and salinity acted synergistically to accelerate the transition into the VBNC state. In both treatments, total cell numbers remained relatively stable throughout the incubation period, which was consistent with previous observations of VBNC formation (Xie et al. 2021; Zhang et al. 2025). Although salinity levels ranging from 50 to 150 g/L have been reported for several industrial saline wastewaters (Lefebvre and Moletta 2006), concentrations approaching 80 g/L NaCl exceed those commonly encountered in most full‐scale biological MBR systems. Therefore, this salinity was intentionally selected not to reproduce a specific engineering condition but to impose a sufficiently stringent and reproducible stress capable of driving the majority of the bacterial population into the VBNC state. Accordingly, the selected condition should be regarded as an experimentally accelerated stress model designed to facilitate mechanistic investigations of VBNC induction and subsequent resuscitation rather than a direct simulation of full‐scale wastewater treatment processes.

FIGURE 1.

Evidence for the transition of the quorum‐quenching (QQ) strain Brucella sp. ZJ1 into the viable but nonculturable (VBNC) state under salinity stress alone (A) and combined phenol‐salinity stress (B). Values represent the means of triplicate experiments (n = 3), and shaded areas indicate standard deviations calculated from three biological replicates.

To further characterise VBNC formation, intracellular indicators associated with energy metabolism and oxidative stress were evaluated. As shown in Figure 2A,B, Na⁺/K⁺‐ATPase activity, a key indicator of membrane‐associated energy metabolism, declined progressively under both stress conditions. By the VBNC stage, ATPase activity decreased from approximately 13.0 U/mg protein in normal cells to 3.46 ± 0.04 and 3.43 ± 0.11 U/mg protein under salinity and phenol‐salinity stress, respectively, corresponding to reductions of about 74%–75%. These observations indicate a substantial suppression of cellular energy metabolism, consistent with previous reports that VBNC cells minimise ATP consumption to enhance long‐term survival under environmental stress (Meng et al. 2022; Pu et al. 2019). Similar reductions in intracellular ATP availability have also been reported during VBNC formation in E. coli and Staphylococcus aureus (Li et al. 2024; Liao et al. 2020), suggesting that metabolic downregulation represents a conserved feature of the VBNC phenotype.

FIGURE 2.

Changes in enzymatic activities of Brucella sp. ZJ1 during viable but nonculturable (VBNC) induction under salinity and combined phenol‐salinity stress. (A, B) Na⁺/K⁺‐ATPase activity normalised to total cellular protein; (C, D) fold change of reactive oxygen species (ROS) and malondialdehyde (MDA); (E, F) superoxide dismutase (SOD) activity and catalase (CAT) activity normalised to total cellular protein. Panels A, C and E show results under salinity stress, while panels B, D and F show results under combined stress. Data are presented as mean ± SD from three independent biological replicates (n = 3). Different lowercase letters indicate significant differences in each enzyme's activities in triplicate experiments over time (p < 0.05).

Because oxidative stress is considered a major driver of VBNC formation, intracellular ROS, MDA and antioxidant enzyme activities were simultaneously monitored. As shown in Figure 2B,C, ROS accumulated continuously during exposure to both stress conditions, reaching maxima of 2.23‐fold (Day 6) under salinity stress and 2.47‐fold (24 h) under combined phenol‐salinity stress relative to initial levels. Correspondingly, intracellular MDA levels increased progressively throughout the induction process, indicating enhanced membrane lipid peroxidation and cumulative oxidative damage. Both ROS accumulation and MDA production were more pronounced under combined phenol‐salinity stress, demonstrating that the simultaneous osmotic and phenolic stresses imposed a substantially greater oxidative burden than salinity alone.

Consistent with this oxidative challenge, antioxidant defence mechanisms were transiently activated during the early stages of stress exposure. Under salinity stress, SOD and CAT activities peaked at 1379 ± 86 U/mg protein and 598 ± 35 U/mg protein, respectively, before declining to 616 ± 99 U/mg protein and 91 ± 8 U/mg protein upon VBNC formation (Figure 2C,E). A similar response was observed under combined phenol‐salinity stress, where SOD and CAT activities initially reached 2161 ± 25 U/mg protein and 550 ± 10 U/mg protein, respectively, but subsequently decreased sharply to 1105 ± 22 U/mg protein and 20 ± 2 U/mg protein at the VBNC stage (Figure 2D,F). The transient elevation of antioxidant enzyme activities likely represents an adaptive response to counteract excessive ROS accumulation and maintain intracellular redox homeostasis (Zhang et al. 2025). However, the persistent increase in ROS and MDA, together with the subsequent collapse of antioxidant enzyme activities, indicates that prolonged stress ultimately overwhelmed the cellular detoxification capacity. The resulting oxidative imbalance is expected to compromise membrane integrity, disrupt energy metabolism and impair essential cellular functions, thereby driving the transition into the VBNC state (Shi et al. 2024; Yang, Zhang, et al. 2024). Collectively, these findings suggest that VBNC formation in Brucella sp. ZJ1 is closely associated with a coordinated physiological shift characterised by metabolic repression, progressive oxidative damage and eventual failure of antioxidant defences under sustained environmental stress.

3.2. Rpf Facilitates Resuscitation of VBNC ZJ1 and Restores Its QQ Activity

The ability to regain culturability upon environmental improvement is a defining characteristic of the VBNC state and serves as a key criterion for its confirmation (Yu et al. 2025). Accordingly, the resuscitation potential of Brucella sp. ZJ1 cells that had entered the VBNC state under salinity or combined phenol‐salinity stress was evaluated in the presence or absence of Rpf. As shown in Figure 3A,B, negligible cell growth was observed during the first 12 h in all VBNC treatments. Following supplementation with biologically active Rpf, cell growth became apparent after approximately 14 h and entered the exponential phase by 16 h. In contrast, VBNC cells without Rpf supplementation failed to recover under phenol‐salinity stress and displayed only delayed recovery under salinity stress, with exponential growth initiating at approximately 24 h. Previous studies have shown that heat‐inactivated Rpf produces recovery patterns indistinguishable from those of untreated controls, confirming that the biological activity of Rpf is essential for resuscitation (Su et al. 2018; Zhou et al. 2023). Given the low dosage of purified Rpf applied in this study (about 5.25 mg protein/L), nonspecific nutritional effects are unlikely to have contributed substantially to the observed recovery. By comparison, normal ZJ1 cells entered the exponential growth within 6 h and reached an OD₆₀₀ of approximately 1.61 after 12 h (Figure S2). These results demonstrate that the observed increase in cell density originated from genuine resuscitation and subsequent proliferation of VBNC cells, rather than the outgrowth of residual culturable cells (Zhang et al. 2025). These observations further confirm that ZJ1 entered the VBNC state under both stress conditions. Although simple removal of salinity stress allowed spontaneous recovery after an extended lag phase, VBNC cells induced by combined phenol‐salinity stress could only be resuscitated following supplementation with biologically active Rpf, suggesting that the combined stress imposed physiological damage beyond the threshold for spontaneous recovery (Yu et al. 2025). Similar observations have been reported for E. coli O157:H7, where increased stress progressively reduced the capacity for spontaneous resuscitation (Zhao et al. 2013). Rpf proteins have been proposed to promote recovery through complementary mechanisms, functioning both as cytokine‐like signalling molecules and as muralytic enzymes capable of remodelling peptidoglycan to facilitate re‐entry into active growth (Mukamolova et al. 2002). Although the present results clearly demonstrate that biologically active Rpf accelerated the resuscitation of VBNC ZJ1 cells, the relative contribution of these signalling and enzymatic mechanisms remains to be elucidated.

FIGURE 3.

Resuscitation of viable but nonculturable (VBNC) Brucella sp. ZJ1 cells and restoration of quorum‐quenching (QQ) activity under salinity alone and combined phenol‐salinity stress with or without resuscitation‐promoting factor (Rpf) treatment. (A, B) Growth recovery of VBNC cells under salinity stress (A) and combined phenol‐salinity stress (B) following treatment with Rpf or without Rpf supplementation. (C, D) QQ activities of normal, VBNC and Rpf‐resuscitated cells under salinity stress (C) and combined phenol‐salinity stress (D). QQ activity was expressed as biomass‐normalised C6‐HSL degradation rate with time relative to its initial concentration (C/C ₀), with biomass normalised based on total cellular protein content. Cell growth data are presented as mean ± SD from three independent biological replicates (n = 3). QQ activities data are represented by the means of triplicate experiments (n = 3), and shaded areas indicate standard deviations. Asterisks indicate statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

QQ activity was subsequently evaluated using C6‐HSL degradation assays. To avoid potential bias arising from the altered morphology and optical properties of VBNC cells, all samples were normalised according to total cellular protein content, ensuring that the observed differences reflected biomass‐normalised QQ activity rather than differences in cell abundance. As shown in Figure 3C,D, normal ZJ1 cells rapidly degraded approximately 67% of C6‐HSL within 60 min and more than 82% after 120 min. In contrast, VBNC cells displayed substantially reduced QQ activity, degrading only 37% and 21% of C6‐HSL after 120 min under salinity and combined phenol‐salinity stress, respectively. The greater loss of activity under combined stress was consistent with the more severe impairment of energy metabolism, antioxidant defence and cellular integrity observed during VBNC induction. These findings indicate that transition into the VBNC state compromises not only culturability but also the specific functional capacity of the extracellular QQ system (Zhang et al. 2025). Following Rpf‐mediated resuscitation, C6‐HSL degradation recovered to 74% and 67% under salinity and combined stress, respectively, approaching the activity of normal cells. This recovery suggests that the molecular machinery responsible for AHL degradation remains largely preserved during dormancy and can be functionally reactivated upon resuscitation, highlighting the potential of Rpf‐assisted strategies to restore long‐term QQ performance in MBRs subjected to persistent salinity and phenolic stress.

To determine whether recovery of QQ activity translated into functional biofilm inhibition, crystal violet staining assays were conducted using Pseudomonas sp. HR1 (Table S2). In the absence of QQ bacteria, HR1 formed dense biofilms (OD₅₇₀ = 1.27). Co‐culture with normal ZJ1 significantly suppressed biofilm formation (OD₅₇₀ = 0.61), whereas VBNC cells exhibited markedly weaker inhibition (OD₅₇₀ = 1.10 and 1.12 under salinity and combined stress, respectively). Following Rpf‐mediated resuscitation, biofilm inhibition was largely restored, with OD₅₇₀ decreasing to 0.66 and 0.72, respectively. These results demonstrate that Rpf not only restores culturability but also re‐establishes the ecological function of ZJ1 in suppressing AHL‐mediated biofilm formation. Such functional recovery reinforces the potential application of Rpf‐assisted QQ bacteria for maintaining stable biofouling control in industrial MBR systems exposed to prolonged environmental stress (Huang et al. 2025; Zhang et al. 2025).

It should be noted, however, that biofilm inhibition was evaluated using a single indicator organism, Pseudomonas sp. HR1 and therefore represents a proof‐of‐concept assessment of the QQ function. Activated sludge communities harbour multiple QS systems, including AHL‐, AI‐2‐, CAI‐1‐, DSF‐ and AIP‐mediated signalling (Sun et al. 2026). However, Gram‐negative bacteria generally dominate activated sludge, and AHL‐dependent communication remains the principal mechanism governing EPS production and biofilm development. Because ZJ1 exerts its QQ activity through extracellular enzymatic degradation of AHL molecules rather than species‐specific interactions, the restored QQ activity observed here is expected to be applicable to a broad range of Gram‐negative bacteria. Moreover, Rpf may further improve process resilience by resuscitating other dormant or stressed functional microorganisms within the microbial community (Su et al. 2023). Nevertheless, interspecies interactions, signalling redundancy and community‐level ecological dynamics may influence the performance of resuscitated ZJ1 in full‐scale activated sludge systems. Consequently, validation in mixed‐species biofilms and complex microbial communities will be essential to determine the ecological robustness and engineering applicability of this Rpf‐assisted QQ strategy.

3.3. Morphological and Physiological Shifts Associated With the VBNC State

Morphological and physiological changes accompanying VBNC formation in Brucella sp. ZJ1 was characterised using SEM, 3D‐EEM and FT‐IR analysis. SEM observations revealed pronounced structural deterioration following exposure to both salinity and combined phenol‐salinity stress (Figure 4B,D). Compared with normal cells (Figure 4A), VBNC cells exhibited obvious morphological damage, including cell shrinkage, surface roughening and reduced intracellular content. These changes were more pronounced under combined phenol‐salinity stress, suggesting that the synergistic effects of osmotic and phenolic stress imposed more severe damage than salinity alone. Following Rpf‐mediated resuscitation, cells from both treatments partially recovered their characteristic rod‐shaped morphology (Figure 4C,E). Nevertheless, complete restoration of cellular ultrastructure was not achieved, indicating that although Rpf effectively reactivated dormant cells, structural repair lagged behind the recovery of culturability. Similar observations have been reported in other VBNC bacteria, where membrane integrity and cellular architecture remained only partially restored during the early stages of resuscitation, potentially constraining full physiological recovery (Zhu et al. 2022).

FIGURE 4.

Morphological and physiological characterisation of Brucella sp. ZJ1 during viable but nonculturable (VBNC) induction and resuscitation. (A–E) Scanning electron microscopy (SEM) images; (F–J) three‐dimensional excitation‐emission matrix (3D‐EEM) spectra; (K–L) Fourier transform infrared (FT‐IR) spectra and (M–O) second derivative spectra in the amide I region. Panels A and F depict normal cells; panels B and G show VBNC cells under salinity stress; panels C and H represent resuscitated cells following salinity stress; panels D and I display VBNC cells under phenol‐salinity stress; panels E and J show resuscitated cells following phenol‐salinity stress. Scale bar = 500 nm. Panels K and L show FT‐IR spectra of cells subjected to salinity and combined phenol‐salinity stress, respectively. Panels M–O show second‐derivative spectra of the amide I region for normal cells, salinity‐induced VBNC cells and combined‐stress‐induced VBNC cells, respectively. Peaks 1–5 represent fulvic‐like substances, humic‐like substances, fulvic acid‐like substances, amino acid‐like substances and tyrosine‐like substances, respectively.

To further investigate biochemical changes associated with VBNC formation, intracellular fluorescent components were analysed (Figure 4F–J). Five characteristic fluorescence peaks were identified, corresponding to fulvic‐like (Peak 1, Ex/Em = 325/400 nm), humic‐like (Peak 2, 360/435 nm), fulvic acid‐like (Peak 3, 245/400 nm), amino acid‐like (Peak 4, 260/300 nm) and tyrosine‐like substances (Peak 5, 280/290 nm) (Chen et al. 2003; Yang, Zhang, et al. 2024). Fulvic‐ and humic‐like substances were readily detected in normal and resuscitated cells but were absent in the VBNC state, indicating substantial suppression of cellular metabolic activity. In contrast, fulvic acid‐like fluorescence emerged exclusively after resuscitation, suggesting metabolic reactivation during recovery. Fulvic acid has been reported to serve as both carbon and electron donors, thereby promoting microbial repair and regeneration (Hatano et al. 2023), consistent with the enhanced metabolic activity observed following Rpf treatment. Conversely, the appearance of amino acid‐ and tyrosine‐like fluorescence exclusively in VBNC cells likely reflects stress‐induced accumulation of proteinaceous EPS components involved in cellular protection. Such compounds have been widely recognised as important constituents of the extracellular matrix that enhance bacterial tolerance to environmental stress (Gao et al. 2023).

Physiological alterations in extracellular macromolecules were further examined using FT‐IR spectroscopy. As shown in Figure 4K,L, VBNC cells displayed markedly reduced absorbance at characteristic bands corresponding to O═C═O stretching (2365 cm⁻¹), amide I (1700–1600 cm⁻¹), amide II (1540 cm⁻¹) and C═O deformation (1300 cm⁻¹) relative to normal and resuscitated cells. These spectral changes indicate a substantial reduction in protein‐ and polysaccharide‐associated functional groups, suggesting extensive remodelling of EPS composition during VBNC formation. Interestingly, VBNC cells induced by salinity alone displayed lower overall spectral intensities than those subjected to combined phenol‐salinity stress. This observation implies that, although combined stress accelerated VBNC formation, exposure to salinity alone may have induced more extensive depletion of extracellular macromolecules, whereas the additional phenolic stress triggered compensatory physiological responses that partially preserved specific structural components.

Because the amide I region is highly sensitive to protein secondary structure (Zhang et al. 2025), second‐derivative and deconvolution analyses were performed to quantify the changes in α‐helices, β‐sheets and other structural components (Figure 4M–O, Figure S3 and Table S3). β‐sheet structures predominated in all treatments, which was consistent with previous reports on EPS proteins from E. coli and Serratia marcescens (Badireddy et al. 2008). Compared to normal cells, VBNC cells exhibited elevated β‐sheet content accompanied by a reduction in α‐helical structures, resulting in the lowest α‐helix/(β‐sheet + random coil) ratio. This structural transition is generally associated with enhanced protein rigidity and stability under environmental stress, representing a protective adaptation that helps preserve essential protein function while reducing metabolic activity (Zhang et al. 2025). In contrast, random coil structures, which were absent in both normal and VBNC cells, appeared following resuscitation, accounting for 22.2% and 17.6% of the protein secondary structure under salinity and combined phenol‐salinity stress, respectively. The emergence of these more flexible conformations likely reflects active protein synthesis and structural remodelling during recovery. Similar reductions in protein compactness have previously been linked to increased protein turnover and metabolic reactivation (Huang et al. 2022). Overall, these morphological, spectroscopic and structural analyses demonstrate that VBNC formation in ZJ1 is accompanied by extensive cellular remodelling involving membrane damage, alterations in EPS composition and reorganisation of protein secondary structure. Although Rpf substantially restored cellular morphology and physiological characteristics, recovery remained incomplete, underscoring the complexity of bacterial adaptation and the need for targeted strategies to restore functional performance in biological treatment systems.

3.4. Gene Expression Changes Driving VBNC Formation Under Combined Stress

To elucidate the molecular mechanisms underlying VBNC formation under combined phenol‐salinity stress, comparative transcriptomic analysis was performed between normal and VBNC Brucella sp. ZJ1 cells. Sequencing quality metrics, including raw and clean read numbers together with Q20 and Q30 values, demonstrated the high quality and reliability of the RNA‐seq datasets (Table S4). Principal component analysis (PCA) revealed that the first principal component (PC1) explained 87.65% of the total variance, indicating the good reproducibility among biological replicates and a clear transcriptional separation between normal and VBNC cells (Figure S4). Hierarchical clustering analysis further confirmed distinct global gene expression profiles under the two physiological states (Figure 5A).

FIGURE 5.

Transcriptomic responses of viable but nonculturable (VBNC) Brucella sp. ZJ1 cells induced by combined phenol‐salinity stress. (A) Hierarchical cluster analysis of transcriptomic profiles. (B) Volcano plot of differentially expressed genes (DEGs), where red and blue dots represent significantly upregulated and downregulated genes, respectively, and grey dots indicate non‐significant genes. (C, D) Significantly enriched gene ontology (GO) categories for upregulated (C) and downregulated (D) DEGs, respectively. (E, F) Significantly enriched KEGG pathways for upregulated (E) and downregulated (F) DEGs, respectively. RNA‐seq analysis was performed using three independent biological replicates per treatment (n = 3). DEGs were defined as genes with |log₂(fold change)| ≥ 1 and an adjusted p < 0.001. GO terms and KEGG pathways with an adjusted p < 0.05 were considered significantly enriched.

A total of 1194 DEGs were identified in VBNC cells relative to normal cells, including 690 upregulated and 504 downregulated genes (Figure 5B). GO classification assigned these DEGs to 26 functional categories across biological processes (BP), cellular components (CC) and molecular functions (MF), with enrichment in cellular and metabolic processes, protein‐containing complexes and catalytic and transporter activities (Figure S5). Notably, 182 downregulated and 257 upregulated genes were significantly enriched in distinct functional categories. Upregulated genes were predominantly enriched in membrane transport, localisation, NADH binding and oxidoreductase activity, whereas downregulated DEGs were mainly associated with biosynthesis, ribosomal structure, organelle function, structural molecule activity and carbohydrate metabolism (Figure 5C,D), indicating extensive metabolic reprogramming during VBNC formation. KEGG annotation further supported this metabolic shift (Figure S6). KEGG enrichment analysis revealed that 228 downregulated and 358 upregulated DEGs were significantly enriched (adjusted p < 0.05) in 6 and 11 KEGG pathways, respectively (Figure 5E,F). Downregulated genes were primarily enriched in key metabolic pathways, including purine metabolism, amino acid metabolism, the tricarboxylic acid (TCA) cycle, oxidative phosphorylation and the phosphotransferase system (PTS), indicating a global reduction in energy generation and biosynthetic capacity. In contrast, upregulated genes were related to ABC transporters, QS, exopolysaccharide biosynthesis and RNA polymerase, suggesting that VBNC cells redirect cellular resources from proliferation towards stress adaptation and environmental survival.

To validate the RNA‐seq results, eight representative DEGs involved in metabolism and stress responses were selected for RT‐qPCR analysis. The strong agreement between RT‐qPCR and RNA‐seq data (Pearson r = 0.997; Figure S7; Table S5) confirmed the robustness of the transcriptomic analysis. Integrating these data with physiological measurements enabled the construction of a working model describing phenol‐salinity‐induced VBNC formation (Figure 6). As illustrated in Figure 6A, genes encoding key components of the electron transport chain complexes I–V (e.g., NADH dehydrogenase and ATP synthase), as well as TCA cycle genes (e.g., aceF and pdhABD), were markedly downregulated. Suppression of these pathways would be expected to impair oxidative phosphorylation and ATP production, consistent with the substantial decline in Na⁺/K⁺‐ATPase activity observed during VBNC formation (Figure 2B) (Dahout‐Gonzalez et al. 2006). Similarly, reduced expression of the PTS genes gatA and srlE suggests diminished carbohydrate uptake, further restricting energy metabolism and reinforcing the low‐metabolic state characteristic of VBNC cells.

FIGURE 6.

Proposed mechanistic model describing the transition of Brucella sp. ZJ1 into the viable but nonculturable (VBNC) state under combined phenol‐salinity stress. The model summarises transcriptional and physiological responses associated with (A) central metabolic processes, (B) DNA replication and transcription, (C) oxidative stress response, (D) osmotic adaptation and (E) biofilm formation and membrane transport. Numbers within red and green circles indicate the proportions of downregulated and upregulated genes, respectively. Genes validated by RT‐qPCR are highlighted in red (downregulated) and green (upregulated).

A similar suppression was observed in nucleotide metabolism and protein synthesis (Figure 6B). Downregulation of genes involved in DNA replication and repair, including dnaX, recA and recF, indicates reduced genomic maintenance during dormancy. Simultaneously, repression of multiple ribosomal protein genes (rpsA, rplA, rpsC and rplX) suggests inhibition of translational activity, thereby minimising the energetic costs associated with growth. Together, these transcriptional changes support the concept that VBNC formation represents an active physiological adaptation rather than passive cellular deterioration.

Oxidative stress responses were also extensively remodelled (Figure 6C). Genes encoding SOD and glutathione S‐transferase (GST) were markedly downregulated in VBNC cells, in agreement with the progressive decline in antioxidant enzyme activities and the concomitant accumulation of intracellular ROS and MDA (Figure 2). These findings indicate that prolonged phenol‐salinity stress ultimately overwhelms the antioxidant defence system, resulting in oxidative damage that likely contributes to VBNC induction (de Azevedo Neto et al. 2006). Meanwhile, upregulation of the proline metabolism genes ooxA and codA (Figure 6D) suggests enhanced synthesis of compatible solutes to maintain intracellular osmotic homeostasis under hypersaline conditions, representing another important adaptive strategy during dormancy (Hoffmann et al. 2012).

Another notable feature of the transcriptomic response was the coordinated upregulation of genes involved in ABC transporters, QS pathways and exopolysaccharide biosynthesis (Figure 6E). ABC transporter systems, including OpuBABC, OccMPQ, TauBC, RbsAC and RhaPQST, have been reported to participate in membrane protein and environmental adaptation during VBNC formation (Deng et al. 2022; Yang, Zhang, et al. 2024). Likewise, increased expression of genes involved in QS and exopolysaccharide biosynthesis suggests activation of protective regulatory networks that enhance environmental sensing, EPS production and collective survival under adverse conditions. Importantly, this transcriptional response does not contradict the observed decline in QQ activity. The QS‐associated genes identified here are components of the bacterium's intrinsic signalling network that regulate its own physiological adaptation, rather than genes encoding extracellular AHL‐degrading enzymes responsible for QQ. In contrast, QQ activity relies primarily on the production and activity of extracellular AHL‐degrading enzymes (e.g., lactonases and acylases), which disrupts QS signalling in neighbouring bacteria and thereby suppresses biofilm formation (Lee et al. 2016). Although endogenous QS‐associated pathways were activated during VBNC formation, biomass‐normalised QQ activity decreased markedly (Figure 3C,D), suggesting that the expression and/or catalytic activity of AHL‐degrading enzymes was impaired under prolonged stress.

It should also be noted that transcriptomic analysis in the present study was performed only for normal and VBNC cells exposed to combined phenol‐salinity stress. Consequently, the transcriptional profiles of Rpf‐resuscitated cells remain unknown. Although Rpf treatment effectively restored culturability and substantially recovered QQ activity, it cannot be concluded that the global transcriptome returned completely to the normal physiological state. Previous studies have suggested that resuscitated cells may retain transcriptional signatures associated with prior stress exposure despite recovering culturability and metabolic activity (Pinto et al. 2015). Consequently, the functional recovery observed in the present study likely reflects restoration of key physiological processes rather than complete reversal of all stress‐induced transcriptional changes. Future transcriptomic analyses of Rpf‐resuscitated cells will be essential to distinguish partial from complete recovery and identify the regulatory pathways specifically targeted during resuscitation.

Overall, the integrated physiological and transcriptomic analyses indicate that phenol‐salinity stress drives VBNC formation in Brucella sp. ZJ1 through coordinated repression of energy metabolism, DNA replication and protein synthesis, accompanied by activation of osmotic protection, membrane transport and endogenous stress‐response pathways. These adaptive responses promote survival under extreme environmental conditions but are associated with a substantial loss of ecological QQ function. Rpf‐mediated resuscitation restores this function at the physiological level, although whether it fully re‐establishes the original transcriptional state remains to be determined. Collectively, these findings provide new mechanistic insights into VBNC formation and functional recovery in QQ bacteria and offer a theoretical basis for improving the long‐term stability of QQ‐assisted biofouling control in industrial wastewater treatment systems.

4. Conclusions

This study demonstrated that combined phenol‐salinity stress accelerated VBNC formation in the QQ bacterium Brucella sp. ZJ1 and caused more severe physiological impairment and loss of QQ activity than salinity stress alone. Integrated physiological and transcriptomic analyses indicated that VBNC formation is an adaptive survival strategy involving coordinated metabolic suppression and activation of stress‐response pathways. Although QQ activity was markedly reduced in VBNC cells, it was largely restored following Rpf‐mediated resuscitation together with recovery of biofilm inhibition capacity. These findings improve the understanding of VBNC formation and functional recovery in QQ bacteria and provide a basis for developing strategies to enhance the stability of QQ‐based biofouling control in MBR systems treating high‐strength industrial wastewaters.

Author Contributions

Xiaomei Su: investigation, conceptualization, funding acquisition, project administration, writing – original draft, supervision. Xiao Xiao: methodology, resources. Tianqi Song: investigation, writing – original draft. Faqian Sun: conceptualization, funding acquisition, project administration, supervision, writing – review and editing. Chaofeng Shen: writing – review and editing. Feng Dong: software, methodology. Qian Zhang: investigation, writing – original draft.

Funding

This work was supported by the National Natural Science Foundation of China (52470094, 42277025) and the Natural Science Foundation of Zhejiang Province (LY21D010006).

Ethics Statement

All the authors approved the manuscript and this submission. This manuscript describes an original work, which has not been published before and is not under consideration by any other journal.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Gene‐specific primers used in this study.

Table S2: Quantitative analysis of Pseudomonas sp. HR1 biofilm formation affected by QQ bacteria in different states under salinity alone and combined phenol‐salinity stress.

Table S3: Relative content of protein secondary structures in three different states under salinity alone and combined phenol‐salinity stress. A “–” indicates that the random coil secondary structure was not detectable in both normal and VBNC cells under salinity alone and phenol‐salinity stress at that position.

Table S4: An overview of the Illumina high throughput RNA‐sequencing statistics.

Table S5: Reverse transcription quantitative PCR (RT‐qPCR) validation analysis of RNA‐seq results with selected eight genes.

Figure S1: Cell growth (A) and quantification (B) of Brucella sp. ZJ1 under different salinity concentrations. CFU, colony‐forming units. Data are presented as mean ± SD from three independent biological replicates (n = 3).

Figure S2: The cell growth of the QQ strain Brucella sp. ZJ1 in LB medium. Data are presented as mean ± SD from three independent biological replicates (n = 3).

Figure S3: Analysis of the derivative spectra in the amide I region of resuscitated cells under salinity alone (A) and combined phenol‐salinity (B) stress.

Figure S4: Principal component analysis (PCA) was performed on differentially expressed genes (DEGs) between normal cells and VBNC cells. VBNC and normal cells are represented by grey and green markers, respectively, with all groups analysed in biological triplicates. The first two principal components, PC1 and PC2, explained 87.65% and 3.91% of the total variance, respectively.

Figure S5: GO annotation analysis of DEGs, where red and blue markers represent up‐regulated DEGs and down‐regulated DEGs, respectively.

Figure S6: KEGG annotation of DEGs, where red and blue markers represent up‐regulated DEGs and down‐regulated DEGs, respectively.

Figure S7: Reverse transcription quantitative PCR (RT‐qPCR) validation of RNA‐seq results was performed using eight selected genes.

Data Availability Statement

Transcriptome data have been deposited in the National Center for Biotechnology Information's (NCBI) Sequence Read Archive (SRA) as under accession numbers SRR33798558 to SRR33798563. All other data of this study are available from the corresponding author upon reasonable request.