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Biotechnology & Applied Microbiology
Bioremediation is an effective cleaning technique for removing toxic waste from polluted environments that is gaining popularity. Various microorganisms, including aerobes and anaerobes, are used in bioremediation to treat contaminated sites. Microorganisms play a major role in bioremediation, given that it is a process in which hazardous wastes and pollutants are eliminated, degraded, detoxified, and immobilized. Pollutants are degraded and converted to less toxic forms, which is a primary goal of bioremediation. Ex situ or in situ bioremediation can be used, depending on a variety of factors, such as cost, pollutant types, and concentration. As a result, a suitable bioremediation method has been chosen.
- bioremediation
- microbes
- pollutants
- environment
1. Introduction
Pollution of the environment, freshwater, and topsoil has evolved from global industrialization. Water quality has worsened as a result of human activity, such as due to mining and ultimate removal of toxic metal effluents from steel mills, battery companies, and electricity generation, posing major environmental concerns. Effluents like petroleum, polythenes, and trace metals harm the environment. Heavy metals are pollutants that exist in nature in the Earth’s crust and are difficult to decompose. They exist as ores in rocks and are recovered as minerals. High-level exposures can release heavy metals into the environment. Once in the environment, they remain toxic for much longer [1]. Many of these pollutants are mutagenic to both humans along with their surroundings. Absorbing heavy metals accumulates in the brain, liver, and kidney. Other effects on animals include cancer, nervous system damage, stunted growth, and even death [2]. Heavy metals in soils reduce food quality and quantity by inhibiting nutrient absorption, plant growth, and physiological metabolic processes. Metal-contaminated soils are being remedied using chemical, biological, and physical methods. However, physicochemical methods produce a lot of waste and pollution, so they are not valued [3]. Bioremediation is a cost-effective and practical solution for removing environmental contaminants [4]. Plant growth promotion, insect control, soil conservation, nutrient recycling, and pollutant reduction are all key functions of soil microorganisms [5]. Bioremediation has come a long way in terms of efficiency, cost, and social acceptability [6]. Bioremediation research has largely focused on bacterial processes, which have numerous applications. Archaea are known to play a role in bioremediation in many applications where bacteria are involved. Many hostile situations have degraded, requiring bioremediation. Microbes can also assist in the elimination of pollutants from hyperthermal, acidic, hypersaline, or basic industrial waste [7,8].
A bioremediation approach requires the use of microbial enzymes to break down hydrocarbons into less harmful compounds. The widespread use of genetically-modified microorganisms that can also help to eliminate petroleum, naphthalene, toluene, benzene, and other xenobiotic chemicals is now being studied [16]. Several factors, such as temperature of the surrounding environment, aerobic or anaerobic conditions, and nutrient availability, all influence bioremediation for better outcomes. Emerging environmental pollutants, such as persistent organic compounds, heavy metals, toxins, and air pollutants that are of synthetic or natural origin, reach ecosystems mainly through anthropogenic activities and pose adverse threats to lifeforms like plants, animals, and humans [17]. One of the most economical and environmentally favorable biotechnological innovations is bioremediation. Waste management mainly relies on bioremediation. It can remove persistent organic pollutants, which are hard to breakdown and are thought to be heterologous biological substances.
2. Principle of Bioremediation
When organic wastes are biologically degraded under controlled conditions, “bioremediation” is the term used to describe this process. Using bioremediation, harmful substances can be degraded or detoxified by providing the organisms with the nutrients and other chemicals they need to function optimally. Enzymes play a critical role in every stage of the metabolic process [24,43]. It is part of the family of oxidoreductases, lyases, transferases, and hydrolases. Non-specific and specific substrate affinities allow many enzymes to degrade a wide range of substrates. There must be enzymatic action on the pollutants in order for bioremediation to be successful. In order to speed up microbial growth and degradation, environmental parameters must often be manipulated during bioremediation [38,43]. This is because bioremediation only works when the environment is right for microbes to grow and move around.
Living organisms and fertilizers can aid in the process of bioremediation, which occurs naturally and is encouraged. Biodegradation is a key component of bioremediation technology. It’s the process of converting harmful organic pollutants like carbon dioxide and water into non-toxic or naturally-occurring inorganic compounds that are safe for use by humans, plants, animals, and aquatic life [44].
3. Types of Bioremediations
3.1. Biopile
In bioremediation, aeration and nutrient supplementation are used to enhance microbial metabolic activities in the piled-up polluted soil above ground. Aeration, nutrients, irrigation, leachate collection, and treatment bed systems are all included in this procedure. When it comes to ex situ biodegradation, this method is becoming increasingly popular because of its cost-effectiveness and useful features, such as pH and nutrient control. Using the biopile to clean up polluted cold environments and treat low-molecular-weight volatile pollutants is an option [15,45]. The biopile’s adaptability allows for a reduction in remediation time by increasing microbial activity and contaminant availability while also increasing biodegradation rate. When warm air is introduced into the biopile system to provide air and heat simultaneously, bioremediation is improved. The biopile’s remediation process has been helped by the addition of bulking agents like straw, sawdust, or wood chips. To replenish the air supply to polluted piled soil in biopiles, ex situ bioremediation techniques such as land farming, biosparging, and bioventing can be applied [46]. However, these techniques are expensive to implement and require a power supply at remote locations. Bioremediation may be slowed down by extreme air temperatures that dry soil and make it more likely to be vaporized than to be broken down by living organisms [47]. Bio-available organic carbon (BOC) plays an important role in bioremediation through the biopile method. Petroleum contaminated soil has been bioremediated using mesophillic conditions (30 °C–40 °C) and a low aeration rate for the removal of total petroleum hydrocarbon (TPH) using alpha, beta, and gamma proteobacteria [48]. Biopile systems have also been utilized for treating the diesel contaminated soil of the sub-Antarctic region. A total of 93% of the total petroleum hydrocarbon (TPH) was removed using the biopile system within one year [49].
3.2. Windrows
Windrows boosts bioremediation by enhancing the biodegradation processes of native and transitory hydrocarbon plastic found in the contaminated soils when spinning the heaped contaminated soils. The aeration, mineralization, and biotransformation of toxic soil can be performed through acclimation, biological treatment, and mineralization [50], can speed up bioremediation. The biopile approach can remove more hydrocarbons from soil than windrow treatment [15,51], which was more efficient in terms of hydrocarbon removal. The periodic rotation connected with windrow remediation is not a better selection approach for the bioremediation of soil affected by harmful volatile chemicals. Windrow treatment is a source of greenhouse gas (CH4) due to the anaerobic system generated inside the heaped contaminated soil [52]. The windrow method of has been applied for the bioremediation of the Gurugram–Faridabad dumpsite in Bandhwari, India by forming terraces and windrows and utilizing bio-culture, and the results showed a decrease in the garbage [53].
3.3. Land Farming
Land farming is the most significant and simple bioremediation method because of its low operating costs and lack of specialized equipment [54]. Ex situ bioremediation is the most common method, but it can also occur with in situ bioremediation. The reason for this is the location of the treatment. It is common practice in land farming to remove and till polluted soils on a regular basis, and the location of treatment dictates the type of bioremediation. On-site treatment is classified as in situ, whereas ex situ bioremediation approaches are used for the treatment of the contaminated soil [55]. Extracted contaminated soils are usually placed on a permanent layer of substrate well above Earth’s surface to permit native microorganisms to aerobically degrade contaminants [56]. Land bioremediation of polluted soil using land farming bioremediation technology is a reasonably simple process that takes little capital, has little ecological footprint, and uses very little energy [57].
3.4. Bioreactor
Following a series of biological reactions, bioreactors transform raw materials into specific products. Bioremediation thrives in a bioreactor, which provides the ideal conditions for growth [58]. The remediation samples are placed in a bioreactor. There are a number of advantages to using a bioreactor to treat contaminated soil as opposed to ex situ bioremediation methods. An efficient bioremediation process based on bioreactors that can precisely regulate pH, agitation, temperature, aeration, substrate concentration, and inoculum concentration significantly reduce the time required for bioremediation [59]. Biological reactions can take place when the bioreactor can be controlled and manipulated. Given their adaptability, bioreactor designs are able to maximize microbial degradation while abiotic losses are kept to a minimum.
3.5. Bioventing
Bioventing is a technique that uses controlled airflow to increase the activity of indigenous microbes for bioremediation by delivering oxygen to the unsaturated zone. The bioremediation process is aided by the addition of nutrients and moisture during the bioventing process. This will lead to the microbial transformation of pollutants into harmless substances. Other in situ bioremediation methods have flocked to this one in recent years [69]. Bioventing is a technique that helps in stimulating the indigenous microflora through ample amounts of aeration to enhance the biodegradation ability of the various microbes and promote decontamination of the heavy metal pollutants by precipitation [70].
3.6. Bioslurping
A direct oxygen supply and stimulation of contaminant biodegradation are used in conjunction with vacuum-assisted pumping, bioventing, and soil vapour extraction (SVE) in order to reach soil and groundwater levels for restoration [71]. This approach can be used to recover unsaturated and saturated zones as well as light non-aqueous phase liquids (LNAPLs). This technology can be used to remediate soils contaminated with flammable and moderately-flammable organic substances. Liquid is drawn from the free product layer by means of a “slurp” that spreads into the layer. LNAPLs are lifted to the surface by the pumping machine, where they are separated from the surrounding air and water [72]. To reduce microbial activity, soil moisture is used in this technique to reduce air permeability and oxygen transfer rate. Given that it uses less groundwater, this method saves money on storage, disposal, and treatment, even though it’s not ideal for remediation in low-permeable soils. Bioslurping requires 25 feet of digging below the ground surface and then the contaminants floating on the water can be removed. It combines both the approaches of bioventing, which utilize aerobic bioremediation of contaminated soil in situ. Free product is recovered by a vacuum-enhanced system that utilizes LNAPLs from the capillary fringe [73]. Free product is “slurped” up the bioslurping tube into a trap or oil–water separator for further treatment after the bioslurping tube is vacuumed. When the LNAPL is removed, the height of the LNAPL drops, which encourages the flow of LNAPL from distant locations into the bioslurping well. The bioslurping tube starts to remove vapours from the unsaturated zone when the fluid level in the bioslurping well decreases as a result of the vacuum extraction of LNAPL. This vapour extraction encourages soil gas movement, which in turn boosts aerobic biodegradation and aeration [74].
3.7. Biosparging
Air is introduced into the soil’s core, just like bioventing, to encourage microbiological activity, which in turn removes pollutants from polluted sites. As an alternative to conventional biodegradation methods, bioventing involves injecting air into a saturated zone in order to encourage the movement of flammable organic chemicals upward to an unsaturated zone nearby [75]. The success of biosparging is dependent on soil porosity and contaminant biodegradability. When it comes to bioventing and soil vapour extraction (SVE), in situ air sparging (IAS) uses high air-flow rates to volatilize contaminants, while biosparging encourages microbial degradation [76]. It is common practice to use biosparging to remove diesel and kerosene from water supplies. In order to hasten the biodegradation processes, oxygen is supplied into microorganisms during enhanced bioremediation [77]. The removal of organic pollutants (BTEX) can be accomplished using a variety of technologies, including adsorption, microbial degradation, biosparging, PRBs, and the use of modified or synthesized zeolites. However, there aren’t many investigations on readily available, inexpensive materials like natural zeolite for BTEX adsorption [78].
3.8. Phytoremediation
Contaminated soils can be cleaned up using phytoremediation. In contaminated areas, this method uses plant interactions at the physical, biological, chemical, biochemical, and microbiological levels to reduce pollutant toxicity. Depending on the quantity and form of the pollutant, phytoremediation employs a variety of processes [79]. Extraction, sequestration, and transformation are common methods for removing pollutants like heavy metals. When using plants like willow or alfalfa, the decay, immobilization, rhizoremediation, and evaporation of organic contaminants such as oils and chloro-compounds is feasible [80]. Tap root system or fibrous root system, penetration, toxicity levels, adaptability to the harsh environmental conditions of the contaminants, plant annual growth, supervision, and, notably, the time needed to reach standard of cleanliness are all important factors in plants that serve as phytoremediators. The plant must also be disease and insect resistant [81]. An important part of phytoremediation is removing pollutants from the roots and shoots. The movement of water and nutrients is also dependent on transpiration and partitioning [82]. When it comes to contaminants and plant nature, it is possible to alter this process. Phytoremediation can be accomplished with the help of the majority of the plants present at a polluted site. In polluted environments, native plants can be bioaugmented by natural or anthropogenic plants, or a combination of both. Phytomining, the process of extracting precious metals from polluted sites with plants, is one of them [83].
4. Bioremediation of Various Pollutants
4.1. Bioremediation for Organic Pollutants
Organic compounds (OCs) such as biocides and flame retardants have been widely used and are now considered a threat to nearly all forms of life on the planet because of the widespread and massive use of these chemicals in the environment. Most OCs, such as polychlorinated biphenyls (PCBs), polybrominated biphenyl ethers (PBEs), and polycyclic aromatic hydrocarbons (PAHs), can be degraded in the environment by microbes. Biodegradation is the process by which microbes break down organic compounds into less toxic or entirely non-toxic residues [91]. In order to obtain organic carbons and energy, the microbes consume the organic substrate. Isolated from other microbes, an individual microbial species usually does not degrade any organic substrate [92] and does well in a community. As a result of community microbe interactions, resistance, chemical-degrading ability, and tolerance are all conferred by the exchange of genetic information among microbial species. Many OC-degrading microorganisms are misidentified due to a lack of internationally agreed-upon methods and protocols for microbial identification [93]. This underlines the significance of studies into microbial consortiums using metagenomics tools and conventional genetic engineering protocols. Bacteria and other microorganisms have the ability to degrade a wide range of organic compounds, depending on the chromosomal genes, as well as the extracellular enzymatic activity (in the case of bacteria) (fungal degradation process). The varying environmental conditions that affect the microbe growth pattern further complicate these processes [94].
A successfully bioengineered microbe requires the identification of the relevant species and strains for each substrate. A viable alternative to the recombinant degradation of resistant organic compounds is biodegradation by microbes using readily-available organic carbon and energy sources in the surrounding environment. Microbes use the fluctuation in chemical gradients in their environment to determine the most favourable conditions for growth. This allows them to thrive in an optimal environment [95]. Microbial consortia and microbial fuel cells (MFCs and bioreactors) are two new developments in microbiological bioremediation that are being used to degrade recalcitrant organic compounds. Toxic organics can be remedied more effectively using fungi rather than bacteria because the latter cannot grow at high concentrations of toxic organics [96]. For example, the enzymes, laccase (LAC), lignin peroxidase (Lip), and manganese decarboxylase (MDA), are active in the metabolism of lignocellulosic compounds by the white rot fungus Phanerochaete chrysosporium [97].
4.2. Bioremediation for Inorganic Pollutants
Toxic heavy metals and their compounds resulting from mining, power plants, metallurgy, and chemical manufacturing processes are among the most common inorganic contaminants [98]. One of the main concerns of environmentalists is toxic elemental pollution because the disposal of toxic metals to soils and waters on or below the surface causes unacceptable health risks [99]. Microbes cannot degrade metal ions; it is essential to know that they are only capable of changing the oxidation states of the metals to stabilize them [100]. They can metabolize and detoxify metals like any other nutrient in the cells. Several microorganisms have been reported for the bioremediation of organic and inorganic pollutants. Microbes that release chelating agents and acids, as well as those that alter physicochemical properties such as redox potential in their environment can cause significant changes in the environment by increasing the bioavailability of metal ions [101]. Physical adsorption, biosorption, and ion complexation are the first steps in the interaction between metals and microbial cells [102]. Enzymes for oxidation, methylation, reduction, precipitation, and dealkylation are involved in the biochemical transformation of metal ions by microorganisms. The adaptability of microbes to heavy metals, such as iron, zinc, chrome, magnesium, mercury, and barium in textile waste, was demonstrated in the multidrug-resistant Pseudomonas aeruginosa T-3 isolate from tannery effluent [67,83]. This shows that microbes can adapt to changing environmental conditions. A plasmid-encoded copper and cadmium metal resistance gene in the bacteria, Pseudomonas putida PhCN, has also been discovered [103]. Plasmid-encoded biochemical information and genetic engineering techniques were used to create recombinant Escherichia coli that expresses the metallothionein gene (Neurospora crasa) for Cd uptake, resulting in significantly faster Cd uptake than the donor microbe [104]. A poly-histidyl peptide was introduced into Staphylococcus xylosus and Staphylococcus carnosus that encoded genes that allowed these microbes to bind nickel [105].
5. Recent Advancement and Challenges in Bioremediation
5.1. Bioinformatics Approaches in Bioremediation
When it comes to waste management, bioremediation is a useful technique that can be used to remove waste from contaminated areas and sites. It is particularly concerned with the utilization of organisms to consume or neutralize pollutants [20]. Using data from various biological databases, such as databases of chemical structure and composition, RNA/protein expression, organic compounds, catalytic enzymes, microbial degradation pathways, and comparative genomics to interpret the underlying degradation mechanism carried out by a particular organism for a specific pollutant is the goal of bioremediation [133]. A variety of bioinformatics tools are used to interpret all of these sources in order to study bioremediation in order to develop more effective environmental cleaning technology. There has been a scarcity of data on the factors that control the growth and metabolism of microbes with bioremediation potential, which has resulted in a limited number of bioremediation applications [134]. These microorganisms with bioremediation capabilities have been profiled and their mineralization pathways and mechanisms have been mapped out using bioinformatics [135]. The use of proteomic approaches such as two-dimensional polyacrylamide gel electrophoresis, microarrays, and mass spectrometry is also critical in the investigation of bioremediation methods and technologies. It significantly improves the structural characterization of microbial proteins that have contaminant-degradable properties, according to the researchers [135]. The structural characterization of microbial proteins capable of degrading contaminants has greatly improved. Research in this field crosses the boundaries between computer science and biology. For example, computers are used to store, manipulate, and retrieve information linked to the DNA, RNA, and proteins of the genome [133,135].
5.2. Bioremediation Using Nanotechnological Methods
A nanometer is the smallest unit of measurement used in nanotechnology. Many toxic substances can be removed with their help because of their unique abilities against various recalcitrant contaminants. Technology such as water treatment has been given a new perspective by nanotechnology. Techniques that are good for the environment can now be categorized as nanofiltration [151].
5.3. Genetic and Metabolic Engineering
“Gene editing” refers to scientific technical developments that enable rational genetically-created fragments at genome level to provide exact addition, deletion, or substitution of pieces of DNA molecules. Transcription activators are utilized in a variety of gene editing methods, including TALENs, ZFNs, and CRISPRs, which are widely used in research. CRISPR-Cas has been dubbed the most efficient and straightforward gene editing tool [157]. A DNA-binding element in TALEN is complementary to the sequence of the host DNA. When TALEN attaches to DNA and exposes sticky ends for stabilization, it creates double-stranded breaks (DSBs). ZFNs also have a DNA-binding domain made up of 30 amino acids. At the target location of the host DNA, the Fok1 cleavage domain causes DSBs. A novel perspective on composite endonuclease comprising TALENs and ZFN nucleases was required to solve molecular problems [158,159]. Two of the CRISPR-Cas system’s unique properties are sequence similarity complementarity and simultaneous gene editing [160,161]. The bacteria, Streptococcus pyogenes, provides this unique ability as a sort of virus resistance. In the CRISPR-Cas system, guide RNA connects crisper-derived RNA (crRNA) and trans-acting antisense RNA (trcRNA). The Cas9 enzyme is able to carry out the requisite DSB when gRNA recognizes the target DNA sequence. These gene editing tools’ knock-in and knock-out effects are being analyzed for usage in bioremediation investigations [161]. In model organisms like Pseudomonas and Escherichia coli, the CRISPR-Cas system has been widely accepted by researchers [138]. In non-model species (such as Rhodococcus ruber TH, Achromobacter sp. HZ01, and Comamonas testosteroni), the area of bioremediation is also exploring new insights into CRISPR toolkits and the synthesis of gRNA for the production of remediation-specific genes [162].
Pollutant-tolerant bacteria are the greatest choices for genetic manipulation and biochemical pathways since they are accustomed to tolerating and storing a variety of toxic, refractory, and non-degradable xenobiotic compounds under harsh circumstances. Furthermore, recognizing biochemical functions is critical for analyzing microbiological bioremediation, such as the bioremediation of harmful pollutants through the production of haloalkane dehalogenases and the disposal of pyrethrins from land through the anaerobic decomposition pathway of fenpropathrin studied in Bacillus sp. DG-02 [163]. The bioremediation process can be improved by metabolic engineering, which alters the existing pathway. The likelihood of obtaining recombinant enzymes increases significantly when using a genetic approach. Some extracellular enzymes have been found to play a role in enzymatic bioremediation, according to some studies. PAHs are degraded by LiPs ((lignin peroxidase) from P. chrysosporium that encode hemoproteins [164]. Even though contaminants can be consumed by microbes as substrates or intermediates in biological pathways, incomplete or partial degradation leads to simpler, non-toxic degradable compounds [136]. For example, LiP can degrade benzopyrene into three quinine compounds, namely 1,6-quinol, 3,6-quinine, and 6,12-quinine [165]. MnP (Mn (II) peroxidase) can also oxidise organic compounds in the presence of MnP (Manganese peroxidase) [166]. As well, laccase, glutathione S transferase, and cytochrome P450 are involved in the biodegradation of recalcitrant compounds [167]. It has been shown that the immobilization of enzymes significantly increases enzyme stability, activity, and stability. Enzymatic bioremediation is a simple, environmentally-friendly, and fast method for removing and degrading persistent xenobiotic compounds by microorganisms [134,145]. Enzyme-producing microorganisms have been isolated and characterized with the limitation of low productivity. Insecticides’ main ingredients, organophosphates (OP) and organochlorines (OC), are found in agricultural soil and run-off into waterways.
Genetically-engineered microorganisms have demonstrated successful bioremediation of hexachlorocyclohexane and methyl parathion [135,146]. Genetically-modified P. putida KT2440 was used in organophosphate and pyrethroid bioremediation experiments [168]. The degradation and catabolism of a variety of persistent compounds has been documented since the advent of metabolic engineering. Sphingobium japonicum and Pseudomonas sp. WBC-3 showed bioremediation of methyl parathion and -hexachlorocyclohexane degradation pathways [169]. When three enzymes from two different microorganisms are combined in E. coli, a persistent fumigant called 1-, 2-, 3-trichloropropane is released into the environment via heterologous catabolism [137,148]. To do this, microbes can be used to turn persistent compounds into minerals [49].
5.4. Designing the Synthetic Microbial Communities
Synthetic biology advancements have had a significant impact on environmental issues in recent years. Toxic compounds, pesticides, and xenobiotics can be removed from the environment by using genetically-modified organisms (GMOs). Natural microbial communities must be understood in order to create a synthetic one [170]. Identifying which species are participating in bioremediation is difficult in a natural community. Through the use of a synthetic microbial community, the development of an artificial microbiome with functionally specific species is possible. Model systems for studying functional and structural characteristics can be found in these communities. Synthetic communities were formed by the co-culturing of two distinct microorganisms under precisely defined conditions, which were based on their interactions and functions [171]. The community’s dynamics and structure are determined by these variables; it is based on the discovery of bacterial processes and behaviors. Metabolism drives these patterns of microbial interaction, which in turn facilitates communication within communities [172]. Interactions between two microbial populations are social in nature (such as mutualism, competition, and cooperation). Cooperation is said to be a key factor in community structure and operation. Cooperation in community dynamics is influenced by the creation of synthetic communities [173] and it was found that modifying environmental conditions, such as deleting genes, could be used to engineer cooperation between two microbial strains. In addition to this, the synthetic community’s engineered microbial species have been examined for other patterns of interaction. Bioremediation strategies frequently make use of this type of engineered interaction [174]. It is possible to sustain the existence of microorganisms in a large population by using synthetic biology.
This entry is adapted from the peer-reviewed paper 10.3390/toxics10080484
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