Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 3406-3425

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 10 (2020) Journal homepage: http://www.ijcmas.com

Review Article https://doi.org/10.20546/ijcmas.2020.910.394

Biosensors and Bioremediation as Biotechnological Tools for Environmental Monitoring and Protection

Haleema Bano1, Shayesta Islam1, Faisal Noor2 and M. Ashraf Bhat3*

1Division of Environmental Sciences, 2Division of Vegetable Science, 3Division of Genetics and Plant Breeding, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar- 190 025, Srinagar, Jammu and Kashmir, India

*Corresponding author

ABSTRACT

Environmental biotechnology is the integration of natural sciences and engineering in order to achieve the application of organisms, cells, parts thereof and molecular analogues for the protection and restoration of the quality of our environment. Biotechnological K e yw or ds processes to protect the environment have been used for almost a century now, even longer than the term “biotechnology” exists. Biotechnological techniques to treat waste before or Biosensor , after it has been brought into the environment are components of environmental Phytoremediation , biotechnological tools. Biotechnology can also be applied industrially for use in Phytostabilization , developing products and processes that generate less waste and use less nonrenewable

Enzymes, Optical resources and consume less energy. A biosensor is an analytical device that integrates a Biosensors , biological sensing element (e.g., an enzyme or an antibody) with a physical (e.g., optical, Microorganisms , Antibodies mass, or electrochemical) transducer, whereby the interaction between the target and the bio-recognition molecules is translated into a measurable electrical signal. Potent alternatives to conventional analytical techniques are Optical biosensors that exploit light Article Info absorption, fluorescence, luminescence, reflectance, Raman scattering and refractive

Accepted: index. Devoid of any time-consuming sample concentration and or prior sample pre-

26 September 2020 treatment steps these biosensors provide rapid, highly sensitive, real-time, and high- Available Online: frequency monitoring. Although optical biosensors have great potential applications in the 10 October 2020 areas of environmental monitoring, food safety, drug development, biomedical research, and diagnosis. Their use in fields of environmental pollution control and early warning is still in the early stages. Biosensors are classified according to their transduction principle such as optical , electrochemical and piezoelectric or based on their recognition element as immunosensors, apt sensors, genosensors, and enzymatic biosensors, when antibodies, aptamers, nucleic acids, and enzymes are, respectively, used.

Introduction it more practical and suitable for our product genetically modified strains as well as desired Biotechnology is the use of living organisms strains to get desired products can be chosen. such as bacteria or plants or other unicellular The entire biological system has been being or simple multi-cellular organisms. To make affected due to human activities which have

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polluted the environment with harmful and Environmental biotechnology poisonous contaminants from past few decades. As a result ecological balance is Environmental biotechnology is the severely disturbed (Khalilian et al., 2015). integration of natural sciences and Conventional goods are manufacture by engineering in order to achieve the manipulating the hereditary bases of living application of organisms, cells, parts thereof organisms using biotechnological tools. (Chen and molecular analogues for the protection et al., 2005) clearly elaborated that and restoration of the quality of our environmental attractiveness is progressed environment. Biotechnological processes to through the use of micro-organisms (Chen et protect the environment have been used for al., 2005). With the advent of advance almost a century now, even longer than the technologies from molecular and microbial term “biotechnology” exists. About the turn biology has enabled researchers to solve of the century for purification of town gas environmental problems e.g. Municipal sewage treatment plants and filters degradation/detoxification of waste by the use were developed. Even though, little was well- of living organisms (Bharagava et al., 2020). known about the biological principles Bioremediation is branch of biotechnology underlying their function at that time, still which can remove pollutants and hazardous they proved effective. Since that time, our material from water and soil through knowledge base has amplified extremely. microorganisms and also protects ecosystem. Biotechnological techniques to treat waste The easily disposed simple forms of before or after it has been brought into the compounds into natural environment or used environment are components of as manure for soil improvement are the environmental biotechnological tools. conversion products of complex organic Biotechnology can also be applied industrially compounds using anaerobic microbe based for use in developing products and processes process called composting. that generate less waste and use less nonrenewable resources and consume less Technologies involving the use of genetically energy. In this respect biotechnology is well modified organisms microorganisms or positioned to contribute to the development of plants; they could also involve different a more sustainable society through a strains of yeast. Insects and mammalian cell sustainable environment. Recombinant DNA lines could be useful too. The most technology has improved the possibilities for outstandingly used are bacteria and its the prevention of pollution and holds a different strains. Because these can be grown promise for a further development of in large quantities and their genome study is bioremediation. What this means for not a tough job. These can be easily modified. environmental biotechnology is that it is These techniques include Landfill, futuristic and limitless in application and Biosensors, Bioremediation, Compositing, Oil usage. (European Federation of eating bugs, Biodegradation of xenobiotic, Biotechnology 1999). Bio mining, Designer bugs, Pollution control, Treating industrial waste, Bio scrubbing, Pest Biosensors control and bio pesticides, Weed control, Restoration of denuded areas, Viral A biosensor is an analytical device that pesticides, Bio fertilizers, Bio diversity and integrates a biological sensing element (e.g., conservation technologies etc. an enzyme or an antibody) with a physical (e.g., optical, mass, or electrochemical)

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transducer, whereby the interaction between Biosensors on the basis of recognition the target and the bio-recognition molecules is elements translated into a measurable electrical signal (Thevenot et al., 2001). Potent alternatives to Enzymes: These are biological molecules that conventional analytical techniques are Optical catalyze specific chemical reactions because of biosensors that exploit light absorption, specificity for substrate. One of the fluorescence, luminescence, reflectance, tremendously important properties of enzymes Raman scattering and refractive index. is immobilization on solid substrate, because the Devoid of any time-consuming sample immobilization method can enhance the concentration and/or prior sample pre- working lifetime and sensitivity of the treatment steps these biosensors provide biosensors. The optical transducers of enzyme- rapid, highly sensitive, real-time, and high- based biosensors are at the heart of the frequency monitoring. Although optical development of compact, self-contained devices biosensors have great potential applications in for environmental monitoring. ChE biosensors the areas of environmental monitoring, food are of fastidious interest in the area of universal safety, drug development, biomedical toxicity monitoring because Cholinesterase research, and diagnosis (Borisov et al., 2008, (ChE) enzymes can be inhibited by several Shankaran et al., 2007) their use in fields of toxic chemicals such as organophosphates and environmental pollution control and early pesticides, heavy metals, and toxins. (Borisov et warning is still in the early stages. al., 2008, Ispas et al., 2012, Luckham et al., 2010]. Considering that different pollutants Biosensors can be classified according to their inhibit enzyme activity in various ways, multi- transduction principle such as optical analyte detection can be achieved using enzyme (including optical fibre and surface plasmon sensors. For example, pesticides and heavy resonance biosensors), electrochemical metal ions can be detected simultaneously in a (including amperometric, and impedance sample solution through the inhibition of biosensors), and piezoelectric (including butyrylcholine esterase by pesticides and urease quartz crystal microbalance biosensors) or by heavy metals ions (Borisov et al., 2008, based on their recognition element as Ligler et al., 2009, Malitest et al., 2005). immunosensors, apt sensors, genosensors, and Toluene ortho-monooxygenase is used as enzymatic biosensors, when antibodies, biorecognition constituent, and an oxygen- aptamers, nucleic acids, and enzymes are, responsive ruthenium-based phosphorescent respectively, used. dye serve as transducer for the measurement of Toluene in aqueous solution in enzymatic In environmental monitoring, the majority of biosensor (Zhongn et al., 2011). The biosensors are identified as immunosensors phosphorescence intensity of the oxygen- and enzymatic biosensors, but recently the receptive probe is changed due to the enzyme development of apt sensors has been catalyzed expenditure of Oxygen which helps in increased, due to the advantageous the determination of Toluene. Although the characteristics of aptamers such as easiness to enzymatic biosensor can detect toluene in modify, thermal stability, in-vitro synthesis, wastewater with a limit of detection (LOD) of 3 and possibility to design their structure, to μM and a linear signal range up to 100 μM, the distinguish targets with different functional response time is long (1 h), and the activity groups, and tore hybridize (Justino et al., decreases with each measurement and with 2015). storage time.

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Antibodies: Antibodies against haptens, such because of their unique character. Aptamers as pesticides, persistent organic pollutants to target small organic and inorganic (POPs), and endocrine disrupting chemicals compounds such as proteins, peptides, amino (EDCs), are prepared by synthesizing acids, nucleotides, drugs, and heavy metal immunogens from the covalent binding of the ions can be produced. The chemical synthesis hapten to a carrier protein and then is aptamers is easy and purification steps immunizing them into animals. The creation required are simple and cheap, which of the chemical binding of the hapten to the eliminates the batch-to-batch variation found carrier protein, called complete antigen when using antibodies. Furthermore, Stability, determines the specificity and quality of affinity, and specificity of the molecules are antibody which is important for immunoassay improved by modification of aptamers (Dorst et al., 2010).The most frequent and through chemical synthesis. In addition, most toxic hepatotoxin, microcystin-LR (MC- aptamers are more stable, and more resistant LR), is detected by complete antigen (MC- to denaturation and degradation than LR-BSA) which is synthesized by introducing antibodies (Mehta et al., 2012, Jo et al., a primary amino group in the seventh N- 2011). methyl dehydro alanine residue of MC-LR. The product amino ethyl-MC-LR was then DNAzymes:-DNAzymes (catalytic DNAs or coupled to bovine serum albumin (BSA) with deoxyribozymes) are functional nucleic acids. glutaraldehyde. A monoclonal antibody These bind to specific targets by folding into (Clone MC8C10) against MC-LR was a well defined three-dimensional structure. produced by immunization with MC-LR- (Hollenstein et al., 2008, Li et al., 2009, BSA. MCs in waters are detected due to the Wang et al., 2013, Xiang et al., 2009) establishment of indirect competitive enzyme- DNAzymes can generally be obtained through linked immunosorbent assay (ic-ELISA) with in vitro selection, allowing them to function MC8C10, which show high specificity with a in the presence of a specific target of choice. detection bound of 0.1 µg/L for MC-LR Allosteric DNAzymes or aptazymes are class (Sheng et al., 2007).Aptamers: An aptamer, a of functional nucleic acids generated by single-stranded DNA or RNA sequence combination of DNAzymes that can perform composed of about 50-80 nucleotides selected chemical modifications on nucleic acids, and by Systematic Evolution of Ligands by aptamers that can bind with a broad range of Exponential enrichment (SELEX), binding of molecules (Hollenstein et al., 2008). aptamer to its target is very selective, through DNAzymes provide clear-cut recognition and folding into a complex three-dimensional exact quantification of environmental structure (Borisov et al., 2008, Ligler et al., pollutants, ranging from low-molecular- 2009, Dorst et al., 2010, Ellington et al., weight organic or inorganic substrates and 1990, Turek et al., 990). The interaction macromolecules to metal ions through between the aptamer and the target includes combination of nano-biological recognition structure compatibility, stacking of aromatic probes and the sensitivity of laser-based rings, electrostatic and Vander Waals optical detection (Li et al., 2009, Wang et al., interactions, hydrogen bonding, or a 2013, Xiang et al., 2009).The extensive combination of all these effects (Borisov et application of RNA-cleaving DNAzymes is al., 2008, Ligler et al., 2009, Dorst et al., because of their simple reaction conditions, 2010). Aptamers are a useful alternative to fast turnover rates, and significant possible antibodies as sensing molecules, thus modifications of their substrate lengths introducing a new era of affinity biosensing (Xiang et al., 2009). The high selectivity of

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DNAzymes toward specific targets makes value. The detection of inhibitory effect of them ideal biorecognition molecules for toxic metal ions on the cellular activity of E. biosensing. Numerous DNAzyme-based coli and Pseudomonas putida was then optical biosensors have been developed for achieved. Amaro et al., reported a whole-cell the detection of various heavy metal ions, biosensor for the detection of heavy metals such as Mg2 +, Ca2 +, Zn2 +, Pb2+, Cu2+, based on metallothionein promoters from Co2 +, Mn2 +, Hg2 +, and Ag + because of Tetrahymena thermophila. Two gene their facile operation, high sensitivity, and constructs using the Tetrahymena easily detectable signals (Hollenstein et al., thermophila MTT1 and MTT5 2008, Li et al., 2009, Wang et al., 2013, metallothionein promoters linked with the Xiang et al., 2009). Given the tremendous eukaryotic luciferase gene, regarded as a advances made in the areas of functional reporter. This kind of biosensor appears to be DNA and nanotechnology, DNAzymes and the most sensitive eukaryotic metal biosensor aptazymes have already been applied to among other published cell biosensors. Using almost every aspect of DNA nanotechnology, bioluminescent bacteria immobilized in an resulting in new materials and devices that alginate matrix on the bottom of the wells in a may be employed in the environmental 96-well microplate. Air toxicity is monitored monitoring field (Hollenstein et al., 2008, Li using fiber-optic biosensor developed by; et al., 2009, Wang et al., 2013, Xiang et al., Eltzov et al., Bioluminescence was 2009, Long et al., 2013). suppressed when the biosensor was exposed to toxic compounds present in air Chloroform Whole Cells: Whole cells are outstanding could be detected by this method with a LOD indicators of toxic compounds. The of 6.6 ppb. The same group developed a flow- bioluminescent light production or through fiber-optic sensing system by fluorescence properties of large number of immobilizing two other bacterial strains for microbial-based optical biosensors have been the online monitoring of toxic pollutants in used to detect toxicity and pollutants. Heavy water (Eltzov et al., 2009, Amaro et al., 2011, metals and inorganic pollutants in wastewater Arain et al., 2006, Eltzov et al., 2011, were rapidly and effectively monitored using Olaniran et al., 2011). The sensor could detect whole-cell bacterial biosensors developed by pollutants in flowing tap water and surface Olaniran et al., when Shigellasonnei and water within 24 h, but a loss of functionality Escherichia coli, were used the biosensors of the bacteria was observed after longer were found to be receptive to the toxicity of periods. wastewater effluents. With increase in concentration of heavy metals and inorganic Optical Biosensors pollutants in water bioluminescence increases with a correlation coefficient (r2) as high as Evanescent wave fiber-optic 0.995 and 0.997, respectively. Achieving immunosensors (EWFI): Extensive range of rapid, sensitive and cost effective detection of pollutants, such as TNT, 2, 4-D, atrazine, E. wastewater quality is the capability of coli O157:H7, and Staphylococcal enter toxin bacterial biosensors. For pH and oxygen an B have been detected using EWFI, which are integrated fluorescence-based sensor, was swift, exact, receptive, money-making and reported by Arain et al., in which bacterial appropriate for real-time on-site detection (Li respiratory activity was monitored via the et al., 2009). Conventional EWFI is not a decrease in the oxygen partial pressure of the transportable device because it requires closed system and also via the decrease in pH critical optical configuration and is costly

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because of having the large size with light reflected from the optical instrument. numerous optic components (e.g., chopper, Real-time detection of miniature changes in off-axis parabolic reflector, and biconvex the refractive index is permitted by SPR silica lens). Evanescent wave all-fiber biosensors through the fastening of biosensor (EWAB) based on a single-multi- biorecognition molecules (e.g., antibodies) fiber optic coupler which is simple, compact immobilized on a transducer surface with and portable was developed for simultaneous their biospecific targets (e.g., analytes) in detection of 2, 4, -D and MC-LR. (Long et solution. SPR biosensors have been used al., 2009). Significant signal enhancement extensively for applications including clinical was achieved by plummeting the optical diagnosis, drug discovery, food analysis, components and configuration with the use of environmental monitoring, since their single-multi-fiber optic coupler, which introduction in early 1990 (Shankaran et al., enables both the transmission of the excitation 2007). In general, a SPR biosensor is light and the collection and transmission of comprised of several important components: a fluorescence. Combination tapered fiber light source, a detector, a transduction surface probes were produced by the tube-etching (e.g., gold-film), a prism, biorecognition method and modified by covalent attachment molecule (e.g., antibody/antigen, DNA and of the MC-LR-OVA (recognition element) to aptamer) and a flow system. The use of SPR a self-assembled monolayer formed onto the to detect environmental contaminants, probe. This probe is highly resistive to non- including atrazine, Dichloro-Diphenyl- specific binding of proteins and can be reused Trichloroethane (DDT), 2, 3, 7, 8- more than 150 times with a LOD of 0.03 µg/L tetrachlorodibenzo-p-dioxin, carbaryl, 2, 4-D, and a LOD of 0.07 µg/L for MC-LR and 2, 4- benzo[a]pyrene (BaP), biphenyl derivatives, D, respectively (Long et al., 2008). and trinitrotoluene (TNT), has recently gained considerable interest (Cooper 2002, SPR Biosensors: When the surface of a thin Shankaran et al., 2007, Long et al., 2011, metal film is excited by an incident light Long et al., 2010, Miura et al., 2003) An SPR under total internal reflection conditions, it immunosensor for BaP, a carcinogenic generates the evanescent electromagnetic endocrine disrupting chemical, was reported field with which a surface-sensitive optical to have a LOD of 10 ppt (Miura et al., technique is associated called Surface 2003).In natural water samples for the Plasmon Resonance (SPR) (Cooper 2002). analysis of carbaryl a portable SPR-based Due to the verity the evanescent field immunosensor was developed. (Mauriz et al., diminishes exponentially with escalating 2006) Based on a binding inhibition distance of penetration from the interface, the immunoassay format, this immunosensor has monitoring of only surface-confined a LOD of 1.38 μg/L. The sensor surface molecular interactions occurring on the covalently modified by the analyte derivative transducer surface is promoted by SPR. Most allows the reuse for more than 220 of the SPR instruments use a Kretchmann regeneration cycles. The immunoassay configuration working at attenuated total performance of the biosensor was validated reflectance (ATR) for excitation of surface with respect to conventional high- plasmons, which can detect a small refractive performance liquid chromatography-mass index change at the metal/analyte interface, spectrometry, and the correlation between and the information of the molecular methods was in good agreement (r2 > 0.998) interactions can be obtained by measuring the for real water samples. Kim et al., fabricated optical intensity (or phase/polarization) of the sensing surface of the SPR immunosensor

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simply by covalent amide binding of 2, 4-D- suitable environmental growth conditions, and BSA conjugate on the Au-thiolate self- appropriate levels of nutrients and assembly. A LOD of 0.1 ppb 2, 4-D is contaminants are important site factors established with a response time of only 4 required for successful bioremediation. min. One of the advantages is that the immunoaffinity interactions of anti-2, 4-D Pollution from air or from aquatic or antibody with the 2, 4-D-BSA sensor surface terrestrial systems is also reduced using and 2, 4-D in solution could be significantly biological systems (EFB 1999), modulated by the control immobilization of 2, bioremediation also involves lessening of 4-D-BSA on the SAM surface. As a result, toxic components by extracting a microbe the sensitivity of the SPR immunosensor is from the environment and exposing it to a enhanced by about 10-fold to 10 ppt without target contaminant. (Vallero, 2010) Thus, the using any high-molecular-weight labels. purpose of bioremediation is the employment Localized surface Plasmon resonance (LSPR) of biosystems such as microbes, higher effect using AuNP for signal amplification organisms like plants (phytoremediation) and was also investigated (Kim et al., 2008, animals to reduce the potential toxicity of Kawaguchi et al., 2008).The amplification chemical contaminants in the environment by method of indirect competitive inhibition and degrading, transforming, and immobilizing LSPR were integrated for the fabrication of an these unwanted compounds. immunosurface using AuNP. The detection range of TNT using this immunosurface was Biodegradation is the use of living organisms from 10 ppt to 100 ppb. to enzymatically and otherwise attack numerous organic chemicals and break them Bioremediation: The term of bioremediation down to lesser toxic chemical species. has been made of two parts: “bios” means life Biotechnologists and bioengineers classify and refers to living organisms and “to pollutants with respect to the ease of remediate” that means to solve a problem. degradation and types of processes that are “Bioremediate” refers to the use biological responsible for this degradation, sometimes organisms to solve an environmental problem referred to as treatability (Vallero, 2010).Most such as contaminated soil or groundwater. commonly occurring bioremediation option is Environmental pollutants are degraded or the use of microorganisms for biodegradation. pollution is prevented by the use of living Microorganisms meet their growth and energy micro organisms through bioremediation. In needs by break down of most compounds. other words, it is a technology for removing These biodegradation processes may or may pollutants from the environment thus not need air. In some cases, the metabolic restoring the original natural surroundings and pathways normally used by organisms for preventing further pollution (Sasikumar et al., growth and energy supply may also be used to 2003). Bioremediation enables utilization of break down pollutant molecules. In these diverse metabolic abilities of microorganisms cases, known as co metabolisms, the to convert contaminants to risk-free products microorganism does not benefit directly. by mineralization, generation of carbon (IV) Researchers have taken advantage of this oxide and water, or by conversion into phenomenon and used it for bioremediation microbial biomass, so as to clean the purposes (EFB 1999). Detoxification of contaminated environment (Baggot et al., pollutants by mineralizing pollutants to 1993, Mentzer et al., 1996). Presence of carbon dioxide (CO2), water (H2O), and metabolically capable microbial populations, harmless inorganic salts is called the complete

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biodegradation (EFB 1999). Incomplete be degraded into less toxic compounds;(3) biodegradation (i.e., mineralization) will sufficient numbers of microorganisms to produce compounds that are usually simpler make bioremediation successful;(4) the (e.g., cleared rings, removal of halogens), but microbial environment must be habitable for with physical and chemical characteristics the microbes to thrive.(Vallero, 2010). different from the parent compound. In Sometimes, environment becomes toxic to addition, side reactions can produce microbial population due to elevated compounds with varying levels of toxicity concentrations of compounds. Therefore, the and mobility in the environment (Vallero, bioengineer must either use a method other 2010). Biodegradation may occur than bioremediation or modify the spontaneously, in which case the expressions environment (e.g., dilution, change of pH, “intrinsic bioremediation” or “natural pumped Oxygen, adding organic matter, etc.) attenuation” is often used (EFB 19994). In to make it habitable. An imperative many cases for natural attenuation to take amendment is the elimination of non- place the natural circumstances may not be aqueous-phase liquids (NAPLs) since the favorable due to inadequate nutrients, oxygen, microbes’ biofilm and other mechanisms or suitable bacteria. Supplying of one or more usually work best when the microbe is of the missing/inadequate environmental attached to a particle; thus, most of the factors help in improving such situations. NAPLs need to be removed, by vapour Extra nutrients (EFB 19994) were extraction (Vallero, 2010). Liquids (water, disseminated to speed up the breakdown of solutes, and nutrients) are difficult to pump the oil spilled on 1000 miles of Alaskan through low permeability soils, like clays and shoreline by the super tanker Exxon Valdez in hence these systems are difficult to treat. 1989. Sandy soils allow mobility and greater likelihood of contact between the microbes According to Vallero, there are millions of and the contaminant, so bioremediation works indigenous species of microbes living at any best in these soils. Therefore, an given time within many soil environments. understanding of the environmental The bioengineer simply needs to create an conditions sets the stage for problem environment where those microbes are able to formulation (i.e., identification of the factors use a particular compound as their energy at work and the resulting threats to health and source (Vallero, 2010). Biodegradation environmental quality) and risk management processes is not new, it had been observed (i.e., what the various options available to empirically for centuries, but putting them to address these factors are and how difficult it use as a distinct field of bioremediation began will be to overcome obstacles or to enhance with the work of Raymond et al., It was those factors; that make remediation studied that the addition of nutrients to soil successful). In other words, bioremediation is increases the abundance of bacteria that was a process of optimization by selecting options associated with a proportional degradation of among a number of biological, chemical and hydrocarbons, and the hydrocarbons under physical factors these include correctly consideration were petroleum by-products matching the degrading microbes to (Raymond et al., 1975). Bioremediation conditions, understanding and controlling the success depends on the following:(1)the movement of the contaminant (microbial growth and survival of microbial populations; food) so as to come into contact with and (2) the ability of these organisms to come microbes, and characterizing the abiotic into contact with the substances that need to conditions controlling both of these factors.

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Optimization can vary among options, such as metal. The heavy metal removal by the artificially adding microbial populations bacteria Pseudomonas was attributed to the known to break down the compounds of cellular growth of these organisms. The concern. Only a few species can break down reactive intermediates and Cr (III) are formed certain organic compounds (Vallero, 2010). through the biotransformation of Chromium Toxicity to the microbial population and (VI) by undergoing enzymatic reduction. inherent biodegradability of the compound are (Seng et al., 2002). Toxic Cr (VI) from two major limiting factors of any contaminated environment can be reduced biodegradation process. using chromium resistant bacteria isolated from the soil. The mechanism of Cr-tolerance Bioremediation of heavy metal or resistance of selected microbes is of contaminated soil and waste water using particular importance in both bioremediation micro organisms:-Microorganisms play a and waste water treatment technology significant and vital role in bioremediation of (Polisak et al., 2010). Algae such as heavy metal contaminated soil and Charophyte, Nitella pseudo flabellate is wastewater. Indigenous soil microbes appear applicable in the remediation of Cr (VI) well suited for Cr (VI) transformation in contaminated water at different calcifying highly contaminated soil. Very stable final potentials (Gomes et al., 2009). Hexavalent chromium forms can be achieved as a result chromium can be reduced into its trivalent of microbial activity, with minimal risk of re- form by Bacillus species. These bacteria release of Cr (VI). In liquid treatment system, could reduce 91% of Chromium from the biotransformation of Cr (VI) by pure culture medium after 96 hours and was also capable has been studied under a range of redox of reducing 84% chromium from the (aerobic and anaerobic), temperature (10- industrial effluents in Lahore after 144 hours 45oC) and pH (6.5-9.5 conditions). (Rehman et al., 2008). Morales et al., wanted Escherichia coli ATCC 33456 transformed Cr to segregate and examine chromium-resistant (VI) faster at 10oC to 45oC under anaerobic microorganisms suitable for bioremediation. than at 10oC to 35oC unders aerobic They found that Streptomyces sp was condition. The phenomenon of bio-absorption efficient at removing Cr (VI) by promoting and bioaccumulation of chromium in metal reduction to Cr (III) and regardless of its and chromium contaminated soil and water negative effect on growth and development help in bioremediation using many genera of tolerated heavy metals and elevated levels of microbes like Bacillus, Enterobacter, chromium. Hence chromium-resistant Escherichia, Pseudomonas and also some microorganisms are a promising candidate for yeasts and fungi. (Bader et al., 1999, Bopp et detoxification of sites containing heavy al., 1988, Cifuentes et al., 1996, Garbisu et metals (Morales et al., 2007). More data of al., 1998, Ishibashi et al., 1990, James et al., chromium reduction was obtained by 1983, Kotas et al., 2000, Losi et al., 1994, scientists from Pseudomonas aeruginosa Nies et al., 1999, Philip et al., 1998, Shen et (Ganguli et al., 2002), Bacillus sp (Camargo al., 1994, Wang et al., 1989).The et al., 2003, Meghraj et al., 2003), bioaccumulation of Cr (VI) and ability to Streptomyces (Amorosa et al., 2001), from remove heavy metal like Cr (VI) from tannery Pseudomonas fluorescens (Appanna et al., effluents by the heavy metal resistant fungi 1996, Khan and Ahmad 2006), from yeasts and bacteria isolated from the soil samples of like Pichiguilliermondii (Ksheminska et al., tanning industries were found. The pH of the 2003) also from Micrococcus sp and effluent was significant in removing the Aspergillus sp in Tamil Nadu (Congeevaram

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et al., 2007]. Aspergillus niger biomass has having the potential of carrying out an angular been found to be very effective in bio dioxygenation, that is, oxygenation at a pair absorption of Cr (III) and Cr (VI) in spent of adjoining carbon atoms, one of which is chrome liquor. For hazardous material concerned in one of the bridges between the degradation not only microorganism are two benzene rings was dioxin dioxygenase of involved, plants are also involved in Sphingomonas sp. strain RW1. The preferred breakdown of complex molecules and genes are now characterized and whereas immobilize waste materials from soil/water genes coding for multi component this process is known as phytoremediation. It dioxygenase are usually gathered, those has been reported that in Arabidopsis thaliana coding for dioxin dioxygenase were (Flowering plant), arsenate reductase and unpredictably scattered throughout the gamma- glutamyl cysteine synthase are coded chromosome (Sharma 2012). Another by two bacterial genes (arsC and gECS1) comparatively identification is made for respectively, the over expression of these lineage of ring activating dioxygenases has genes enables this plant to capture arsenate been modified genetically to contain, between and degrade it (Hooker and Skeen 1999). It is the genes coding for the electron transport postulated that; environmental security is chain of a naphthalene dioxygenase, genes contributed appreciably by transgenic plants coding for subunits of an enzyme catalyzing a (Frerot et al., 2006). Biotechnology somehow separate step in the pathway, that is, salicylate solves these problems and plays a significant 5-hydroxylase. Even other unrelated role in environmental remediation by helping naphthalene and phenanthrene systems from us to clean environmental pollution and Rhodococcus sp. NCIMB 12038 and maintain natural ecosystem. Burkholderia sp. strain RP007 have been described, but no substrate specificity profiles Genetically engineered microorganisms in have been given. There are so many examples environmental remediation: A variety of of using microorganisms for bioremediation. organisms reside in the soil. They range from The organisms can easily be manipulated and complex as insects to simple organisms like by modifying their genome everything can be earth worms. They could be evident as former made from these organisms because their and could be indistinguishable, but genome does not need so many regulators as performing a great role in maintenance and eukaryotic genome require, fertility of soil, like bacteria. Energy is not transported to the cells vital to run their Phyto remediation: -Phytoremediation is metabolism proficiently during most of the defined as utilizing the natural abilities of enzymatic reactions involving substrate and is plants to uptake, accumulate, and degrade unintentional. So human made contaminants constituents of contaminants in soil and water such as chlorobiphenyls which are present in to clean up the environment. the environment by somehow being a part of Phytoremediation is applicable to a wide our metabolism (Bayat et al., 2015). These variety of contaminants including plentiful enzymes stay in study for decades because metals and radio nuclides, different organic they have a very complex structure and compounds (such as chlorinated solvents, contain many different substrates for their BTEX, PCBs, PAHs, pesticides/insecticides, performance. So serious amendment is made explosives, nutrients, and surfactants as is in these enzymes by changing their respective reported in results of various research and genes in genome of different organisms development. According to information mostly bacteria. The first enzymes considered reviewed, general site conditions best suited

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for potential use of phytoremediation include required (usually several growing seasons), large areas of low to moderate surface soil (0 depth limitations (3 feet for soil and 10 feet to 3 feet) contamination or large volumes of for groundwater), and the likelihood of water with low-level contamination subject to contaminant entrance into the food chain low (stringent) treatment standards. Depth to through animal consumption of plant groundwater for in situ treatment is limited to material. about 10 feet, but ex situ treatment in constructed troughs or wetlands has also been Types of phyto remediation investigated (Brown 1995, Carricaburu and Lisa. 1996, Dushenkov et al., 1995, Schnoor Rhizofiltration: Rhizofiltration of surface et al., 1995).There are five basic types of water may be conducted in situ by growing phytoremediation techniques:- plants directly in the contaminated water body, rhizofiltration of groundwater can also 1) Rhizofiltration, is a water remediation be done in situ if ground water is positioned technique in which plant roots uptake the within the Rhizosphere (root contaminants. zone).Alternately, in rhizofiltration contaminated groundwater is pumped into 2) Phyto extraction, is a soil remediation troughs filled with the large root systems of technique involving uptake of contaminants appropriate plant species. Efficient absorption from soil of metals from the contaminated groundwater into root tissues is permitted by the large 3) Phytotransformation, is applicable to both surface areas of root systems. Metals from soil and water, in which contaminants are ground water are also detached through degraded through plant metabolism precipitation caused by exudates (liquids released from plant tissues) in addition to 4) phyto-stimulation or plant-assisted removal through absorption. When the bioremediation, also used for both soil and groundwater passes through the plant troughs, water, in which microbial biodegradation is the precipitates are filtered out and then stimulated through the activities of plants in treated water is removed from the process the root zone, and loop. Roots are harvested, and depending on the species of plant used, shoots may be 5) Phytostabilization, the mobility and transplanted to grow new roots. Plants can be migration potential of contaminants is replaced in the system to ensure constant reduced using in soil. (Brown. 1995, operation results. Rhizofiltration using Carricaburu and Lisa. 1996). sunflowers has been used in the remediation of radio nuclides from surface water near Phytoremediation technologies are Chernobyl (strontium and cesium) and in advantageous as compared to traditional water using a rhizofiltration system, as remediation technologies, chief advantages described above, at a DOE facility in Ohio include likelihood of generating less (Brown. 1995, Dushenkov et al., 1995, Salt et secondary wastes, minimal connected al., 1995, Vance 1996). environmental disturbance, and the capability to leave soils in place and in an exploitable Phytotransformation: Phytotransformation condition following treatment. In spite of can be accomplished for in-situ remediation advantages, it has certain disadvantages of both surface and ground water, Surface which include the extended lengths of time water includes ponds or wetlands.

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Groundwater can be remediated Insitu if the water table is within the zone tapped by deep- Soil remediation methods rooted plants such as poplars or ex situ by pumping water to troughs or constructed Phytoextraction: In this process plant tissues wetlands containing appropriate plants. In the help in removal of metals, radio nuclides, and Phytotransformation process, plants take up certain organic compounds (i.e., petroleum organic contaminants and degrade them to hydrocarbons) by direct uptake. Hyper less toxic or non-toxic compounds (Brown. accumulators of one or more species of the 1995, Schnoor et al., 1995, Vance. 1996, concerned contaminants are planted for the Black. 1996).This technique is being tested on implementation of phyto-extraction program. explosives-contaminated groundwater (TNT It has been determined from preliminary field and RDX) at Milan Army Ammunition Plant testing, that certain amendments (i.e., in Tennessee by the U. S. Army Corps of fertilizer, water, etc.) may be required to Engineers Waterways Experimental Station ensure successful plant growth. Lengths of (WES) (Brown 1995, Schnoor et al., 1995, time before harvesting the plants are also Salt et al., 1995, Black. 1996). determined from preliminary testing, and after this period of time, plant tissue is removed In addition, an Environmental Security and, if necessary, a new crop of plants are Technology Certification Program (ESTCP) planted. Although testing has focused on project is testing the ability of trees with roots single plants, several species may be used at a tapping groundwater to degrade TCE and site, either at the same time or subsequently, hydrazine present in the aquifer (ESTCP to remove more than one contaminant 1996). The U.S. Air Force is planning to (Schnoor et al., 1995, Salt et al., 1995, Kumar evaluate phytoremediation through field et al., 1995, Newell and parry 1995). studies followed by cell cultures and bio- chamber studies (Edwards and Robert 1996). Plants which have the ability to perform phytoextraction are characterized by (a) Plant-Assisted Bioremediation: This is used Ability to accumulate and tolerate high for conduction of near-surface concentrations of metals in harvestable tissue. bioremediation. Under this technique (2) Rapid growth rate. (3)High biomass appropriate plants are installed in these areas. production (This results in more metal The plants provide carbonaceous material removed per planting) (Salt et al., 1995, from liquids released from roots and through Black 1996, Kumar et al., 1995). the decay of root tissue. Besides this oxygen content in the bioremediation area is DOE field trials involving phytoextraction of increased due to release of oxygen from the metals and radio nuclides from soils are being root system of plants. The microbial activity conducted in association with Cornell increases due to additions from plant activity University, at sites in Montana and Idaho which results in increase in the contaminant (Brown 1995). Also, a study is being degradation. The roots of plants have conducted by the University of Iowa and beneficial effects on rate of Insitu Kansas State University, in association with bioremediation by microorganisms was the Hazardous Substance Research Center studied under ESTCP project (Carricaburu (HSRC) at Kansas State, to determine the and Lisa. 1996, Schnoor et al., 1995, Vance. efficiency of poplars to take up and 1996, Black 1995, ESTCP 1996, Jordahl et accumulate arsenic and cadmium in soils al., 1997). (Schnoor et al., 1995).

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Phytostabilization: Phytostabilization is the Some examples of genetically modified plants reduction in bioavailability of metals In recent years, hairy roots (HRs) have been contaminants and reducing the potential for productively used as research tools for human exposure to these contaminants by the selecting the abilities of different plant use of certain plant species through species to tolerate, accumulate, and/or remove absorption and precipitation. In areas where environmental pollutants, such as TNT, natural vegetation is lacking due to high PCBs, pharmaceutical, dyes from textile, metals concentrations in surface soils or phenolics, radionuclides, and heavy metals. physical disturbances to surficial materials The plants are associated with contaminants this technique is helpful for re-establishment through their root system and have model of vegetation in these areas. The potential system this makes plants more advantageous migration of contamination through wind and beneficial. The hairy rooted plants use erosion and transport of exposed surface soils metabolic and catalytic pathways for uptake and leaching of soil contamination to of pollutants, change their morphology and groundwater is decreased by metal tolerant store them in different plant organs and species, so as to restore vegetation to these conjugation. Plant roots entrap and degrade sites. (Erosion and leaching are common in the pollutants by the liberation of chemicals unvegetated areas). Plants appropriate for such as peroxidases and laccases which act as Phytostabilization are tolerant to high levels enzymes. Biotechnology modifies the genome of contaminants concentration, have the of plants in such a way that they can tolerate ability to immobilize the contaminants and disintegrate heavy non degradable through uptake, precipitation, or reduction pollutants. (Samanta et al., 2002). due to high root biomass production, have the Agrobacterium-Ti plasmid transformation tendency to retain contaminants in shoots increase the chance of efficient embryo rather than transferring to shoots, to avoid formation with a 1000-fold reasonable speed special handling and disposal of shoots. in Pinusabies. This happens by disarming the (Brown 1995, Schnoor et al., 1995, Black Agrobacterium tumifaciens strains and by 1996, Munshower. 1996). Phytostabilization modifying its vir genes. Embryonic field studies are being conducted at the Pinustaeda culture is able to get GUS University of Iowa and Kansas State expression 10 folds higher than normal. Co- University, in conjunction with the HSRC at cultivation of P. strobus embryogenic tissue Kansas State. These tests involve the re with A. tumefaciens carrying a 35S-35S- vegetation of a mine tailings site in Kansas AMVgus: nptII fusion also resulted in the containing, elevated levels of cadmium, lead, regeneration of efficiently transformed and zinc, to reduce wind and water erosion somatic embryos compounds produced by (Banks et al., 1994). industrial waste (Poupin and Johnson 2005). Pinus, abies, Oak, scots pine has improved Plant-Assisted Bioremediation: Plant- soil property by accumulating heavy metals. assisted bioremediation techniques for soil remediation are same as groundwater Phytoremediation of Heavy Metals application. This technique is being tested at a Chevron site in Ogden, Utah using alfalfa to Phytoremediation technology involves uptake address fuel contamination (Carricabura and of heavy metals in different amounts from the Lisa. 1996) and at the University of Iowa soil and storing them in harvestable parts of using poplar trees to address atrazine plant, it is a part of promising green contamination (Licht and Schnoor. 1996). technology (Chaturvedi et al., 2016).The

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changes in soil properties enable the plants to quantities still they are more suitable for tolerate pollutants (Mench et al., 2009). phytoremediation because of their greater root Phytoremediation can be easily used for the system and biomass. This issue could be treatment of soils containing heavy meals, and solved by using fast-growing plants instead of the biomass that is formed throughout the trees (Da Conceicao Gomes et al., 2016, He et process can be further applied in biodiesel al., 2010). For example, the Poplusalba is a production. The plants suitable for this deciduous tree, which can accumulate zinc purpose have the capability to accumulate (Zn) in different plant parts such as leaves, toxic heavy metals, these are called bio stems, and roots. An increasing trend of Zn energy crops example Brassica species. Some accumulation was observed in the leaves of plants can accumulate pollutants in tissues Poplusalba with the application of SDS (Munir and Faisal 2016). Among the Energy (Pierattini et al., 2010). The ability of the crop that is used for the phytoremediation of plants to assemble amplified levels of toxic soils contaminated by heavy metals, the most metals within their tissues influences the suitable for phyto- extraction is Jerusalem phytoremediation process (Ma et al., 2016). artichoke (Helianthus tuberosus L.). The The enzymes from some plants can break highest heavy metal uptake was observed at a down a number of organic compounds but dose of 60 Mg DMha−1 in the Jerusalem cannot degrade inorganic pollutants. Thus, artichoke (Antonkiewicz et al., 2018). Hyper there is a need to ensure that inorganic accumulating plants are fit to grow on soils pollutants are less available in soils or contaminated with heavy metals and can be extracted and accumulated in different parts of used to eradicate pollutants (Da Conceicao the plants and also reduce volatile versions of Gomes et al., 2016). Plants that contain inorganic pollutants (Ribierodesouza 2012). greater than 10, 000 mg/kg dry weight of Zn Energy recovery strategies can be used to or Mn or more than 1000 mg/kg dry weight of produce bioenergy from plant biomass, such Ni, Cu, or Pb or greater than 100 mg/kg dry as to form biodiesel. Fuel gas, char, and bio- weight of Cd in contaminated areas is oil can be produced via Pyrolysis, during considered as hyper accumulating plants. which the biomass undergoes thermal Important plants of families such as degradation without oxygen (Lievens et al., Lamiaceae, Fabaceae, Scrophulariaceae, 2013). Soils greatly polluted with toxic heavy Asteraceae, Euphorbiaceae, and Brassicaceae, metals can be remediated easily by growing are known to hyper accumulate toxic heavy plants that are tolerant to more than one heavy metal are frequently used in the metal, can produce a good amount of phytoremediation processes. Other than that, harvestable biomass with enhanced growth there have been reports of heavy metal hyper rates, and are highly competitive (Jeffre et al., accumulation in about 500 plant species 2013). (Jaffre et al., 2013). Plants with greater hyper accumulating abilities include Alyssum References bertolonii, Thlaspicaerulescens, Calendula officinalis, and Tageteserecta (Glick 2012). Agraeber, K., Nakabayashi, K., Miatton, E., Higher concentrations of Ni, Zn, and Cd are Metzger, G. L. and Soppe, W. J. 2012. best gathered by Thlaspicaerulescens Molecular mechanisms of seed dormancy. (Assuncao et al., 2003). This plant can Plant Cell Environment. 35(10): 1769- accumulate 500–52, 000 mg kg−1 of Zn and 1786. 0.3–1020 mg kg−1 of Cd. Trees took more Khalilian, M., Zolfaghari, M.R., Soleimani, M. time in accumulating metals, even in low 2015. High potential application in bioremediation of Selenate by Proteus

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How to cite this article:

Haleema Bano, Shayesta Islam, Faisal Noor and Ashraf Bhat, M. 2020. Biosensors and Bioremediation as Biotechnological Tools for Environmental Monitoring and Protection. Int.J.Curr.Microbiol.App.Sci. 9(10): 3406-3425. doi: https://doi.org/10.20546/ijcmas.2020.910.394

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