Naturally occurring hypersaline ecosystems of terrestrial as well as marine origin are an active, pivotal and vital part of planet earth. The interplay of biological, geological and chemical processes in this distinctive ecosystem not only impact the environment and society, but also have an effect on the economy (Shadrin and Anufriieva, 2013). Many hypersaline niches including naturally occurring saline water bodies, salt pans and salt marshes are frequently polluted due to the entry of elevated concentrations of metals from anthropogenic sources during the high tide. In solar salterns, when salt crystallizes the water evaporates and gets dense and metal concentrations increase. Unfortunately, heavy metals cannot be destroyed or degraded, unlike organic matter which can be easily converted to harmless end products. In such environments the native halophiles play a major role in mitigation of metal toxicity since they are able to survive under polyextremophilic conditions of salt stress, metal stress, alkalinophilic as well as thermophilic conditions (Ventosa et al., 1998). Some well-known examples of metal-tolerant bacteria are those belonging to the genera Flavobacterium, Alcaligenes, Moraxella, Pseudomonas, Vibrio, Xanthomonas, Micrococcus, Aeromonas, Acinetobacter, and Spirochaeta (DasSarma and DasSarma, 2012). Bacteria, fungi and algae from such environments can accumulate metal ions in their cells to concentrations much higher than the background concentrations of these metals (Coral et al., 2005). Zhang and Majidi (1994) evaluated microbes from extreme environments exposed to high salinity and metal stress and showed that the tolerance of microorganisms to heavy metals depended on their exposure to metals as well as their inherent physiological and/or genetic response mechanisms (Nagel and Voigt, 1989; Devars et al., 1998).

The Ribandar solar saltern from Goa, India, is one such classic example of a dual stressed environment subjected to high salt as well as high metal concentrations. It is located on the banks of the Mandovi estuary which receives an input of agricultural, industrial and domestic wastes, as well as an influx of spilled ferro-manganese ore during barge transport. Pollution studies of this estuary have been carried out by several researchers which indicate a continuous exposure of the saltern organisms to heavy metals (Kamat and Sankaranarayanan, 1975; Zingde et al., 1976; Alagarsamy, 2006; Krishnan et al., 2007). Kuppusamy and Hussain (2021) in their book chapter “Seasonal Variation for Trace Metals Contamination of Groundwater Using GIS Technology in Pissurlem, Sonshi, Cudnem, Velguem, Surla Watersheds, North Goa District, Goa State, India” reported findings of Mn in groundwater of villages in North Goa and observed that the Mn content in ground water increased during the summer season. Rodrigues et al. (2021) reported elevated levels of Mn in the Zuari estuary which is a part of the Mandovi Estuary in the State of Goa. Values as high as 4058 ppm at the surface and 3488 ppm at the bottom were reported. On similar lines, metal levels in sediments were 4.72%, 850 ppm, 95 ppm, and 45 ppm for Fe, Mn, Zn, and Cu, respectively, which exceeded average shale values, indicating anthropogenic pollution. Further, bioaccumulation of Fe, Mn, Zn, and Cu was reported in the edible bivalve Saccostrea cucullata, impacting their growth and reproductive ability. The prevalence of metals, such as Fe, Mn, and Zn, in hard shells of bivalves above the permissible limits was also attributed to anthropogenic pollution in the Zuari Estuary, due to iron ore mining. This has resulted in the emergence of bacterial strains that have acclimatized and developed tolerance to high concentrations of metals (Pereira et al., 2012). In our earlier studies we showed that this Mandovi estuary falls in the moderately contaminated category according to the geoaccumulation index calculation (Pereira et al., 2013). The increase in Fe concentration in this estuary was reported to be 8–17 times higher in the salt making season, whereas the increase in Mn was 34–54 times more, with a resultant amplification in the number of metal tolerant bacteria in the adjoining saltern. Based on the physiological importance of metals they may be categorized as: (1) non-toxic, e.g., Ca and Mg; (2) toxic, e.g., Hg and Cd; and (3) essential in small amounts but harmful above a certain level, e.g., Mn. The concentration of Mn in the Mandovi estuary falls in the harmful category. Mitigation of such toxic levels can be brought about by manganese tolerant bacteria. Swanson et al. (2023) observed that anoxic conditions resulting from overgrowth of algae impacted metal mobility by influencing the oxidation states and bonding capability of Fe and Mn at the interface of water and sediment at depths above 2.5 cm. In our study, the Ribandar Salt pan is inundated with microbes and algae during the non-salt-making season, creating an anoxic environment which would be conducive to alteration of the oxidation states of Fe and Mn, thereby causing temporary release of Fe and Mn into the surrounding which could influence metal-microbe bond formation and complexation.

Palmer et al. (1986) and Bargar et al. (2005) reported strains belonging to the genera Brevibacillus, Arthrobacter, and Sphingomonas capable of manganese oxidation. Manganese tolerant bacteria such as Bacillus species capable of Manganese oxidation have been isolated from various environments including coastal areas (Francis and Tebo, 2002), deep-sea vents (Dick et al., 2006), and the oxygen limited zone of the Black Sea (Stewart et al., 2007). Manganese oxidation by Halomonas sp. has been reported in the Carlsberg ridge (Fernandes et al., 2005). The marine Pseudomonas sp. strain S-36 was reported to use manganese oxidation as a means for obtaining energy for CO₂ fixation (Kepkay and Nealson, 1987).

Hence, we hypothesize that indigenous manganese tolerant bacteria could play an important role in mitigation of manganese toxicity. Further, these microorganisms could be harnessed for biosynthesis of metal oxides, since green chemistry is increasingly being sought as a low-cost, eco-friendly, functionally effective option. Metal oxides such as manganese oxide have many applications, in being used as bio-catalysts in the chemical industry (Kratošová et al., 2019). Synthesis of n-butyl acetate is normally carried out using metal oxides or mineral acids as catalysts (Nagvenkar et al., 2015). The n-butyl acetate, a colorless ester with a fruity odor finds applications in the food industry as a fruity flavoring agent in the manufacture of ice creams, baked goods and confectionery. It is used in agriculture as a natural trap for insects because it has odor mimicking alarm pheromones. It is a highly versatile solvent, capable of reducing viscosity and improving solvency and so it has many pronounced industrial and commercial applications, viz as an extraction solvent for lacquers and paints, in pharmaceutical and chemical industries. It finds uses as an ingredient in cosmetics, perfumes, cleaning products and car care products. It is also used in the leather industry, and for manufacture of adhesives and hardened coatings. PBLFs (Petroleum-based liquid fuels) require blending with biofuels in order to stem the emissions of particulates and minimize the utilization of PBLFs. In this regard, n-butyl acetate is considered to be the best candidate as reported in the literature (Wang et al., 2023). The n-butyl acetate is prepared by refluxing acetic acid and butan-1-ol, utilizing inorganic liquid or solid acid catalysts such as sulfuric acid or p-toluene sulphonic acid (Gangadwala et al., 2003; El-Sharkawy and Al-Shihry, 2004; Blagov et al., 2006; Martins et al., 2011). However, synthesis of n-butyl acetate by chemical methods though economical is not environment friendly, resulting in other problems such as environmental pollution and corrosion (Jermy and Pandurangan, 2006). Biological synthesis methods by the use of microorganisms offers a permanent solution to environmental pollution due to their safety and eco-friendly characteristics.

In the present study we have isolated and characterized manganese tolerant bacteria capable of manganese oxidation, and evaluated their potential in Mn removal, as well as utilized the manganese oxide formed as a biocatalyst. This is the first study to show that a moderately halophilic Chromohalobacter sp. from a marine saltern is capable of Mn oxidation and bioremoval of Mn. The study gains further relevance due to the fact that oxides of manganese (MnO_x) are extremely reactive (Tebo et al., 2004) and may therefore adsorb other metals, thereby regulating the high concentration of other metals in the salterns by chemisorption, thus augmenting the overall bioremoval of the other metals from the environment. Additionally in our present study, taking into account the varied applications of n–butyl acetate, we have synthesized and characterized n–butyl acetate using a biocatalytic route employing Chromohalobacter sp. as a catalyst.

Environments continuously exposed to elevated concentrations of heavy metals are known to promote metal tolerance in bacteria (Clausen, 2000; Nosalova et al., 2023). The toxicity and solubility of metals is also known to be influenced by several physico-chemical parameters of that particular area as shown in Table 1, which plays an important role in developing metal tolerance in the indigenous microbial population (Shi et al., 2013; Gupta et al., 2023). This study was thus aimed at exploiting this hypothesis, for the isolation and screening of metal tolerant bacteria as potential isolates for bioremediation and other industrial applications. The State of Goa, in India is known to be a major exporter of Ferro-manganese ore, with a continuous influx of Fe and Mn into its estuaries during iron ore transport by barges. As shown in Figure 1A, the location of the Ribandar solar saltern is such that it is adjacent to and fed by the Mandovi estuary which faces the threat of metal contamination. Concentrations of Fe as high as 17.2 ± 2.8 to 26.3 ± 6.7 % and Mn at 0.6 ± 0.2 to 0.9 ± 0.2 % have been previously reported by Pereira et al. (2013) from the Ribandar saltern sediments. Metal-tolerance in bacteria is also influenced by metal speciation (Gadd, 1990; Li et al., 2022). In this study, the solubility of Mn in the saltern was found to be influenced by salinity, Eh and pH (Table 1). When the salinity of the water in the saltern increased, there was an increase in flocculation and coagulation of particulate material in suspension which in turn favored the quick settling of the particulates. This could be the reason why the Mn concentration obtained in this study was generally higher in the sediment than the overlying water. In the overlying water the pH varied from 7.0 to 8.5 and in the sediment pH varied between 5.7 and 7.4. A lower pH in the sediment could be responsible for the dissolution of the sedimented metals, thereby making them bioavailable to the surrounding microorganisms (Atkinson et al., 2007). Cyclic changes in pH and salinity in the overlying water are known to considerably influence metal liberation from estuarine sediments, thereby increasing the bioavailability of metals to the microbes, which subsequently results in a larger population of metal tolerant bacteria (Hong et al., 2011; Pereira et al., 2013; Swanson et al., 2023). Olaniran et al. (2013) demonstrated that even a small fluctuation in pH could influence the solubility and bioavailability of metals significantly, wherein they showed how the solubility of cadmium reduced by a factor>8 with an increase in pH from 6 to 7. In the present study, we observed that when the pH in the saltern increased from 7.4 to 8.4, it resulted in an approximately twofold decrease in the dissolved Mn. Largely positive values of Eh are known to favor the existence of oxidized metal species, whereas low values are known to favor the existence of reduced species. In the Ribandar saltern, the Eh values obtained were as low as −10, to as high as 122 mV. Positive values of Eh, coupled with low pH values may have resulted in higher levels of free ionic species of metals in the sediment. In the water, the detected concentrations of metal were lower and pH values were higher; resulting in negligible metal availability to the microbes. According to Grybos et al. (2022) the mobility of metals and its bioavailability to heterotrophic bacteria was greatly influenced by pH, Eh, temperature and the strain of microorganism under consideration. They reported a 40 times increase in Fe, Pb, and Mn concentration in the aquatic phase when pH decreased from 3.7 to 2.1. Thus this interplay of several factors could have influenced the heavy metal availability and the degree of metal toxicity in the saltern, resulting in the emergence of the multi-metal tolerant Chromohalobacter sp. Nosalova et al. (2023) in their study on mine tailings from a lead, silver and zinc mining heap noticed that the bacterial counts were low but showed exceptionally high levels of resistance to heavy metals. Our study showed a similar scenario as reported earlier (Pereira et al., 2013).

Growth rate kinetic analysis of bacteria was performed to understand the cellular processes that link two or more cellular functions, for example, the relationships between growth and cell doubling time, fitted using empirical functions by mathematical modeling based on multiple steps involved in the progression of cell growth (Tiwari et al., 2021). The experimental measurements requires the analysis of parameter statistics to estimate the precision of the results by applying descriptive methods. Virtually, all of the models developed for kinetic analysis are non-linear (Qu et al., 2004). These mathematical models describe the number of organisms or logarithm of the number of organisms as a function of time.

Growth curves obtained in UV-visible experiments can be fitted using various growth functions such as logistic and Gompertz model (Zwietering et al., 1990; Sprouffske and Wagner, 2016). The experimental optical density (O.D.) data is a function of time for control without any treatment. Bacterial growth often shows a phase in which the specific growth rate starts at a value of zero and then reaches a maximal value (μ_m) in a certain period, resulting in a lag time (λ). The growth progress also contains a final phase in which the rate decreases and finally reaches zero, so that an asymptote (A) is reached or maximum O.D. As shown in Table 2, we fitted the growth curve of Chromohalobacter sp., treated with Manganese at varying salinities, employing the logistic and Gompertz function using Mathematica 9 software (Williams et al., 2012). These functions were found to be useful to obtain easily interpretable metrics such as population kinetics, the growth rate, the initial population size, and doubling time to summarize microbial growth curve data. In our study the changes in the growth rate showed sigmoidal behavior, with a lag phase immediately after t = 0, followed by an exponential phase and then a stationary phase.

The growth rate parameter (μ_m), which controls the steepness of the function, is known to be the primary variable that describes the cellular growth. In our study, the growth rate parameter (μ_m) showed a significant increase compared to the control, when salinity was enhanced to 10% (Supplementary Figure 2). With further increase in salinity to 20% there was a decrease in growth (Supplementary Figure 2).

Ventosa et al. (1998) reported that the requirement for salt and the limits of salt tolerance vary from species to species according to growth conditions, temperature and composition of media. In our study, Chromohalobacter sp. failed to grow in NaCl-deficient medium. Increase in the salinity to 2% (w/v) led to a greater tolerance to Mn. Further increase in salinity to 20 % enhanced tolerance to Mn from 0.01 to 5 mM. High salt tolerance of Chromohalobacter sp. could be due to the intrinsic adaptability of the organism to coastal saline soils (Ventosa et al., 1998). Similar findings were reported by Amoozegar et al. (2005). Osmotic stress was reported to trigger inhibition of ectoine transport at low salinity in C. salexigens Tn1732-induced mutant (CHR95) resulting in slower growth of the organism (Rodríguez-Moya et al., 2010). Enhanced growth of our Chromohalobacter sp. in the presence of manganese, as well as its potential to show multi-metal tolerance at high salinity could well be an indicator that our isolate had adapted to extremes of salinity and metal stress.

The Mn tolerant isolate FSK3 was identified as Chromohalobacter sp by 16 S rRNA sequencing (Table 3) Chromohalobacter spp. are known to be halophilic and capable of growing optimally at salinities of 8–10% NaCl, Our Chromohalobacter sp. was a borderline halophile capable of growing at salt concentrations up to 20%. It could grow over a temperature range of 25–55°C and pH 5.5–8.5, respectively. The preferred carbohydrate substrates utilized by Chromohalobacter sp, in the absence of Mn were Fructose, Gentobiose and Glucose, whereas the substrate utilization pattern changed with Mn amendments to D-Melibiose, Gentobiose and N Acetyl D-Glucosamine. It could not utilize lactose, xylose, maltose and mannitol. It was oxidase, catalase and urease positive. The organism produced a brown precipitate on Mn amended media, characteristic of it being a Manganese oxidizer. The first report of salt loving Chromohalobacter sp, was by T. Hof. It was isolated from fermented salted beans preserved in brine (Peçonek et al., 2006). The present study is the first report of a halophilic metal tolerant Chromohalobacter species from a solar saltern.

In the current study, metal tolerance in Chromohalobacter sp. could be due to an interplay of diverse mechanisms such as the secretion of EPS (Figure 4B), reduction in cell size (Figure 5B) and expression of Mnx genes (Supplementary Figure 4), resulting in the precipitation of Mn as MnO (Figure 6B). Bacterial polysaccharides have been reported by other authors to rapidly adsorb a range of metal ions such as Cd(II), Cu(II), Ni(II), and Zn(II) (Geddie and Sutherland, 1993). The anionic carboxyl and hydroxyl groups of EPS, a secondary metabolite, have powerful cation chelating properties. Further, the present Chromohalobacter sp. showed the presence of MnxG genes in the chromosome implying a complexity of genes involving vertical inheritance and horizontal gene transfer. Haritha et al. (2009) suggested a strong possibility of acute metal stress triggering transposition of metal tolerance genes from plasmid to chromosome, thereby disseminating metal resistance traits. Chromosomal resistance therefore is expected due to continuous metal exposure. Dick et al. (2008) tentatively identified the putative enzymes multi-copper oxidases (MCOs) encoded by the Mnx gene to trigger Mn(II) oxidation in Bacillus, Pseudomonas, and Leptothrix spp., suggesting a universal mechanism of bacterial Mn(II) oxidation. MCOs are a family of enzymes that utilize four Copper ion cofactors to catalytically oxidize a wide range of substrates such as metals and organic compounds (Sakurai and Kataoka, 2007). In the present study, the oxide produced by Chromohalobacter sp. could be of Mn(IV) oxide, since it is more likely to be deposited on the bacterial surface on prolonged incubation (Ehrlich, 1999). Similar to this study, Francis and Tebo (2002) had reported oxidation of manganese by a spore forming marine Bacillus sp. strain SG-1 where Mn(II) oxidation was catalyzed by a multi-copper oxidase, MnxG. Zhang et al. (2014) demonstrated the role of Fe and Mn oxides in the sorption of other toxic elements like arsenic and elucidated that they could be highly efficient, economical, and environment friendly oxidants of toxic metals capable of environmental remediation. Our Chromohalobacter sp. showed ability to remove metals, well above the range of metal concentrations encountered in the saltern water (Figure 3A). Maximum bioremoval of Mn (76%) by Chromohalobacter sp. was at 0.1 mM at a rate of 8 mg Mn/g/day. Similar bioremediation of water contaminated with metals such as Fe, Mn, Cu, As, and Zn, by species from the Ralstonia family, i.e., Ralstonia eutropha, phylogenetically related to Cupriavidus was reported by Mondal et al. (2008), with removals of up to 65.2, 72.7, 98.6, 8, and 99.3%,respectively. Mn(III, IV) oxides and Mn complexes which are soluble are known to be the strongest oxidizing agents in the environment followed by oxygen and thereby play a key role in the biogeochemical cycles of major (C and S) and trace elements (Fe, Co, Pb, Cu, Cd, and Cr) (Laha and Luthy, 1990; Dick et al., 2008). Literature reveals biogeochemical cycling of Mn oxides to control the distribution of many trace elements, resulting in the adsorption and concentration of diverse metals. This is due to the highly charged nature of Mn minerals (Huang, 1991). In aerobic natural waters (pH 6 to 8), chemical oxidation of Mn (II) is slow, but in the presence of Mn (II)-oxidizing microorganisms, the rate is 4 to 5 times higher (Nealson et al., 1988; Wehrli et al., 1995; Stewart et al., 2007). Consequently, Mn oxidation could provide several pathways for transformation/sequestration of metal and organic contaminants. Hence, it is relevant that Chromohalobacter sp. capable of precipitating manganese as oxides, could also be capable of adsorbing or precipitating metals other than Mn, thus regulating and detoxifying the concentration of other metals as well in the salterns. Chromohalobacter sp. therefore could find applicability as an ideal model organism for manganese removal from ferro-manganese contaminated sites in estuaries and hypersaline environments. Wani et al. (2023) conducted in vitro studies and documented the use of heterotrophic bacteria as bio-inoculants beneficial in the removal of metal pollutants.

Further studies on mechanism of metal tolerance using AFM with Mn and crude salt also indicated binding of metal to the bacterial surface, as evidenced by the outer membrane roughness in the height profile (amplitude imaging) compared to the control (Figure 7). The exact mechanism of how these particles interact with the cell surface either directly or indirectly still remains unexplored. This study needs to be extended further to identify the exact concentration and other parameters.

The IR data confirmed presence of Mn (Figure 8) Chromohalobacter sp. grown at Mn amendments of 20 mM (sample A), and 2 mM (sample B), signifying that there is metal present in these samples which is absent in the other two samples. The existence of the peak at 718 cm^–1 indicated the presence of metal–oxygen stretching in tetrahedral environment. This peak was absent in samples C and D, confirming that these samples did not have metal oxide. Presence of hydroxyl, amidic and carbonyl functionalities assist in the metal binding and in the formation of metal oxides. Swanson et al. (2023) demonstrated that alteration in the oxidation states of Fe and Mn, as would be the case in the Ribandar Saltern due to fluctuations in pH, temperature and organic matter, would cause temporary release of Fe and Mn into the surrounding, resulting in a larger population of metal tolerant bacteria capable of immobilizing Mn as manganese oxide. Thus, it can be seen that our Chromohalobacter sp. being capable of extracellular deposition of Mn, biosorption by EPS as well as oxidation of Mn could well be playing a crucial role in the remediation of metal pollutants in the saltern.

Another noteworthy application of this Chromohalobacter sp. is as a biocatalyst in the synthesis of n-butyl acetate from n-butyl alcohol. It is significant considering that this hypersaline, metal tolerant and high temperature tolerant organism could act as a biocatalyst capable of 50% conversion which is similar to that reported using chemical catalysts, but the noteworthy thing is that it could do so at a temperature less than 70°C, where Chen et al. (2011) reported the requirement of 1.5 g chemical catalyst and a temperature of 135°C for 4.5 h for the conversion, though the reported yield was higher. Our bio-catalyst was better than that reported by Nagvenkar et al. (2015) who used ZnO and Hβ zeolite as a catalyst but were able to obtain less than 50% conversion. Therefore, our Chromohalobacter sp. could be an excellent candidate for use as a bio-catalyst, since it exhibited catalytic activity for the synthesis of n-butyl acetate.

Heavy metal accumulation in Ribandar salterns has affected the estuaries, as well as human health. Exposure to metals has resulted in emergence of metal tolerant bacteria. Multiple mechanisms have been suggested to get adjusted with fluctuations in metal concentrations. Presence of functional manganese tolerance genes could be used as biomarkers for Mn contamination. Since the Chromohalobacter sp. was capable of tolerating and immobilizing high concentration of Mn in this hypersaline ecosystem, it could be a candidate organism for scavenging Mn ions from polluted euryhaline environments. Existence of such metal tolerant bacteria in the Ribandar salterns could thus provide a solution to the environmental problems. Chromohalobacter sp. showed a catalytic activity of ∼50% conversion for the synthesis of n-butyl acetate, which is comparable to the percentage conversions shown by other catalysts in the literature. Thus this halophilic Chromohalobacter species from estuarine coastal waters could serve as an excellent detoxifier of Manganese, as well as a novel bio-catalyst for synthesis of n-butyl acetate.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

FP: conceptualization, investigation, lab experiment, data analysis, writing—original draft, and revision of manuscript. SK: conceptualization, supervision, resources, and project administration. DD: lab experiment, data analysis, and writing—original draft. VG: AFM experiment and data analysis. All authors provided critical feedback, helped to shape the research, analyzed the manuscript, discussed the results, and contributed to the final version of the manuscript.