Desulfovibrio vulgaris Hildenborough (Heidelberg et al., 2004) and Desulfobacterium corrodens (DSM 15630) were propagated in either saline or freshwater media recommended by Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures. D. vulgaris strain used in this study harbors its 200 kbp native plasmid pDV1, whose presence has been reported to be crucial in its biofilm formation and maintenance (Clark et al., 2007). Saline medium (modified DSMZ medium 141) had the following composition (concentration in g/L) (salinity = 25.9g/L): KCl, 0.34; MgCl₂.6H₂O, 4.00; NH₄Cl, 0.25; CaCl₂.2H₂O, 0.14; K₂HPO₄, 0.14; NaCl, 20; yeast extract, 1; tryptone, 1. Dissolved ingredients were initially autoclaved and then supplemented with 5 g/L NaHCO₃ and 10 mL/L of DSMZ-141 vitamin solution (10×) and DSMZ-141 trace element solution (10×) from their respective filter-sterilized stock solutions (Bajracharya et al., 2015, 2017). Freshwater medium (modified DSMZ 641) had the following composition (concentration in g/L) (salinity = 4.17 g/L): MgCl₂, 2; K₂HPO₄, 0.50; NH₄Cl, 1; CaCl₂, 0.75; yeast extract, 1; tryptone, 1. The freshwater medium was autoclaved and further supplemented with 5 g/L NaHCO₃, 10 mL/L of DSMZ-141 vitamin solution (10×) and 1 mL/L of SL-10 trace element solution (10×) (from DSMZ medium 503) (Kádár et al., 2003; Ünal et al., 2012). Sodium lactate and Na₂SO₄ at final respective concentrations of 20 mM (2.24 g/L) and 10 mM (1.42 g/L) were added to both media to serve as electron donor and acceptor, respectively (Bryant et al., 1977; McInerney and Bryant, 1981; Krumholz et al., 2015). The pH of the saline and freshwater media was adjusted to 7.30, and both media were filtered through 0.25 μm syringe filter prior transferring to sterile autoclaved anaerobic tubes. All the tubes were sealed with butyl rubber stoppers and then maintained under anaerobic environment by purging the media with N₂ for 10 min. Further, 0.50 g/L Na₂S.9H₂O was added to saline and freshwater media inside anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI, United States). All cultures were incubated at 30°C on a rotary shaker.

Sulfate concentration in extracted samples (diluted 100×) were quantified using a Dionex ICS-1600 Ion Chromatography system (Dionex Corp., Sunnyvale, CA, United States) equipped with a high-pressure pump, a sample auto-injector, a guard and separator column, a chemical suppressor, a conductivity cell and a data collection system with KOH as the eluent. Data collection and processing were regulated by software Chromeleon 7.0 (Dionex Corp., Sunnyvale, CA, United States) (Altland and Locke, 2012; Uzhel et al., 2016).

Cell density of D. vulgaris and Db. corrodens propagated in saline and freshwater media were measured using Accuri C6 Flow cytometry system (BD Bioscience, Franklin Lakes, NJ, United States) using protocols described earlier (Cheng et al., 2016). Cell pellets harvested through centrifugation (12,000 × g, 15 min) was washed (two times) with 0.9% NaCl. Prior to flow cytometry, diluted cell suspensions (10⁵–10⁶ cells/mL) were stained with SYBR green (Invitrogen AG, Bazel, Switzerland), diluted 10⁴-times from stock concentration (10⁴-fold concentrated in DMSO) (Marie et al., 1997; Noble and Fuhrman, 1998). After staining, cells were incubated at room temperature under dark conditions for 15 min. About 50 μL of stained cells were injected at a flow rate of 35 μL/min to Accuri C6 Flow cytometry system and then excited at 488 nm to enumerate the cell density. In order to evaluate differences in morphological changes between saline and freshwater media, black spots within the flow cytometry gating region were observed. No significant change was observed, which suggested no change in morphology between saline and freshwater media.

For extraction of total AHLs, cell-free extracts of both SRB grown in saline and freshwater media were collected by centrifugation (12,000 × g, 15 min). Cell-free extracts were re-concentrated initially through lyophilization and then by resuspending the lyophilized fraction to 1/10th of the initial extracted volume in autoclaved H₂O (pH 6.7). Total AHLs were quantitatively determined using a bioassay with beta-glo (Promega, United States) as luminescence substrate (Kawaguchi et al., 2008; Decho et al., 2009). Bioluminescence assay was conducted using a flat white 96-well plate (Greiner Bio-One, Sigma-Aldrich, MI, United States). Briefly, 20 μL of samples were mixed with 80 μL of Agrobacterium (A.) tumefaciens NT1 biosensor prepared in AT medium (Kawaguchi et al., 2008). After incubation for 90 min at 30°C, 100 μL of Beta-Glo reagent were added into each well of the 96-well plate. After incubation for 30 min at room temperature, bioluminescence intensity of each sample was recorded using a microplate reader (TECAN M200, M200, Männedorf, Switzerland) (Kawaguchi et al., 2008; Decho et al., 2009). Biosensor A. tumefaciens NT1 harbors ß-galactosidase enzyme, whose expression is regulated by the presence of AHLs. Beta-galactosidase cleaves beta-glo substrate to form luciferin in the presence of AHLs, which generates luminescence (Kawaguchi et al., 2008). Each bioassay was conducted in triplicate to assess reproducibility. Different AHLs such as N-butanoyl-homoserine lactone (C4-HSL), N-hexanoyl-homoserine lactone (C6-HSL), N-octanoyl-homoserine lactone (C8-HSL), N-decanoyl-homoserine lactone (C10-HSL), N-dodecanoyl-homoserine lactone (C12-HSL), N-tetradecanoyl homoserine lactone (C14-HSL), N-hexadecanoyl homoserine lactone (C16-HSL) and N-octadecanoyl homoserine lactone (C18-HSL) were used to prepare standard curves to optimize the bioluminescence assay (Supplementary Figure S1A). Dominant AHLs produced by SRBs were analyzed using liquid chromatography (LC) – mass spectrometry (MS)/MS (Agilent Technologies, Santa Clara, CA, United States) using protocols described elsewhere (Ortori et al., 2011) (Supplementary Figure S1B). For LC-MS/MS analysis, a part of cell-free extract was mixed with equal volumes of dichloromethane. AHL extraction procedure was repeated three times, and the organic solvent was evaporated to complete dryness using anhydrous Na₂SO₄. Dried samples were re-dissolved in methanol prior to analysis (McClean et al., 1997).

To elucidate effects of salinity on planktonic cells, D. vulgaris and Db. corrodens were propagated in saline and freshwater media using lactate and Na₂SO₄ as electron donor and electron acceptor respectively. Test conditions and media composition were as described earlier. A working volume of 22 mL was maintained in each anaerobic tube. Three biological replicates were used for each test conditions. About 1 mL of culture was extracted from anaerobic tubes every 24 h, and then centrifuged at 12,000 × g for 15 min. Harvested cell pellets were used to enumerate cell density with flow cytometry (Cheng et al., 2016). Cell-free supernatant was used to quantify sulfate concentration (100× diluted) and total AHLs produced, corresponding to each time interval. The effects of salinity were quantified in terms of specific sulfate reduction rate and specific AHL production rate (Fründ and Cohen, 1992; Detmers et al., 2001; Flodgaard et al., 2003; Bruhn et al., 2004). Specific sulfate reduction rate was defined in terms of total amount (μmoles) of sulfate reduced per cell per unit time (μmoles of sulfate/cell/h), whereas specific AHL production rate was expressed in terms of the total amount (nmoles) of AHLs synthesized per cells per unit time (nmoles of AHLs/cell/h).

To elucidate effects of salinity on SRB biofilm formation, a static biofilm assay was conducted on D. vulgaris and Db. corrodens biofilms cultivated on a polystyrene flat bottom 96-well plate (Costar, Corning Inc., Corning, NY, United States). A total of eight biological replicates, with three technical replicates per each biological replicate, were used for this study. The biofilms were propagated using both saline and freshwater media. Biofilms were incubated at 30°C for 144 h within anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI, United States). After 8 days, planktonic cells were removed and cells attached to the bottom of wells were washed with sterile 0.9% NaCl. Attached cells were then stained with 100 μL of 1% crystal violet (CV) reagent. After staining, cells were incubated at room temperature for 15 min. Excess CV was removed from each well, which was then air dried. Attached cells were then resuspended in 100 μL of 96% ethanol. Biofilm biomass was quantified in terms of OD₅₉₀ using a microplate reader (SpectraMax 340PC384, Molecular Devices, CA, United States).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based approach was selected to quantify the expression of target genes associated with sulfate utilization, carbon and energy metabolism as well as biofilm formation in D. vulgaris. The complete list of selected genes with their annotated functions and primers are listed in Supplementary Table S1. A detailed explanation for the selection of these target genes are also provided for as Supplementary Information 1. D. vulgaris biofilms were propagated in anaerobic serum bottles (working volume of 120 mL) using both saline and freshwater media, with three biological replicates each. Submerged fed-batch biofilm reactors were used to propagate biofilms on cellulose acetate (CA) coupons (5 mm × 5 mm) fastened together on a sterile 4” 22G needle (Air-Tite, Virginia Beach, VA, United States) and each reactor had three of such networks (five coupons per network) of cellulose acetate coupons. Tests were conducted in three phases, with each phase lasting for 7 days. At the end of each phase, half of the spent medium was replaced with fresh medium. At the end of the final phase, planktonic cells and CA membrane coupon-bound biofilms in the reactor were harvested separately. Briefly, coupons from each reactor were placed in 10 mL 0.9% NaCl and then individually subjected to ultrasonication at 25% amplitude with 2 s pulsating intervals for 3 min using a water-bath sonicator (Q500, Qsonica, Newton, CT, United States) (Cheng et al., 2016). Dispersed biofilm cells as well as freely suspended planktonic cells in the reactor were harvested through centrifugation (8,000 × g, 10 min) and used for extracting RNA after treatment with RNA protect (Qiagen, Hilden, Germany). The cell-RNA protect mixture was incubated for 5 min at room temperature and then centrifuged at 8,000 × g for 15 min. RNA-protected treated cell pellets were then stored at −80°C until RNA extraction. RNA extraction was performed using RNeasy Mini kits (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and RNA concentration was quantified using the Qubit 2.0 fluorometer (Thermo Fisher Scientific, San Jose, CA, United States) (Jumat et al., 2018). 1 μg of RNA extracts from biofilms were used as template for the synthesis of complementary DNA (cDNA) for RT-qPCR based on previously described protocols (Jumat et al., 2018). Target genes were amplified from the D. vulgaris genome using polymerase chain reaction (PCR). PCR products were cloned to pCR2.1 cloning vectors (Thermo Fisher Scientific, San Jose, CA, United States) and then transformed to E. coli TOP10 cells (Thermo Fisher Scientific, San Jose, CA, United States). The plasmids encoding each respective target gene were extracted using Plasmid Miniprep protocol (Promega, Madison, WI, United States). Based on the empirical relationship between plasmid DNA concentration, insert and vector size, the plasmid copy number was calculated. Plasmid DNA were then subjected to successive 10-fold serial dilutions to prepare the standard curve between the threshold cycle (C_T) and plasmid DNA copy number. The amplification efficiency and regression coefficient (R²) corresponding to standard curves for each target gene are provided in Supplementary Table S1. The volumes of reagents used for RT-qPCR were as follows: Fast SYBR Green Master Mix (Thermo Fisher Scientific, San Jose, CA, United States), 10 μL; forward and reverse primers, 0.4 μL each; cDNA template, 1 μL and PCR grade H₂O, 8.2 μL. RT-qPCR was conducted using Applied Biosystems^® QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific, San Jose, CA, United States). RT-qPCR cycle also included a melting curve analysis through an increase in temperature from 60 to 95°C for 5 s at 0.5°C interval. The copy numbers of each target gene estimated from the RT-qPCR standard curve were normalized against the single copy housekeeping gene Recombinase A recA (DVU1090). recA has displayed lower levels of gene expression heterogeneity in D. vulgaris biofilm growth mode, compared to other internal reference gene such as 16S rRNA (DV16SA) and glyceraldehyde 3-phosphate dehydrogenase (DVU0565), in accordance with previous studies (Zhang et al., 2007; Clark et al., 2012; Qi et al., 2014, 2016). Threshold cycle data values of recA extracted from D. vulgaris planktonic cells and biofilms are shown in Supplementary Table S2, and further demonstrated a low level of gene expression heterogeneity in this study.

Extracellular polysaccharides and proteins (EPS) from cellulose acetate membrane coupons-attached biofilms were detached through ultrasonication in 10 mL 0.9% NaCl, as mentioned in the previous section. After ultrasonication, the suspension harboring detached cells and EPS from CA membrane coupons was centrifuged (8,000 × g, 10 min). 0.25 μm syringe-filtered cell-free supernatant was used for quantification of EPS using liquid chromatography with organic carbon detector (LC-OCD) model-8 (DOC-Labor, Germany) equipped with a Toyopearl size exclusion chromatography column TSK HW50S (Tosoh, Japan) (Dimension: 250 mm × 20 mm, particle size: 20–40 μm). Polysaccharides and proteins from total EPS were fractionated and resolved using organic carbon detector (OCD) and organic nitrogen detector (OND). ChromCALC uni software was used to determine the concentration of each fraction in organic matter below the curve based on the integration of the defined area (Huber et al., 2011; Stewart et al., 2013).

To gain a better understanding of the role of QS in events leading to biocorrosion, three quorum sensing inhibitors (QSIs), (5Z)-4-bromo-5-(bromoethylene)-3-butyl-Z(5H)-furanone (bromofuranone), 3-oxo-D12-N-(2-oxocyclohexyl) dodecanamide (3-oxo-N) and γ-aminobutyric acid (GABA) (Sigma-Aldrich, MI, United States) were added to saline medium harboring D. vulgaris and Db. corrodens planktonic cells at different concentrations. Sub-inhibitory concentrations of QSI were selected from previous studies based on the effect of QSI on growth kinetics and biofilm formation (Hentzer et al., 2003; Smith et al., 2003; Ren and Wood, 2004; Chevrot et al., 2006). QSIs were deployed at sub-inhibitory concentrations (bromofuranone, 40 μM; 3-oxo-N, 20 μM; and GABA, 1 mM) and also at concentrations higher than sub-inhibitory concentrations, as follows: Bromofuranone (μM) – 80, 120, 160; 3-oxo-N (μM) – 40, 80, 120, 160; GABA (mM) – 1, 2, 5, 10, 20, 50. Planktonic SRB cells in saline medium without the addition of any QSI were used as a control in this study. Three biological replicates were maintained for each test condition, in a working volume of 22 mL. The effects of QSI on sulfate reduction, AHL production and specific growth rate were quantified. All concentrations of QSI described above were also used for biofilms to determine their effects on SRB biofilm formation.

All statistical assays were performed using Data Analysis tool on Microsoft Excel 2017. The degree of correlation in kinetics involving sulfate reduction, AHL production and cell density for all test conditions was measured in terms of Spearman’s rank correlation coefficient. The statistical significance tests were performed using two-tailed t-test on Microsoft Excel 2017.

D. vulgaris exhibited similar specific growth rates under saline and freshwater conditions, respectively (saline, 0.17 ± 0.02/d; freshwater, 0.14 ± 0.02/d; p = 0.15) (Supplementary Figure S2A). Similar trend was also observed for Db. corrodens (saline, 0.17 ± 0.03/d; freshwater, 0.16 ± 0.02/d; p = 0.32) (Supplementary Figure S2B). However, salinity significantly improved biofilm formation by D. vulgaris and Db. corrodens. Under saline conditions, D. vulgaris produced 1.5-times higher biofilm biomass than freshwater conditions (saline, OD₅₉₀ = 1.80 ± 0.48; freshwater, OD₅₉₀ = 1.17 ± 0.38; p = 7.65 × 10⁻⁶). Similarly, salinity also enhanced biofilm biomass of Db. corrodens by1.6-times (saline, OD₅₉₀ = 1.64 ± 0.43; freshwater, OD₅₉₀ = 1.03 ± 0.18; p = 2.77 × 10⁻⁶). In addition, higher polysaccharide to protein ratio for both D. vulgaris (2.05-folds) (saline, 0.76 ± 0.11 μg/μg; freshwater, 0.37 ± 0.05 μg/μg; p = 0.02) and Db. corrodens (2.0-folds) (saline, 0.56 ± 0.01 μg/μg; freshwater, 0.28 ± 0.06 μg/μg, p = 0.03) biofilms was observed under saline conditions.

Under saline conditions, D. vulgaris displayed significantly higher specific sulfate reduction rate (ca. 1.4- to 2.5-times, p < 0.05) during early (24 h), middle (48–72 h) and late exponential phases (96–120 h) as well as stationary phase (144–168 h) (Figure 1A) compared to that observed under freshwater conditions. Similarly, high specific sulfate reduction rates (ca. 1.3- to 2.3-times, p < 0.05) were observed for Db. corrodens under saline conditions during exponential phases (Figure 1B). However, Db. corrodens exhibited similar specific sulfate reduction rates during stationary phase (144–168 h, p > 0.05) under saline and freshwater conditions (Figure 1B).

In saline medium, total AHLs for D. vulgaris ranged from 20 to 27 nM (Supplementary Figure S2C) and 6 to 10 nM for Db. corrodens during exponential and stationary phases. This amount is higher, when compared to freshwater conditions (D. vulgaris, 12–14 nM; Db. corrodens, 4–6 nM) (Supplementary Figures S2C,D). Under saline conditions, D. vulgaris exhibited ca. three to four times higher specific AHL production rate for all growth phases as compared to freshwater conditions (p < 0.05, Figure 1C). In the case of Db. corrodens, an increase in specific AHL production rate by ca. 1.5- to 2-times was observed between early to mid-exponential phases in saline medium (p < 0.05) but the significant difference was no longer apparent in the latter growth phases (Figure 1D).

A higher correlation between specific sulfate reduction rate and specific AHL production rate was observed for D. vulgaris (R² = 0.87; p = 0.01) under saline conditions compared to freshwater conditions (R² = 0.75; p = 0.01). Similarly, Db. corrodens exhibited higher correlation between specific sulfate reduction rate and specific AHL production rate in saline medium (R² = 0.93; p = 0.03) compared to freshwater medium (R² = 0.73; p = 0.01). In addition, both D. vulgaris and Db. corrodens displayed a higher correlation between sulfate reduction and AHL production during early and mid-exponential phase (R²≥ 0.79; p < 0.05) compared to late exponential and stationary phases (R²≤ 0.63; p < 0.05) in saline medium.

RT-qPCR was conducted to quantify the abundance of specific genes related to sulfate reduction, carbon metabolism, hydrogenases and cytochromes, exopolysaccharide synthesis and signal response regulator in D. vulgaris biofilms propagated under saline and freshwater conditions. Key functions of all the respective genes are listed in Supplementary Table S1. Compared to freshwater conditions, expression levels of genes related to lactate metabolism like lactate dehydrogenase ldh (2.04-folds; p = 0.03) and pyruvate: ferredoxin oxidoreductase DVU3025 (2.19-folds; p = 0.04) were significantly upregulated under saline conditions (Figure 2A). High relative expression of genes involved in pyruvate and formate cycling such as pyruvate formate lyase DVU2272 (4.96-folds; p = 0.02) and formate dehydrogenases DVU0588 (7.71-folds; p = 0.02) was also detected under saline conditions (Figure 2A). The expression of all dissimilatory sulfite reductase subunits such as dissimilatory sulfite reductase alpha subunit dsrA (25-folds; p = 0.02), dsrB (1.92-folds; p = 0.05) and dsrC (2.49-folds; p = 0.05) was upregulated in saline conditions in D. vulgaris biofilms (Figure 2B). Although, no significant induction was detected for adenosine 5′-phosphosulfate reductase aprA and aprB (p > 0.05) (Figure 2B), sulfate adenyltransferase Sat, a key player in sulfate reduction was significantly upregulated (3.50-folds; p = 0.03) under saline conditions (Figure 2B). High abundance of periplasmic Fe hydrogenase alpha subunit hydA (2.73-folds; p = 7.40 × 10⁻⁵), NiFe hydrogenase alpha subunit hynA-1 (5.61-folds; p = 2.20 × 10⁻³) and NiFeSe hydrogenase hysA-1 (4.12-folds; p = 0.02) as well as Ech hydrogenases echE (14.88-folds; p = 0.03), echF (7.45-folds; p = 0.03) and cytochrome c553 DVU1817 (5.22-folds; p = 6.30 × 10⁻³) was also observed under saline conditions (Figure 2C). In addition, c3-type cytochromes harboring heme groups such as DVU3171 (5.14-folds; p = 7.07 × 10⁻³), DVU2524 (4.92-folds; p = 8.03 × 10⁻³) and DVU2809 (8.57-folds; p = 0.04) were also found to be significantly upregulated in the presence of salinity (Figure 2C). Lastly, salinity enhanced the expression of DVU0281 (3.58-folds; p = 0.04), which encodes for exopolysaccharide synthesis and DVU3062 (2.47-folds; p = 9.60 × 10⁻³), a histidine kinase involved in intracellular communication (Figure 2D). Apparently, expression of target genes was also found to be upregulated in case of D. vulgaris planktonic cells extracted from the biofilm reactor (Supplementary Figure S3).

To further comprehend and establish the linkage between QS and sulfate reduction in SRB, D. vulgaris and Db. corrodens were propagated in saline media in the presence and absence of QSIs. Figure 3 shows specific growth rate and biofilm biomass of D. vulgaris and Db. corrodens in saline medium in the presence and absence of QSI. Specific growth rate of D. vulgaris decreased significantly (ca. 1.41 to 2.65-times; p < 0.05) in the presence of bromofuranone ≥ 80 μM compared to the control (Figure 3A). The addition of 3-oxo-N ≥ 40 μM (ca. 1.72- to 2.71-times; p < 0.05) (Figure 3B) and GABA ≥ 5 mM (ca. 1.43- to 2.08-times; p < 0.05) (Figure 3C) resulted in similar decrease of specific growth rates of D. vulgaris. Likewise, at similar inhibitory concentrations, bromofuranone (ca. 2.35-times; p < 0.05), 3-oxo-N (ca. 1.33 to 2.32-times; p < 0.05) and GABA (ca. 1.84 to 2.70-times; p < 0.05) significantly decreased the specific growth rate of Db. corrodens (Figure 3C). Biofilm formation by D. vulgaris and Db. corrodens was compromised (p < 0.05) even at bromofuranone ≤ 40 μM, 3-oxo-N ≤ 20 μM and GABA ≤ 2 mM, as illustrated by the sharp decrease in biofilm biomass compared to control (Figure 3).

The effect of QSI with increasing concentrations on specific sulfate reduction rate during early, middle, late exponential and stationary phases is shown in Figure 4. Addition of bromofuranone ≥ 80 μM significantly decreased the specific sulfate reduction rate of D. vulgaris to ca. 0.52- to 0.71-times that of control (p < 0.05) (Figure 4A and Supplementary Table S3), while specific reduction rate of Db. corrodens decreased to ca. 0.72- to 0.84-times that of control (p < 0.05) during exponential phase (Figure 4B and Supplementary Table S3). In the presence of 3-oxo-N ≥ 40 μM, the specific sulfate reduction rate of D. vulgaris dropped to ca. 0.62 to 0.78-times of control (p < 0.05) (Figure 4C and Supplementary Table S3). The same is observed for Db. corrodens in the presence of 3-oxo-N during exponential phase (Figure 4D and Supplementary Table S3). Similarly, the specific sulfate reduction rate of D. vulgaris was ca. 0.58 to 0.83-times of control (p < 0.05) (Figure 4E and Supplementary Table S3) and that of Db. corrodens was ca. 0.60 to 0.84-times of control (p < 0.05) (Figure 4F and Supplementary Table S3) when exposed to GABA ≥ 5 mM. During stationary phase, decrease in specific sulfate reduction rate displayed by QSI-treated D. vulgaris and Db. corrodens was marginal (ca. 0.75- to 0.95-times of control; p > 0.05) compared to exponential phase (Figure 4 and Supplementary Table S3).

Addition of bromofuranone (≥80 μM), 3-oxo-N (≥40 μM), and GABA (≥5 mM) to D. vulgaris reduced the specific AHL production rate to ca. 0.2- to 0.40-times of control during middle and late-exponential phases (p < 0.05) and to ca. <0.25-times of control during stationary phase (p < 0.05) (Figure 5A,C,E and Supplementary Table S4). Similarly, bromofuranone (≥80 μM), 3-oxo-N (≥40 μM) and GABA (>5 mM) considerably decreased the specific AHL production rate of Db. corrodens during middle and late exponential phases (ca. <0.50-times of control; p < 0.05) and stationary phase (ca. <0.40-times of control; p < 0.05) (Figure 5B,D,F and Supplementary Table S4).

Increasing concentrations of QSIs (bromofuranone ≥ 80 μM; 3-oxo-N ≥ 40 μM and GABA ≥ 5 mM) decreased the overall correlation (R²) between specific sulfate reduction rate and specific AHL production rate from 0.87 to 0.57–0.78 for D. vulgaris (p < 0.05) and to 0.54–0.73 for Db. corrodens (p < 0.05). Likewise, QSIs also decreased the correlation between specific sulfate reduction rate and specific AHL production rate during early and mid-exponential phases from 0.79 to a range of 0.27–0.56. This decrease in correlation was more apparent in the exponential phases compared to that observed during stationary phase.