Biotechnological Applications of Microbes for the

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biotechnology applications, like DNA microarray, environmental biosensors, ... environmental biotechnology has experienced the exponential growth and.

7 Biotechnological Applications of Microbes for the Remediation of Environmental Pollution MANVI SINGH1,2*, PANKAJ KUMAR SRIVASTAVA1, VIRENDRA KUMAR JAISWAL1 AND RAVINDRA NATH KHARWAR2

ABSTRACT

Ever-increasing urbanization and industrialization are responsible for intensifying different pollutants in the environment. Biotechnology provides many solutions to combat these pollutants. Biotechnology involving potential of microbes is offering remediation of wide array of pollutants (viz. heavy metals, organic wastewater and solid wastes, radionuclides, polycyclic aromatic hydrocarbons, explosives, dioxins, etc.). Microbes-based biotechnological applications are focusing on improving their inherent capabilities and mechanisms to deal with different pollutants for their further decontamination. Bioaugmentation and biostimulation are the two prime process of biotechnology-based bioremediation. Recently, advanced biotechnology applications, like DNA microarray, environmental biosensors, metagenomics, proteomics, etc., have been applied in the field of environmental pollution. Key words: Environmental biotechnology, Bioremediation, Biosorption, Bioaugmentation, Heavy metal, Waste water, Microbial fuel cells.

1

CSIR–National Botanical Research Institute, Rana Pratap Marg, Lucknow - 226001, India. 2 Department of Botany, Banaras Hindu University, Varanasi - 221005, India. *Corresponding author: E-mail: [email protected]

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1. ENVIRONMENTAL BIOTECHNOLOGY

1.1. Introduction The Hungarian agricultural and economist Károly Ereky coined the word “Bio-technology” in Hungary during the 19th century to discover a novel technique based on converting raw materials into a more useful products. In 1988, the European Federation of Biotechnology has described the term “Bio-technology” more specifically, as the “integrated approach of biochemistry, microbiology and engineering sciences, in order to achieve application of capabilities of microorganisms, cultured animal cells or plant cells or part of thereof in industries, agriculture, health care and environmental processes”. The start of the 21st century seems to be the emergent century for biotechnology, as a key technology, for sustainable environmental protection(1–3). Rapid industrialization and urbanization has led to magnify many human activities which intensifying the air pollution (with CO 2, NOx, SO 2, greenhouse gasses, etc.), water pollution (with chemical and biological pollutant materials, nutrients, leachate, oil spills, etc.), and soil pollution (due to hazardous waste, use of pesticides, non-biodegradable materials, chemical fertilizers)[4]. Although, biotechnology offers a platform that has enabled many countries to tackle their environmental problems and named as Environmental Biotechnology. Gavrilescu [4] has discussed the core opportunities offered by biotechnology in respect to environment protection;  Biological treatment of wastes from agriculture, industries, domestic, wastewaters.  Application of bioremediation technology for cleaning the contamination from soil and water.  Identification of various species their preservation and conservation of biodiversity.  Monitoring environmental pollutants through biological organisms.  Sustainable manufacturing with low pollution and less waste products.  Energy generation from organic biomass.  Genetic engineering for better crop production and yield at contaminated sites. Therefore, Environmental bio-technology can be defined as “the multidisciplinary field with the integration of sciences and engineering in order to utilize biochemical potential of microorganisms, plants and parts thereof for the restoration and preservation of the environment and for the sustainable use of its resources”. Marousek et al. [5] has described the term “Environmental bio-technology” as the use of biological systems from bacteria

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to plants, by applying their bio-remediation strategies, monitoring of contaminants using biosensors, transforming organic wastes to generate green-energy, production of biosurfactants for improving and protecting environmental health. In the last 25 years, researchers working on environmental biotechnology has experienced the exponential growth and have been awarded with novel discoveries by the development of analytical and molecular biology tools.

1.2. Role of Biotechnology in Microbial Manipulation for Combating Environmental Pollution Microbes are the fundamental element for the basic research in biotechnology to develop a wide range of products and in maintaining and improving the environment. So, it would not wrong to say that without microbes, biotechnology would be an extremely limited science. Microbes are microscopic organisms such as fungi (which include yeasts), bacteria and viruses. The increased scientific approach has led to the development of screening and isolation processes of various microbes for their specific utilization for solving the environmental issues. Microbes able to degrade the contaminant and increases their population, and when the contaminant is degraded, pollutant free zone/area generates. The by-products of this treatment are carbon dioxide, water, and cell biomass are usually harmless for environmental conditions. This microbial bio-technology, allows us to do detailed studies on their genomic, proteomic, metatranscriptomics, metagenomics parts for developing improved microbial agents to resist environmental pollution. The microbial engineering generates microbial agents for bioremediation of soil and water contaminated by agricultural runoff, development of new transgenic crops, waste water minimization, energy production, production of biosensors etc. A diagrammatic representation of various applications of environmental biotechnology showed in Fig. 1. All forms of life can be considered having a potential function in environmental biotechnology. With the help of genetic engineering and recombinant DNA technologies, the scientists are able to manipulate at the genomic levels of living organism. Followings are the benefits of genetic engineering in plants with the help of microbes: (a) (b) (c) (d)

Improved resistance to pests Improved resistance to disease Improved plants for phytoremediation Improved tolerance to abiotic stress like high temperature, drought, floods, etc.

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Fig. 1:

Applications of environmental bio-technology in various fields.

2. BIOREMEDIATION

The term Bioremediation composed of two words: “bios” means living organisms and “remediate” means to treat a problem. Bioremediation is an efficient technology/process which extracts the potential of biological organisms to degrade environmental pollution and thus helps environment in maintaining natural surroundings. The bioremediation method has been classified as ex situ and in situ. The process where contaminant is treated at its native site under natural environmental conditions is called as in situ, and the ex situ process involves the removal of the contaminant and to be treated far away from their native site e.g., under laboratory conditions[6]. In March 1989, a huge and devastating oil spill accident occurred, by the tanker named as Exxon Valdez, spilled approximately 41,000 m3 of crude oil into in the gulf of Alaska, which contaminated about 2000 km of coastline. This is the period when bioremediation came into limelight with its usefulness and accelerated with its development in the clean-up of environment. There are many other such accidents happened like the Union-Carbide (Dow) Bhopal disaster, occurred due to discharge of extremely harmful radioactive material in the Chernobyl accident, and most recently the crises resulted from crude oil pollution of Mexico gulf waters and the leakage of the radioactive materials from Fukushima reactor in Japan. From this era, the bioremediation technology has become as focus of attention and well demonstrated for its usefulness, as a fairly complete solution to oil contamination. Using the

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potential of biodiversity, focus on protecting the environmental pollution with the microbial consortia along with advanced techniques like genomics, proteomics, metabolomics, and computational techniques as well as the generation of micropollutant sensors, will give rise to reliable and innovative options for cheaper decontamination. Bioremediation is environmentfriendly, low maintenance, low-cost, less input and sustainable approach for the cleanup of polluted sites. The use of biological organisms for the contaminant removal is based on the concept that organisms feeds on the contaminant for their own growth and metabolism an d th us co uld remov e su bstan ces from the environment[1,7,8]. Various microorganisms, like bacteria and fungi are very efficient in degrading the complex molecules, and the resultant products are generally safe for the environment. In comparison, fungi can digest large complex organic compounds using their hyphaes that are normally not degraded by other organisms. Similarly, others biological organisms like protozoa, algae and plants are also found suitable to absorb nitrogen, phosphorus, sulphur, and many minerals and metals from the contaminated environments. Biotreatment/bioremediation methods are recommended as “end-of-pipe processes” as the technology is efficiently and successfully helps in removing, degrading and detoxifying the pollutants in water, soil and solid waste[9]. There are different types of in-situ bioremediation process available (i) bioattenuation- a natural process of degradation by inducing the growth of indigenous microbes, (ii) biostimulation, by changing chemical reactions within the contaminated soils/site by adding water, nutrient, electron acceptors or donors, and (iii) bioaugmentation is a process where the microbes having potential of degrading or transforming the pollutants into less toxic form, are added into contaminated soil matrices after their culturing in the laboratory for speeding up their growth and metabolism[10]. The strategies usually adopted to act on the contaminant are discussed below [11–13] 1. Removal: A physical process of removing the contaminant from the site without disturbing the host medium; 2. Separation: A process that separates the contaminant from its host medium; 3. Degradation: A chemical or biological process to transforms the contaminant to produce less toxic compounds; 4. Immobilization: A process where contaminant is locked by microbes and hence prevent their access to plants or crops.

2.1. Bioaugmentaion Bioaugmentaion is a new method of employing the potential of diverse microorganisms at their native places to clean up the contaminant from

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the polluted sites. The method uses isolation of native microorganisms (fungus, bacteria, etc.) and their subsequent addition after culturing them in the conditions ex-situ to the contaminated sites to enhance their population and functional metabolism to remediate contaminants like metal (loid)[14] . Bioaugmentation can be applied as: (a) by isolating native microorganisms from the target contaminated site and their subsequent culturing under laboratory conditions and further re-inoculation; (b) Isolates are not necessarily inoculated to the source of the original culture; and (c) isolates can be applied with help of constructed and mutated microorganisms to the site[15] . A number of studies have been done for cleaning the contaminant from the environment using bioaugmentation technology. Gouda et al.[16] has observed the efficiency of a bacterial strain Pseudomonas sp. CK upon bioaugmentation for twenty days resulting in kerosene degradation by 86%. Jacques et al.[17] has evaluated the potential of microbial consortium using five bacteria: Mycobacterium fortuitum, Bacillus cereus, Gordonia polyisoprenivorans, Microbacterium sp, Microbacteriaceae bacterium which feeds on napthalene; and a fungus Fusarium oxysporum; which found to degrade phenanthrene, pyrene and anthracene present in the contaminated soil. The authors also reported that this microbial consortium can mineralized upto an average of 98% of PAHs in the soil approximately after 70 days. Similarly, Qiao et al [18] also applied a microbial consortium in bioaugmentation treatment of aromatic compounds, using bacterial species (Pseudomonas fluorescens, Streptococcus faecalis, Bacillus subtilis) and a yeast (Candida tropicalis). It has been noticed that most of bioaugmentation studies employed using gram-negative bacteria especially belonging to the species of Flavobacterium, Pseudomonas, Achromobacte, Alcaligenes and Sphingomonas[19]. An effort has been made by Mrozik and Seget[20] to check the bioaugmentation potential of some gram-positive bacteria species also, such as Mycobacterium, Bacillus and Rhodococcus and some useful fungal species belong to genus Aspergillus, Absidia, Penicillium, Acremonium, Mucor and Verticillium. The application of biotechnological tools in collaboration with bioaugmentation technology has marked with some significant results in the field of bioremediation. For a successive bioaugmentaion within soil matrix, it is important to monitor the amount and activity of microbes inoculated soil either as a single strain or as consortia. In the 2003, Jansson[17] used reporter genes and fluorescent and luminescent markers like green fluroscent protein (gfp), lux, etc. to detect phenotypes of microbial cells. According to his studies, Fluorescent in-situ hybridization (FISH) technique allows analysis of microbial population and their specific function along with their relative abundance within the target soil. A new report was given by Li et al.[21] where they have incorporated fluorescence staining technique with soil thin section technology to procure the images of microbial populations within the inoculated soil matrix. The RT-qPCR (reverse

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transcription polymerase chain reaction) and qPCR (quantitative polymerase chain reaction), are another methods applied for quantitative and qualitative assessment of microbial population. In bioaugmentation, these methods have been applied to check the abundance of Nocardia sp. H17-1, an oil degrading bacterium from oil contaminated soil[22] and also quantified a genetically engineered Pseudomonas putida bacterial strain labelled with green fluorescent protein during degradation activity of 2-chlorobenzoate in soil[23].

2.2. Biostimulation To speed up the metabolism of a competent microbe for degrading the contaminant efficiently, favourable changes in some parameters e.g., oxygen, water, change in pH, available phosphorous and nitrogen sources are needed to be applied in order to improve the bioremediation process. These strategies involve the addition of nutrients i.e. biostimulation, holds the promise of accelerating the degradation rates. Perfumo et al.[24] described biostimulation as the addition of nutrients, oxygen, changes in pH and temperature, etc. to the contaminated site for enhancing the mass activity of native microorganisms available for bioremediation. Thus, biostimulation boost up the rate of degradation on the polluted sites, by adding the limiting nutrients to the system of the inhabiting microbial population. Biostimulation has a significant role in providing favourable conditions, especially in activating microbial population which acclimatized themselves in their habitats due to the constant exposure of pollutant at the contaminated sites. Some examples of Bioaugmentation and Biostimulation in fields: 1. Application of bioaugmentation and biostimulation with enriched microbial culture for crude-oil degradation on Zvulon beach in Israel in stimulating the site using hydrophobic fertilizer (F-1) as Phosphorus and Nitrogen[25]. 2. Bioaugmentation of microbial inoculation and biostimulation with inorganic mineral nutrients to degrade crude oil contaminating a beach in Delaware, USA[26]. 3. MECHANISMS OF MICROBES AND POLLUTANT INTERACTION

3.1. Biosorption and Bioaccumualtion Biosorption is defined as a physiochemical interaction between metal species and microbial biomass where the passive adsorption of dissolved metals on the microbial biomass occurs due to their chemical activities, providing the strength for the new biosorption technology for metal removal and recovery[27,28]. The fungal cell wall has some important components like

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chitin (polymer of N-acetyl-D-glucosamine) and chitosan (-1, 4-polymer of D-glucosamine) and acts as barrier to toxic metal species through adsorption[28,29]. Biosorption capability of fungal biomass is found effective towards several metals[30] in comparison to bacteria. Melanins (fungal pigments found interior or exterior to cell wall) are also playing an important role in binding with toxic metals as it contains phenolic group, aliphatic hydrocarbons, peptides, carbohydrates, and fatty acids and these groups carrying many metal binding sites[31]. These metal binding active sites can be identified using some techniques like scanning electron microscopy (SEM), electron dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy, transmission electron microscopy (TEM), Xray diffraction analysis (XRD), nuclear magnetic resonance (NMR). The bioaccumulation of heavy metals by fungi has received more attention in recent years. Many of studies are being taking place in concern with environmental protection and recovery of metals by using hidden potential of diverse fungi. The biological mechanism of fungi for removing metals can has three ways to detoxify metals: (1) bio-sorption of metal ions on the cell wall of fungi; (2) intracellular uptake of metal ions and their sequestration into vacuoles and (3) biological transformation of metal ions by redox reactions catalysed by fungi. The marine fungi Monodictys pelagica and Corollospora lacera have been found to bioaccumulate cadmium and lead extracellularly into their mycelia[32]. The studies on genetic engineering of biosorbents have been conducted involving the cloning of metallothioneins(MTs) proteins for their intracellular expression in bacteria. Metallothioneins (MTs), is a family of metal-chelating low molecular mass proteins and are able to well coordinate and chelate a variety of heavy metal ions. These proteins are thought to play the most imoportant role in offering the protective mechanism to eukaryotes by protecting th emselves from the c ontaminants. The cloning of metallothioneins from yeast are fused to glutathione S-transferase in E. coli, together with a nickel (Ni) transporter from bacterium Helicobacter pylori, where the results produced nickel accumulation has increased by three times in respect to cells expressing metallothioneins but not the transporter [33] . Similarly, an enhanced mercury bioaccumulation was reported in genetically engineered bacteria, which was created for simultaneously expressing the MerT-MerP mercury transporters, together with the metal-binding peptides within the cytoplasm than on their cell surface[34,35]. 3.1.1. Complexation The removal of metals from solution by a complex formation between metal and the active groups present on the cell wall of microbes, is another

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mechanism. The groups which are actively involved and present on cell wall are carboxyl, thiol, amino, hydroxyl, phosphate, etc., can interact in a coordinated way with heavy metal ions[36]. A complex compound is a molecule consisting of one or more metal ions present in the centre surrounded by other atoms or groups, usually of negative or neutral charges (ligands) and this process of complexation plays an important role in sorbate (heavy metal ions)–sorbent (microbial biomass) interactions. 3.1.2. Precipitation Precipitation is a result of the bioreaction between metal(s) and metabolites produced by microbes as a result of their defence system against a heavy metal pollutant. In this case, microorganisms sense the toxicity of metals and start producing compounds/metabolites that favour the metal precipitation process in the form of a compound[37]. 3.1.3. Redox reaction The toxicity and mobility of a metalloid within the soil is dependent on various chemical and physical parameters along with the redox state of metal. Oxidation and reduction reactions catalysed by microbes are also responsible for metal speciation. Depending on the metal species, redox reactions may also result in metal immobilization. For example, chromium species Cr(VI) is dominant species in the aqueous environment between pH 6.5–9 and is highly toxic exhibiting carcinogenic and mutagenic effects on living organisms due to their strong oxidising nature, whereas Cr(III) is found to be less toxic and less mobile below pH 5 as insoluble forms of oxides and hydroxides. The enzymatic reactions mediated by various microbes like Desulfovibrio desulfuricans, Pseudomonas sp., Bacillus sp. and E. coli responsible for catalyzing reduction of soluble Cr(VI). This aerobic reduction process is considered as detoxification mechanism of microbial cells which uses their soluble enzymes to reduce Cr(VI) to Cr(III) either internally or externally to the plasma membrane. Similary, reduction of Se(VI) to elemental selenium[38]; reduction of U(VI) to U(IV)[39]; and reduction of Hg(II) to Hg(0)[40] are some well proved detoxification mechanisms carried out by microorganisms.

3.2. Biomethylation/Biovolatilization Describes the mechanism of forming both volatile and non-volatile methylated compounds of metals and metalloids. Biomethylation of heavy metals can be mediated by a range of microbes[41,42] and it is thought to be a detoxification mechanism in case of arsenic a heavy metalloid[43,44].

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Microbial methylation plays a crucial role in the biogeochemical cycle of metals, where toxic metal species can be converted to less toxic forms and are volatile in nature. For example, biomethylation of mercury [Hg(II)] is reported by many bacterial species (e.g., Bacillus sp., Pseudomonas sp., Escherichia sp., and Clostridium sp.) to gaseous phase methylated product i.e., methylmercury[45]. The fungi, Alternaria alternata also found to volatilize selenium to the dimethylselenide[46]. Methylation of arsenic in fungi is carried out by S-adenosylmethyltransferase using the (S-adenosyl methionine) SAM as methyl group donor. The product of this reaction is a pentavalent monomethylarsonic acid MMA(V), which is reduced by an arsenate reductase enzyme into trivalent monomethylarsenous acid MMA(III) with thiols (e.g., glutathione). The continuous methylation and reduction steps produce di and tri-methyl compounds including formation of dimethylarsinic acid DMA(V), dimethylarsinic acid DMA(III), trimethylarsine oxide (TMAO) and finally trimethylarsine (TMA) as the final product which is highly volatile in nature[47]. The identification of genes encoding for homologs of As (III) methyltransferase (ArsM) in fungi e.g. in Westerdykella aurantiaca has been done. This ArsM gene has been previously isolated from the soil bacterium Rhodopseudomonas palustris and has shown As (III) resistance when expressed in arsenic sensitive strain of E. coli[48]. Moreover, Meng et al.[44] put forward an interesting fact that phylogenetic analysis has shown that bacterial ArsM is more closely related to fungal ArsM. Also, arsenic biomethylation has been found in other bacteria, such as Pseudomonas sp and Halobacterium sp. NRC-1[49]. 4. APPLICATIONS OF MICROBES COUPLED WITH BIOTECHNOLOGY FOR REMEDIATION OF ENVIRONMENTAL POLLUTION

4.1. Heavy Metals The term “heavy metals” designates to an element that has high density greater than 5 g/cm3 and can be toxic or poisonous even at very low concentrations [50–52] . Heavy metals are persistent environmental contaminants since they are non-biodegradable and occur naturally in the earth crust. There are a number of anthropogenic activities exists due to rapid industrialization like a disposal of heavy metal, fertilizers, paints, sewage sludge, waste water from agricultural run-off, coal combustion residues, spillage of petrochemicals, and atmospheric deposition. The most common heavy metals found at contaminating the environment are lead (Pb), arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), zinc (Zn) and nickel (Ni). Heavy metal contamination of soil and water, raising health hazards to humans and the ecosystem through: direct ingestion with contaminated water and in directly through agricultural soils

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and to the food chain. It is directly or in directly increasing toxicity levels in drinking water, irrigation groundwater, agricultural soil, food chain, crops, etc. marking a big question on food safety, reduction in food quality, economical issues around the world[50]. Therefore, the development and implementation of cost-effective strategies for the removal of these heavy metals are essentially required to minimize the environmental pollution. Several studies reporting the potential of both living and dead microbial biomass for accumulating heavy metals by adopting different mechanism. Dead biomass has more advantages than living biomass, as it can be obtained easily and inexpensively as waste, which is not subjected to metal toxicity. Some of examples are given in the Table 1. Table 1: Microbes with their heavy metal removal efficiency. Microbial biomass

Heavy metal

Removal efficiency (%)

References

Bacillus sphaericus

Cu(II) Ni(II) Cr(VI) Cr(VI) Pb(II) Pb(II) As(V) Zn(II) Pb(II) Zn(II)

82.20% 59.46% 76.50% 89.87% 79.40% 80.70% 65.81% 12% 98.85% 73%

[55]

Bacillus cereus Aspergillus niger Trichoderma reesei Lasiodiplodia sp. FA-13 Fusarium sp. Penicillium austurianum Streptomyces ciscaucasicus

[56] [57] [57] [58] [59] [57] [60]

The advancement of biotechnological applications has led the possibility of engineering within the genomes of promising microbes for more effective bioremoval of target toxic metals. Recently, Liu et al.[53] in their studies have demonstrated that arsenic volatilization can be done by employing genetically engineered bacteria, whose genome is modified by Sadenosylmethionine methyltransferase gene (arsM) from the contaminated soils. The gene has been found to methylate inorganic arsenic (AsIII, AsV, etc.) by adding a methyl group (CH3), converting into less toxic volatile trimethylarsine (TMA). These findings suggested that over-expression of arsM genes in Bacillus idriensis and Sphingomonas desiccabilis resulted into many fold increase in the release of methylated arsenic gas, which is less toxic than other inorganic arsenic species when compared wild type strain activities. Similarly, the recombinant Caulobacter crescentus strain JS4022/p723-6H, expressing RsaA-6His fusion protein was able to remove 94.3–99.9% of the Cd(II), whereas the control strain could remove only 11.4– 37.0%[54]. As discussed before on enhanced nickel accumulation reported by Krishnasamy et al.[33] where they constructed an E. coli strain incorporating nixA gene which encodes for nickel transport system for Ni2+ accumulation from Helicobacter pyroli into E. coli JM109 that expressed S-transferasemetallothionein fusion protein.

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4.2. Waste Water The era of industrial revolution is one of the most key factor responsible for generating environmental pollutants. Due to this industrialization and urbanization, the concern has been increased around the world regarding environmental pollution. Wastewater can originate from a combination of domestic, industrial, commercial or agricultural activities, surface runoff or storm water, and from sewer inflow or infiltration. A number of methods available that can be used to clean up wastewaters depending their types and level of contamination. It can be easily treated in Wastewater Treatment Plants which include physical, chemical and biological treatment processes. The main objectives of waste water treatment processes are summarized as follows: 1. 2. 3. 4. 5.

Reduction of biodegradable organic contents (BOD) Removal of recalcitrant organics Removal of toxic heavy metals Removal and inactivation of pathogenic microorganism and parasites. Removal of compounds containing nitrates and phosphates.

Apart from applying traditional methods, recent advances and development in the fields microbiology, biochemistry and biotechnology have changed the way of treating wastewater targeting at eco-friendly low cost services e.g., wastewater bioreactors. Microbial fuel cells (MFCs) presents a novel method for treating the wastewaster generating from various industries, refineries, for simultaneous production of bioelectricity. A MFC is a bioreactor that converts chemical energy from organic compounds present in wastewater into electrical energy catalysed by microorganisms under anaerobic conditions. In this system, bacterial cells are being used as a catalyst which oxidizes the organic and inorganic matter from wastewater. Here, the negatively charged electrons are produced by the bacteria using glucose as substrates and are transferred towards the cathode (positive terminal) from the anode (negative terminal). Conventionally, current flows from the positive to the negative terminal, in a direction opposite to that of electron flow through resistor[61]. Detailed working of MFC is shown in the following figure (Fig. 2). Bacteria feeds on organic matter (oxidaton) in the anode chamber producing and transferring electrons through mediator to the anode electrode. During electron production, protons are also produced in excess and migrate towards the cathode chamber through ion exchange membrane. Aerobic conditions maintained in cathode chamber by supplying oxygen where electrons, protons and oxygen combine to form water. The mediator job is to gather the electrons produced by bacteria from their lipid membrane and then shuttles them to the anode.

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Schematic diagram of Microbial Fuel Cell

The present global scenario reflecting that the world is facing severe energy crisis which has led academic researchers to develope their interests in production of MFCs, as a direct way to generate electric power (green power) from bacterial biomass using glucose as substrate without a net carbon emission into the ecosystem[62,63]. Mosqsud et al.[64] generated, a compost-based microbial fuel cell that generates bio-electricity by biodegradation of organic matter. The applied genetic engineering methods for heavy metal removal from wastewater has aroused with great interest for researchers to deal with the industrial wastewater in sustainable way. A gram-negative bacillus, Alcaligenes eutrophus strain AE104 (pEBZ141) has been reported for removing chromium from industrial wastewater[65]. In a similar way mercury (Hg2+) removal from heavy metal wastewater using recombinant photosynthetic bacterium, Rhodopseudomonas palustris was also reported. Sriprang et al.[66] has introduced gene for enhancing phytochelatin synthase enzyme (PCS; PCSAt) activity into Mesorhizobium huakuii sp. and which produces phytochelatins on sensing cadmium metal toxicity. Formation and sequestration of phytochelatin and cadmium complex occurs under the control of bacteroid specific promoter, the nifH gene. Paper and pulp mills, textiles, tanneries and distilleries are the major industries, which releasing highly coloured wastewaters effluents containing recalcitrant dyes. The contamination of waterbodies with these industrial effluents containg dyes (xenobiotic compounds-comprised of known carcinogens, such as benzidine and other aromatic compounds), posing a serious environmental problem and a public health concern. Environmental

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pollution due to dyes has increased, spreading their toxicity and carcinogenicity within living organisms. Various microbes including bacteria, yeasts, algae and fungi, have been used for decolourization and degradation of synthetic dyes. Microbes not only decolorizes the synthetic dyes but also helps in breakdown of dye molecules into simpler forms. Therefore, herewith “Bio-degradation can be defined as an energy-dependent process, involving the breakdown of dye into various by-products through the action of various enzymes like laccase, oxidoreductases, azoreductase, horse radish peroxidases”. Malachite Green was used as source of carbon in the decolorization medium. Alternaria solani and Aspergillus flavus fungi can decolourized a solution contaminating with a dye at 30 µM concentration by 99.78% and 91.72% respectively, within 6 days[67], Aspergillus niger decolorized direct red 81 and reactive red 120[68] Fusarium solani decolourized crystal violet[69], Phanerochaete chrysosporium can decolourized multiple dyes upto 93 to 100%[70], Penicillium ochrochloron decolourized cotton blue [71] . Similarly, there are many bacteria also reported for dye decolourization for example Crystal violet, Basic fuchsin, Brilliant green, Malachite green, Great red GR which have been reported to be decolorized by Aeromonas hydrophila[72], Methyl red, Congo red, Malachite green, Crystal violet, Gentian violet by Citrobacter sp. [73] , Scarlet R by Micrococcus glutamicus[74], Crystal violet by Pseudomonas putida[75]. The dyes Reactive brilliant red K-2BP, Acid mordant red S-8 and Reactive brilliant blue X-BR are found to decolorized by yeasts Candida krusei and Pseudozyma rugulosa[76], Crystal violet, Malachite green, Methyl violet, Navy blue HER by yeast Trichosporon beigelii[74].

4.3. Bioconversion of Organic Wastes The bioconversion of organic waste materials is considered to be of great importance in aspect of environmental pollution. It accounts for the development of sustainable biotechnology processes in the near future because of its favourable economics, low capital and energy cost, minimizing environmental pollution/problems. The production of usable products from agricultural and industrial waste is therefore, a feasible and favourable option. A high quantity of waste materials is being released through various industrial activities such as those in the forestry, agriculture and municipal sectors. These agro-industrial wastes substrates include distillery and whey wastes, plant oil extracts, oil mill effluent, kitchen waste and cassava wastewater that can be used as feedstock for biosurfactants production. 4.3.1. Production of biosurfactants The high rates of bio-degradation can be achieved frequently on using microbes in consortia form than as individual. As soon as, the microbes

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senses the organic material, their metabolic mechanism starts working for degrading the wastes. While performing their jobs, these microbes enhance their hidden potential to reach the contaminant more efficiently by secreting surface active agents that reduces the surface tension. Universally a typical, biosurfactant is consisting of two parts, a polar (hydrophilic) group like polysaccharides, proteins and a non-polar (hydrophobic) group saturated, unsaturated and hydroxylated fatty acids. Biosurfactants have a wide range of properties like lowering of surface and interfacial tension of liquids, the ability to form micelles and micro-emulsions between two different phases, the ability to increase the surface area of water-insoluble substances, and thus increase the water bioavailability of such substances. According to Banat et al.[77] the best-studied biosurfactants are glycolipids like rhamnose lipids, sophorolipids, trehalolipids and mannosylerythritol lipids (MELs). Where rhamnose lipids are reported to be produced by Pseudomonas aeruginosa, which has been used up by Jeneil Biosurfactant, USA as fungicidal additive to enhance bioremediation in agriculture. They have also metioned about genetic manipulation within Bacillus sp. and Acinetobacter sp. which may able to grow on various substrates producing high amounts of biosurfactants. Biosurfactants increases the bio-availability of PAHs resulting in enhanced growth of the microorganism feeding on organic contaminant. Berg et al.[78] reported Pseudomonas aeruginosa UG2 producing biosurfactant and thus inc reases the solubility of an aromatic c ompound hexachlorobiphenyl which was added to the soil, resulting in the compound recovery by 31%. Churchill et al.[79] discussed the properties of rhamnose lipids biosurfactants produced by bacteria, applying in combination with the oleophilic fertilizer, Inipol EAP-22 (responsible for carbon-dioxide evolution and increasing chemoheterotrophs population), accelerates the rate of degradation of benzene, hexadecane, toluene and naphthalene in the aqueous-phase bioreactors and in the contaminated soil.

4.4. Radionucleolides A radionuclide is an isotope with an unstable nucleus having large amount of energy available to be transferred either to a newly created radiation particle within the nucleus or via internal conversions. The commonly encountered radionuclides in the environment includes radium-226 (226Ra), cobalt-60 (60Co), technetium-99 (99Tc), plutonium-239 (239Pu), radon-222 (222Rn), and thorium-232 (232Th). Exposure to these radionuclides causes acute health effects that begin with some primary symptoms like vomiting, nausea and headaches. On increasing radionuclide exposure, a person may show symptoms like weakness, fatigue, fever, disorientation and diarrhoea, elevated risk of leukaemia, hair loss, blood in stool, leucopenia, kidney and

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genetic damages, which can result into lethal health problems and can be transferred to new generation and ultimately can cause death. Foetuses are also affected by radiations at the cellular level, which can result in small brain size, abnormal growth, poorly formed eyes and mental retardation[80,81]. Bioremediation of radionuclides from soil, sediments and water aquifers can be achieved biologically by using active microbes. Radionuclides can be immobilized by direct or indirect enzymatic reactions through oxidation– reduction, changes in pH, biosorption by cell biomass, biodegradation of radionuclide[82] . Wildung et al. [83] reported an enzymatic reduction of Uranium (VI) on the surface of the microorganism Shewanella putrefaciens. A c-type cytochrome of 9.6 kDa mass was observed in the periplasm of Shewanella putrefaciens required for U(VI) reduction. Khani et al.[84] uses the adsorption ability of brown marine alga Cystoseira indica of radionuclide U(VI) and also discussed the enhanced adsorption efficiency of radionuclides upon pre-treatment of the alga with calcium. Several microbes including Citrobacter freundii and Firmicutes have been reported as radionuclide biosorbents[86]. Bioremediation of Uranium(VI) with ethanol-biostimulation on active microbial site is an alternative approach that can effectively reduce about 87% of U(VI)[86]. An extremophilic bacterium Deinococcus radiodurans is well known as “radiation resistant” have been studied to detoxify Cr(VI), U(VI) and Tc(VII) by reduction reactions within soil[87] . A genetically engineered Deinococcus radiodurans strain was developed by cloning gene merA shows ability to extract and utilize energy from the catabolism of mercury and toluene as substrate for its growth[88]. In order to identify the roles of genes, enzymes and proteins in the bioremediation of radionuclides, it is important to study the functional and structural interactions of proteins with other metabolites using advanced genomics and proteomics techniques[89]. Recent advances in next-generation sequencing, the study of genomics and proteomics has become more accessible and allows the expression of proteins of interest into an organism on radionuclide stress for bioremediation practice. The 2DE and MALDI-TOF MS analysis of this radioresitent bacterium, D. radiodurans, 31 radiation sensitive proteins were significantly found upregulated, including PprA and RecA, which are well known for DNA repair[90] and conferring resistance. In a comparative genomics study, the organism Thermococcus gammatolerans was observed to be radioresistant among archaea, expressing a thioredoxin reductase (tgo180), peroxiredoxins (tg1253 and tg1220) and glutaredoxin-like protein (tg1302) which allowed the organism to resist radionuclides stress[91].

4.5. Polycyclic Aromatic Hydrocarbons (PAH) Polycyclic aromatic hydrocarbons (PAH) are persistent organic pollutants with adverse effects on the environment. They have been detected in several

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terrestrial and aquatic ecosystems as persistent xenobiotics. PAH are mainly consist of more than three benzene rings arranged in angular, linear or cluster pattern. Due to their strong negative resonance energy, they are thermodynamically stable. They have a tendency to adsorb and accumulate in sediments also due to their non-polar nature and low-vapour pressure. It has been found that high-molecular-weight PAH are more recalcitrant to microbial attack than low molecular-weight PAH. The major sources of PAH contamination in sediments include petroleum transportation, atmospheric deposition, refineries, marine seeps of petroleum. Several researchers have discussed the role of various species of bacteria like Alcaligenes, Rhodococcus, Staphylococcus, Pseudomonas, Beijerinckia, Mycobacterium, Arthrobacter, Gordona and Nocardia in potential degradation of high-molecular-weight PAHs. Bacterial degradation of PAHs have been reported by Brezna et al.[93] using Mycobacterium sp. PYR1. Recently, Vila et al.[93] have isolated a methylotrophic bacteria Methylophaga strain AF3, from a beach contaminated by the oil spill. It showed enhanced growth in seawater medium amended with polycyclic aromatic hydrocarbons (PAH) as as energy substrate. Researchers also discussed the potential of algal and cyanobacterial metabolism in PAH degradation[94,95]. A report on red (Porphyridium cruentum), green (Chlorella autotrophica strain 580) and brown (Petalonia fascia) algae and cyanobacteria (Oscillatoria, Nostoc, etc ) are found to oxidize naphthalene to 1-naphthol[96]. A marine cyanobacterium Agmenellum quadruplicatum PR-6, has also reported for the transformation of phenanthrene to different metabolites like 9,10-dihydrophenanthrene,1methoxyphenanthrene and trans-9,10-dihydroxy[97]. Benzo(a)pyrene has been completely metabolized by green algae Scenedesmus acutus, Selenastrum capricornutum and Ankistrodesmus braunii to dihydrodiols under white light[98]. While degrading PAH, bacteria are falls apart as they are unable to degrade PAH having more than four aromatic rings whereas fungi have the highest capability to degrade and mineralize PAH[99] due to their mycelial mobilization towards the contaminant. A diverse group of fungi has been reported to oxidize PAH ranging from two to six aromatic rings. Like the ascomycetes: Penicillium sp. and Aspergillus niger and the zygomycete: Cunninghamella elegans and the white-rot basidiomycetes: Trametes versicolo, Phanerochaete chrysosporium, Bjerkandera sp. and Pleurotus ostreatus. It was reported by Brodkorb and Legge[100], that P. chrysosporium can be used as supplement for enhanced in situ biodegradation of PAH in oil tar-contaminated soil obtained from a former oil gasification plant. Llanos and Kjoller[101] have observed that after the amendment of soil with oil-waste, the native fungi increase their mycelial biomass and become dominant by increasing their population specially belonging to the genera:

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Graphium, Fusarium, and Penicillium. Fernandez-Luqueno et al.[102] reported that expression levels of genes that code for degradative enzymes from host microbes increases after their exposure to hydrocarbon pollution, and that these genes are selected during adaptation to the new PAH-rich environment. Peng et al.[99] suggested that a consortia of white rot fungi and bacteria could establish and function well at PAH contaminated sites for its degradation, as the fungi breaks down the larger molecules of PAH into low molecular weight PAH, on which bacteria can easily act for their further degradation.

4.6. Explosives 2, 4, 6-trinitrotoluene (TNT) and hexahydro-1, 3, 5-trinitro-1,3,5-triazine (RDX) are major explosives and major environmental pollutants. US Environmental Protection Agency has classified it as a Class 1 carcinogen. TNT has toxicity towards all organisms and exerts its toxicity in soils by binding to the organic matter present in it which is recalcitrant to degradation. Similarly, the widespread presence of RDX, as a common explosive to be used in military exists and has been raised a concern because of its toxicity and recalcitrance to degradation. However, RDX, unlike TNT, is comparatively mobile in the soil, which leaches into the groundwater, resulting in the building up of RDX in aquifers, with high rates of accumulation, contaminating the drinking water supplies in military sites. In order to investigate the potential of bio-organisms like plants to remove RDX from contaminated soil and water, Rylot et al.[103] has opted for an engineering technology where they have used Arabidopsis thaliana as a model plant to express a gene xplA encoding for RDX-degrading cytochrome P450 isolated from bacteria. They demonstrated that the P450 domain of xplA is fused to a flavodoxin redox partner and which catalyses the degradation of RDX in anaerobic conditions. Their results also showed an important finding that A. thaliana expressing xplA was grown in RDXcontaminated soil, producing more shoot and root biomasses than of wildtype plants. Thus, suggesting that expression of xplA in A. thaliana plants may provide a suitable remediation strategy for sites contaminated by RDX. The gene xplA has been also traced in Microbacterium sp, Rhodococcus and Gordonia sp.[104]. TNT (trinitrotoluene) is another explosive having both toxic and mutagenic effects on organisms. These are highly oxidized molecules and reduction of the nitro groups of TNT was undertaken by microorganisms in aerobic and anaerobic conditions. Enzymes from “Old Yellow Enzyme” family of flavoproteins such as pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2 and the xenobiotic reductase XenB from Pseudomonas fluorescens I-C are found to act as nitroreductases and also helps in biotransformation of TNT with the release of nitrite. A new approach of genome shuffling has been used to improve biotransformation rate of TNT in Stenotrophomonas maltophilia by Lee et al.[105].

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4.7. Dioxins and PCBs Dioxins are environmental pollutants and they belong to a group of dangerous chemicals known as persistent organic pollutants (POPs). They are known to be called “dirty-dozen” because of their highly toxic potential. Their half-life in the body is estimated to be persist upto 7–11 years. Polychlorinated dibenzo dioxins (PCDDs) and furans (PCDFs) are chlorinated, planar, aromatic compounds containing two benzene rings and polychlorinated biphenyls (PCBs) are examples of persistent organic pollutant (POPs). PCDD/Fs are formed as a result of combustion processes of municipal wastes and are also by-products of some chemical reactions such as chlorine bleaching of paper and pulp. Manufacturing of some herbicides, chlorinated pesticides, wood preservatives, fungicides and textile dyes are also sources of PCDD/Fs[106]. Removal of these pollutants from the environment has become a difficult task due to their persistent and ubiquitous nature. Biological degradation (biodegradation) is considered as a feasible approach to remove dioxins and also a low-cost alternative to other expensive physico-chemical approaches. There are several microbial mechanisms found responsible for biodegradation of dioxins, including oxidative degradation by dioxygenase-containing aerobic bacteria, bacterial and fungal cytochrome P-450, fungal lignolytic enzymes, reductive dechlorination by anaerobic bacteria, and direct ether ring cleavage by fungi containing etherase-like enzymes. A group of authors have deeply studied biodegradation of chlorinated PCDDs/PCDFs by various strains of bacteria, such as Psudomonas veronii PH-03, Rhodococus opacus SAO101, Bacillus megaterium AL4V, Beijerinckia sp. B8/36, Psudomonas sp. HH69, EE41, CA10, Sphingomonas sp. HL7 and RWI[107–108]. The gram-negative bacterium Sphingomonas wittichii RW1 possess the bioremediation ability to biotransform toxic polychlorinated dioxins by utilizing dibenzofuran and dibenzo-p-dioxin as a growth substrate[109]. The changes in protein expressions of Sphingomonas wittichii RW1 was observed on dioxin metabolism using difference gel electrophoresis (DIGE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)[110]. Yoshida et al.[111] also observed that Dehalococcoides like bacteria were responsible for the complete dechlorination of dioxins in a microbial consortium. Polychlorinated biphenyls (PCBs) are another threat to environment and to human health due to their persistent and bioaccumulative properties. They were mainly used as coolants and lubricants in electrical equipment. According to the Stockholm Convention on Persistent Organic Pollutants, PCBs are currently targeted for worldwide elimination and strongly recommended to be disposed by 2028. The conventional methods for PCB degradation requires a high-heat, pressure and basic conditions[112]. The

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first data on the aerobic bio-degradation of PCBs was reported by Ahmed and Focht[113] where conversion of biphenyl and monochlorobiphenyl to chlorobenzoic acid was achieved by two species of Achromobacter. In case of PCBs, pyrosequencing analysis showed that the bacterial community belonging to the genera Sphingomonas, Bulkholderia, Arthrobacter, Bacillus have been reported for PCBs removal abilities. The fungal community was mostly represented by Basidiomycota and Ascomycota, which accounted for >80% of all the sequences detected in the three soils. Fungal taxa for PCBs biodegradation potential belong to genera Paxillus, Cryptococcus, Phoma, Mortierella[114]. Panellus stypticus strain 99–334 is found as new effective dioxin degrader due to its 100% reported efficiency to decrease in 2,7-DCDD levels after 40 days of exposure[115]. Nakamiya et al.[116] have isolated a fungus Acrymonium sp. strain 622 (later identified as Pseudallescheria boydii) from denitrifying activated sludge. This strain grew under aerobic conditions and effectively degraded highly chlorinated dioxins [TCDD-OCDD] by oxidation of aromatic rings followed by dechlorination. Some dechlorinating bacteria Desulfomonile tiedje, Dehalobacter restrictus, Dehalospirillum multivorans, Desulfuromonas chloroethenica, Desulfitobacterium dehalogenans and Dehalococcoides ethenogenes are capable of dehalogenating PCB which can be used in consortia form with some potential fungi. Phanerochaete chrysosporium is also one of the most characterized white-rot fungi studied for dioxin biodegradation potential using 2,4,7,8-TCDD[117] along with some other genera Aleurodiscus, Bjerkandera, Ceriporia, Panellus, Phlebia and Pleurotus. Phytoremediation is another efficient technique to remove or degrade various pollutants using plants or their parts in soils, water and sediments, and provides some new possibilities for reducing toxicity due to dioxins and PCBs as reviewed by Campanella et al.[118]. Recently, Sonoki et al.[119] developed a transgenic plant capable of degrading dioxins. They cloned genes responsible for laccase and lignin degradation from white-rot fungi Phanerochaete chrysosporium and Trametes versicolor and recombinantly expressed the gene products in Arabidopsis thaliana which is useful in biodegradation of PCBs.

4.8. Cyanide Cyanide is highly toxic chemical and exerts its toxicity to most of the living organisms through inhalation, dermal absorption, ingestion. It attacks living cells by binding strongly to the metalloproteins present within it[120]. Majorily, it has two forms, namely as a hydrogen cyanide (HCN) which disintegrates into an anionic cyanide molecule (CN) in the solution. The environmental

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sources of cyanide are through release of large amounts of cyanide via industrial activities, such as production of organic nitriles, acrylic plastics, nylon, paints, dyes, etc. and the use of cyanide in gold mining and the metal and jewellery industries. Chemical treatment of cyanide removal from environment are very expensive and generate toxic products, therefore, cyanide biodegradation can be an alternative and eco-friendly treatment. Bacteria such as Rhodococcus sp. and Nocardia sp; fungi such as Fusarium sp. and Aspergillus sp; algae such as Arthrospira maxima and Scenedesmus obliquus, possess enzymatic mechanisms to detoxify and bioremediate free cyanide and other cyanide complexes[121,122]. Kunz et al.[123] have discussed the mechanism of cyanide degradation by bacterial strain Pseudomonas fluorescens NCIMB 11764 by releasing of pyruvate and 2-oxoglutarate to the medium, and formation of their cyanohydrins via cyanide oxygenase enzyme. LuqueAlmagro et al.[124] also reported that an alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 which has potential to grow with cyanide, consuming it as the nitrogen source for catalysing the production of oxaloacetate from L-malate. 5. ADVANCED BIOTECHNOLOGICAL APPLICATIONS FOR ENVIRONMENTAL POLLUTION

5.1. DNA Microarray Microarray technology is a developing technology used to study the gene expressions about how organisms and cells adapt themselves to external environmental changes. The technology has a unique way to read expressions of genes by putting them in known locations on a glass slide called a gene chip. A nucleic acid sample containing DNA or RNA is placed on the gene chip and hybridization occurs (complementary base pairing) between the target sample and the gene sequences on the chip producing fluorescence, that can be measured. The fluorescence producing locations on the gene chip may indicate the expressed genes within the sample. Best results can be obtained if fluorescent dyes can be chemically or enzymatically incorporated into the sample which get hybridized; and therefore, the readout of the microarray is based on the detection of a fluorescence signal. The microarray technology is potentially well suited for identifying microbes population in natural environments. Lemarchand et al.[125] have used DNA microarray technology for microbial monitoring of water and also to detect the presence of pathogens. Using glass-based two-dimensional microarrays, Small et al.[126] has detected metal-reducing bacteria, such as Desulfovibrio desulfuricans and Geobacter chapellei. Momose and Iwahashi[127] obtained useful information about cadmium toxicity and observed that all genes corresponding to sulfur metabolism in bacteria were up-regulated by cadmium.

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5.2. Environmental Biosensors A bio-sensor is an analytical device formulated by the synergistic interaction of two fields i.e., biotechnology and microelectronic where it is composed of a biosensing component with a transducing element that produces measurable signals. According to the International Union of Pure and Applied Chemistry (IUPAC), a biosensor is an integrated device that is capable of providing analytical information using a biological recognition element (e.g., biochemical receptors), which is connected to a transduction element. It basically comprises of three main components: a biocomponent (e.g., enzymes, immunoaffinity recognition elements, whole cells of microorganisms, plants or animals, or DNA fragments), a transducer (electrochemical, optical, piezoelectrical or thermal) and an amplification unit. Applicability of biosensors is that it can be developed for various environmental pollutants like xenobiotics, heavy metals, etc. and even can also applied for detecting human health issues. Pesticides, heavy metals and food contaminants have been majorily focused target for biosensor construction[128]. The use of biosensors for environmental purposes have made a significant advancement towards monitoring of pollutants at contaminated sites as they have the capability to detect the pollutants by interaction with the biological systems through highly sensitive biorecognition processes (signals). The contamination due to heavy metal toxicity has been reported as major environmental and human health threat, as these are non-biodegradable. The metal which is mostly involved in contaminating the environment are: lead, zinc, chromium, copper, mercury, arsenic[129]. In recent scenario, researchers focus has been shifted towards the development of biosensors from microbes like bacteria, for analysis of heavy metals in environmental samples (Table 2). The use of specific genes as biological receptors which confer resistance to bacteria against heavy metals contamination. A number of bacterial strains have been reported conferring resistant to heavy metals like mercury, cobalt, zinc, tin, copper and silver. Ravikumar et al.[129] has worked on developing a molecular biosensor for zinc and copper. They applied the use of specific zraP and cusC promoter, whose expression is sensitive to zinc and copper concentration at 200 µM and greater. The promoters can be turned on or off with the help of specific molecules, hence they provide the required specificity in signal generation. The generated signals are directly proportional to the expression of the promoter. Jansson et al.[130] reported an introduction of a biomarker or marker gene, into an organism, which provides variations in genotypic or phenotypic traits to their facilitate monitoring in a given environment. Brayner et al.[131] has discussed microalgae based biosensors in a review. He mentioned about three groups of micro-algae, which are chlorophyta, cyanobacteria, and diatoms. Durrieu and Tran-Minh[132] has developed an optical biosensor to detect heavy metals like cadmium and lead by inhibiting the activity of alkaline phosphatase

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enzyme in Chlorella vulgaris microalgae, as biological recognition element present on the external membrane. Naturally occurring bioluminescent fungi like Mycena citricolor and Armillaria mellea were also used to develop a bioluminescence-based biosensor for detecting the toxicity levels of copper, zinc, (3,5-dichlorophenol (DCP) and pentachlorophenol (PCP)[133]. Chen et al. [134] has developed a biosensor for the determination of nitrite contamination in the environment. They used the sensitivity of cytochromec nitrite reductase (ccNiR) enzyme present in sulphate reducing bacterium Desulfovibrio desulfuricans.The process involves by immobilizing the bacterium and its attachment to a carbon electrode by entrapment into redox active (ZnCr-AQS) double layered hydroxide containing anthraquinone2-sulfonate (AQS). This instrument/arrangement was very sensitive to nitrite and responded within 5 seconds with a linear range of nitrite concentration at 0.015 and 2.35 µM and a detection limit of 4 nM. Table 2: Some bio-sensors developed for environmental contaminants Environmental contaminant

Biocomponent

Transducing element

Ref. (s)

Cadmium

DNA and phytochelatins

Electrochemical

Zinc, Copper, Cadmium and Nickel

Pseudomonas fluorescens 10586s Optical (luminometer)

[137]

Mercury arsenite

Pseudomonas fluorescens (with sensors plasmids)

Optical (bioluminescence)

[138]

Cadmium, Copper and Mercury

Enzyme (acetyl cholinesteraseAChE and urease)

Electrochemical and optical signal

[139]

Nickel ions

Bacillus sphaericus strain MTCC5100

Electrochemical

[140]

Arsenic

luxAB gene from Vibrio harveyi Optical (Bioluminein E. coli DH5 (pJAMA-arsR) scence)

[135,136]

Dioxine and dioxine- Recombinant hepatome cells like chemicals

Optical (luminescence)

[141]

Daunomycin PCBs, Aflatoxin

DNA

Electrochemical

[142]

PCBs

Antibodies

Optical (fluorescence)

[143]

BOD

E. coli with Vibrio fisheri genes lux AE.

Electrochemical

[144

Polycyclic aromatic Sphingomonas yanoikuyae B1 hydrocarbons (PAHs) with E. coli

Electrochemical (amperometric)

[145]

Benzene and its derivatives

Recombinant E. coli

Optical (luminescence)

[146]

Nitrate

Recombinant E. coli

Optical (fluorescence)

[147]

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5.3. Metagenomics Metagenomics is one of the advanced methods to study genomes of the microbes without culturing them under laboratory conditions. This metagenomic technique has emerged as powerful tool and provides a way for genomic analysis of microbial DNA directly extracted from microbial communities of environmental samples. This advanced technique has overcome almost all the limitations of culture dependent studies, as it relies on extracting the nucleic acids directly from environmental samples which may constitute the entire set of microbial genomes present within the environmental sample. These methods can be used for monitoring and identification of enzymes, mechanisms and functions of microbes involving in degradation process of herbicides, xenobiotics and other pollutants[148]. The construction of these metagenomic libraries are processed by cloning of DNA fragments extracted from environmental samples in a suitable vectors like plasmid, cosmid, bacterial artificial chromosomes (BAC), followed by transformation into a suitable host strain. Now, target DNA incorporated into the vector genome, isolated and amplified and analyzed using sequence screening procedures like next-generation sequencing. Martin et al.[149] has constructed metagenomic libraries for microbial population involved in enhanced biological phosphate removal (EBPR) systems. This helps in identifying the metabolic function of these microorganism. Suenaga et al.[150] also constructed fosmid libraries from metagenomic DNA fragements recovered from sludge samples. The results obtained for microbes having extradiol dioxygenases (EDOs) activities, using catechol as a substrate. Some of the following researches revealed the successful results of metagenomic studies for environment al pollution.  Pyrosequencing of 16S rRNA gene sequences was done for bacterial communities isolated from oil contaminated samples. The sequences showed that bacteria mainly belongs to the Proteobacteria phylum, mostly of the Gamma, Delta and Alpha-proteobacteria classes[151]. The authors have also obtained the significant results for bacterial genera like Alcanivorax, Marinobacter, Clostridium, Fusibacter and Marinobacterium found to be associated in hydrocarbon degradation mechanism in environments. On the other hand, sequences showed a decline in the genera Helia commun ity after hydr ocarbon contamination indicating that this group is highly sensitive to oil pollution.  Wery et al.[152] studied bacterial species belonging to groups like Clostridiales, Bacterioidales, Bifidobacteria and the BacillusStreptococcus-Lactobacillus (BSL) dominant clade was found from wastewater treatment plant effluents in pyrosequencing results. The bacterial diversity from the resulting profile of Bacteroides,

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Clostridiales, Bifidobacterium and BSL cluster is very similar. Results indicating that B. caccae, B. adolescentis and H. filiformis as potential bioindicators of human fecal contamination in aquatic environments.  A recent research by Jia et al.[153] also characterized the heavy metal copper and cadmium resistance genes in activated sludge of tannery plant using metagenomic analysis.

5.4. Proteomics Proteomics is a well-known technique for the analysis of the protein components within the living organisms. The technique allows analysing the different levels of proteins at intracellular, extracellular, memberane and organelle in an organism. The protein gels produce the expressions of proteins and their alterations in response to changes in various biotic and abiotic stresses. These protein based investigations have been useful for identifying key proteins and subsequently their functions involved in the physiological response of microorganisms when exposed to pollutants or environmental stress. Kim et al. [154] has applied two-dimensional gel electrophoresis (2DE) method to identify PAH-induced proteins in the bacterial strain of Mycobacterium vanbaalenii PYR-1, having a potential to use polycyclic aromatic hydrocarbons for their growth. The proteomic profiles showed the enzymes involve in PAH degradation like dioxygenases, catalaseperoxidase, putative monooxygenases and aldehyde dehydrogenase that were induced in M. vanbaalenii PYR-1. In another similar study by Yun et al.[155]. The catabolic pathways for aromatic hydrocarbons in bacterial strain Pseudomonas putida KT2440 was analyzed using a combined proteomic approach of 2DE with Mass spectroscopy and cleavable isotope-coded affinity tag analysis. This work produced about eighty unique proteins including different thiolases, hydrolases, dioxygenases which were found to induce in the presence of aromatic compounds like vanilline, benzoate and phydroxybenzoate. Benndorf et al.[156] conducted a functional metaproteomics analysis on microbes isolated from soils contaminated with 2,4dichlorophenoxy acetic acid (2,4-D) pesticide and ground water contaminated from cholorobenzene, where alkaline phenol treatment was given to separate metaproteomes from humic soil matrices and were subjected to 2DE analysis, and spots were identified by LC-ESI-MS. The study reported chlorocatechol dioxygenases, 2, 4-dichlorophenoxy acetate dioxygenase, molecular chaperons and transcription factors from chlorophenoxy aciddegrading and chlorobenze-degrading microbial communities. Wilmes et al.[157] also put forward the utilization of 2-DE in combination with MALDITOF, to identify highly expressed proteins during biological transformations conducted by microbes from activated sludge of an Enhanced biological Phosphorus Removal (EBPR) sewage treatment system. In this study, 638

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protein spots were generated in 2DE gels from the phosphate removing sludges. Of these, 46 proteins were found similar to proteins secreted by betaproteobacteria “Candidatus accumulibacter phosphatis”, a polyphosphate accumulating organism suggesting their roles in the EBPR sewage treatment.

5.5. Stable Isotope Probing DNA stable-isotope probing (DNA-SIP) is one of the emerging and powerful tool for detecting the microbes whose functions is to assimilate carbon substrates and nutrients into their cellular biomass. This novel SIP technique is becoming more famous among environmental microbiologists and being used to determine carbon fixation pathways of plant species. This Isotope based molecular technique allows researchers to analyse the flow of atoms through lar ge microbial communities into metabolically active microorganisms[158]. The basic principle of SIP involves the introduction of a substrate, labelled with a stable isotope (e.g., 13C, 15N, and 18O) into microbial biomarkers like DNA, phospholipids fatty acids (PLFAs) and ribosomal RNA (rRNA) and further identification of these microbes from source areas. Mostly, carbon sources in the nature are 12C-based therefore, the nucleic acids labelled with stable isotope easily indicates the genomes or transcriptomes from microorganisms that have mineralized and assimilated the stable-isotope carbon substrate into their biomass. After the extraction of these biomaterials, it has become possible to separate heavy and light fractions using ultracentrifugation technique[159]. DeRito and Madsen[160] has a report on soil fungus which has been found to involve in biodegradation of phenol from agricultural field using field-based DNA-SIP of the 18S–28S(ITS) region. They informed about Trichosporon multisporum fungi possessing the capability to metabolize phenol in field based soil experiments using SIP technique. Triclosan, a ubiquitous pollutant prevalent in hospitals scrubs, soaps, shampoos, cleaning supplies and also detected in environmental samples. Lolas et al.[161] have discussed and identify, a Triclosan degrading bacteria using DNA-SIP with microautoradiography-fluorescence in situ hybridization (MAR-FISH) technique together in a culture amended with activated sludge. Mishamandani et al.[162] have used the SIP tool to link the phylogenetic variations and functions of hydrocarbon-degrading microbes isolated from coastal region of North Carolina sea USA, which is uniformly labelled with (13C) n-hexadecane. The sequences extracted from from 13C-enriched bacterial DNA were identified to belong to the genus Alcanivorax and Methylophaga. Recently, Verastegui et al.[163] have coupled DNA stable-

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isotope probing (DNA-SIP) with functional metagenomics, using carbon substrates derived from plants and soils to characterize active bacterial communities and their glycoside hydrolase genes, which have industrial applications. Prosser et al. [164] have studied plant-microbe interactions using SIP method, where plants were labelled with 13CO2. From the rhizospheric zone of plant, a 13C enriched ribosomal RNA was recovered suggesting, the conversion of 13CO2 by photosynthesis mechanism into plant sugars and root exudates are metabolized by the rhizospheric microorganisms. 6. ACKNOWLEDGEMENTS

Authors are thankful to the Department of Biotechnology, Govt. of India for the financial support, and to the director, CSIR – National Botanical Research Institute, Lucknow, India for providing the institutional support. Ms. Manvi Singh, UGC-SRF (ref. no. 3185/NET-DEC-2011) is thankful to UGC for financial support to her as Senior Research Fellowship. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

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