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American Journal of Public Health Research
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The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
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The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
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Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
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The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

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Crude Oil Bioremediation - Genetically Modified Microorganisms for Poly-Aromatic Hydrocarbon Degradation

Sadhana S*, JaiVarshini E, Nikita Reddy S, Shruthi S

Department of Biotechnology, Kumaraguru College of Technology, Coimbatore, Tamil Nadu, India

Abstract: The process of Bioremediation uses the microorganisms/their enzymes to aid the degradation and removal of contaminants present in the environment. The microbial metabolic ability is used for degradation and removal of environmental pollutants providing an economically safer alternative compared to the rest of physicochemical methodologies. One of the hazardous organic priority pollutants are the Polyaromatic hydrocarbons (PAHs). There are a lot of public concerns and critical environmental challenges in the world because of being carcinogenically toxic and mutagenic properties, their ubiquitous distribution, recalcitrance and their environmental presence. The understanding about harmful effects of PAHs on ecosystems and human health has resulted in an interest of researchers on their degradation. Many types of microbes like bacteria, fungi, and algae are capable to use PAHs as carbon and energy source under aerobic and anaerobic conditions leading to their degradation or transformation. Microbial genetic makeup having genes conceal catabolic enzymes results in PAH-degradation mechanism. From the past twenty years, PAH- biodegradation mechanism, catabolic gene system encoding catabolic enzymes, and adaptation through genetics and regulations have been investigated in detail. This review is to attain an overview of the present knowledge of the genetically modified organisms in crude oil spills using PAH degradation mechanism.

Keywords: PAHs, bioremediation, microbial degradation, degradative enzymes, genetically modified microbes

Received July 12, 2021; Revised August 17, 2021; Accepted August 26, 2021

Citation: Sadhana S, JaiVarshini E, Nikita Reddy S, Shruthi S, Crude Oil Bioremediation - Genetically Modified Microorganisms for Poly-Aromatic Hydrocarbon Degradation. Applied Ecology and Environmental Sciences, 9 (8). 769-785. January 2021. doi:10.12691/aees-9-8-8

1. Introduction

Crude oil spillages cause perilous consequences to living and non-living things (soil or water) with the growth of petroleum production. Most of these spillages are due to anthropogenic activities [1,2]. Crude oil petroleum is one of the important pollutants consisting of hydrocarbons of varying molecular weight and about 17,000 organic compounds, classified into four classes -saturates, aromatics, asphaltenes, and resins [3] out of which 30% are PAHs (polyaromatic hydrocarbons) [4]. These compounds proving hazardous to terrestrial and aquatic lifeforms are leading to bioaccumulation. 16 PAHs compounds have been designated by the U.S.EPA as priority pollutants [5]. Most PAHs are carcinogenic, mutagenic in nature and also potent immunosuppressants that affect the immune system growth, humoral immunity, and resistances of the host [4,6]. Approximately 1.3 million liters of crude oil is released into the environment every year [7]. To prevent the adverse effects of crude oil, degradation of this compound is mandatory. There are several techniques to remove or isolate or modify contaminants in the environment. These techniques have expanded to remediate PAHs contaminated areas [8]. PAH remediation can be done by different means. Physically it can be done by solvent extraction, soil washing, air sparging techniques, etc., chemically by chemical oxidation, photocatalytic degradation, etc., thermally by thermal desorption, microwave frequency, etc. But these techniques are expensive and complicated [9].

In recent years’ biological techniques have picked up steam as it is economically efficient and non-invasive. These techniques include bioremediation and phytoremediation. In bioremediation, different microorganisms (bacteria, fungi, and algae) help in degrading PAHs. In phytoremediation, various plants help to degrade/stabilize PAH contamination [8,10].

Microorganisms can degrade complex compounds into simple compounds that are environmentally friendly. Several hydrogen degrading microorganisms are isolated, which comprise 22 genera of bacteria and 31 genera of fungi, being isolated from the soil environment and 27 genera of bacteria and 27 genera of fungi isolated from the marine environment, which can degrade hydrocarbons [11].

Microbial adaptation has a crucial role in microbial degradation to enhance it. Gene modification is done to increase the degradation process efficiently [8]. The application of genetically modified microorganisms in PAHs degradation has gained attention as these organisms are modified to have high degradation capacity and strive through harsh conditions [12]. This review article explains how microbially degrading the PAHs with the help of genetically modified microorganisms can be helpful along with the challenges faced.

2. Crude Oil and Its Impacts on Environment

2.1. Crude Oil

Crude oil, a complex mixture is a non-renewable fossil fuel. Crude oil is one of the most important energy sources in the world. It is used to produce diesel, asphalt, propane, gasoline, aviation fuel, etc. Crude oils can be in the form of light volatile to highly viscous. Most crude oils are dark brown or black. Also, it can exist in yellow, green or, red color [13]. Crude oil is categorized into heavy (that possess resins and asphaltenes in higher amounts than saturated hydrocarbons) and light crude oil (which comprises saturated hydrocarbons and PAHs) [1]. The composition and physicochemical behavior of crude oil can vary from one source to other. Also, crude oil extracted from the same source but during different times can vary in chemical composition. Naphthene is found in crude oils up to 50%. Crude oils rarely contain aromatic compounds of more than 15% [14]. Crude oil is a complex mixture containing aromatic hydrocarbons (PAHs), non-aromatic hydrocarbons, non-hydrocarbons, organic compounds and, trace elements (iron, nickel, copper, and, vanadium) [13,15]. Crude oil leakages can be caused through natural (volcanic eruptions, etc) or, anthropogenic sources (oil refineries, oil transportations, etc) [1]. The leakage of crude oils has vandalized exposed ecosystems nearly all over the world [16].

2.2. Crude Oil Spillages Impact in Marine Environment

Crude oil is toxic to many organisms physically or biochemically. Toxicity of crude oil depends on its composition (Total Petroleum Hydrocarbon (TPH), WAF, Polycyclic Aromatic Hydrocarbon (PAH) content), its amount and its duration in the environment [17], properties of the oil leaked, the sensitivity of the organisms and their habitation exposed and the surrounding conditions of the environment exposed [16]. In crude oil, LMW hydrocarbons are more toxic than HMW hydrocarbons as HMW hydrocarbons have less solubility and less bioavailability [17]. Exposure to crude oil has impacts on phytoplankton (which are important components of the food web in the sea) abundance and composition [15]. Crude oil toxicity primarily targets developing heart in organisms [18] and changes its structure and its function in the embryonic stage [15]. Than adults, marine invertebrate larvae are more sensitive to crude oil exposure. Based on studies, it was known that exposure of many species of marine organisms to crude oil for 48 hours, can reduce their swimming performance in the future. The Organic compounds, which are volatile and known as VOCs like toluene, benzene, xylene and, ethylbenzene known as BTEX, are found in crude oil [19] are toxic. These are PAHs [15]. Many PAHs are toxic, cancer-causing agents and/or mutagens. PAHs are highly lipid-soluble and persistent in the environment [20]. The persistence of PAHs is due to the presence of dense pi electrons clouds in aromatic rings, which provide resistance against nucleophilic attack [10,21]. In fishes, crude oil exposure causes changes in heart structure, heart rate and, rhythm in the embryonic stage. PAHs affect the heart development of fishes in several mechanisms. Some PAHs activate aryl hydrocarbon-receptor(AhR), which detoxifies PAHs. Despite this protective function, activation of AhR by some PAHs and others such as dioxins and PCBs (Polychlorinated Biphenyl) in water-soluble crude oils, which lack PAHs, disrupt usual cardiac morphogenesis in fishes [22]. Some PAHs are phototoxic. Phototoxic PAHs undergo bioaccumulation, which is the first and also rate-limiting step of phototoxicity [23]. These exposed to UV light, produce excited singlet and triplet state molecules [24]. These excited molecules produce reactive oxygen species by transferring outer valence electrons to oxygen or biomolecules close to them [23]. The produced reactive oxygen species cause oxidative stress, tissue damage and, also raised mortality [24]. A leakage of nearly five million crude oil barrels in the Gulf of Mexico underwater in April 2010, has minimized the biodiversity of vertebrates and also metazoan meiofauna species. Also, crude oil leakage in Spain has wiped out almost 66% of the whole species (comprising of crustaceans, insects, mollusks, and polychaetes) [1]. Also it was reported that crude oil exploitation in Niger Delta, has raised deforestation, which resulted in changes in animal habitation, loss in biodiversity, and also vegetation fragmentation [25].

2.3. Crude Oil Spillages Impact in Terrestrial Environment

The impacts of crude oil spills depend on the type of oil spilled, its amount, season and, many other factors. More oil spill cases were recorded on land than in water. In agricultural lands, exposure to petroleum hydrocarbons caused many chronic effects on agricultural products. Lighter crude oil exhibits more toxicity to plants than heavier crude oils. Exposure of crude oil in plants in the presence of light is more toxic than its exposure at night. Because in light, stomata in plants are opened, so crude oil exposure may even lead to the death of the plant [26]. Soil contaminated with crude oil has reduced fertility and increased soil pH [27]. Petroleum hydrocarbons produced by crude oil have both positive and negative effects on plants. Carr [174] observed improved growth in soybeans when 0.75% w/w of crude oil is added to the soil. Adieze et al [175] noticed increased shoot height and weight when 1% w/w of crude oil is added to the soil. Many negative effects in plants were also reported such as reduction in plant growth, reduced germination due to absence of viable seeds, embryo killing, yellowing and death of oiled leaves, etc. [26]. Oil spills that occurred in Nigeria, between the years 1978 and 1979 made negative impacts on farming lands of rice, maize, yams, cassava and, petroleum hydrocarbons have sterilized the soil [28]. Crude oil can be exposed to wildlife, either directly or indirectly. Oil can interact with organisms through inhalation, physical contact, ingestion and, adsorption [29]. When exposed, crude oil can bind to the fur of mammals, quills of marine birds, which can cause the death of the organism when cleaning themselves as a result of ingesting more hydrocarbons [1]. Also, when crude oil binds to the fur or feathers of organisms, they lose their capability to fly, swim and thermoregulate. These effects can result in a decrease in body temperature of organisms, drowning and, deaths [29]. Human health is affected by both the chemical exposure of crude oils and their physiological and economic impacts. Humans can be exposed to crude oil through inhalation, consuming food & water contaminated with crude oil, contact with beach sand and, dermal contact. Effects of PAHs in crude oil on human health depend upon the toxicity of PAHs, the concentration of PAHs and, its exposure route in humans [30]. PAHs are chemically inert and to exhibit their toxicity, mutagenic effect, etc. they need metabolic activation. Exposure to UV radiation is one of those activations. When human skin which is exposed to PAHs, is photo irradiated it can contribute to skin cancer [31]. Based on much research, it was known that people who are exposed to crude oil spills have suffered skin rashes, shortness of breath, cough, fatigue, anxiety, depression, etc. Cheong and co-researchers studied the effects of crude oil on the physical health of people in the Hebei Spirit-laden oil spill, which occurred on December 7, 2007. They analysed urinary metabolites and found VOCs and PAHs in 154 people, who were exposed to oil spills. The result was found that 8 weeks after the disaster, people suffered from nose irritation (83.3%), headache (84.7%), irritation in the dermis of the skin (81.7%), memory disturbance (62.5%), fatigue/fever (83.3%), sore throats (73.6%), abdominal pain (50%), palpitation (56.3%), visual disturbances (61.1%) and many other symptoms [32]. PAHs found in crude oil are carcinogenic, cause mutations, and suppress the immune system. PAHs are genotoxic in rodents’ Chronic effects of PAHs of crude oil include cataracts, kidney and, liver damage, asthma, etc. in humans. Naphthalene, Polycyclic Aromatic Hydrocarbon (PAH) in crude oil causes the breakdown of Red Blood Cells (RBCs) in humans when inhaled or ingested [30]. Naphthalene and anthracene are allergic skin irritants in humans and other animals [30].

3. Microbial Hydrocarbon Degradation

Micro-organisms play a significant role in maintaining biosphere and ecosystem to sustain our environment [33]. Bioremediation is a process that uses micro-organisms and/or the metabolites of micro-organisms, like enzymes to degrade pollutants [34,35] into innocuous substances, requiring biostimulation or bioaugmentation or both [36]. It was reported that the presence of microorganisms with microorganisms of suitable metabolic abilities to degrade pollutants influence the victory of bioremediation [37]. Biostimulation is the process of adding limiting nutrients and also electron acceptors/oxygen to the contaminated environment to stimulate the growth of native oil-degrading micro-organisms [38]. Nitrogen and phosphorus are crucial growth-limiting supplements for the growth of micro-organisms [37]. This enhances the process of bioremediation. Bioaugmentation is the process of introducing selective micro-organisms with specific enzymes [38], to supplement the indigenous micro-organisms in a contaminated environment for more effective degradation of contaminants [39,40,41]. Bioaugmentation is a preferable option when the crude oil polluted soil has a low population of native hydrocarbon-degrading microorganisms, and when the pollutants are toxic to the indigenous micro-organisms. Also, it was reported that although bioaugmentation can degrade total petroleum hydrocarbons (TPH) of petroleum more effectively than biostimulation, biostimulation showed greater efficiency in remediation of petroleum polluted soil than bioaugmentation. This is because the dominant inoculants in bioaugmentation have suppressed the native micro-organisms during remediation, this showed less diversity of micro-organism species than biostimulation. Biodiversity is influenced by the richness, also evenness of the species [37]. Also, it was reported that native micro-organisms can adapt easily, and respond to environmental conditions [42]. Contaminants of an environment can be degraded naturally by their native micro-organisms by the process known as natural attenuation or bio attenuation. When this process is not enough to degrade the pollutants, biostimulation or/and bioaugmentation is used [40].

Bioremediation is very much used in reducing the risks of hydrocarbon contaminants [43]. The susceptibility of hydrocarbons to microbial attack differs in the order: n-Alkanes > Branched alkanes > low-molecular-weight aromatic compounds > cyclic alkanes > polyaromatic hydrocarbons [44]. Immobilization is a technique which is extensively used to keep the microbes or enzymes of them immobilized in a required space in marine oil spills by using the physical/chemical method [33]. Immobilization makes the bioremediation process more effective against environmental barriers [38]. As hydrocarbons are insoluble and hydrophobic, they restrict to be absorbed by the microbial cells and are also toxic to cells. Hydrocarbons also disturb the microbial cell membrane fluidity, so exposed micro-organisms want to change their composition of the cell membrane, to stabilize the phospholipid bilayer structure according to the exposed hydrocarbon type [45].

Micro-organisms capable of degrading hydrocarbons are found all-round in our natural environments [46]. Hydrocarbon contaminants found in an environment is primarily degraded by bacteria, fungi [43] and yeast [41]. Protozoa and Algae participation in biodegradation reports were scanty [40]. It was reported that a consortium of micro-organisms was more effective than individuals [47,40]. Prior exposure of hydrocarbon-degrading bacteria to hydrocarbons can help the microbial community to adapt and utilize hydrocarbons as carbon and energy sources [43]. This is the rate-determining step of biodegradation [48]. The three methods by which microbial adaptation can occur are (1) Specific enzyme induction and/or depression, (2) Genetic modifications that can affect metabolic pathways, (3) Selective enrichment of hydrocarbon-degrading microbes. Bacteria perform the above methods more than other microbes in soil [43]. In the degradation of hydrocarbons, bacteria are the most active degraders [41], and they play various roles than fungi [43]. Some marine bacteria (that consume PAHs as carbon and energy source) like Alcanivoras, Neptunomonas, Pseudomonas, Pseudoaltermonas, Altermonas, etc play a censorious role in metabolizing and completely oxidizing PAHs and many other petroleum hydrocarbons. This prevents PAHs from pile forming. So, these bacteria are important for the remediation of crude oil spills in the marine environment [49]. The extent to which crude oil biodegradation occurs directly influences the crude oil quality. Bacteria make use of petroleum compounds as a source of carbon and energy. So during this process, a progressive reduction of light hydrocarbons takes place, then degradation of saturated hydrocarbons and aromatics occur. This biodegradation of crude oil by bacteria can increase oil viscosity because it leaves the oil enriched with heavy petroleum compounds. Sometimes, this may make mining less efficient. Also in extreme cases, it is unreasonable to extract the crude oil degraded by microbes. As the activity of microbes is limited by high temperatures, crude oils rich in LMW compounds are present in higher depths underground (>3.5 Km), where the temperature is higher than 80 degrees Celcius [50]. Some hydrocarbon degradation abilities in bacteria are having degrading plasmids and more mobile genetic components, surfactant production and having certain catabolic enzymes (Oxygenases, Hydroxylases). Hyphal structures and extended surface area in fungi helps them in exposure to hydrocarbons and better penetration. Extracellular enzymes of fungi like oxidases also improve their hydrocarbon-degrading activity in soil [34]. It was reported that in ligninolytic fungi, enzymes in lignin systems like lignin peroxidases, H202 producing enzymes and phenol oxidases (laccases & tyrosinases) can degrade PAHs [47]. Factors that influence microbial hydrocarbon degradation is temperature, nutrients (e.g., nitrogen, phosphorus, iron) [41], pH, type and concentration of the contaminant, type of the soil [48], and electron acceptors in aerobic biodegradation [33].

3.1. Enzymes Involved in Microbial Degradation

Enzymes are biological catalysts that help in the conversion of substrates into products by providing conditions favourable for lowering the activation energy of the reaction. An enzyme can be either a protein or a glycoprotein [51]. In both cases, it consists of at least one polypeptide moiety. Enzymes act as catalysts for many biochemical reactions happening in microbial degradation. The feasibility of bioremediation decreases when the process of degradation occurs only with the help of microorganisms since it is a slow process. In the last few years, microbial enzymes separated from their cells have been used for bioremediation as compared to using whole microorganisms [52]. Sometimes, pollutants can be toxic to microbes consuming them. This gives rise to the usage of metabolic enzymes of microbes in bioremediation instead of a whole microbial cell, which was termed as “enzymatic remediation” [53]. Enzyme bioremediation is independent of the microbial survival in the environment and depends on the efficiency of the enzyme. This process is safe as it does not form any toxic compounds during remediation, and this method is even applicable to soils without any adequate nutrients. Also, it is possible to bring more stable and active enzymes for remediation at lower costs and higher scales through recombinant DNA technology. This makes the usage of enzymes more favorable and advantageous [53]. But, the enzyme activity can be very easily affected by external environmental factors. To overcome this issue, methods like immobilization are used [54]. The enzymes involved in bioremediation are of two types i.e., Oxidoreductase and Hydrolases respectively. These enzymes have the potential to convert aromatic hydrocarbons, aromatic amines, phenols, and many other pollutants to simpler less reactive forms [35]. Hydrolase is a class of enzymes that commonly perform as biochemical catalysts that use water to break a chemical bond, which typically results in dividing a larger molecule into smaller molecules [55]. For the conversion of pollutants to harmless inert substances, a sequence of reactions with many enzymes is necessary, which demands many types of micro-organisms for a good result. Micro-organisms require optimum environmental conditions for growth. So, the usage of enzyme extracts of micro-organisms would be a good alternative method for the bioremediation of complex compounds [35].

3.1.1. Oxidoreductase

An oxidoreductase (EC class 1) is an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. Oxidoreductases catalyze the detoxification of synthetic organic substances such as azo rings, aniline substances, and phenolics [56]. Oxidoreductases are produced by fungi, bacteria, also higher plants. So, these organisms can detoxify natural and artificial pollutants, also can reverse xenobiotic toxicity. Two types of oxidoreductases are oxygenases (monooxygenases, dioxygenases, and peroxidases) and oxidases (laccases) [35].

Oxygenases

Oxygenases (EC 1.13, 1.14) catalyzes the transfer of oxygen from molecular oxygen(O2) to inorganic or organic substrates using coenzymes line FAD, NADH, and NADPH. Oxygenases are classified based on the usage of the number of oxygen atoms. There are two types of oxygenases, monooxygenases (EC 1.14.14) (they transfer only one oxygen atom and electrons from coenzyme NADPH or NADH), dioxygenases (1.13.11) (they transfer two oxygen atoms), and peroxidases (EC 1.11.1.7) (they catalyze peroxide reduction processes, through oxidation of electron donor) [35,57]. Oxygenases break the aromatic rings in compounds by the addition of one/two molecules of oxygen [35]. Oxygenases catalyze the process of oxidation of aromatic compounds such as chlorinated biphenyls, aliphatic olefins making them prone to further transformation and mineralization [52]. Oxygenases degrade the organic compounds by rising the reactivity or water solubility of compounds, or by breakage of aromatic rings. Generally, the addition of oxygen atoms to compounds breaks their aromatic rings. Monooxygenases catalyze aliphatic and aromatic compounds by undergoing desulfurization, denitrification, hydroxylation, biodegradation, dehalogenation, ammonification, and biotransformation. The most distinguishing monooxygenase enzyme is methane monooxygenase, which is capable of degrading alkanes, alkenes, methanes, haloalkenes, cycloalkanes, ethers, aromatics, and heterocyclic hydrocarbons. P450 monooxygenase enzyme produced by bacterium Bacillus metaterium can catalyze the degradation of aromatic compounds [57]. Dioxygenases catalyze the transformation of aromatics to aliphatic compounds. And, they are used mainly for the bioremediation of pharmaceutical wastes, chemical wastes, and colorants [35]. They are categorized into aromatic ring hydroxylation dioxygenases (ARHDs) and aromatic ring cleavage dioxygenases (ARCDs) was based on the method they break the bonds on aromatic compounds. Toluene (VOC) degradation can be catalyzed by Toluene Dioxygenase (TOD) produced in Pseudomonas putida [57]. Peroxidases such as hydrogen peroxide (H2O2) catalyze the reduction reactions and generate reactive free radicals after the oxidation of organic compounds [52]. Peroxidases (oxidizing agents) [56] that contain hydrogen peroxide oxidoreductase donors are widespread in the environment [57]. They are divided into heme and non-heme peroxidases. Heme peroxidases are divided into two groups, the first group comprises peroxidases in animals and the second group comprises heme peroxidases in plants, prokaryotes, and fungi. Non-heme peroxidases are divided into three classes. In mammals, peroxidases participate in hormone regulation, immune systems, and other biological processes. In plants, they are involved in cell elongation, auxin metabolism, etc [57]. Peroxidases are used in bioremediation, because they are thermostable, and they can oxidize a broad range of substrates. Peroxidases are produced by plants, animals, fungi, and bacteria [35].

Oxidases (Laccases)

Laccases are polyphenol oxidases, that contain four ions of copper [37]. Laccases catalyze the cleaving of rings present in aromatic compounds by reducing one molecule of oxygen in the water and producing free radicals [52]. They are widespread and found scattered in bacteria, higher plants, fungi, and insects. They are the oldest and majorly studied enzyme systems. They are polymeric. Laccases oxidize methoxy-phenolic acids and phenolics and also decarboxylates their methoxy groups [57]. Intracellular and extracellular laccases produced by many micro-organisms catalyze the oxidation of polyphenols, polyamines, ortho-diphenols, para-diphenols, aminophenols, aryl diamines, and lignins, also some of the inorganic ions [56,57]. Among other biological enzymes, laccases show promising roles in bioremediation and biotechnical applications. Many reagents like halides (except iodine) can inhibit laccases. Different laccases have different tolerance capacities on inhibitors. Recombinant laccases could be made homologously or heterologously. Immobilization of laccases improves their stability, enzyme half-life, and resistivity to proteases. Laccase produced by Trametes Versicolor fungus immobilized on glass beads containing pores, showed good bioremediation of many pollutants, like phenolic compounds, heterocyclic aromatic compounds, and amine-containing aromatics [57].

3.1.2. Hydrolases

Hydrolases are biochemical catalysts that use water to break chemical bonds in toxic compounds, dividing larger molecules into smaller molecules, thus reducing toxicity [52,57]. There are mainly five types of Hydrolases involved in bioremediation. These are lipases, cellulases, carboxylesterases, phosphotriesterases, and haloalkane dehalogenases. Lipases help in breaking triglycerol into glycerol and fatty acid and are used for wastewater treatment, polyaromatic hydrocarbon degradation (PAHs), etc. [52]. Lipases deteriorate hydrocarbon concentration in polluted soils. It was reported that lipase can be produced from P.aeruginosa, a fungal species, and can be utilized for crude oil bioremediation [53]. Esterases are capable of degrading alkanes, also aromatic rings in fungal and bacterial isolates. It was reported that lipases and esterases can be used as a biological indicator to observe total petroleum hydrocarbon degradation. In the presence of hydrocarbons, lipases and esterases are induced [58]. Cellulases perform the breakdown of complex cellulosic materials into simple sugars. They are commonly used in the treatment of agricultural residues like cotton waste, sawdust of Khaya ivorensis, and rice straw. Carboxylesterases help in the catalyzes of the hydrolysis of carboxyl ester bonds present in synthetic pesticides such as organophosphates with the addition of water. Phosphotriesterase catalyzes the hydrolysis of phosphotriester, the main components of organophosphorus compounds used worldwide in pesticides, causing severe poisoning and death. Haloalkane dehalogenases are used for biodegradation of halogenated aliphatic compounds such as 1,2,3-Trichloropropane [52]. Cellulase, hemicellulase, and glycosidase actively degrade biomass. Advantages of utilizing these hydrolases are their non-selectivity, good tolerability, its availability, and cofactor stereo-selectivity lack [56]. It is an effective biodegradation technique in case of oil spillages [57]. It was reported that entrapment of organophosphorus hydrolase in the bacterial membrane has improved the catalytic rate of enzymes for parathion degradation [53].

Bioremediation elicited from purified and partially purified enzymes do not depend on the growth of a particular microorganism in a polluted environment but it depends upon the catalytic activity of the enzyme secreted by microbes. In soil that is nutrient deficient, bioremediation can be done by using a purified enzyme. Microbial biotransformation produces toxic side products which are not produced by environment-friendly enzymatic biotransformation. Enzymes are more specific to their substrate than microbes. Their smaller size makes them mobile in nature.

3.2. Mechanism

Breakdown of hydrocarbon pollutants microbially is a very slow process at times, but optimum biodegradation could be achieved if environmental conditions such as PH, temperature, nutrients, and relevant microbial consortia are made possible [59]. We are going to discuss the degradation process which can be taken up by either bacteria or yeast. Hydrocarbon-degrading bacteria are widespread in marine, freshwater, soil habitats and they are exploited for their ability to degrade/detoxify organic contaminants, hence being recognized as an efficient, economical, versatile, and environmentally sound treatment [60]. Bacteria can exploit carbon compounds in oil considering it as a necessary source of energy [61]. Taking the metabolic diversity demonstrated for yeasts into account, yeasts contribute to the environmental hydrocarbon biodegradation more than previously expected [62]. The isolate of yeast also shows a high potency in petroleum oil biodegradation of some hydrocarbons [63]. The biodegradation of petroleum compounds happens through three processes. First, the absorption of petroleum takes place on microbial surfaces. Second, these compounds are transferred to microbial cell membranes degrading into the microbial cell after that. Third, microorganisms degrade these compounds into various small molecules [64]. Petroleum components with different structures have different pathways of degradation such as saturated, aromatic, resin, and asphaltene fractions hydrocarbon. Depending on the chain length, enzyme systems need to introduce oxygen in the substrate to start the process of biodegradation. Higher eukaryotes generally contain several different P450 families that consist of a large number of individual P450 forms that may contribute as an ensemble of isoforms to the metabolic conversion of the given substrate. In microorganisms, such P450 multiplicity can only be found in a few species [65]. Results of studies also suggest that Rhodococcus sp., Trichoderma tomentosum, and Fusarium oxysporum may effectively enhance the biodegradation of PAH compounds in the category of bacterial degradation [66].

3.2.1. Degrading Process of Polycyclic Aromatic Hydrocarbons (PAHs)

Polycyclic aromatic hydrocarbons (PAHs) are mainly high carcinogenic, mutagenic, and teratogenic substances, which have drawn much attention to their degradation mechanism [67]. PAHs have high boiling points and are lipophilic, so easily spread and accumulate in aquatic ecosystems [68]. PAHs contamination in our environment increases with the increase in the production and transport of petroleum oils. PAHs exist in many structural varieties than other non-halogenated compounds in the biosphere. The fate of these PAHs in the ecosystem includes adsorption on the soil particles, volatilization, photo-oxidation, oxidation by chemicals, and leaching. PAHs are highly stable [54] and difficult to degrade naturally. It was reported that bioremediation proves as a good solution for PAH degradation [69]. PAHs are of intense public concern owing to their presence in the environment and deleterious effects on human health [65]. Laccases perform well in PAH degradation. Also, it was reported that the reverse micelle system, that uses laccase, when operated under optimum conditions for 24 hours has been shown to degrade PAHs (51.2% benzo(a)anthracene degradation, 50.3% phenanthrene degradation, 68.2% anthracene, 68.9% benzo(alpha) fluoranthrene) [54]. It was reported that the pyruvate dehydrogenase enzyme complex plays a crucial role in PAH compounds degradation. Microalgae can be used as a biosorbent for PAH sorption and degradation can be done by the usage of enzymes. Genetic engineering is a promising tool for enhancing the adsorption process and remediation of organic compounds. Also, can improve microalgae tolerance in these organic compounds [68]. It was reported that Rhodococcus wratislaviensis strain 9, can degrade both LMW, and HMW PAHs efficiently as it has genes that code for the enzymes 2,3-dihydroxy biphenyl 1,2-dioxygenase (bphC), oxygenase component (nphA1), naphthalene dioxygenase, 4-nitrophenol 2-monooxygenase component B (npcB), 4-hydroxybenzoate 3-monooxygenase (phbH), and extradiol dioxygenase (edo) [70]. Generally, during degradation, the PAHs undergo gradual degradation into epoxide, trans diol, phenol, and trans dihydro 2 phenol by yeast monooxygenase enzyme. Another method for PAH degradation is that PAHs undergo gradual degradation by dioxygenase enzyme into epoxide, cis diol, etc. In both PAH degradation pathways, the final products are water and carbon dioxide. The overall steps of biodegradation of PAHs are demonstrated in Figure 1 [64]. PAHs are different to be degraded, and the degraded level of PAHs should be graded in order of solubility, the number of benzene rings, the species, and the number of substituent species, the properties of heterocyclic atoms, etc. Also, asphalt has the most complicated structure, which is very much difficult to be decomposed by biodegradation. These results have common implications for bioremediation that’s, nature harbors diverse microbial populations capable of pollutant degradation from which a couple of pollutant-degrading populations are selected consistent with bioremediation strategies.

Figure 1. PAH Degradation by Yeast and Bacteria (Modified from ):

Table 1. Microbes Involved in Aerobic Biodegradation of Hydrocarbons

Table 2. Microbes Involved in Anaerobic Biodegradation of Hydrocarbons

3.3. Some Examples of Microorganisms Involved in Biodegradation of Hydrocarbons

3.3.1. Microbes Involved in Aerobic Biodegradation of Hydrocarbons

Some examples of microbes involved in aerobic biodegradation of hydrocarbon are given in Table 1.

3.3.2. Microbes Involved in Anaerobic Biodegradation of Hydrocarbons

Some examples of microbes involved in anaerobic biodegradation of hydrocarbons are given in Table 2.

4. Genetically Modified Microbes for PAH Degradation

PAH degradation studies have gained importance in bioremediation but occasionally the process becomes very slow due to some abiotic and biotic factors [71]. Microbes that help in PAH degradation utilizes PAHs as the carbon source for their catabolic regulation [72]. In laboratory conditions, microbes show ideal activity for PAH degradation but it shows the lesser capability to degrade PAH in field conditions.

These microbe’s enzymatic activities can be modified by altering their genetic composition in order to enhance their potential to degrade PAHs in field conditions. Using genetic engineering the microbes can be modified so that they can have high salt tolerance, withstand toxic chemicals and produce biosurfactants to degrade different kinds of PAHs [12].

4.1. Genes Responsible for PAH Degradation

Anaerobic and aerobic PAH degradation is studied and a few catabolic genes which are responsible for PAH degradation was identified [78]. Gram-positive bacteria like Rhodococcus, Mycobacterium, and Nocardioides help in the degradation of PAHs have catabolic genes like nar, phd, nid and pdo. Gram-negative bacteria like Pseudomonas, Ralstonia, Burkholderia, Sphingomonas, and Polaromonas have catabolic genes like nag, nah, ndo, phn and pah to degrade PAHs. Some plants species also possess genes that are responsible in PAH degradation. Species and genes responsible for PAH degradation are mentioned in Table 3 [71].

4.2. Genetic Engineering Approaches for PAH Bioremediation

There are different approaches to modify microorganisms genetically to increase the efficiency of PAH bioremediation. General genetic engineering approaches used in bioremediation are directed evolution, saturation mutagenesis, metabolic engineering, rational designing [79].

4.2.1. Directed Evolution

Directed evolution is a laboratory approach involving the evolution of biological macromolecules by Darwinian principles of selection and mutation [126]. It is a strong approach to explore, alter the enzyme-substrate specificity and improve bio-catalysis. This strategy enables to alteration of the enzyme with desired characteristics by site-directed mutagenesis [79]. Directed evolution does not require more detailed analyses about the protein structure and in vitro, it imitates natural methods like sexual recombination and random mutagenesis which accelerated in order to bring desirable changes [127]. A directed evolution approach is can be used to engineer improved or new enzymes or remodel metabolic pathways or engineer the whole genome for any biological applications [128]. Error-prone PCR is used to reconstruct the DNA fragments of different lengths around 10-300 bps to construct the desired gene where the random mutants can be inserted in any DNA fragments. In error-prone PCR the polymerase is not extremely specific it is modulated by the alteration of the composition, So the polymerase can make mistakes in the base pairing and synthesis of a new complementary DNA strand. By controlling the buffer concentration, the rate of error introduced can be regulated [129]. Homologous recombination is done to assemble the DNA fragments. The polymerase extension is done using dNTPs and the full length of the mutated gene is obtained using a nested PCR methodology [127]. The enzymes or metabolic pathways responsible for PAH degradation can be altered using this approach so that the efficiency of the degradation process can be enhanced. For example, Laccases enzymes are multicopper enzymes that help in catalyzing the four-electron reduction by four electron pathways to reduce oxygen to water and laccase also catalyzes one-electron oxidation of reducing substratum. These are extensively found in white-rot fungi [130]. This fungal laccase enzyme can be helpful in the degradation of PAH. For effective bioremediation of PAHs organic co-solvents are required in high concentrations. But this organic co-solvent can decrease the stability and activity of the laccases enzyme. Therefore, laccase enzyme from Myceliophthora thermophila (MtLT2) well expressed in Saccharomyces cerevisiae is utilized as the parent type to perform directed evolution using error-prone PCR to improve activity in organic co-solvent high concentration and to maintain the stability of the enzymes in those conditions [131,132].

Table 3. Genes responsible for PAH degradation

4.2.2. Rational Designing

By comparing homologous genetic sources, the key sites of the gene are identified and those specific sites are mutated by site-directed mutagenesis, in which the amino acid at a specific region is mutated replacing another amino acid, this approach is known as rational designing [79]. To perform a rational designing molecular approach prior information of the 3d structure, dynamics, mechanics of the protein to be modified, is required. This is because every protein has fine folding kinetics any mutation in the undesired sites can lead to disruption of its confirmation resulting in reduced catalytic activity [133]. The main advantage of this strategy is it can be successful even in the absence of a high throughput assay system [134]. Many techniques like functional bioinformatics techniques, x-ray crystallography, etc. are used to identify the structural properties of the biomolecule which is modified by rational designing. X-ray crystallography or functional bioinformatics techniques provides the structural information about the biomolecule and this information is mathematically and graphically displayed in the computer for further analysis a model depicting the biomolecule is created in the computer and this allows to predict the effect of the mutation at a specific site [135].

The main element of rational designing is that the rDNA technique should be used in order to make native protein variants. The protein responsible for PAH degradation can be altered in this approach so as to perform the desired function in the degradation of PAH. For example, the oxidation activity efficiency of the p450 monooxygenase enzyme is improved for PAH bioremediation purposes. Six sets of fungal cytochrome p450 monooxygenase (pc-pah1--pc-pah6) were identified in Phanerochaete chrysosporium. Out of which pc-pah4 (CYP5136A3) has the ability to oxidizes PAH compounds like pyrene and phenanthrene [136]. Mammalian and microbial CYP enzymes show less stability, reduced expression of the enzyme, and low activity in field systems due to unfavourable conditions. These major limitations are the cause to engineer this enzyme to improve its oxidation efficiency [137]. The main residues involved in substrate recognition trp129 and leu324 were identified and located at SRS1 and SRS4 respectively [138]. Rational engineering is done by alternative mutation of the specific site (W129L AND L324G) to increase the catalytic activity [139].

Figure 2. Schematic for Directed Evolution (modified from [79])

4.2.3. Saturation Mutagenesis

Saturation mutagenesis involves iterative mutations of the gene at specific sites which are chosen rationally to bring desired characteristics to the gene [79]. The target sites undergo an iterative mutation cycle till the gene property of the particular gene is enhanced. The gene mutated in the first cycle is used as the template for the next mutation cycle in the respective other sites and the process is repeated until the desired feature of the gene is obtained [140]. Saturated mutagenesis has numerous applications like enhancing enzymatic activity, stereoselectivity, and stability, manipulating transcriptional factors, and binding properties of the antibodies [141]. Using saturation mutagenesis, it is possible to generate a library of mutants which comprises all possible mutation at predestined target sites [142].

Most of the homologous enzymes have different catalytic activity but have common structural properties. So change in few residues result in a change in its function. Performing saturation mutagenesis on those target sites helps to enhance the geometry of the binding sites in the improved or new enzyme [143]. This approach comprises both rational designing and directed evolution (random mutation) approach to obtain the desired gene characteristics. Like rational designing, the key sites are identified using the spatial arrangement of the protein which is determined by x-ray crystallography, NMR techniques. The iterative mutation is done likewise in directed evolution to obtain a high-quality library of mutants that is easier to screen [144]. This saturation mutagenesis approach can be used to enhance the degradation capacity of the enzymes for PAH remediation. For example, previously we understood the cytochrome p450 monooxygenase enzymatic activity towards PAHs oxidation, and the enzymatic properties were enhanced by rational designing [139] likewise the same enzyme can be engineered by using the saturation mutagenesis approach. The iterative mutation is done at the key sites until the enzymatic activity towards PAH is enhanced. Using this approach library of mutants can be constructed and the variant active toward PAH degradation, enhanced enzymatic activity, and has more stability can be screened using the library [145].

Figure 3. Schematic for Rational Designing modified from [79]

Figure 4. Schematic for Saturation Mutagenesis [79]

4.2.4. Metabolic Engineering

Metabolic engineering involves enhancing the production of a particular cellular compound in an organism by genetic and regulatory alteration [146]. The metabolic pathway of a microorganism is modified to change the cellular property of the cell such that it can produce desired cellular compounds. This approach offers a technological framework to increase the creation of newly synthesized metabolic enzymes and pathways or altering the existing metabolic pathway in order to increase cellular activity or cellular compound production [147]. Metabolic engineering can be employed for enhancing the production of chemicals which are naturally inherent. This approach is also utilized to produce artificial-non inherent, natural but non-inherent, and artificially created chemicals in an organism. The efficiency of natural- inherent chemical production can be enhanced by directly altering the host strain’s existing pathway to overproduce the required chemicals or biomolecules. Whereas to produce artificial non-inherent, natural but non-inherent, and artificially created chemicals which are not produced by the host’s existing pathway is initiated with engineering a suitable synthetic metabolic pathway designed heterologous or combinational expression of new or known genes [148]. After the designed pathway is constructed the host strain is further engineered to increase the efficiency of the production of these compounds [149]. The type of host organism, the desired product and the type of metabolic engineering approach to be used should be analysed and determined to obtain successful metabolic engineering [150]. This approach can be used for PAH bioremediation purposes. In order to enhance the degradation process, the metabolic activity of the organism can be enhanced by combining the metabolic pathway of the different organisms in one organism [79]. For example, Arabidopsis is an autotrophic plant variety it can thrive in PAH contaminated soil conditions. But it lacks genes responsible for PAH degradation. So engineering bacterial pathways into this plant by metabolic engineering approach can be helpful for efficient degradation of PAH present in the contaminated soil. For this purpose, Mycobacterium vanbaalenii PYR-1 naphthalene dehydrogenase system is incorporated into Arabidopsis plants for the initial transformation of three aromatic rings PAHs. M.vanbaalenii PYR-1 genes encoding for electron transport chain was undetermined hence suitable electron transport chain from Pseudomonas putida G7 naphthalene dehydrogenase system was incorporated into nib genes of the mycobacterium. This co-transformed mycobacterium is introduced into the plants and the desired gene for detoxifying PAH is produced [151].

4.3. Some Examples of Genetically Engineered Microbes for PAH Bioremediation

PAHs present in the environment can be degraded efficiently by modifying the genetic makeover of the microbes. For this purpose, many microbes are genetically modified to check their degradation efficiency. Modified microbial species and their respective genetic modification method, desired outcome from the modified microbial species are explained in Table 4.

Table 4. Examples of GEMs used in PAH bioremediation

5. Regulatory Challenges for Gem:

When it comes to the development of Genetically Engineered microorganisms that could be deployed for the purpose of bioremediation and other different environmental applications, there arise ethical arguments relating to possible outcomes of risk when they are used in the field. The slow progress of Genetically engineered microbes over the past decade and strict regulations involved in their deployment is referenced to legislative constraints and safety concerns sensed by the public in general [79,152]. The regulatory agencies that oversee the regulations of Genetically modified organisms are the United Nations Environmental Programme (UNEP) and the Organisation for Economic Co-operation and Development (OECD). (OECD 1992, 1994) [153]. Genetically modified organisms are most probable of creating adverse effects on the environment as well as human health. The effects caused depends on the characteristics of the GMO and the nature of the environment. With reference to [154], it has been prescribed by the regulatory agents that the genetic makeup of the mutated microorganisms need to be compared to their wild-type counterparts with all the necessary field conditions before being deployed [79]. The main obstacle in deterring the use of genetically modified organisms is containment [167]. The rate of survival and horizontal gene transfer ability of genetically modified organisms is also a serious issue to consider. More often, genetically engineered organisms don’t exploit the target substrate as expected even if it is a well-defined biochemical pathway. It leads to unintended side reactions resulting in the formation of toxins which makes determining the possible impacts on the diversity of the ecosystem challenging [168]. On the whole, designing a microorganism that shows satisfactory affinity to ecological diversity is one of the prime strategies [169].

6. Future Aspects

The progress in protein engineering, molecular biology and genetic engineering has led to novel ways of redefining the containment system of genetically engineered microbes [170]. The extended presence of genetically engineered organisms in the ecosystem after the desired purpose is attributed to the physical and chemical stability of selective markers, recombinant genes and plasmids [171]. To avoid this possibility, we could refrain from using selective markers without antibiotic resistance and certain transposons and plasmids [168,172].

The unintended damage to the ecological diversity can be nipped by using containment strategies such as suicide mechanisms, which is based on killer-anti-killer gene mechanism where the microbes are activated only in the presence of the target substrate and the cells are prone to autolysis upon successful degradation of the target substrate [169]. Development of these suicidal engineered genetically modified microorganisms will be a very relevant technology for the future as it tackles both the regulatory challenges associated with genetically modified organisms as well as preventing ecological damage.

7. Conclusion

Crude oil spillages are a threat to our environment. Among them, PAHs causes dangerous health hazards to many organisms in our environment. So, degradation of these contaminants can cause a considerable effect in lowering the toxicity of crude oil. As earlier, micro-organisms play a notable role in bioremediation. Genetically modifying the micro-organisms allows us to modify the micro-organisms, with required biodegrading capabilities, which are more effective than natural micro-organisms in degrading toxic wastes. Many reports have shown the potential of genetically engineered micro-organisms (GEMs) to degrade pollutants in soil, groundwater and sludge. The survival of GEMs in the contaminated environment is non-problematic even in harsh environmental conditions. Also, they persist even after the degradation of pollutants. This can be a disadvantage. In future, we may be able to see more safe GEMs to remove toxic pollutants from our environment.

Acknowledgements

This review paper on the:

CRUDE OIL BIOREMEDIATION - GENETICALLY MODIFIED MICROORGANISMS FOR POLY-AROMATIC HYDROCARBON DEGRADATION is immensely supported by Ré - The research and exploratory cell of Kumaraguru College of Technology. We sincerely thank our mentors from Ré for their extraordinary support and guidance throughout the course of the completion of the review paper.

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