Exploring water hardness removal potential of halotolerant bacteria isolated and characterized from brine lemon pickle

Halotolerant microorganisms capable of thriving in high salt concentrations are of significant interest due to their ecological roles and potential industrial applications. This study aimed to isolate and characterize a halotolerant bacterial strain from a traditional fermented food source. A bacterial strain designated SS1 was isolated from brine lemon pickle. The isolate was characterized using morphological and biochemical tests, antibiotic susceptibility profiling, and 16S rDNA sequencing. Growth was assessed across varying NaCl concentrations, and biofilm formation was evaluated under high-salt conditions. The strain’s potential application in hard water treatment was also examined. The isolate was Gram-positive, coccoid, and showed remarkable tolerance to salt, growing at NaCl concentrations up to 20%, with optimal growth between 0% and 10% (doubling time 25–35 min). The colonies on agar were cream-coloured, circular, glistering, and entire-edged. Based on morphological attributes and biochemical characterization, the isolate exhibited characteristics consistent with those of the Staphylococcus genus. Partial sequencing of 16s rDNA also revealed that strain SS1 was similar to Staphylococcus gallinarum. The strain exhibited susceptibility to all tested antibiotics except cefonicid and cloxacillin. Biofilm formation was observed only at a high NaCl concentration (20%). The isolated strain treated hard water and showed a 45% hardness removal efficiency on the 15th day. With its unique halotolerant and biochemical profile, strain SS1 presents promising potential for future exploration and application in environmental remediation, hard water treatment, and other industries.

Water hardness is a prevalent issue affecting water quality across the globe. Hard water is characterized by high levels of calcium and magnesium ions (Hori et al., 2021; Worku and Sahu, 2014). About 70% of the Earth’s surface is covered by water, with 97% being seawater, which is unsuitable for drinking. Of the 2.5% freshwater available, only 0.5% is accessible for human use. Over 85% of available freshwater sources are hard water, as it accumulates minerals like calcium and magnesium from rocks and soil (Ahn et al., 2018). Hard water can cause significant issues in industrial settings, thus monitored closely to prevent costly damage and breakdown in equipment such as boilers and cooling towers, where mineral deposits can impair functionality. Consuming very hard water over extended periods can pose health risks, including cardiovascular problems, diabetes, kidney stones, and Alzheimer’s disease, due to the high concentrations of calcium and magnesium (Sengupta 2013; Bykowska-Derda et al., 2023). According to the drinking water quality standards (IS 10500 2012), the permissible limit of hardness in water is 200 mg/L, with levels above 500 mg/L are unacceptable (Madhusudhan et al., 2023). Conventional methods for addressing water hardness include ion exchange, reverse osmosis, filtration, and chemical precipitation. Despite their effectiveness, these methods often have drawbacks such as high costs, environmental impact, and operational complexity, driving the search for more sustainable alternatives (Padmapriya et al., 2015). One promising avenue for sustainable water treatment involves biological methods.

Halotolerant bacteria, which thrive in hypersaline environments with extremely high concentrations of salts, have shown potential in various bioremediation applications (Dutta and Bandopadhyay, 2022; Kumar and Gummadi, 2009). Halotolerant microorganisms tolerate salt stress by regulating osmotic pressure within their cells (Mokashe et al., 2018). They have evolved two different strategies for adapting to salt stress - salt in/out cytoplasm and compatible solute strategies (Agu et al., 2017; Wang et al., 2023). These adapting strategies enable them to thrive in saline environments such as salted food products, saline soils, and marine habitats (Rahman et al., 2017). Scientists are fascinated by exploring halotolerant microbes in saline environments due to their significant biotechnological potential in industry, agriculture, and medicine, as they harbor valuable enzymes and salt-tolerance genes (Edbeib et al., 2016). Halotolerant bacteria found in saline environments hold the potential to enhance plant growth under stressful conditions (Albdaiwi et al., 2019; Banik et al., 2018; Haque et al., 2022; Hernández-Canseco et al., 2022). Halotolerant microorganisms have the potential to be utilized in bioremediation, effectively degrading organic pollutants in saline environments (Al Farraj et al., 2020; Guo et al., 2015; Muralidharan et al., 2023; Ravikumar et al., 2011) and purifying industrial wastewater with high levels of inorganic salts and organic pollutants (McGenity et al., 2012).

Despite showing greater versatility under salt conditions compared to ordinary microorganisms, their capabilities are yet to be fully exploited. Numerous underexplored diversities of halotolerant bacteria exist in hypersaline environments. With limited research on exploring halotolerant in harsh conditions, the untapped potential of these extremophiles has garnered the attention of researchers. Consequently, there is a growing emphasis on isolating and studying halotolerant bacteria from environments, aiming to discover new species, elucidate their adaptation mechanism, and harness their potential application in water treatment. The present research reports the first instance of isolating a halotolerant bacteria, Staphylococcus gallinarum (strain SS1), from brine lemon pickles, followed by screening and characterizing through morphological, biochemical, and molecular methodologies. The study also evaluates the efficiency of the strain SS1 in reducing the total hardness of the water. Future investigations will explore the potential application of halotolerant bacteria in various fields, such as bioremediation and wastewater treatment.

Isolation and screening of halotolerant bacteria

A loopful of samples was taken from brine lemon pickles and streaked onto nutrient agar plates (Himedia) supplemented with 3% (w/v) NaCl. The plates were incubated for 24–48 h at 37 °C to isolate halotolerant bacteria. Further, the cultures were transferred gradually to media containing higher concentrations of up to 25%. The salt tolerance ability of the isolated strain was subsequently assessed through plate assay.

Morphological and biochemical characterization

Isolated Strain SS1 was morphologically characterized by colony morphology, Gram-staining, and scanning electron microscope (SEM) imaging (Sigma 500 VP Gemini 1, Zeiss). Strain SS1 was examined for colony morphology, emphasising color, elevation, size, margin, form, opacity, and surface. Biochemical tests such as urease (Brink, 2010) and catalase (Reiner, 2010) were carried out, and the capacity to utilize different carbohydrates was tested using a carbohydrate utilization kit (HiCarboTM kit KB009, Himedia). Additionally, tests for the production of industrially important enzymes, including amylase test (Yassin et al., 2021), β-galactosidase test (Kolev et al., 2022), cellulase test (Lisdiyanti et al., 2012), laccase test (Dhiman and Shirkot, 2015), protease test (Khan et al., 2011), and xylanase test (Singh et al., 2010) were performed.

Growth curve at varying salt concentrations

The growth kinetics of strain SS1 was monitored by inoculating 1% (v/v) of an overnight grown culture in nutrient broth containing NaCl in different concentrations of 5%, 10%, 15%, and 20% (w/v). The nutrient broth without NaCl was used as a control. Growth was determined at intervals of 4 h for 72 h at 150 rpm by measuring absorbance at 600 nm with a spectrophotometer (UV-1800, Shimadzu).

Effects of various pH levels

The isolated strain was screened for tolerance to low pH in nutrient broth and adjusted to pH levels ranging from 3.0 to 6.0, with pH 7.0 as the control. Each pH-adjusted media was inoculated with an overnight grown inoculum 1% (v/v) and incubated for 24 h at 37 °C and 150 rpm. Cell growth was assessed by measuring absorbance at 600 nm with a spectrophotometer.

Molecular characterization of isolate SS1

The molecular identification of bacterial strain SS1 was performed through 16s rDNA gene sequencing (Biokart, India). Following the purification of genomic DNA, the 16s rDNA gene fragment was amplified using PCR with a pair of Universal primers: forward primer (5′-GGA​TGA​GCC​CGC​GGC​CTA-3′) and reverse primer (5′-CGG​TGT​GTA​CAA​GGC​CCG​G-3′). The PCR protocol consisted of an initial denaturation for 3 min at 94 °C, followed by 30 cycles of denaturation for 1 min at 94 °C, primer annealing for 1 min at 50 °C, and extension for 2 min at 72 °C, with a final extension step of 7 min at 72 °C. The sequencing of amplified genes was performed using the ABI 3130 Genetic Analyzer, and the sequencing chemistry kit used was Big Dye Terminator version 3.1.

The 16s rDNA sequence obtained was aligned and compared for similarities in the NCBI database using BLAST software (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The top ten sequences were chosen based on the highest identity scores and aligned using ClustalW, and a phylogenetic tree was constructed using MEGA 11 software (Tamura et al., 2021). The phylogenetic tree was built from unambiguously aligned nucleotides using the neighbor-joining algorithm with bootstrap analysis using 1,000 replicates. Furthermore, the sequence result was submitted to GenBank at NCBI.

Antibiotic susceptibility

The antibiotic susceptibility profile of strain SS1 was determined using the Kirby-Bauer disc diffusion method, following the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) (Clinical and Laboratory Standards Institute, 2026). The test was performed using nine antibiotics, namely, ampicillin (10 µg), amoxicillin (20 µg), tetracycline (30 µg), chloramphenicol (30 µg), rifampicin (5 µg), sulphafurazole (300 µg), cefonicid (30 µg), cloxacillin (1 µg), and vancomycin (30 µg); (Himedia). Nutrient agar plates were spread with 100 µl of overnight-grown culture, and antibiotic discs were placed on them at appropriate distances under sterile conditions. After incubation for 24 h at 37 °C, the growth of the isolate was observed. Results were interpreted by measuring the diameter (mm) of the clearance zone and expressed as either resistant or susceptible.

Static biofilm formation assay

The static biofilm formation ability of strain SS1 was determined according to a standard protocol, with some modifications (O’Toole, 2011). Strain SS1 was cultured in nutrient broth containing different NaCl concentrations (0%, 5%, 10%, 15%, and 20%) and distributed in the 96 well microtiter plates, with eight replicate wells for each NaCl concentration and incubated for 48 h at 37 °C under static condition. The medium was decanted after incubation, and wells were washed with distilled water. Subsequently, wells were filled with 120 µl of 0.1% crystal violet, kept for 15 min, and washed with distilled water thrice. The plate was left overnight to air dry, following which the dye-bound cells were solubilized with 30% acetic acid, and OD was measured at 595 nm using a microplate reader.

Reduction of hardness

The isolated strain SS1 was assessed for its ability to reduce hardness in an artificial hard water sample. The sample was prepared by modifying methods (Ahn et al., 2018; Hettiarachchi et al., 2017) supplemented nutrient broth with a solution containing magnesium sulfate and calcium chloride to achieve a total hardness of 200 ppm, further inoculated with an overnight grown inoculum (1% v/v). Following inoculation, samples were incubated at 37 °C for 20 days. Initial optical density and sample hardness were measured. Total hardness was determined by the EDTA (ethylene diamine tetra acetic acid) titration method (Baird et al., 2017). Further, the reduction in total hardness was subsequentially monitored by titration every alternate day for 20 days according to the standard operating procedure.

Statistical analysis

Experiments were conducted in triplicates, and the data were analyzed using GraphPad Prism software version 9. Error bars represent the standard error of the mean from three replicates. Significant differences were calculated using one-way ANOVA followed by Bonferroni post-tests.

Isolation and morphological characterization

The bacterial strain SS1 was isolated from the brine pickle prepared in the laboratory. SS1 could fully grow up to 20% NaCl, indicating its halotolerant nature. Thereafter, the bacterial strain exhibited very limited growth at 22% NaCl, and further, no growth was observed beyond 22% on agar plates. Microscopic examination showed that strain SS1 is a Gram-positive bacterium with a cocci morphology (Figure 1).

Further characterization through Scanning Electron Microscopy (SEM) confirmed the spherical shape of the bacterial cells. Table 1 shows the morphological characteristics of the isolated strain SS1.

Biochemical characterization

The isolated strain was further biochemically characterized. The presence of catalase was confirmed by observing bubbles’ formation upon adding H 2 O 2 to the bacteria. The isolated strain tested positive for catalase. Additionally, a positive urease test was indicated by the development of a pink color after incubation. The carbohydrate utilization pattern revealed that the isolates utilized a range of sugars, as detailed in Table 2. The isolate was evaluated for its ability to produce industrially important enzymes. It showed high production levels for amylase, cellulase, and β-galactosidase, moderate protease production, and did not produce laccase and xylanase (Table 3).

Molecular identification

Based on comparing 16s rDNA sequencing data with NCBI BlastN, the strain SS1 was identified as Staphylococcus gallinarum. A phylogenetic tree was constructed using MEGA 11 software based on 16s rRNA gene sequences, employing a neighbor joining (NJ) method with 1,000 bootstrap replications. This tree elucidates the precise phylogenetic position of Staphylococcus gallinarum SS1 among 10 Staphylococcus species (Figure 2). The sequence result was submitted to GenBank at NCBI and assigned an accession number PP474738.

Growth curve studies

Strain SS1 was grown under different NaCl concentrations, ranging from 0% to 20%. Samples were taken at distinct time intervals, and a growth curve was constructed. Analysis of the growth curve at different salt concentrations indicated that with an increase in salt concentration (>10%), the growth of the bacteria slowed down, as evident from the increased duration of lag and log phase (Figure 3). Especially, at the highest salt concentration of 20%, there is a prolonged lag phase. At a low salt concentration of 5%, the exponential phase was around 24 h before transitioning into the stationary phase at 28 h. However, at the highest salt concentration of 20%, the exponential phase increased to 40 h before entering the stationary phase after 48 h. Salt concentration ranging from 0% to 10% was found in the optimal range for strain SS1, where it grows efficiently with a doubling time of 28–37 min. Consequentially, the doubling time was extended to 55 min and 72 min for 15% and 20% salt concentrations, respectively.

Effects of various pH levels

A pH tolerance assay assessed the halotolerant bacterial strain’s adaptability to acidic conditions. Growth was observed only at pH 6.0. In contrast, no detectable growth was observed at lower pH levels of 3.0, 4.0, and 5.0, and the observations are kept in the supplementary file (Supplementary Figure S1). Bacterial strain adaptability to acidic environments is constrained, with growth only feasible in mildly acidic to neutral conditions, indicating a limited range of pH tolerance.

Antibiotic susceptibility

The isolated strain exhibited sensitivity to all the selected antibiotics, as evidenced by the presence of inhibition zones except vancomycin (intermediate susceptible), Cefonicid (resistant), and Cloxacillin (resistant). The antibiotic susceptibility profile of the isolated strain, determined through the disc diffusion method, is presented in Table 4.

Static biofilm formation assay

A static biofilm formation assay was conducted to evaluate the effect of varying NaCl concentration (0%, 5%, 10%, 15%, and 20%) on biofilm development by isolated bacteria; it was found that biofilm formation was induced explicitly at the highest tested concentration of 20% NaCl. This result was evident by violet coloration at the bottom of the wells containing the 20% NaCl after 48 h of incubation, indicative of biofilm presence. In contrast, wells containing lower NaCl concentration and the control wells without bacteria showed no coloration, indicating the absence of biofilm formation. Absorbance was recorded at 595nm, and a graph was plotted against different salt concentrations (Figure 4).

Reduction of total hardness

The reduction of total hardness by strain SS1 was carried out for 20 days. The maximum total hardness removal was 45% in 15 days of the experiment, which remained unchanged until day 20. Reductions in total hardness of 3%, 12%, 23%, 32%, 40%, and 43% were observed on days 1, 3, 5, 7, 9, and 11, respectively (Figure 5).

In the present study, a halotolerant strain of Staphylococcus gallinarum, designated as SS1, was isolated from a brine pickle and subjected to further characterization. Previously, Staphylococcus gallinarum has been isolated mainly from poultry sources such as cows (de Oliveira et al., 2022), skin and nares of chicken (Devriese et al., 1983), breast muscle of chicken (Islam et al., 2023), the gut of the insect Bombyx mori (Barretto and Vootla, 2018), immunocompromised patients (Morfin-Otero et al., 2012) and infected wounds of human patients (Kolawole and Shittu, 1997). There are reports also available on isolation from food sources like fermented coconut water (Dhanya Raj et al., 2023), spoilt butter (Kocharekar and Dukhande, 2017), and Japanese fermented foods, like miso (Onda et al., 2003) and soybean paste (Kim et al., 2010). However, to the best of our knowledge, this study is the first to report the isolation of halotolerant S. gallinarum from a food source. Our findings revealed that the isolated strain SS1 demonstrated remarkable halotolerance, capable of growing fully up to 20% NaCl concentration, with limited growth observed at 22% NaCl on agar plates. Notably, other species within the Staphylococcus genus have been known to grow up to 25% concentrations of NaCl (Komaratat and Kates, 1975).

Phenotypically, the strain SS1 was identified as Gram-positive cocci, forming small, cream-colored, circular, glistening surfaces and entire edge colonies similar to those described for the S. gallinarum strain (Devriese et al., 1983). Biochemical analysis revealed that the strain was catalase and urease positive, capable of utilizing a range of sugars, producing cellulase, amylase, β-galactosidase, and moderate protease production, aligning with the Staphylococcus genus characteristics (Dhanya Raj et al., 2023; Kary and Alizadeh, 2016). Molecular analysis confirmed the identity of a strain as Staphylococcus gallinarum, and phylogenetic analysis supported its close association with other S. gallinarum strains, according to tree topology.

In control samples without the addition of salt, S. gallinarum SS1 showed a doubling time of 28 min, which is comparable with previous reports where the wild-type strain of S. gallinarum had a generation time of 27.6 min (Ebner et al., 2018). The doubling time was 34.5 min and 37 min at 5% and 10% NaCl concentration, respectively, suggesting that strain SS1 maintained a high growth even in a moderately high salt level. However, growth of SS1 slows at higher salt concentrations of 15% and 20%. A previous study on halotolerant Bacillus licheniformis MHN 12 also showed that as salt concentration was increased from 0.5% to 15%, growth gradually slowed down (Bhutani et al., 2022). In the present study, there is also an increase in the duration of both the lag phase and log phase of the growth curve at 20% salt concentration. This could be mostly due to osmotic shock on the bacteria, resulting in growth suppression in a stressed environment. During the lag phase, bacteria adapt to high salt concentrations by synthesizing and accumulating compatible solutes, organic molecules that maintain cellular osmotic balance. This process requires energy and resources, which prolongs the lag phase. Strain SS1 thrives in mildly acidic to neutral conditions, exhibiting growth only at pH levels 6 and 7. Below pH 6, the acidity might disrupt crucial cellular functions, inhibiting growth.

Strain SS1, which exhibits tolerance to high salt concentration and can produce industrially important enzymes such as cellulase and amylase, represents a promising reservoir of salt-tolerant enzymes. These enzymes could be used for diverse biotechnological applications, especially in industries where high salt concentrations hinder enzymatic reactions (Ruginescu et al., 2020). The strain’s ability to utilize various sugars enhances its versatility and potential applications. This characteristic indicates metabolic flexibility, enabling the strain to thrive in diverse environments and optimize resource utilization. The isolate showed susceptibility to most antibiotics tested except cefonicid and cloxacillin, which is a vital trait for potential future applications. The susceptibility simplifies bioremediation processes by minimizing the risk of spreading antibiotic resistance genes, ensuring the strain poses no risk when applied in environmental settings.

Biofilms protect microorganisms from extreme environmental conditions, and Staphylococcus species are known for their ability to form biofilm at high salt levels (Rachid et al., 2000). The biofilm formation ability of strain SS1 was assessed at various salt concentrations. The results indicated no biofilm formation within the 0%–15% NaCl concentration range, but significant biofilm formation was observed at 20% NaCl concentration. These findings suggest a distinct adaptive response of bacteria to high salt stress, potentially signaling a threshold mechanism. A previous study showed that rbf gene regulates biofilm formation in the presence of NaCl as biofilm production plateaued at 1.6% NaCl in Staphylococcus aureus, suggesting that above a threshold of 1.6% NaCl, biofilm formation in response to NaCl is controlled by rbf (Lim et al., 2004). Similarly, the increase from 15% to 20% NaCl might surpass the threshold, activating a pathway in strain SS1 that promotes biofilm formation. At lower concentrations, bacteria likely cope with stress through other mechanisms, such as the efflux of ions or the accumulation of compatible solutes that balance the osmotic pressure. Conversely, at high concentrations, strain SS1 used biofilm formation as an alternative strategy for survival. In another study, researchers investigated the impact of NaCl on biofilm formation in Staphylococcus aureus, and their findings revealed an increase in biofilm formation with increasing NaCl concentration (Lee et al., 2014). This emphasizes the role of high NaCl levels in stimulating biofilm formation, corroborating our findings.

Strain SS1, further utilized for hardness removal, showed a remarkable reduction of 45%. To the best of our knowledge, this is the first reported instance of a halotolerant Staphylococcus bacterium being employed for hardness removal. Algae have been previously reported for the treatment of hard water, with Chara being one of them utilized explicitly due to its ability to adsorb calcium and magnesium (Hamdona et al., 2022; Worku and Sahu, 2014). Previously, salt-tolerant Staphylococcus xylosus has been reported for biological treatment of highly saline wastewater containing 72% salinity, achieving a significant removal of COD up to 88% (Abou-Elela et al., 2010) Exploring SS1 for bioremediation of hard water represents a promising avenue for sustainable water treatment. Future research will optimize treatment strategies for long-term water quality management. Using bacteria for hardness removal presents a natural, cost-effective, and eco-friendly alternative to synthetic chemicals.

Strain SS1 holds immense potential across various sectors, including food industries, water treatment, salinity removal, and bioremediation. The continued exploration and testing of this strain are poised to unlock more innovative applications in the future. In future studies, the pathways governing halotolerance and gene regulation will be investigated through metagenomic and transcriptomic analyses, and the hardness removal treatment method will be optimized.

Isolation and characterization of strain SS1, a halotolerant strain of Staphylococcus gallinarum from brine pickle, and its hardness removal potential was highlighted in this study. SS1 showed remarkable adaptability to high salt concentration, thriving up to 20% NaCl, a characteristic previously not reported for S. gallinarum. Its morphological and biochemical profile aligns with attributes of the Staphylococcus genus, further confirmed by molecular analysis. Isolate SS1 had a doubling time of 28–37 min under a concentration of 0%–10% NaCl, although the doubling time extended to 72 as salt concentration increased to 20%. Strain SS1 exhibited susceptibility to most antibiotics except cefonicid and cloxacillin. The observation of biofilm formation at 20% NaCl concentration suggests a unique adaptive response to extreme salt stress. The isolated strain was employed to treat hard water and showed a 45% hardness removal efficiency on the 15th day. The promising results can be used to study the application of strain SS1 in treating high-salinity wastewater, food industries, and bioremediation. Future research will be conducted to understand the molecular mechanism underlying SS1 halotolerance, hardness removal and biofilm formation, identifying essential genes and pathways.