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Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review

Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A... fmicb-07-01369 August 31, 2016 Time: 16:47 # 1 REVIEW published: 31 August 2016 doi: 10.3389/fmicb.2016.01369 Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review 1 2 3 1,2 Debajyoti Ghosal , Shreya Ghosh , Tapan K. Dutta * and Youngho Ahn * Environmental Engineering Laboratory, Department of Civil Engineering, Yeungnam University, Gyeongsan, South Korea, 2 3 Disasters Prevention Research Institute, Yeungnam University, Gyeongsan, South Korea, Department of Microbiology, Bose Institute, Kolkata, India Polycyclic aromatic hydrocarbons (PAHs) include a group of organic priority pollutants of critical environmental and public health concern due to their toxic, genotoxic, mutagenic and/or carcinogenic properties and their ubiquitous occurrence as well as recalcitrance. The increased awareness of their various adverse effects on ecosystem and human health has led to a dramatic increase in research aimed toward removing PAHs from the environment. PAHs may undergo adsorption, volatilization, photolysis, and chemical oxidation, although transformation by microorganisms is the major neutralization Edited by: process of PAH-contaminated sites in an ecologically accepted manner. Microbial Pankaj Kumar Arora, degradation of PAHs depends on various environmental conditions, such as nutrients, M. J. P. Rohilkhand University, India number and kind of the microorganisms, nature as well as chemical property of the PAH Reviewed by: being degraded. A wide variety of bacterial, fungal and algal species have the potential Matthias E. Kaestner, Helmholtz Centre for Environmental to degrade/transform PAHs, among which bacteria and fungi mediated degradation Research – UFZ, Germany has been studied most extensively. In last few decades microbial community analysis, Eric D. Van Hullebusch, University of Paris-Est, France biochemical pathway for PAHs degradation, gene organization, enzyme system, *Correspondence: genetic regulation for PAH degradation have been explored in great detail. Although, Tapan K. Dutta xenobiotic-degrading microorganisms have incredible potential to restore contaminated tapan@jcbose.ac.in environments inexpensively yet effectively, but new advancements are required to Youngho Ahn yhahn@ynu.ac.kr make such microbes effective and more powerful in removing those compounds, which were once thought to be recalcitrant. Recent analytical chemistry and genetic Specialty section: This article was submitted to engineering tools might help to improve the efficiency of degradation of PAHs by Microbiotechnology, Ecotoxicology microorganisms, and minimize uncertainties of successful bioremediation. However, and Bioremediation, appropriate implementation of the potential of naturally occurring microorganisms for a section of the journal Frontiers in Microbiology field bioremediation could be considerably enhanced by optimizing certain factors Received: 02 May 2016 such as bioavailability, adsorption and mass transfer of PAHs. The main purpose Accepted: 18 August 2016 of this review is to provide an overview of current knowledge of bacteria, halophilic Published: 31 August 2016 archaea, fungi and algae mediated degradation/transformation of PAHs. In addition, Citation: factors affecting PAHs degradation in the environment, recent advancement in genetic, Ghosal D, Ghosh S, Dutta TK and Ahn Y (2016) Current State genomic, proteomic and metabolomic techniques are also highlighted with an aim of Knowledge in Microbial to facilitate the development of a new insight into the bioremediation of PAH in the Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review. environment. Front. Microbiol. 7:1369. doi: 10.3389/fmicb.2016.01369 Keywords: biodegradation, polycyclic aromatic hydrocarbons (PAHs), bacteria, fungi, algae Frontiers in Microbiology | www.frontiersin.org 1 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 2 Ghosal et al. PAH Biodegradation by Microbes 2000; Marston et al., 2001; Xue and Warshawsky, 2005). On INTRODUCTION the basis of abundance and toxicity, 16 PAHs are already Over the last few decades, with an increasing global awareness enlisted as priority environmental pollutants by the United about the potential adverse effects of pollutants on public health States Environmental Protection Agency (US EPA), (Agency and environment, remediation and renovation of environment for Toxic Substances and Disease Registry [ATSDR], 1990; Liu contaminated with hazardous materials have received increasing et al., 2001) which are depicted in Figure 1. Various physical- attention. Among others, polycyclic aromatic hydrocarbons chemical properties and some relevant information of 16 PAHs (PAHs) include a group of priority organic pollutants of enlisted as priority pollutants by US EPA are depicted in significant concern due to their toxic, genotoxic, mutagenic Table 1. and/or carcinogenic properties (WHO, 1983; Cerniglia, 1992; In their pure chemical form, PAHs generally exist as Mastrangelo et al., 1996; Schützendübel et al., 1999). PAHs colorless, white, or pale yellow-green solids having a faint, are composed of fused aromatic rings in linear, angular, or pleasant odor. They are basically non-polar organic compounds, cluster arrangements. Generally, the electrochemical stability, characteristically composed of carbon and hydrogen atoms. persistency, resistance toward biodegradation and carcinogenic Mostly incomplete combustion of organic materials like coal, index of PAHs increase with an increase in the number of tar, oil and gas, automobile exhaust, tobacco or smoked food, aromatic rings, structural angularity, and hydrophobicity, while either during industrial and other human activities or during volatility tends to decrease with increasing molecular weight geothermal reactions associated with the production of fossil- (Mackay and Callcott, 1998; Marston et al., 2001). The PAHs fuels and minerals, result in PAH formation. In nature, they have a natural potential for bioaccumulation in various food are formed during forest fires, volcanic eruptions, or by plant chains, which make their presence in the environment quite and bacterial reactions (Blumer, 1976; Wilson and Jones, 1993). alarming (Morehead et al., 1986; Xue and Warshawsky, 2005), Diverse types of combustion yield different distributions of PAHs and are therefore being considered as substances of potential in both relative amounts of individual PAHs as well as their human health hazards (Mastrangelo et al., 1996; Binkova et al., isomers. In nature, they are formed during forest fires, volcanic FIGURE 1 | Structure of the16 PAHs enlisted as priority pollutants by US EPA. Frontiers in Microbiology | www.frontiersin.org 2 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 3 Ghosal et al. PAH Biodegradation by Microbes Frontiers in Microbiology | www.frontiersin.org 3 August 2016 | Volume 7 | Article 1369 TABLE 1 | Physical-chemical properties and some relevant information of 16 PAHs enlisted as priority pollutants by US EPA. Name Molecular Cas registry Physical chemical properties Toxicology Biodegradation formula no. a b c d B. Pt. ( C) M.Pt ( C) V.P. (mmHg at 25 C) Solubility (mg/L) TEF IARC EPA Estimated Measured half-lives e f half-lives (days) (days) Naphthalene C H 91-20-3 218 80.2 8.5  10 31 n.d. 2B C 5.66 n.d. 10 8 Acenaphthene C H 83-32-9 279 93.4 2.5  10 3.93 0.001 3 D 18.77 n.d. 12 10 Acenaphthylene C H 208-96-8 280 91.8 6.68  10 1.93 0.001 n.c. D 30.7 n.d. 12 8 Anthracene C H 120-12-7 342 216.4 6.53  10 0.076 0.01 3 D 123 2.7 14 10 Phenanthrene C H 85-01-8 340 100.5 1.2  10 1.20 0.001 3 D 14.97 5 14 10 Fluorene C H 86-73-7 295 116-7 6.0  10 1.68–1.98 0.001 3 D 15.14 n.d. 13 10 Fluoranthene C H 206-44-0 375 108.8 9.22  10 0.20–0.26 0.001 3 D 191.4 9.2 16 10 Benzo[a]anthracene C H 56-55-3 438 158 4.11  10 0.010 0.1 2B B2 343.8 > 182 18 12 9 3 Chrysene C H 218-01-9 448 254 6.23  10 1.5  10 0.010 2B B2 343.8 n.d. 18 12 Pyrene C H 129-00-0 150.4 393 4.5  10 0.132 0.001 3 D 283.4 151 16 10 9 3 Benzo[a]pyrene C H 50-32-8 495 179 5.49  10 3.8  10 1.0 1 B2 421.6 11 20 12 Benzo[b]fluoranthene C H 205-99-2 481 168.3 5.0  10 0.0012 n.d. 2B B2 284.7 n.d. 20 12 10 4 Benzo[k]fluoranthene C H 207-08-9 480 215.7 9.7  10 7.6  10 0.1 2B B2 284.7 n.d. 20 12 10 4 Dibenzo[a,h]anthracene C H 53-70-3 524 262 9.55  10 5.0  10 n.d. 2A B2 511.4 n.d. 22 14 10 5 Benzo[g,h,i]perylene C H 191-24-2 500 277 1.0  10 2.6  10 n.d. 3 D 517.1 n.d. 22 12 Indenol[1,2,3-cd]pyrene C H 193-39-5 536 161-3 1.25  10 0.062 n.d. 2B B2 349.2 n.d. 22 12 a b c (Mackay and Shiu, 1977). Toxic equivalent factor relatively to Benzo[a]pyrene (Chang et al., 2014). International Agency for Research on Cancer Classification Monographs Volume 1-111 updated 18 February 2015 (1, carcinogenic to humans; 2A, probably carcinogenic to humans; 2B, possibly carcinogenic to humans; 3, not classifiable as carcinogenic to humans; n.c., not classified). EPA carcinogenic classification: A, human carcinogenic; B1 and B2: probable human carcinogenic; C, possible human carcinogenic; D, not Classifiable as to human carcinogenicity; E, evidence of non-carcinogenicity for humans. Estimation using BioHCwin software v1.01 on EPI Suite software develop by (Howard et al., 2005). (Comber et al., 2012); n.d., not determined. fmicb-07-01369 August 31, 2016 Time: 16:47 # 4 Ghosal et al. PAH Biodegradation by Microbes eruptions, or by plant and bacterial reactions (Blumer, 1976; Consequently, cleaning of such polluted places has been Wilson and Jones, 1993). Nevertheless, the anthropogenic input thought to be one of the most essential alternatives for of PAHs to the environment far exceeds the natural sources restoring environmental damage. Several physical and chemical (National Academy of Sceinces [NAS], 1971). treatment methods including incineration, base-catalyzed de- Polycyclic aromatic hydrocarbons are formed whenever chlorination, UV oxidation, fixation, solvent extraction etc. are organic substances are exposed to high temperatures (pyrolysis), already in practice (Norris et al., 1993; Gan et al., 2009), but and the composition of the products thus formed depends have several drawbacks including cost, complexity, regulatory largely on the nature of the starting material as well as the burden etc. Moreover, these conventional techniques, in many transformation temperature (Blumer, 1976; Cerniglia, 1992). cases, do not destroy the contaminating compounds completely, Fossil fuels also contain huge amounts of PAHs which are but instead transfer them from one environment or form to released into the environment during incomplete combustion another. In order to solve this burning problem, researchers or by accidental discharge during transport, use, or disposal have devised an efficient and eco-friendly clean-up technique of petroleum products or as a result of uncontrolled emissions known as bioremediation, which is being progressively refined (Cerniglia, 1992; Wilson and Jones, 1993; Johansson and van to fight pollution. This technique utilizes and manipulates the Bavel, 2003). PAHs are widely present as contaminants in air, detoxification abilities of living organisms to convert hazardous soil, aquatic environments, sediments, surface water as well as organic wastes including xenobiotics into harmless products, in ground water (Huntley et al., 1993; Van Brummelen et al., often carbon dioxide and water (Cerniglia and Heitkamp, 1989; 1996; Boxall and Maltby, 1997; Holman et al., 1999; Lim et al., Mueller et al., 1996; Bamforth and Singleton, 2005; Johnsen 1999; Ohkouchi et al., 1999). Natural and anthropogenic sources et al., 2005). Bioremediation addresses the limitations associated of PAHs, in combination with global transport phenomena, with most of the physicochemical processes by destroying many result in their worldwide distribution and consequently, PAHs organic contaminants at reduced cost, under ambient conditions get dispersed from the atmosphere to vegetation, ultimately and thus, has now become a popular remedial alternative for leading to bioaccumulation in various food chains (Edwards, pollutant removal including PAHs (Young and Cerniglia, 1996; 1983; Morehead et al., 1986; Wagrowski and Hites, 1997). Apart Juhasz and Naidu, 2000; Kastner, 2000; Lovley, 2001; Andreoni from biodegradation, the fate of PAH in nature varies depending and Gianfreda, 2007; Jorgensen, 2007; Megharaj et al., 2011; on the environment, for example, in air, PAH can undergo Abdel-Shafy and Mansour, 2016). photo-oxidation, whereas in the case of soil and water, they In addition, many natural habitats (e.g., aquifers, aquatic can undergo both photo-oxidation and chemical oxidation while sediments) contaminated with a huge amount of aromatic some PAHs like naphthalene and alkyl naphthalene are partly lost pollutants are often anoxic. In these environments, the anaerobic by volatilization (Cerniglia, 1992). degradation of aromatic compounds by microorganisms plays The toxicity of PAH was first recognized in 1761 by John Hill, a major role in the removal of contaminants, recycling of a physician who documented a high incidence of nasal cancer in carbon and sustainable development of the ecosystem. Reports tobacco snuff consumers (Cerniglia, 1984). The low-molecular- on anaerobic biodegradation of PAHs are relatively recent, weight (LMW) PAHs (containing two or three aromatic rings) and only a limited number of preliminary studies have are acutely toxic while the high-molecular-weight (HMW) PAHs demonstrated the anaerobic degradation of PAHs including (containing four or more rings) are largely considered as naphthalene, anthracene, phenanthrene, fluorene, acenaphthene genotoxic (Cerniglia, 1992; Mueller et al., 1996; Abdel-Shafy and and fluoranthene (Foght, 2008; Carmona et al., 2009; Mallick Mansour, 2016). It is a known fact that PAH can covalently et al., 2011). However, detailed information on anaerobic bind to DNA, RNA and proteins, but it is the amount of degradation of PAHs under sulfate-reducing and nitrate- covalent interaction between PAHs and DNA that correlates reducing conditions is scarce and very little is known about best with carcinogenicity (Marston et al., 2001; Santarelli et al., their degradation pathways, catabolic genes/enzymes and/or 2008). In addition, the transformation products of some PAHs regulatory mechanisms, but this emerging field is ready to are more toxic than parent PAHs and can lead to critical blossom in various aspects of biotechnological applications. cellular effects (Schnitz et al., 1993). In humans cytochrome P450 Compared to HMW PAHs, LMW PAHs are reasonably monooxygenase group of enzymes oxidize PAHs to epoxides, more volatile and more soluble in water and consequently some of which are highly reactive (such as “bay-region” diol more susceptible to biodegradation (Pannu et al., 2003). LMW epoxides) and known as ultimate carcinogens. They can bind PAHs like naphthalene, anthracene and phenanthrene are to DNA and have the ability to transform normal cells to widely present throughout the environment and designated malignant one (Milo and Casto, 1992; Schnitz et al., 1993; as prototypic PAHs and serve as signature compounds to Marston et al., 2001). Studies have shown that processing food detect PAH contamination. Naphthalene represents the simplest at high temperatures, like grilling or barbecuing result in high PAH whereas the chemical structures of anthracene and levels of PAHs in cooked meat and smoked fish (Morehead et al., phenanthrene are found in many carcinogenic PAHs (such as 1986). The concern associated with PAHs further increased due in benzo[a]pyrene, benz[a]anthracene etc.), and phenanthrene to their ability to interfere with hormone metabolizing enzymes represents the smallest PAH to have both the bay and K regions of the thyroid glands, and their adverse effects on reproductive (Figure 2). Thus, they are often used as a model substrate for as well as immune system (Veraldi et al., 2006; Oostingh et al., studies on the metabolism of carcinogenic PAHs (Mallick et al., 2008). 2011) (and the references therein). In line with the advances in Frontiers in Microbiology | www.frontiersin.org 4 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 5 Ghosal et al. PAH Biodegradation by Microbes (Foght, 2008; Carmona et al., 2009). While the aerobic catabolism of aromatic compounds has been studied for several decades, the anaerobic degradation of aromatic compounds is a more recently discovered microbial capacity that still awaits a deeper understanding. However, anoxic conditions dominate in many natural habitats and contaminated sites (e.g., aquifers, aquatic sediments and submerged soils) where biodegradation is carried out by anaerobes using alternative final electron acceptors such as nitrate, sulfate or ferric ions (Foght, 2008; Carmona et al., 2009). Principally, bacteria favor aerobic conditions for degradation of PAHs via oxygenase-mediated metabolism (involving either FIGURE 2 | Structure of phenanthrene, the simplest PAH containing monooxygenase or dioxygenase enzymes). Usually, the first step bay and K region. in the aerobic bacterial degradation of PAHs is the hydroxylation of an aromatic ring via a dioxygenase, with the formation of a cis-dihydrodiol, which gets rearomatized to a diol intermediate the knowledge of bacterial diversity within an ecosystem, many by the action of a dehydrogenase. These diol intermediates unique metabolic pathways on degradation of the PAHs are may then be cleaved by intradiol or extradiol ring-cleaving reported and scattered in literatures, which would collectively dioxygenases through either an ortho-cleavage or meta-cleavage deepen the understanding on the range of catabolism as well pathway, leading to intermediates such as catechols that are as the biochemical and genetic diversities. Timely collective ultimately converted to TCA cycle intermediates (Evans et al., update of literature is most important to address current status 1965; Cerniglia, 1992; Eaton and Chapman, 1992; Mueller et al., of subjects related to our life and surrounding environment 1996; Mallick et al., 2011). Dioxygenase is a multicomponent where the ongoing threat due to PAH-mediated pollution is enzyme generally consisting of reductase, ferredoxin, and no exception rather an issue that deserves top priority. The terminal oxygenase subunits (Mallick et al., 2011). Bacteria can present review provides an overview of the current knowledge also degrade PAH via the cytochrome P450-mediated pathway, of microbial degradation/transformation of PAHs. Moreover, with the production of trans-dihydrodiols (Sutherland et al., factors affecting biodegradation of PAHs, recent advancement 1990; Moody et al., 2004) or under anaerobic conditions, e.g., in genetic, genomic, proteomic and metabolomic techniques under nitrate-reducing conditions (Foght, 2008; Carmona et al., and their application in bioremediation of PAHs have also been 2009). described. Naphthalene degrading bacteria are ubiquitous in nature and there are enormous numbers of reports documenting the bacterial degradation of naphthalene including the elucidation PAH DEGRADATION BY BACTERIA AND of the biochemical pathways, enzymatic mechanisms and genetic HALOPHILIC ARCHAEA regulations (Cerniglia, 1992; Peng et al., 2008; Seo et al., 2009; Lu et al., 2011; Mallick et al., 2011). The naphthalene catabolic Bacterial Catabolism of PAHs genes present in the plasmid NAH7 in Pseudomonas putida Bacteria, which have evolved more than three billion years ago, G7 are well characterized (Simon et al., 1993). In the plasmid have developed strategies for obtaining energy from virtually NAH7, the naphthalene catabolic genes (nah) are organized every compound and have been considered as nature’s ultimate into two operons: the nal operon containing the genes for the scavengers. Because of their quick adaptability, bacteria have upper pathway enzymes involved in conversion of naphthalene largely been used to degrade or remediate environmental hazards. to salicylate, and the sal operon containing the genes for the Various bacteria have been found to degrade PAHs, in which lower pathway enzymes involved in the conversion of salicylate degradation of naphthalene and phenanthrene has been most to pyruvate and acetaldehyde (Simon et al., 1993). The operons widely studied. Numerous unique metabolic pathways for the are positively regulated by a common regulator NahR, a LysR bacterial degradation of PAHs have been well documented in a type of positive transcriptional regulator and are widely dispersed number of excellent review articles (Cerniglia, 1992; Peng et al., in bacteria. NahR is induced in presence of salicylate leading 2008; Seo et al., 2009; Mallick et al., 2011). So, the biochemical to high-level expression of the nah genes in bacteria (Yen and pathways for bacterial degradation of PAHs will not be discussed Gunsalus, 1985; Peng et al., 2008). There are several reports on in details in this communication. Nevertheless, there are two the nucleotide sequences of genes encoding the upper pathway major strategies to degrade PAHs depending on the presence enzymes in different Pseudomonas strains, and the genes are more or absence of oxygen. In the aerobic catabolism of aromatics, than 90% identical (Menn et al., 1993; Simon et al., 1993; Yang the oxygen is not only the final electron acceptor but also a co- et al., 1994; Bosch et al., 2000; Peng et al., 2008). Moreover, substrate for the hydroxylation and oxygenolytic ring cleavage in Ralstonia sp. U2, the naphthalene catabolic operon (nag) of the aromatic ring. In contrast, the anaerobic catabolism of contains all of the upper pathway genes similar to that of the aromatic compounds uses a completely different strategy to classical nah genes of Pseudomonas strains in the same order, attack the aromatic ring, primarily based on reductive reactions with the exception of two extra genes named nagG and nagH, Frontiers in Microbiology | www.frontiersin.org 5 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 6 Ghosal et al. PAH Biodegradation by Microbes which are structural subunits of salicylate-5-hydroxylase enzyme 2005). The narAa and narAb genes codes for the a- and b- required for the conversion of naphthalene to gentisate (Zhou subunit of the naphthalene dioxygenase (NDO). Both subunits et al., 2001). Additionally, in Comamonas testeroni strain GZ42, of NDO in Rhodococcus sp. strain NCIMB12038 showed only the naphthalene catabolic genes are similar to that found in 30% amino acid identity to the corresponding P. putida NDO Ralstonia sp. U2 (Goyal and Zylstra, 1997). The lower pathway subunits. In addition, there is no gene in Rhodococcus strains genes for the naphthalene catabolism are also similar in different which is similar to the genes encoding the electron transport Pseudomonas strains like P. putida G7, NCIB9816-4, ND6, and components reductase and ferredoxin of NDO in Pseudomonas P. stutzeri AN10 (Habe and Omori, 2003; Peng et al., 2008). In strains (Kulakov et al., 2005; Larkin et al., 2005). Moreover, those organisms, lower pathway of naphthalene operon contains the narA and narB genes are transcribed as a single unit 11 genes present in the order nahGTHINLOMKJY, in which nahY through different start sites, and their transcription is induced represent a naphthalene chemotaxis gene. However, in AN10 in the presence of naphthalene in contrast to salicylate-inducible and ND6 strains another salicylate hydroxylase gene (nahW ) naphthalene catabolic genes in Pseudomonas species. There are has been found to be present outside, but near to the sal two putative regulatory genes (narR1 and narR2, GntR-like operon. transcriptional regulator) which are shown to be transcribed as a Along with Pseudomonas, a high proportion of the PAH- single mRNA in naphthalene-induced cells (Kulakov et al., 2005; degrading isolates belong to the sphingomonads, which comprise Larkin et al., 2005). Figure 3 represent the gene organization a physiologically versatile group within the Alphaproteobacteria: of the nar gene cluster of various naphthalene degrading mainly Sphingomonas, Sphingobium and Novosphingobium that Rhodococcus sp. are frequently found as aromatic degraders. Species belonging to Along with naphthalene, a number of reports on phenan- these genera show great catabolic versatility, capable of degrading threne degradation by various Gram negative and Gram a wide range of natural and xenobiotic compounds including positive bacterial species have been reported (Peng et al., 2008; HMW PAHs (Basta et al., 2005; Peng et al., 2008; Stolz, 2009; Seo et al., 2009; Mallick et al., 2011). In a study, Mallick Vila et al., 2015). In a few reports, it has been shown that et al. (2007) reported the degradation of phenanthrene by the catabolic versatility of sphingomonads relies on the large Staphylococcus sp. strain PN/Y, by initiating the dioxygenation plasmids present in those organisms (Basta et al., 2005; Peng specifically at the 1,2-position followed by meta-cleavage of et al., 2008). However, the plasmid-encoded degradative genes phenanthrene-1,2-diol, leading to the formation of 2-hydroxy- have been found to be largely scattered, or they are not organized 1-naphthoic acid as the metabolic intermediate; while the in coordinately regulated operons (Romine et al., 1999). It may be ortho-cleavage could yield the naphthalene-1,2-dicarboxylic acid. possible that this kind of ‘flexible’ gene organization i.e., different Authors also reported that 2-hydroxy-1-naphthoic acid was combinations of conserved gene clusters allows sphingomonads metabolized by a meta-cleavage enzyme 2-hydroxy-1-naphthoate to adapt easily and proficiently to degrade various aromatic dioxygenase leading to the formation of trans-2,3-dioxo-5- compounds including xenobiotics (Basta et al., 2005; Peng et al., (2 -hydroxyphenyl)-pent-4-enoic acid, a novel metabolite in 2008). Recently an illustrative report on the catabolic versatility the phenanthrene degradation pathway, and was subsequently of sphingomonads has shown that strain PNB, acting on diverse degraded via salicylic acid and catechol (Mallick et al., 2007). monoaromatic and polyaromatic compounds, contains seven Later on, Ghosal et al. (2010) reported the assimilation of sets of ring-hydroxylating oxygenases (RHO) with different phenanthrene by Ochrobactrum sp. strain PWTJD, isolated from substrate specificities (Khara et al., 2014). In general, it has municipal waste contaminated soil sample using phenanthrene been seen that mobile genetic elements (MGEs) like plasmids as a sole source of carbon and energy. The strain PWTJD and transposons play a crucial role in the biodegradation of could also degrade phenanthrene via 2-hydroxy-1-naphthoic organic pollutants like PAHs. The presence of foreign compounds acid, salicylic acid and catechol. The strain PWTJD was can often lead to the selection of mutant bacteria that are found to utilize 2-hydroxy-1-naphthoic acid and salicylic acid capable of metabolizing them. Apart from vertical gene transfer, as sole carbon sources, while the former was metabolized aromatics catabolic genes are often harbored by MGEs that by a ferric-dependent meta-cleavage dioxygenase. In the successfully disseminate the catabolic traits to phylogenetically lower pathway, salicylic acid was metabolized to catechol diverse bacteria by horizontal gene transfer (Top and Springael, and was further degraded by catechol 2,3-dioxygenase to 2003; Nojiri et al., 2004). 2-hydroxymuconoaldehyde acid, ultimately leading to TCA Among PAHs degrading bacteria the genus Rhodococcus cycle intermediates. The metabolic pathway involved in the is very unique, having an enormous catabolic versatility. In degradation of phenanthrene by Ochrobactrum sp. strain PWTJD contrast to Pseudomonas and other Gram-negative bacteria is shown in Figure 4 (Ghosal et al., 2010). This was the first whose naphthalene catabolic genes are usually clustered, the report of the complete degradation of a PAH molecule by Gram-positive Rhodococcus strains usually exhibit only three Gram-negative Ochrobactrum sp. describing the involvement of structural genes required for naphthalene degradation (narAa, the meta-cleavage pathway of 2-hydroxy-1-naphthoic acid in narAb and narB) (Kulakov et al., 2005; Larkin et al., 2005). It phenanthrene assimilation. has been seen that the nar region is not arranged into a single Other LMW PAHs like anthracene, fluorene, acenaphthene operon, and there are several homologous transcription units and acenaphthylene are also found in high quantities in PAH- in different Rhodococcus strains separated by non-homologous polluted sites and various bacterial species have the capability sequences containing direct and inverted repeats (Larkin et al., to utilize these compounds individually as sole carbon and Frontiers in Microbiology | www.frontiersin.org 6 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 7 Ghosal et al. PAH Biodegradation by Microbes FIGURE 3 | Gene organization of the nar gene cluster of various naphthalene degrading Rhodococcus sp. The respective Rhodococcus sp. along with the accession numbers of their corresponding naphthalene degrading gene cluster: Rhodococcus opacus R7 (accession no. DQ846881), Rhodococcus sp. NCIMB12038 (accession no. AF082663), Rhodococcus opacus CIR2 (accession no. AB024936), Rhodococcus sp. P200 (accession no. AY392424), Rhodococcus sp. P400 (accession no. AY392423), Rhodococcus sp. I24 (accession no. AF121905). The genes and their corresponding function of the gene products: narR1- GntR-like regulator protein; narR2- XylR-like regulator protein; rub1 and 2- Rubredoxin; narAa- Naphthalene dioxygenase large subunit; narAb- Naphthalene dioxygenase small subunit; narB- Naphthalene dihydrodiol dehydrogenase (adapted from Di Gennaro et al., 2010). energy sources (Moody et al., 2001; Peng et al., 2008; Seo Harayama, 2000; Peng et al., 2008; Seo et al., 2009). However, et al., 2009; Mallick et al., 2011). Lately, Ghosal et al. (2013) further investigations are prerequisite in several areas of HMW reported assimilation of acenaphthene and acenaphthylene by PAH biodegradation, namely, research related to the regulatory the Acinetobacter sp. strain AGAT-W, isolated from municipal mechanisms of HMW PAH biodegradation, biodegradation of waste contaminated soil sample using acenaphthene as a sole HMW PAH associated with other hydrocarbons in mixtures; source of carbon and energy. The strain AGAT-W could and the interactions of complex microbial community during degrade acenaphthene via 1-acenaphthenol, naphthalene-1,8- HMW PAH degradation can be exploited in more details. dicarboxylic acid, 1-naphthoic acid, salicylic acid and cis, cis These will enrich our understanding on the microbial ecology muconic acid ultimately leading to TCA cycle intermediates. The of HMW PAH-degrading communities and the mechanisms metabolic pathways involved in the degradation of acenaphthene by which HMW PAH biodegradation occur. In addition, the and acenaphthylene by Acinetobacter sp. strain AGAT-W is outcome will also help in predicting the environmental fate of shown in Figure 5 (Ghosal et al., 2013). This was the first these recalcitrant compounds and aid for the development of report on the complete degradation of acenaphthene and convenient as well as cost-effective bioremediation strategies in acenaphthylene individually by strain AGAT-W belonging to the near future. genus Acinetobacter. Polycyclic aromatic hydrocarbons with more than three rings Degradation of PAHs by viz. pyrene, benzo[a] pyrene (Bap), are generally referred to as Halophilic/Halotolerant Bacteria and HMW PAHs (Figure 1), which are of significant environmental concern due to their long persistence and high toxicity as well as Archaea their mutagenic and/or carcinogenic properties (Cerniglia, 1992; Environmental pollution due to anthropogenic activity has Kanaly and Harayama, 2000; Peng et al., 2008; Seo et al., 2009). spread to all types of ecosystems and marine ecosystem is In the last few decades research on microbial degradation of not excluded from this list. Hypersaline environments are HMW PAHs has advanced significantly and a number of HMW regularly being polluted with organic pollutants including PAHs, PAH-degrading isolates have been reported (Cerniglia, 1992; through industrial and municipal effluents. Industrial effluents, Kanaly and Harayama, 2000, 2010; Peng et al., 2008; Seo et al., specifically, petroleum industry where one of the main sources 2009). Biodegradation of HMW PAHs by microorganisms is of contaminants in the waste waters are aromatic hydrocarbons discussed adequately in many excellent reviews and the pathways including PAHs. Contamination and biodegradation in extreme for their degradation have also been depicted (Kanaly and environments like high salinity has been receiving increased Frontiers in Microbiology | www.frontiersin.org 7 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 8 Ghosal et al. PAH Biodegradation by Microbes FIGURE 4 | Proposed pathway for the degradation of phenanthrene by Ochrobactrum sp. strain PWTJD. The transient intermediates, which were not detected, are shown in parentheses. Filled arrows indicate mineralization; open arrows indicate a dead-end metabolite; and dotted arrows indicate multiple steps. Chemical designations: (I) phenanthrene; (II) cis-1,2-phenanthrenedihydrodiol; (III) 1,2-dihydroxyphenanthrene; (IV) 5,6-benzocoumarin; (V) cis-2-oxo-4-(20-hydroxynaphthyl)-but-3-enoic acid; (VI) 2-hydroxy-1-naphthoic acid; (VII) trans-2,3-dioxo-5-(20-hydroxyphenyl)- pent-4-enoic acid; (VIII) 2,3-dioxo-5-hydroxy-5-(20-hydroxyphenyl)-pentanoic acid; (XI) salicylaldehyde; (X) salicylic acid; (XI) catechol; (XII) 2-hydroxymuconaldehyde acid. Adapted from Ghosal et al. (2010). attention in recent times (Oren et al., 1992; Margesin and Schinner, 2001a,b; Le Borgne et al., 2008; Bose et al., 2013; Cravo- FIGURE 5 | Proposed pathway for the degradation of acenaphthene by Laureau and Duran, 2014; Elango et al., 2014; Fathepure, 2014; Acinetobacter sp. strain AGAT-W. Chemical designations: (I) acenaphthene; (II) acenaphthylene; (III) 1-acenaphthenol; (IV) Genovese et al., 2014; Kappell et al., 2014; Kostka et al., 2014; 1-acenaphthenone; (V) 1-hydroxy-2-ketoacenaphthene; (VI) Thomas et al., 2014; Torlapati and Boufadel, 2014). In addition, a 1,2-dihydroxyacenaphthylene; (VII) acenaphthenequinone; (VIII) survey of relevant literatures indicates that along with numerous naphthalene-1,8-dicarboxylic acid; (IX) 1,8-naphthalic anhydride; (X) biotechnological applications, halophilic microorganisms have 1-naphthoic acid; (XI) salicylaldehyde; (XII) salicylic acid; (XIII) catechol. more extended catabolic versatility than previously thought Adapted from Ghosal et al. (2013). about (Margesin and Schinner, 2001b; Garcia et al., 2005; Smith et al., 2013; Kappell et al., 2014; Lamendella et al., 2014; Roling and Bodegom, 2014; Scott et al., 2014; Singh et al., 2014). deal with hypersaline environments that are contaminated with The biological treatment of industrial hypersaline waste waters organic pollutants (Margesin and Schinner, 2001a; Mellado and and the bioremediation of polluted hypersaline environments Ventosa, 2003; Peyton et al., 2004; Garcia et al., 2005; Cui are not possible with conventional microorganisms because et al., 2008; Mnif et al., 2009; Zhao et al., 2009; Bonfa et al., they are incapable to function efficiently at salinities that of 2011; Smith et al., 2013; Fathepure, 2014; Kappell et al., 2014; seawater or above. Thus, halophilic microorganisms are the best Lamendella et al., 2014; Singh et al., 2014; Thomas et al., 2014). alternatives to overcome this problem (Oren et al., 1992; Garcia Some halophiles that have shown PAHs degrading property et al., 2005; Fathepure, 2014; Kostka et al., 2014). In the last are depicted in Supplementary Table S1. However, studies decade there has been an increasing interest in the development concerning the ability of this group of microbes to degrade and optimization of bioremediation processes via halophiles to PAHs are still in their infancy. Although in the last few years, Frontiers in Microbiology | www.frontiersin.org 8 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 9 Ghosal et al. PAH Biodegradation by Microbes a few studies on the degradation of PAHs by halophiles have bacterial/archaeal (halophilic) strains involved in the degradation been published. In this context, Gauthier et al. (1992) described of various PAHs. Figure 6 illustrates the 16S rRNA gene-based the isolation of Marinobacter hydrocarbonoclasticus which can phylogenetic analysis of all the bacterial/archaeal strains cited in degrade various organic compounds including naphthalene Supplementary Table S1. It has been seen that bacterial/archaeal (Gauthier et al., 1992). The presence of naphthalene dioxygenase, strains having PAHs catabolic property are distributed mainly similar to those from Pseudomonas and Burkholderia spp. was in six major groups: Alphaproteobacteria, Betaproteobacteria, reported in a naphthalene-degrading Marinobacter sp. strain Gammaproteobacteria, Actinomycetes, Firmicutes and Archaea NCE312 (Hedlund et al., 2001). Again, based on the analysis (Halophiles). It may be mentioned here that many strains listed of the shoreline sand and rocks (Costa da Morte, northwestern in Supplementary Table S1 have not been cultured in laboratory. Spain) affected by the Prestige oil spill, unveiled the high relative Some PAHs degrading isolates represent novel strains which are abundances of Sphingomonadaceae and Mycobacterium that isolated from various geographically diverse habitats. Thus, all could be associated with PAH degradation (Alonso-Gutierrez this information illustrate that PAH-degrading machinery is not et al., 2009). In a similar study, Vila et al. (2010) reported confined to a few particular genus reported so far but is more the isolation of a marine microbial consortium from a beach likely to be distributed widely in the prokaryotic kingdom found polluted with the Prestige oil spill which is efficient in removing in diverse geographical niches. different hydrocarbon present in heavy fuel oil including three to five-ring PAHs (for example anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, and BaP) (Vila et al., 2010). FUNGAL DEGRADATION OF PAHs In addition based on community dynamics analysis, it has been contemplated that Alphaproteobacteria (Maricaulis and The biodegradation of PAHs by fungi has been studied Roseovarius), could be associated with the degradation of extensively in last several years and numerous fungal species LMW PAHs, whereas Gammaproteobacteria (Marinobacter and have been reported to metabolize different PAHs (Cerniglia, Methylophaga), could be associated with the degradation of 1992; Cerniglia and Sutherland, 2010). Most fungi cannot use HMW PAHs. A similar bacterial community associated with PAHs as sole sources of carbon and energy; however, they may the degradation of various organic compounds was found in a co-metabolize PAHs to a wide variety of oxidized products beach affected by the Deepwater Horizon oil spill (Kostka et al., and sometimes to CO . Bacterial PAHs degradation mainly 2011; Lamendella et al., 2014). Recently, Gallego et al. (2014) involves dioxygenase enzymes and partially monooxygenase reported the isolation of a marine pyrene-degrading microbial mediated reactions and the same is valid for algae. For consortium from a beach polluted by an oil spill and found example, the extent of dioxygenase vis-à-vis monooxygenase that an uncultured Gordonia sp. is the key pyrene degrader in catalyzed transformation of naphthalene by Mycobacterium sp. the consortium based on community structure and PAH ring- was found to be in the ratio of around 25:1 (Kelley et al., hydroxylating genes analyses (Gallego et al., 2014). Nevertheless, 1990). On the other hand, fungal PAHs degradation mainly detailed information on the bioremediation of PAHs under high involves monooxygenase enzymes (Cerniglia and Sutherland, salinity by halophilic/halotolerant bacteria and archaea is still in 2010) (and the references therein). However, the transformation its initial stage of exploration, but it is expected that this emerging of PAHs by fungi involves several enzymatic pathways and field is ready to flourish in near future. depends on the particular species and growth conditions. The fungi involved in PAHs biodegradation are mainly of two types- ligninolytic fungi or white-rot fungi (they have the Occurrence of PAH-Degrading ability to produce enzymes including lignin peroxidase (LiP), Machinery in Diverse manganese peroxidase (MnP) and laccases to degrade the lignin Bacterial/Halophilic Archaeal Genera in wood) and non-ligninolytic fungi (those who do not produce The community analysis of indigenous microorganisms peroxidases or laccases but instead produce cytochrome P450 capable of degrading various aromatic compounds in diverse monooxygenase like enzymes) (Hofrichter, 2002; Tortella et al., environments has been of great interest in the recent years 2005; Cerniglia and Sutherland, 2010; Li et al., 2010). Although, (Watanabe et al., 2002; Brakstad and Lodeng, 2005; Gerdes various ligninolytic fungi such as Phanerochaete chrysosporium et al., 2005; Yakimov et al., 2005; Coulon et al., 2007; Cui et al., and Pleurotus ostreatus can secrete both ligninolytic and non- 2008) and different strategies have been developed for the study ligninolytic type of enzymes, but it is uncertain to what extent of associated microbial communities (Widada et al., 2002). each enzyme participates in the degradation of the PAHs (Bezalel Previously it was thought that PAH degradation capabilities et al., 1997). On the other hand, another type of ligninolytic may be associated with certain genera or groups of bacteria fungi, known as brown-rot fungi, mainly produces hydrogen independent of the origin of the source sample (Kastner et al., peroxide for degrading hemicelluloses and cellulose. Although, 1994; Mueller et al., 1997). But consequently, with time it has only limited data is available about PAH metabolism by brown been reported that PAH-degrading bacteria are far more diverse rot fungi, some brown-rot fungi such as Laetiporus sulphureus (Widada et al., 2002; Andreoni et al., 2004; Cui et al., 2008; and Flammulina velutipes have been shown to metabolize Hilyard et al., 2008). Currently, there are enormous reports PAHs like phenanthrene, fluoranthene and fluorine (Sack et al., of various bacterial as well as some archaeal genera capable 1997; Cerniglia and Sutherland, 2010) (and the references of degrading PAHs. Supplementary Table S1 represents the therein). Frontiers in Microbiology | www.frontiersin.org 9 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 10 Ghosal et al. PAH Biodegradation by Microbes FIGURE 6 | Phylogenetic tree based on 16S rRNA gene sequence of organisms enlisted in Supplementary Table S1. Organism designations are according to those given in Supplementary Table S1. However, for organisms devoid of 16S rRNA gene sequence information, sequence of representative type strain has been considered. Accession numbers are given within parentheses. 16S rRNA gene sequence of Thermus aquaticus strain ATCC 25104 was used as outgroup. Numbers at nodes indicate levels of bootstrap support based on neighbor-joining analysis of 100 resampled datasets. Bootstrap values below 70% are not shown. Bar represents 0.05 substitutions per nucleotide position. Catabolism of PAHs by Non-ligninolytic Fungi Polycyclic aromatic hydrocarbons metabolic pathway of non- ligninolytic fungi is similar to those formed by mammalian enzymes. In this case, the predominant pathway of initial oxidation of PAHs by non-ligninolytic fungi involves the activity of the cytochrome P450 monooxygenase enzymes. These enzymes catalyze a ring epoxidation to form an unstable arene oxide, which is further transformed to trans-dihydrodiol via an epoxide-hydrolase catalyzed reaction (Jerina, 1983; Sutherland et al., 1995). Non-ligninolytic fungi such as Cunninghamella elegans and ligninolytic fungi such as Pleurotus ostreatus, metabolize PAHs via this pathway (Bezalel et al., 1996; Tortella et al., 2005). For example, the transformation of fluoranthene by C. elegans produce fluoranthene trans-2,3-dihydrodiol, 8- and 9-hydroxyfluoranthene trans-2,3-dihydrodiols (Tortella et al., 2005). Similarly, P. ostreatus metabolizes pyrene into pyrene trans-4,5-dihydrodiol (Bezalel et al., 1996). Arene oxide produced by cytochrome P450 can also be rearranged to phenol derivatives by non-enzymatic reactions and are subsequently conjugated with sulfate, xylose, glucoronic acid, or glucose (Mueller et al., 1996; Pothuluri et al., 1996). In some fungi, cytochrome P450 monooxygenases oxidize PAHs to epoxides and dihydrodiols which are potent carcinogens and more toxic than the respective parent PAHs, while on the other hand peroxidase-mediated oxidation of PAHs produces quinines which are less toxic than the parent PAHs (Cerniglia and Sutherland, 2010). This is why oxidation of PAHs by ligninolytic enzymes could be a more logical strategy for the detoxification of PAHs contaminated environment. Catabolism of PAHs by Ligninolytic Fungi White-rot fungi are ubiquitous in nature and can produce ligninolytic enzymes which are secreted extracellularly. These enzymes can degrade lignin present in wood and other organic substances. Ligninolytic enzymes are mainly of two types, peroxidases and laccases. On the basis of reducing substrate types, peroxidase enzyme can be classified again into two types, lignin peroxidase and manganese peroxidase. Both types of peroxidases have the ability to oxidize PAHs (Hammel, 1995; Cerniglia and Sutherland, 2010). Laccases, the phenol oxidase enzymes, also have the ability to oxidize PAHs (Cerniglia and Sutherland, 2010). Compared to bacterial PAHs degrading enzymes, ligninolytic enzymes are not induced in the presence of PAHs or by FIGURE 6 | Continued its degradation products (Verdin et al., 2004). As ligninolytic Frontiers in Microbiology | www.frontiersin.org 10 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 11 Ghosal et al. PAH Biodegradation by Microbes enzymes are secreted extracellularly, they are able to diffuse et al., 2003). Oxidation of pyrene, anthracene, fluorene and toward the immobile PAHs, and that is why they are more BaP to the corresponding quinines by lignin peroxidase and useful than bacterial intracellular enzymes in making initial manganese peroxidase was reported by the white-rot fungus attack on PAHs in soil. In addition compared to bacterial PAH- Phanerochaete chrysosporium (Bogan et al., 1996a,b). In another degrading enzymes, ligninolytic enzymes have broad substrate study, complete mineralization of BaP (HMW PAHs) by the specificity and are therefore able to transform a wide range of white-rot fungus P. chrysosporium was reported in a two-stage substrates, even those which are most recalcitrant (Tortella et al., pilot scale reactor (May et al., 1997). Clemente et al. (2001) 2005; Cerniglia and Sutherland, 2010). Ligninolytic enzymes studied PAHs degradation by thirteen ligninolytic fungal strains can transform PAHs by producing hydroxyl free radicals by and reported that the rate of degradation varies with a variation of the donation of one electron, which oxidizes the PAH ring lignolytic enzymes (Clemente et al., 2001). In this study, highest (Sutherland et al., 1995). As a result, PAH-quinones and acids are naphthalene degradation (69%) was reported by the strain 984 formed instead of dihydrodiols. It has been seen that ligninolytic which have Mn-peroxidase activity, followed by strain 870 (17%) fungi mineralize PAHs by a combination of ligninolytic enzymes, having lignin peroxidase and laccase activities. The ability of cytochrome P450 monooxygenases, and epoxide hydrolases soil fungi to degrade PAHs that produce ligninolytic enzymes (Bezalel et al., 1997). Numerous reports have been documented was also studied under microaerobic and very-low-oxygen on the degradation of PAHs by white-rot fungi (Tortella et al., conditions (Silva et al., 2009). It was reported that Aspergillus sp., 2005; Cerniglia and Sutherland, 2010). The metabolic pathway Trichocladium canadense, and Fusarium oxysporum can degrade for the degradation of phenanthrene by the ligninolytic fungus LMW PAHs (2–3 ring compounds) most extensively, whereas Pleurotus ostreatus is illustrated in Figure 7 (Bezalel et al., 1997). highest degradation of HMW PAHs (4–7 rings) was observed in PAHs degradative potential of wood-rotting fungi Plrurotus T. canadense, Aspergillus sp., Verticillium sp., and Achremonium ostreatus and Antrodia vaillantii was also examined in soil, sp. These results suggest that, along with bacteria, fungi can be artificially contaminated with fluorene, phenanthrene, pyrene, exploited as a valuable endeavor for the bioremediation of PAH and benz[a]anthracene (Andersson et al., 2003). It has been contaminated sites. reported that although P. ostreatus significantly increased the degradation of PAHs in soil, but in the process, accumulated toxic PAH metabolites. It has also been seen that this white- MICROALGAL DEGRADATION OF PAHs rot fungus inhibits the indigenous microbial populations in the soil, which may have prohibited the complete mineralization Compared to bacteria and fungi, relatively little attention of the PAHs. Conversely, the brown-rot fungus A. vaillantii has been paid to the biodegradation of PAHs by microalgae did not generate any dead-end metabolites, although its PAHs (cyanobacteria, diatoms etc.). Microalgae are one of the major degradation rate was similar to that of P. ostreatus (Andersson primary producers in aquatic ecosystems, and play vital roles FIGURE 7 | Proposed phenanthrene degradation pathway by the ligninolytic fungus Pleurotus ostreatus (adapted from Bezalel et al., 1997). Frontiers in Microbiology | www.frontiersin.org 11 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 12 Ghosal et al. PAH Biodegradation by Microbes in the fate of PAHs in those environments. Several strains Table S2 represents different microalgal strains involved in of microalgae are known to metabolize/transform naphthalene, the bioremediation of various PAHs. Figure 8 illustrates the phenanthrene, anthracene, BaP and other PAHs (Cerniglia rubisco large subunit gene-based phylogenetic analysis of all the et al., 1979, 1980a,b; Narro et al., 1992a,b; Safonova et al., organisms enlisted in Supplementary Table S2. Figure 9 shows 2005; Chan et al., 2006; El-Sheekh et al., 2012). Supplementary the biotransformation pathway of naphthalene by microalgae FIGURE 8 | Phylogenetic tree based on ribulose 1, 5-bisphosphate carboxylase (rubisco) gene sequence (large subunit) of organisms enlisted in Supplementary Table S2. Organism designations are according to those given in Supplementary Table S2. rubisco gene sequences are taken from representative strains present in NCBI and the respective accession numbers are given within parentheses. rubisco gene sequence (large subunit) of organism Nicotiana tabacum was used as outgroup. Numbers at nodes indicate levels of bootstrap support based on neighbor-joining analysis of 100 resampled datasets. Bootstrap values below 60% are not shown. Bar represents 0.05 substitutions per nucleotide position. Frontiers in Microbiology | www.frontiersin.org 12 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 13 Ghosal et al. PAH Biodegradation by Microbes incubated with mixture of aliphatic hydrocarbons and PAHs as substrates. Though the immobilized organism can degrade n-alkanes but it’s PAH degradation rate is very less, however, PAHs accumulation did not impair the degradation of PAHs; whereas in case of free-living cells, the organisms can reduce the concentration of both PAHs and n-alkanes satisfactorily (Ueno et al., 2006, 2008). Hong et al. (2008) have examined the accumulation and biodegradation of phenanthrene (Phn) and fluoranthene (Fla) by the two diatoms Skeletonema costatum and Nitzschia sp., enriched from a mangrove aquatic ecosystem. It was seen that the strains were capable of accumulating and degrading phenanthrene and fluoranthene simultaneously and the PAHs accumulation and degradation capability of Nitzschia sp. were higher than those of S. costatum. Further, it had been observed that the degradation of fluoranthene by the two diatoms was slower, compared to phenanthrene. The strains also showed similar or higher efficiency in the removal of the Phn–Fla mixture than Phn or Fla alone (Hong et al., 2008). In another study, the efficiency of seven microalgal species to remove pyrene from solution was reported (Lei FIGURE 9 | Proposed naphthalene transformation pathway by the et al., 2002). In a recent study removal of benzo(a)pyrene cyanobacteria, Oscillatoria sp. strain JCM (adapted from Cerniglia (BaP) by sorption and degradation was determined by two et al., 1980a). microalgal species Selenastrum capricornutum and Scenedesmus acutus (Garcia de Llasera et al., 2016). It has been seen that S. capricornutum can remove 99% of BaP after 15 h of Oscillatoria sp., strain JCM (Cerniglia et al., 1980a). Under exposure, whereas S. acutus can remove 95% after 72 h of photoautotrophic growth conditions, strain JCM has been exposure. In a separate study, the effects of metals on biosorption reported to oxidize naphthalene to 1-naphthol, whereas marine and biodegradation of fluorene, phenanthrene, fluoranthrene, cyanobacterium Agmenellum quadruplicatum strain PR-6 can pyrene and benzo[a]pyrene by Selenastrum capricornutum were convert phenanthrene to phenanthrene trans-9,10-dihydrodiol investigated (Ke et al., 2010). It has been shown that both metal and 1-methoxyphenanthrene (Narro et al., 1992a,b). In a dosage and exposure time yielded a significant effect on the separate study, oxidation of BaP by the microalgae Selanastum ability of removal of low molecular weight PAHs like fluorene capricornutum has been evaluated. Transformation of BaP and phenanthrene by the alga, whereas for high molecular resulted in the formation of cis-4,5-, 7,8-, 9,10- and 11,12- weight PAHs like fluoranthrene, pyrene and BaP, the removal BaP-dihydrodiols, involving a dioxygenase system, similar efficiency was not affected by the presence of metals. Patel to bacterial PAH degradation systems but unlike those of et al. (2016) recently reported the biodegradation of anthracene eukaryotic organisms (like fungi) which involve monooxygenase and pyrene by Anabaena fertilissima (Patel et al., 2016), while systems (Warshawsky et al., 1988). In another study, the Takácov ˇ á et al. (2014) reported the biodegradation of BaP by effects of gold, white or UV-A fluorescent lights for the the microalgae Chlorella kessleri. Removal and transformation biotransformation of BaP and phototoxicity of carcinogenic of seven high molecular weight PAHs in water was reported PAHs in different algal systems was determined. It has been by live and dead cells of a freshwater microalga, Selenastrum found that algae like S. capricornutum, Scenedesmus acutus capricornutum under gold and white light irradiation. The and Ankistrodesmus braunii are able to degrade BaP to removal efficiency of PAHs, and the effectiveness of live and dihydrodiols, and the degradation varies with the kind and dead cells, was found to be predominantly PAH dependent intensity of light sources (Warshawsky et al., 1995). The removal (Ke et al., 2010; Luo L. et al., 2014; Luo S. et al., 2014). efficiency for either fluoranthene or pyrene, or a mixture of The first study on the potential of algal–bacterial microcosms fluoranthene and pyrene were also determined using Chlorella was reported for the biodegradation of aromatic pollutants vulgaris, Scenedesmus platydiscus, Scenedesmus quadricauda, and comprising salicylate, phenol and phenanthrene in a one- Selenastrum capricornutum microalgal species (Lei et al., 2007). stage treatment (Borde et al., 2003). The green alga Chlorella After 7 days of treatment PAHs removal by S. capricornutum sorokiniana was grown with those three aromatics at different and C. vulgaris was 78 and 48% respectively. The removal rate of concentrations, showing increasing inhibitory effects in the order fluoranthene and pyrene in a mixture was found to be similar, or salicylate < phenol < phenanthrene. However, a satisfactory higher than the respective single compound, which indicates that removal (>85%) was achieved only in the system having the presence of one PAH acts synergistically in the removal of the both bacteria and algae, incubated under continuous lighting, other PAH (Lei et al., 2007). In another study, microalgal strain indicating the synergistic relationship between the algal–bacterial Prototheca zopfii immobilized in polyurethane foam has been microcosms in the removal of organic pollutants. An algal– reported to accumulate a mixture of PAHs in the matrix, when bacterial consortium consisting of Chlorella sorokiniana and Frontiers in Microbiology | www.frontiersin.org 13 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 14 Ghosal et al. PAH Biodegradation by Microbes Pseudomonas migulae was also reported for the degradation of Various white-rot fungi can metabolize PAHs along with phenanthrene under photosynthetic conditions and without an bacteria in consortium by improving bioavailability of target external source of oxygen (Munoz et al., 2003). In another study, compounds. Due to lack of suitable enzymes, generally fungi accelerated pyrene degradation by a bacterial-algal consortium cannot degrade HMW PAHs completely, but can transform them was reported under photosynthetic condition (Luo L. et al., 2014; into comparatively polar metabolite(s) with their extracellular Luo S. et al., 2014). These studies indicate that microalgae can enzymes, which can further be degraded by bacteria and other be used singly or along with bacteria as a potential candidate microbes (Sutherland, 1992). Meulenberg et al. (1997) reported for biodegradation of PAHs. However, more research is needed that the degradation product of anthracene by white-rot fungi for optimizing their efficiency to apply them successfully in the can be mineralized by indigenous mixed bacterial cultures (e.g., bioremediation of PAH-contaminated environment. activated sludge or soil) more quickly than anthracene itself (Meulenberg et al., 1997). It has been seen that, inoculation of fungal–bacterial co-cultures into the soils contaminated with DEGRADATION OF PAHs BY MICROBIAL PAHs, can significantly enhance degradation of HMW PAHs, like chrysene, benzo-[a]anthracene and dibenzo[a,h]anthracene CONSORTIA AND CO-METABOLISM (Boonchan et al., 2000). Consequently, it is assumed that Occasionally it has been observed that a particular microor- PAH degradation in nature is a result of coordinate steps ganism does not have all the genes required for the complete mediated by fungi and bacteria, with the fungi playing the initial mineralization of a particular organic pollutant like PAH. oxidation step (Meulenberg et al., 1997; Boonchan et al., 2000; Therefore, researchers are developing microbial consortia for Cerniglia and Sutherland, 2010). Along with bacteria and fungi, complete degradation of such pollutants. In those consortia, each various microalgal strains have been used for the degradation microorganism has specialized role in certain degradation steps of PAHs in a consortium. Microalgae can be exploited as where, intermediates produced by certain microorganisms are a potential candidate for degradation of PAHs specifically in utilized by other members. On the other hand, cometabolism is aquatic environments. Metabolic competition is another feature a phenomenon by which a recalcitrant compound is degraded of biodegradation when a combinations of two individually in the presence of an analogous degradable compound. degradable PAHs are present in the medium (Bouchez et al., Several microorganisms can co-metabolize PAHs, and it is 1995). For example, when LMW PAHs such as phenanthrene a very complex phenomenon. As PAHs in the environment and fluorene present in the medium, they can inhibit the are present as a mixture, co-metabolism plays a very crucial degradation of fluoranthene and pyrene (Dean-Ross et al., role for bioremediation of PAHs contaminated sites. Co- 2002). Nevertheless, another cause of inhibition is due to the metabolism of one PAH could have a synergistic effect on the formation of dead-end products that result from co-metabolic degradation of other PAHs, specifically for the degradation degradation of non-growth substrates (Stringfellow and Aitken, of HMW PAHs (van Herwijnen et al., 2003). In a report, 1995). Rhodococcus sp. strain S1 when grown on anthracene has shown to cometabolize phenanthrene to phenanthrene trans-9,10- FACTORS AFFECTING THE dihydrodiol (Tongpim and Pickard, 1999). The cometabolic degradation of phenanthrene, fluoranthene, anthracene and BIOREMEDIATION OF PAHs dibenzothiophene was reported by the fluorene grown The effectiveness of bioremediation has been mainly investigated Sphingomonas sp. LB126 (van Herwijnen et al., 2003). In a under ideal laboratory conditions, having a circum-neutral separate study, cometabolic degradation of a creosote-PAHs pH and ambient mesophilic temperature. However, in the mixture including phenanthrene, fluoranthene, and pyrene, by real situation, bioremediation can be effective only at sites the pyrene-degrading strain Mycobacterium sp. AP1 was reported (Lopez et al., 2008). However, it has been observed that the rate where environmental conditions permit microbial growth and express associated enzyme activity so that microorganisms can of degradation of individual PAHs was related to their aqueous enzymatically attack the pollutants converting them to harmless solubility, for example, the biodegradation rate of fluoranthene products. Numerous abiotic and biotic factors (such as pH, and pyrene are significantly lower than that of phenanthrene nutrient availability and the bioavailability of the pollutants) can (Lopez et al., 2008). In another study, cometabolism of apparently differ from site to site, which in turn can influence acenaphthene and acenaphthylene by the succinate grown the process of bioremediation in those environments either by Beijerinckia sp. and one of its mutant strain, Beijerinckia sp. inhibiting or accelerating the growth of the pollutant-degrading strain B8/36 was reported (Schocken and Gibson, 1984). Both microorganisms. Figure 10 illustrates the various abiotic and the wild type and the mutant strains cometabolize acenaphthene biotic factors influencing PAHs degradation in soil. The main to 1-acenaphthenol, 1-acenaphthenone, 1,2-acenaphthenediol, environmental factors that could affect the rate of biodegradation acenaphthenequinone, and 1,2-dihydroxyacenaphthylene. of PAHs in the environment are summarized below. Furthermore, Sphingobium sp. strain PNB was observed to co-metabolize fluoranthene, acenaphthene, benz[a]anthracene, pyrene and benzo[a]pyrene, in presence of phenanthrene Temperature indicating metabolic robustness of the strain (Roy et al., Temperature has a profound effect on the biodegradation 2013). of PAHs in contaminated sites since those places are not Frontiers in Microbiology | www.frontiersin.org 14 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 15 Ghosal et al. PAH Biodegradation by Microbes FIGURE 10 | Abiotic and biotic factors influencing the degradation of PAHs in soil. always at ambient temperature for activity of the inhabitant most of them for their normal activity. However, it has been microorganisms. When temperature increases, the solubility of seen that in many PAHs contaminated sites, pH is very far PAHs also increases, which in turn increases the bioavailability of from neutral settings. For example, some abandoned gasworks PAH molecules. On the other hand, with increasing temperature, sites frequently contain a huge amount of demolition waste like dissolved oxygen level decreases, as a result of which the concrete and brick. Leaching of these materials can result in the metabolic activity of aerobic mesophilic microorganisms reduces. increase of pH of the native soil. Moreover, the leaching of some There are some microorganisms called psychrophiles, which can material like coal spoil can generate an acidic environment due to operate at low temperature, and are in contrast to thermophiles the oxidation of sulfides. These acidic or alkaline conditions can that function at high temperature. At high temperature, some create adverse conditions for microbial activities, which in turn pollutants can get transformed into a new compound and often decrease the biodegradation of PAHs in those sites. Hence, it is the daughter compound appears to be more toxic than the parent recommended to adjust the pH at those sites by adding chemicals: compound, which in turn inhibits its biodegradation rate (Müller basic soils can be treated with ammonium sulfate or ammonium et al., 1998). Generally in most of the studies biodegradation nitrate while acidic soils can be treated by liming with calcium or of PAHs have been examined under moderate temperatures, magnesium carbonate (Bowlen and Kosson, 1995) to generate an however, there are reports on PAHs biodegradation under environment for effective biodegradation. extreme temperatures. For example, biodegradation of petroleum hydrocarbons including PAHs was reported in seawater at low Oxygen temperatures (0–5 C) (Brakstad and Bonaunet, 2006) while the Biodegradation of organic contaminants including PAHs can biodegradation of PAHs along with long chain alkanes was proceed under both aerobic and anaerobic conditions; however, documented at very high temperature (60–70 C) by Thermus and most of the studies focus bioremediation of PAHs under aerobic Bacillus spp (Feitkenhauer et al., 2003). condition, where oxygen acts as a co-substrate and a rate limiting factor. During aerobic biodegradation of PAHs, oxygen pH is required for the action of both mono- and dioxygenase pH also plays a significant role in biodegradation processes enzymes in the initial oxidation of the aromatic rings. For including that of PAHs. Usually, microorganisms are pH- in situ bioremediation of contaminants, sometimes oxygen sensitive and near neutral conditions (6.5–7.5) are favored by is added from an external source. The addition of oxygen Frontiers in Microbiology | www.frontiersin.org 15 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 16 Ghosal et al. PAH Biodegradation by Microbes externally may be achieved by drainage, tilling, and addition is the chemical and biological extractability of the contaminants, of chemicals that release oxygen or by injection of air into the which is known as ‘aging’ of the contaminant (Alexander, 2000; contaminated site (Pardieck et al., 1992; Atlas and Unterman, Luo et al., 2012). It has been reported that extractability and 1999; Bewley and Webb, 2001). In a study, enhanced intrinsic bioavailability of PAHs decrease significantly with time in aging bioremediation of BTEX compounds in aquifer using an oxygen processes which can be significantly rate-limiting for in situ releasing compound (magnesium peroxide; ORC ) was reported bioremediation (Alexander, 2000; Megharaj et al., 2002; Luo et al., (Odencrantz et al., 1996). Again, in a separate study, in 2012; Abdel-Shafy and Mansour, 2016). situ bioremediation of an aquifer contaminated with various pollutants, including phenols, BTEX compounds and PAHs was Inhibition Due to Excess Substrate or reported, in which sodium nitrate was circulated through the End Product aquifer as the oxygen source, via a series of injection and The major goal of bioremediation is the removal or detoxification abstraction wells (Bewley and Webb, 2001). of contaminants from a given environment. However, sometimes it is possible that the contaminant gets transformed into a more Nutrients toxic dead end product. This is why it is essential to confirm Availability of nutrients is another rate-limiting factor for that the pollutant is completely mineralized at the end of the successful bioremediation of PAHs contaminated environments. treatment (Mendonca and Picado, 2002; Lundstedt et al., 2003). Along with easily metabolized carbon source, microorganisms A study using a bioreactor to treat PAH-contaminated gasworks require various minerals like nitrogen, phosphorus, potassium, soil using in situ bioremediation monitored both the removal of and iron for normal metabolism and growth. Thus, PAHs and the accumulation of oxy-PAHs, such as PAH-ketones, supplementation of nutrients, in the poor nutrient content quinines and coumarins as dead end products (Lundstedt et al., pollutant contaminated sites is required to stimulate the growth 2003). As oxy-PAHs are more toxic than the parent PAHs, this of autochthonous microorganisms, to enhance bioremediation of study highlights the significance of monitoring the metabolites pollutants (Atagana et al., 2003). In marine environments, poor during bioremediation, specifically for toxic dead-end products, biodegradation of petroleum hydrocarbon is due to a low level of and determining the toxicity of the metabolites both before and nitrogen and phosphorous (Floodgate, 1984). On the other hand, after treatment (Lundstedt et al., 2003). excessive nutrient availability can also inhibit the bioremediation Since PAHs in the environment are present as mixtures, of pollutants (Chaillan et al., 2006). There are several reports on the effect of substrate interaction during biodegradation is the negative effects of high nutrient levels on the biodegradation crucial in determining the fate of PAHs in nature. It has been of organic pollutants especially PAHs (Carmichael and Pfaender, reported that, when present as a mixture, the high molecular 1997; Chaîneau et al., 2005). weight PAHs are degraded followed by the degradation of low molecular weight PAHs, by the bacterial community (Mueller Bioavailability et al., 1989). It has been also observed that a high concentration In case of a biological system, the term bioavailability is defined of naphthalene have inhibitory effect on the degradation of other as the fraction of a chemical that can be taken up or transformed PAHs, by a defined bacterial coculture (Bouchez et al., 1995). by living organisms during the course of the experiment. This Similarly, a competitive inhibition of phenanthrene degradation fraction can vary under the influence of mass transfer parameters, by naphthalene, methylnaphthalene and fluorene in binary which include physicochemical processes governing dissolution, mixtures using two pure cultures was reported (Stringfellow and desorption and diffusion, hydrological processes like mixing and, Aitken, 1995). Thus during in situ bioremediation of PAHs, finally, biological processes, such as uptake and metabolism concentration of PAHs in the contaminated sites, and a chance (Bosma et al., 1997; Semple et al., 2003). Bioavailability is for the formation of the toxic dead end product(s) need to be regarded as one of the most crucial factors in bioremediation considered for effective process development. of pollutants. Biodegradation of PAHs in the environment is often limited, due to their low aqueous solubility and their strong tendency to sorbs to mineral surfaces (like clays) and RECENT ADVANCEMENT IN organic matters (like black carbon, coal tar humic acids) in MOLECULAR TECHNIQUES IN the soil matrix, processes which reduce bioavailability. There UNDERSTANDING BACTERIAL are numerous reports on poor or unsuccessful remediation of PAHs contaminated sites due to absorption of PAHs in black DEGRADATION OF PAHS AND FUTURE carbon, coal-tar substances which decreases bioavailability of DIRECTIONS PAHs (Volkering et al., 1992; Luthy et al., 1993, 1994; Nelson et al., 1996; Northcott and Jones, 2001; Hong et al., 2003; Bucheli Nowadays, bioremediation has become an intensive area et al., 2004; Cornelissen et al., 2005; Liu et al., 2009; Benhabib of research. However, more advancement has to be made et al., 2010; Rein et al., 2016). Again, the aqueous solubility in developing practically efficient microbial bioremediation of PAHs decreases with increasing molecular weight, which in processes. Although microorganisms have the capability to use turn reduces the bioavailability of PAHs. It has been seen that numerous organic pollutants as their carbon and energy sources, longer any hydrophobic organic contaminants (like PAHs) is in their proficiency at eliminating such pollutants might not be contact with soil, the more irreversible the sorption, and lesser of top class level for cleaning up present-day pollution. In fact, Frontiers in Microbiology | www.frontiersin.org 16 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 17 Ghosal et al. PAH Biodegradation by Microbes microorganisms have evolved in the direction of ecological details (Nierman and Nelson, 2002; Buermans and den Dunnen, fitness rather than biotechnological efficacy (Schmid et al., 2014). Presently many complete or nearly complete genome 2001). To enhance the catabolic efficiency of a microorganism sequences of cultivable microorganisms are available which have for bioremediation, bioengineering is a prerequisite. Recent potential catabolic activity. However, unfortunately by using advancement in genetic, genomic, proteomic and metabolomic modern microbiological and molecular techniques over the past technologies, which are applied to study bioremediation of 40 years so far only an extremely small fraction (1%) of the organic pollutants, have contributed considerably to enrich total microbial diversity has been cultivated from all habitats our knowledge on various aspects of the physiology, ecology, investigated (Hugenholtz and Pace, 1996; Hugenholtz et al., biochemistry and regulatory mechanisms of the microbial 1998). While culturing novel environmental microorganisms is catabolic pathways. Hence, practical utilization of that indispensable (Zinder, 2002), perhaps the greatest promise to knowledge is essential to manipulate or reconstruct as well date comes from metagenomic approaches, which are the only as enhance the natural processes, thereby making strategies ones currently available to adequately utilize the tremendous for the development of more competent biocatalysts for microbial diversity- one of the richest sources on earth various biotechnological applications including bioremediation (Handelsman et al., 1998). Although there are some associated of culprit organic pollutants in the contaminated sites and challenges in metagenome-based approaches, scientists already transformation of toxic chemical into harmless product or overcame many of those problems (Liles et al., 2008; Mareckova other specialty chemicals (Schmid et al., 2001; Carmona et al., et al., 2008). The progressively decreasing cost and increasing 2009). speed of DNA sequencing has made it possible to sequence A considerable advancement in microbial ecology was billions of bases of metagenomic DNA. Culturable aromatics achieved based on the identification of conserved sequences degrading bacteria are being isolated mostly on the basis of their present in a particular group of microorganisms, most notably ability to utilize aromatic compound as their sole carbon and the 16S rRNA or 18S rRNA genes which could provide a energy sources and by genome sequencing, scientist can identify phylogenetic characterization of the microbial population all the genes necessary to completely degrade the compound. present in a particular habitat (Pace et al., 1986; Amann et al., Therefore, inclusive knowledge of the catabolic potential of 1995). This approach is a significant achievement in the field a contaminated site virtually remains unknown by using of bioremediation because by determining the microbiota in culture-dependent techniques. High throughput metagenomics a polluted environment it is possible to identify particular reduces that bottleneck. However, at present, relatively little microorganisms inhabiting those environments, thereby resources have been spent for the sequencing of soil or marine predicting possible bioremediation potential. With the advent metagenomes contaminated with aromatic pollutants, compared of Denaturing Gradient Gel Electrophoresis (DGGE), it is now to those committed to the human microbiome (Blow, 2008). That possible to analyze the microbial community structure and is why there is a scarcity of information about the xenobiotics dynamics of a particular habitat, more precisely (Gallego et al., degrading novel bacteria that are difficult to culture in the 2014; Vila et al., 2015). Development of fluorescence in situ laboratory. Sites contaminated with toxic chemicals have hybridization (FISH), which is another technique is quite useful become biotechnological gold mines because the indigenous in this field and has often been practiced (Amann et al., 2001). microorganisms may have evolved the necessary enzymes, to Generally, there is a positive link between the relative degrade or transform those toxic contaminants to an inanimate abundance of the genes associated with pollutant removal and one, quite different from those found in common cultivable the efficiency of bioremediation. However, sometimes it is microorganisms (Handelsman et al., 1998; Galvao et al., 2005; possible that the genes associated with pollutant removal can Boubakri et al., 2006). In addition, sequencing of the soil or be present but not expressed. So, there is an increasing interest aquatic metagenome will also provide insights into the ecology in quantifying the mRNA for key catabolic genes via real-time of microorganisms which in turn will help to identify who the PCR (Schneegurt and Kulpa, 1998; Debruyn and Sayler, 2009). dominant and rare community members are and what are their In addition transcriptomics, DNA-based stable isotope probing, probable roles in the degradation of recalcitrant molecules like single cell genomics and DNA microarray techniques are also PAHs and other related xenobiotics. emerging for application in the field of bioremediation (Wilson Recently another two techniques metaproteomics and et al., 1999; Denef et al., 2005, 2006; Chain et al., 2006; Parnell metabolomics have been utilized to unfold various aspects of et al., 2006; Macaulay and Voet, 2014; Mason et al., 2014; environmental microbiology and have shown their promise in Mishamandani et al., 2014). DNA microarray which is a high- the field of bioremediation (Nesatyy and Suter, 2007; Keum throughput version of DNA hybridization technique can detect et al., 2008). Proteomics is an efficient technology to recognize an enormous number of genes in a single test. One of the proteins and their roles associated with catabolism of PAHs recent applications of microarray in the PAHs biodegradation while metabolomics can be exploited to identify metabolites is the construction of GeoChip (He et al., 2010; Nostrand et al., produced during PAHs biodegradation. Figure 11 illustrates a 2012). summarized representation of the molecular techniques involved The advancement in genome sequencing technology in studying microbial degradation of PAH. In near future, practically revolutionized the field of bioremediation. By whole functional metagenomics, metaproteomics, metabolomics, genome sequencing, it is now possible to study the physiology metatranscriptomics and DNA microarrays will become crucial of microorganisms associated with pollutant removal in more tools to elucidate the mechanisms of biodegradation of PAHs Frontiers in Microbiology | www.frontiersin.org 17 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 18 Ghosal et al. PAH Biodegradation by Microbes FIGURE 11 | Schematic diagram summarizing different molecular techniques as future directions for better understanding of the microbial PAH degradation and ecologically sustainable bioremediation of sites contaminated with PAHs. in the environment, and will offer more information on as-yet- the bioremediation potential of the indigenous micro flora uncultured organisms associated with PAHs bioremediation dwelling at the site contaminated with pollutants. These (Wilson et al., 1999; Amann et al., 2001; Fromin et al., 2002; Liang, techniques provide the treatment at the site itself avoiding 2002; Zhou and Thompson, 2002; Baldwin et al., 2003; Ginige excavation and transport of contaminants, which makes them et al., 2004). Moreover, in silico biology is being progressively the most desirable options due to lower cost and fewer applied in different aspects in the field of bioremediation (Kweon disturbances. On the other hand extrinsic bioremediation et al., 2010; Chakraborty et al., 2012; Khara et al., 2014). Thus, (also called ‘ex-situ’ bioremediation) mainly involves the it is expected that emerging molecular biological, analytical physical removal of the contaminated material to another and computational methods which can predict the activity of location for treatment (Carberry and Wik, 2001). While microorganisms involved in biodegradation, should transform in the case of “biostimulation” process, it is generally the bioremediation field from a largely empirical practice into a possible to stimulate the indigenous microorganisms to use branch of modern science. the contaminants as a food source at a much greater rate by compensating limiting parameters, for example, by introducing additional oxygen or nutrients to the indigenous population APPROACHES TO THE (Straube et al., 2003). Another method, “bioaugmentation,” involves introduction of exogenous microorganisms into the BIOREMEDIATION OF contaminated environment which are capable of degrading the PAH-CONTAMINATED ENVIRONMENTS target pollutants, either with or without additional nutrients (Straube et al., 2003). Whereas, “humification” is a process Various types of bioremediation technologies can be employed by which strongly persistent substances in the polluted for contaminant removal. Intrinsic bioremediation (also called environment are incorporated into the humic substances mostly natural attenuation or ‘in situ’ bioremediation) depends on Frontiers in Microbiology | www.frontiersin.org 18 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 19 Ghosal et al. PAH Biodegradation by Microbes by means of enzymatic reactions (Megharaj et al., 2011). In bioremediation procedures. However, due to the high cost addition, “phytoremediation” involves removal of environmental associated with the extraction and shipment, ex situ treatment contaminants using plants (Haritash and Kaushik, 2009; of contaminated groundwater is not generally performed. Megharaj et al., 2011). Another bioremediation technology called Few reports are documented on the treatment of PAH ‘landfarming,’ is used to stimulate indigenous biodegradative contaminated water. Bewley and Webb (2001) reported the microorganisms by providing nutrients, water and oxygen, in in situ bioremediation of an aquifer which was contaminated order to facilitate aerobic degradation of contaminants (Megharaj with diverse pollutants, including phenols, BTEX compounds et al., 2011). While ‘composting’ involves the degradation and PAHs. The site was bioremediated using a procedure of pollutant along with other agricultural wastes such as involving a combination of bioaugmentation and biostimulation. manure etc. (Semple et al., 2001). Hence, it becomes clear that Nutrients (a commercial mixture of urea and diammonium regardless of the method chosen, an ideal bioremediation strategy phosphate), a commercially available phenol-degrading mixed can be designed only on the basis of knowledge about the bacterial inoculum (PHENOBAC, Microbac Ltd, Durham), and microorganisms and the contaminant present within the polluted sodium nitrate (an oxygen source) were circulated through the environments, along with their catabolic potential and response aquifer via a series of injection and abstraction wells. After to changes in the environmental conditions. treatment for over two and half years, the mean concentration 1 1 of PAHs was reduced to 0.9 mgL from 11 mgL along with Treatment of Soils and Sediments reduction of other contaminant (Bewley and Webb, 2001). Apart There are some reports on the treatment of PAH contaminated from that, groundwater remediation technology of petroleum- soils and sediments by in situ or ex situ bioremediation methods. derived compounds (PDCs) including PAHs based on enhanced Straube et al. (2003) reported that a pilot-scale landfarming solubility of PDCs in humic acid was also reported (Van treatment of PAH-contaminated soil from a wood treatment Stempvoort et al., 2002). Zein et al. (2006) reported the treatment facility was achieved by biostimulation of the soil with water, of groundwater contaminated with PAHs, gasoline hydrocarbons, ground rice hulls as a bulking agent, and palletized dried blood and methyl tert-butyl ether using an ex situ aerobic biotreatment as a nitrogen source and bioaugmentation of the microbial system and it was observed that after 10 months of treatment, the community with an inoculum of Pseudomonas aeruginosa strain concentration of PAHs was reduced to >99% along with other 64 (Straube et al., 2003). It has been seen that within 1 year pollutants (Zein et al., 2006). of treatment 86% of total PAHs were removed from the soil including a moderate reduction in HMW PAHs such as pyrene, Treatment with Genetically Engineered BaP etc. In a separate study on the treatment of an aged gasworks Microorganisms (GEMs) soil contaminated with aromatic compounds including PAHs, it has been observed that LMW PAHs and heterocyclic compounds The bioremediation of PAHs contaminated site is generally were degraded more quickly than the HMW counterparts very slow because there are a number of biotic and abiotic (Lundstedt et al., 2003). In addition, the unsubstituted PAHs factors responsible for successful bioremediation. In addition, were degraded faster than the related alkyl-PAHs as well as the incomplete success rates might be due to the fact that some nitrogen-containing heterocyclics. Treatment of soil at a tar places are heavily contaminated, and hence, the microorganisms contaminated site via composting along with conventional land are incapable to grow and degrade the contaminant at the treatment process revealed that composting led to more extensive same rate at which they are introduced into the environment. PAH removal than did by two different land treatment processes Although there is very few information on the use of GEMs (Guerin, 2000). Sasek et al. (2003) reported the remediation in bioremediation, they can be a promising candidate for of a manufactured-gas plant soil contaminated with PAHs via such processes (Ang et al., 2005; Singh et al., 2008; Megharaj composting. In this study, treatment of soil was performed et al., 2011). Using genetic engineering it is possible to in a thermally insulated chamber using mushroom compost enhance the activity or broad substrate specificity of certain containing wheat straw, chicken manure and gypsum, where at enzymes associated with PAH-degrading pathways, which in the end of 54 days, removal of 20–60% of individual PAHs turn will improve the mineralization of those pollutants in the was reported, while 37–80% of individual PAHs degradation environment (Timmis et al., 1994; Timmis and Pieper, 1999; Ang was observed after another 100 days of composting (Sasek et al., et al., 2005; Singh et al., 2008). In recent times, various molecular 2003). Moreover, studies on the removal of PAHs present in biology tools such as gene conversion, gene duplication, and contaminated soil using the associated bacterial communities in transposon or plasmids mediated gene delivery are available, two aerobic, lab-scale, slurry-phase bioreactors, which were run which might play vital roles to boost up the biodegrading semi-continuously and fed either on weekly or monthly basis, potential of naturally occurring microorganisms (Timmis et al., showed that most of the PAHs, including HMW PAHs were 1994; Timmis and Pieper, 1999; Ang et al., 2005; Singh et al., 2008; biodegraded to a greater extent in the weekly fed bioreactor for Megharaj et al., 2011). However, it is crucial to ensure the stability up to 76% (Singleton et al., 2011). of GEMs prior to their field application since the catabolic activity of released GEM is associated largely with the stability of the Treatment of Waters recombinant plasmid introduced into the organism (Brunel et al., Polycyclic aromatic hydrocarbons-polluted groundwater can 1988; Shaw et al., 1992; Samanta et al., 2002). As GEMs that also be bioremediated through both in situ and ex situ are released into a contaminated site can spread to other places Frontiers in Microbiology | www.frontiersin.org 19 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 20 Ghosal et al. PAH Biodegradation by Microbes and multiply under favorable conditions, so before releasing into soil (Gandolfi et al., 2010; Loick et al., 2012; Lukic ´ et al., 2016; the environment there should be a clear understanding of the Rein et al., 2016). So adjustment of organic matter content of possible side effects associated with GEMs, which would possibly a polluted site with exogenous addition of compost or other guide the restriction of their application in pollutant abatement substances like buffalo manure, food and vegetables waste may (Samanta et al., 2002; Ang et al., 2005; Singh et al., 2008; Megharaj enhance the bioremediation efficiency of PAH polluted site. et al., 2011). In addition, instead of treating with a single microorganism, a defined microbial consortium may give a better result in some cases for successful remediation of a contaminated site. CONCLUSION So, more research is required on the catabolic capabilities of PAHs degrading microbial consortia, which will further In last few decades, there has been a great deal of progress in the deepen our understanding on the microbial consortia-mediated study of the bioremediation of PAHs. Numerous microorganisms remediation of contaminated environments, and will help to have been isolated and characterized having PAHs catabolic develop potential microbial consortium having robust pollutant potentials. In addition, many unique enzymes with different degrading potential. It is also possible to treat the contaminated catabolic efficiency associated with PAHs degradation have site sequentially with fungi, bacteria and algae or in combination been purified and different novel biochemical pathways for for more efficient pollutant removal. PAHs degradation have been elucidated. Moreover, many PAH In addition, genetic engineering can be employed to boost the catabolic operons have been sequenced, and their regulatory catabolic efficiency of microorganisms used in bioremediation. mechanism for PAH degradation has been determined. The Today, scientists are capable to create unique PAHs metabolic advancement in genetic, genomic, proteomic and metabolomic pathways by recombining different catabolic genes from different approaches, which are employed to study catabolism of organisms in a single host cell. In this way it is possible to organic pollutants have contributed remarkably in understanding enhance the substrate specificity of a catabolic pathway to the physiology, ecology, biochemistry of PAHs degrading degrade new substrates, complete partial pathways and also to microorganisms. However, more detail research is a prerequisite construct novel pathways not found in nature, which may permit to determine exactly what is going on in PAH-contaminated the mineralization of highly recalcitrant compounds and avoid environment. In addition, there are still various aspects of the accumulation of toxic dead end product. However, before bioremediation of PAHs that remain unknown or otherwise have releasing such GMOs into the environment, it is necessary to insufficient information, which requires future attention. There is check thoroughly the possibility of any unwanted side effects very scarce knowledge on genes, enzymes as well as the molecular produced by those GMOs. In addition, authorities should be mechanism of PAHs degradation in high salinity environments, convinced that the GMOs are safer, cheaper, and more efficient or anaerobic environments. Also, there is very little information than the present alternatives. Thus, it is anticipated that the on the transmembrane trafficking of PAHs and their metabolites. compiled information present in this manuscript will open up Various transporters have been assumed to be participating in the new avenues to the researchers, and the field of bioremediation transport of PAHs into microorganisms, but till date, none has will revolutionize in near future. been characterized. There are various factors which may affect bioremediation of PAHs in a contaminated environment. Adjustment of AUTHOR CONTRIBUTIONS oxygen concentration, pH, temperature, nutrient availability and improvement of bioavailability may increase PAHs degradation. DG translated the concept and wrote the manuscript. DG and It is seen that in contaminated soils and sediments, PAHs get SG analyzed the data, edited and formatted the manuscript. TD entrapped in coal tar or black carbon particles, which results in and YA generated the concept and ideas, critically revised the unsuccessful remediation due to decrease in PAHs bioavailability manuscript and approved the final version for publication. (Bucheli et al., 2004). This phenomenon is a major bottleneck for successful remediation of PAHs contaminated environments (Ortega-Calvo and Alexander, 1994). Some microorganisms ACKNOWLEDGMENTS are known to excrete biosurfactants which enhance the bioavailability of organic pollutants. Many microorganisms This study was supported by the Yeungnam University Research exhibit chemotaxis toward pollutants. These strategies lead to Grant (2015). enhanced degradation of organic pollutants. The addition of small amount of biosurfactant, which increases the bioavailability of PAHs, or some merely toxic chemicals, like salicylic acid, which SUPPLEMENTARY MATERIAL induce PAHs catabolic operons may enhance biodegradation of PAHs in the environment. It has been seen that organic The Supplementary Material for this article can be found amendments influence the indigenous microbial community as online at: http://journal.frontiersin.org/article/10.3389/fmicb. well as efficiency of bioremediation of PAHs in contaminated 2016.01369 Frontiers in Microbiology | www.frontiersin.org 20 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 21 Ghosal et al. PAH Biodegradation by Microbes Binkova, B., Giguere, Y., Rossner, P. Jr., Dostal, M., and Sram, R. J. (2000). REFERENCES The effect of dibenzo[a,1]pyrene and benzo[a]pyrene on human diploid lung Abdel-Shafy, H. I., and Mansour, M. S. M. (2016). 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Hum. 32, The use, distribution or reproduction in other forums is permitted, provided the 1–453. original author(s) or licensor are credited and that the original publication in this Widada, J., Nojiri, H., Kasuga, K., Yoshida, T., Habe, H., and Omori, T. journal is cited, in accordance with accepted academic practice. No use, distribution (2002). Molecular detection and diversity of polycyclic aromatic or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 27 August 2016 | Volume 7 | Article 1369 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Microbiology Pubmed Central

Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review

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fmicb-07-01369 August 31, 2016 Time: 16:47 # 1 REVIEW published: 31 August 2016 doi: 10.3389/fmicb.2016.01369 Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review 1 2 3 1,2 Debajyoti Ghosal , Shreya Ghosh , Tapan K. Dutta * and Youngho Ahn * Environmental Engineering Laboratory, Department of Civil Engineering, Yeungnam University, Gyeongsan, South Korea, 2 3 Disasters Prevention Research Institute, Yeungnam University, Gyeongsan, South Korea, Department of Microbiology, Bose Institute, Kolkata, India Polycyclic aromatic hydrocarbons (PAHs) include a group of organic priority pollutants of critical environmental and public health concern due to their toxic, genotoxic, mutagenic and/or carcinogenic properties and their ubiquitous occurrence as well as recalcitrance. The increased awareness of their various adverse effects on ecosystem and human health has led to a dramatic increase in research aimed toward removing PAHs from the environment. PAHs may undergo adsorption, volatilization, photolysis, and chemical oxidation, although transformation by microorganisms is the major neutralization Edited by: process of PAH-contaminated sites in an ecologically accepted manner. Microbial Pankaj Kumar Arora, degradation of PAHs depends on various environmental conditions, such as nutrients, M. J. P. Rohilkhand University, India number and kind of the microorganisms, nature as well as chemical property of the PAH Reviewed by: being degraded. A wide variety of bacterial, fungal and algal species have the potential Matthias E. Kaestner, Helmholtz Centre for Environmental to degrade/transform PAHs, among which bacteria and fungi mediated degradation Research – UFZ, Germany has been studied most extensively. In last few decades microbial community analysis, Eric D. Van Hullebusch, University of Paris-Est, France biochemical pathway for PAHs degradation, gene organization, enzyme system, *Correspondence: genetic regulation for PAH degradation have been explored in great detail. Although, Tapan K. Dutta xenobiotic-degrading microorganisms have incredible potential to restore contaminated tapan@jcbose.ac.in environments inexpensively yet effectively, but new advancements are required to Youngho Ahn yhahn@ynu.ac.kr make such microbes effective and more powerful in removing those compounds, which were once thought to be recalcitrant. Recent analytical chemistry and genetic Specialty section: This article was submitted to engineering tools might help to improve the efficiency of degradation of PAHs by Microbiotechnology, Ecotoxicology microorganisms, and minimize uncertainties of successful bioremediation. However, and Bioremediation, appropriate implementation of the potential of naturally occurring microorganisms for a section of the journal Frontiers in Microbiology field bioremediation could be considerably enhanced by optimizing certain factors Received: 02 May 2016 such as bioavailability, adsorption and mass transfer of PAHs. The main purpose Accepted: 18 August 2016 of this review is to provide an overview of current knowledge of bacteria, halophilic Published: 31 August 2016 archaea, fungi and algae mediated degradation/transformation of PAHs. In addition, Citation: factors affecting PAHs degradation in the environment, recent advancement in genetic, Ghosal D, Ghosh S, Dutta TK and Ahn Y (2016) Current State genomic, proteomic and metabolomic techniques are also highlighted with an aim of Knowledge in Microbial to facilitate the development of a new insight into the bioremediation of PAH in the Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review. environment. Front. Microbiol. 7:1369. doi: 10.3389/fmicb.2016.01369 Keywords: biodegradation, polycyclic aromatic hydrocarbons (PAHs), bacteria, fungi, algae Frontiers in Microbiology | www.frontiersin.org 1 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 2 Ghosal et al. PAH Biodegradation by Microbes 2000; Marston et al., 2001; Xue and Warshawsky, 2005). On INTRODUCTION the basis of abundance and toxicity, 16 PAHs are already Over the last few decades, with an increasing global awareness enlisted as priority environmental pollutants by the United about the potential adverse effects of pollutants on public health States Environmental Protection Agency (US EPA), (Agency and environment, remediation and renovation of environment for Toxic Substances and Disease Registry [ATSDR], 1990; Liu contaminated with hazardous materials have received increasing et al., 2001) which are depicted in Figure 1. Various physical- attention. Among others, polycyclic aromatic hydrocarbons chemical properties and some relevant information of 16 PAHs (PAHs) include a group of priority organic pollutants of enlisted as priority pollutants by US EPA are depicted in significant concern due to their toxic, genotoxic, mutagenic Table 1. and/or carcinogenic properties (WHO, 1983; Cerniglia, 1992; In their pure chemical form, PAHs generally exist as Mastrangelo et al., 1996; Schützendübel et al., 1999). PAHs colorless, white, or pale yellow-green solids having a faint, are composed of fused aromatic rings in linear, angular, or pleasant odor. They are basically non-polar organic compounds, cluster arrangements. Generally, the electrochemical stability, characteristically composed of carbon and hydrogen atoms. persistency, resistance toward biodegradation and carcinogenic Mostly incomplete combustion of organic materials like coal, index of PAHs increase with an increase in the number of tar, oil and gas, automobile exhaust, tobacco or smoked food, aromatic rings, structural angularity, and hydrophobicity, while either during industrial and other human activities or during volatility tends to decrease with increasing molecular weight geothermal reactions associated with the production of fossil- (Mackay and Callcott, 1998; Marston et al., 2001). The PAHs fuels and minerals, result in PAH formation. In nature, they have a natural potential for bioaccumulation in various food are formed during forest fires, volcanic eruptions, or by plant chains, which make their presence in the environment quite and bacterial reactions (Blumer, 1976; Wilson and Jones, 1993). alarming (Morehead et al., 1986; Xue and Warshawsky, 2005), Diverse types of combustion yield different distributions of PAHs and are therefore being considered as substances of potential in both relative amounts of individual PAHs as well as their human health hazards (Mastrangelo et al., 1996; Binkova et al., isomers. In nature, they are formed during forest fires, volcanic FIGURE 1 | Structure of the16 PAHs enlisted as priority pollutants by US EPA. Frontiers in Microbiology | www.frontiersin.org 2 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 3 Ghosal et al. PAH Biodegradation by Microbes Frontiers in Microbiology | www.frontiersin.org 3 August 2016 | Volume 7 | Article 1369 TABLE 1 | Physical-chemical properties and some relevant information of 16 PAHs enlisted as priority pollutants by US EPA. Name Molecular Cas registry Physical chemical properties Toxicology Biodegradation formula no. a b c d B. Pt. ( C) M.Pt ( C) V.P. (mmHg at 25 C) Solubility (mg/L) TEF IARC EPA Estimated Measured half-lives e f half-lives (days) (days) Naphthalene C H 91-20-3 218 80.2 8.5  10 31 n.d. 2B C 5.66 n.d. 10 8 Acenaphthene C H 83-32-9 279 93.4 2.5  10 3.93 0.001 3 D 18.77 n.d. 12 10 Acenaphthylene C H 208-96-8 280 91.8 6.68  10 1.93 0.001 n.c. D 30.7 n.d. 12 8 Anthracene C H 120-12-7 342 216.4 6.53  10 0.076 0.01 3 D 123 2.7 14 10 Phenanthrene C H 85-01-8 340 100.5 1.2  10 1.20 0.001 3 D 14.97 5 14 10 Fluorene C H 86-73-7 295 116-7 6.0  10 1.68–1.98 0.001 3 D 15.14 n.d. 13 10 Fluoranthene C H 206-44-0 375 108.8 9.22  10 0.20–0.26 0.001 3 D 191.4 9.2 16 10 Benzo[a]anthracene C H 56-55-3 438 158 4.11  10 0.010 0.1 2B B2 343.8 > 182 18 12 9 3 Chrysene C H 218-01-9 448 254 6.23  10 1.5  10 0.010 2B B2 343.8 n.d. 18 12 Pyrene C H 129-00-0 150.4 393 4.5  10 0.132 0.001 3 D 283.4 151 16 10 9 3 Benzo[a]pyrene C H 50-32-8 495 179 5.49  10 3.8  10 1.0 1 B2 421.6 11 20 12 Benzo[b]fluoranthene C H 205-99-2 481 168.3 5.0  10 0.0012 n.d. 2B B2 284.7 n.d. 20 12 10 4 Benzo[k]fluoranthene C H 207-08-9 480 215.7 9.7  10 7.6  10 0.1 2B B2 284.7 n.d. 20 12 10 4 Dibenzo[a,h]anthracene C H 53-70-3 524 262 9.55  10 5.0  10 n.d. 2A B2 511.4 n.d. 22 14 10 5 Benzo[g,h,i]perylene C H 191-24-2 500 277 1.0  10 2.6  10 n.d. 3 D 517.1 n.d. 22 12 Indenol[1,2,3-cd]pyrene C H 193-39-5 536 161-3 1.25  10 0.062 n.d. 2B B2 349.2 n.d. 22 12 a b c (Mackay and Shiu, 1977). Toxic equivalent factor relatively to Benzo[a]pyrene (Chang et al., 2014). International Agency for Research on Cancer Classification Monographs Volume 1-111 updated 18 February 2015 (1, carcinogenic to humans; 2A, probably carcinogenic to humans; 2B, possibly carcinogenic to humans; 3, not classifiable as carcinogenic to humans; n.c., not classified). EPA carcinogenic classification: A, human carcinogenic; B1 and B2: probable human carcinogenic; C, possible human carcinogenic; D, not Classifiable as to human carcinogenicity; E, evidence of non-carcinogenicity for humans. Estimation using BioHCwin software v1.01 on EPI Suite software develop by (Howard et al., 2005). (Comber et al., 2012); n.d., not determined. fmicb-07-01369 August 31, 2016 Time: 16:47 # 4 Ghosal et al. PAH Biodegradation by Microbes eruptions, or by plant and bacterial reactions (Blumer, 1976; Consequently, cleaning of such polluted places has been Wilson and Jones, 1993). Nevertheless, the anthropogenic input thought to be one of the most essential alternatives for of PAHs to the environment far exceeds the natural sources restoring environmental damage. Several physical and chemical (National Academy of Sceinces [NAS], 1971). treatment methods including incineration, base-catalyzed de- Polycyclic aromatic hydrocarbons are formed whenever chlorination, UV oxidation, fixation, solvent extraction etc. are organic substances are exposed to high temperatures (pyrolysis), already in practice (Norris et al., 1993; Gan et al., 2009), but and the composition of the products thus formed depends have several drawbacks including cost, complexity, regulatory largely on the nature of the starting material as well as the burden etc. Moreover, these conventional techniques, in many transformation temperature (Blumer, 1976; Cerniglia, 1992). cases, do not destroy the contaminating compounds completely, Fossil fuels also contain huge amounts of PAHs which are but instead transfer them from one environment or form to released into the environment during incomplete combustion another. In order to solve this burning problem, researchers or by accidental discharge during transport, use, or disposal have devised an efficient and eco-friendly clean-up technique of petroleum products or as a result of uncontrolled emissions known as bioremediation, which is being progressively refined (Cerniglia, 1992; Wilson and Jones, 1993; Johansson and van to fight pollution. This technique utilizes and manipulates the Bavel, 2003). PAHs are widely present as contaminants in air, detoxification abilities of living organisms to convert hazardous soil, aquatic environments, sediments, surface water as well as organic wastes including xenobiotics into harmless products, in ground water (Huntley et al., 1993; Van Brummelen et al., often carbon dioxide and water (Cerniglia and Heitkamp, 1989; 1996; Boxall and Maltby, 1997; Holman et al., 1999; Lim et al., Mueller et al., 1996; Bamforth and Singleton, 2005; Johnsen 1999; Ohkouchi et al., 1999). Natural and anthropogenic sources et al., 2005). Bioremediation addresses the limitations associated of PAHs, in combination with global transport phenomena, with most of the physicochemical processes by destroying many result in their worldwide distribution and consequently, PAHs organic contaminants at reduced cost, under ambient conditions get dispersed from the atmosphere to vegetation, ultimately and thus, has now become a popular remedial alternative for leading to bioaccumulation in various food chains (Edwards, pollutant removal including PAHs (Young and Cerniglia, 1996; 1983; Morehead et al., 1986; Wagrowski and Hites, 1997). Apart Juhasz and Naidu, 2000; Kastner, 2000; Lovley, 2001; Andreoni from biodegradation, the fate of PAH in nature varies depending and Gianfreda, 2007; Jorgensen, 2007; Megharaj et al., 2011; on the environment, for example, in air, PAH can undergo Abdel-Shafy and Mansour, 2016). photo-oxidation, whereas in the case of soil and water, they In addition, many natural habitats (e.g., aquifers, aquatic can undergo both photo-oxidation and chemical oxidation while sediments) contaminated with a huge amount of aromatic some PAHs like naphthalene and alkyl naphthalene are partly lost pollutants are often anoxic. In these environments, the anaerobic by volatilization (Cerniglia, 1992). degradation of aromatic compounds by microorganisms plays The toxicity of PAH was first recognized in 1761 by John Hill, a major role in the removal of contaminants, recycling of a physician who documented a high incidence of nasal cancer in carbon and sustainable development of the ecosystem. Reports tobacco snuff consumers (Cerniglia, 1984). The low-molecular- on anaerobic biodegradation of PAHs are relatively recent, weight (LMW) PAHs (containing two or three aromatic rings) and only a limited number of preliminary studies have are acutely toxic while the high-molecular-weight (HMW) PAHs demonstrated the anaerobic degradation of PAHs including (containing four or more rings) are largely considered as naphthalene, anthracene, phenanthrene, fluorene, acenaphthene genotoxic (Cerniglia, 1992; Mueller et al., 1996; Abdel-Shafy and and fluoranthene (Foght, 2008; Carmona et al., 2009; Mallick Mansour, 2016). It is a known fact that PAH can covalently et al., 2011). However, detailed information on anaerobic bind to DNA, RNA and proteins, but it is the amount of degradation of PAHs under sulfate-reducing and nitrate- covalent interaction between PAHs and DNA that correlates reducing conditions is scarce and very little is known about best with carcinogenicity (Marston et al., 2001; Santarelli et al., their degradation pathways, catabolic genes/enzymes and/or 2008). In addition, the transformation products of some PAHs regulatory mechanisms, but this emerging field is ready to are more toxic than parent PAHs and can lead to critical blossom in various aspects of biotechnological applications. cellular effects (Schnitz et al., 1993). In humans cytochrome P450 Compared to HMW PAHs, LMW PAHs are reasonably monooxygenase group of enzymes oxidize PAHs to epoxides, more volatile and more soluble in water and consequently some of which are highly reactive (such as “bay-region” diol more susceptible to biodegradation (Pannu et al., 2003). LMW epoxides) and known as ultimate carcinogens. They can bind PAHs like naphthalene, anthracene and phenanthrene are to DNA and have the ability to transform normal cells to widely present throughout the environment and designated malignant one (Milo and Casto, 1992; Schnitz et al., 1993; as prototypic PAHs and serve as signature compounds to Marston et al., 2001). Studies have shown that processing food detect PAH contamination. Naphthalene represents the simplest at high temperatures, like grilling or barbecuing result in high PAH whereas the chemical structures of anthracene and levels of PAHs in cooked meat and smoked fish (Morehead et al., phenanthrene are found in many carcinogenic PAHs (such as 1986). The concern associated with PAHs further increased due in benzo[a]pyrene, benz[a]anthracene etc.), and phenanthrene to their ability to interfere with hormone metabolizing enzymes represents the smallest PAH to have both the bay and K regions of the thyroid glands, and their adverse effects on reproductive (Figure 2). Thus, they are often used as a model substrate for as well as immune system (Veraldi et al., 2006; Oostingh et al., studies on the metabolism of carcinogenic PAHs (Mallick et al., 2008). 2011) (and the references therein). In line with the advances in Frontiers in Microbiology | www.frontiersin.org 4 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 5 Ghosal et al. PAH Biodegradation by Microbes (Foght, 2008; Carmona et al., 2009). While the aerobic catabolism of aromatic compounds has been studied for several decades, the anaerobic degradation of aromatic compounds is a more recently discovered microbial capacity that still awaits a deeper understanding. However, anoxic conditions dominate in many natural habitats and contaminated sites (e.g., aquifers, aquatic sediments and submerged soils) where biodegradation is carried out by anaerobes using alternative final electron acceptors such as nitrate, sulfate or ferric ions (Foght, 2008; Carmona et al., 2009). Principally, bacteria favor aerobic conditions for degradation of PAHs via oxygenase-mediated metabolism (involving either FIGURE 2 | Structure of phenanthrene, the simplest PAH containing monooxygenase or dioxygenase enzymes). Usually, the first step bay and K region. in the aerobic bacterial degradation of PAHs is the hydroxylation of an aromatic ring via a dioxygenase, with the formation of a cis-dihydrodiol, which gets rearomatized to a diol intermediate the knowledge of bacterial diversity within an ecosystem, many by the action of a dehydrogenase. These diol intermediates unique metabolic pathways on degradation of the PAHs are may then be cleaved by intradiol or extradiol ring-cleaving reported and scattered in literatures, which would collectively dioxygenases through either an ortho-cleavage or meta-cleavage deepen the understanding on the range of catabolism as well pathway, leading to intermediates such as catechols that are as the biochemical and genetic diversities. Timely collective ultimately converted to TCA cycle intermediates (Evans et al., update of literature is most important to address current status 1965; Cerniglia, 1992; Eaton and Chapman, 1992; Mueller et al., of subjects related to our life and surrounding environment 1996; Mallick et al., 2011). Dioxygenase is a multicomponent where the ongoing threat due to PAH-mediated pollution is enzyme generally consisting of reductase, ferredoxin, and no exception rather an issue that deserves top priority. The terminal oxygenase subunits (Mallick et al., 2011). Bacteria can present review provides an overview of the current knowledge also degrade PAH via the cytochrome P450-mediated pathway, of microbial degradation/transformation of PAHs. Moreover, with the production of trans-dihydrodiols (Sutherland et al., factors affecting biodegradation of PAHs, recent advancement 1990; Moody et al., 2004) or under anaerobic conditions, e.g., in genetic, genomic, proteomic and metabolomic techniques under nitrate-reducing conditions (Foght, 2008; Carmona et al., and their application in bioremediation of PAHs have also been 2009). described. Naphthalene degrading bacteria are ubiquitous in nature and there are enormous numbers of reports documenting the bacterial degradation of naphthalene including the elucidation PAH DEGRADATION BY BACTERIA AND of the biochemical pathways, enzymatic mechanisms and genetic HALOPHILIC ARCHAEA regulations (Cerniglia, 1992; Peng et al., 2008; Seo et al., 2009; Lu et al., 2011; Mallick et al., 2011). The naphthalene catabolic Bacterial Catabolism of PAHs genes present in the plasmid NAH7 in Pseudomonas putida Bacteria, which have evolved more than three billion years ago, G7 are well characterized (Simon et al., 1993). In the plasmid have developed strategies for obtaining energy from virtually NAH7, the naphthalene catabolic genes (nah) are organized every compound and have been considered as nature’s ultimate into two operons: the nal operon containing the genes for the scavengers. Because of their quick adaptability, bacteria have upper pathway enzymes involved in conversion of naphthalene largely been used to degrade or remediate environmental hazards. to salicylate, and the sal operon containing the genes for the Various bacteria have been found to degrade PAHs, in which lower pathway enzymes involved in the conversion of salicylate degradation of naphthalene and phenanthrene has been most to pyruvate and acetaldehyde (Simon et al., 1993). The operons widely studied. Numerous unique metabolic pathways for the are positively regulated by a common regulator NahR, a LysR bacterial degradation of PAHs have been well documented in a type of positive transcriptional regulator and are widely dispersed number of excellent review articles (Cerniglia, 1992; Peng et al., in bacteria. NahR is induced in presence of salicylate leading 2008; Seo et al., 2009; Mallick et al., 2011). So, the biochemical to high-level expression of the nah genes in bacteria (Yen and pathways for bacterial degradation of PAHs will not be discussed Gunsalus, 1985; Peng et al., 2008). There are several reports on in details in this communication. Nevertheless, there are two the nucleotide sequences of genes encoding the upper pathway major strategies to degrade PAHs depending on the presence enzymes in different Pseudomonas strains, and the genes are more or absence of oxygen. In the aerobic catabolism of aromatics, than 90% identical (Menn et al., 1993; Simon et al., 1993; Yang the oxygen is not only the final electron acceptor but also a co- et al., 1994; Bosch et al., 2000; Peng et al., 2008). Moreover, substrate for the hydroxylation and oxygenolytic ring cleavage in Ralstonia sp. U2, the naphthalene catabolic operon (nag) of the aromatic ring. In contrast, the anaerobic catabolism of contains all of the upper pathway genes similar to that of the aromatic compounds uses a completely different strategy to classical nah genes of Pseudomonas strains in the same order, attack the aromatic ring, primarily based on reductive reactions with the exception of two extra genes named nagG and nagH, Frontiers in Microbiology | www.frontiersin.org 5 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 6 Ghosal et al. PAH Biodegradation by Microbes which are structural subunits of salicylate-5-hydroxylase enzyme 2005). The narAa and narAb genes codes for the a- and b- required for the conversion of naphthalene to gentisate (Zhou subunit of the naphthalene dioxygenase (NDO). Both subunits et al., 2001). Additionally, in Comamonas testeroni strain GZ42, of NDO in Rhodococcus sp. strain NCIMB12038 showed only the naphthalene catabolic genes are similar to that found in 30% amino acid identity to the corresponding P. putida NDO Ralstonia sp. U2 (Goyal and Zylstra, 1997). The lower pathway subunits. In addition, there is no gene in Rhodococcus strains genes for the naphthalene catabolism are also similar in different which is similar to the genes encoding the electron transport Pseudomonas strains like P. putida G7, NCIB9816-4, ND6, and components reductase and ferredoxin of NDO in Pseudomonas P. stutzeri AN10 (Habe and Omori, 2003; Peng et al., 2008). In strains (Kulakov et al., 2005; Larkin et al., 2005). Moreover, those organisms, lower pathway of naphthalene operon contains the narA and narB genes are transcribed as a single unit 11 genes present in the order nahGTHINLOMKJY, in which nahY through different start sites, and their transcription is induced represent a naphthalene chemotaxis gene. However, in AN10 in the presence of naphthalene in contrast to salicylate-inducible and ND6 strains another salicylate hydroxylase gene (nahW ) naphthalene catabolic genes in Pseudomonas species. There are has been found to be present outside, but near to the sal two putative regulatory genes (narR1 and narR2, GntR-like operon. transcriptional regulator) which are shown to be transcribed as a Along with Pseudomonas, a high proportion of the PAH- single mRNA in naphthalene-induced cells (Kulakov et al., 2005; degrading isolates belong to the sphingomonads, which comprise Larkin et al., 2005). Figure 3 represent the gene organization a physiologically versatile group within the Alphaproteobacteria: of the nar gene cluster of various naphthalene degrading mainly Sphingomonas, Sphingobium and Novosphingobium that Rhodococcus sp. are frequently found as aromatic degraders. Species belonging to Along with naphthalene, a number of reports on phenan- these genera show great catabolic versatility, capable of degrading threne degradation by various Gram negative and Gram a wide range of natural and xenobiotic compounds including positive bacterial species have been reported (Peng et al., 2008; HMW PAHs (Basta et al., 2005; Peng et al., 2008; Stolz, 2009; Seo et al., 2009; Mallick et al., 2011). In a study, Mallick Vila et al., 2015). In a few reports, it has been shown that et al. (2007) reported the degradation of phenanthrene by the catabolic versatility of sphingomonads relies on the large Staphylococcus sp. strain PN/Y, by initiating the dioxygenation plasmids present in those organisms (Basta et al., 2005; Peng specifically at the 1,2-position followed by meta-cleavage of et al., 2008). However, the plasmid-encoded degradative genes phenanthrene-1,2-diol, leading to the formation of 2-hydroxy- have been found to be largely scattered, or they are not organized 1-naphthoic acid as the metabolic intermediate; while the in coordinately regulated operons (Romine et al., 1999). It may be ortho-cleavage could yield the naphthalene-1,2-dicarboxylic acid. possible that this kind of ‘flexible’ gene organization i.e., different Authors also reported that 2-hydroxy-1-naphthoic acid was combinations of conserved gene clusters allows sphingomonads metabolized by a meta-cleavage enzyme 2-hydroxy-1-naphthoate to adapt easily and proficiently to degrade various aromatic dioxygenase leading to the formation of trans-2,3-dioxo-5- compounds including xenobiotics (Basta et al., 2005; Peng et al., (2 -hydroxyphenyl)-pent-4-enoic acid, a novel metabolite in 2008). Recently an illustrative report on the catabolic versatility the phenanthrene degradation pathway, and was subsequently of sphingomonads has shown that strain PNB, acting on diverse degraded via salicylic acid and catechol (Mallick et al., 2007). monoaromatic and polyaromatic compounds, contains seven Later on, Ghosal et al. (2010) reported the assimilation of sets of ring-hydroxylating oxygenases (RHO) with different phenanthrene by Ochrobactrum sp. strain PWTJD, isolated from substrate specificities (Khara et al., 2014). In general, it has municipal waste contaminated soil sample using phenanthrene been seen that mobile genetic elements (MGEs) like plasmids as a sole source of carbon and energy. The strain PWTJD and transposons play a crucial role in the biodegradation of could also degrade phenanthrene via 2-hydroxy-1-naphthoic organic pollutants like PAHs. The presence of foreign compounds acid, salicylic acid and catechol. The strain PWTJD was can often lead to the selection of mutant bacteria that are found to utilize 2-hydroxy-1-naphthoic acid and salicylic acid capable of metabolizing them. Apart from vertical gene transfer, as sole carbon sources, while the former was metabolized aromatics catabolic genes are often harbored by MGEs that by a ferric-dependent meta-cleavage dioxygenase. In the successfully disseminate the catabolic traits to phylogenetically lower pathway, salicylic acid was metabolized to catechol diverse bacteria by horizontal gene transfer (Top and Springael, and was further degraded by catechol 2,3-dioxygenase to 2003; Nojiri et al., 2004). 2-hydroxymuconoaldehyde acid, ultimately leading to TCA Among PAHs degrading bacteria the genus Rhodococcus cycle intermediates. The metabolic pathway involved in the is very unique, having an enormous catabolic versatility. In degradation of phenanthrene by Ochrobactrum sp. strain PWTJD contrast to Pseudomonas and other Gram-negative bacteria is shown in Figure 4 (Ghosal et al., 2010). This was the first whose naphthalene catabolic genes are usually clustered, the report of the complete degradation of a PAH molecule by Gram-positive Rhodococcus strains usually exhibit only three Gram-negative Ochrobactrum sp. describing the involvement of structural genes required for naphthalene degradation (narAa, the meta-cleavage pathway of 2-hydroxy-1-naphthoic acid in narAb and narB) (Kulakov et al., 2005; Larkin et al., 2005). It phenanthrene assimilation. has been seen that the nar region is not arranged into a single Other LMW PAHs like anthracene, fluorene, acenaphthene operon, and there are several homologous transcription units and acenaphthylene are also found in high quantities in PAH- in different Rhodococcus strains separated by non-homologous polluted sites and various bacterial species have the capability sequences containing direct and inverted repeats (Larkin et al., to utilize these compounds individually as sole carbon and Frontiers in Microbiology | www.frontiersin.org 6 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 7 Ghosal et al. PAH Biodegradation by Microbes FIGURE 3 | Gene organization of the nar gene cluster of various naphthalene degrading Rhodococcus sp. The respective Rhodococcus sp. along with the accession numbers of their corresponding naphthalene degrading gene cluster: Rhodococcus opacus R7 (accession no. DQ846881), Rhodococcus sp. NCIMB12038 (accession no. AF082663), Rhodococcus opacus CIR2 (accession no. AB024936), Rhodococcus sp. P200 (accession no. AY392424), Rhodococcus sp. P400 (accession no. AY392423), Rhodococcus sp. I24 (accession no. AF121905). The genes and their corresponding function of the gene products: narR1- GntR-like regulator protein; narR2- XylR-like regulator protein; rub1 and 2- Rubredoxin; narAa- Naphthalene dioxygenase large subunit; narAb- Naphthalene dioxygenase small subunit; narB- Naphthalene dihydrodiol dehydrogenase (adapted from Di Gennaro et al., 2010). energy sources (Moody et al., 2001; Peng et al., 2008; Seo Harayama, 2000; Peng et al., 2008; Seo et al., 2009). However, et al., 2009; Mallick et al., 2011). Lately, Ghosal et al. (2013) further investigations are prerequisite in several areas of HMW reported assimilation of acenaphthene and acenaphthylene by PAH biodegradation, namely, research related to the regulatory the Acinetobacter sp. strain AGAT-W, isolated from municipal mechanisms of HMW PAH biodegradation, biodegradation of waste contaminated soil sample using acenaphthene as a sole HMW PAH associated with other hydrocarbons in mixtures; source of carbon and energy. The strain AGAT-W could and the interactions of complex microbial community during degrade acenaphthene via 1-acenaphthenol, naphthalene-1,8- HMW PAH degradation can be exploited in more details. dicarboxylic acid, 1-naphthoic acid, salicylic acid and cis, cis These will enrich our understanding on the microbial ecology muconic acid ultimately leading to TCA cycle intermediates. The of HMW PAH-degrading communities and the mechanisms metabolic pathways involved in the degradation of acenaphthene by which HMW PAH biodegradation occur. In addition, the and acenaphthylene by Acinetobacter sp. strain AGAT-W is outcome will also help in predicting the environmental fate of shown in Figure 5 (Ghosal et al., 2013). This was the first these recalcitrant compounds and aid for the development of report on the complete degradation of acenaphthene and convenient as well as cost-effective bioremediation strategies in acenaphthylene individually by strain AGAT-W belonging to the near future. genus Acinetobacter. Polycyclic aromatic hydrocarbons with more than three rings Degradation of PAHs by viz. pyrene, benzo[a] pyrene (Bap), are generally referred to as Halophilic/Halotolerant Bacteria and HMW PAHs (Figure 1), which are of significant environmental concern due to their long persistence and high toxicity as well as Archaea their mutagenic and/or carcinogenic properties (Cerniglia, 1992; Environmental pollution due to anthropogenic activity has Kanaly and Harayama, 2000; Peng et al., 2008; Seo et al., 2009). spread to all types of ecosystems and marine ecosystem is In the last few decades research on microbial degradation of not excluded from this list. Hypersaline environments are HMW PAHs has advanced significantly and a number of HMW regularly being polluted with organic pollutants including PAHs, PAH-degrading isolates have been reported (Cerniglia, 1992; through industrial and municipal effluents. Industrial effluents, Kanaly and Harayama, 2000, 2010; Peng et al., 2008; Seo et al., specifically, petroleum industry where one of the main sources 2009). Biodegradation of HMW PAHs by microorganisms is of contaminants in the waste waters are aromatic hydrocarbons discussed adequately in many excellent reviews and the pathways including PAHs. Contamination and biodegradation in extreme for their degradation have also been depicted (Kanaly and environments like high salinity has been receiving increased Frontiers in Microbiology | www.frontiersin.org 7 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 8 Ghosal et al. PAH Biodegradation by Microbes FIGURE 4 | Proposed pathway for the degradation of phenanthrene by Ochrobactrum sp. strain PWTJD. The transient intermediates, which were not detected, are shown in parentheses. Filled arrows indicate mineralization; open arrows indicate a dead-end metabolite; and dotted arrows indicate multiple steps. Chemical designations: (I) phenanthrene; (II) cis-1,2-phenanthrenedihydrodiol; (III) 1,2-dihydroxyphenanthrene; (IV) 5,6-benzocoumarin; (V) cis-2-oxo-4-(20-hydroxynaphthyl)-but-3-enoic acid; (VI) 2-hydroxy-1-naphthoic acid; (VII) trans-2,3-dioxo-5-(20-hydroxyphenyl)- pent-4-enoic acid; (VIII) 2,3-dioxo-5-hydroxy-5-(20-hydroxyphenyl)-pentanoic acid; (XI) salicylaldehyde; (X) salicylic acid; (XI) catechol; (XII) 2-hydroxymuconaldehyde acid. Adapted from Ghosal et al. (2010). attention in recent times (Oren et al., 1992; Margesin and Schinner, 2001a,b; Le Borgne et al., 2008; Bose et al., 2013; Cravo- FIGURE 5 | Proposed pathway for the degradation of acenaphthene by Laureau and Duran, 2014; Elango et al., 2014; Fathepure, 2014; Acinetobacter sp. strain AGAT-W. Chemical designations: (I) acenaphthene; (II) acenaphthylene; (III) 1-acenaphthenol; (IV) Genovese et al., 2014; Kappell et al., 2014; Kostka et al., 2014; 1-acenaphthenone; (V) 1-hydroxy-2-ketoacenaphthene; (VI) Thomas et al., 2014; Torlapati and Boufadel, 2014). In addition, a 1,2-dihydroxyacenaphthylene; (VII) acenaphthenequinone; (VIII) survey of relevant literatures indicates that along with numerous naphthalene-1,8-dicarboxylic acid; (IX) 1,8-naphthalic anhydride; (X) biotechnological applications, halophilic microorganisms have 1-naphthoic acid; (XI) salicylaldehyde; (XII) salicylic acid; (XIII) catechol. more extended catabolic versatility than previously thought Adapted from Ghosal et al. (2013). about (Margesin and Schinner, 2001b; Garcia et al., 2005; Smith et al., 2013; Kappell et al., 2014; Lamendella et al., 2014; Roling and Bodegom, 2014; Scott et al., 2014; Singh et al., 2014). deal with hypersaline environments that are contaminated with The biological treatment of industrial hypersaline waste waters organic pollutants (Margesin and Schinner, 2001a; Mellado and and the bioremediation of polluted hypersaline environments Ventosa, 2003; Peyton et al., 2004; Garcia et al., 2005; Cui are not possible with conventional microorganisms because et al., 2008; Mnif et al., 2009; Zhao et al., 2009; Bonfa et al., they are incapable to function efficiently at salinities that of 2011; Smith et al., 2013; Fathepure, 2014; Kappell et al., 2014; seawater or above. Thus, halophilic microorganisms are the best Lamendella et al., 2014; Singh et al., 2014; Thomas et al., 2014). alternatives to overcome this problem (Oren et al., 1992; Garcia Some halophiles that have shown PAHs degrading property et al., 2005; Fathepure, 2014; Kostka et al., 2014). In the last are depicted in Supplementary Table S1. However, studies decade there has been an increasing interest in the development concerning the ability of this group of microbes to degrade and optimization of bioremediation processes via halophiles to PAHs are still in their infancy. Although in the last few years, Frontiers in Microbiology | www.frontiersin.org 8 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 9 Ghosal et al. PAH Biodegradation by Microbes a few studies on the degradation of PAHs by halophiles have bacterial/archaeal (halophilic) strains involved in the degradation been published. In this context, Gauthier et al. (1992) described of various PAHs. Figure 6 illustrates the 16S rRNA gene-based the isolation of Marinobacter hydrocarbonoclasticus which can phylogenetic analysis of all the bacterial/archaeal strains cited in degrade various organic compounds including naphthalene Supplementary Table S1. It has been seen that bacterial/archaeal (Gauthier et al., 1992). The presence of naphthalene dioxygenase, strains having PAHs catabolic property are distributed mainly similar to those from Pseudomonas and Burkholderia spp. was in six major groups: Alphaproteobacteria, Betaproteobacteria, reported in a naphthalene-degrading Marinobacter sp. strain Gammaproteobacteria, Actinomycetes, Firmicutes and Archaea NCE312 (Hedlund et al., 2001). Again, based on the analysis (Halophiles). It may be mentioned here that many strains listed of the shoreline sand and rocks (Costa da Morte, northwestern in Supplementary Table S1 have not been cultured in laboratory. Spain) affected by the Prestige oil spill, unveiled the high relative Some PAHs degrading isolates represent novel strains which are abundances of Sphingomonadaceae and Mycobacterium that isolated from various geographically diverse habitats. Thus, all could be associated with PAH degradation (Alonso-Gutierrez this information illustrate that PAH-degrading machinery is not et al., 2009). In a similar study, Vila et al. (2010) reported confined to a few particular genus reported so far but is more the isolation of a marine microbial consortium from a beach likely to be distributed widely in the prokaryotic kingdom found polluted with the Prestige oil spill which is efficient in removing in diverse geographical niches. different hydrocarbon present in heavy fuel oil including three to five-ring PAHs (for example anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, and BaP) (Vila et al., 2010). FUNGAL DEGRADATION OF PAHs In addition based on community dynamics analysis, it has been contemplated that Alphaproteobacteria (Maricaulis and The biodegradation of PAHs by fungi has been studied Roseovarius), could be associated with the degradation of extensively in last several years and numerous fungal species LMW PAHs, whereas Gammaproteobacteria (Marinobacter and have been reported to metabolize different PAHs (Cerniglia, Methylophaga), could be associated with the degradation of 1992; Cerniglia and Sutherland, 2010). Most fungi cannot use HMW PAHs. A similar bacterial community associated with PAHs as sole sources of carbon and energy; however, they may the degradation of various organic compounds was found in a co-metabolize PAHs to a wide variety of oxidized products beach affected by the Deepwater Horizon oil spill (Kostka et al., and sometimes to CO . Bacterial PAHs degradation mainly 2011; Lamendella et al., 2014). Recently, Gallego et al. (2014) involves dioxygenase enzymes and partially monooxygenase reported the isolation of a marine pyrene-degrading microbial mediated reactions and the same is valid for algae. For consortium from a beach polluted by an oil spill and found example, the extent of dioxygenase vis-à-vis monooxygenase that an uncultured Gordonia sp. is the key pyrene degrader in catalyzed transformation of naphthalene by Mycobacterium sp. the consortium based on community structure and PAH ring- was found to be in the ratio of around 25:1 (Kelley et al., hydroxylating genes analyses (Gallego et al., 2014). Nevertheless, 1990). On the other hand, fungal PAHs degradation mainly detailed information on the bioremediation of PAHs under high involves monooxygenase enzymes (Cerniglia and Sutherland, salinity by halophilic/halotolerant bacteria and archaea is still in 2010) (and the references therein). However, the transformation its initial stage of exploration, but it is expected that this emerging of PAHs by fungi involves several enzymatic pathways and field is ready to flourish in near future. depends on the particular species and growth conditions. The fungi involved in PAHs biodegradation are mainly of two types- ligninolytic fungi or white-rot fungi (they have the Occurrence of PAH-Degrading ability to produce enzymes including lignin peroxidase (LiP), Machinery in Diverse manganese peroxidase (MnP) and laccases to degrade the lignin Bacterial/Halophilic Archaeal Genera in wood) and non-ligninolytic fungi (those who do not produce The community analysis of indigenous microorganisms peroxidases or laccases but instead produce cytochrome P450 capable of degrading various aromatic compounds in diverse monooxygenase like enzymes) (Hofrichter, 2002; Tortella et al., environments has been of great interest in the recent years 2005; Cerniglia and Sutherland, 2010; Li et al., 2010). Although, (Watanabe et al., 2002; Brakstad and Lodeng, 2005; Gerdes various ligninolytic fungi such as Phanerochaete chrysosporium et al., 2005; Yakimov et al., 2005; Coulon et al., 2007; Cui et al., and Pleurotus ostreatus can secrete both ligninolytic and non- 2008) and different strategies have been developed for the study ligninolytic type of enzymes, but it is uncertain to what extent of associated microbial communities (Widada et al., 2002). each enzyme participates in the degradation of the PAHs (Bezalel Previously it was thought that PAH degradation capabilities et al., 1997). On the other hand, another type of ligninolytic may be associated with certain genera or groups of bacteria fungi, known as brown-rot fungi, mainly produces hydrogen independent of the origin of the source sample (Kastner et al., peroxide for degrading hemicelluloses and cellulose. Although, 1994; Mueller et al., 1997). But consequently, with time it has only limited data is available about PAH metabolism by brown been reported that PAH-degrading bacteria are far more diverse rot fungi, some brown-rot fungi such as Laetiporus sulphureus (Widada et al., 2002; Andreoni et al., 2004; Cui et al., 2008; and Flammulina velutipes have been shown to metabolize Hilyard et al., 2008). Currently, there are enormous reports PAHs like phenanthrene, fluoranthene and fluorine (Sack et al., of various bacterial as well as some archaeal genera capable 1997; Cerniglia and Sutherland, 2010) (and the references of degrading PAHs. Supplementary Table S1 represents the therein). Frontiers in Microbiology | www.frontiersin.org 9 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 10 Ghosal et al. PAH Biodegradation by Microbes FIGURE 6 | Phylogenetic tree based on 16S rRNA gene sequence of organisms enlisted in Supplementary Table S1. Organism designations are according to those given in Supplementary Table S1. However, for organisms devoid of 16S rRNA gene sequence information, sequence of representative type strain has been considered. Accession numbers are given within parentheses. 16S rRNA gene sequence of Thermus aquaticus strain ATCC 25104 was used as outgroup. Numbers at nodes indicate levels of bootstrap support based on neighbor-joining analysis of 100 resampled datasets. Bootstrap values below 70% are not shown. Bar represents 0.05 substitutions per nucleotide position. Catabolism of PAHs by Non-ligninolytic Fungi Polycyclic aromatic hydrocarbons metabolic pathway of non- ligninolytic fungi is similar to those formed by mammalian enzymes. In this case, the predominant pathway of initial oxidation of PAHs by non-ligninolytic fungi involves the activity of the cytochrome P450 monooxygenase enzymes. These enzymes catalyze a ring epoxidation to form an unstable arene oxide, which is further transformed to trans-dihydrodiol via an epoxide-hydrolase catalyzed reaction (Jerina, 1983; Sutherland et al., 1995). Non-ligninolytic fungi such as Cunninghamella elegans and ligninolytic fungi such as Pleurotus ostreatus, metabolize PAHs via this pathway (Bezalel et al., 1996; Tortella et al., 2005). For example, the transformation of fluoranthene by C. elegans produce fluoranthene trans-2,3-dihydrodiol, 8- and 9-hydroxyfluoranthene trans-2,3-dihydrodiols (Tortella et al., 2005). Similarly, P. ostreatus metabolizes pyrene into pyrene trans-4,5-dihydrodiol (Bezalel et al., 1996). Arene oxide produced by cytochrome P450 can also be rearranged to phenol derivatives by non-enzymatic reactions and are subsequently conjugated with sulfate, xylose, glucoronic acid, or glucose (Mueller et al., 1996; Pothuluri et al., 1996). In some fungi, cytochrome P450 monooxygenases oxidize PAHs to epoxides and dihydrodiols which are potent carcinogens and more toxic than the respective parent PAHs, while on the other hand peroxidase-mediated oxidation of PAHs produces quinines which are less toxic than the parent PAHs (Cerniglia and Sutherland, 2010). This is why oxidation of PAHs by ligninolytic enzymes could be a more logical strategy for the detoxification of PAHs contaminated environment. Catabolism of PAHs by Ligninolytic Fungi White-rot fungi are ubiquitous in nature and can produce ligninolytic enzymes which are secreted extracellularly. These enzymes can degrade lignin present in wood and other organic substances. Ligninolytic enzymes are mainly of two types, peroxidases and laccases. On the basis of reducing substrate types, peroxidase enzyme can be classified again into two types, lignin peroxidase and manganese peroxidase. Both types of peroxidases have the ability to oxidize PAHs (Hammel, 1995; Cerniglia and Sutherland, 2010). Laccases, the phenol oxidase enzymes, also have the ability to oxidize PAHs (Cerniglia and Sutherland, 2010). Compared to bacterial PAHs degrading enzymes, ligninolytic enzymes are not induced in the presence of PAHs or by FIGURE 6 | Continued its degradation products (Verdin et al., 2004). As ligninolytic Frontiers in Microbiology | www.frontiersin.org 10 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 11 Ghosal et al. PAH Biodegradation by Microbes enzymes are secreted extracellularly, they are able to diffuse et al., 2003). Oxidation of pyrene, anthracene, fluorene and toward the immobile PAHs, and that is why they are more BaP to the corresponding quinines by lignin peroxidase and useful than bacterial intracellular enzymes in making initial manganese peroxidase was reported by the white-rot fungus attack on PAHs in soil. In addition compared to bacterial PAH- Phanerochaete chrysosporium (Bogan et al., 1996a,b). In another degrading enzymes, ligninolytic enzymes have broad substrate study, complete mineralization of BaP (HMW PAHs) by the specificity and are therefore able to transform a wide range of white-rot fungus P. chrysosporium was reported in a two-stage substrates, even those which are most recalcitrant (Tortella et al., pilot scale reactor (May et al., 1997). Clemente et al. (2001) 2005; Cerniglia and Sutherland, 2010). Ligninolytic enzymes studied PAHs degradation by thirteen ligninolytic fungal strains can transform PAHs by producing hydroxyl free radicals by and reported that the rate of degradation varies with a variation of the donation of one electron, which oxidizes the PAH ring lignolytic enzymes (Clemente et al., 2001). In this study, highest (Sutherland et al., 1995). As a result, PAH-quinones and acids are naphthalene degradation (69%) was reported by the strain 984 formed instead of dihydrodiols. It has been seen that ligninolytic which have Mn-peroxidase activity, followed by strain 870 (17%) fungi mineralize PAHs by a combination of ligninolytic enzymes, having lignin peroxidase and laccase activities. The ability of cytochrome P450 monooxygenases, and epoxide hydrolases soil fungi to degrade PAHs that produce ligninolytic enzymes (Bezalel et al., 1997). Numerous reports have been documented was also studied under microaerobic and very-low-oxygen on the degradation of PAHs by white-rot fungi (Tortella et al., conditions (Silva et al., 2009). It was reported that Aspergillus sp., 2005; Cerniglia and Sutherland, 2010). The metabolic pathway Trichocladium canadense, and Fusarium oxysporum can degrade for the degradation of phenanthrene by the ligninolytic fungus LMW PAHs (2–3 ring compounds) most extensively, whereas Pleurotus ostreatus is illustrated in Figure 7 (Bezalel et al., 1997). highest degradation of HMW PAHs (4–7 rings) was observed in PAHs degradative potential of wood-rotting fungi Plrurotus T. canadense, Aspergillus sp., Verticillium sp., and Achremonium ostreatus and Antrodia vaillantii was also examined in soil, sp. These results suggest that, along with bacteria, fungi can be artificially contaminated with fluorene, phenanthrene, pyrene, exploited as a valuable endeavor for the bioremediation of PAH and benz[a]anthracene (Andersson et al., 2003). It has been contaminated sites. reported that although P. ostreatus significantly increased the degradation of PAHs in soil, but in the process, accumulated toxic PAH metabolites. It has also been seen that this white- MICROALGAL DEGRADATION OF PAHs rot fungus inhibits the indigenous microbial populations in the soil, which may have prohibited the complete mineralization Compared to bacteria and fungi, relatively little attention of the PAHs. Conversely, the brown-rot fungus A. vaillantii has been paid to the biodegradation of PAHs by microalgae did not generate any dead-end metabolites, although its PAHs (cyanobacteria, diatoms etc.). Microalgae are one of the major degradation rate was similar to that of P. ostreatus (Andersson primary producers in aquatic ecosystems, and play vital roles FIGURE 7 | Proposed phenanthrene degradation pathway by the ligninolytic fungus Pleurotus ostreatus (adapted from Bezalel et al., 1997). Frontiers in Microbiology | www.frontiersin.org 11 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 12 Ghosal et al. PAH Biodegradation by Microbes in the fate of PAHs in those environments. Several strains Table S2 represents different microalgal strains involved in of microalgae are known to metabolize/transform naphthalene, the bioremediation of various PAHs. Figure 8 illustrates the phenanthrene, anthracene, BaP and other PAHs (Cerniglia rubisco large subunit gene-based phylogenetic analysis of all the et al., 1979, 1980a,b; Narro et al., 1992a,b; Safonova et al., organisms enlisted in Supplementary Table S2. Figure 9 shows 2005; Chan et al., 2006; El-Sheekh et al., 2012). Supplementary the biotransformation pathway of naphthalene by microalgae FIGURE 8 | Phylogenetic tree based on ribulose 1, 5-bisphosphate carboxylase (rubisco) gene sequence (large subunit) of organisms enlisted in Supplementary Table S2. Organism designations are according to those given in Supplementary Table S2. rubisco gene sequences are taken from representative strains present in NCBI and the respective accession numbers are given within parentheses. rubisco gene sequence (large subunit) of organism Nicotiana tabacum was used as outgroup. Numbers at nodes indicate levels of bootstrap support based on neighbor-joining analysis of 100 resampled datasets. Bootstrap values below 60% are not shown. Bar represents 0.05 substitutions per nucleotide position. Frontiers in Microbiology | www.frontiersin.org 12 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 13 Ghosal et al. PAH Biodegradation by Microbes incubated with mixture of aliphatic hydrocarbons and PAHs as substrates. Though the immobilized organism can degrade n-alkanes but it’s PAH degradation rate is very less, however, PAHs accumulation did not impair the degradation of PAHs; whereas in case of free-living cells, the organisms can reduce the concentration of both PAHs and n-alkanes satisfactorily (Ueno et al., 2006, 2008). Hong et al. (2008) have examined the accumulation and biodegradation of phenanthrene (Phn) and fluoranthene (Fla) by the two diatoms Skeletonema costatum and Nitzschia sp., enriched from a mangrove aquatic ecosystem. It was seen that the strains were capable of accumulating and degrading phenanthrene and fluoranthene simultaneously and the PAHs accumulation and degradation capability of Nitzschia sp. were higher than those of S. costatum. Further, it had been observed that the degradation of fluoranthene by the two diatoms was slower, compared to phenanthrene. The strains also showed similar or higher efficiency in the removal of the Phn–Fla mixture than Phn or Fla alone (Hong et al., 2008). In another study, the efficiency of seven microalgal species to remove pyrene from solution was reported (Lei FIGURE 9 | Proposed naphthalene transformation pathway by the et al., 2002). In a recent study removal of benzo(a)pyrene cyanobacteria, Oscillatoria sp. strain JCM (adapted from Cerniglia (BaP) by sorption and degradation was determined by two et al., 1980a). microalgal species Selenastrum capricornutum and Scenedesmus acutus (Garcia de Llasera et al., 2016). It has been seen that S. capricornutum can remove 99% of BaP after 15 h of Oscillatoria sp., strain JCM (Cerniglia et al., 1980a). Under exposure, whereas S. acutus can remove 95% after 72 h of photoautotrophic growth conditions, strain JCM has been exposure. In a separate study, the effects of metals on biosorption reported to oxidize naphthalene to 1-naphthol, whereas marine and biodegradation of fluorene, phenanthrene, fluoranthrene, cyanobacterium Agmenellum quadruplicatum strain PR-6 can pyrene and benzo[a]pyrene by Selenastrum capricornutum were convert phenanthrene to phenanthrene trans-9,10-dihydrodiol investigated (Ke et al., 2010). It has been shown that both metal and 1-methoxyphenanthrene (Narro et al., 1992a,b). In a dosage and exposure time yielded a significant effect on the separate study, oxidation of BaP by the microalgae Selanastum ability of removal of low molecular weight PAHs like fluorene capricornutum has been evaluated. Transformation of BaP and phenanthrene by the alga, whereas for high molecular resulted in the formation of cis-4,5-, 7,8-, 9,10- and 11,12- weight PAHs like fluoranthrene, pyrene and BaP, the removal BaP-dihydrodiols, involving a dioxygenase system, similar efficiency was not affected by the presence of metals. Patel to bacterial PAH degradation systems but unlike those of et al. (2016) recently reported the biodegradation of anthracene eukaryotic organisms (like fungi) which involve monooxygenase and pyrene by Anabaena fertilissima (Patel et al., 2016), while systems (Warshawsky et al., 1988). In another study, the Takácov ˇ á et al. (2014) reported the biodegradation of BaP by effects of gold, white or UV-A fluorescent lights for the the microalgae Chlorella kessleri. Removal and transformation biotransformation of BaP and phototoxicity of carcinogenic of seven high molecular weight PAHs in water was reported PAHs in different algal systems was determined. It has been by live and dead cells of a freshwater microalga, Selenastrum found that algae like S. capricornutum, Scenedesmus acutus capricornutum under gold and white light irradiation. The and Ankistrodesmus braunii are able to degrade BaP to removal efficiency of PAHs, and the effectiveness of live and dihydrodiols, and the degradation varies with the kind and dead cells, was found to be predominantly PAH dependent intensity of light sources (Warshawsky et al., 1995). The removal (Ke et al., 2010; Luo L. et al., 2014; Luo S. et al., 2014). efficiency for either fluoranthene or pyrene, or a mixture of The first study on the potential of algal–bacterial microcosms fluoranthene and pyrene were also determined using Chlorella was reported for the biodegradation of aromatic pollutants vulgaris, Scenedesmus platydiscus, Scenedesmus quadricauda, and comprising salicylate, phenol and phenanthrene in a one- Selenastrum capricornutum microalgal species (Lei et al., 2007). stage treatment (Borde et al., 2003). The green alga Chlorella After 7 days of treatment PAHs removal by S. capricornutum sorokiniana was grown with those three aromatics at different and C. vulgaris was 78 and 48% respectively. The removal rate of concentrations, showing increasing inhibitory effects in the order fluoranthene and pyrene in a mixture was found to be similar, or salicylate < phenol < phenanthrene. However, a satisfactory higher than the respective single compound, which indicates that removal (>85%) was achieved only in the system having the presence of one PAH acts synergistically in the removal of the both bacteria and algae, incubated under continuous lighting, other PAH (Lei et al., 2007). In another study, microalgal strain indicating the synergistic relationship between the algal–bacterial Prototheca zopfii immobilized in polyurethane foam has been microcosms in the removal of organic pollutants. An algal– reported to accumulate a mixture of PAHs in the matrix, when bacterial consortium consisting of Chlorella sorokiniana and Frontiers in Microbiology | www.frontiersin.org 13 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 14 Ghosal et al. PAH Biodegradation by Microbes Pseudomonas migulae was also reported for the degradation of Various white-rot fungi can metabolize PAHs along with phenanthrene under photosynthetic conditions and without an bacteria in consortium by improving bioavailability of target external source of oxygen (Munoz et al., 2003). In another study, compounds. Due to lack of suitable enzymes, generally fungi accelerated pyrene degradation by a bacterial-algal consortium cannot degrade HMW PAHs completely, but can transform them was reported under photosynthetic condition (Luo L. et al., 2014; into comparatively polar metabolite(s) with their extracellular Luo S. et al., 2014). These studies indicate that microalgae can enzymes, which can further be degraded by bacteria and other be used singly or along with bacteria as a potential candidate microbes (Sutherland, 1992). Meulenberg et al. (1997) reported for biodegradation of PAHs. However, more research is needed that the degradation product of anthracene by white-rot fungi for optimizing their efficiency to apply them successfully in the can be mineralized by indigenous mixed bacterial cultures (e.g., bioremediation of PAH-contaminated environment. activated sludge or soil) more quickly than anthracene itself (Meulenberg et al., 1997). It has been seen that, inoculation of fungal–bacterial co-cultures into the soils contaminated with DEGRADATION OF PAHs BY MICROBIAL PAHs, can significantly enhance degradation of HMW PAHs, like chrysene, benzo-[a]anthracene and dibenzo[a,h]anthracene CONSORTIA AND CO-METABOLISM (Boonchan et al., 2000). Consequently, it is assumed that Occasionally it has been observed that a particular microor- PAH degradation in nature is a result of coordinate steps ganism does not have all the genes required for the complete mediated by fungi and bacteria, with the fungi playing the initial mineralization of a particular organic pollutant like PAH. oxidation step (Meulenberg et al., 1997; Boonchan et al., 2000; Therefore, researchers are developing microbial consortia for Cerniglia and Sutherland, 2010). Along with bacteria and fungi, complete degradation of such pollutants. In those consortia, each various microalgal strains have been used for the degradation microorganism has specialized role in certain degradation steps of PAHs in a consortium. Microalgae can be exploited as where, intermediates produced by certain microorganisms are a potential candidate for degradation of PAHs specifically in utilized by other members. On the other hand, cometabolism is aquatic environments. Metabolic competition is another feature a phenomenon by which a recalcitrant compound is degraded of biodegradation when a combinations of two individually in the presence of an analogous degradable compound. degradable PAHs are present in the medium (Bouchez et al., Several microorganisms can co-metabolize PAHs, and it is 1995). For example, when LMW PAHs such as phenanthrene a very complex phenomenon. As PAHs in the environment and fluorene present in the medium, they can inhibit the are present as a mixture, co-metabolism plays a very crucial degradation of fluoranthene and pyrene (Dean-Ross et al., role for bioremediation of PAHs contaminated sites. Co- 2002). Nevertheless, another cause of inhibition is due to the metabolism of one PAH could have a synergistic effect on the formation of dead-end products that result from co-metabolic degradation of other PAHs, specifically for the degradation degradation of non-growth substrates (Stringfellow and Aitken, of HMW PAHs (van Herwijnen et al., 2003). In a report, 1995). Rhodococcus sp. strain S1 when grown on anthracene has shown to cometabolize phenanthrene to phenanthrene trans-9,10- FACTORS AFFECTING THE dihydrodiol (Tongpim and Pickard, 1999). The cometabolic degradation of phenanthrene, fluoranthene, anthracene and BIOREMEDIATION OF PAHs dibenzothiophene was reported by the fluorene grown The effectiveness of bioremediation has been mainly investigated Sphingomonas sp. LB126 (van Herwijnen et al., 2003). In a under ideal laboratory conditions, having a circum-neutral separate study, cometabolic degradation of a creosote-PAHs pH and ambient mesophilic temperature. However, in the mixture including phenanthrene, fluoranthene, and pyrene, by real situation, bioremediation can be effective only at sites the pyrene-degrading strain Mycobacterium sp. AP1 was reported (Lopez et al., 2008). However, it has been observed that the rate where environmental conditions permit microbial growth and express associated enzyme activity so that microorganisms can of degradation of individual PAHs was related to their aqueous enzymatically attack the pollutants converting them to harmless solubility, for example, the biodegradation rate of fluoranthene products. Numerous abiotic and biotic factors (such as pH, and pyrene are significantly lower than that of phenanthrene nutrient availability and the bioavailability of the pollutants) can (Lopez et al., 2008). In another study, cometabolism of apparently differ from site to site, which in turn can influence acenaphthene and acenaphthylene by the succinate grown the process of bioremediation in those environments either by Beijerinckia sp. and one of its mutant strain, Beijerinckia sp. inhibiting or accelerating the growth of the pollutant-degrading strain B8/36 was reported (Schocken and Gibson, 1984). Both microorganisms. Figure 10 illustrates the various abiotic and the wild type and the mutant strains cometabolize acenaphthene biotic factors influencing PAHs degradation in soil. The main to 1-acenaphthenol, 1-acenaphthenone, 1,2-acenaphthenediol, environmental factors that could affect the rate of biodegradation acenaphthenequinone, and 1,2-dihydroxyacenaphthylene. of PAHs in the environment are summarized below. Furthermore, Sphingobium sp. strain PNB was observed to co-metabolize fluoranthene, acenaphthene, benz[a]anthracene, pyrene and benzo[a]pyrene, in presence of phenanthrene Temperature indicating metabolic robustness of the strain (Roy et al., Temperature has a profound effect on the biodegradation 2013). of PAHs in contaminated sites since those places are not Frontiers in Microbiology | www.frontiersin.org 14 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 15 Ghosal et al. PAH Biodegradation by Microbes FIGURE 10 | Abiotic and biotic factors influencing the degradation of PAHs in soil. always at ambient temperature for activity of the inhabitant most of them for their normal activity. However, it has been microorganisms. When temperature increases, the solubility of seen that in many PAHs contaminated sites, pH is very far PAHs also increases, which in turn increases the bioavailability of from neutral settings. For example, some abandoned gasworks PAH molecules. On the other hand, with increasing temperature, sites frequently contain a huge amount of demolition waste like dissolved oxygen level decreases, as a result of which the concrete and brick. Leaching of these materials can result in the metabolic activity of aerobic mesophilic microorganisms reduces. increase of pH of the native soil. Moreover, the leaching of some There are some microorganisms called psychrophiles, which can material like coal spoil can generate an acidic environment due to operate at low temperature, and are in contrast to thermophiles the oxidation of sulfides. These acidic or alkaline conditions can that function at high temperature. At high temperature, some create adverse conditions for microbial activities, which in turn pollutants can get transformed into a new compound and often decrease the biodegradation of PAHs in those sites. Hence, it is the daughter compound appears to be more toxic than the parent recommended to adjust the pH at those sites by adding chemicals: compound, which in turn inhibits its biodegradation rate (Müller basic soils can be treated with ammonium sulfate or ammonium et al., 1998). Generally in most of the studies biodegradation nitrate while acidic soils can be treated by liming with calcium or of PAHs have been examined under moderate temperatures, magnesium carbonate (Bowlen and Kosson, 1995) to generate an however, there are reports on PAHs biodegradation under environment for effective biodegradation. extreme temperatures. For example, biodegradation of petroleum hydrocarbons including PAHs was reported in seawater at low Oxygen temperatures (0–5 C) (Brakstad and Bonaunet, 2006) while the Biodegradation of organic contaminants including PAHs can biodegradation of PAHs along with long chain alkanes was proceed under both aerobic and anaerobic conditions; however, documented at very high temperature (60–70 C) by Thermus and most of the studies focus bioremediation of PAHs under aerobic Bacillus spp (Feitkenhauer et al., 2003). condition, where oxygen acts as a co-substrate and a rate limiting factor. During aerobic biodegradation of PAHs, oxygen pH is required for the action of both mono- and dioxygenase pH also plays a significant role in biodegradation processes enzymes in the initial oxidation of the aromatic rings. For including that of PAHs. Usually, microorganisms are pH- in situ bioremediation of contaminants, sometimes oxygen sensitive and near neutral conditions (6.5–7.5) are favored by is added from an external source. The addition of oxygen Frontiers in Microbiology | www.frontiersin.org 15 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 16 Ghosal et al. PAH Biodegradation by Microbes externally may be achieved by drainage, tilling, and addition is the chemical and biological extractability of the contaminants, of chemicals that release oxygen or by injection of air into the which is known as ‘aging’ of the contaminant (Alexander, 2000; contaminated site (Pardieck et al., 1992; Atlas and Unterman, Luo et al., 2012). It has been reported that extractability and 1999; Bewley and Webb, 2001). In a study, enhanced intrinsic bioavailability of PAHs decrease significantly with time in aging bioremediation of BTEX compounds in aquifer using an oxygen processes which can be significantly rate-limiting for in situ releasing compound (magnesium peroxide; ORC ) was reported bioremediation (Alexander, 2000; Megharaj et al., 2002; Luo et al., (Odencrantz et al., 1996). Again, in a separate study, in 2012; Abdel-Shafy and Mansour, 2016). situ bioremediation of an aquifer contaminated with various pollutants, including phenols, BTEX compounds and PAHs was Inhibition Due to Excess Substrate or reported, in which sodium nitrate was circulated through the End Product aquifer as the oxygen source, via a series of injection and The major goal of bioremediation is the removal or detoxification abstraction wells (Bewley and Webb, 2001). of contaminants from a given environment. However, sometimes it is possible that the contaminant gets transformed into a more Nutrients toxic dead end product. This is why it is essential to confirm Availability of nutrients is another rate-limiting factor for that the pollutant is completely mineralized at the end of the successful bioremediation of PAHs contaminated environments. treatment (Mendonca and Picado, 2002; Lundstedt et al., 2003). Along with easily metabolized carbon source, microorganisms A study using a bioreactor to treat PAH-contaminated gasworks require various minerals like nitrogen, phosphorus, potassium, soil using in situ bioremediation monitored both the removal of and iron for normal metabolism and growth. Thus, PAHs and the accumulation of oxy-PAHs, such as PAH-ketones, supplementation of nutrients, in the poor nutrient content quinines and coumarins as dead end products (Lundstedt et al., pollutant contaminated sites is required to stimulate the growth 2003). As oxy-PAHs are more toxic than the parent PAHs, this of autochthonous microorganisms, to enhance bioremediation of study highlights the significance of monitoring the metabolites pollutants (Atagana et al., 2003). In marine environments, poor during bioremediation, specifically for toxic dead-end products, biodegradation of petroleum hydrocarbon is due to a low level of and determining the toxicity of the metabolites both before and nitrogen and phosphorous (Floodgate, 1984). On the other hand, after treatment (Lundstedt et al., 2003). excessive nutrient availability can also inhibit the bioremediation Since PAHs in the environment are present as mixtures, of pollutants (Chaillan et al., 2006). There are several reports on the effect of substrate interaction during biodegradation is the negative effects of high nutrient levels on the biodegradation crucial in determining the fate of PAHs in nature. It has been of organic pollutants especially PAHs (Carmichael and Pfaender, reported that, when present as a mixture, the high molecular 1997; Chaîneau et al., 2005). weight PAHs are degraded followed by the degradation of low molecular weight PAHs, by the bacterial community (Mueller Bioavailability et al., 1989). It has been also observed that a high concentration In case of a biological system, the term bioavailability is defined of naphthalene have inhibitory effect on the degradation of other as the fraction of a chemical that can be taken up or transformed PAHs, by a defined bacterial coculture (Bouchez et al., 1995). by living organisms during the course of the experiment. This Similarly, a competitive inhibition of phenanthrene degradation fraction can vary under the influence of mass transfer parameters, by naphthalene, methylnaphthalene and fluorene in binary which include physicochemical processes governing dissolution, mixtures using two pure cultures was reported (Stringfellow and desorption and diffusion, hydrological processes like mixing and, Aitken, 1995). Thus during in situ bioremediation of PAHs, finally, biological processes, such as uptake and metabolism concentration of PAHs in the contaminated sites, and a chance (Bosma et al., 1997; Semple et al., 2003). Bioavailability is for the formation of the toxic dead end product(s) need to be regarded as one of the most crucial factors in bioremediation considered for effective process development. of pollutants. Biodegradation of PAHs in the environment is often limited, due to their low aqueous solubility and their strong tendency to sorbs to mineral surfaces (like clays) and RECENT ADVANCEMENT IN organic matters (like black carbon, coal tar humic acids) in MOLECULAR TECHNIQUES IN the soil matrix, processes which reduce bioavailability. There UNDERSTANDING BACTERIAL are numerous reports on poor or unsuccessful remediation of PAHs contaminated sites due to absorption of PAHs in black DEGRADATION OF PAHS AND FUTURE carbon, coal-tar substances which decreases bioavailability of DIRECTIONS PAHs (Volkering et al., 1992; Luthy et al., 1993, 1994; Nelson et al., 1996; Northcott and Jones, 2001; Hong et al., 2003; Bucheli Nowadays, bioremediation has become an intensive area et al., 2004; Cornelissen et al., 2005; Liu et al., 2009; Benhabib of research. However, more advancement has to be made et al., 2010; Rein et al., 2016). Again, the aqueous solubility in developing practically efficient microbial bioremediation of PAHs decreases with increasing molecular weight, which in processes. Although microorganisms have the capability to use turn reduces the bioavailability of PAHs. It has been seen that numerous organic pollutants as their carbon and energy sources, longer any hydrophobic organic contaminants (like PAHs) is in their proficiency at eliminating such pollutants might not be contact with soil, the more irreversible the sorption, and lesser of top class level for cleaning up present-day pollution. In fact, Frontiers in Microbiology | www.frontiersin.org 16 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 17 Ghosal et al. PAH Biodegradation by Microbes microorganisms have evolved in the direction of ecological details (Nierman and Nelson, 2002; Buermans and den Dunnen, fitness rather than biotechnological efficacy (Schmid et al., 2014). Presently many complete or nearly complete genome 2001). To enhance the catabolic efficiency of a microorganism sequences of cultivable microorganisms are available which have for bioremediation, bioengineering is a prerequisite. Recent potential catabolic activity. However, unfortunately by using advancement in genetic, genomic, proteomic and metabolomic modern microbiological and molecular techniques over the past technologies, which are applied to study bioremediation of 40 years so far only an extremely small fraction (1%) of the organic pollutants, have contributed considerably to enrich total microbial diversity has been cultivated from all habitats our knowledge on various aspects of the physiology, ecology, investigated (Hugenholtz and Pace, 1996; Hugenholtz et al., biochemistry and regulatory mechanisms of the microbial 1998). While culturing novel environmental microorganisms is catabolic pathways. Hence, practical utilization of that indispensable (Zinder, 2002), perhaps the greatest promise to knowledge is essential to manipulate or reconstruct as well date comes from metagenomic approaches, which are the only as enhance the natural processes, thereby making strategies ones currently available to adequately utilize the tremendous for the development of more competent biocatalysts for microbial diversity- one of the richest sources on earth various biotechnological applications including bioremediation (Handelsman et al., 1998). Although there are some associated of culprit organic pollutants in the contaminated sites and challenges in metagenome-based approaches, scientists already transformation of toxic chemical into harmless product or overcame many of those problems (Liles et al., 2008; Mareckova other specialty chemicals (Schmid et al., 2001; Carmona et al., et al., 2008). The progressively decreasing cost and increasing 2009). speed of DNA sequencing has made it possible to sequence A considerable advancement in microbial ecology was billions of bases of metagenomic DNA. Culturable aromatics achieved based on the identification of conserved sequences degrading bacteria are being isolated mostly on the basis of their present in a particular group of microorganisms, most notably ability to utilize aromatic compound as their sole carbon and the 16S rRNA or 18S rRNA genes which could provide a energy sources and by genome sequencing, scientist can identify phylogenetic characterization of the microbial population all the genes necessary to completely degrade the compound. present in a particular habitat (Pace et al., 1986; Amann et al., Therefore, inclusive knowledge of the catabolic potential of 1995). This approach is a significant achievement in the field a contaminated site virtually remains unknown by using of bioremediation because by determining the microbiota in culture-dependent techniques. High throughput metagenomics a polluted environment it is possible to identify particular reduces that bottleneck. However, at present, relatively little microorganisms inhabiting those environments, thereby resources have been spent for the sequencing of soil or marine predicting possible bioremediation potential. With the advent metagenomes contaminated with aromatic pollutants, compared of Denaturing Gradient Gel Electrophoresis (DGGE), it is now to those committed to the human microbiome (Blow, 2008). That possible to analyze the microbial community structure and is why there is a scarcity of information about the xenobiotics dynamics of a particular habitat, more precisely (Gallego et al., degrading novel bacteria that are difficult to culture in the 2014; Vila et al., 2015). Development of fluorescence in situ laboratory. Sites contaminated with toxic chemicals have hybridization (FISH), which is another technique is quite useful become biotechnological gold mines because the indigenous in this field and has often been practiced (Amann et al., 2001). microorganisms may have evolved the necessary enzymes, to Generally, there is a positive link between the relative degrade or transform those toxic contaminants to an inanimate abundance of the genes associated with pollutant removal and one, quite different from those found in common cultivable the efficiency of bioremediation. However, sometimes it is microorganisms (Handelsman et al., 1998; Galvao et al., 2005; possible that the genes associated with pollutant removal can Boubakri et al., 2006). In addition, sequencing of the soil or be present but not expressed. So, there is an increasing interest aquatic metagenome will also provide insights into the ecology in quantifying the mRNA for key catabolic genes via real-time of microorganisms which in turn will help to identify who the PCR (Schneegurt and Kulpa, 1998; Debruyn and Sayler, 2009). dominant and rare community members are and what are their In addition transcriptomics, DNA-based stable isotope probing, probable roles in the degradation of recalcitrant molecules like single cell genomics and DNA microarray techniques are also PAHs and other related xenobiotics. emerging for application in the field of bioremediation (Wilson Recently another two techniques metaproteomics and et al., 1999; Denef et al., 2005, 2006; Chain et al., 2006; Parnell metabolomics have been utilized to unfold various aspects of et al., 2006; Macaulay and Voet, 2014; Mason et al., 2014; environmental microbiology and have shown their promise in Mishamandani et al., 2014). DNA microarray which is a high- the field of bioremediation (Nesatyy and Suter, 2007; Keum throughput version of DNA hybridization technique can detect et al., 2008). Proteomics is an efficient technology to recognize an enormous number of genes in a single test. One of the proteins and their roles associated with catabolism of PAHs recent applications of microarray in the PAHs biodegradation while metabolomics can be exploited to identify metabolites is the construction of GeoChip (He et al., 2010; Nostrand et al., produced during PAHs biodegradation. Figure 11 illustrates a 2012). summarized representation of the molecular techniques involved The advancement in genome sequencing technology in studying microbial degradation of PAH. In near future, practically revolutionized the field of bioremediation. By whole functional metagenomics, metaproteomics, metabolomics, genome sequencing, it is now possible to study the physiology metatranscriptomics and DNA microarrays will become crucial of microorganisms associated with pollutant removal in more tools to elucidate the mechanisms of biodegradation of PAHs Frontiers in Microbiology | www.frontiersin.org 17 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 18 Ghosal et al. PAH Biodegradation by Microbes FIGURE 11 | Schematic diagram summarizing different molecular techniques as future directions for better understanding of the microbial PAH degradation and ecologically sustainable bioremediation of sites contaminated with PAHs. in the environment, and will offer more information on as-yet- the bioremediation potential of the indigenous micro flora uncultured organisms associated with PAHs bioremediation dwelling at the site contaminated with pollutants. These (Wilson et al., 1999; Amann et al., 2001; Fromin et al., 2002; Liang, techniques provide the treatment at the site itself avoiding 2002; Zhou and Thompson, 2002; Baldwin et al., 2003; Ginige excavation and transport of contaminants, which makes them et al., 2004). Moreover, in silico biology is being progressively the most desirable options due to lower cost and fewer applied in different aspects in the field of bioremediation (Kweon disturbances. On the other hand extrinsic bioremediation et al., 2010; Chakraborty et al., 2012; Khara et al., 2014). Thus, (also called ‘ex-situ’ bioremediation) mainly involves the it is expected that emerging molecular biological, analytical physical removal of the contaminated material to another and computational methods which can predict the activity of location for treatment (Carberry and Wik, 2001). While microorganisms involved in biodegradation, should transform in the case of “biostimulation” process, it is generally the bioremediation field from a largely empirical practice into a possible to stimulate the indigenous microorganisms to use branch of modern science. the contaminants as a food source at a much greater rate by compensating limiting parameters, for example, by introducing additional oxygen or nutrients to the indigenous population APPROACHES TO THE (Straube et al., 2003). Another method, “bioaugmentation,” involves introduction of exogenous microorganisms into the BIOREMEDIATION OF contaminated environment which are capable of degrading the PAH-CONTAMINATED ENVIRONMENTS target pollutants, either with or without additional nutrients (Straube et al., 2003). Whereas, “humification” is a process Various types of bioremediation technologies can be employed by which strongly persistent substances in the polluted for contaminant removal. Intrinsic bioremediation (also called environment are incorporated into the humic substances mostly natural attenuation or ‘in situ’ bioremediation) depends on Frontiers in Microbiology | www.frontiersin.org 18 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 19 Ghosal et al. PAH Biodegradation by Microbes by means of enzymatic reactions (Megharaj et al., 2011). In bioremediation procedures. However, due to the high cost addition, “phytoremediation” involves removal of environmental associated with the extraction and shipment, ex situ treatment contaminants using plants (Haritash and Kaushik, 2009; of contaminated groundwater is not generally performed. Megharaj et al., 2011). Another bioremediation technology called Few reports are documented on the treatment of PAH ‘landfarming,’ is used to stimulate indigenous biodegradative contaminated water. Bewley and Webb (2001) reported the microorganisms by providing nutrients, water and oxygen, in in situ bioremediation of an aquifer which was contaminated order to facilitate aerobic degradation of contaminants (Megharaj with diverse pollutants, including phenols, BTEX compounds et al., 2011). While ‘composting’ involves the degradation and PAHs. The site was bioremediated using a procedure of pollutant along with other agricultural wastes such as involving a combination of bioaugmentation and biostimulation. manure etc. (Semple et al., 2001). Hence, it becomes clear that Nutrients (a commercial mixture of urea and diammonium regardless of the method chosen, an ideal bioremediation strategy phosphate), a commercially available phenol-degrading mixed can be designed only on the basis of knowledge about the bacterial inoculum (PHENOBAC, Microbac Ltd, Durham), and microorganisms and the contaminant present within the polluted sodium nitrate (an oxygen source) were circulated through the environments, along with their catabolic potential and response aquifer via a series of injection and abstraction wells. After to changes in the environmental conditions. treatment for over two and half years, the mean concentration 1 1 of PAHs was reduced to 0.9 mgL from 11 mgL along with Treatment of Soils and Sediments reduction of other contaminant (Bewley and Webb, 2001). Apart There are some reports on the treatment of PAH contaminated from that, groundwater remediation technology of petroleum- soils and sediments by in situ or ex situ bioremediation methods. derived compounds (PDCs) including PAHs based on enhanced Straube et al. (2003) reported that a pilot-scale landfarming solubility of PDCs in humic acid was also reported (Van treatment of PAH-contaminated soil from a wood treatment Stempvoort et al., 2002). Zein et al. (2006) reported the treatment facility was achieved by biostimulation of the soil with water, of groundwater contaminated with PAHs, gasoline hydrocarbons, ground rice hulls as a bulking agent, and palletized dried blood and methyl tert-butyl ether using an ex situ aerobic biotreatment as a nitrogen source and bioaugmentation of the microbial system and it was observed that after 10 months of treatment, the community with an inoculum of Pseudomonas aeruginosa strain concentration of PAHs was reduced to >99% along with other 64 (Straube et al., 2003). It has been seen that within 1 year pollutants (Zein et al., 2006). of treatment 86% of total PAHs were removed from the soil including a moderate reduction in HMW PAHs such as pyrene, Treatment with Genetically Engineered BaP etc. In a separate study on the treatment of an aged gasworks Microorganisms (GEMs) soil contaminated with aromatic compounds including PAHs, it has been observed that LMW PAHs and heterocyclic compounds The bioremediation of PAHs contaminated site is generally were degraded more quickly than the HMW counterparts very slow because there are a number of biotic and abiotic (Lundstedt et al., 2003). In addition, the unsubstituted PAHs factors responsible for successful bioremediation. In addition, were degraded faster than the related alkyl-PAHs as well as the incomplete success rates might be due to the fact that some nitrogen-containing heterocyclics. Treatment of soil at a tar places are heavily contaminated, and hence, the microorganisms contaminated site via composting along with conventional land are incapable to grow and degrade the contaminant at the treatment process revealed that composting led to more extensive same rate at which they are introduced into the environment. PAH removal than did by two different land treatment processes Although there is very few information on the use of GEMs (Guerin, 2000). Sasek et al. (2003) reported the remediation in bioremediation, they can be a promising candidate for of a manufactured-gas plant soil contaminated with PAHs via such processes (Ang et al., 2005; Singh et al., 2008; Megharaj composting. In this study, treatment of soil was performed et al., 2011). Using genetic engineering it is possible to in a thermally insulated chamber using mushroom compost enhance the activity or broad substrate specificity of certain containing wheat straw, chicken manure and gypsum, where at enzymes associated with PAH-degrading pathways, which in the end of 54 days, removal of 20–60% of individual PAHs turn will improve the mineralization of those pollutants in the was reported, while 37–80% of individual PAHs degradation environment (Timmis et al., 1994; Timmis and Pieper, 1999; Ang was observed after another 100 days of composting (Sasek et al., et al., 2005; Singh et al., 2008). In recent times, various molecular 2003). Moreover, studies on the removal of PAHs present in biology tools such as gene conversion, gene duplication, and contaminated soil using the associated bacterial communities in transposon or plasmids mediated gene delivery are available, two aerobic, lab-scale, slurry-phase bioreactors, which were run which might play vital roles to boost up the biodegrading semi-continuously and fed either on weekly or monthly basis, potential of naturally occurring microorganisms (Timmis et al., showed that most of the PAHs, including HMW PAHs were 1994; Timmis and Pieper, 1999; Ang et al., 2005; Singh et al., 2008; biodegraded to a greater extent in the weekly fed bioreactor for Megharaj et al., 2011). However, it is crucial to ensure the stability up to 76% (Singleton et al., 2011). of GEMs prior to their field application since the catabolic activity of released GEM is associated largely with the stability of the Treatment of Waters recombinant plasmid introduced into the organism (Brunel et al., Polycyclic aromatic hydrocarbons-polluted groundwater can 1988; Shaw et al., 1992; Samanta et al., 2002). As GEMs that also be bioremediated through both in situ and ex situ are released into a contaminated site can spread to other places Frontiers in Microbiology | www.frontiersin.org 19 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 20 Ghosal et al. PAH Biodegradation by Microbes and multiply under favorable conditions, so before releasing into soil (Gandolfi et al., 2010; Loick et al., 2012; Lukic ´ et al., 2016; the environment there should be a clear understanding of the Rein et al., 2016). So adjustment of organic matter content of possible side effects associated with GEMs, which would possibly a polluted site with exogenous addition of compost or other guide the restriction of their application in pollutant abatement substances like buffalo manure, food and vegetables waste may (Samanta et al., 2002; Ang et al., 2005; Singh et al., 2008; Megharaj enhance the bioremediation efficiency of PAH polluted site. et al., 2011). In addition, instead of treating with a single microorganism, a defined microbial consortium may give a better result in some cases for successful remediation of a contaminated site. CONCLUSION So, more research is required on the catabolic capabilities of PAHs degrading microbial consortia, which will further In last few decades, there has been a great deal of progress in the deepen our understanding on the microbial consortia-mediated study of the bioremediation of PAHs. Numerous microorganisms remediation of contaminated environments, and will help to have been isolated and characterized having PAHs catabolic develop potential microbial consortium having robust pollutant potentials. In addition, many unique enzymes with different degrading potential. It is also possible to treat the contaminated catabolic efficiency associated with PAHs degradation have site sequentially with fungi, bacteria and algae or in combination been purified and different novel biochemical pathways for for more efficient pollutant removal. PAHs degradation have been elucidated. Moreover, many PAH In addition, genetic engineering can be employed to boost the catabolic operons have been sequenced, and their regulatory catabolic efficiency of microorganisms used in bioremediation. mechanism for PAH degradation has been determined. The Today, scientists are capable to create unique PAHs metabolic advancement in genetic, genomic, proteomic and metabolomic pathways by recombining different catabolic genes from different approaches, which are employed to study catabolism of organisms in a single host cell. In this way it is possible to organic pollutants have contributed remarkably in understanding enhance the substrate specificity of a catabolic pathway to the physiology, ecology, biochemistry of PAHs degrading degrade new substrates, complete partial pathways and also to microorganisms. However, more detail research is a prerequisite construct novel pathways not found in nature, which may permit to determine exactly what is going on in PAH-contaminated the mineralization of highly recalcitrant compounds and avoid environment. In addition, there are still various aspects of the accumulation of toxic dead end product. However, before bioremediation of PAHs that remain unknown or otherwise have releasing such GMOs into the environment, it is necessary to insufficient information, which requires future attention. There is check thoroughly the possibility of any unwanted side effects very scarce knowledge on genes, enzymes as well as the molecular produced by those GMOs. In addition, authorities should be mechanism of PAHs degradation in high salinity environments, convinced that the GMOs are safer, cheaper, and more efficient or anaerobic environments. Also, there is very little information than the present alternatives. Thus, it is anticipated that the on the transmembrane trafficking of PAHs and their metabolites. compiled information present in this manuscript will open up Various transporters have been assumed to be participating in the new avenues to the researchers, and the field of bioremediation transport of PAHs into microorganisms, but till date, none has will revolutionize in near future. been characterized. There are various factors which may affect bioremediation of PAHs in a contaminated environment. Adjustment of AUTHOR CONTRIBUTIONS oxygen concentration, pH, temperature, nutrient availability and improvement of bioavailability may increase PAHs degradation. DG translated the concept and wrote the manuscript. DG and It is seen that in contaminated soils and sediments, PAHs get SG analyzed the data, edited and formatted the manuscript. TD entrapped in coal tar or black carbon particles, which results in and YA generated the concept and ideas, critically revised the unsuccessful remediation due to decrease in PAHs bioavailability manuscript and approved the final version for publication. (Bucheli et al., 2004). This phenomenon is a major bottleneck for successful remediation of PAHs contaminated environments (Ortega-Calvo and Alexander, 1994). Some microorganisms ACKNOWLEDGMENTS are known to excrete biosurfactants which enhance the bioavailability of organic pollutants. Many microorganisms This study was supported by the Yeungnam University Research exhibit chemotaxis toward pollutants. These strategies lead to Grant (2015). enhanced degradation of organic pollutants. The addition of small amount of biosurfactant, which increases the bioavailability of PAHs, or some merely toxic chemicals, like salicylic acid, which SUPPLEMENTARY MATERIAL induce PAHs catabolic operons may enhance biodegradation of PAHs in the environment. It has been seen that organic The Supplementary Material for this article can be found amendments influence the indigenous microbial community as online at: http://journal.frontiersin.org/article/10.3389/fmicb. well as efficiency of bioremediation of PAHs in contaminated 2016.01369 Frontiers in Microbiology | www.frontiersin.org 20 August 2016 | Volume 7 | Article 1369 fmicb-07-01369 August 31, 2016 Time: 16:47 # 21 Ghosal et al. PAH Biodegradation by Microbes Binkova, B., Giguere, Y., Rossner, P. Jr., Dostal, M., and Sram, R. J. (2000). REFERENCES The effect of dibenzo[a,1]pyrene and benzo[a]pyrene on human diploid lung Abdel-Shafy, H. I., and Mansour, M. S. M. (2016). 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Hum. 32, The use, distribution or reproduction in other forums is permitted, provided the 1–453. original author(s) or licensor are credited and that the original publication in this Widada, J., Nojiri, H., Kasuga, K., Yoshida, T., Habe, H., and Omori, T. journal is cited, in accordance with accepted academic practice. No use, distribution (2002). Molecular detection and diversity of polycyclic aromatic or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 27 August 2016 | Volume 7 | Article 1369

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