Table of Contents
What is Bioremediation?
Bioremediation is a process used to clean up and detoxify polluted sites by utilizing microorganisms, plants, or their enzymes to break down and degrade environmental contaminants. It involves the natural or deliberate introduction of these biological agents to enhance the rate of degradation and transformation of pollutants.
The concept of bioremediation includes biodegradation, which refers to the breakdown and detoxification of contaminants by microorganisms and plants. This natural process can be enhanced through various methods:
- Biostimulation: This involves providing nutrients, carbon sources, or electron donors to stimulate the growth and activity of indigenous microorganisms. By supplementing the natural microbial community, biostimulation enhances their ability to degrade pollutants.
- Biorestoration: Similar to biostimulation, biorestoration involves creating favorable conditions for the indigenous microorganisms to degrade contaminants. This can include optimizing temperature, pH, oxygen levels, and moisture content in the environment.
- Bioaugmentation: In bioaugmentation, an enriched culture of microorganisms with specific characteristics is introduced to the polluted site. These specialized microorganisms have the ability to degrade the targeted contaminants at a faster rate than the indigenous microorganisms. This method can be particularly effective when the indigenous microbial community lacks the necessary capabilities to degrade certain pollutants.
The goal of bioremediation is to reduce pollutant levels to undetectable, nontoxic, or acceptable levels set by regulatory agencies. Ideally, the contaminants are completely transformed into harmless byproducts such as carbon dioxide, water, and mineral salts through a process called mineralization.
Bioremediation offers a sustainable and environmentally friendly approach to address pollution issues, as it relies on natural biological processes to remediate contaminated sites. It can be applied to various types of pollution, including organic pollutants like petroleum hydrocarbons, pesticides, and solvents, as well as inorganic contaminants such as heavy metals and nitrates.
Definition of Bioremediation
Bioremediation is the use of microorganisms, plants, or their enzymes to clean up and detoxify polluted environments by breaking down and degrading contaminants.
Principle of Bioremediation
The principle of bioremediation is based on harnessing the natural capabilities of certain microorganisms to break down and degrade environmental contaminants. The process involves the following key elements:
- Contaminant Utilization: Bioremediation relies on specific microbes that can use contaminants as a source of food and energy. These microorganisms have the ability to metabolize and transform pollutants, such as oil, solvents, and pesticides, into simpler, less harmful substances.
- Microbial Conversion: The selected microbes consume the contaminants and convert them into smaller amounts of harmless byproducts, primarily water and gases like carbon dioxide. This conversion process reduces the toxicity and concentration of the contaminants, effectively cleaning up the polluted site.
- Optimal Conditions: To ensure effective bioremediation, the right conditions must be provided for the growth and activity of the target microbes. This includes maintaining the appropriate temperature, supplying necessary nutrients and food sources for the microbes, and ensuring adequate oxygen levels.
- Environmental Amendments: If the natural conditions are not conducive to bioremediation, amendments can be added to the environment. These amendments, such as molasses, vegetable oil, or increased aeration, create optimal conditions for the microbes to thrive and carry out the bioremediation process more efficiently.
- Timeframe: The duration of the bioremediation process can vary significantly depending on various factors. The size of the contaminated area, the concentration of contaminants, temperature, soil density, and the choice between in situ (on-site) or ex situ (off-site) bioremediation methods all contribute to the overall timeline. Bioremediation can range from a few months to several years.
By understanding and applying the principles of bioremediation, it is possible to harness the natural abilities of microorganisms and create conditions that facilitate the breakdown and removal of environmental contaminants. This approach offers a sustainable and environmentally friendly solution to clean up polluted sites.
Types of Bioremediation (Classification of Bioremediation)
Bioremediation methods can be classified into different types based on the approach used and the location of the remediation process:
- Natural Attenuation or Intrinsic Bioremediation: This method relies on the natural processes and capabilities of microorganisms present in the environment to degrade and attenuate contaminants without any additional intervention. The existing microbial populations break down the pollutants over time.
- Biostimulation: Biostimulation involves the enhancement of bioremediation by stimulating the growth and activity of indigenous microorganisms. This is achieved by adding nutrients, such as fertilizers, to the contaminated medium. The added nutrients increase the bioavailability of contaminants and promote microbial degradation.
- In Situ Bioremediation: In situ bioremediation refers to the treatment of contaminated materials directly at the site where they are located. It avoids the need for excavation or removal of the polluted materials. In this approach, bioremediation processes are conducted in the subsurface, groundwater, or soil, depending on the nature and extent of contamination.
- Ex Situ Bioremediation: Ex situ bioremediation involves the removal of contaminated materials from their original location and their treatment at a different location. This method is often used when the contamination is widespread, the site is inaccessible, or the contaminants are highly concentrated. The polluted materials are excavated and transported to a controlled facility or bioreactor for treatment.
Both in situ and ex situ bioremediation methods have their advantages and limitations, and the selection of the appropriate method depends on factors such as the type and extent of contamination, site characteristics, regulatory requirements, and cost considerations.
In Situ Bioremediation
- These methods involve treating polluted substances at the source of pollution.
- It requires no excavation and minimal or no soil disturbance during construction.
- These procedures ought to be more cost-effective than ex-situ bioremediation techniques.
- Some in-situ bioremediation procedures, such as bioventing, biosparging, and phytoremediation, may be improved, while others, such as intrinsic bioremediation and natural attenuation, may continue without modification.
- Sites polluted with chlorinated solvents, heavy metals, dyes, and hydrocarbons have been satisfactorily remedied using in-situ bioremediation approaches.
Advantages of in-situ bioremediation
- Methods of in-situ bioremediation do not involve the excavation of contaminated soil.
- This approach treats both dissolved and solid pollutants using volumetric treatment.
- Frequently, expedited in-situ bioremediation can cure subsurface pollutants more quickly than pump-and-treat techniques.
- It may be possible to convert all organic pollutants into harmless substances such as carbon dioxide, water, and ethane.
- It is a cost-effective solution because site disruption is minimised.
Disadvantages of Advantages of in-situ bioremediation
- Some toxins may not be completely turned into safe compounds depending on the place.
- If transformation ceases at an intermediate component, the intermediate may be more hazardous and/or mobile than the original chemical; refractory pollutants are not biodegradable.
- Due to the combination of nutrients, electron donor, and electron acceptor, injection wells may get clogged with abundant microbiological growth if administered improperly.
- Concentrations of heavy metals and organic chemicals impede the action of indigenous microorganisms.
- In-situ bioremediation typically requires acclimatisation of microorganisms, which may not occur for spills and stubborn substances.
Types of in-situ bioremediation
There are two types of in-situ bioremediation: intrinsic and engineered bioremediation.
1. Intrinsic bioremediation
- Intrinsic bioremediation, also known as natural reduction, is an in-situ bioremediation method involving the passive remediation of polluted environments, without the need of external force (human intervention).
- This procedure involves the promotion of the indigenous or native microbial population.
- Using both aerobic and anaerobic microbial mechanisms to biodegrade contaminating elements, including those that are resistant.
- Due to the lack of external force, the procedure is less expensive than other in-situ techniques.
2. Engineered in-situ bioremediation
- The second method involves introducing certain microorganisms to the contaminated region.
- Genetically Engineered microorganisms used in in-situ bioremediation expedite the degradation process by promoting the proliferation of microorganisms by increasing the physicochemical conditions.
Methods of In Situ Remediation
- Bioaugmentation refers to the introduction of specialised microorganisms, either naturally occurring or genetically created, into polluted soil or water for their remarkable ability to breakdown or detoxify a specific contaminant or contaminant group.
- To expedite the remediation process, microorganisms having the potential to utilise or detoxify pollutants are typically identified, cultivated in the laboratory, and then delivered to contaminated locations.
- Dehalococcoides sp., which dechlorinates trichloroethylene (TCE) to ethene, has the potential to be used as a bioaugmentation agent in TCE-contaminated groundwater rehabilitation.
- Bioremediation of BTEX-contaminated soils is effective when inoculated with microbial populations capable of metabolising benzene, toluene, ethylbenzene, and xylene (BTEX).
- Bioaugmentation has proven successful with groupings of microorganisms from the same or different taxonomic groups (e.g., microbial mats and assemblages of bacteria–microalgae/cyanobacteria).
Limitations of Bioaugmentation
- Extensive studies of treatability and site characterisation may be required.
- The risk of contaminant leakage into groundwater must be mitigated due to the increased mobility of contaminants.
- Bioventing is a promising new method that provides oxygen to promote the natural in situ aerobic biodegradation of pollutants by microorganisms already present at the site.
- Popular groundwater pollutants, BTEX chemicals, are readily biodegradable by aerobic microorganisms; hence, the addition of oxygen to contaminated aquifers to induce aerobic degradation has been regarded as a common bioremediation approach.
- In order to provide enough oxygen for the sustaining of microbial activity, only modest air flow rates are maintained in the vadose zone.
- Commonly, oxygen is injected directly into soil containing residual pollution. Due to the sluggish movement of vapours through biologically active soil, not only the adsorbed fuel residuals but also the volatile compounds are biodegraded as a result of bioventing.
- Soils contaminated with petroleum hydrocarbons, non-chlorinated solvents, pesticides, wood preservatives, and other organic contaminants have been successfully remedied by bioventing.
The following reasons may hinder the effectiveness of bioventing:
- Soils with poor permeability
- Vapor accumulation in basements inside the influence radius of air injection wells.
- Extremely low moisture content.
- monitoring of soil surface emissions.
- In certain situations, a remediation procedure could be slowed by the low temperature.
3. Nitrate-Enhanced In Situ Bioremediation
- To oxidise a substrate, the microbial metabolic process requires an electron giver and an electron acceptor.
- Nitrate enhancement is yet another new bioremediation approach in which nitrate acts as an alternative electron acceptor for microbial activity, hence accelerating the breakdown of organic molecules.
- This approach employs the circulation of nitrate throughout the contaminated area of the groundwater in order to accelerate the breakdown of the contaminant.
- This technique is efficient for treating groundwater contaminated with BTEX.
Among the variables that may limit the efficacy of this technology are the following:
- Subsurface heterogeneity.
- Requirement for a groundwater circulation system to prevent contaminants from escaping the biodegradation zone.
- Since nitrate is regulated by drinking water standards, regulatory acceptance.
4. Cometabolic Process
- Some microbes may not utilise the intended contamination for their growth, but they can cometabolize the contaminant while utilising another molecule for growth.
- For instance, methane monooxygenase, an enzyme produced by methanotrophic bacteria during the oxidation of methane, can convert chlorinated solvents such as TCE.
- Cometabolic process is an innovative in situ bioremediation approach utilised for nonpetroleum hydrocarbons, such as chlorinated solvents, by utilising enzymes generated during the decomposition of certain chemicals.
- Cometabolism is contingent upon the presence of a suitable substrate whose metabolism can result in the transformation of the target pollutant.
- Water containing methane and oxygen is pumped into groundwater to boost the methanotrophic bacteria’ capacity for cometabolic breakdown of chlorinated organic solvents.
- Since it is difficult to circulate methane solution across each portion of a contaminated zone, the efficacy of the cometabolic process depends on the subsurface’s homogeneity.
5. Monitored Natural Attenuation
- Monitored natural attenuation (MNA) entails dependence on natural processes to obtain pollutant cleanup.
- Consideration of MNA for remediation of contaminated aquifers and groundwater systems typically necessitates modelling and evaluation of contaminant degradation rates, exposure pathways, impacts on sensitive receptors, and prediction of contaminant concentrations downgradient to the migrating contaminant plume.
- Typically, the appropriateness of MNA is evaluated on a case-by-case basis. Evaluation of MNA is not an easy task; it requires multidisciplinary skills in microbiology, chemistry, hydrogeology, and geochemistry, among others.
- MNA has been effective, particularly for hydrocarbon fuels. Typically, fuel and volatile organic compounds containing halogen are examined for MNA.
Among the problems that may restrict the use and efficacy of MNA is the need for site-specific data for modelling.
- not being suitable for locations with impending dangers.
- It is possible for the rate of pollutant migration to exceed the rate of contaminant degradation.
- Products of degradation may be more hazardous and mobile.
- It may take more time than an active cleanup approach.
- When many contaminants are included in a spill, some of the toxins may not decompose in the subsoil.
- Before considering MNA, the necessary must designate contamination both horizontally and vertically.
- Over time, the geochemical and hydrologic conditions that are conducive to MNA may alter and remobilize the stable pollutants.
- necessity for contaminated source elimination prior to MNA adoption.
- Long-term surveillance and related expenditures.
- This method combines vacuum-enhanced pumping, soil vapour extraction, and bioventing to remediate soil and ground water by indirect oxygenation and promotion of pollutant biodegradation.
- This technology is intended for the recovery of products from capillary, light non-aqueous phase liquids (LNAPLs), unsaturated and saturated zones during remediation.
- This method is utilised to decontaminate soils contaminated with volatile and semi-volatile organic pollutants.
- The approach employs a “slurp” that spreads into the layer of free product and draws liquids from this layer.
- By ascending, the pumping machine takes LNAPLs to the surface, where they are isolated from air and water. In this method, soil moisture limits air permeability and decreases the oxygen transfer rate, hence decreasing microbial activity.
- Although this method is unsuitable for low-permeable soil restoration, it is a cost-effective operation process since it uses less ground water and reduces storage, treatment, and disposal expenses.
- This method is similar to bioventing in that air is pumped into the subsurface of the soil to promote microbial activity and accelerate the removal of pollutants from polluted locations.
- Bioventing, on the other hand, involves injecting air into the saturated zone, which facilitates the upward migration of volatile organic molecules into the unsaturated zone, so accelerating the biodegradation process.
- The effectiveness of biospraying depends on two primary aspects, namely soil permeability and the biodegradability of pollutants.
- In bioventing and soil vapour extraction (SVE), biosparging is closely related to the in-situ air sparging (IAS) technology, which relies on high air-flow rates for pollutant volatilization, whereas biosparging promotes biodegradation.
- Biosparging has often been employed to treat diesel and kerosene-contaminated groundwater.
- Phytoremediation decontaminates polluted soils. This technique utilises physical, chemical, biological, microbiological, and biochemical plant interactions to reduce the toxicity of pollutants at contaminated locations.
- Involved in phytoremediation are a number of mechanisms, including as extraction, degradation, filtration, accumulation, stability, and volatilization, which are dependent on pollutant quantity and kind.
- Heavy metals and radionuclides are typically eliminated through extraction, transformation, and sequestration.
- Organic pollutants such as hydrocarbons and chlorinated chemicals are typically eliminated through degradation, rhizoremediation, stabilisation, and volatilization; however, mineralization is possible when certain plants such as willow and alfalfa are utilised.
- The root system, which may be fibrous or tap depending on the depth of the pollutant, the above-ground biomass, the toxicity of the pollutant to the plant, the existence of the plant and its adaptability to the prevailing environmental conditions, the plant’s growth rate, site monitoring, and, most importantly, the time required to achieve the desired level of cleanliness are all important factors of plant as a phytoremediator.
- Additionally, the plant must be disease- and insect-resistant. Pollutant removal in phytoremediation involves uptake and transfer from roots to shoots. Additionally, translocation and accumulation are contingent upon transpiration and partitioning.
- However, the method may be altered based on variables such as the nature of the pollutant and the facility.
- The majority of plants growing in contaminated areas are effective phytoremediators. Therefore, the effectiveness of any phytoremediation strategy rests primarily on enhancing the remediation potentials of native plants growing on polluted areas, either by bioaugmentation using endogenous or foreign plant material.
- Some precious metals can bioaccumulate in some plants and be recovered after remediation, a process known as phytomining. This is one of the key benefits of utilising plants to rehabilitate polluted sites.
9. Permeable reactive barrier (PRB)
- As a physical method for remediating polluted groundwater, this technique is frequently observed.
- However, the biological mechanisms used in the PRB approach for pollution removal are precipitation degradation and sorption.
- To incorporate the biotechnology and bioremediation aspects of the technique, the words biological PRB, bio-enhanced PRB, and passive bioreactive barrier have been proposed as substitutes.
- In general, PRB is an in-situ technology used to remove heavy metals and chlorinated chemicals from polluted groundwater.
Ex Situ Bioremediation
- Ex-situ bioremediation approaches entail excavating pollutants from contaminated locations and moving them to a new location for treatment.
- Ex-situ bioremediation procedures are frequently evaluated based on the depth of contamination, kind of pollutant, level of contamination, cost of treatment, and geographic location of the contaminated site.
- Additionally, performance guidelines govern the selection of ex-situ bioremediation procedures.
Types of Ex-situ bioremediation
There are two types of Ex-situ bioremediation;
1. Solid-phase treatment
- Solid-phase bioremediation is an ex-situ process that involves the excavation and piling of contaminated soil.
- It also comprises domestic, industrial, and municipal trash, as well as organic wastes such as leaves, animal dung, and agricultural wastes.
- Pipes are utilised to transport bacterial growth throughout the piles.
- Ventilation and microbial respiration require air to flow through the pipes.
- In comparison to slurry-phase procedures, solid-phase systems necessitate a vast amount of area and require more time to clean up.
- Solid-phase treatment methods include biopiles, windrows, land farming, and composting, among others.
2. Slurry-phase bioremediation
- Slurry-phase bioremediation is a somewhat faster treatment method than the others.
- In the bioreactor, contaminated soil is blended with water, nutrients, and oxygen to produce the optimal environment for microorganisms to breakdown soil pollutants.
- This procedure involves separating rocks and debris from contaminated soil.
- The concentration of additional water is dependent on the quantity of contaminants, the rate of biodegradation, and the physicochemical parameters of the soil.
- Using vacuum filters, pressure filters, and centrifuges, the soil is extracted and dehydrated following this procedure.
- The succeeding steps involve disposing of the soil and treating the resulting fluids in advance.
Methods Ex Situ Remediation
- Biopiles is a treatment technology in which excavated soils are combined with soil amendments and placed in above-ground enclosures equipped with an aeration system and a leachate collection system.
- Biodegradation is frequently utilised to remediate petroleum hydrocarbons in excavated soils.
- In order to decrease the possibility of contaminants seeping into groundwater or uncontaminated soil, the treatment area is typically lined with an impermeable membrane. Diverse fertiliser and supplement formulas are utilised to increase microbial activity in biopiles.
- Dirt piles can reach a height of up to 6 metres, although the optimum height is 2–3 metres. Typically, a vacuum or positive pressure air distribution system is constructed beneath the soil and maintained. The biopiles are covered with a plastic sheet to reduce runoff, evaporation, and volatilization, which can also result in increased solar heating.
- If there are volatile organic compounds (VOCs) in the soil, it may be necessary to clean the air leaving the soil prior to its release into the sky. The operation of biopiles might take anywhere between a few weeks and several months.
- Biopile treatment has been demonstrated to be effective for fuel hydrocarbons and non-halogenated VOCs. This method has also been used to treat halogenated VOCs and pesticides; however, the success rate will vary and some chemicals may be inapplicable.
Among the issues that may limit the efficacy of biopile treatment are:
- The demand for soil excavation.
- Tests of treatability to measure oxygenation and nutrient loading.
- Unlike methods involving periodic mixing, static treatment processes do not result in uniform treatment.
- Composting (windrows) is a controlled biological process in which excavated contaminated soil is mixed with bulking agents and organic amendments (wood chips, hay, manure, green waste, etc.) in a proper proportion to provide the proper balance of carbon and nitrogen required for thermophilic microbial activity.
- Under aerobic and anaerobic conditions, microbial activities convert organic pollutants (such as polycyclic aromatic hydrocarbons (PAHs) and 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT)) into harmless stable compounds.
- During the composting process, the heat produced by indigenous microorganisms during the decomposition of organic materials will result in a thermophilic phase (55–65 degrees Celsius), which is essential for the transformation of hazardous pollutants.
- Maintaining adequate oxygenation (through windrow rotation), moisture content (by irrigation), and temperature can increase degradation efficiency.
- The various composting designs include (1) aerated static piles, in which compost piles are aerated through blowers or vacuum pumps, (2) in-vessel composting with mechanical agitation, in which compost is placed in a reactor vessel and mixed and aerated, and (3) windrow composting, a more cost-effective method in which compost is placed in long piles called windrows and mixed periodically with mobile equipment.
- Using the composting process, biodegradable pollutants in soils and sediments can be removed. Pilot and large-scale operations have provided evidence that aerobic, thermophilic composting can reduce the content of explosives such as trinitrotoluene (TNT), RDX, HMX, and ammonium picrate to tolerable levels.
- Also suitable to PAHs and DDT residues is this method.
Limitations of the composting process include the need for a large amount of area.
- any uncontrolled VOC emissions related with soil excavation.
- Due to the need for additions, there is a rise in material quantity.
3. Land Farming
- Land farming is a large-scale bioremediation technique in which excavated contaminated soil is deposited on lined beds of a predefined depth and aerated by periodic turning or tilling (plow depth about 4–12 in).
- During this procedure, soil parameters such as moisture content, aeration, pH, and amendments such as soil bulking agents, fertilisers, etc. are manipulated to achieve the highest possible rate of pollutant breakdown.
- This mechanism enables aerobic microbial digestion through the availability of oxygen, nutrients, and moisture.
- Land cultivation has been effective for treating petroleum hydrocarbons. More chlorinated and nitrated substances decay slowly.
- Diesel fuel, fuel oils, oil sludge, wood preservation wastes, coke wastes, and insecticides are also successfully treated.
- The need for a big amount of area is one of the variables that may limit the efficiency of land farming.
- Some of the elements governing microbial proliferation and decomposition, such as precipitation and temperature, are out of control and may lengthen the duration of degradation.
- need for handling volatile substances to avoid these gases from migrating off-site and into the environment.
- required for the construction of a facility for collecting and monitoring runoff debris.
- For optimal facility design, it is necessary to analyse the site’s topography, erosion, climate, and permeability, among other factors.
4. Slurry-Phase Bioreactors
- Slurry-phase biological treatment is combining excavated contaminated soil or sediment with water and other additives in a bioreactor under regulated conditions to produce aqueous slurry.
- The amount of water given to soil is dependent on the concentration of the pollutant, its rate of biodegradation, and the soil’s physicochemical qualities.
- During the treatment process, the solids are kept in slurry suspension in the reactor and combined with nutrients and oxygen to bring microorganisms into contact with soil constituents.
- If an appropriate indigenous population of microorganisms capable of degrading specific pollutants is not present in the soil, it is possible to introduce such organisms.
- The pH will be adjusted to the desired level, if necessary, in the reactor vessel. After biodegradation is complete, the slurry can be dewatered and the treated soil can be discarded.
- It has been demonstrated that slurry-phase bioreactors are effective for remediating soils and sediments contaminated with petroleum hydrocarbons, explosives, solvents, pesticides, and other contaminants.
- When treating diverse and impermeable soils, as well as when faster treatments are necessary, bioreactors are preferable to in situ biological methods.
Among the constraints of slurry-phase biotreatment are:
- The obligation to excavate polluted soil.
- The amount of soil that can be introduced to the reactor, especially when treating huge quantities of contaminated soil.
- The expense associated with dewatering treated soil.
- locating a safe and appropriate method of wastewater disposal.
5. Fungal Remediation
- The metabolism of fungi has been linked to the breakdown of numerous organic pollutants, particularly hydrocarbons.
- One type of fungi, specifically white-rot fungus (Phanerochaete chrysosporium), may decompose a wide range of organic pollutants, such as PCBs, PAHs, and explosives.
- These enzymes, lignin peroxidases, are generated by these fungi and are responsible for their wide biodegradability.
- It has been established that white-rot fungus may decompose chlorinated hydrocarbons, PAHs, PCBs, polychlorinated(p)dioxins, pesticides (lindane and DDT), and some azodyes.
- Also, white-rot fungi have been demonstrated to degrade PAHs such as benzo(a)pyrene, pyrene, fluorene, and phenanthrene; however, breakdown is favoured under nitrogen-limited and low pH circumstances.
Among the conditions that impede fungal remediation are:
- Their awareness of biological processes.
- their incapacity to effectively grow in suspension systems.
- mixing’s deleterious influence on enzyme synthesis.
- The inability of fungi to adhere to fixed media.
- chemical adhesion
- conflict with native microorganisms
- Transformation ability that is sluggish.
Advantages of ex-situ bioremediation
- Compatible with a wide variety of pollutants
- Suitability is reasonably straightforward to evaluate using site investigation data.
- The contaminated environment is more manageable, controllable, and predictable in a bioreactor system than in solid-phase systems.
Disadvantages of ex-situ bioremediation
- Not applicable to contamination with heavy metals or chlorinated hydrocarbons like trichloroethylene.
- Non-permeable soil requires further preparation.
- Before introducing a contamination into a bioreactor, the contaminant can be removed from the soil through soil washing or physical extraction.
Factors affecting the bioremediation
Various environmental factors, such as temperature, salinity, pH, and oxygen availability, have the potential to influence petrochemical waste bioremediation. There is an inverse link between salinity and the solubility of petroleum hydrocarbons, since an increase in salt increases aromatic hydrocarbon absorption. It has been documented for pyrene in several types of sediments, with salting out effects identified in both solution and solid phases as a likely source for this increase in degradation. Moreover, in some instances, hypersalinity inhibits microbial growth and, consequently, metabolic processes, while promoting the growth of unidentified halophytic archaea during biodegradation. Several other environmental variables are discussed in detail below:
- Temperature exerts a crucial influence on both in situ and ex situ microbial metabolism and hydrocarbon breakdown.
- At low temperatures, microbial growth and multiplication decrease, resulting in a sluggish rate of petrochemical degradation.
- In contrast, numerous studies have demonstrated that a rise in temperature enhances the solubility of hydrocarbons in the medium, making petrochemical hydrocarbons readily accessible to microbes.
- The breakdown rate of petrochemical wastes is typically rapid at temperatures between 30 and 40 degrees Celsius in soil and 15 and 20 degrees Celsius in aquatic or marine environments.
- However, some thermophilic bacteria (e.g., Bacillus thermoleovorans) have been shown to change phenanthrene, naphthalene, and anthracene efficiently even at higher temperatures.
- Variations in pH conditions significantly influenced the microbial decomposition of petrochemical wastes in soil or aquatic medium.
- Diverse research support the advantageous mineralization of petroleum hydrocarbons close to pH neutrality.
- A little change in pH can have a substantial effect on the overall biological degradation processes in an aquatic environment.
- It has been found that certain fungi and acidophilic microorganisms can grow and have biodegradation capacity in highly acidic settings.
3. Oxygen availability
- The presence of oxygen determines whether a given environmental condition is aerobic or anaerobic.
- It has been noted that the degradation of petrochemical hydrocarbons occurs predominantly in aerobic conditions and occasionally in anaerobic environments.
- Biodegradation in anaerobic conditions occurs primarily in aquifers and submerged marine sediments with a negligible degradation rate, and is primarily limited to halogenated aromatics.
- Oxygen is important for the activity of mono- and dioxygenase enzymes during aromatic ring oxidation, which is required for the aerobic breakdown of petrochemicals. To promote aromatic compound oxidation in an anaerobic environment, substituted electron acceptors such as ferrous iron, nitrate, and sulphate are important.
- However, the reduction of electron acceptors such as ferric iron, nitrate, and sulphate under anaerobic circumstances releases substantial quantities of phosphorus and ferrous iron, further contaminating the natural ecosystem.
- In addition, under anaerobic breakdown of petrochemical hydrocarbons, greenhouse gas emissions (CH4, NO2, etc.) and an increase in pH have been reported.
- Thus, oxygen availability plays a crucial role in aromatic chemical bioremediation.
4. Concentration of pollutant
- Individual contaminants’ degradation rates may be affected by the interactions between substrates.
- Due to its probable impact in bacterial sensitivity and metabolism, the evaluation of substrate-substrate interactions at varying concentrations has been deemed a crucial factor.
- The synergistic interactions between a contaminant’s many components can enhance breakdown rates and catabolic enzyme activities.
- In a batch culture investigation, it was revealed that the growth rate of Pseudomonas putida was reduced at high substrate concentrations.
- Due to their complicated interactions, BTEX chemicals (at specific doses) had an inhibiting influence on microbiological processes.
5. Nutrients availability
- Nutrients (such as carbon, nitrogen, phosphorus, potassium, and calcium) are essential for microbial development and activity; hence, their availability plays a crucial role in regulating the breakdown rates of various pollutants.
- In addition, relative nutrient availability has a significant regulatory effect on pollutant degradation. At a contaminated site with higher levels of organic carbon in the contaminants, for instance, the microbial activity is reported to be significantly higher, resulting in the rapid depletion of bioavailable nutrients such as nitrogen, phosphorus, potassium, and iron in the early phase, followed by a decrease in degradation as a result of the depletion of these essential elements.
- On the contrary, increased availability of N, P, and K has been observed to have a deleterious effect on aromatic hydrocarbon decomposition.
6. Microbial adaptation (acclimatization)
- Petroleum waste decomposition is potentially influenced by microbial adaptability.
- The adaptation of microbial populations to aromatic organic compounds increases their efficiency of breakdown.
- Alcaligenes xylosoxidans Y234, for instance, decomposed benzene and toluene more effectively under adapted conditions than under non-adapted ones.
- Non-acclimatized microorganisms exhibit the quickest biodegradation rates in both single-component and multicomponent-based confinement systems.
- Accessibility of organic contaminants to microorganisms is one of the most important determinants of biodegradation rate.
- The bioavailability of hydrocarbons depends on their physical properties and chemical composition.
- Some compounds generated from bacteria may operate as additives that accelerate the breakdown of petroleum hydrocarbons.
- Biosurfactants, a rich source of carbon, produced by diverse microbes (e.g., bacteria and fungus), have the potential to contribute to the uptake and mineralization of petroleum hydrocarbons.
8. Redox Potential
- Redox Potential and oxygen content are indicative of oxidising or reducing circumstances, respectively.
- The presence of electron acceptors such as nitrate, manganese oxides, iron oxides, and sulphate influences redox potential (ICSS 2006).
Microorganisms used in bioremediation
Microorganisms play a crucial role in nutritional chains, which are an integral aspect of the biological equilibrium of life. Bioremediation is the process of removing contaminated materials using bacteria, fungi, algae, and yeast. In the presence of toxic substances or any waste stream, microorganisms can flourish at temperatures below zero and at high temperatures. The adaptability and biological systems of microorganisms make them useful for the cleanup process. Carbon is the essential component for microbial action. In various conditions, a consortium of microorganisms performed bioremediation. These microbes include Achromobacter, Arthrobacter, Alcaligenes, Bacillus, Corynebacterium, Pseudomonas, Flavobacterium, Mycobacterium, Nitrosomonas, and Xanthobacter, among others.
There are various microbe groups utilised in bioremediation, including:
- Aerobic: Aerobic bacteria such as Pseudomonas, Acinetobacter, Sphingomonas, Nocardia, Flavobacterium, Rhodococcus, and Mycobacterium have the ability to breakdown complex substances. According to reports, these bacteria breakdown insecticides, hydrocarbons, alkanes, and polyaromatic chemicals. Numerous of these bacteria utilise the pollutants as a source of carbon and energy.
- Anaerobic: Anaerobic bacteria are utilised less frequently than aerobic bacteria. Aerobic bacteria utilised for bioremediation of chlorinated aromatic chemicals, polychlorinated biphenyls, and dechlorination of the solvent trichloroethylene and chloroform, decomposing and converting contaminants to less hazardous forms, are gaining growing attention.
Bioremediation approaches used for dye degradation
Dye decolorization is initiated by an anaerobic reduction reaction performed by azo-reductases or azo-bonds breaking under aerobic or anaerobic conditions, which results in the creation of aromatic amines due to the physiological and metabolic activities of the mixed bacterial community. The following is a detailed description of various dye degradation processes:
1. Aerobic treatment
- There are very few reports on the bacterial degradation of azo-dyes; yet, several microorganisms have demonstrated their capacity for dye reduction.
- Pseudomonas aeruginosa has been shown to degrade the commercially used textile and tannery dye Navitan Fast Blue SSR in an aerobic medium including glucose as a carbon source.
2. Anaerobic treatment
- Under anaerobic conditions, azo-dye reduction is accomplished through the dissolution of azo-bonds.
- Under anaerobic conditions, dyes are cleaved, producing poisonous aromatic amines via bacterial metabolism.
3. Anoxic treatment
- Various studies show the anaerobic degradation of various colours by facultative anaerobic and mixed aerobic bacteria.
- Although a number of bacteria are capable of flourishing in an aerobic environment, the dye is only destroyed in anaerobic conditions.
- A number of pure bacterial cultures, including Pseudomonas luteola, Aeromonas hydrophila, Bacillus subtilis, and Proteus mirabilis, are known to digest azo-dyes anaerobically.
4. Sequential degradation of dyes
- It has been proposed that aromatic amines generated by the anaerobic decomposition of azo-dyes can be degraded in an aerobic environment.
- First, the applicability of this method was demonstrated for the sulfonated azo-dye Mordant Yellow. Following aeration, full microbial mineralization of amine is seen.
Different Categories of Bioremediation
Bioremediation can be categorized into two main types: microbial remediation and phytoremediation.
a. Microbial Remediation:
Microbial remediation involves the use of microorganisms such as bacteria, fungi, yeasts, and actinomycetes to break down and remove contaminants from the environment. These microorganisms have the ability to degrade a wide range of organic compounds and absorb inorganic substances. Key points about microbial remediation include:
- Microorganisms are diverse, abundant, and readily available for use in bioremediation processes.
- Different microbial systems can be employed for the removal of toxic and other contaminants from the environment.
- Microorganisms can be used in both in situ (on-site) and ex situ (off-site) conditions, and they can thrive in extreme environmental conditions.
- Synergistic interactions among mixed cultures of microorganisms are often beneficial in bioremediation, particularly for the degradation of crude oil in soil.
- Various bacteria, including Pseudomonas, Aeromonas, Moraxella, Nocardia, Bacillus, and Cyanobacteria, are commonly used for the removal of petroleum hydrocarbon contaminants from soil.
Phytoremediation is a bioremediation process that utilizes plants to remove, transfer, stabilize, or destroy contaminants in the soil and groundwater. Different mechanisms are involved in phytoremediation, including:
- Rhizosphere biodegradation: Plants release natural substances through their roots, providing nutrients to soil microorganisms, which enhance the biological degradation of contaminants.
- Phyto-stabilization: Chemical compounds produced by plants immobilize contaminants, preventing their movement and reducing their toxicity.
- Phyto-accumulation (phytoextraction): Plant roots absorb contaminants, along with other nutrients and water, which accumulate in the shoots and leaves. This method is primarily used for contaminants containing metals.
- Hydroponic systems for treating water streams (rhizofiltration): Plants are grown in greenhouses with their roots in water, allowing the plants to absorb contaminants from the water. The saturated roots are harvested and disposed of.
- Phyto-volatilization: Plants absorb water containing organic contaminants and release the contaminants into the air through their leaves.
- Phyto-degradation: Plants metabolize and destroy contaminants within their tissues.
- Hydraulic control: Trees indirectly remediate by controlling the movement of groundwater. The dense root mass of trees takes up large quantities of water, helping to regulate the water table and limit the spread of contaminants.
Overall, microbial remediation and phytoremediation offer valuable approaches for the cleanup of contaminated environments, utilizing the natural capabilities of microorganisms and plants to mitigate pollution.
What is Slurry phase bioremediation?
- Slurry phase bioremediation is a batch treatment approach in which excavated soil or sediments are combined with water and treated in bioreactor vessels or ponds.
- To reduce the viscosity of contaminated soil, removal of stones and rubbles from the soil is required during soil processing.
- The soil is then combined with a specific quantity of water to create slurry.
- Thus, slurry phase treatment is a three-phase system consisting of water, air, and suspended particulate matter, including the desired microorganism.
- The amount of water added depends on the quantity of contaminants, the rate of biodegradation, and the soil’s physical characteristics (USEPA, 2006). In addition to ensuring appropriate aeration and mixing, nutrients are always provided, along with surfactants or dispersants as necessary.
- In bioreactor vessels, optimal pH and temperature conditions are also provided.
- With slurry phase systems, soil and sediments contaminated with a wide variety of organic chemicals, such as pesticides, petroleum hydrocarbons, pentachlorophenol, polychlorinated biphenyls (PCBs), etc., have undergone effective bioremediation.
- There are three biologically distinct types of slurry phase bioreactors: aerated lagoons, lowshear airlift reactors, and fluidized-bed soil reactors.
- A slurry bioreactor is a vessel and apparatus used to create a three-phase (solid, liquid, gas) mixing condition to increase the rate of bioremediation of soil-bound and water-soluble pollutants as a water slurry of contaminated soil and biomass (typically indigenous microorganisms) capable of degrading target contaminants.
- In general, the pace and amount of biodegradation are larger in a bioreactor system than in situ or solid-phase systems because the enclosed environment is more manageable and, thus, more predictable.
- Despite the benefits of bioreactor systems, there are downsides, such as the need to pre-treat contaminated soil (e.g., excavation) or remove contaminants from the soil via soil washing or physical extraction (e.g., vacuum extraction) prior to placing the soil in a bioreactor.
What is Solid phase bioremediation?
- The solid phase system consists of organic wastes, manures, sewage sludge, and municipal solid wastes.
- Traditional cleanup methods include the informal processing of organic debris and the generation of compost, which can be used as a soil amendment.
- Solid phase bioremediation involves the excavation and piling of contaminated soil.
- A network of pipes that are dispersed throughout the piles stimulates bacterial development. By drawing air through pipes, microbial respiration is given with the necessary ventilation.
- Spraying the soil with water introduces moisture. Solid-phase systems demand a substantial amount of area, and cleanups take longer than slurry-phase procedures.
- Land farming chemical groups on mineral surfaces, reactive organic chemicals, and inorganic metals are some solid-phase treatment techniques.
- The precise method by which microorganisms respond to insecticides is not understood. It is possible for microorganisms to acquire genetic material encoding the biochemical pathways required to handle a potential substrate.
- The process of microbial bioremediation can occur under both aerobic and anaerobic environments. In an aerobic environment, bacteria use air oxygen for their metabolic processes in order to produce carbon dioxide and water through pesticide breakdown.
- In the absence of oxygen, however, bacteria use these chemical compounds in the soil as substrate, breaking them down to obtain the energy they require.
Applications of Bioremediation
Bioremediation has a wide range of applications in the cleanup of various contaminants and polluted sites. Some key applications of bioremediation include:
- Remediation of Metals, Radionuclides, and Pesticides: Bioremediation can be used to address contamination by heavy metals, radioactive substances, and pesticides. Specific microbial or plant-based strategies can target these contaminants and facilitate their degradation or immobilization.
- Treatment of Explosives, Fuels, VOCs, and SVOCs: Bioremediation is effective in treating pollutants such as explosives (e.g., TNT), fuels (e.g., gasoline, diesel), volatile organic compounds (VOCs), and semi-volatile organic compounds (SVOCs). Microorganisms can break down these contaminants into less harmful substances.
- Phytoremediation of Perchlorate: Phytoremediation, a form of bioremediation using plants, shows promise in addressing perchlorate contamination in surface and groundwater systems. Research is being conducted to understand the role of plants in removing this persistent contaminant.
- Cleanup of Soil and Groundwater: Bioremediation is commonly used to clean up contaminants found in soil and groundwater. Microorganisms or plants are introduced to the polluted sites to break down or remove the pollutants, restoring the environmental quality.
- Remediation of Petroleum Products: Bioremediation is highly effective in treating petroleum products, such as gasoline and diesel fuel, which may contaminate soil and groundwater. Microorganisms can metabolize and degrade these hydrocarbons.
- Treatment of Production-related Chemicals: Bioremediation can address contamination caused by leaked or released production-related chemicals, including both difficult-to-remediate heavy metals and minor pollutants. Certain microorganisms have the ability to biologically eliminate these pollutants.
- Landfill Remediation: Overfilled, leaking, or closed landfills can benefit from bioremediation. This approach can help regulate methane gas, a common byproduct of landfill waste, through air stripping and scrubbing techniques.
- Remediation of Over-Fertilized Farms: Bioremediation can be applied to farms that have been excessively fertilized, addressing both synthetic fertilizers and animal waste. Microorganisms can break down the excess nutrients and restore the balance in the soil.
- Cleanup of Wood Preservative Contamination: Bioremediation efforts can remove pollutants from lumber processing yards, where wood preservatives have seeped into the soil and groundwater. Microorganisms can break down these contaminants.
- Treatment of Failed Septic Tanks and Disposal Fields: Onsite sewage systems that fail and contaminate the soil and groundwater can be effectively treated using bioremediation. Biological treatment methods can help restore the affected areas.
- Remediation of Mine Tailings: Toxic mine tailings can be remediated through bioremediation techniques. Efforts to decontaminate old mine quarries and pits using bioremediation have shown promising results.
- Cleanup of Chemical Leaks along Traffic Routes: Accidental chemical spills, including road salts and petroleum spills along traffic routes, can be remedied through biological treatments. Bioremediation helps break down and remove these pollutants.
Advantage of bioremediation
Bioremediation offers several advantages over other cleanup methods, making it a favorable choice for addressing environmental contamination:
- Environmentally Friendly: Bioremediation is a natural process that utilizes the existing microbial and plant-based mechanisms in the environment. It causes less damage to ecosystems and is considered a green method of cleanup.
- Minimal Disruption: Bioremediation can be conducted underground, allowing amendments and microbes to be pumped into groundwater and soil without causing significant disruption to nearby communities or surface activities.
- Safe and Harmless Byproducts: The process of bioremediation converts contaminants and pollutants into harmless byproducts such as water and carbon dioxide. It generates few harmful byproducts, reducing the risk of creating additional environmental hazards.
- Cost-Effective: Bioremediation is generally a cost-effective method compared to other cleanup methods. It requires minimal equipment and labor, making it an affordable option for remediation projects.
- Tailored Approach: Bioremediation can be tailored to the specific needs of the polluted site. The selection of appropriate microbes and the optimization of growth-promoting factors allow for targeted degradation of pollutants.
- Minimal Effort and On-Site Treatment: Bioremediation can be performed on-site with minimal effort. It does not require the transfer of contaminated material off-site, reducing transportation costs and potential risks associated with waste disposal.
- Complete Breakdown of Contaminants: Bioremediation promotes the complete breakdown of contaminants. Hazardous substances can be converted into less harmful products, ensuring the effective removal and disposal of pollutants.
- No Hazardous Chemicals: Bioremediation does not rely on the use of hazardous chemicals. Instead, it often involves the addition of nutrients or fertilizers to stimulate microbial growth, resulting in the conversion of contaminants into harmless substances.
- Sustainable and Eco-Friendly: Bioremediation methods align with sustainable and eco-friendly practices. They utilize natural processes and minimize the need for external interventions, contributing to environmental preservation.
Overall, the advantages of bioremediation make it a viable and preferred approach for the cleanup of contaminated sites, offering an environmentally friendly, cost-effective, and efficient solution to environmental pollution.
Disadvantage of bioremediation
Bioremediation, while offering numerous advantages, also has certain disadvantages that should be considered:
- Unknown Toxicity and Bioavailability: The toxicity and bioavailability of by-products generated during biodegradation processes are not always well understood. It is important to assess the potential risks associated with these by-products to ensure they do not pose a threat to ecosystems or human health.
- Mobilization and Bioaccumulation: Degradation by-products may be mobilized in groundwater or bio-accumulated in animals, potentially leading to the transfer of contaminants through the food chain. The fate and potential impacts of these accumulated contaminants need to be carefully evaluated.
- Fate of Contaminants in Plant Metabolic Cycle: Further research is required to determine the fate of contaminants within the plant metabolic cycle. It is crucial to understand whether plant droppings, leaves, or wood release harmful chemicals into the environment, particularly when they are utilized as mulch or firewood.
- Disposal of Harvested Plants: The disposal of harvested plants that contain high levels of heavy metals can be challenging. Proper handling and disposal methods must be implemented to prevent the release of contaminants into the environment.
- Treatment Limitations: Bioremediation, including phytoremediation, is often limited to shallow soils, streams, and groundwater. The depth of contaminants can restrict the effectiveness of treatment in deeper soil layers.
- Seasonal and Climatic Influences: The success of phytoremediation can be affected by seasonal variations and climatic factors. Different plants may exhibit varying levels of effectiveness depending on the location and specific environmental conditions.
- Ecological Ramifications: Introducing new plant species for remediation purposes can have widespread ecological consequences. The potential impacts on native flora and fauna should be thoroughly studied and monitored to avoid unintended ecological ramifications.
- Plant Survival and Contaminant Concentrations: High concentrations of contaminants may inhibit plant growth and survival. If contaminant levels are too high, the plants may not be able to thrive or effectively remediate the site.
- Media Transfers: Phytoremediation may transfer contaminants across different environmental media, such as from soil to air, posing challenges in managing and controlling the movement of contaminants.
- Land Requirement: Phytoremediation often requires a large surface area of land to achieve effective remediation. This can limit its applicability, particularly in densely populated areas or sites with limited available land.
Considering these disadvantages, careful assessment and monitoring are essential when implementing bioremediation strategies to ensure the effectiveness of the remediation process while minimizing potential risks to the environment and human health.
What is Phytoremediation?
- Phytoremediation is a type of bioremediation that uses plants to remove toxins by healing and rebuilding the soil and ground and surface water.
- The plants utilised in the method absorb the toxins from the soil, store them inside their plant tissues, and bind them until they are decomposed by the roots.
- The plants extract pollutants from the soil through their roots, which then accumulate in the stems. Plants absorb toxic substances from the soil and release them into the atmosphere via transpiration and evaporation.
- Metals, pesticides, chlorinated solvents, polychlorinated biphenyls, and petroleum hydrocarbons are just a few of the contaminants that plants may eliminate.
- Indian Mustard, Indian Grass, Brown Mustard, Sunflower plants, Barley Grass, Pumpkin, Poplar trees, Pine trees, and White Willows are examples of plants that can be utilised for phytoremediation.
- These possess renewing and energising properties that aid the procedure.
What is Mycoremediation?
- Fungi are recognised as the decomposers of nature. They decompose the majority of the plant and woody debris on Earth, resulting in the renewal of the soil.
- Using their metabolic enzymes, fungi breakdown substances such as metals and diverse insecticides.
- By breaking down bigger hydrocarbon chains into smaller ones, fungi operate as a catalyst for microbes and plants, making their processes simpler.
- The fungi absorb the chemicals by degrading them with the aid of enzymes and then store the nutrients in the fleshy portions, which are known as mushrooms.
What is bioremediation?
Bioremediation is a process that uses living organisms, such as bacteria, fungi, and plants, to degrade or detoxify contaminants in the environment.
What types of contaminants can bioremediation address?
Bioremediation can be used to address a wide range of contaminants, including petroleum hydrocarbons, chlorinated solvents, pesticides, and heavy metals.
How does bioremediation work?
Bioremediation works by using microorganisms to break down contaminants into less harmful substances. This can be done through different mechanisms, such as metabolic transformation or adsorption.
What are the benefits of bioremediation?
Bioremediation offers several benefits, such as being cost-effective, environmentally friendly, and producing minimal waste. It can also be used in situ, meaning it can be performed on site, reducing the need for transportation and disposal of contaminated materials.
What are the limitations of bioremediation?
Bioremediation has some limitations, such as the need for specific environmental conditions and the length of time required for the process to work effectively. Additionally, some contaminants may be resistant to biodegradation.
What are some examples of successful bioremediation projects?
Some examples of successful bioremediation projects include the cleanup of the Exxon Valdez oil spill in Alaska, the restoration of a contaminated industrial site in Spain, and the treatment of groundwater contaminated with chlorinated solvents in California.
What are the different types of bioremediation?
There are several types of bioremediation, including natural attenuation, bioaugmentation, biostimulation, and phytoremediation.
What is natural attenuation?
Natural attenuation is a type of bioremediation that relies on naturally occurring microorganisms to degrade contaminants without human intervention.
What is phytoremediation?
Phytoremediation is a type of bioremediation that uses plants to absorb, degrade, or stabilize contaminants in the soil or water. Different plants have different abilities to remediate different contaminants.
Is bioremediation safe?
Bioremediation is generally considered safe, as it relies on naturally occurring processes and does not involve the use of harsh chemicals. However, as with any environmental remediation process, precautions should be taken to ensure the safety of workers and the surrounding community.
- Megharaj, M., Venkateswarlu, K., & Naidu, R. (2014). Bioremediation. Encyclopedia of Toxicology, 485–489. doi:10.1016/b978-0-12-386454-3.01001-0
- Singh, P., Singh, V. K., Singh, R., Borthakur, A., Madhav, S., Ahamad, A., … Mishra, P. K. (2020). Bioremediation. Abatement of Environmental Pollutants, 1–23. doi:10.1016/b978-0-12-818095-2.00001-1
- Azubuike CC, Chikere CB, Okpokwasili GC. Bioremediation techniques-classification based on site of application: principles, advantages, limitations and prospects. World J Microbiol Biotechnol. 2016 Nov;32(11):180. doi: 10.1007/s11274-016-2137-x. Epub 2016 Sep 16. PMID: 27638318; PMCID: PMC5026719.
- Hlihor, R. M., Gavrilescu, M., Tavares, T., Favier, L., & Olivieri, G. (2017). Bioremediation: An Overview on Current Practices, Advances, and New Perspectives in Environmental Pollution Treatment. BioMed Research International, 2017, 1–2. doi:10.1155/2017/6327610
- Sharma, I. (2019). Bioremediation Techniques for Polluted Environment: Concept, Advantages, Limitations, and Prospects. In M. A. Murillo-Tovar, H. Saldarriaga-Noreña, & A. Saeid (Eds.), Trace Metals in the Environment – New Approaches and Recent Advances. IntechOpen. https://doi.org/10.5772/intechopen.90453