Bioremediation in the Marine Environment: Challenges and Prospective Methods for Enhancement

. Bioremediation is a low-cost, clean, and environmentally friendly method in managing marine pollution. Despite its great potential, marine bioremediation has its own challenges. As an open system, limited nutrients and fluctuating environmental conditions in the ocean affect the metabolism of degrading microorganisms, thus influencing the biodegradation rate. Multiple strategies have been employed to enhance the bioremediation rate at varying degrees of success. This review discusses these strategies from the perspective of experimental studies under controlled conditions and their potential applications for bioremediation. The addition of nutrients or other electron acceptors (biostimulation), as well as competent microbes to the contaminated site (bioaugmentation), have been reported to enhance pollutant degradation rate. Further modifications, such as using immobilized cells and genetic engineering have been employed to enhance the effectiveness of bioaugmentation. It is possible to combine more than one of these strategies to complement each other. However, one should note that all the reports to date were mostly done at the laboratory scale. Further studies need to be conducted by considering other factors such as climate, location, and types of pollutants, for the improvement of pollutant removal from the marine environment as a whole.


Introduction
The aromatic compounds, whether it is natural or xenobiotics, play a major role in our daily life and industries.Some examples of these aromatic compounds are petroleum hydrocarbon, phenol, alkanes, and polycyclic aromatic hydrocarbons.They are used in oil refineries, manufacturing paints, pharmaceuticals, pesticides, explosives, and as a precursor to many processes in industries.Nevertheless, they also represent a major cause of marine pollution [1,2].
Various physical and chemical methods have been employed to remove phenol from the contaminated site, such as adsorption, solvent extraction, chlorination, and oxidation [3,4].However, these methods will produce various intermediates and by-products that are even more toxic than the substance of origin [5].Bioremediation, which involves the use of pollutant-degrading microorganisms, is an efficient method to remove pollutants from the ocean since it is low-cost, clean, and environmentally friendly [6].From the early 1990s to date, many researchers have successfully isolated degrading bacteria from various contaminated sites in the ocean and studied their properties [7,8].
Even though it has a great potential for further development, marine bioremediation has its own challenges.Since the ocean is an open and highly mobile system, it is hard to keep the conditions under control, such as providing the appropriate and necessary amount of nutrients for microbial growth [9].Moreover, an open system can cause the dispersal of degrading strains, thus decreasing the effectiveness of bioremediation [6].In addition, pollutants are generally introduced to the environment at high concentrations, creating an environment that is too toxic for the microbes to grow and degrade [10].

Challenges for marine bioremediation
To design an efficient bioremediation strategy, one should consider any critical factors that may interfere with the process.Being an open and highly mobile system, it is quite challenging to carry out practical bioremediation and restoration in the ocean considering the width of the area [11].This can cause the loss of effective strains that were able to biodegrade the pollutants, thus decreasing the biodegradation rate [6,12].The openness of the marine system generally makes it hard to contain additional nutrients or to control essential conditions that affect the bioremediation process, such as temperature, pH, oxygen, and bioavailability [13].
The addition of nutrients such as nitrogen (N) and phosphorus (P) has a major role in marine bioremediation since the N and P levels are limited in seawater.Both affect the growth and activities of degrading microorganisms in the marine environment, thus stimulating the biodegradation process [13].This has been applied as early as in the 90s to clean up oil spills following the Exxon Valdez incident in Alaska and has been proved to successfully stimulate the biodegradation process [1].Despite that, specific strategies to introduce nutrients and prolong their bioavailability in seawater are still under investigation [9].
In addition, the ocean is exposed to constant temperature changes, in which case it may be extreme due to climate change [12].Temperature affects the metabolic rate of degrading microorganisms, hence it influences the biodegradation rate.It also affects the solubility and viscosity of pollutants [13].Delille et al. observed that low temperature could inhibit petroleum degradation in seawater [14].As temperature or environmental changes cannot be controlled in an open system like the ocean, the use of microbes that are adaptable to such fluctuation is of importance [6,14].

Biostimulation
Biodegradation using the natural population of microorganisms is perceived as the most reliable mechanism to eliminate both natural and xenobiotic pollutants within a habitat [15].There are several factors that affect biodegradation activities by microorganisms, such as , 00038 (2023) https://doi.org/10.1051/e3sconf/202337400038E3S Web of Conferences 374 3 r d NRLS the availability of nutrients, compound toxicity, pH, oxygen, and temperature [11].As an open system, the ocean is exposed to fluctuating environmental conditions and it lacks necessary nutrients such as inorganic nitrogen and phosphorus [16].Biostimulation is considerably the mechanism that can solve this problem.
Biostimulation is the modification of environmental conditions and available nutrients to stimulate the growth of microbes and thus enhance their degrading activity.It involves adding various forms of limiting nutrients and electron acceptors such as nitrogen, phosphorus, oxygen, carbon to support microbial [17].The addition of one or more limited nutrients or other electrons to the site has been reported to increase the activity of the naturally-occurring microbial population, thereby accelerating the degradation rate [18].The use of native microbes is of benefit as they are well-suited and well-distributed spatially to the subsurface environment [19].However, the delivery of additive nutrients or biostimulants so that they are readily available for existing microbes may be challenging.In this case, an open system like the ocean leads to rapid dilution of nutrients, thereby affecting the effectiveness of biostimulation [9].
To overcome the above challenge, the application of various forms of biostimulants has been tested (Table 1).Ideally, biostimulants should target microbes around the pollutant droplets and not easily get diluted in the ocean.In this sense, slow-release nutrients are well-suited for a highly mobile environment since they are dispersed in phases over time [17].The effect of slow-release inorganic NPK on the bioremediation of crude oil in the marine environment has been previously evaluated [20].This study showed that addition of slow-release inorganic NPK led to up to 94.87 % crude oil degradation [20].
Similarly, Delille et al. used a commercial oleophilic fertilizer (Inipol® EAP 22, C:N:P = 62:7.4:0.7) for biostimulation and reported a 50 % increase in diesel oil degradation rate in the sub-Antarctic coastal sea [14].A large amount of oleic acid in such fertilizer may serve as an alternative carbon source, hence increasing the amount of C/N ratio in the environment.Upon contact with water, the fertilizer emulsion will break and release urea, yet it may not always be readily available for microbes [21].Furthermore, there is risk of eutrophication due to the increased concentration of N and P, leading to algal blooming and consequently result in oxygen depletion in the seawater [17].

Combination of washing agent and nutrients
Crude oil 59 % of oil removal [19] Several researchers have used organic nutrients as biostimulants.Umanu et al. studied the effects of cow dung on biodegrading of motor oil in lagoon water [22].In 10 wk, the degradation percentage has reached up to 88.37 % [22].Using the solid waste date as a biostimulant, the heavy and light crude oil in the collected seawater were degraded by 79.49 % and 94.15 % in 14 d, respectively [23].Knezevichetal studied petroleum degradation by bacteria in a simulated open seawater system using guano or uric acid [9].Guano was considered an approachable method for biostimulation since it has low solubility in water and binds to hydrocarbon.Within 14 d, 70 % of hydrocarbon was degraded [9].
Besides adding the limiting nutrients such as N and P, there is a conducted study regarding biostimulation of hydrocarbon using halophilic bacteria by adding the amended cation salts such as K + , Ca 2+ , Mg 2+ , Fe 3+ [24].The authors simulated the condition by adding mineral salts and oil into the sampled water from the hypersaline coastal area.It is said the percentage removal exceeded 75 %.The trivalent (Fe 3+ ), as well as divalent (Ca 2+ , Mg 2+ ), are more effective than monovalent (K + ) at enhancing microbial activities under salt stress.It has also been said that the cations combined with pure vitamins enhanced the microbial numbers thus increasing the biodegradation rate [24].
In order to serve sufficient bioavailability to the microorganisms, there have been studies about the use of biosurfactants or washing agent to enhance the biodegradation rate.The washing agents act as a chemical dispersant that increases pollutant availability to microbes [19,25].A combination of biosurfactants with N and P enabled a faster degradation rate of and adaptation to pollutants by native microbial populations [25].In contrast, Crisafi et al. compared the effect of a combination of washing agents as well as nutrients in biostimulation treatment to treat oil-spill in Taranto Gulf, Italy [19].The addition of washing agents appear to result in a lower degradation rate, in which the removal of petroleum with and without washing agents was 59 % and 73 %, respectively [19].

Bioaugmentation
Bioaugmentation is the addition of a highly concentrated or specialized population of microorganisms to the contaminated site.The introduced microorganisms must be specific and competent in order to improve the biodegradation process [26].The most common options for bioaugmentation involve either the addition of pre-adapted pure bacterial strain, a consortium of microorganisms, or genetically engineered bacteria.This technique is suitable for contaminated sites that may be limited in microorganisms with the ability to metabolize specific pollutants [27,28].
Despite that, initial screening should be based on prior knowledge regarding native microorganisms in the contaminated site.This may include the metabolic potential of microorganisms and the essential features that help microbial cells to adapt and be functionally active under the desired environment [28].Bhoodevi et al. used Actinobacteria from marine sediments for the biodegradation of toxic textile dyes in the industry [29].Actinobacteria are suitable because they are highly stable in different conditions and tolerant to toxic textile dyes [29].
The use of microbial consortia is considered a more effective strategy than using pure cultures for bioaugmentation.Microbial consortia represent more diverse metabolic capabilities, thus increasing the potential for degradation [30].This was demonstrated in a case study in which a microbial consortium was used to degrade alkane in the oil spill in Taranto Gulf, Italy, resulting in a success rate of degradation up to 79 % [19].
Unfortunately, in some real cases, the microorganisms started decreasing in number soon after they were introduced to the contaminated site [31][32][33].This indicates the failure to adapt and/or compete with pre-existing microbial population.To overcome such problems regarding microbes' adaptation, some literature suggested looking for microorganisms from similar environmental conditions [27].Li et al. enriched the indigenous microbial consortia under aerobic conditions by using different xenobiotic compounds to produce a robust consortium for xenobiotic bioremediation [34].
It appears that the addition of microorganisms alone is generally insufficient and rather it should be accompanied by the environmental adjustment that is suitable for microbial growth preference.According to Hassanshahian et al., a combination of bioaugmentation and biostimulation could accelerate the biodegradation rate [15].However, such intervention should be evaluated thoroughly, including the applicability and limitations of each method in actual conditions such as at the contaminated site [27].

Enhancement of biodegradation
Attempts to introduce bioremediating microbes directly to the contaminated site may fail due to environmental stresses such as extreme temperature, pH, salinity, and pollutant toxicity [13].These issues may be overcome by modifying the selected microorganisms to enhance the effectiveness of bioaugmentation [27].Two forms of modifications, i.e. cell immobilization and genetic engineering, will be further discussed in this section.

Cell Immobilization
Immobilization of microbial cells involving carrier materials is one of the strategies to enhance the delivery of bioremediating strains to the natural environment.It provides a physical barrier to protect biomass, moisture, and aeration [35].A good carrier should provide ideal conditions for microbe survival, thus extending their shelf-life and improving their activity.This enables faster and more efficient biodegradation compared to that by free-living cells [10].Moreover, immobilized cells are favored for their high cell concentration and low microbial loss [6].Immobilized cells, in general, are also more stable in various temperature, pH, salinity, and crude oil concentrations, therefore making them more tolerant to environmental changes [6].
The carrier material for bioremediation should be non-toxic, environmentally friendly, stable, and low-cost.It should also be biodegradable in the natural environment to prevent accumulation issues once the introduced microorganisms have accomplished biodegradation.Chitin and chitosan flakes extracted from shrimp waste have been used as alternative carriers to immobilize degrading microorganisms [10].Both can potentially be mass-produced as the sources are abundant in nature, renewable, and affordable.Bacterial cells that were cultured in the presence of chitin and chitosan attached to the surface of these carriers through the formation of biofilm [10].The biodegradation rate of , 00038 (2023) https://doi.org/10.1051/e3sconf/202337400038E3S Web of Conferences 374 3 r d NRLS hydrocarbon has reached up to 60 % when the cells have been immobilized with chitin and chitosan flakes, whereas the free-living cells are only able to degrade just about 30 % of hydrocarbon.The authors noted that biofilm formation is a key process in protecting microbial cells from environmental stressors thus extending their survival rate and enhancing the efficiency of bioremediation [10].
Encapsulation is a method that is widely used for cell immobilization.It is well known for its simple operation, low probability of microbial leakage, reusability, stability, and high immobilization efficiency [6].Moreover, encapsulation protects the cells by reducing exposure to toxic compounds [6].It also minimizes cell membrane damage and lowers the amount of toxicity in the microenvironment of the cells [36].
Common materials to encapsulate the introduced microorganisms include gellan gum, calcium alginate, gelatin, and polyvinyl alcohol-boric acid.These materials have been well studied and tested in several experiments (Table 2).
Some researchers used polyvinyl alcohol-boric acid for encapsulation due to its mechanical strength, hence a much longer life [6].Unfortunately, the use of polyvinyl alcohol-boric acid for encapsulation may reduce the activity of microorganisms due to the toxicity of boric acid and agglomeration of the beads [6,37].To eliminate this problem, sodium sulfate has been used as a substitute for boric acid to cross-link with polyvinyl alcohol [37].Similarly, Liu et al., showed that the replacement of boric acid with phosphate buffer from the polyvinyl alcohol beads, with the inclusion of activated carbon, enhanced the efficiency of diesel degradation to 47 % [38].The highest degradation rate at 92 % was achieved using polyvinyl alcohol as a carrier [38].
Calcium alginate encapsulation is also common due to its simple preparation, mild reaction conditions, non-toxicity, and higher activity of encapsulated microorganisms.However, calcium alginate produced beads with poor mechanical strength and durability, therefore, providing less protection for the cells compared to other carriers [37].Chen et al. showed an 11 % improvement in crude oil degradation due to the addition of activated carbon to the calcium alginate-immobilized cells [6].

Enhancement with genetic engineering
Despite that genetically engineered microorganisms have been used to enhance the bioremediation potential as well as metabolic activity of the bacteria, to date, there are limited reports that are specifically targeted for marine bioremediation.Attempts to construct recombinant strains have been made to improve microbial ability to utilize both hydrocarbon pollutants and xenobiotics.This may be done through the insertion of new genes into the target genome, the introduction of a new, engineered plasmid and the alteration of metabolic pathways [39].
, 00038 (2023) https://doi.org/10.1051/e3sconf/202337400038E3S Web of Conferences 374 3 r d NRLS Modifications may be made by inserting new gene(s) to create a recombinant strain with more efficient pollutant catabolism.The Antarctic bacteria Pseudoalteromonas haloplanktis TAC125 was engineered to express the toluene-o-xylene monooxygenase (ToMO) gene of the mesophilic bacteria Pseudomonas sp.OX1 [40].The study showed that this enzyme is able to convert several aromatic compounds such as phenol, cresols, dimethylphenols, toluene, and o-xylene into the related catechols [40].However, the increasing amount of catechol from phenol oxidation may interfere with cell growth.Parrilli et al. improved the metabolic capability of Pseudoalteromonas haloplanktis TAC125 by combining the action of ToMO with the endogenous Pseudoalteromonas haloplanktis TAC125 laccase-like protein, a catechol oxidizer [41].
Other genes not directly related to the catabolism of pollutants may also be introduced to improve biodegradation efficiency.The Vitreoscilla hemoglobin gene (vgb) has been introduced to a wide variety of pollutant-degrading bacteria such as Burkholderia sp.DNT and Pseudomonas aeruginosa [34,42].The gene enhances respiration, growth and productivity, overall enabling recombinant bacteria to achieve better oxygen uptake and to degrade aromatic compounds more effectively under hypoxic conditions compared to the wild-type strains [42,43].
Biosensor using recombinant bioluminescent bacteria has also been explored for detecting and monitoring polycyclic aromatic hydrocarbons (PAHs) level in marine environments [44].Cho et al., developed a recombinant E. coli strain that could produce red color in the presence of PAHs.The recombinant E. coli carried nahR::lacZ fusion gene [45].The nahR gene-encoded regulatory protein for naphthalene degradation, whereas lacZ encoded for β-D-galactosidase.In the presence of chlorophenol red-β-D-galactopyranoside, which is the substrate of β-D-galactosidase enzyme, the recombinant E. coli will produce a visible red color.In this study, the authors demonstrated that the recombinant E. coli was able to selectively detect naphthalene and salicylate better than other PAHs [45].
Despite its great prospects, it should be highlighted that most of the studies were being done on a laboratory scale.The actualization of field studies requires lengthy procedures and authorization.Moreover, it is difficult to determine the exact extent to which genetically engineered microorganisms contribute to pollutant degradation in the environment.In addition, a highly diverse distribution of contaminants hampers the determination of biodegradation efficiency precisely [28,46].

Conclusion and future opportunities
Bioremediation is a promising approach to remove pollutants from the ocean since it is low-cost, clean, and environmentally friendly.This strategy may involve the addition of nutrients or other growth requirements for pollutant-degrading microbes (biostimulation) or the microbes themselves (bioaugmentation) in situ.In addition, various studies have demonstrated the use of cell immobilization and genetic engineering to improve bioremediation efficiency.It is also possible to combine the above strategies to complement each other.This prompts for further studies to pinpoint the most optimal conditions for pollutant removal, especially considering that the majority of research related to marine bioremediation has only been done at the laboratory scale.More efforts need to be done to bring such knowledge and findings to a larger scale by considering other factors such as climate, location, and types of pollutants, resulting in more efficient strategies for marine bioremediation as a whole.

Table 1 .
Recent studies on the application of biostimulants to remove pollutants.

Table 2 .
Studies on immobilized cells for marine bioremediation.