Innovative approaches in bioremediation: the role of halophilic microorganisms in mitigating hydrocarbons, toxic metals, and microplastics in hypersaline environments | Microbial Cell Factories

Despite the microbial diversity, most hypersaline environments have been regarded as unproductive regions with minimal commercial value. Consequently, there are few laws and awareness programs in place to protect these environments [18]. These environments have been harmed by human activities, including salinization, water diversion, mineral extraction, sewage discharge, and pollution from agricultural and industrial runoff such as pesticides, fertilizers, and excess nutrients [18, 25].

Lakes with salty water and freshwater have different hydrological systems that significantly impact pollutant distribution. Freshwater lakes are open systems, whereas saline lakes are generally closed and experience low precipitation and high evaporation. This combination leads to significant pollutant accumulation and rapid dispersal in saline lakes compared to freshwater systems [18, 25]. Saline lakes are mostly polluted by agricultural wastewater and domestic and industrial organic and inorganic wastes [25].

Furthermore, the discharge of untreated high-salinity wastewater can lead to significant environmental pollution and harm both aquatic and terrestrial ecosystems. Many industrial processes, including the chemical coal industry, agricultural by-product processing, and pharmacology, generate significant amounts of high-salinity wastewater containing complex contaminants. It contains inorganic contaminants such as chloride, toxic metals, sulfate, sulfide, and nitrogen [26, 27].

One of the most significant contaminants in these ecosystems is petroleum hydrocarbons and crude oil. High-salinity ecosystems are exposed to hydrocarbons and improper drilling operations and processing can lead to remarkable hydrocarbon releases into these environments [28]. Additionally, wastewater from the petroleum industry contains numerous toxic organic contaminants, including polycyclic aromatic hydrocarbons and halocarbons, as well as inorganic contaminants such as lead and mercury [27]. The accumulation of toxic metals and metalloids in water and soil is another major problem that should be considered. These metals pose significant global health risks, as they are toxic and persist in the ecosystem [29,30,31]. Metal toxicity depends on both the exposure dose and the specific metallic chemical species, which affect their mobility and bioavailability in the organisms and environment. The most soluble and bioavailable metallic species have the greatest toxicity [32]. Therefore, it is crucial to eliminate these hazardous pollutants.

Another important challenge in saline and hypersaline environments is pollution caused by microplastics (MPs). Annually, approximately 8 million tons of plastic waste enter the ocean, the world’s largest saline water resource, contributing to increasing anthropogenic pollution in marine environments. Saline soils can also be affected by MPs from pollution sources such as erosion, wind, contaminated water, and plastic disposal. Their low self-purification capacity and poor biological activity make these environments more susceptible to retaining MPs for longer periods [33]. Given the remarkable metabolic capabilities and powerful enzymes of halophilic microbes, these organisms offer a promising solution to remove these three significant contaminants in extreme habitats [11, 30].

Hydrocarbon pollution in hypersaline ecosystems

Naturally occurring compounds that consist of hydrogen and carbon and serve as the fundamental components of natural gas are known as hydrocarbons. The majority of hydrocarbons are produced through the processes of thermal cracking and fractional distillation of crude oil [29]. Among the hydrocarbons, Petroleum Hydrocarbons (PHs) pose the greatest environmental threat. PHs are persistent and harmful pollutants in the environment that affect all living organisms. These hydrocarbons can be classified into four chemical fractions: alkanes, aromatics, resins, and asphaltenes [20].

The presence of oil is often accompanied by high salt concentrations. Crude oil is formed in areas with high salinity and evaporation rates, and it is linked to hypersaline brines. Drilling operations enhance the risk of exposing oily brines to the environment. To maintain an osmotic barrier and prevent swelling in drilled rocks, large amounts of salt are added to drilling mud, which can be discharged into the ecosystem [34]. Moreover, high-salinity ecosystems are often exposed to hydrocarbons, and it is clear that bioremediation plays a crucial role in naturally cleansing such environments [35]. Many hypersaline environments like salt flats, natural saline lakes, estuaries, beaches, inland lakes, oilfields, saline industrial effluents, and salt marshes have been found to contain high levels of petroleum hydrocarbons [36].

The petroleum industry also generates a large amount of oily effluents containing at least 10% salinity after oil separation from reservoir water. Additionally, there is always the potential risk of pollution in any place where oil is being extracted. Currently, between 600,000 and 1,750,000 tons of oil are being released into the sea annually through shipping and delivery operations [21]. The biological removal of crude oil and refined products in seawater (3–3.5% salinity) has been extensively investigated, but there is less understanding of hydrocarbon fate at higher salinities [4].

In hypersaline ecosystems, different hydrocarbons display varying levels of resistance to being broken down by microorganisms. The preferred order for breaking down PHC substrates from most to least resistant is polyaromatic hydrocarbons, low molecular weight aromatics, cyclic alkanes, branched alkanes, and n-alkanes. The components, such as octane, pristane, pentacosane, and tetracosane, are known to persist in these environments. Polycyclic aromatic hydrocarbons like naphthalene, salicylate, phenanthrene, biphenyl, o-phthalate, anthracene, 9-methylanthracene, and phenol are also widespread in hypersaline environments. Moreover, benzene, toluene, ethylbenzene, and xylenes are common in these environments [21, 34, 37]. The slow rates of hydrocarbon degradation under extreme conditions are influenced by the substrate’s metabolism, including uptake, transport, and enzyme activities [4]. High concentrations of salt can reduce hydrocarbon and oxygen solubility, disrupt cell membrane function, denature enzymes, and dehydrate cells, hence limiting microbial metabolic rates [38]. However, halophiles generate more hydrophobic cells to adapt to high salinities, enhancing their ability to consume more hydrocarbons. Thus, the understanding of halophilic and halotolerant microbes capable of degrading PHCs has significantly advanced, and research has revealed a much broader microbial diversity that can effectively remediate these contaminants across a wide range of salinity [21].

Furthermore, using enzymes that are stable in extreme conditions is remarkably important for biodegradation [34]. Halophiles’ enzymes are non-toxic, biodegradable, and effective catalysts. However, some of them are unstable under industrial process conditions. Thus, we need to understand the metabolic mechanisms of halophilic microbes for waste remediation and environmental processes [39].

The biodegradation of petroleum compounds typically involves three main steps: adsorption to microbial surfaces, transfer through cell membranes, and degradation by microbial enzymes [40]. Numerous studies have investigated various metabolic pathways involved in the removal of petroleum hydrocarbons under aerobic and anaerobic conditions [29, 30]. Recent studies indicate that hydrocarbon degradation at high salinity occurs via enzymes known to function in many non-halophiles (like mono-oxygenase and di-oxygenases) [34, 41]. For example, mono-oxygenase enzymes serve a dual function in the biochemical pathway: they facilitate both the initial oxidation of alkanes and the final stage of ω-hydroxy fatty acid oxidation in crude oil substrates [41]. However, there is limited information on the precise removal mechanisms of petroleum hydrocarbons in saline environments. In one study, Wang et al. (2018) investigated the phenanthrene degradation pathway in the halophilic consortium CY-1, initiated by dioxygenation at the C1 and C2 positions. In another study, Kadri et al. (2018) exhibited the ability of Alcanivorax borkumensis to utilize motor oil, hexane, and hexadecane, producing high levels of alkane hydroxylase, esterase, and lipase enzymes [34]. For more details and examples, see Rezaei Somee et al. (2020) and Pereira et al. (2024) [34, 41].

Different studies have demonstrated that many halophiles and halotolerants like Haloarcula, Haloferax, Marinobacter, Acinetobacter, Pseudomonas, Enterobacter, Ochrobactrum, Stenotrophomonas, Achromobacter, Cycloclasticusare, Rhodococcus, Rhodanobacter, Chromohalobacter, Micrococcus, Thalassospira, Arthrobacter, Bacillus, Rhodanobacter, Alcanivorax can use aromatic and aliphatic compounds as the sole carbon source [29, 41, 42]. Table 1 lists halophilic and halotolerant microorganisms capable of eliminating petroleum hydrocarbons. Based on the table, halotolerant microbes are notably effective at low to moderate salinities. For instance, Staphylococcus sp. CO100 achieved 72% degradation of aliphatic hydrocarbons at 10% NaCl over 20 days. Additionally, a bacterial consortium was able to remove 56.24% of Total Petroleum Hydrocarbons (TPH) at 4.5% salinity within 30 days. Other isolates, such as Pseudomonas aeruginosa NAPH6 and Bacillus kochii AHV-KH14, demonstrated near-complete hydrocarbon degradation within one week at lower salinities (0.03–1.5%).

In contrast, halophilic microorganisms perform better at high salinities (≥ 10%), often showing enhanced degradation. For example, a consortium including Marinobacter and Rhodococcus removed up to 100% hexadecane at 0.5–2% salinity and up to 97% fluorene at 8–12% NaCl within 6–18 days. Similarly, Hortaea B15 achieved 100% phenanthrene degradation at 10% salinity in just 7 days. Thus, while halotolerant microorganisms are well-suited for bioremediation in moderately saline environments, halophilic microorganisms exhibit broader adaptability and often higher degradation efficiencies, making them particularly suitable for bioremediation in hypersaline conditions such as salt lakes, saline soils, and oil-contaminated brines.

Some halotolerant or halophilic microbes have developed mechanisms for synthesizing different types of biosurfactants to utilize complex hydrocarbon pollutants in hypersaline environments. These biosurfactants reduce interfacial and surface tension by forming emulsions, improving solubility and mobility. Data on microorganisms associated with oil hydrocarbon biodegradation is extensive; however, reports on biosurfactant producers are limited [21]. In one study, fifty-five biosurfactant producers belonging to the genera Alcanivorax, Halomonas, Bacillus, Rhodococcus, Streptomyces, Acinetobacter, Exiguobacterium, and Pseudomonas were isolated, and their emulsification abilities were investigated. The result indicated that Acinetobacter and Rhodococcus were able to emulsify n-hexadecane at low temperatures and high saline conditions. The strain Acinetobacter P1-1 A exhibited the highest emulsification ability at 62.5% [43]. In another study, a halotolerant strain of Bacillus licheniformis produced the highest amount of biosurfactant at a 5% NaCl concentration [44].

Halophilic microbes also produce Extracellular Polymeric Substances (EPS), which promote surface adhesion and biofilm formation. These exopolysaccharides function as biosurfactants, aiding in oil aggregation, hydrocarbon emulsification, and providing cellular resistance to toxic heavy metals. In one study, EPS-producing Halomonas was applied for biodegradation at a hydrocarbon-contaminated site from the Deepwater Horizon spill. The extracted EPS enhanced the solubilization of aromatic hydrocarbons and increased their degradation rate [45, 46]. Other halophiles and halotolerants, such as Vibrio parahaemolyticus and Bacillus licheniformis, also produce EPS that aid in hydrocarbon emulsification and degradation [46].

In contaminated hypersaline environments, successful petroleum hydrocarbon degradation depends on the ability of indigenous microbes to tolerate varying salt concentrations. Generally, salt content impacts the biological removal of petroleum hydrocarbons, as the types and number of substrates used decrease with increasing salinity. There is considerable interest in using Bacillus and Pseudomonas microbes to remove contaminants from hypersaline environments, even though they are not known to be halophiles but can still tolerate high salt concentrations [21].

In order to bioremediate high-salt environments lacking effective halophilic or halotolerant strains, two strategies can be employed to manage salinity. In the first strategy, salt concentrations can be reduced by diluting with fresh or diluted seawater, or by using reverse osmosis, electrodialysis, or ion exchange [36]. The alternative approach involves using engineered halophilic oil-degrading bacteria that thrive in high salt concentrations, as well as bioaugmentation with foreign microbial consortia or biostimulation to enhance the metabolic activity of native species [8, 36]. Owing to the cost implications of diluting high salt concentrations, using engineered halotolerant or halophilic microorganisms appears to be a promising approach [36]. However, introducing external microorganisms is discouraged as they may harm the ecosystem [1]. On the other hand, studies have reported that biostimulation through improved environmental conditions effectively accelerates the growth of native microorganisms. Oil spills release a large amount of hydrocarbons, disrupting the normal carbon-to-nitrogen and phosphorus ratio in marine ecosystems and hindering the growth of oil-degrading microbes. Adding nitrogen and phosphorus compounds can help restore this balance and promote the growth of native microbes [34]. Other microbial-assisted technologies, such as using biosurfactant-producer organisms, biofilm formation, and immobilized microbial technology, have been discussed in detail by Jimoh et al. (2022) [21].

Toxic metals in hypersaline ecosystems

Industrialization and technological advancement have increasingly burdened the environment with hazardous waste, including toxic metals such as chromium (Cr), cadmium (Cd), lead (Pb), arsenic (As), selenite (Se), tellurium (Te) and antimony (Sb) [47,48,49,50,51]. Some toxic metals (Cr, As, Cu (copper), Al (aluminum), and Fe (iron)) primarily come from untreated industrial effluents discharged into nearby water bodies, especially in developing countries. Toxic metals like Pb, Cd, Ni (nickel), and Zn (zinc) are also released from vehicle exhaust and accumulate in roadside soils, entering aquatic systems during rainfall. Additionally, antifouling paints from ships contribute to marine metal pollution [50, 51].

Essential trace metals like Fe, Zn are crucial for life and must be present in specific amounts. They function as enzyme cofactors, regulators of osmotic pressure, and stabilizers of molecules. However, excess amounts can be toxic, depending on their availability and the dose absorbed by the body [30]. Exposure to these toxic metals is associated with mutagenicity, cancer, and disorders of the neurological, circulatory, endocrine, and immune systems. Consequently, these substances induce molecular and cellular changes, including oxidative stress, genotoxicity, enzymatic inhibition, and modulation of gene expression. The diverse chemical properties of metals hinder the attribution of a single toxicity mechanism, and several mechanisms have been identified to date [30, 32, 52]. Various methods, such as sludge filtration, adsorption, chemical oxidation, and reverse osmosis, are applied to remediate metal-contaminated environments. However, these techniques can be costly, especially when metal concentrations are very low [53]. The high solubility of most toxic metal salts also makes separation through chemical and physical techniques challenging [30]. Furthermore, the effectiveness of the current chemical removal of toxic metals under extreme conditions is limited by their poor accessibility [45]. Therefore, there is a need to explore alternative methods that are appropriate for local conditions.

Some microbes have acquired resistance mechanisms to adapt to these pollutants, making them suitable candidates for bioremediation [5, 30, 32, 54]. These microbes assist in detoxifying hazardous components through natural processes, which can be enhanced by adding nutrients and electron acceptors. Toxic metals can be detoxified through vacuole compartmentalization, volatilization, and metal binding [30, 32]. Various studies have explained these mechanisms and related enzymes in detail with examples [5, 30, 32, 54]. In general, microorganisms employ techniques such as bioabsorption, bioaccumulation, biotransformation, and biomineralization to survive in metal-polluted environments. The microbial removal of toxic metals is influenced by different factors, such as pollutant concentration, metal bioavailability, pH, oxygen levels, electron acceptors, temperature, nutrients, and osmotic pressure [55].

Many extremophiles effectively remove toxic metals [11, 29]. They can use metals in their metabolism or synthesize enzymes and biomolecules that enable them to bypass metal toxicity and utilize these metals for survival. The defense mechanisms of extremophiles against toxic metals are significantly important to researchers. They aim to utilize these mechanisms for deploying microbes that can thrive in less extreme conditions to remediate environmental contaminants [29]. Therefore, the sustainable development of bioremediation methods using extremophilic microorganisms has been explored over the past several decades [45]. For more details and examples, see Giovanella et al. (2019) [32].

Halophiles possess various transport systems, enzymes, and regulatory factors that facilitate the bioremediation of metals [5]. Halotolerant and halophilic microbes have been observed to biotransform toxic metals in high-salinity water bodies [29]. Additionally, some halophilic microbes have the capacity to absorb metals effectively [5, 30]. For example, Vibrio harveyi demonstrated a strong ability to accumulate cadmium cations, achieving a high adsorption rate of up to 23.3 mg Cd²⁺/g of dry cells [45]. In another study, the isolated Halomonas sp. WQL9 demonstrated a 94% reduction in Pb levels (at a concentration of 1 mM) at extreme conditions (10% NaCl and low pH) within 6 h [56]. Table 2 lists other halophilic microorganisms that can tolerate and remove toxic metals. As shown in the table, halotolerant microorganisms generally exhibit faster and more efficient activity under moderate saline conditions. For instance, Bacillus, Oceanobacillus, and Salinicoccus species demonstrated high removal efficiencies of Pb (up to 98.8%) and Ni (up to 76%) at 10% NaCl within 30–40 h. Similarly, Bacillus pumilus removed 96% of Pb in just 24 h at 5% NaCl. In contrast, halophiles maintain robust performance even at higher salinities, indicating their potential for bioremediation in hypersaline, metal-contaminated environments. Halophilic microorganisms such as Exiguobacterium sp. and Desulfovibrio halophilus operated effectively at higher salinities (10–12%), achieving Pb and Fe removal rates of 89% and 85.3%, respectively.

It should be noted that while metal combinations are often highly toxic, some halophiles are unaffected and can even tolerate and remove them from the environment. This ability is especially notable when compared to non-halophilic organisms. This may be attributed to the various toxic metal resistance mechanisms that halophiles utilize, including the regulation of transport across membranes, sequestration by biopolymers, and detoxification or biotransformation into less toxic forms [57, 58]. Genes that encode toxic metal multi-resistance (including Ni, Cr, Cu, Zn, As, Co, and Cd) were identified in the genome of slightly halophilic microbes, such as Marinobacter manganoxydans MnI7-9 [57].

Some studies have investigated the multi-metal-resistant ability of halophilic microbes [59,60,61,62]. For instance, in one research, multi-metal-resistant halophiles (Halobacterium saccharovorum, Hbt. sodomense, and Hbt. salinarium) were isolated from Tamil Nadu, India. These organisms exhibited resistance to Zn, Ni, Cd, Al, As, and Hg. The authors demonstrated that this ability is directly controlled by chromosomal DNA and might be related to metal-binding proteins [63]. In another study, researchers isolated a plasmid (pGIAK1) from the halotolerant endospore-forming bacilli strain JMAK1 and indicated that this plasmid carries the genes for arsenic and cadmium resistance [64].

However, understanding the mechanisms of microbial activity and metal metabolism in extreme conditions needs further clarification before effective site-specific treatments can be implemented.

Microplastics in hypersaline ecosystems

Nowadays, plastic has revolutionized industries with its versatility and durability [65]. Common synthetic plastics include Polyethylene (PE), Polypropylene (PP), Polystyrene (PS), Polyethylene terephthalate (PET), and Polyvinyl chloride (PVC) [19]. Microplastics (smaller than 5 mm) are small particles that originate from the breakdown of larger plastic debris, known as secondary microplastics. Primary microplastics, on the other hand, are produced by human activities and include beads, pellets, fibers, and powders found in cosmetics, clothing, and industrial materials [66]. Due to the widespread use of plastics, significant amounts of them have been released into the environment, leading to serious ecological issues. The toxicity of microplastics increases further when they adsorb different pollutants (organic and inorganic contaminants) from the environment. They have shown an affinity toward heavy metals [67,68,69,70]. Also, hydrophobic particles, such as microplastics, with a rough surface structure, can adsorb crude oil, affecting the effectiveness of oil dispersion [66]. The rate at which toxic contaminants are adsorbed onto microplastics depends on the type of polymer [51].

Many niches contaminated with plastic are often characterized by extreme conditions like low or high temperatures, acidic or alkaline pH levels, high salinity, or elevated pressure [71]. Various studies have reported the presence of plastics in saline and hypersaline environments. For example, two different studies examined the sources, concentration, and distribution of microplastics in the hypersaline Maharloo Lake in Iran. Both studies showed that PP, PE, and PET were the most common types of microplastics in this lake. The highest concentration of microplastics was detected in the northwestern region of the lake, where urban, industrial, and agricultural wastewater is discharged [72, 73]. In another report, Pashaei et al. confirmed the presence of microplastics in Urmia Lake, located in northwestern Iran [74].

Using extremophilic microbes and their enzymes presents a promising solution to this issue. Despite their relatively short evolutionary time, many extremophilic microbes have adapted to thrive in these ecosystems through plastic removal, playing a crucial role in the biodegradation of contaminated habitats. Extreme conditions enhance plastic degradation by extremophiles due to increased enzyme activity, which softens the plastics and disrupts their mechanical integrity. Additionally, extremophilic microbes are producers of various potentially useful hydrolytic enzymes. Thermophilic and halophilic enzymes have a slower aging process, allowing for room-temperature storage and extending their half-life in commercial preparations. This longevity helps maintain enzymatic activity in time-consuming processes like plastic remediation [71, 75].

Several investigations revealed the presence of halophilic microorganisms on microplastic surfaces and their capacity to form biofilms. It is reported that in extreme environments contaminated with plastics, the most halophilic microbes are found to be slight or moderate extremophiles, with Erythrobacter species predominating [76, 77]. For instance, Dussud et al. (2018) studied various niches of the Western Mediterranean Sea with a salinity of 3.87% and found a higher number and density of bacteria attached to microplastic particles compared to free bacteria and those attached to organic particles. They demonstrated that the plastic fragments mainly consisted of PE, PP, and PS. The identified strains primarily included Calothrix sp. and Pleurocapsa, with a notable presence of the moderate halophiles Hyphomonas and Phorimidium [77]. In another study, authors compared microbial diversity on PS microplastics incubated in Black Sea water (1.86% salinity) and industrial effluent (0.1% salinity) and indicated that the significant salinity difference may influence the growth of weak halophilic microorganisms and microbial community formation. Analysis of 16 S rRNA showed that Erythrobacter dominated during the two months of incubation in Black Sea water, contributing to PS degradation [76].

However, research on the ability of halophilic and halotolerant strains to degrade microplastics in high-salinity environments is limited. In one study in 2016, Krasowska et al. investigated the removal of polyurethane by microorganisms isolated from the Baltic Sea (salt level 1.86%, temperature 10 °C, and pH 8). In this study, changes in polymer structure, polymer weight, and strength were investigated after 12 months, and about 4–19% weight loss in polymers was reported [78]. In another study, Adithama et al. (2023) isolated two moderately halotolerant bacteria that were able to grow in a medium containing 10% NaCl from saline sludge in Indonesia (Stenotrophomonas maltophilia and Enterococcus sp.). They reported that these bacteria were able to remove 5% and 6% of Low-Density Polyethylene (LDPE), respectively, within 90 days [79]. Moreover, Singh et al. (2023) tested the biodegradation of LDPE by using three novel halophilic bacterial isolates, Brevundimonas naejangsanensis MGS1, Arthrobacter crystallopoietes SW4, and Arthrobacter pokkalii PD2, and demonstrated that all the isolates reduced the weight of LDPE and modified its chemical structure (maximum weight reduction: 4.64 ± 0.5%) [80].

Therefore, the search for new microorganisms in extreme environments is promising due to their unique adaptations. However, their industrial use is limited by challenges such as difficult cultivation, lower biomass, and lower productivity yields. As mentioned, extremophilic degraders mainly include slight and modest extremophiles. This is due to the fact that harsh conditions reduce biological diversity and hinder the evolution of plastic-degrading microorganisms. Furthermore, low growth rates in these environments make it difficult for bacteria to thrive on hard-to-degrade polymers [71].