2014_bioremediation of petroleum hydrocarbons catabolic genes, microbial communities, and...

8/16/2019 2014_Bioremediation of Petroleum Hydrocarbons Catabolic Genes, Microbial Communities, And Applications

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MINI-REVIEW

Bioremediation of petroleum hydrocarbons: catabolic genes,microbial communities, and applications

Sebastián Fuentes & Valentina Méndez & Patricia Aguila &

Michael Seeger

Received: 30 January 2014 /Revised: 10 March 2014 /Accepted: 11 March 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Bioremediation is an environmental sustainableand cost-effect ive technology for the cleanup of hydrocarbon-polluted soils and coasts. In spite of that longer times are usually required compared with physicochemicalstrategies, complete degradation of the pollutant can beachieved, and no further confinement of polluted matrix isneeded. Microbial aerobic degradation is achieved by theincorporation of molecular oxygen into the inert hydrocarbonmolecule and funneling intermediates into central catabolic pathways. Several families of alkane monooxygenases andring hydroxylating dioxygenases are distributed mainlyamong Proteobacteria , Actinobacteria , Firmicutes and Fungi strains. Catabolic routes, regulatory networks, andtolerance/resistance mechanisms have been characterized inmodel hydrocarbon-degrading bacteria to understand andoptimize their metabolic capabilities, providing the basis toenhance microbial fitness in order to improve hydrocarbonremoval. However, microbial communities taken as a whole play a key role in hydrocarbon pollution events. Microbialcommunity dynamics during biodegradation is crucial for understanding how they respond and adapt to pollution andremediation. Several strategies have been applied worldwidefor the recovery of sites contaminated with persistent organic pollutants, such as polycyclic aromatic hydrocarbons and petroleum derivatives. Common strategies include controllingenvironmental variables (e.g., oxygen availability, hydrocarbon solubility, nutrient balance) and managing hydrocarbon-degrading microorganisms, in order to overcome the rate-limiting factors that slow down hydrocarbon biodegradation.

Keywords Petroleum . Hydrocarbon . Bioremediation .Biodegradation . Microbial community . Catabolic genes

Introduction

Petroleum is a natural resource confined in large deposits inthe Earth crust. Accidental petroleum spills alter the impactedenvironment and trigger the development and implementationof remediation strategies for cleaning up the polluted sites. Oilspills became an international concern in 1967, when~120,000 tons of crude oil was released by the TorreyCanyon supertanker into the English Channel. This first large-scale oil spill forced UNO ’ s International MaritimeOrganization to create in 1973 the International Conventionfor the Prevention of Pollution from Ships MARPOL with theaim of designing emergency protocols and strategies towardoil spills. Since then, there have been a number of significant marine oil spills, even only the emblematic spills usually alert the public opinion. Oil spills are difficult to avoid during the petroleum processing and delivery.

Petroleum is mainly composed by three hydrocarbon frac-tions. Paraffin is usually the most abundant fraction andcontains linear and branched aliphatic hydrocarbons. Naphthenes are alicyclic hydrocarbons composed by one or more saturated rings with or without lateral aliphatic branches.The aromatic fraction is composed by hydrocarbons contain-ing at least one aromatic ring. Hydrocarbons can possess fromfew up to >60 carbons. A higher molecule size correlates witha higher boiling point. Petroleum-derived products are obtain-ed by fractional distillation, by which different fractions areenriched according to its boiling range (Speight 2001).

For the cleanup of hydrocarbon-polluted sites, diverse physicochemical and bioremediation treatments have beenapplied. Bioremediation techniques are cost-effective, envi-ronmental sustainable, and can achieve complete pollutant

S. Fuentes : V. Méndez : P. Aguila : M. Seeger (* )Laboratorio de Microbiología Molecular y Biotecnología Ambiental,Departamento de Química & Centro de Biotecnología & Center of Nanotechnology and Systems Biology, Universidad Técnica Federico Santa María, Valparaíso, Chilee-mail: [email protected]

Appl Microbiol BiotechnolDOI 10.1007/s00253-014-5684-9


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degradation. Microorganisms are the main biocatalysts for hydrocarbon bioremediation. Thus, microbial degradationhas to be optimized in order to improve bioremediation. Inaddition, the study of the microbial community dynamics in polluted sites is useful for the management of the process. Theaims of this review are to update bioremediation strategies for hydrocarbon-polluted sites, hydrocarbon catabolic pathwaysand genes, and microbial dynamics in oil-polluted soils and tohighlight main challenges for bioremediation technologies.

Hydrocarbons as persistent pollutants

Petroleum hydrocarbons are organic pollutants of major con-cern due to their wide distribution, persistence, complex com- position, and toxicity. The most common petroleum hydro-carbons include aliphatic, branched, and cycloaliphatic al-kanes, as well as monocyclic and polycyclic aromatic hydro-carbons (PAHs). PAHs include naphthalene, fluorene, phen-anthrene, anthracene, fluoranthene, pyrene, benzo[ a ]-anthracene, and benzo[ a]pyrene. Combined cycloaliphatic –

aromatic structures can also be found in crude oil. Each petroleum fraction is usually composed by hundreds of dif-ferent hydrocarbon molecules rather than a defined composi-tion. Thus, fractions are dissimilar in terms of volatility, bio-availability, toxicity, degradability, and persistence (Table 1).This complex array of compounds depicts the tremendouschallenge for designing effective bioremediation strategies,which can be illustrated by the effects of major contaminationevents in the past (Atlas and Hazen 2011).

Once petroleum hydrocarbons reach an environment, dam-age can be the result of several causes. Primary biologicalimpact is due to the blocking effect of oil layer to water,nutrients, oxygen, and light access. Cytotoxic and mutageniceffects of hydrocarbons are behind the long-term pollutionconsequences. A more bioavailable toxic compound not onlyshows increased noxious effects but also has higher accessi- bility for biodegradation. In contrast, strongly adsorbed frac-tion is less toxic but more recalcitrant. This general ruleis relevant for designing biological strategies for the cleanupof polluted soils or sediments because petroleum hydrocarbons tend to tightly adsorb to these matrices (Baboshin andGolovleva 2012).

Two hydrocarbon fractions are present in solid particles(i.e., soil, sediment). The fraction that remains irreversiblyadsorbed to particles is considered non-toxic and non- biodegradable because it is not bioavailable. The reversibly bound portion able to desorb and diffuse into water phaseconstitutes the bioavailable fraction. Nonetheless, bioavail-ability is always limited due to the low water solubility of hydrocarbons. Main hydrocarbon degraders are bacteria, fila-mentous fungi, and yeasts (van Beilen and Funhoff 2007;Wentzel et al. 2007). To overcome low bioavailability,

bacteria can get access to hydrophobic substrates by reducingsurface tension or by direct contact with hydrophobic droplet (Wentzel et al. 2007). Surface tension reduction can beachieved by the secretion of surfactants, molecules that dis- perse hydrocarbons into small droplets (Baboshin andGolovleva 2012). Biosurfactants can be glycolipids, phospho-lipids, lipopeptides, lipoproteins, fatty acids, neutral lipids, polymeric lipids, and high molecular weight biopolymers(Atlas and Philp 2005; Das et al. 2008). Direct attachment tohydrocarbon droplet interface can be achieved by increasingthe hydrophobicity of cell surface. This includes the synthesisof adhesion structures like proteins, lipopolysaccharides,mycolic acids, and other hydrophobic exopolymers(Abbasnezhad et al. 2011 ). Acinetobacter venetianus RAG-1synthesizes emulsan and a hydrophobic fimbriae, enhancingthe attachment to the droplet (Rosenberg et al. 1982). Rhodococcus erythropolis 20S-E1-c has an external hydrophobic layer composed by mycolic acids. Despite both outer structures facilitate the access to hydrophobic surfaces, they possess different dynamic and mechanical properties for at-tachment to droplets (Abbasnezhad et al. 2011). In closecontact to bacteria, hydrocarbon molecules enter the bacterialcell, where the catabolic machinery achieves their breakdown.Uptake apparently occurs by a lateral diffusion mechanism,where the hydrocarbon molecule diffuses from the trans- porter ’ s lumen laterally into the outer membrane (Hearnet al. 2009).

Microorganisms possess evolved mechanisms to activatehydrocarbons, generating metabolic intermediates that funnelto central metabolic pathways. By oxidizing these substrates,microorganisms can take advantage in nutrient-limited niches.Addition of one or two hydroxyl groups to the hydrocarbonskeleton seems to be the ubiquitous first step during aerobiccatabolism (Figs. 1 and 2). The key enzymes in hydrocarbondegradation pathways are oxygenases, which catalyze theaddition of molecular oxygen to the substrate (Rojo 2009).Dioxygenases catalyze the addition of two hydroxyl groups,whereas monooxygenases catalyze introduction of one atomof oxygen into the hydrocarbon. During anaerobic degrada-tion, activation is achieved coupling CO 2 or fumarate tohydrocarbons (Fig. 2), and sulfate and nitrate are used asterminal electron acceptors (Callaghan et al. 2012; So et al.2003 ). However, anaerobic degradation of alkanes occurs at lower rates compared with aerobic microbial catabolism(Wentzel et al. 2007).

Bacterial metabolism toward alkane hydrocarbondegradation

Unbranched intermediate C 5 – C11 chain-length n-alkanes arethe major hydrocarbon constituents of petroleum. The initialaerobic alkane degradation step is oxidation via an alkane

Appl Microbiol Biotechnol

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oxygenase, carried out by either a multimeric monooxygenaseor a cytochrome P450 monooxygenase (van Beilen andFunhoff 2007). Monooxygenases belong to a widely distrib-uted protein family based upon functional properties (i.e.,alkane hydroxylation), but exhibiting significant structuraldifferences (van Beilen and Funhoff 2007). These catalyticsystems could contain at their active site iron – sulfur, di-iron,heme, and copper (van Beilen and Funhoff 2007; Wang andShao 2012).

Aerobic alkane degradation pathways first oxidize analkane into an alcohol. Commonly a primary alcohol is produced, but subterminal oxidation has also been reported (Rojo 2009). The primary alcohol is oxidizedinto an aldehyde, which is subsequently transformedvia oxidation into a fatty acid, which is funneled intoβ -oxidation (Fig. 1). Initial reaction of alkane degrada-tion pathway is mediated by an integral-membrane non-heme di-iron monooxygenase that hydroxylates the sub-strate at terminal position. This monooxygenase complex cons ists of a par ticulate membrane-bound

hydroxylase (pAH), a rubredoxin, and a rubredoxinreductase. For the reduction step, electrons are subse-quently transferred from reduced NADH via its cofactor FAD to rubredoxin reductase (AlkT), soluble rubredoxin(AlkG), pAH, and finally into the alkane. AlkB from Pseudomonas putida GPo1 is a well-characterizedmodel pAH enzyme that oxidizes propane, n-butane,and C 5 – C13 alkanes. The alkB-like genes are widelydistributed in Proteobacteria and Actinobacteria , includ-ing environmental, opportunistic, and pathogenic strains(van Beilen et al. 2006). Differences in substrate rangeand specific ity among pAHs from Burkholderia , Acinetobacter , Pseudomonas , Alcanivorax , Oleiphilus , M y c ob a c t e r i um , R h o d o c o c c u s , Nocard ia , an d Prauserell a genera have been reported. Nonetheless,structural and catalytic motifs are conserved amongthem (van Beilen and Funhoff 2007). Strains able todegrade intermediate C 5 – C11 and long C 12 n-alkanesg e n er a l ly p o s s es s A l k B- l i ke m o n oo x y ge n a se s(Rojo 2009).

Table 1 Physicochemical and toxicological properties of representative hydrocarbons from different crude oil fractions

Hydrocarbon Molecular formula Melting point a (°C) Boiling point a (°C) Water solubility a,b

(mg L−1)

log K OWa,c Toxicity d, e

(mg kg−1)

Aliphatic

n-Hexane C 6H14 −95.4 68.7 9.5 – 262 2.9 – 4.3 25,000n-Octane C 8H18 −56.8 125.7 0.5 – 14.0 4.0 – 5.6 nd

n-Hexadecane C 16 H34 18.1 286.9 2×10−6

– 6×10−3

7.3 – 8.3 ndPristane C 19 H40 −100 f 68f nd nd 980Cyclohexane C 6H12 6.6 80.7 50.2 – 88.8 2.5 – 3.7 nd

BTEXBenzene C 6H6 5.5 80.1 1,402 – 2,167 1.6 – 2.5 930Toluene C 7H8 −95 110.6 155 – 739 2.1 – 3.0 636Ethylbenzene C 8H10 −95 136.2 131 – 208 3.1 – 3.5 3,500

PAH Naphthalene C 10 H8 80.3 217.9 12.5 – 38.4 3.0 – 3.8 533Anthracene C 14 H10 215.8 339.9 0.03 – 0.09 3.5 – 5.34 4,900

Pyrene C 16 H10 150.6 404 0.03 – 1.6 4.5 – 5.5 800Benzo[ a]pyrene C 20 H12 181.1 495 4×10

−5 – 6×10−3 5.1 – 8.0 300

DistillateKerosene – – 205-300 g 4.8-10.4 g 3.3->6 g 15,000

nd not determineda Physicochemical data from Mackay et al. ( 2006) unless otherwise indicated b At 25 °C. Values depend on the used methodc Octanol – water partition coefficient d Lethal dose 50 tested in adult rat (mouse for PAH)e Material Safety Data Sheets (MSDS) available onlinef Sigma-Aldrich product informationg American Petroleum Institute, Petroleum HPV Testing Group ( 2010 )



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Substrate range and alk genes organization differ among alkane-degrading bacteria. Metabolic enzymes for the conversion of n-alkanes into fatty acids are encoded inalkBFGHJKL and alkST operons located in the OCT plasmi d from P. puti da GPo1 (Rojo 2009 ) . T healkBFGHJKL operon encodes the enzymes for alkaneconversion into acetyl-CoA. The alkST cluster encodesthe rubredoxin reductase AlkT and the transcriptionalactivator AlkS (van Beilen and Funhoff 2007 ) . In Acinetobacter baylyi ADP1, long C 12 – C36 n-alkane hy-droxylation is mediated by the AlkB-l ike alkanemonooxygenase AlkM. Interestingly, AlkR-mediatedalkM gene expression is induced by non-substrates C 7 –

C11 n-alkanes (Rojo 2009). Acinetobacter sp. M1 is ableto use C 20 – C44 alkanes and possesses two n-alkanehydroxylase-encoding genes ( alkMa and alkMb ).Expression of alkMa gene is induced by >C 22 chain-

length n-alkanes, whereas alkMb gene expression is pref-erentially induced by C 16 – C22 n-alkanes. Pseudomonasaeruginosa strains PAO1 and RR1 possess AlkB1 andAlkB2 hydroxylases that probably play different physiologi-cal roles. While alkB2 gene expression is higher in earlyexponential phase, alkB1 gene is preferentially expressedduring stationary phase (Rojo 2009). Long chain n-alkanesare oxidized by alkane hydroxylase AlmA from Acinetobacter sp. DSM17874 that degrades C 32 or longer alkanes and alkanemonooxygenase LadA from Geobacillus thermodenitrificans NG80-2 that degrades up to C 36 long-chain alkanes (Li et al.2008 ; Throne-Holst et al. 2007).

Two additional hydroxylation systems include methanemonooxygenases (MMOs) and cytochrome P450 protein su- perfamily. MMOs are found primarily in methanotrophic bac-teria. Cytoplasmic soluble form of methane monooxygenase(sMMO) is present in Methylococcus , Methylosinus ,

Fig.1 Bacterial n-alkane degradation pathways. Aerobic pathways( a , b)are shown in the left panel , whereas anaerobic pathways ( c, d ) are shownin the right panel. a Aerobic alk -like degradation pathways oxidizealkanes into fatty acids. In some cases, ω-hydroxylation generates a dicarboxylic acid. b Sub-terminal oxidation of n-alkanes in some Rhodococcus , Mycobacterium , and Pseudonocardia strains yields a pri-mary alcohol two carbons shorter than the original alkane which is further

oxidized as shown in a (dotted line ). c Anaerobic degradation in D. oleovorans Hxd3 includes the loss of two terminal carbon atoms via an unknown process. d Anaerobic degradation in D. alkenivorans AK-01

achieved via conjugation with fumarate at C-2 position followed by a decarboxylation. In all cases, the fatty acid is conjugated with coenzymeA (CoA) andfunneled into β -oxidation. In strain AK-01, the second cycleof β -oxidation yields propionate instead of acetate. Propionate can re-generate fumarate via methylmalonyl-CoA pathway. AH alkane hydrox-ylase, AD alcohol dehydrogenase, ALD aldehyde dehydrogenase, ASS alkylsuccinate synthase, ACS acyl-CoAsynthetase, BVM Baeyer – Villiger

monooxygenase, E esterase, β -Ox β -oxidation cycle, C? putative carbox-ylase, ? unknown enzyme



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Methylocys tis , Methylomonas , Methylomicrobium , and Methylocella genera. The membrane-bound particulate form(pMMO) has also been identified in all methanotrophs except in Methylocella (McDonald et al. 2006). The sMMOs possessa [2Fe-2S] catalytic center and utilize FAD and NADH ascofactors and oxidize C 1 – C8 chain-length alkanes, halogenat-ed alkanes, alkenes, and cycloalkanes. The pMMOs have a mono- or di-copper center and hydroxylate only C 1 – C5 sub-strates such as alkanes, halogenated alkanes, and alkenes (vanBeilen and Funhoff 2007).

Cytochrome P450 comprises several oxidase systems in Bacteria , Archaea , and Eukarya . Soluble cytochromes P450involved in alkane oxidation have been described in Proteobacteria and Actinobacteria (van Beilen et al. 2006).Seven Rhodococcus erythropolis strains possess two cyto-chromes CYP153 and three to five AlkB-like pAHs, whereas both Alcanivorax borkumensis strains AP1 and SK2 harbor two CYP153 and two AlkB homologs.

Anaerobic alkane degradation has been described insulfate-reducing Deltaproteobacteria . In Desulfococcusoleovorans Hxd3, n-alkanes are activated by carboxylationat C-3 position and elimination of the two adjacent terminalcarbons to yield a carboxylic acid that contains one carbonless than the initial n-alkane (Fig. 1) (So et al. 2003). Hxd3enzymes involved in anaerobic alkane catabolic pathwayshave not been identified. Desulfatibacillum alkenivoransAK-01 is able to grow on C 13 – C18 chain-length n-alkanes,1-hexadecene, and 1-pentadecene. Alkane activation isachieved via conjugation with fumarate at C-2 position bythe alkyl succinate synthase (Ass) to yield (1-methyl-alkyl)succinate. After carbon rearrangement and decarboxyl-ation, a methylated fatty acid that is two carbons larger thanthe original n-alkane is formed (Callaghan et al. 2006). Theresulting fatty acid is funneled into β -oxidation pathway(Fig. 1). Although the genome of Desulfatibacillumalkenivorans strain AK-01 has been sequenced, mechanismsfor (1-methyl-alkyl)succinate carbon rearrangement and fu-marate regeneration remains poorly known (Callaghan et al.2012 ).

Bacterial aromatic hydrocarbon degradation

Aerobic microbial metabolism for aromatic hydrocarbonssuch as biphenyl, naphthalene, phenanthrene, and pyrene(Fig. 2) involves an initial dioxygenation reaction by a ring-hydroxylating dioxygenase (RHD) (Kim et al. 2007; Pagnout et al. 2007; Peng et al. 2008; Seo et al. 2012). In contrast, thexylene/toluene pathway from P. putida mt-2 ( P. putidaKT2440 (pWW0)) starts with a monooxygenation of a methylgroup (Fig. 2). PAH-RHD catalytic sites share a Rieske [2Fe-2S] non-heme iron catalytic center and are less diverse thanalkane oxygenases (Iwai et al. 2011; Kweon et al. 2010). The

cis -diol is rearomatized by a dihydrodiol dehydrogenase.Depending on the catabolic pathway, the dihydroxylated aro-matic ring can undergo fission in meta - or ortho -position(Peng et al. 2008 ). After ring cleavage, aromatichydrocarbonswith at least two rings give rise to different carboxy-aromaticmetabolic intermediates, which are further transformed intointermediates that enter central metabolism, including cate-chol, salicylate, gentisate, homogentisate, and protocatechuatecentral pathways (Chain et al. 2006; Méndez et al. 2011;Romero-Silva et al. 2013). Eventually, activation of the orig-inal aromatic substrate generates molecules with even higher toxicity, leading to dead-end metabolic pathways (Cámara et al. 2004; Pieper and Seeger 2008).

Benzene, toluene, ethylbenzene, and xylenes (BTEX) aremain components of the volatile organic fraction from crudeoil. For toluene degradation, five aerobic pathways and oneanaerobic route have been reported (Shinoda et al. 2004). P. putida mt-2 oxidizes toluene at the methyl group by thexylene monooxygenase into benzaldehyde (Fig. 2). P. putidaF1 oxidizes toluene by the toluene dioxygenase TodC1C2BAinto cis -toluene dihydrodiol. Burkholderia cepacia G4, Ralstonia pickettii PKO1, and Pseudomonas mendocinaKR1 oxidize toluene using specific monooxygenases into o-,m-, and p-cresol, respectively. The anaerobic pathway for toluene degradation from Thauera aromatica K172 and Azoarcus sp. T involves an initial addition of fumarate by benzylsuccinate synthase to the methyl group of toluene.Degradation of toluene by P. putida mt-2 has been extensivelystudied. The enzymes for toluene ( m-xylene, p-xylene) deg-radation are encoded by the xyl genes from pWW0 plasmidfrom P. putida mt-2 (Domínguez-Cuevas et al. 2006). Theupper operon xylUWCMABN encodes the enzymes for theoxidation of the methyl group of toluene, m-xylene, and p-xylene into the corresponding (alkyl)benzoate. Oxidation isfollowed by the dioxygenation of the (alkyl)benzoate and themeta -cleavage of the resulting (methyl)catechol, which isfunneled into the Krebs cycle. This second set of reactions iscatalyzed by the meta TOL gene products from the xylXYZLTEGFJQKIH gene cluster. Positive transcriptionalregulators XylR and XylS orchestrate the coordinated expres-sion of the upperandlowerpathways. The xylE geneencodingcatechol 2,3-dioxygenase enzyme of the toluene/xylene deg-radation pathway has been detected in novel hydrocarbon-degrading Acinetobacter , Kocuria , and Pseudomonas strainsisolated from a crude oil-contaminated soil (Méndez et al.2010 ).

The naphthalene catabolic pathway encoded by the nahgenes from the NAH7 plasmid in P. putida G7 shares notori-ous resemblances with xyl catabolic route. The upper catabolicoperon nahAaAbAcAdBFCED encodes enzymes for the con-version of naphthalene into salicylate. Enzymes encoded bythe lower operon nahGTHINLOMKJ transform salicylate via meta -cleavage into pyruvate and acetaldehyde. The


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ids_by_a_Sulfate-Reducing_Bacterium_Strain_Hxd3?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==


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naphthalene dioxygenase NahAaAbAcAd from P. putida G7incorporates two hydroxyl groups in cis-position. The nahgenes-encoded enzymes in the NAH7-like plasmid pKA1 from Pseudomonas fluorescens 5R oxidize also anthracene and phenanthrene into hydroxynaphthoic acid (Peng et al. 2008 ).

Low substrate specificity seems to be a common feature in bacteria that degrade several PAHs. Bacteriaable to use a widerange of compounds for growth should have an advantageover bacteria able to use only few compounds. In evolution,this feature is favored and is more likely to be maintained intime (Copley 2000). The low substrate specificity allowsRHDs to oxidize related compounds usually found together in nature, but with different conversion rates. Mycobacteriumvanbaalenii PYR-1 possesses the NidAB and NidA3B3 en-zymes involved in the mono- and dioxygenation of toluene,m-xylene, naphthalene, phenanthrene, anthracene, fluoran-thene, pyrene, benz[ a ]anthracene, benzo[ a ]pyrene, and thenon-hydrocarbons carbazole and dibenzothiophene (Kweonet al. 2010). NidAB showed higher conversion rates toward pyrene, whereas NidA3B3 presents higher conversion ratesfor phenanthrene and fluoranthene. Their low substrate spec-ificity correlates with larger binding pocket sizes in the activesite (Kweon et al. 2010).

Biphenyl is present naturally in crude oil and is of specialconcern because it is the starting molecule for the synthesis of polychlorobiphenyls (PCBs). Biphenyl and PCB degradation by enzy me s encod ed by th e bph genes occurs in P s e u d o m o n a s , B u r k h o l d e r i a , S p h i n g o m o n a s , Achromobacter , and Rhodococcus strains (Chain et al. 2006;Pieper and Seeger 2008; Seeger et al. 1997). Biphenyl-2,3-dioxygenase (BDO) catalyzes the initial cis-hydroxylation of one ring (Seeger and Pieper 2009). The BDO of Burkholderia xenovorans LB400 has an unusual wide substrate range frommono- to hexachlorobiphenyls (Seeger et al. 1999 ; Seeger andPieper 2009). Biphenyls with fluoro, bromo, nitro, and hy-droxy substituents, dibenzofuran, dibenzodioxin, and naturaland synthetic isoflavonoids are also accepted as substrates bythe LB400 BDO (Seeger et al. 2001, 2003; Overwin et al.2012 ). A wide array of dioxygenases and hydroxylases-encoding genes from strain LB400 accounts for its unusualcatabolic versatility toward aromatic compounds (Méndezet al. 2011 ; Romero-Silva et al. 2013). In addition to therelaxed substrate range, protein derivatives generated by com- bination of gene segments (Cámara et al. 2007) or randommutagenesis (Zielinski et al. 2006) can give rise to enzymesthat oxidize novel substrates, which could be useful for bio-remediation and biotransformation processes. Improved PCB bioremediation was observed with the genetically modifiedCupriavidus necator JMS34 in which the bph locus of B. xenovorans LB400 that encodes the PCB-degradationpath-ways was incorporated (Saavedra et al. 2010).

An aromatic hydrocarbon can be a carbon source as well asa toxic signal for bacteria. In response to hydrocarbons,

P. putida mt-2 induces a solvent extrusion system and exhibitsa tolerance response. Efflux pumps and several stress-response related proteins are up-regulated in presence of bothtoluene and the non-substrate o-xylene (Domínguez-Cuevaset al. 2006). In B. xenovorans LB400, stress responses due tothe presence of toxic aromatic compounds such as biphenyland PCBs include the induction of general stress and oxidativestress proteins (Agulló et al. 2007). Bacteria possess sophisti-cated mechanisms to counteract aromatic toxicity, inducinggenes involved in stress response and degradation. Bacterialisolates from crude oil-polluted environments showed in-creased catalaseand peroxidase activities. Enzymatic defenseswere crucial for the degradation of pollutants but also tocounteract reactive oxygen species (Bu č ková et al. 2010).Induction of components of the OxyR regulon was observedduring toluene degradation in P. putida KT2440 (pWW0)(Domínguez-Cuevas et al. 2006). Antioxidant enzymes fromthe OxyR regulon such as alkylhydroperoxide reductase andcatalase possess a key role in cellular response to oxidativedamage. Bacterial adaptation to toxic compounds compriseschanges at genome and physiological levels (Agulló et al.2007 ; Segura et al. 2012).

Designing bioremediation strategies

Choosing an appropriate remediation strategy relies on the physicochemical properties of the polluted matrix and on thedegree and age of the spill. The aim of bioremediation is toovercome the limiting factors that slow down biodegradationrates. Bioremediation can be accomplished either by in situ or ex situ treatments. During in situ applications, the pollution istreated at the site. The ex situ technologies involve the trans- port of the polluted soil to a place where a suitable treatment system can be engineered (Table 2).

Table 2 Estimated costs of remediation strategies for hydrocarbon- polluted soils and sediments

Remediation strategy Treating site Cost (US $/m 3)a

Biological

Biostimulation In situ 30 – 100Bioaugmentation In situ 30 – 100Bioventing In situ 79 – 970Biopiles Ex situ 130 – 260Composting Ex situ 630 – 757

Landfarming Ex situ 30 – 70Physicochemical

Vapor extraction In situ 405 – 1,485

Thermal desorption Ex situ 44 – 252

a Data from the US Federal Remediation Technologies Roundtable ( 2014).Additional costs for laboratory and pilot scale experiments are not included


https://www.researchgate.net/publication/23132938_Microbial_biodegradation_of_polyaromatic_hydrocarbons_FEMS_Microbiol_Rev?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/23132938_Microbial_biodegradation_of_polyaromatic_hydrocarbons_FEMS_Microbiol_Rev?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/23132938_Microbial_biodegradation_of_polyaromatic_hydrocarbons_FEMS_Microbiol_Rev?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/12478851_Evolution_of_a_metabolic_pathway_for_degradation_of_a_toxic_xenobiotic_the_patchwork_approach_Trends_Biochem_Sci_25_261-265?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/12478851_Evolution_of_a_metabolic_pathway_for_degradation_of_a_toxic_xenobiotic_the_patchwork_approach_Trends_Biochem_Sci_25_261-265?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/12478851_Evolution_of_a_metabolic_pathway_for_degradation_of_a_toxic_xenobiotic_the_patchwork_approach_Trends_Biochem_Sci_25_261-265?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/45660722_Substrate_Specificity_and_Structural_Characteristics_of_the_Novel_Rieske_Nonheme_Iron_Aromatic_Ring-Hydroxylating_Oxygenases_NidAB_and_NidA3B3_from_Mycobacterium_vanbaalenii_PYR-1?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/45660722_Substrate_Specificity_and_Structural_Characteristics_of_the_Novel_Rieske_Nonheme_Iron_Aromatic_Ring-Hydroxylating_Oxygenases_NidAB_and_NidA3B3_from_Mycobacterium_vanbaalenii_PYR-1?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/45660722_Substrate_Specificity_and_Structural_Characteristics_of_the_Novel_Rieske_Nonheme_Iron_Aromatic_Ring-Hydroxylating_Oxygenases_NidAB_and_NidA3B3_from_Mycobacterium_vanbaalenii_PYR-1?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/6484598_Generation_by_a_Widely_Applicable_Approach_of_a_Hybrid_Dioxygenase_Showing_Improved_Oxidation_of_Polychlorobiphenyls?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/6484598_Generation_by_a_Widely_Applicable_Approach_of_a_Hybrid_Dioxygenase_Showing_Improved_Oxidation_of_Polychlorobiphenyls?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7259772_Generation_of_Novel-Substrate-Accepting_Biphenyl_Dioxygenases_through_Segmental_Random_Mutagenesis_and_Identification_of_Residues_Involved_in_Enzyme_Specificity?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7259772_Generation_of_Novel-Substrate-Accepting_Biphenyl_Dioxygenases_through_Segmental_Random_Mutagenesis_and_Identification_of_Residues_Involved_in_Enzyme_Specificity?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7259772_Generation_of_Novel-Substrate-Accepting_Biphenyl_Dioxygenases_through_Segmental_Random_Mutagenesis_and_Identification_of_Residues_Involved_in_Enzyme_Specificity?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/43300163_Mineralization_of_PCBs_by_the_genetically_modified_strain_Cupriavidus_necator_JMS34_and_its_application_for_bioremediation_of_PCB_in_soil?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/43300163_Mineralization_of_PCBs_by_the_genetically_modified_strain_Cupriavidus_necator_JMS34_and_its_application_for_bioremediation_of_PCB_in_soil?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/43300163_Mineralization_of_PCBs_by_the_genetically_modified_strain_Cupriavidus_necator_JMS34_and_its_application_for_bioremediation_of_PCB_in_soil?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/6634166_Response_to_chlorobiphenyls_of_the_polychlorobiphenyl-degrader_Burkholderia_xenovorans_LB400_involves_stress_proteins_also_induced_by_heat_shock_and_oxidative_stress_FEMS_Microbiol_Lett?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/6634166_Response_to_chlorobiphenyls_of_the_polychlorobiphenyl-degrader_Burkholderia_xenovorans_LB400_involves_stress_proteins_also_induced_by_heat_shock_and_oxidative_stress_FEMS_Microbiol_Lett?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/6634166_Response_to_chlorobiphenyls_of_the_polychlorobiphenyl-degrader_Burkholderia_xenovorans_LB400_involves_stress_proteins_also_induced_by_heat_shock_and_oxidative_stress_FEMS_Microbiol_Lett?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/12478851_Evolution_of_a_metabolic_pathway_for_degradation_of_a_toxic_xenobiotic_the_patchwork_approach_Trends_Biochem_Sci_25_261-265?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/45660722_Substrate_Specificity_and_Structural_Characteristics_of_the_Novel_Rieske_Nonheme_Iron_Aromatic_Ring-Hydroxylating_Oxygenases_NidAB_and_NidA3B3_from_Mycobacterium_vanbaalenii_PYR-1?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/43300163_Mineralization_of_PCBs_by_the_genetically_modified_strain_Cupriavidus_necator_JMS34_and_its_application_for_bioremediation_of_PCB_in_soil?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/23132938_Microbial_biodegradation_of_polyaromatic_hydrocarbons_FEMS_Microbiol_Rev?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/6484598_Generation_by_a_Widely_Applicable_Approach_of_a_Hybrid_Dioxygenase_Showing_Improved_Oxidation_of_Polychlorobiphenyls?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/6634166_Response_to_chlorobiphenyls_of_the_polychlorobiphenyl-degrader_Burkholderia_xenovorans_LB400_involves_stress_proteins_also_induced_by_heat_shock_and_oxidative_stress_FEMS_Microbiol_Lett?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7259772_Generation_of_Novel-Substrate-Accepting_Biphenyl_Dioxygenases_through_Segmental_Random_Mutagenesis_and_Identification_of_Residues_Involved_in_Enzyme_Specificity?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==


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Strategy selection depends on rate-limiting factors and the pollutant ’ s chemical nature. For petroleum hydrocarbons, four well-known scenarios arise: (1) The excess of carbon sourcedue to hydrocarbon input results in limitation of other nutri-ents. Nitrogen and phosphorus amendments can be used torestore balance and thus increase biodegradation rates; (2)insufficient oxygen availability decreases biodegradationrates. Air injection or simple stirring can overcome oxygenlimitation during aerobic hydrocarbon degradation; (3) low bioavailability of hydrocarbons. Addition of surfactants canimprove solubility and thus bioavailability of hydrocarbons.The application of biosurfactants (i.e., surfactants producedbymicroorganisms or plants) is an environmentally friendly al-ternative, as they are non-toxic and biodegradable; and (4)non-efficient catabolic machinery from native microbial com-munities. An input of hydrocarbon-degrading microorganismsas pure culture or microbial consortium can enhance degrada-tion rates.

Bioremediation costs are commonly lower than physico-chemical treatments and depend largely on the amounts of soilto remediate the degree and depth of pollution and the soiltype (Table 2). In general, costs per soil volume increase whenthe site is small, the pollution is deep, and the soil particles aresmall, i.e., clay/silt soil is more difficult to remediate thansandy soils (US Federal Remediation TechnologiesRoundtable 2014).

Main in situ remediation strategies for hydrocarbon- polluted soils are biostimulation, bioaugmentat ion, and bioventing. Biostimulation involves the enhancement of native microorganisms ’ metabolism by management of en-vironmental factors and nutrients. Bioaugmentation isach ieved by the add i t ion of na t ive or exogenoushydrocarbon-degrading microorganisms, when native mi-crobial communities lack the desired catabolic capabilities.Bioventing is based on air delivery by a network of slotted pipes, either passively or by forced aeration in order toenhance aerobic metabolism.

The most common ex situ bioremediation technologies for oil-polluted soil treatments are biopiles, composting, andlandfarming. The aim is to speed up hydrocarbon degradation by adding low-cost nutrients and oxygen. Biopiles and wind-row composting involve the mixing of polluted soil withorganic material as a bulking agent. This mixture promotesmicrobial activity by improving soil texture, aeration, andmoisture maintenance (Jørgensen et al. 2000). Organic matter can be also a substrate for microbial growth or even a microbial source (Pèrez-Armendáriz et al. 2004). Main difference between both strategies is the aeration methodology.Composts are aerated by turning the soil/bulking agent mix-ture periodically with a modified windrow turner, whereas in biopiles a pipe network delivers air. Biopiles and windrowshave been successfully used for the remediation of a widerange of contaminants (Namkoong et al. 2002; Van Gestel

et al. 2003). Landfarming is based on the controlled spreadingof organic waste on the soil surface to allow native microor-ganisms to aerobically degrade pollutants. Main features of these strategies are summarized in Table 3.

A key aspect to take into account for designing and scalingup a bioremediation technique is the technology cost. Organicmaterial from industrial residues allows to scale up applica-tions with low-associated costs. Moreover, it has the ecologicaladvantage of minimizing organic industrial waste. Some illus-trative examples include orange peel addition, which resultedas an effective strategy to enhance hydrocarbon degradation. Ina pilot scale with high total petroleum hydrocarbon (TPH)concentration (58,000 mg kg

−1), higher degradation rates cor-related with increasing amounts of peel added (Roldán-Martínet al. 2006). The addition of coffee beans on compostingindicated that higher degradation rates were achieved withlower amounts of coffee beans (Roldán-Martín et al. 2007).Increasing coffee proportions may lower pH to a range that isdetrimental for hydrocarbon-degrading microorganisms.Besides being used as carbon source, fruit residues can coun-teract oxidative stress during degradation of aromatic compounds. Improved degradat ion rates by B. xenovoransLB400 was obtained with the addition of berry extract toPCB-contaminated soils (Ponce et al. 2011). Organic materialthat possesses antioxidant properties can increase bacterialtolerance to the toxicity of aromatics and the oxidative stressduring their degradation (Ponce et al. 2011).

Sugarcane bagasse addition improved microbial oil degrada-tion in polluted soils. Increased degradation rates were observedwith both bagasse and soil microorganisms, compared witheither bagasse or soil microorganisms. Thus, biostimulationand bioaugmentation approaches can be contributing to biodeg-radation (Pèrez-Armendáriz et al. 2004). For bioaugmentation, bagasse can also be used as a low-cost non-toxic carbon sourcefor microbial growth. Bacteria and Fungi grown in bagassedegraded phenanthrene in soil, probably due to preadaptationto use aromatic compounds present in lignocellulosic residues.As a rich nutrient source, bagasse allows to exploit synergicinteractions by co-culturing different strains in one step.Different co-culture combinations among four bacterial and four fungal strains resulted in degradation rates ranging between 4.4and >73 % (Chávez-Gómez et al. 2003). These results highlight the fact that not all microbial interactions are synergistic or beneficial. However, this study reveals also the utility of usinglignocellulose residues for inoculum growth instead of conven-tional and usually more expensive carbon sources.

Lessons from an emblematic oil spill

During the supertanker Exxon Valdez oil spill (EVOS) inci-dent in 1989, ~37,000 crude oil tons was released into thePrince William Sound in Alaska (Atlas and Hazen 2011).


https://www.researchgate.net/publication/51244072_Oil_Biodegradation_and_Bioremediation_A_Tale_of_the_Two_Worst_Spills_in_US_History?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/51244072_Oil_Biodegradation_and_Bioremediation_A_Tale_of_the_Two_Worst_Spills_in_US_History?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/51244072_Oil_Biodegradation_and_Bioremediation_A_Tale_of_the_Two_Worst_Spills_in_US_History?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/51244072_Oil_Biodegradation_and_Bioremediation_A_Tale_of_the_Two_Worst_Spills_in_US_History?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==


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surveys due to their key role in degradation, wide phyloge-netic distribution, and high sequence divergence (Iwai et al.2011 ; Wang et al. 2010). These genes usually becomeenriched after hydrocarbon input. Different RHD gene shiftswere observed in response to diverse PAHs. After spiking thesame soil with naphthalene, phenanthrene, or pyrene, nahAc -related genes from Pseudomonas became enriched innaphthalene-polluted soil (Ní Chadhain et al. 2006). During phenanthrene degradation, abundance, diversity, and phylo-genetic identity of enriched RHD genes were soil-type depen-dent (Ding et al. 2010 ). Similar results were observed in Arcticsoils after long-term (i.e., 1 year) diesel pollution. Levels of RHD genes changed significantly in two soil-types during bioreme diation. Ad ditionally, 16S rRNA, alkanemonooxygenase, and nitrate reductase encoding genesshowed different dynamics at both soils (Yergeau et al.2009 ). Similar shifts have been reported in seawater, wherealkB gene copy number increased up to 100-fold in less than1 week after pollution (Sei et al. 2003). Genes encodingenzymes catalyzing downstream reactions seem to behave ina similar way as RHD genes. For example, levels of catechol2,3-dioxygenase xylE gene from (methyl)toluene degradation pathway correlate with degradation rates in hydrocarbon pol-luted soils. A positive correlation between hydrocarbon deg-radation rate and functional alkB, xylE and nahAc genesabundance was observed (Salminen et al. 2008). Therefore,catabolic gene quantification can be an adequate approach for monitoring bioremediation processes.

Bioremediation in two Arctic diesel-polluted soils revealedsome interesting trends. At low temperatures, nutrient amend-ments with nitrogen and phosphorous achieved significant degradation rates. In the northern Alert site, 58 % degradationwas achieved just 1 month after nutrient amendment, and91 %degradation was observed after 1 year. In the southern Eureka site, 47 % of diesel was degraded after 1 month, but thedegradation increased up to only 52 % after 1 year (Yergeauet al. 2009). These dissimilar results can be explained bydifferences in microbial communities at both sites. RHD genesfromGram-negative bacteria were higher in Alert site, whereasRHD from Gram-positive strains were higher in Eureka site.Probably Gram-negative strains responded more efficiently tohydrocarbon pollution. In a previous survey on Arctic andAntarctic soils, alkB genes from Rhodococcus genus weredominant in pristine and hydrocarbon-contaminated soils,whereas alkM genes from Acinetobacter genus were less abun-dant. However, alkB genes from Pseudomonas showed themain increase in response to hydrocarbons (Whyte et al.2002 ). These studies indicated that Gram-negative bacteria seem to play a key role facing hydrocarbon degradation incold soils. Metagenomic assembly of Alert site microbialcommunities showed that Gammaproteobacteria were pre-dominant at higher degradation rates. After 1 year, abundanceof these taxa as well as degradation rates decreased. After

1 month, alkane hydroxylase and catechol 2,3-dioxygenasegenes from Gammaproteobacteria and cytochrome P450genes from Actinobacteria were enriched, whereas protocatechuate 3,4-dioxygenase genes from both taxa co-dominated (Yergeau et al. 2012). Other surveys carried out inAntarctic soils support the role of Gammaproteobacteria inhydrocarbon degradation, especially Pseudomonas strains(Ruberto et al. 2003).

Hydrocarbon degradation at high altitude Alpian coldsoils showed that N – P – K fertilization achieved higher die-sel degradation (Margesin and Schinner 2001). Higher deg-radation occurred at first 78 days during summer. Nonetheless, degradation was less effective during the sec-ond summer even after a second nutrient amendment (Margesin and Schinner 2001). During the first year, pHdecreased ~0.5, whereas after the second year, only a ~0.1 pH reduction was observed. Higher diesel decay was ob-served after the first 3 months. Both acidification and dieselaging may have contributed to lower degradation efficiencyat the second year. Gamma - and Betaproteobacteria are present at higher abundances in polluted soils (Labbéet al. 2007). Additionally, levels of alkB, xylE , and ndoBgenes from Pseudomonas , as well as alkM genes from Acinetobacter , were higher in polluted soils than in pristinesites (Margesin et al. 2003). These results suggest that Proteobacteria , and particularly Gammaproteobacteria , arethe best-adapted phylogenetic group facing hydrocarbon pollution in cold environments. It is worth noting that in both cases, degradation rates were higher during summer.In addition to higher metabolic activity at higher tempera-tures during summer, the reduced bioavailability is maybean overlooked factor contributing to lower degradation ratesin winter. Stable isotope probing (SIP) and metagenomicanalyses also indicated that Proteobacteria dominated intwo long-term aromatic-contaminated soils after additionof biphenyl, naphthalene, or benzoate (Uhlik et al. 2012).

The ecology of a special group of ubiquitous marinehydrocarbon-degrading bacteria offers a model of microbialdynamics after oils spills. Obligate hydrocarbonoclastic bac-teria (OHCB) are an unusual ecological group that plays a keyrole in biodegradation of petroleum hydrocarbons in marineenvironments. OHCB are able to grow on a reduced spectrumof hydrocarbons. In non-polluted marine environments,OHCB are found in low numbers, but after an oil pollutionevent, members of this group undergo a bloom (Atlas andPhilp 2005 ; Yakimov et al. 2004 ). OHCB growth is favored inhydrocarbon-polluted seawater because they can breakdownsubstrates that are useless for most bacteria (Yakimov et al.2007 ). The model OHCB Alcanivorax borkumensis SK2 isable to degrade n-alkanes of chain-length up to C 32 , long-chain isoprenoids, phytane, pristine, and alkyl-aromatic hy-drocarbons and to syn thes ize b iosur fac tan ts andexopolysaccharides probably involved in biofilm formation


https://www.researchgate.net/publication/7030507_Microbial_Dioxygenase_Gene_Population_Shifts_during_Polycyclic_Aromatic_Hydrocarbon_Biodegradation?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7030507_Microbial_Dioxygenase_Gene_Population_Shifts_during_Polycyclic_Aromatic_Hydrocarbon_Biodegradation?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7030507_Microbial_Dioxygenase_Gene_Population_Shifts_during_Polycyclic_Aromatic_Hydrocarbon_Biodegradation?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7188601_Sei_K_Sugimoto_Y_Mori_K_Maki_H_Kohno_T_Monitoring_of_alkane-degrading_bacteria_in_a_sea-water_microcosm_during_crude_oil_degradation_by_polymerase_chain_reaction_based_on_alkane-catabolic_genes_Enviro?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7188601_Sei_K_Sugimoto_Y_Mori_K_Maki_H_Kohno_T_Monitoring_of_alkane-degrading_bacteria_in_a_sea-water_microcosm_during_crude_oil_degradation_by_polymerase_chain_reaction_based_on_alkane-catabolic_genes_Enviro?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7188601_Sei_K_Sugimoto_Y_Mori_K_Maki_H_Kohno_T_Monitoring_of_alkane-degrading_bacteria_in_a_sea-water_microcosm_during_crude_oil_degradation_by_polymerase_chain_reaction_based_on_alkane-catabolic_genes_Enviro?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/221755777_Metagenomic_Analysis_of_the_Bioremediation_of_Diesel-Contaminated_Canadian_High_Arctic_Soils?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/221755777_Metagenomic_Analysis_of_the_Bioremediation_of_Diesel-Contaminated_Canadian_High_Arctic_Soils?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/221755777_Metagenomic_Analysis_of_the_Bioremediation_of_Diesel-Contaminated_Canadian_High_Arctic_Soils?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/223055410_Effectiveness_of_the_Natural_Bacteria_Xora_Biostimulation_and_Bioaugmentation_on_the_Bioremediation_of_a_Hydrocarbon-Contaminated_Antarctic_Soil?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/223055410_Effectiveness_of_the_Natural_Bacteria_Xora_Biostimulation_and_Bioaugmentation_on_the_Bioremediation_of_a_Hydrocarbon-Contaminated_Antarctic_Soil?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/223055410_Effectiveness_of_the_Natural_Bacteria_Xora_Biostimulation_and_Bioaugmentation_on_the_Bioremediation_of_a_Hydrocarbon-Contaminated_Antarctic_Soil?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/10723209_Characterization_of_Hydrocarbon-Degrading_Microbial_Populations_in_Contaminated_and_Pristine_Alpine_Soils?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/10723209_Characterization_of_Hydrocarbon-Degrading_Microbial_Populations_in_Contaminated_and_Pristine_Alpine_Soils?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/10723209_Characterization_of_Hydrocarbon-Degrading_Microbial_Populations_in_Contaminated_and_Pristine_Alpine_Soils?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7030507_Microbial_Dioxygenase_Gene_Population_Shifts_during_Polycyclic_Aromatic_Hydrocarbon_Biodegradation?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/223055410_Effectiveness_of_the_Natural_Bacteria_Xora_Biostimulation_and_Bioaugmentation_on_the_Bioremediation_of_a_Hydrocarbon-Contaminated_Antarctic_Soil?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/7188601_Sei_K_Sugimoto_Y_Mori_K_Maki_H_Kohno_T_Monitoring_of_alkane-degrading_bacteria_in_a_sea-water_microcosm_during_crude_oil_degradation_by_polymerase_chain_reaction_based_on_alkane-catabolic_genes_Enviro?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/10723209_Characterization_of_Hydrocarbon-Degrading_Microbial_Populations_in_Contaminated_and_Pristine_Alpine_Soils?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==https://www.researchgate.net/publication/221755777_Metagenomic_Analysis_of_the_Bioremediation_of_Diesel-Contaminated_Canadian_High_Arctic_Soils?el=1_x_8&enrichId=rgreq-06ea8840-c2c9-49b2-b871-09c6a83c0580&enrichSource=Y292ZXJQYWdlOzI2MTI5MzU3MDtBUzoxNTA3MDAxMzg5NjI5NDRAMTQxMjk0MTEyMTc2Mw==


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(Schneiker et al. 2006). SK2 genome revealed multiple sys-tems for hydrocarbon degradation pathways, i.e., two alkanehydroxylase systems, AlkB1 and AlkB2, and three P450cytochromes. The absence in strain SK2 of genes belongingto glucose breakdown pathways highlights its amazing metabolic specialization (Schneiker et al. 2006).

Concluding remarks and perspectives

Hydrocarbon breakdown in nature seems to be dominated byaerobic processes. Hydroxylation is the main aerobic strategyfor hydrocarbon activation. High diversity of mono- anddioxygenases for alkane hydroxylation has been reported.Aromatic hydrocarbons are oxidized by RHDs, which are lessdiverse than alkane oxygenases. Hydrocarbon degradation pathways expand the microbial metabolic versatility and thecarbon source range for growth. In alkane degradation, suc-cessive oxidations produce carboxylic acids that can be de-graded by the β -oxidation pathway. In PAH degradation,metabolic intermediates are channeled into central aromaticroutes such as catechol, gentisate, and protocatechuate pathways.

In nature, hydrocarbons are mainly present as complexmixtures. Relaxed substrate range enables a given enzyme totransform various structurally related compounds. In addition,same bacterial strains harbor several enzymes, which overlapin substrate range or are expressed in different conditions.This expands the substrate range and provides an additionallevel of robustness and versatility to the system. A conserved panorama making sense with evolution theory has been ob-served: Bacterial shifts occur anytime the selective pressurecomes, where bacteria capable to tolerate and use hydrocarbonas carbon source will be selected. Bioremediation can alsoimpact microbial communities, favoring the growth of select-ed phylogenetic groups.

The genetic and biochemical basis of bacterial degradationof aliphatic and aromatic hydrocarbons has been elucidated inmodel bacteria. The role of biosurfactants on microbial deg-radation of hydrocarbons has been highlighted, pushing fur-ther studies to reveal the structure and the biosynthetic path-ways of novel surfactants.

Culture-dependent methods indicate that three phyla de-grade successful ly hydrocarbons: Proteobacteria , Actinobacteria and Firmicutes . Interestingly, culture-independent environmental surveys tend to confirm that main-ly these bacterial groups respond to oil spills. The character-ization of the microbial communities involved in hydrocarbonremoval by modern approaches such as next-generation se-quencing techniques, PCR, and community fingerprinting or clone libraries, FISH, qPCR, and SIP provides critical knowl-edge on hydrocarbon degradation. Bioremediation is usuallycase-specific; however, some general rules can be pointed out.

Management of environmental conditions such as dissolvedoxygen, pH, temperature, nutrient availability, and water con-tent will increase the bioremediation. The knowledge of thesystem will be the best advisor for choosing the adequate bioremediation strategy. Improved bioremediation strategiesare required for an efficient removal of hydrocarbons fromincreasing polluted environments.

Acknowledgments The authors gratefully acknowledge Conicyt PhD(SF, VM), Mecesup FMS0710 PhD (PA, SF), and Fulbright (SF) fellow-ships. MS acknowledges financial support of FONDECYT (1110992 and1070507) ( http://www.fondecyt.cl ), Conicyt-BMBF, Center for Nano-technology and Systems Biology ( http://www.usm.cl ), and USM(131342, 131109, 130948) ( http://www.usm.cl ) grants. The funders hadno role in study design, data collection and analyses, decision to publish,or preparation of the manuscript.

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McDonald IR, Miguez CB, Rogge G, B

2014_bioremediation of petroleum hydrocarbons catabolic genes, microbial communities, and...

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