Keywords

7.1 Introduction

Release of petroleum by-products, crude oil, xenobiotic compounds, heavy metals, radioactive substances, and emission of greenhouse gases into the environment has exponentially increased over the last two decades owing to global industrialization, urbanization, unsafe agricultural practices, mining activities, population growth, and civil war. It causes lasting impacts on the environment and human health depending on the pollutant and degree of pollution (Bai et al. 2017; Power et al. 2018; Wang 2018). Many different strategies were employed over time to restore these degraded landscapes and to minimize the impact of effluents emitted to the environment. Genetically modified organisms hold a small fraction in these interventions, but holds a great promise for the future.

As an industry, crude oil holds the largest market share where they are used in industries, transportation, and household activities as a major source of energy. It is a mixture of naturally occurring hydrocarbons and the process of extraction can cause small to large-scale petroleum contaminations that are regularly reported worldwide during exploration, refining, transportation, and storage. Air pollution occurs via hydrocarbon gas liquids and soil and water pollution occur via liquid and solid hydrocarbons. Hydrocarbon contamination is greatly affected in particular ecosystems (Wang et al. 2011). However, the estimation of the impact on aquatic and terrestrial environments and on human health is not feasible. Soil, fresh water bodies, and plants are affected mainly through industrial effluents and seepage of oil storage tanks (Moubasher et al. 2015). Marine environments are greatly affected owing to accidental and intentional oil spills during transportation, mining and in periods of civil war (Linden et al. 2004; Saadoun 2016) (Fig. 7.1).

Fig. 7.1
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Deepwater Horizon in flames 20th April 2010

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Accumulation of xenobiotic compounds in ecosystems with the release of synthetic compounds from medicine, food, agriculture, textile, and other products has been increasing with technological advancements (Giri et al. 2017a, b; Atashgahi et al. 2018a, b). Xenobiotic compounds persist in ecosystems due to less bioavailability and create toxic effects upon exposure to humans and other organisms (Atashgahi et al. 2018a, b).

A large amount of heavy metals are being unearthed and dispersed with uncontrolled applications in pesticides, fertilizers, and industrial practices. However, heavy metals have received special emphasis due to their high toxicity even at trace amounts. They can accumulate in the human body through food chains and direct absorption through the skin since those are not metabolized in the body. In addition, reduction in crop production and food quality is the other emerging problem behind heavy metal pollution (Tarekegn et al. 2020).

Radioactive waste always reminds us of high energy radiation is discharged through nuclear explosions, testing of nuclear weapons, accidents at nuclear power plants, volcanic eruption, and mining of radioactive ores (Nies 2018). Since radioactive substances emit ionizing radiation, they cause great impact on human health and the surrounding environment. Exposure to radiation causes skin irritation, genetic defects, cancer, and fatal effects to humans. Furthermore, they can destroy wildlife and marine life, and cause soil infertility (Pollution 2014; Nies 2018). The emerging problems are with the radioactive substances can trace to being ubiquitous with the technological developments and they produce a low level of radiation. The effect of such long-term exposure to low level radiation may not be visible for many generations to come.

It is a known fact that the greenhouse gas emission have increased exponentially after late 1700s owing to the industrial revolution. This has caused many impacts on ecosystems and human health. World health organization reports show that seven million deaths occur every year due to exposure to ambient and household air pollution (WHO 2021). In addition, it was estimated that half-of the world’s population will be living in water stressed areas by 2025. However, human and ecological health are increasingly threatened and it is a great concern worldwide (Albert et al. 2018).

Restoring ecological health and improving human health is a mainstream discussion in every forum. Turning to nature to find solutions to this problem by harnessing what nature has to offer us and responsible utilization becomes the theme of this chapter.

7.2 Bioremediation

The perspective of an environmental scientist for sustainable environmental remediation is the implementation of rapid restoration strategies at the contaminated sites in an eco-friendly and economically viable framework. Because of the exorbitant costs involved and adverse effects of physiochemical methods such as addition of chemicals and coagulants, filtration, ion exchange methods, reverse osmosis, physical removal etc., they are of limited usage.

Bioremediation on the other hand employs the use of bacteria, fungus, plants, and enzymes for removing and detoxifying pollutants (Dash et al. 2021; Kumar et al. 2021; Kour et al. 2021). This is considered a better alternative owing to its capabilities of complete mineralization, lower liability, low cost, feasibility in in situ applications, and eco-friendliness. However, the rate of biodegradation depends on the concentration of the contaminant and the number of catalytic organisms as well as the number of catalytic enzymes produced by each organism, amount of oxygen, amount of nutrients, pH, temperature, moisture, etc. Therefore, natural degradation is a slow process compared to pollution accretion.

Depending on the state of the contaminant to be remediated, two bioremediation techniques named; in-situ and ex-situ are used. The application of non-pathogenic microorganisms that has the capability of converting toxic pollutants into less toxic compounds is called in-situ bioremediation. This is widely used for the remediation of petroleum contaminated sites. However, supplement of nutrients such as nitrogen, phosphorus, sulphur and oxygen is required for the growth and survival of bacterial species. This a comparatively less expensive and effective method. In addition, the chemotactic ability of the microorganisms is another important factor that aids efficient in-situ bioremediation.

Ex-situ bioremediation refers to treating pollutants after excavating the contaminated site. Slurry phase bioremediation and solid phase bioremediation are the two main types of ex-situ bioremediation. The slurry phase bioremediation is done in a bioreactor providing the optimal growth conditions to the microorganisms. Here, water and other nutrients are mixed with contaminated soil. After the treatment, water is separated from the soil.

Solid phase bioremediation is mainly used for soil contaminated with toxic compounds. Here, the contaminated soil is treated in above–ground treatment areas taking care not to allow pollutants to escape. Conditions required for microbial growth are properly maintained.

Biostimulation and bioaugmentation are other general methods that are widely used for the remediation of contaminated sites. Biostimulation is providing suitable conditions for the growth of indigenous microorganisms present in the contaminated sites. Bioaugmentation is an application of microbial cultures that are cultivated in a laboratory into a contaminated site or a bioreactor.

Even though bioremediation is more advantageous compared to physiochemical methods, there are limitations and disadvantages in bioremediation. Lack of microorganisms evolved to degrade many xenobiotic compounds, biological reactions are specific, bi-products generated are highly toxic than initial compound, there is no acceptable endpoint for bioremediation, treatment time is longer, and difficulties in controlling volatile compounds are the key problems associated with bioremediation. To mitigate these issues, knowledge, innovative ideas, and novel techniques are essential.

With the advances in biotechnology, the use of genetically modified organisms for bioremediation is a rapidly developing field. Thus, organisms that can persist in many different stress conditions owing to pollutant accretion have been developed by using recombinant DNA technology in many laboratories throughout the world. The development of novel genetic engineering methods, identification of extremophiles, and understanding of their genetics, physiology, and metabolic pathways are of utmost importance in developing effective bioremediation strategies (Bhatt et al. 2021). This chapter summarizes recent advances and current research on bioremediation with genetically modified organisms.

7.3 Bioremediation by Extremophilic Organisms

Organisms that have evolved to live in extreme environments such as high or low pH, high temperature, cold conditions, acidic conditions, high salinity, high pressure, nutrient deficiencies, etc. are considered extremophiles. This group of organisms can be categorized as extremophilic organisms and extremotolerant. Organisms in need of one or more extreme conditions to thrive are named extremophilic and the organisms are named as extremotolerant when they grow well in both normal and extreme conditions. Moreover, extremophiles can be categorized further as acidophilic (organisms live at low pH), alkalophilic (organisms live at high pH), thermophilic (organisms live at high temperatures), psychrophilic (organisms live at low temperatures), barophilic (organisms live at high pressure), oligotrophic (organisms live at nutrient deficiencies), etc. according to the particular extreme conditions that they can thrive (Sani and Nawanietha Krishanraj 2017). With the advancement of genetic engineering techniques, extremophiles have acquired special attention owing to their various biotechnological applications. Understanding the metabolic pathways and characterization of proteins that give advantages in extreme environmental conditions are trending topics among environmental scientists (Dumorné et al. 2017).

Deinococcus geothermalis and Deinococcus radiodurans are applied for bioremediation of radioactive wastes at heated environments owing to their capabilities of reducing Cr+6, Hg+2, Tc+7, Fe+3, Mn+3, Mn+4, and U+6. In addition, xenobiotics such as toluene, diethyl sulphate, and ethyl acetated can be degraded by D. geothermalis strain T27. Removal of 99Tc+7 from the contaminated environments is very difficult via chemical methods due to its capability to form complexes. Thermophiles, Thermoterrabacterium ferrireducens, Thermoproteus usoniensis, Pyrobaculum islandicum, has been reported to reduce 99Tc+7 into insoluble 99Tc+4. Shewanella putrefaciens acquired capability to reduce U+6 via c-type cytochrome in the periplasm.

Penicillium chrysogenum, Pseudomonas sp., Acinetobacter sp., Mycobacterium sp., and Basillus subtilis have acquired the ability to utilize many different compounds present in petroleum hydrocarbons as their sole source of carbon and energy. Pseudomonas fluorescens, Pseudomonas aeruginosa, Microbacterium profundi strain Shh49T, Saccharomyces cerevisiae, Geobacter sp., Rhodopseudomonas palustris, Acromonas sp., and Trichoerma sp. possess the capability to utilize heavy metals. Bacillus firmus, Bacillus macerans, Exiguobacterium indium, Acinetobacter baumanii, Penicillium ochrochlorum, and Tremetes trogii have been isolated in industrial effluents with dye compounds and Arthrobactor sp., Enterobacter sp., Photobacterium sp., Bacillus sp., Staphylococcus sp., and Pseudomonas sp. persist on pesticides contaminated sites (Peeples 2014; Shukla and Singh 2020).

7.4 Designer Organisms for a Cleaner Tomorrow

Even though many organisms have been evolving in degrading or converting pollutants, the rate of biodegradation depends on many environmental and biological factors. Accumulation of pollutants affects adversely for almost all organisms including humans. Some of the pollutants (Xenobiotic compounds) are resistant to natural degradation owing to the catabolic pathways that are yet to be resolved. With the rapid development of molecular engineering technology, molecular biologists are trying to construct genetically modified organisms that have the capability in biodegradation. Enhancement of enzyme specificities, substrate affinities, cellular localization, expression, and genetic stability occurs mainly via mutations, the introduction of novel genes, pathways, and regulatory mechanisms via cloning, fusion proteins, into heterologous hosts.

Gene editing is a DNA manipulation technique by using engineered nucleases having a growing popularity among genetic engineers due to the potential advantages. These engineered nucleases are named molecular scissors. Gene editing is used for the manipulation of genes that can convert toxic chemicals into less toxic compounds and for the remediation of xenobiotic compounds (Butt et al. 2018; Hussain et al. 2018). The main gene editing tools are CRISPR-Cas9, Transcription activator-like effector nucleases, and Zinc finger nucleases (Waryah et al. 2018).

7.4.1 Gene Transfer

Molecular cloning, homologous recombination, horizontal gene transfer are used to insert or delete or replace genes. Thus, particular organisms can be engineered to grow on pollutants. Figure 7.2 indicates the types of probable genes that are currently used for the genetic manipulations to enhance biodegradation. However, identification and characterization of novel gene clusters that can improve bioremediation through genetic alterations provide basis for the novel bioremediation strategies.

Fig. 7.2
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Genes that are currently manipulated for the construction of genetically modified bacterial strains

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Owing to anthropogenic activities, agricultural practices, and effluents, arsenic (As) is added into the soil and water. Arsenic adversely affects human health due to its genotoxic and mutagenic effects. Bio volatilization is a process that involve a series of methylation reactions catalysed by enzymes. Soil microbes have evolved to form volatile arsenic (Kumar et al. 2021). The enzyme arsenite S-adenosylmethionine methyltransferase which is encoded by arsM gene can convert arsenicals into volatile arsenic. Liu et al. have cloned the arsM gene in Rhodopseudomonas palustris in the expression vector pET28a and transformed it into the Sphingomonas desiccabilis and Bacillus idriensis. They have found that about 2.2–4.5% of arsenic has been removed from the soil after 30 days of incubation with these genetically modified strains (Liu et al. 2011).

Accumulation of heavy metals in E. coli was enhanced by introducing Arabidopsis thaliana phytochelatin synthase (AtPCS) gene via pBluscript plasmid. Phytochelatin is a metal binding cysteine rich peptide synthesized from glutathione. Here, for the easy detection of AtPCS, it is fused to a C-terminal flag epitope and expressed under the control of the T5 Promoter/lac operator/repressor system. They have shown that intracellular metal content is increased when this genetically modified E. coli strain is grown in a medium supplemented with Cd, Cu, and As form 20, 5, and 50 folds respectively. However, no significant intercellular accumulation of Ag, Zn, and Hg was observed (Sauge-merle et al. 2003).

Sun et al. (2019) identified yeast as an organism that can be improved well for heavy metal remediation. Since metal transporters, storage components, and chelators play a pivotal role in the hyperaccumulation of metal ions in cells, genes that express metal transporters through cell membranes, vacuole and an oxidoreductase (ZRT1, ZRT2, CTR1, CTR3, FTR1, FTE4, SMF1, SMF2, ZRT3, CTR2, SMF3, FET3) have been cloned into 2 μ plasmids which contain GAL1 promoter and were transformed to constructed engineered yeast strains. ZRT1 and 2 selectively transport Zn ions and CTR1 and 3 are selectively transport Cu. The oxidoreductase (FET3) membrane transporter SMF1 were not shown with such selectivity. The natural degradation of SMF1 was reduced by mutating the ubiquitination site by site-directed mutagenesis and SMF1 expression was enhanced by deleting the BSD2 ubiquitin ligase (Sun et al. 2019).

Vitreoscilla stercoraria is a gram-negative bacterium that can live in oxygen limited environments. The first bacterial hemoglobin protein (VHb) was isolated from Vitreoscilla stercoraria (Webster and Hackett 1966). VHb is produced at oxygen stress conditions and plays a key role in binding oxygen at low concentrations and delivering directly to the terminal respiratory oxidases. In addition, they pass the signal to transcriptional regulators in response to oxygen concentrations and control the expression of many genes (Stark et al. 2012). The expression of VHb in different bacterial species enhances cell density, oxidative metabolism, and bioremediation at oxygen stress conditions; i.e. in Burkholderia, the degradation of 2,4,-DNT was increased.

Today, many environments are co-contaminated with different pollutants. Chlorpyrifos is an organophosphate and γ-Hexachlorocyclohexane is an organochlorine found in insecticides. Co-contamination of these compounds and heavy metals in nature contributes to potentiate toxicity. Yang et al. has constructed a genetically engineered Sphingobium japonicum UT26 strain for the remediation of Cd+2, Chlorpyrifos, and γ-Hexachlorocyclohexane (Yang et al. 2015). Sphingobium japonicum UT26 is a gram-negative bacterium isolated from the γ-Hexachlorocyclohexane contaminated site in Japan (Nagata et al. 2010). The capability of degrading γ-Hexachlorocyclohexane in Sphingobium japonicum UT26 is well characterized. In general, phytochelatins are used for metal remediation since they have metal binding moieties (Alkorta et al. 2004). Methyl parathion hydroxylase hydroxylates chlorpyrifos. The access of metals and chlorpyrifos into phytochelatins and methyl parathion hydroxylase is greatly reduced as these proteins are produced in the cytoplasm. Therefore, they have chemically synthesized a fusion gene that contains mpd gene that encodes methyl parathion hydroxylase in Stenotrophomonas sp. (Yang et al. 2006) and a synthetic phytochelatin. This fusion protein is coupled to a truncated ice nucleation protein so that fusion proteins can transport to the cell surface. The fusion gene is cloned to the surface expression vector pVINPEM using E. coli–Pseudomonas shuttle vector pVLT33 (Yang et al. 2008, 2015). The engineered Sphingobium japonicum UT26 strain can be used to simultaneously detoxify heavy metal and pesticide contaminated sites (Cao et al. 2013; Yang et al. 2015).

7.4.2 Genetic Mutations Improve Biodegradation of Pollutants

Gene mutations are involved in genetic variations and contribute to both the survival and the extinction of organisms. Many different environmental and biological factors such as exposure to radiation and chemicals, viral infections, mating patterns, etc. are associated with mutations. Bacteria attribute to high mutation frequencies. Mutations induced owing to pollutants imply organisms mounting a survival strategy to tolerate harmful conditions. Taking cues from nature, organisms are engineered by mutating the genes to improve their capabilities of degrading or detoxifying pollutants according to requirements. Strategies such as random mutagenesis by exposure to mutagens, site directed mutagenesis, PCR based methods, TA strategy are currently used for the construction of genetically modified organisms.

Phenol is an aromatic hydrocarbon that contains a hydroxyl group. Phenolic compounds are widely used in industries to synthesis many different organic chemicals. Phenolic compounds are further grouped into simple phenols, bi-phenols, and poly-phenols depending on the number of phenol rings of a compound. Many industries such as textile, dyes, explosives, oil, gas, and coal utilize phenol as a raw material and phenolic compounds are discharged into the environment through effluents. Phenolic compounds are considered as pollutants owing to long term and short term severe effects on humans and animals. Therefore, phenols are enlisted in the United States Environment Protection Agency (USEPA) and European Union (EU) under harmful chemical categories.

Ozonation, use of UV light, and filtration methods are applied to remove phenols from effluents (Anku et al. 2019). In addition, microorganisms that have the ability to degrade phenolic compounds (Pseudomonas pseudoalcaligenes (Kurzbaum et al. 2010), Acinetobacter sp. (Liu et al. 2009), Candida sp. (Basak et al. 2013), Sphingomonas sp. (Liu et al. 2009) have been reported).

Mao et al. (2015) isolated Pseudochlorobactrum sp. that can degrade 1800 mg/l phenols from active sludge from a wastewater treatment plant. Pseudochlorobactrum sp. strain that has enhanced phenol degradation capability was screened upon subjecting to UV light for 120 s for random mutagenesis. The tolerance to pH and temperature variations and faster phenol degradation had been observed in mutated strain, Pseudochlorobactrum sp. XF1-UV (Mao et al. 2015).

Today, plastics have invaded earth owing to over consumption and low biodegradability. Plastics of various forms became a necessity for the day today life and over 380 million tons of plastics are produced per year where 5% of them are single use plastics (Plastics – the Facts 2019). About 90% of the plastic waste is accumulated in the ocean without a proper waste management strategy (Schmidt et al. 2017). Bacteria and fungi species that have the capability to degrade polythene have evolved and their underlining mechanisms are to be uncovered (Debbarma et al. 2017; Singh et al. 2021). Attachment of microbes on the plastic surfaces and biofilm formation are initial steps in plastic biodegradation (Goel et al. 2008). Pseudomonas aeruginosa (Ogunbayo et al. 2019), Aspergillus niger (Raaman et al. 2012; Ogunbayo et al. 2019), Fusarium lini, Bradyrhizobium japonicum, Pseudomonas sp. AKS2 can efficiently get attached to the plastic surfaces and are identified as plastic degrading organisms (Łabużek et al. 2004). It has been reported that mutations in O-antigen which plays a fundamental role in the attachment of gram-negative bacteria on hydrophilic surfaces can enhance the affinity towards hydrophobic surfaces (Bogino et al. 2013). In addition, by expressing cellulose binding domain (CBD) of the cellobiohydrolase I (CBHI) in Trichoderma reesei in the cell surface of yeast (Saccharomyces cerevisiae) and mycoremediation of lignin have been enhanced by increasing binding affinity to cellulose (Jafari et al. 2013).

Pseudomonas putida strains are utilizing terephthalic acid for growth as a sole source of carbon and for accumulation of medium chain length polyhydroxyalkanoate at limited nitrogen conditions. However, they are not degrading ethylene glycol that is produced in the terephthalic acid degradation as a carbon source. A recent study has demonstrated that a mutated strain of Pseudomonas putida KT2440 that overexpresses glyoxylate carboligase operon and glycolate oxidase operon has acquired the capability of utilizing ethylene glycol as the sole source of carbon (Ann et al. 2018; Li et al. 2019). A mutated P. putida strain constructed by DNA restructuring had also improved the degradation of low density polyethylene (Anantharam and Talkad 2018).

The extracellular enzyme laccase contributes to the oxidation of many xenobiotic compounds such as synthetic dyes, aromatic ammines, polyphenols. Mutant strains constructed by mutating bacillus sp. by substituting the Glutamic acid 188 residues with Lysine, Arginine, and Alanine were exhibited to have thermal stability and thermal activation compared to the wild type. Moreover, mutants constructed by substituting Glutamic acid 188 with Alanine, Leucine, Isoleucine, Valine, Lysine, and Arginine have exhibited tolerance to organic solvents (Rasekh et al. 2014).

Cytochrome P450 monooxygenase in Bacillus megaterium 3 oxidizes aromatic compounds. Mutants generated by error-prone PCR in the gene; cytochrome P450 monooxygenase showed an improved hydroxylation capability towards chrysene and pyrene (Santos et al. 2019).

7.4.3 Genetically Engineered Enzymes in Bioremediation

Enzymes are macromolecules that function as catalysts in biological reactions. In bioremediation, enzymes produced by bacteria, fungi, and plants are utilized as biocatalysts instead of using the whole microorganisms. This technique has been identified as an advantageous method compared to the use of microorganisms in bioremediation. For the reasons that purified or partially purified enzymes can be applied to the contaminated sites. Enzymes can be applied to poor nutrient conditions, enzymatic reactions are specific and generation of toxic by-products is prevented.

Phanerochaete chysosporium, Trametes versicolor, Bjerkandera adjusta and Ceriporiopsis subvermispora are white rot fungi that produce ligninolytic enzymes (laccases, peroxidases). These enzymes convert toxic organic pollutants into nontoxic compounds. Laccases that have low substrate specificities cleave aromatic rings of cyclic hydrocarbons. Peroxidases generate reactive oxygen species initiating the oxidation of pollutants. However, the degradation of pollutants into simpler forms by biocatalysts is achieved through further genetic modifications. The genetically engineered biocatalysts are grown in bioreactors for efficient removal of pollutants (Akhtar and Mannan 2020).

The enzymes that are predominantly involved in the bioremediation include cytochrome P450s, laccases, hydrolases, dehalogenases, dehydrogenases, proteases, and lipases. These enzymes are involved in degradation of polymers, aromatic hydrocarbons, halogenated compounds, dyes, detergents, agrochemical compounds etc. with the use of various mechanisms such as oxidation, reduction, elimination, and ring-opening (Bhandari et al. 2021).

Under natural conditions, the production of these enzymes from its native host is considerably low. Therefore genetic approaches such as isolating and transferring the coding genes of the enzyme into a better expression host, ensures that the enzyme production is enhanced such that biotransformation of compounds occurs more effectively (Sharma et al. 2018).

Using DNA technology and genetic engineering approaches that involves change or modification in the basic amino acid structure of the enzyme, recombinant enzymes can be produced with higher activity and stability, with enhanced shelf-life, substrate range, pH, temperature stability and stress tolerance. So that the engineered enzyme would have a higher capacity to degrade the contaminant under defined environmental conditions. (Sharma et al. 2018).

Nitrobenzene 1,2-dioxygenase is an enzyme that catalyses the conversion of nitrobenzene into catechol and nitrite. It also oxidizes the aromatic rings of mononitotolunes and dinitrotoluenes at the nitro-substituted carbon. The important features of regiospecificity with nitroarene and enantiospecificity with naphthalene is determined by the residues at positions 258, 293, and 350 in the α subunit. Phenylalanine amino acid residue at position 293, near the active site of nitrobenzene 1,2-dioxygenase is modified/substituted with Glutamine such that it results in an increase of 2.5-fold oxidation rate against 2,6 dinitrotoluene (Ju and Parales 2006). Similarly, 2-nitrotoluene dioxygenase which is responsible for the conversion of nitrotoluene to 3-methyl catechol and nitrite was modified at position 258 by site directed mutagenesis (Singh et al. 2008).

Polyethyleneimine (PEI) can bind onto the surface of Horseradish peroxidase (HRP) mainly via hydrophobic interaction and van der Waals interactions. The complex formation between HRP and PEI induces a more compact conformation of the enzyme and thus the hydrophobicity of the microenvironment surrounding the heme pocket is enhanced. The non-planarity of the porphyrin ring in the heme group contributes to an increased degree of exposure to the active centre, significantly enhancing the catalytic efficiency of HRP in the presence of high molecular weight PEIs (Huang et al. 2018).

The technique of site-directed mutagenesis is used to alter genes thereby protein sequences and to explore the structure-function relationship of enzymes. The basis of rational protein design is the combined use of protein crystallography to provide detailed knowledge of the three-dimensional structures of proteins, and elucidating the residues that are of significance to the enzyme catalysis and substrate specificity (Taylor et al. 2011).

Halogenated organic compounds, such as 1,2,3-trichloropropane (TCP), are stable and chemically inert molecules. As a result, their natural degradation is difficult in the environment. Studies conducted using random mutagenesis of haloalkane dehalogenase from Rhodococcus sp. m15–3 (DhaA) has yielded an enzyme eightfold more efficient than the initial protein, significantly improving the degradation of TCP (Bosma et al. 2002).

The key residues function in modulating entrance into the active site buried in haloalkane dehalogenase were determined by error-prone PCR followed by computer modelling, site saturation mutagenesis. The combined rational design and directed evolution strategy employed in the study yielded a mutant haloalkane dehalogenase. Use of mutated haloalkane dehalogenase achieved a 30-fold improvement of TCP degradation and also the resulting substitutions promote the formation of an activated reaction complex. Thus, solvent accessibility to the active site of the mutated haloalkane dehydrogenase decreased. This study demonstrates the substitutions in the “access tunnels” not in the catalytic site can also be critical for engineering proteins to enhance their catalysis (Pavlova et al. 2009).

Studies have applied a directed evolution strategy to the arsenic resistance (ars) operon from Staphylococcus aureus. The ars operon encodes a repressor (arsR), a membrane-efflux protein (arsB), and arsenate reductase (arsC). E. coli is resistant to arsenate and arsenite when ars operon is expressed. A mutant operon that has a 40-fold improvement of arsenate resistance was isolated with three rounds of DNA shuffling; random fragmentation of the operon, followed by mutagenic PCR, and library generation and screening. Interestingly, they have observed ten coding mutations in the arsB protein in final evolved version of the operon. Though there are no mutations in arsC, the activity of arsenate reductase has significantly improved (more than tenfold). This study clearly elicits that directed evolution is capable of improving the function of pathways (Carameri et al. 1997).

Biphenyl oxygenases (BOs) catalyse the oxygenation followed by degradation of polychlorinated biphenyls (PCBs). Biochemical properties of BOs are improved by directed evolution. The gene shuffling experiments confirmed large subunit of the biphenyl oxygenases present in Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400 plays a pivotal role in improving degradation properties for PCB and single aromatic compounds such as benzene, toluene, etc. (Suenaga and Furukawa 2006).

Toluene-ortho monooxygenase (TOM) has the ability to modify its substrate specificity via direct evolution. Different mutant proteins that effectively oxidize chlorinated ethane and naphthalene have been reported and they form dihydroxy and trihydroxy compounds from benzene and toluene, to enhance the oxidation of nitro-aromatics, and the degradation of chlorinated aliphatic compounds (Canada et al. 2002; Ryu and Wood 2005; Leungsakul et al. 2006).

Saturation and random mutagenesis of aniline dioxygenase isolated from Acinetobacter sp. strain YAA have developed a mutated enzyme that harbour the capability to metabolize aniline, 2,4-dimethylaniline (a carcinogenic aromatic amine), and 2-isopropylaniline. Thereby the hydroxylation of aromatic amines is significantly improved (Ang et al. 2009).

The 1,2,3-trichloropropane (TCP) is highly toxic, recalcitrant in the environment and is a ground water pollutant which is frequently detected at sites where chemical waste has been inappropriately disposed. Interestingly, no natural microorganisms are known to degrade TCP under oxic conditions, even though anaerobic and oxidative biotransformations are possible. Studies have used a combination of protein and metabolic engineering to construct bacteria that use TCP as a growth substrate to facilitate biodegradation.

The haloalkane dehalogenase called DhaA from Rhodococcus was used as the starting enzyme which has very low but detectable activity with TCP. Researchers have determined its protein structure and have developed enzyme variants through directed evolution and particularly by inserting mutations in the substrate access/product exit tunnel that connects solvent and active site to have increased activity towards TCP with much more thermal stability (Janssen and Stucki 2020).

7.4.4 Genetically Modified Plants for Improved Phytoremediation

Plants have been used in bioremediation for centuries. Historical records show that the man-made irrigation systems in ancient Sri Lanka promoted growing selected trees around them and are known to be a measure taken for improving water quality. With growing amount of pollutants released, the natural capacity of these plants would not be sufficient to perform this. Hence, genetically modified, fast growing, and high biomass plants are called for phytoremediation (Nedjimi 2021). Phytoextraction (removal of contaminants by using plant roots), phytovolatilization (conversion of non-volatile compounds into volatile compounds by using plants), phytofiltration (absorb or adsorb pollutants by using plant roots or seedlings), phytostabilization (conversation of toxic compounds into less toxic compounds by using plants), phytodegradation (degradation of organic compounds by using plants), and rhizosphere bioremediation (removal of contaminants with the help of rhizosphere microorganisms) are the different types of phytoremediation explored for this purpose (Li et al. 2019; Nedjimi 2021). In addition, soil microbes invaded the roots are activated to degrade or detoxify pollutants. Phytoremediation contributes to the removal of organic pollutant, and metals and prevent soil leaching and it is an attractive strategy today.

Heavy metals are released to the environments mainly due to anthropogenic activities. Since heavy metals are non-degradable, they persist in soil for a long period. Therefore it is a long-term threat to ecosystems (Demarco et al. 2019). In general, heavy metals such as Zn, Cu, Mn, Ni are required for the form and function of plants and are named as essential heavy metals. However, Cd, Hg, Pb, and As are highly toxic and are not required for the physiological and biochemical process of plants (Chaffai and Koyama 2011). These non-essential heavy metals contribute to biomagnification and they are a major threat to human and animal health. Removal of heavy metals from contaminated sites is of utmost importance to prevent them from entering the food chains. Root systems of plants form the rhizosphere ecosystem so that they can increase the bioavailability of metal ions by root exudates and stabilize the soil fertility (Mishra et al. 2017). More than 400 plant species that can be utilized for phytoremediation have been identified (Sciences et al. 2008). These plants are slow growing and require strict nutritional and environmental conditions. To ensure the efficient removal of heavy metals, plant traits and environmental factors that limit the metal remediation should be improved. Recently, chemical assisted phytoremediation by applying synthetic cheaters such as ethylene di-ammine tetra acetic acid (EDTA), Ethylene glycol tetra acetic acids, or organic acids such as acetic acid, citric acid into the contaminated sites to form the metal complexes to enhance the bioavailability has shown promising results (Agnello et al. 2013). Thereby the metal uptake of low accumulator plants is increased. Biochar assisted phytoremediation has also shown promising results (Sun et al. 2018). Microbial assisted phytoremediation can improve heavy metal uptake by altering the soil pH and plant exudates composition. Plant growth promoting bacteria increase the plant growth by alleviating heavy metal stress and toxic effects. Use of transgenic plants i.e. enhancing the phytoremediation by improving plant traits such as pollutant uptake, translocation, sequestration, and tolerance by genetic manipulations, have moved into mainstream research with encouraging results (Buhari et al. 2019; Cherian and Oliveira 2005; Khan 2006; Suman et al. 2018). However, a better understanding of molecular mechanisms of degradation of pollutants is crucial.

For phytoremediation of metals, metal tolerance is a prerequisite. This is achieved either by screening clones in greenhouses or selecting plant species that are naturally grown in contaminated sites. Arabidopsis has been identified as a model organism for genetic manipulations to improve plant traits associated with phytoremediation (Koornneef et al. 2010).

ZIP proteins are metal ion transporters. Novel investigations have been focused on using genes encoding ZIP proteins for phytoremediation. It has been reported in a model study, the uptake of Cd and Zn was improved by 150% upon the overexpression of AtIRT1 gene in Arabidopsis thaliana (Connolly et al. 2002). Noccaea cearulescens is a metal hyperaccumulating plant belongs to Brassicaceae family used to recover metal contaminated sites. Introducing the gene, NcZNT1 that encodes Zn transporter of N. cearulescens into A. thaliana improved the accumulation of Zn and Cd (Lin et al. 2016).

Metal tolerance is increased by inserting metal transporter genes such as ABC transporters improving metal sequestration in the vacuole. Overexpression of ABC genes improves the translocation of heavy metals. Nicotiana tabacum (Tobacco) plant was genetically modified by expressing yeast metallothionein gene to improve cadmium tolerance (Huang et al. 2012; Chen et al. 2015).

Overexpression of genes of cation diffusion facilitator (CDF) family proteins facilitates hyperaccumulation of metals. In addition, proteins encoded by the CDF family are metal tolerance/transport proteins that contribute to maintaining the level of ions in the cytoplasm. It has been reported that the gene OsMTP1 of Oryza sativa L. cv. IR64 in tobacco improved the accumulation of Cd and tolerance to As (Das et al. 2016).

Phytochelatins are metal chelators produced by plants and enzymes where phytochelatin synthase and c-glutamyl cysteine synthetase are involved in the synthesis of phytochelatins. In many transgenic plants, metal tolerance has been enhanced by introducing genes that encode these enzymes. For an instance, Cd and Pb accumulation and Cd tolerance were improved in Nicotina glauca and Nicotina tabacum by introducing phytochelatin synthase gene. However, a recent study reported that the uptake of Cd is improved by overexpressing both genes; phytochelatin synthase and c-glutamyl cysteine gene compared to single gene transformants and wild type (Chen et al. 2015).

Metallothioneins are cysteine rich proteins associated with the homeostasis of essential metals such as Zn, Cu, etc. Overexpression of metallothioneins in transgenic plants has improved metal tolerance. For an instance, tolerance to Cu and Cd of transgenic A. thaliana was improved by introducing and overexpressing metallothionine gene IlMt2a of Iris lcatae var. chinensis into A. thaliana (Gu et al. 2014). Overexpression of SbMt2 gene of Solicornia brachiate in tobacco has improved its metal tolerance as well as Zn translocation (Chaturvedi et al. 2014).

Recent studies have demonstrated that overexpression of genes responsible for transcription and DNA repair enhances metal tolerance (Fae et al. 2014; Charfeddine et al. 2016). Underlining mechanisms attributed are upregulation of genes encoding antioxidant enzymes, metallothioneins, metal transporters, and enzymes of DNA repair systems. Accumulation of reactive oxygen species under stress conditions damages the particular plant species. Improvement of phytoremediation properties by optimizing the activity of antioxidant enzymes and reactive oxygen species (ROS) scavengers is a special concern. However, plant responses observed in transgenes and wild type upon heavy metal stresses are debatable. For an instance, overexpression of superoxide dismutase, ascorbate peroxidase, and catalase generates a lower amount of ROS compared to wild type plants when exposed to Cu, Cd, and As. However, Iannone, Groppa and Benavides, 2014 has reported that catalase is not playing a pivotal role in tobacco plants upon Cd toxicity. Reduction of catalase activity by genetic modifications enhances the expression of alternative defence mechanisms mediated by guaiacol peroxidase and ascorbate peroxidase to Cd stress. Genetic factors affecting the rate of glutathione and phytochelatin production have been determined by introducing gshl gene of E. coli to Brassica juncea. The γ-ECS transgenic seedlings with increased production of phytochelatins, glutathione, nonprotein thiols, γ-GluCys were tolerant to Cd. The genes; AtNramps, AtPcrs, CDA in Arabidopsis thaliana and gshI, gshII, PCS cDNA clone in Brassica juncea contribute to metal uptake and translocation (de Farias and Chaves 2011).

Arabidopsis thaliana harbouring MerA and MerB genes have shown increased tolerance to Hg. In addition, increased metal tolerance was achieved by altering oxidative stress responsive enzymes such as glutathione-S-transferase, peroxidase, and 1-aminocyclopropane-1-carboxylic acid deaminase (de Farias and Chaves 2011). Transgenic Alfalfa plant constructed by co-expressing GST gene of Trichoderma vires and cytochrome P450 2E1 gene in humans has shown improved efficiency in metal accumulation, tolerance to heavy metals; Cd, Hg, and xenobiotic; trichloroethylene (Zhang and Liu 2011).

Transplastomic plants are developed through genetic modifications in plastid DNA instead of genomic DNA. This is achieved mainly by homologous recombination. Thus genetic defects such as gene silencing can be eliminated by precisely modifying the specific sites (Bock 2015; Daniell et al. 2016). In addition, the following advantages are attributed to genetic modifications in plastid DNA.

  1. 1.

    Expression of bacterial genes is not difficult because codon optimization is not required in transgenes (Quesada-vargas et al. 2005)

  2. 2.

    Low risk in transient gene transfer into the environment through pollen (Maliga 2002)

Successful mercury phytoremediation was achieved by using transplastomic tobacco plants constructed by introducing bacterial merA and merB genes into chloroplast genomes. The expression of merA and merB genes induces plant tolerance to a high concentration of phenylmercuric acetate (Ruiz et al. 2003; Hussein et al. 2007). Expression of murine metallothionein gene (MT1) enhances the accumulation of Hg in tobacco cells and translocated actively in leaves.

Successful insertion of three genes that encode antioxidant enzymes; dehydroxy ascorbate reductase (DHAR), glutathione S-transferase (GST), and glutathione reductase (GR) into chloroplast genome contributes to the stimulation of antioxidant system of tobacco plant under Cd and Zn treatment (Martret et al. 2011). However, screening of the most suitable gene is critical for transformation into plastid DNA because some genes are more effective when they are transformed into chromosomes than chloroplast DNA (Martret et al. 2011).

Cadmium (Cd) detoxification by transgenic Arabidopsis sp. was achieved by successful transformation of BrMT1 gene which encodes type 1-metallothionien of Brassica rapa into chloroplasts. In addition, the expression of BrMT1 gene significantly reduces the chlorosis and accumulation of H2O2 (Kim et al. 2007).

Gene silencing is the other strategy used for the enhancement of metal remediation. Here, the small RNA inhibits the translation of target mRNA (Saurabh and Vidyarthi 2014). Arsenic reductase converts arsenate into arsenite in roots. Silencing of arsenate reductase gene via RNA interference in Arabidopsis improved the As translocation to shoot compared to wild type (Dhankher et al. 2006). In rice plants, OsNRAMP5 transporter contributes to uptake Cd, Fe, and Mn. Silencing of the gene OsNRAMP5 increases the translocation of Cd to shoot, however, the content of Cd to root to shoot has reduced. Genetically modified OsNRAMP5 RNAi plant was constructed by using natural Cd accumulating cultivar Anjana Dhan (A5i). Even though the biomass of A5i plant with silenced OsNRAMP5 gene remains unchanged, the Cd accumulation in shoot was twice higher than the wild type plant (Sugimoto et al. 2014).

Use of a combination of transgenic plants and rhizobacteria for pollutant remediation has achieved significant advantages in the removal of pollutants. In a recent study it was reported that the transgenic Arabidopsis thaliana plant constructed by substituting the bacterial gene N-demethylase PdmAB into chloroplast has shown improved tolerance to isoproturon (IPU) while efficiently translocating to leaves. IPU is demethylated to 3-(-4-isoprophylphenyl)-1-methylurea (MDIPU) and can be released to outside. Rhizobacteria Sphingobium sp. strain 1017-1 can further mineralize MDIPU in the rhizosphere. Thereby complete removal of IPU is achieved (Yan et al. 2018). The use of combination of transgenic plants and microorganisms is determined as one of the more efficient approaches to remediate contaminated sites with organic pollutants over individual phytoremediation (Dhankher et al. 2011).

To date, a plethora of genetically modified organisms/biocatalysts have been developed and have successfully applied for the bioremediation. We have summarized few newly modified organisms/biocatalysts in Table 7.1 in addition to the above-mentioned examples for perusal of readers interested in genetic engineering.

Table 7.1 Genetically modified organisms/biocatalysts for efficient bioremediation
Full size table

7.5 Impact and Challenges of Using Genetically Modified Organisms for Bioremediation

Even though the efficiency of bioremediation can be improved using genetically modified organisms, a prior risk assessment is essential before applying to the environment. Genetic manipulations of a particular organism can alter the pattern of gene expression, and copies of a particular gene of interest will integrate into the genome. Thus, potential risks associated with genetically engineered organisms cannot be fully ascertained. According to IUCN, the World Conservation Union has identified following environmental risks related to the release of GMOs into the environment (IUCN 2004).

  1. 1.

    Genetic contamination/inbreeding

  2. 2.

    Competition with natural species

  3. 3.

    Increases selection pressure on target and non-target organisms

  4. 4.

    Ecosystem damage and destruction due to novel species

  5. 5.

    Horizontal transfer of recombinant genes to other organisms

  6. 6.

    Adverse effects on human health (enhanced pathogenicity, emergence of new diseases, etc.)

However, unidentified and indirect effects can be observed and those vary according to species, characteristics of particular organisms, environmental factors, interactions among other species, etc. Major challenge associated with the development of genetically modified organisms for the bioremediation is mitigating potential risks associated with particular organisms (Saxena et al. 2020).

7.6 Future Perspectives

Continuous understanding of the genetic and physiological characteristics of microorganisms, plants, and other organisms is essential to improve organisms towards bioremediation. Identification, characterization, and isolation of novel species and biocatalysts are important for the development of effective bioremediation strategies. The process of rapid identification, characterization, isolation, purification, and genetic manipulations is directly linked to the current advancements in proteomics, metabolomics, genomics, transcriptomics, and phenomics. Studying the regulatory mechanisms of these pathways would turn a new leaf in bioengineering of these organisms. The strategies for which would be a subject for another review. However, synthetic biological approaches with proper regulatory mechanisms may find solutions for some of the drawbacks in engineered organisms. For effective bioremediation, the development of environmentally-safe genetically modified organisms for synergetic approaches is highly recommended.