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Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 80:723736 (2005)DOI: 10.1002/jctb.1276

ReviewBioremediation of polycyclic aromatichydrocarbons: current knowledge andfuture directionsSelina M Bamforth1 and Ian Singleton21School of Civil Engineering and Geosciences, Drummond Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU,UK2School of Biology, Institute for Research on the Environment and Sustainability, Devonshire Building, University of Newcastle upon Tyne,Newcastle upon Tyne, NE1 7RU, UK

Abstract: Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds that haveaccumulated in the natural environment mainly as a result of anthropogenic activities such as thecombustion of fossil fuels. Interest has surrounded the occurrence and distribution of PAHs for manydecades due to their potentially harmful effects to human health. This concern has prompted researchers toaddress ways to detoxify/remove these organic compounds from the natural environment. Bioremediationis one approach that has been used to remediate contaminated land and waters, and promotes the naturalattenuation of the contaminants using the in situ microbial community of the site. This review discussesthe variety of fungi and bacteria that are capable of these transformations, describes the major aerobicand anaerobic breakdown pathways, and highlights some of the bioremediation technologies that arecurrently available. 2005 Society of Chemical Industry

Keywords: polycyclic aromatic hydrocarbons (PAHs); bioremediation; bacteria; fungi; soil; environment

INTRODUCTIONPolycyclic aromatic hydrocarbon contaminationin the environmentSources and occurrence of polycyclic aromatichydrocarbons (PAHs)Polycyclic aromatic hydrocarbons (PAHs) are a classof organic compounds that consist of two or morefused benzene rings and/or pentacyclic molecules thatare arranged in various structural configurations. Theyare highly recalcitrant molecules that can persist inthe environment due to their hydrophobicity andlow water solubility.1 Some representative PAHs areshown in Fig 1.

PAHs are ubiquitous in the natural environment,and originate from two main sources: these are natural(biogenic and geochemical) and anthropogenic.2 It isthe latter source of PAHs that is the major cause ofenvironmental pollution and hence the focus of manybioremediation programmes. PAHs naturally occur infossil fuels such as coal and petroleum, but are alsoformed during the incomplete combustion of organicmaterials such as coal, diesel, wood and vegetation.3,4

This results in airborne PAH contamination, which isthe main route for PAH transport over long distances.5

Point sources of PAHs can originate from petroleumand diesel spills and from industrial processes suchas coal liquefaction and gasification during cokeproduction.6 For example, creosotes and coal tar,which are by-products of coking, contain significantquantities of PAHs (eg creosote contains up to 85%PAHs2). More minor sources of PAHs include tobaccosmoke and burnt food.

Natural processes can also provide a source ofPAHs, such as volcanic eruptions and forest fires.7

In addition, PAHs can have a geochemical origin asthey are formed during pyrolysis, which involves theexposure of sediments to high temperatures duringsediment diagenesis.7

PAHs are widely distributed in soils and sediments,groundwater and the atmosphere. They have beendetected in marine sediments such as San Diego Bay,California8,9 and the Central Pacific ocean,10 intertidalsediments,11 gas works site soils,12,13 sewage sludge-contaminated soils,14 aquifers and groundwater and inatmospheric deposits such as vehicle exhaust fumes.4

Point sources of PAH contamination are themost significant environmental concern. Though theareas contaminated are relatively small in size, the

Correspondence to: Selina M Bamforth, School of Civil Engineering and Geosciences, Drummond Building, University of Newcastle uponTyne, Newcastle upon Tyne, NE1 7RU, UKE-mail: [email protected](Received 19 February 2004; revised version received 29 October 2004; accepted 19 January 2005)Published online 22 April 2005

2005 Society of Chemical Industry. J Chem Technol Biotechnol 02682575/2005/$30.00 723

SM Bamforth, I Singleton

Figure 1. Chemical structures and physical characteristics of somerepresentative polycyclic aromatic hydrocarbons (PAHs).

contaminant concentration at these sites is oftenhigh and associated with co-contaminants suchas benzene, toluene, ethylene and xylene (BTEX)compounds, heavy metals and aliphatic hydrocarbons,which can hinder remediation efforts. Soils can becontaminated with between 1 g kg1 and 300 g kg1PAHs,15 depending on the source of contamination(eg old coal gasification sites have the higher levelsstated). Atmospheric levels of PAHs resulting fromthe incomplete combustion of materials such as coaland wood have been found to be between 60 g m3and 3 mg m3 air.3

Persistence of PAHs in the environmentThe persistence of PAHs in the environment isdependent on a variety of factors, such as the chemicalstructure of the PAH, the concentration and dispersionof the PAH and the bioavailability of the contaminant.In addition, environmental factors such as soil typeand structure, pH and temperature and the presenceof adequate levels of oxygen, nutrients and waterfor the activity of the pollutant-degrading microbialcommunity will control the time that PAHs persistin the environment16 (see later, Factors affecting thebioremediation of PAHs).

In general, the higher the molecular weight ofthe PAH molecule, the higher the hydrophobicityand toxicity, and the longer the environmentalpersistence of the molecule.1 In addition the ageof the contaminant in the soil/sediment matrix playsa significant role in the biodegradability of PAHsin soil.17 A study using phenanthrene as a modelPAH showed that phenanthrene mineralisation andtherefore biodegradability was significantly reducedwith time of ageing.17

The association of PAHs with co-pollutants such ashydrocarbons and heavy metals is another factor thatcan prolong their residence time in the environment.Aliphatic hydrocarbons and BTEX compounds arereadily biodegradable by the in situ microbial commu-nity relative to the more complex chemical structuresof the PAHs. This results in the depletion of avail-able oxygen in the surrounding environment and theonset of anaerobicity. Though recent work has shownthat there is a real potential for the biodegradationof PAHs in the absence of molecular oxygen (seeAnaerobic metabolism of PAHs), details regardingthe efficiency and scale of PAH degradation in anaero-bic environments is still limited, with rates of anaerobicorganic matter oxidation up to an order of magni-tude less than those under aerobic conditions.18 Inaddition, it is possible that the presence of heavy met-als in soil could inhibit microbial growth and hencelimit the metabolism of contaminants under anaerobicconditions.

Toxicity of PAHsIt has long been known that PAHs can haveserious deleterious affects to human health,6 withthe physician John Hill first recognising the linkbetween the use of snuff and nasal cancer in 1761.6

Following this discovery, research into the toxiceffects that PAHs have upon mammalian healthhas continued, with many PAHs displaying acutecarcinogenic, mutagenic and teratogenic properties.Benzo[a]pyrene is recognised as a priority pollutantby the US Environmental Protection Agency19 as thiscompound is known to be one of the most potentlycarcinogenic of all known PAHs.5

When ingested, PAHs are rapidly absorbed into thegastrointestinal tract due to their high lipid solubility.6

A major route of PAH uptake is via dermal absorptionas highlighted by a study of 12 coke-oven workers.20

An estimated 75% of the total absorbed amount ofPAHs (specifically pyrene) entered the body throughthe skin, highlighting this as a major exposure routeof PAHs. The rapid absorption of PAHs by humansresults in a high potential for biomagnification in thefood chain. In general, the greater the number ofbenzene rings, the greater the toxicity of the PAH.1

The relative toxicity of PAHs can be measured usingLD50 values (the lethal dose in 50% of cases). Theseare expressed as milligrams of toxic material perkilogram of the subjects body weight that will causedeath in 50% of cases. It is important to specify theroute by which the toxic material was administered tothe test animal (such as oral or intraperitoneal), andthe animal upon which the toxic material was tested(ie rat, mouse). See Table 1 for the LD50 values ofsome representative PAHs.

PAHs are also suspected carcinogens but arenot thought to be genotoxic unless they are acti-vated by mammalian enzymes to reactive epox-ides and quinones. This occurs via a cytochromeP450 monooxygenase enzyme-mediated reaction that

724 J Chem Technol Biotechnol 80:723736 (2005)

Bioremediation of polycyclic aromatic hydrocarbons

Table 1. LD50 values of some representative PAHs

Material Number of carbon rings LD50 value (mg kg1) Test subject Exposure route

Naphthalene 2 533710 Male/female mice respectively OralPhenanthrene 3 750 Mice OralAnthracene 3 >430 Mice IntraperitonealFluoranthene 4 100 Mice IntravenousPyrene 4 514 Mice IntraperitonealBenzo[a]pyrene 5 232 Mice Intraperitoneal

Generally, toxicity increases with an increase in number of benzene rings, but data should be examined using careful consideration of the exposureroute, etc (data taken from the Risk Assessment Information System (RAIS) http://risk.lsd.ornl.gov).

oxidises the aromatic ring to form epoxide anddiolepoxide reactive intermediates. It is reported thatthese intermediates may undergo one of at least fourdifferent mechanisms of oxidation and/or hydrolysisbefore the intermediates combine with and/or attackDNA to form covalent adducts with DNA. DNAadducts can lead to mutations of the DNA, resultingin tumours.21

The solutionbioremediation?Bioremediation, which is also referred to as biorecla-mation and biorestoration, can be described asthe process whereby organic wastes are biologicallydegraded under controlled conditions to an innocu-ous state.22 The main principle of this techniqueis to remove pollutants from the natural environ-ment and/or convert the pollutants to a less harmfulproduct using the indigenous microbiological com-munity of the contaminated environment. Biore-mediation strategies are developed to promote themicrobial metabolism of contaminants, by adjustingthe water, air and nutrient supply. This is accom-plished by the biostimulation (the addition of abulking agent such as wood chips and/or nutri-ents such as N/P/K) and bioaugmentation (often aninoculum of microorganisms with known pollutanttransformation abilities) of the contaminated environ-ment.

Bioremediation of PAH-contaminated soils, sedi-ments, and water can be accomplished in a vari-ety of ways, eg in situ treatment or ex-situ methodssuch as bio-piling and composting. Specific detailsof bioremediation in relation to PAH treatment arecovered later, see Approaches to the bioremediationof PAH-contaminated environments. Waste can alsobe treated in bioreactors, though this can be morecostly than in situ technologies. It is important forbioremediation to be comparable in cost and successto physical and chemical treatments of contaminatedland, such as landfilling, incineration and soil washing.The applicability of bioremediation can be variable,but this is generally due to unfavourable site con-ditions (see Factors affecting the bioremediation ofPAHs), therefore a thorough understanding of siteconditions will allow optimisation of bioremediationand subsequently more effective results. In commer-cial situations bioremediation of PAH-contaminated

soils is not typically carried out when the site con-tains significant amounts of PAHs that have morethan four rings as the low percentage removal ofPAHs of this molecular weight and the time takenfor successful reduction in PAH concentrations isnot economically viable (Bio-Logic, personal com-munication). The method used is normally nutrientaddition (see Nutrient availability) and aeration byfrequent turning of contaminated soil. Total PAH lev-els during a bioremediation trial are generally reducedfrom approximately 3000mg to 1000 mg total PAHs,per kg.

MICROBIAL METABOLISM OF PAHSThere are three fundamentally different mechanismsin the aerobic metabolism of PAHs by microorganisms(Fig 2) and specific details of bacterial and fungal(ligninolytic and non-ligninolytic) PAH metabolismare discussed below. The basis of these mechanismsis the oxidation of the aromatic ring, followedby the systematic breakdown of the compound toPAH metabolites and/or carbon dioxide. Anaerobicmetabolism of PAHs is thought to occur via thehydrogenation of the aromatic ring with details of theseprocesses given in the section Anaerobic metabolismof PAHs.

PAH-degrading microorganisms are ubiquitouslydistributed in the natural environment, such asin soils (bacteria and non-ligninolytic fungi) andwoody materials (ligninolytic fungi). Many PAH-contaminated soils and sediments host active pop-ulations of PAH-degrading bacteria. For example,phenanthrene-degrading bacteria were isolated fromPAH-contaminated mangrove sediments in HongKong.11 These isolates were able to degrade phenan-threne under a range of salinities both in pure andmixed cultures. Anaerobic environments, eg munici-pal sewage sludges23 and marine sediments,9 can alsohost a diverse array of PAH-degrading bacteria. Unlikenon-ligninolytic fungi, the ligninolytic fungi, such asPhanerochaete chrysosporium, are commonly associatedwith woody materials and are not commonly found insoils. However, these fungi can be enriched in a soilby the addition of straw, wood chips and other lignin-rich substrates. A thorough listing of microorganismscapable of PAH degradation is provided by Muelleret al.2

J Chem Technol Biotechnol 80:723736 (2005) 725


PAH

PAH-QUINONES

CO2

BACTERIAL DEGRADATION Hydroxylation of ring via dioxygenase

enzymes

LIGNOLYTIC FUNGAL DEGRADATIONOxidation of ring by lignin and manganese peroxidase

enzymes

cis-DIHYDRODIOL

CATECHOL

cis, cis-MUCONIC ACID

2-HYDROXYMUCONIC SEMIALDEHYDE

Ring fission

NON-LIGNOLYTIC FUNGAL AND BACTERIAL DEGRADATION

Oxidation of ring via cytochrome P450monoxygenase enzymes

ARENE OXIDE

trans-DIHYDRODIOL

PHENOL

O-GLUCOSIDEO-GLUCURONIDEO-SULFATE

O-XYLOSIDE

O-METHYL

Nonenzymatic rearrangement

Epoxide hydrolasecatalysed reaction

CO2

Dehydrogenase enzymes

Orthofission

Metafission

H

OHH

R

OH

R

COOHCOOH

ROH

CHOCOOHR

H H

R

O

HOH OH

H

R

R

OH

R

OH

OH

Figure 2. The three main pathways for polycyclic aromatic hydrocarbon degradation by fungi and bacteria.1.

Bacterial metabolism of PAHsThe principal mechanism for the aerobic bacterialmetabolism of PAHs is the initial oxidation of thebenzene ring by the action of dioxygenase enzymesto form cis-dihydrodiols. These dihydrodiols aredehydrogenated to form dihydroxylated intermediates,which can then be further metabolised via catecholsto carbon dioxide and water. The metabolic pathwaysand enzymatic reactions involved in the microbialdegradation of naphthalene have been studied indetail with an example pathway of naphthalenetransformation given in Fig 3.

There is a large diversity of bacteria that are ableto oxidise naphthalene using dioxygenase enzymes,including organisms from the genus Pseudomonas andRhodococcus (see Refs 1 and 2 for a full listing). Afew bacteria are also capable of oxidising PAHs by theaction of the cytochrome P450 monoxygenase enzymeto form trans-dihydrodiols such as Mycobacterium sp.24

Rockne and colleagues reported the ability of marinemethanotrophs in degrading PAHs via the actionof the methane monoxygenase gene.25 It is thoughthowever that these are minor mechanisms comparedwith the activity of the dioxygenase enzymes.26 Themechanisms involved in this pathway are detailed inthe next section.

The toxicity of naphthalene metabolites gener-ated during bacterial degradation has been littlestudied. The metabolites of naphthalene, such as

naphthalene dihydrodiols, have a higher water sol-ubility than naphthalene and are therefore poten-tially more bioavailable, and could pose a greatertoxicity than the naphthalene precursor. Naph-thalene 1,2 dihydrodiols show minimal toxicityto human liver cells relative to control sam-ples, whereas the metabolites of 1-naphthol, 1,2-naphthoquinone and 1,4-naphthoquinone, generatedduring the human cytochrome P450-mediated oxi-dation reactions, showed a significant toxicity tohuman liver cells and mononuclear leucocytes.27

These metabolites were considerably more toxicthan the naphthalene precursor.27 In comparison tothe naphthalene metabolites produced by humansand some fungi, naphthalene intermediates gener-ated during the cytochrome P450-mediated oxidationby Mycobacterium sp generate trans-dihydrodiols thatcould reasonably be expected to show minimal toxicityto humans.

Fungal metabolism of PAHsThere are two main types of fungal metabolism ofPAHs; these are mediated by the non-ligninolyticand ligninolytic fungi (also known as the white-rotfungi). The majority of fungi are non-ligninolytic,as they do not grow on wood, and thereforehave no need for the lignin peroxidase enzymesthat are produced by the ligninolytic fungi. How-ever, many ligninolytic fungi such as Phanerochaete



Figure 3. The main pathways in the aerobic degradation of naphthalene by bacteria.85.

chrysosporium28 and Pleurotus ostreatus29 can pro-duce both non-ligninolytic and ligninolytic typeenzymes, but it is unclear to what degree eachenzyme contributes to the breakdown of the PAHmolecule.

Non-ligninolytic fungiThe first step in the metabolism of PAHs by non-ligninolytic fungi is to oxidise the aromatic ringin a cytochrome P450 monoxygenase enzyme catal-ysed reaction to produce an arene oxide.16 Thisroute is similar to the mammalian metabolism ofPAHs. In comparison to the oxidation of the aro-matic ring by dioxygenase enzymes to form cis-dihydrodiols, the monoxygenase enzyme incorporatesonly one oxygen atom onto the ring to form anarene oxide. This is subsequently hydrated via anepoxide-hydrolase catalysed reaction to form a trans-dihydrodiol.30 In addition, phenol derivatives may beproduced from arene oxides by the non-enzymaticrearrangement of the compound, which can act assubstrates for subsequent sulfation or methylation, orconjugation with glucose, xylose, or glucuronic acid.2

Although most non-ligninolytic fungi are not capa-ble of the complete mineralisation of PAHs, thesePAH-conjugates are generally less toxic and more sol-uble than their respective parent compounds. Forexample, Pothuluri and colleagues31 demonstratedthis with the degradation of fluoranthene by the

non-ligninolytic fungal species Cunninghamella elegans.The metabolites 3-fluoranthene--glucopyranoside,3-(8-hydroxy-fluoranthene)--glucopyranoside, fluo-ranthene trans-2,3-dihydrodiol and 8-hydroxy-fluo-ranthene-trans-2,3-dihydrodiol showed no mutageniceffects to a rat liver homogenate fraction, and 9-hydroxy-fluoranthene-trans-2,3-dihydrodiol was con-siderably less toxic than fluoranthene.Chrysosporium pannorum, Cunninghamella elegans

and Aspergillus niger are examples of non-ligninolyticfungi that use a P450 monoxygenase enzyme-mediatedoxidative pathway for PAH degradation. An examplepathway of the cytochrome P450-mediated oxidationof phenanthrene is detailed in steps 1 to 4 of Fig 4.The latter steps (5 and 6) are thought to be mediatedby lignin peroxidase enzymes29 (detailed in the nextsection).

Ligninolytic fungiWhite-rot fungi are a group of fungi that produceligninolytic enzymes involved in the oxidation oflignin present in wood and other organic matter.There are two types of ligninolytic enzymes; thesebeing peroxidases and laccases.32 These enzymes aresecreted extracellularly, and oxidise organic mattervia a non-specific radical based reaction.33 There aretwo main types of peroxidase enzyme depending ontheir reducing substrate type, lignin peroxidase (LP)and manganese peroxidase (MnP), both of which arecapable of oxidising PAHs.32 Laccases, which are



Figure 4. Proposed pathway for the degradation of phenanthrene by the ligninolytic fungus Pleurotus ostreatus.29.

phenol oxidase enzymes, are also capable of oxidisingPAHs.

Under ligninolytic conditions, white-rot fungi canoxidise PAHs by generating free radicals (ie hydroxylfree radicals) by the donation of one electron,16 whichoxidises the PAH ring. This generates a selectionof PAH-quinones and acids rather than dihydrodiols(see steps 5 and 6, Fig 4). There is significantinterest surrounding the use of ligninolytic fungi todegrade PAHs, as they have low substrate specificityand are therefore able to degrade even the mostrecalcitrant of compounds. Also, the enzymes involvedare extracellular, and are theoretically able to diffuseinto the soil/sediment matrix and potentially oxidisePAHs with low bioavailability (see Bioavailabilitysection).

Degradation studies of the ligninolytic fungihave shown that PAHs may be degraded by acombination of ligninolytic enzymes, cytochromeP450 monooxygenases, and epoxide hydrolases thatcan result in the complete mineralisation of thecompound.29 Degradation studies of high molecularweight PAHs such as pyrene and benzo[a]pyrene byligninolytic fungi (ie Phanerochaete chrysosporium andPleurotus ostreatus) have suggested that a combinationof ligninolytic and non-ligninolytic enzymes may be thekey to the complete mineralisation of these recalcitrantcompounds.29

Substantial research has focused upon the potentialof this group of fungi to remediate PAH-contaminatedmaterials. Bioremediation trials that used ligninolyticfungi to remediate PAH-contaminated soils andsediments have shown mixed results. For example,Canet and colleagues34 used four white-rot fungispecies to degrade a coal-tar-contaminated soil.Although the soil in this study was supplemented with

straw (as a substrate for the fungi), the indigenoussoil microorganisms were more successful in PAH-degradation than the introduced fungal species. Inanother study that monitored the potential of white-rotand brown-rot fungi to degrade a PAH-contaminatedsoil, Pleurotus ostreatus and Antrodia vaillantii wereused to inoculate an artificially-contaminated soil todegrade a range of PAHs.35 The P ostreatus fungalinoculum significantly increased the degradationof PAHs relative to the un-amended soils, butresulted in the accumulation of potentially toxic PAHmetabolites. As this white-rot fungus also inhibitedthe in situ microbial populations within the soil,this may have prevented the complete mineralisationof the PAHs, resulting in the accumulation ofPAH metabolites. The authors suggest that afungalbacterial consortium would be beneficial tothe decontamination of this soil. As the brown-rotfungus A vaillantii showed equal if not better PAHdegradation than P ostreatus, and did not generatedead-end PAH metabolites, it was suggested that thisfungus could be exploited as a valuable inoculum inbioremediation trials.

In order to increase the efficiency of white-rotfungi in the remediation of PAH-contaminated soil,May and colleagues36 designed a two-stage pilot-scale reactor that initially extracted PAHs froma contaminated soil and subsequently treated theextracted PAHs in a fungal bioreactor. This fungalbioreactor utilised P chrysosporium, and was successfulin degrading high molecular weight PAHs such asbenzo[a]pyrene.

Anaerobic metabolism of PAHsPAHs are a common contaminant of anaerobicenvironments such as aquifers3739 and marine



sediments.810,40 Even aerobic environments suchas contaminated soils, sediments and groundwatercan develop anaerobic zones.41 This is due to theorganic contaminant stimulating the in situ microbialcommunity, resulting in the depletion of molecularoxygen during aerobic respiration. This oxygen is notreplenished at the same rate as its depletion, whichresults in the formation of anaerobic zones proximalto the contaminant source.

It was not until recently that the potential ofmicroorganisms to degrade PAHs in the absence ofmolecular oxygen has been recognised. Previous stud-ies have tended to focus upon the thermodynamicallymore favourable aerobic processes of bioremediationof recalcitrant organic compounds such as PAHs,whereby molecular oxygen is incorporated into thearomatic ring prior to the dehydrogenation and sub-sequent PAH ring cleavage (see earlier for details ofthe mechanisms of aerobic degradation of PAHs).In the absence of molecular oxygen, alternative elec-tron acceptors such as nitrate, ferrous iron and sulfateare necessary to oxidise these aromatic compounds,with recent research clearly demonstrating that PAHdegradation will occur under both denitrifying18,42 andsulfate-reducing8,9,39,43 anaerobic conditions.

The mechanisms of anaerobic PAH degradation arestill tentative, though recent studies have proposeda mechanism for the anaerobic degradation ofnaphthalene,39,43 which is summarised in Fig 5. Thefirst step is the carboxylation of the aromatic ring to

Figure 5. Simplified proposed pathway for the anaerobic metabolismof naphthalene under sulfate-reducing conditions.39,43.

2-naphthoic acid, which may activate the aromatic ringprior to hydrolysis. Stepwise reduction of 2-naphthoicacid via a series of hydrogenation reactions resultsin decaclin-2-carboxylic acid which is subsequentlyconverted to decahydro-2-naphthoic acid. There maybe other mechanisms for anaerobic naphthalenedegradation, however these have not yet beenelucidated. For example, it is proposed that the initialstep in anaerobic naphthalene degradation undersulfate-reducing conditions occurs via a hydroxylationreaction to form a naphthol intermediate.44

FACTORS AFFECTING THE BIOREMEDIATIONOF PAHSThere are many examples of the successful applicationof bioremediation technologies to contaminated sitesusing approaches such as biopiling and composting.Many published studies have investigated the efficacyof bioremediation on a bench scale and under ideallaboratory conditions, such as a circum-neutral pHand mesophilic temperatures. However, it is apparentthat environmental factors that vary from site tosite (such as soil pH, nutrient availability and thebioavailability of the contaminant) can influence theprocess of bioremediation by inhibiting growth ofthe pollutant-degrading microorganisms. The mainenvironmental factors that could affect the feasibilityof bioremediation are summarised in the following fivesections, whilst the final section addresses the concernof the toxicity of the metabolites formed during thebiodegradation of PAHs.

TemperatureTemperature has a considerable effect on the abilityof the in situ microorganisms to degrade PAHs and,in general, most contaminated sites will not be atthe optimum temperature for bioremediation duringevery season of the year. The solubility of PAHsincreases with an increase in temperature,45 whichincreases the bioavailability of the PAH molecules. Inaddition, oxygen solubility decreases with increasingtemperature, which will reduce the metabolic activityof aerobic microorganisms.

Biodegradation of PAHs can occur over a widetemperature range, however most studies tendto focus on mesophilic temperatures rather thanthe efficiency of transformations at very low orhigh temperatures. However, it is apparent thatmicroorganisms have adapted to metabolise PAHsat extreme temperatures, for example naphthaleneand phenanthrene degradation was reported fromcrude oil in seawater at temperatures as low as0 C.46 In comparison, the laccase and manganeseperoxidase enzymes of ligninolytic fungi were reportedto have a temperature optimum of 50 C and>75 C respectively in spent-mushroom compostduring the degradation of PAHs,47 with over 90%degradation of the contaminating PAHs occurring atthese temperatures.



pHMany sites contaminated with PAHs are not at theoptimal pH for bioremediation. For example, retiredgasworks sites often contain significant quantities ofdemolition waste such as concrete and brick. Leachingof this material will increase the pH of the nativesoil and/or made ground of the site, resulting in lessfavourable conditions for microbial metabolism. Inaddition, the oxidation and leaching of coal spoilwill create an acidic environment by the release andoxidation of sulfides. As the pH of contaminated sitescan often be linked to the pollutant, the indigenousmicroorganisms at the sites will not have the capacityto transform PAHs under acidic or alkaline conditions.Therefore, it is common practice to adjust the pH atthese sites, for example by the addition of lime.48

Phenanthrene degradation in liquid culture has beeninvestigated at a range of pH values (pH 5.57.5) withBurkholderia cocovenenas, an organism isolated froma petroleum-contaminated soil.49 Although bacterialgrowth was not significantly affected by the pH,phenanthrene removal was only 40% at pH 5.5after 16 days, whereas at circum-neutral pH values,phenanthrene removal was 80%. Sphingomonaspaucimobilis (strain BA 2) was however more sensitiveto the pH of growth media, with the degradation ofthe PAHs phenanthrene and anthracene significantlyinhibited at pH 5.2 relative to pH 7.50

PAH degradation has been recorded in an acidicsoil (pH 2) contaminated by coal spoil by theindigenous microorganisms, with the concentrationsof naphthalene, phenanthrene and anthracene reducedover a 28-day period.51 Naphthalene concentrationswere reduced by 50% in soils downstream of anearby coal pile, with phenanthrene and anthracenereduced by between 10 and 20%. The authorsshowed that a consortium of fungi and bacteriaaccomplished this, and suggest the presence andactivity of PAH-degrading acidophilic bacteria. Theseresults suggest that future research would benefitfrom the isolation and characterisation of PAH-degrading microorganisms from both acidic andalkaline environments.

Recent unpublished research from the authorslaboratory, which investigated the presence, activ-ity and diversity of Pseudomonas species from aPAH-contaminated concrete highlighted the possi-bility that microorganisms isolated from alkalophilicenvironments may be able to degrade PAHs atan elevated pH.52 The potential of these Pseu-domonas to degrade naphthalene in liquid culture wascompared with a selection of characterised PAH-degrading Pseudomonas species. It was found thatsome of the environmental isolates were able toboth reduce the pH of the liquid media from 9 to6.5 within 24 h, and also utilise naphthalene as asole source of carbon. In contrast, the naphthalene-degrading microorganismsPseudomonas fredrikbergensis(DSM 13 022) and Pseudomonas fluorescens (DSM6506), were severely inhibited by the elevated pH.

This suggests that the in situ microorganisms ata contaminated site may be not only tolerant ofthe site conditions, but may have the potential tometabolise PAHs in sub-optimal conditions (in thiscase, high pH).

OxygenThough it is now well established that bioremediationof organic contaminants such as PAHs can proceedunder both aerobic and anaerobic conditions (seeearlier section, Anaerobic metabolism of PAHs),most work has tended to concentrate upon thedynamics of aerobic metabolism of PAHs. This isin part due to the ease of study and culture of aerobicmicroorganisms relative to anaerobic microorganisms.During aerobic PAH metabolism, oxygen is integralto the action of mono- and dioxygenase enzymes inthe initial oxidation of the aromatic ring.53 Waysin which to maintain adequate oxygen levels foraerobic metabolism for in situ treatments are discussedbelow and include hydrogen peroxide for sub-surfacecontamination. For surface contamination, simple soiltilling and/or mixing, for example using compostturners, can aerate contaminated material well enoughto allow PAH transformation to proceed.

There is still debate as to whether the benefitsof anaerobic bioremediation are outweighed by thenegatives, with the aeration of contaminated anaer-obic aquifers successfully used to stimulate aerobicmicrobial communities resulting in significant reduc-tions in PAH concentrations in groundwater. Thishas been accomplished using hydrogen peroxide,54

sodium nitrate38 and perchlorate.55 In addition, theaerobic biodegradation of hydrocarbons has beenreported to be up to an order of magnitude higher rel-ative to anaerobic biodegradation.18 However, it hasalso been reported that rates of anaerobic PAH degra-dation under denitrifying conditions were comparableto those under aerobic conditions.56

Though it appears that the future of anaerobicbioremediation is promising, there are several draw-backs to the promotion of anaerobic bioremediation.Not all environments contain an active populationof anaerobes that are able to degrade PAHs. Thishas been shown in a creosote-contaminated sedi-ment, where limited biodegradation of PAHs was seenunder denitrifying, sulfate-reducing and methanogenicconditions,40 even though there was an actively respir-ing anaerobic community present in the sediment.Similar results were found when investigating thepotential for PAH degradation in sediment samplesfrom San Diego Bay, California.9 Metabolism of PAHsin these sediments that had had low levels of previ-ous exposure to PAHs only occurred after a long lagperiod, and was promoted when they were spikedwith PAH-contaminated sediments that contained anactive community of PAH-degraders. This suggeststhat the dominant in situ microbial community didnot consist of PAH-degrading microorganisms andthat bioremediation was limited by low numbers of



PAH-degrading microorganisms rather than adverseenvironmental conditions.9

Another potential disadvantage of the promotionof in situ anaerobic bioremediation is that thegeochemistry of the subsurface will be altered by theimposition of reducing conditions. As an environmentis driven anaerobic, all residual oxygen is depleted,and electron acceptors such as nitrate, ferric iron andsulfate are reduced during respiration.57 This resultsin the mobilisation of ferrous iron, and thereforethe release of phosphate from iron(III)phosphatecomplexes. Both of these are toxic to the environment;iron(II) is rapidly oxidised when exposed to oxygen,causing an orange precipitate in freshwater frequentlyassociated with acid mine drainage58 and excessphosphate in freshwaters can cause eutrophication.In addition, there is often a concomitant increase inpH, which can result in the solubilisation of carbonateminerals and the release of trace metals.59 Respirationwill also produce potentially potent greenhouse gasessuch as H2S, CH4 and N2O.60

It is clear that more research is needed to fullyunderstand the implications associated with thepromotion of anaerobic bioremediation. The discoveryof a wide diversity of pollutant-transforming anaerobesis a significant step forward in understanding theprocesses involved in bioremediation, and the designand application of anaerobic remediation both in situand ex situ to the contaminated site.

Nutrient availabilityIn addition to a readily degradable carbon source,microorganisms require mineral nutrients such asnitrogen, phosphate and potassium (N, P and K)for cellular metabolism and therefore successfulgrowth. In contaminated sites, where organic carbonlevels are often high due to the nature of thepollutant, available nutrients can become rapidlydepleted during microbial metabolism.61 Thereforeit is common practice to supplement contaminatedland with nutrients, generally nitrogen and phosphatesto stimulate the in situ microbial community andtherefore enhance bioremediation.62,63

The amounts of N and P required for optimalmicrobial growth and hence bioremediation havebeen previously estimated from the ratio of C:N:Pin microbial biomass (between 100:15:364 and120:10:165). However, a recent study has shown thatoptimal microbial growth and creosote biodegradationoccurred in soil with a much higher C:N ratio(25:1) than those predicted from the ratio inmicrobial biomass, with lower C:N ratios (5:1) causingno enhancement in microbial growth.63 The levelof nutrients required for PAH transformation aregenerally thought to be similar to those requiredfor other organic pollutants such as petroleumcompounds. However, little work has been doneregarding the most favourable nutrient levels requiredfor the optimal degradation of PAHs, and further workin this area would benefit future bioremediation trials.

It is worth noting that fungi are able to effectivelyrecycle nutrients (specifically nitrogen), and thatexcessively high nutrient loadings may in fact inhibitmicrobial metabolism. In addition, the high molecularweight PAH-oxidising ligninolytic enzymes of thewhite-rot fungi are produced under nutrient deficient(often low nitrogen) conditions.66

It therefore appears imperative that the nutrientstatus of the site is established prior to the sup-plementation of the site with additional nutrients.Even though microbial metabolism may be temporar-ily increased, the long-term inhibition of functionallyimportant organisms may result in the failure of thebioremediation of high molecular weight PAHs (suchas benzo[a]pyrene).

BioavailabilityBioavailability can be defined as the effect of physico-chemical and microbiological factors on the rate andextent of biodegradation2 and is believed to be oneof the most important factors in bioremediation. PAHcompounds have a low bioavailability, and are classedas hydrophobic organic contaminants.67 These arechemicals with low water solubility that are resistantto biological, chemical and photolytic breakdown.67

The larger the molecular weight of the PAH, thelower its solubility (see Fig 1), which in turn reducesthe accessibility of the PAH for metabolism by themicrobial cell.68,69 In addition, PAHs can undergorapid sorption to mineral surfaces (ie clays) andorganic matter (ie humic and fulvic acids) in the soilmatrix. The longer that the PAH is in contact withsoil, the more irreversible the sorption, and the loweris the chemical and biological extractability of thecontaminant.17 This phenomenon is known as ageingof the contaminant. Therefore the bioavailability ofa pollutant is linked to its persistence in a givenenvironment.

Release of PAHs from the surface of mineralsand organic matter can be achieved by the use ofsurface-active agents (also known as surfactants ordetergents). These are compounds that contain both ahydrophobic and hydrophilic moiety, thus providing abridge between the hydrophobic PAH molecule andthe hydrophilic microbial cell. Some microorganismscan produce surfactants (biosurfactants), that canenhance the desorption of PAHs from the soilmatrix.70,71 These are potentially more effective thanusing synthetic surfactants, as they are thought to beless toxic to the in situmicrobial community and do notproduce micelles, which can encapsulate contaminantPAHs and prevent microbial access.71

The bioavailability of PAHs in soil can be assessedusing both chemical and biological methods, thoughit is questionable which type of test(s) are mostrepresentative of the bioavailability of hydrophobicorganic contaminants in soil, as both approacheshave inherent limitations.72 There are many biologicaltechniques to assess the bioavailability of PAHs insoil. These can be based upon the monitoring of



biological function, such as microbial respirationrates (mineralisation) of 14C-labelled contaminants,the bioluminescence of microorganisms such as luxmicroorganisms and/or lux-tagged pollutants thatare in contact with a contaminated material73 andby assessing the degree of dermal diffusion andgastrointestinal sorption of PAHs in earthworms andhence the bioavailability and ecotoxicity of PAHs inthe soil.74 In addition, bioavailability can be measuredby monitoring changes in the expression of genes thatcode for PAH degradation using molecular probes,51

and also by the extraction of soil pollutants using asimulated mouth and gut digestive fluid such as saliva,in order to demonstrate the risk that the contaminantwill pose if ingested.75

Biological assays are often supported by performinga chemical assay of the bioavailability of the con-taminant in the soil, which physically extracts thecontaminant from the soil matrix using a chemicalsolvent. Organic solvents have been traditionally usedto extract organic contaminants during harsh extrac-tion processes (such as Soxhlet extraction), althoughthis does not demonstrate the true bioavailability ofthe contaminant, but the total contaminant concen-tration in the soil. However, a more representativeapproach was used by Hatzinger and Alexander,17

who extracted the contaminants with mild organic sol-vents (such as methanol) to represent the bioavailableproportion in the soil. As microorganisms can mostlyonly access those contaminants that are in the aque-ous phase, water-based solvents are also being usedto more accurately predict the bioavailable fraction oforganic contamination in soil. One such compound ishydroxypropyl--cyclodextrin72 (HPCD), which canencapsulate hydrophobic contaminants. In addition,HPCD does not appear to inhibit lux-type microorgan-isms, allowing for a combined biological and chemicalassessment of bioavailability.

Toxicity of end-productsThe principle of bioremediation is to remove ordetoxify a contaminant from a given environmentusing microorganisms. Most commercial bioreme-diation trials tend to monitor the success of thetreatment by the degree of removal of the parentcontaminant and do not consider the possibility ofthe biological production of more toxic breakdownmetabolites. However, it is important to ensure thatthe contaminated material is suitably detoxified atthe end of the treatment.12,76 A recent study using abioreactor to treat PAH-contaminated gasworks soilmonitored both the removal of PAHs and the accumu-lation of oxy-PAHs, such as PAH-ketones, quinonesand coumarins.12 These compounds are formed dur-ing the microbial metabolism of PAHs (see earlierMicrobial metabolism of PAHs), and can also beformed from chemical oxidation and phototransfor-mation of PAHs.77 Such transformation products canbe equally toxic, if not more toxic, to human healthwhen compared with the parent PAH,21 with many of

the oxy-PAHs formed during the treatment of PAH-contaminated soils more persistent than the parentcompounds.12 In this study, Lundstedt and colleaguesshowed that although there were no new oxy-PAHsformed during the bioremediation of an aged gasworkssoil, the concentrations of 1-acenaphthenone and 4-oxapyrene-5-one increased in the soil by 30% and60% respectively over 30 days of bioslurry treatment.In addition, they showed that some oxy-PAHs actuallyincreased in concentration during treatment, and weresubsequently more persistent to microbial degradationthan their corresponding parent PAH compound. Asoxy-PAHs are more toxic than the parent PAHs, thisstudy highlights the importance of monitoring themetabolites of bioremediation, specifically for toxicdead-end products, and assessing the toxicity of thematerial both before and after treatment.

The Microtox bioassay has been used to performan assessment of the success of composting of agarden soil spiked with a range of high molecularweight PAHs.47 In this study, the authors found thatthe degradation products were far less toxic than theparent PAHs, and concluded that composting thissoil with spent mushroom compost was successful. Inaddition, a multitude of eco-toxicological tests, such asbacterial (Microtox), algal and Daphnia-based testswere performed to ensure that the toxicity of a cokeoven soil was suitably reduced using landfarming.76

They concluded that landfarming resulted in asignificant reduction in toxicity of the coke workssoil.

However, it is important to understand the relevanceof ecotoxicity tests to the overall toxicity of theremediated land. Many of these tests monitor foracute toxicity of compounds (via organism death),whereas it would be more representative, particularlywhen assessing the carcinogenic and mutagenic PAHs,to consider the chronic toxicity of these soils, suchas monitoring for organism DNA damage and theoccurrence of DNA adducts21 (see earlier, Toxicityof PAHs).

APPROACHES TO THE BIOREMEDIATION OFPAH-CONTAMINATED ENVIRONMENTSTreatment of soils and sedimentsSoils and sediments can be treated for PAH con-tamination both by in situ and ex situ methods.48

Landfarming is an in situ treatment for soils, whichfocusses upon stimulating the indigenous microor-ganisms in the soil by providing nutrients, waterand oxygen. For example, a pilot-scale landfarmingtreatment of PAH-contaminated soil from a wood-treatment facility was achieved by biostimulation ofthe soil with water, ground rice hulls (as a bulk-ing agent), and pelletised dried blood (as a nitrogensource) and bioaugmentation of the microbial com-munity with an inoculum of Pseudomonas aeruginosa(strain 64).78 Aeration was provided by tilling of thesoil. The workers found that 86% of total PAHs



were removed from the soil over 1 year, includinga reduction in high molecular weight PAHs such asbenzo[a]anthracene and benzo[a]pyrene (79.5% and11.3% respectively). Biopiling of soil48 and the treat-ment of soil in bioreactors12 are ex situ treatments thatare less cost effective than in situ treatment, howeverex situ treatment benefits from being more subject tomonitoring and control.

Composting, which is also an ex situ treatmentfor PAH-contaminated soil, is an aerobic processwhereby microorganisms degrade organic materialswhich results in thermogenesis and the generationof organic and inorganic compounds.79 The processof composting can be divided into four mainstages according to the temperature of the material;these are mesophilic, thermophilic, cooling andmaturation.80 These four stages are driven by changesin the microbial community, with an increase inthe metabolic activity of the in situ microorganismscreating heat. This allows for the establishmentof thermophilic microorganisms which displace themesophilic organisms in the decomposition of organicmatter. Temperatures decrease as organic matter isdepleted, upon which the composting process entersthe cooling and maturation stages.

The success of bioremediation of PAHs by compost-ing has been reported in several studies.47,8184 Com-posting in bioreactors is a popular option because it ispossible to exert greater control over parameters suchas temperature and oxygen supply during the compost-ing process, and a variety of organic materials as bulk-ing agents can be used. For example, the concentra-tions of PAHs (anthracene, phenanthrene and pyrene)were successfully reduced during a 60-day periodof composting (30 days thermophilic composting,30 days cooling and maturation) in laboratory-scalebioreactors using a variety of municipal wastes such aspaper, grass and food as a carbon source.82

Treatment of watersAs with soils and sediments, contaminated ground-water can be remediated both in situ and ex situ tothe contaminated site. However, it is often not fea-sible to remediate contaminated groundwater ex situdue to the costs involved with abstraction and ship-ping of the contaminated water, and the fact thatmuch of the contamination will be sorbed within theaquifer. Therefore, in situ treatment of aquifers canbe accomplished by the biostimulation, and possi-bly the bioaugmentation of the indigenous aquifercommunity.

The in situ treatment of an aquifer contaminatedwith a range of organic pollutants, including phenols,BTEX compounds and PAHs was carried out ona site that produces flooring, damp proofing androofing materials manufactured from bitumen andsynthetic resins.38 In addition, records suggest thatthe site was formerly an oil works and used for coalgasification. This has resulted in the aquifer belowthe site containing a multitude of organic pollutants,with a mean concentration of 11 g L1 PAHs in thegroundwater.

The site was remediated using a combination ofbioaugmentation and biostimulation over a 21/2-yearperiod. Nutrients (Purisol 100, supplies nitrogen andphosphate; ICI Chance and Hunt), a commerciallyavailable bacterial inoculum (PHENOBAC, MicrobacLtd, Durham) and oxygen (supplied by the reductionof sodium nitrate to gaseous oxides of nitrogen) werecirculated through the aquifer by means of a series ofinjection and abstraction wells (see Fig 6). The in situremediation of this site was successful, even thoughthe contaminants had undergone significant ageing inthe aquifer. PAHs were reduced to a concentrationof 0.7 g L1, and co-contaminants such as phenolsreduced from 1100 g L1 to 12 g L1.

CONTAMINANTPLUME

WATER TABLE

CONCRETE HARDSTANDING

RIVER

NUTRIENTS

INJECTION WELLABSTRACTION WELL

DIRECTION OFGROUNDWATER

MOVEMENT

INOCULUM OXYGEN SOURCE (NO3)

Figure 6. Cross-section of the in situ remediation of contaminated groundwater using an injection and abstraction well to circulate an oxygensource, inoculum and nutrients through a hydrocarbon contaminated aquifer.38.



CONCLUSIONSThe persistence and toxicity problems associatedwith PAHs in the environment have resulted ina large amount of laboratory-based work that hasconcentrated on the ability of a variety of microbes(fungi and bacteria) to transform these complexaromatic molecules. The pathways of aerobic PAHtransformation have been established and it isknown that many environments contain microbescapable of reducing PAH concentrations. Thesefactors have led to an interest in the potentialuse of microbes to remediate PAH-contaminatedsoils and more recent work has established thatit is possible to use microbial-based processes toremediate PAH-contaminated soil. These processes,eg land-farming and biopiling, are effective on shallowcontamination but when PAH contamination is atdepth then the use of bioremediation becomesmore problematical. However, a recent field studyhas shown that bioremediation of contaminatedaquifers is possible by the introduction of aerationto the subsurface. In addition, the potential of thebiodegradation of PAHs under anaerobic conditionsis promising, allowing further advances for the in situtreatment of the contaminated subsurface. Overall, wefeel that the bioremediation of PAH-contaminatedsites is feasible given the breadth of our currentknowledge. Although the inherent limitations of thebioremediation of PAH-contaminated environmentsare known, further research is required to testthese limitations, and exploit the potential of thein situ microbial communities to metabolise PAHs(particularly the larger molecular weight PAHs) inthose sites with sub-optimal conditions, such asextreme pH and/or temperatures. In addition, furtherresearch is required to develop potential anaerobicremediation technologies that can be applied toremediate the numerous subsurface sites that arecontaminated with PAHs.

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source of pahs

representative pahs

significantquantities

distribution of pahs

point sources of pahs

minor sources of pahs

natural environment

university of newcastle