Appl Microbiol Biotechnol

DOI 10.1007/s00253-014-5684-9

MINI-REVIEW

Bioremediation of petroleum hydrocarbons: catabolic genes,

microbial communities, and applications
Sebastin Fuentes & Valentina Mndez & 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 sustainable

and cost-effective technology for the cleanup of
hydrocarbon-polluted soils and coasts. In spite of that longer
times are usually required compared with physicochemical
strategies, complete degradation of the pollutant can be
achieved, and no further confinement of polluted matrix is
needed. Microbial aerobic degradation is achieved by the
incorporation of molecular oxygen into the inert hydrocarbon
molecule and funneling intermediates into central catabolic
pathways. Several families of alkane monooxygenases and
ring hydroxylating dioxygenases are distributed mainly
among Proteobacteria, Actinobacteria, Firmicutes and
Fungi strains. Catabolic routes, regulatory networks, and
tolerance/resistance mechanisms have been characterized in
model hydrocarbon-degrading bacteria to understand and
optimize their metabolic capabilities, providing the basis to
enhance microbial fitness in order to improve hydrocarbon
removal. However, microbial communities taken as a whole
play a key role in hydrocarbon pollution events. Microbial
community dynamics during biodegradation is crucial for
understanding how they respond and adapt to pollution and
remediation. Several strategies have been applied worldwide
for the recovery of sites contaminated with persistent organic
pollutants, such as polycyclic aromatic hydrocarbons and
petroleum derivatives. Common strategies include controlling
environmental variables (e.g., oxygen availability, hydrocarbon solubility, nutrient balance) and managing hydrocarbondegrading microorganisms, in order to overcome the ratelimiting factors that slow down hydrocarbon biodegradation.
S. Fuentes : V. Mndez : P. Aguila : M. Seeger (*)
Laboratorio de Microbiologa Molecular y Biotecnologa Ambiental,
Departamento de Qumica & Centro de Biotecnologa & Center of
Nanotechnology and Systems Biology, Universidad Tcnica
Federico Santa Mara, Valparaso, Chile
e-mail: michael.seeger@usm.cl

Keywords Petroleum . Hydrocarbon . Bioremediation .

Biodegradation . Microbial community . Catabolic genes

Introduction
Petroleum is a natural resource confined in large deposits in
the Earth crust. Accidental petroleum spills alter the impacted
environment and trigger the development and implementation
of remediation strategies for cleaning up the polluted sites. Oil
spills became an international concern in 1967, when
~120,000 tons of crude oil was released by the Torrey
Canyon supertanker into the English Channel. This first
large-scale oil spill forced UNOs International Maritime
Organization to create in 1973 the International Convention
for the Prevention of Pollution from Ships MARPOL with the
aim of designing emergency protocols and strategies toward
oil 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 fractions. Paraffin is usually the most abundant fraction and
contains 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 containing at least one aromatic ring. Hydrocarbons can possess from
few up to >60 carbons. A higher molecule size correlates with
a higher boiling point. Petroleum-derived products are obtained by fractional distillation, by which different fractions are
enriched according to its boiling range (Speight 2001).
For the cleanup of hydrocarbon-polluted sites, diverse
physicochemical and bioremediation treatments have been
applied. Bioremediation techniques are cost-effective, environmental sustainable, and can achieve complete pollutant

Appl Microbiol Biotechnol

degradation. Microorganisms are the main biocatalysts for

hydrocarbon bioremediation. Thus, microbial degradation
has to be optimized in order to improve bioremediation. In
addition, the study of the microbial community dynamics in
polluted sites is useful for the management of the process. The
aims of this review are to update bioremediation strategies for
hydrocarbon-polluted sites, hydrocarbon catabolic pathways
and genes, and microbial dynamics in oil-polluted soils and to
highlight main challenges for bioremediation technologies.

Hydrocarbons as persistent pollutants

Petroleum hydrocarbons are organic pollutants of major concern due to their wide distribution, persistence, complex composition, and toxicity. The most common petroleum hydrocarbons include aliphatic, branched, and cycloaliphatic alkanes, as well as monocyclic and polycyclic aromatic hydrocarbons (PAHs). PAHs include naphthalene, fluorene, phenanthrene, 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 different hydrocarbon molecules rather than a defined composition. Thus, fractions are dissimilar in terms of volatility, bioavailability, toxicity, degradability, and persistence (Table 1).
This complex array of compounds depicts the tremendous
challenge for designing effective bioremediation strategies,
which can be illustrated by the effects of major contamination
events in the past (Atlas and Hazen 2011).
Once petroleum hydrocarbons reach an environment, damage can be the result of several causes. Primary biological
impact is due to the blocking effect of oil layer to water,
nutrients, oxygen, and light access. Cytotoxic and mutagenic
effects of hydrocarbons are behind the long-term pollution
consequences. A more bioavailable toxic compound not only
shows increased noxious effects but also has higher accessibility for biodegradation. In contrast, strongly adsorbed fraction is less toxic but more recalcitrant. This general rule
is relevant for designing biological strategies for the cleanup
of polluted soils or sediments because petroleum hydrocarbons tend to tightly adsorb to these matrices (Baboshin and
Golovleva 2012).
Two hydrocarbon fractions are present in solid particles
(i.e., soil, sediment). The fraction that remains irreversibly
adsorbed to particles is considered non-toxic and nonbiodegradable because it is not bioavailable. The reversibly
bound portion able to desorb and diffuse into water phase
constitutes the bioavailable fraction. Nonetheless, bioavailability is always limited due to the low water solubility of
hydrocarbons. Main hydrocarbon degraders are bacteria, filamentous fungi, and yeasts (van Beilen and Funhoff 2007;
Wentzel et al. 2007). To overcome low bioavailability,

bacteria can get access to hydrophobic substrates by reducing

surface tension or by direct contact with hydrophobic droplet
(Wentzel et al. 2007). Surface tension reduction can be
achieved by the secretion of surfactants, molecules that disperse hydrocarbons into small droplets (Baboshin and
Golovleva 2012). Biosurfactants can be glycolipids, phospholipids, lipopeptides, lipoproteins, fatty acids, neutral lipids,
polymeric lipids, and high molecular weight biopolymers
(Atlas and Philp 2005; Das et al. 2008). Direct attachment to
hydrocarbon droplet interface can be achieved by increasing
the hydrophobicity of cell surface. This includes the synthesis
of adhesion structures like proteins, lipopolysaccharides,
mycolic acids, and other hydrophobic exopolymers
(Abbasnezhad et al. 2011). Acinetobacter venetianus RAG-1
synthesizes emulsan and a hydrophobic fimbriae, enhancing
the 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 attachment to droplets (Abbasnezhad et al. 2011). In close
contact to bacteria, hydrocarbon molecules enter the bacterial
cell, where the catabolic machinery achieves their breakdown.
Uptake apparently occurs by a lateral diffusion mechanism,
where the hydrocarbon molecule diffuses from the transporters lumen laterally into the outer membrane (Hearn
et al. 2009).
Microorganisms possess evolved mechanisms to activate
hydrocarbons, generating metabolic intermediates that funnel
to central metabolic pathways. By oxidizing these substrates,
microorganisms can take advantage in nutrient-limited niches.
Addition of one or two hydroxyl groups to the hydrocarbon
skeleton seems to be the ubiquitous first step during aerobic
catabolism (Figs. 1 and 2). The key enzymes in hydrocarbon
degradation pathways are oxygenases, which catalyze the
addition of molecular oxygen to the substrate (Rojo 2009).
Dioxygenases catalyze the addition of two hydroxyl groups,
whereas monooxygenases catalyze introduction of one atom
of oxygen into the hydrocarbon. During anaerobic degradation, activation is achieved coupling CO2 or fumarate to
hydrocarbons (Fig. 2), and sulfate and nitrate are used as
terminal 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 hydrocarbon

degradation
Unbranched intermediate C5C11 chain-length n-alkanes are
the major hydrocarbon constituents of petroleum. The initial
aerobic alkane degradation step is oxidation via an alkane

Appl Microbiol Biotechnol

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

Molecular formula

Melting pointa (C)

Boiling pointa (C)

Water solubilitya,b
(mg L1)

log KOWa,c

Toxicityd, e
(mg kg1)

Aliphatic
n-Hexane
n-Octane
n-Hexadecane

C6H14
C8H18
C16H34

95.4
56.8
18.1

68.7
125.7
286.9

9.5262
0.514.0
21066103

2.94.3
4.05.6
7.38.3

25,000
nd
nd

C19H40
C6H12

100f
6.6

68f
80.7

nd
50.288.8

nd
2.53.7

980
nd

C6H6
C7H8
C8H10

5.5
95
95

80.1
110.6
136.2

1,4022,167
155739
131208

1.62.5
2.13.0
3.13.5

930
636
3,500

C10H8
C14H10
C16H10
C20H12

80.3
215.8
150.6
181.1

217.9
339.9
404
495

12.538.4
0.030.09
0.031.6
41056103

3.03.8
3.55.34
4.55.5
5.18.0

533
4,900
800
300

205-300g

4.8-10.4g

3.3->6g

15,000

Pristane
Cyclohexane
BTEX
Benzene
Toluene
Ethylbenzene
PAH
Naphthalene
Anthracene
Pyrene
Benzo[a]pyrene
Distillate
Kerosene
nd not determined
a

Physicochemical data from Mackay et al. (2006) unless otherwise indicated

At 25 C. Values depend on the used method

Octanolwater partition coefficient

Lethal dose 50 tested in adult rat (mouse for PAH)

Material Safety Data Sheets (MSDS) available online

Sigma-Aldrich product information

American Petroleum Institute, Petroleum HPV Testing Group (2010)

oxygenase, carried out by either a multimeric monooxygenase

or a cytochrome P450 monooxygenase (van Beilen and
Funhoff 2007). Monooxygenases belong to a widely distributed protein family based upon functional properties (i.e.,
alkane hydroxylation), but exhibiting significant structural
differences (van Beilen and Funhoff 2007). These catalytic
systems could contain at their active site ironsulfur, di-iron,
heme, and copper (van Beilen and Funhoff 2007; Wang and
Shao 2012).
Aerobic alkane degradation pathways first oxidize an
alkane into an alcohol. Commonly a primary alcohol is
produced, but subterminal oxidation has also been reported (Rojo 2009). The primary alcohol is oxidized
into an aldehyde, which is subsequently transformed
via oxidation into a fatty acid, which is funneled into
-oxidation (Fig. 1). Initial reaction of alkane degradation pathway is mediated by an integral-membrane nonheme di-iron monooxygenase that hydroxylates the substrate at terminal position. This monooxygenase complex consists of a particulate membrane-bound

hydroxylase (pAH), a rubredoxin, and a rubredoxin

reductase. For the reduction step, electrons are subsequently 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-characterized
model pAH enzyme that oxidizes propane, n-butane,
and C5C13 alkanes. The alkB-like genes are widely
distributed in Proteobacteria and Actinobacteria, including environmental, opportunistic, and pathogenic strains
(van Beilen et al. 2006). Differences in substrate range
and specificity among pAHs from Burkholderia,
Acinetobacter, Pseudomonas, Alcanivorax, Oleiphilus,
M y c o b a c t e r i u m , R h o d o c o c c u s , N o c a rd i a , a n d
Prauserella genera have been reported. Nonetheless,
structural and catalytic motifs are conserved among
them (van Beilen and Funhoff 2007). Strains able to
degrade intermediate C5C11 and long C12 n-alkanes
generally possess AlkB-like monooxygenases
(Rojo 2009).

Appl Microbiol Biotechnol

Fig. 1 Bacterial n-alkane degradation pathways. Aerobic pathways (a, b)

are shown in the left panel, whereas anaerobic pathways (c, d) are shown
in the right panel. a Aerobic alk-like degradation pathways oxidize
alkanes into fatty acids. In some cases, -hydroxylation generates a
dicarboxylic acid. b Sub-terminal oxidation of n-alkanes in some
Rhodococcus, Mycobacterium, and Pseudonocardia strains yields a primary 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 coenzyme
A (CoA) and funneled into -oxidation. In strain AK-01, the second cycle
of -oxidation yields propionate instead of acetate. Propionate can regenerate fumarate via methylmalonyl-CoA pathway. AH alkane hydroxylase, AD alcohol dehydrogenase, ALD aldehyde dehydrogenase, ASS
alkylsuccinate synthase, ACS acyl-CoA synthetase, BVM BaeyerVilliger
monooxygenase, E esterase, -Ox -oxidation cycle, C? putative carboxylase, ? unknown enzyme

Substrate range and alk genes organization differ

among alkane-degrading bacteria. Metabolic enzymes for
the conversion of n-alkanes into fatty acids are encoded in
alkBFGHJKL and alkST operons located in the OCT
plasmid from P. putida GPo1 (Rojo 2009). The
alkBFGHJKL operon encodes the enzymes for alkane
conversion into acetyl-CoA. The alkST cluster encodes
the rubredoxin reductase AlkT and the transcriptional
activator AlkS (van Beilen and Funhoff 2007). In
Acinetobacter baylyi ADP1, long C12C36 n-alkane hydroxylation is mediated by the AlkB-like alkane
monooxygenase AlkM. Interestingly, AlkR-mediated
alkM gene expression is induced by non-substrates C7
C11 n-alkanes (Rojo 2009). Acinetobacter sp. M1 is able
to use C 20 C 44 alkanes and possesses two n-alkane
hydroxylase-encoding genes (alkMa and alkMb).
Expression of alkMa gene is induced by >C22 chain-

length n-alkanes, whereas alkMb gene expression is preferentially induced by C16C22 n-alkanes. Pseudomonas
aeruginosa strains PAO1 and RR1 possess AlkB1 and
AlkB2 hydroxylases that probably play different physiological roles. While alkB2 gene expression is higher in early
exponential phase, alkB1 gene is preferentially expressed
during stationary phase (Rojo 2009). Long chain n-alkanes
are oxidized by alkane hydroxylase AlmA from Acinetobacter
sp. DSM17874 that degrades C32 or longer alkanes and alkane
monooxygenase LadA from Geobacillus thermodenitrificans
NG80-2 that degrades up to C36 long-chain alkanes (Li et al.
2008; Throne-Holst et al. 2007).
Two additional hydroxylation systems include methane
monooxygenases (MMOs) and cytochrome P450 protein superfamily. MMOs are found primarily in methanotrophic bacteria. Cytoplasmic soluble form of methane monooxygenase
(sMMO) is present in Methylococcus, Methylosinus,

Appl Microbiol Biotechnol

Fig. 2 Bacterial aerobic aromatic
hydrocarbon degradation
pathways overlap in substrates
and metabolites. Pyrene and
phenanthrene degradation
pathways from M. vanbaalenii
PYR-1 and M. aromativorans
JS19b1 and naphthalene (nah)
degradation pathway from
P. putida G7 (NAH7 plasmid) are
shown. Biphenyl (bph) and
toluene/xylene (xyl) degradation
pathway from B. xenovorans
LB400 and P. putida mt-2 are
represented. In general, initial
aromatic ring activation yields a
cis-dihydroxylated ring, which is
re-aromatized resulting in a
catechol-like orthodihydroxylated ring. Ring
cleavage at ortho (intradiol) or
meta (extradiol) position depends
on the catabolic route. NDO
naphthalene dioxygenase, DHD
dihydrodiol dehydrogenase,
EXDO extradiol dioxygenase,
INDO intradiol dioxygenase; IS
isomerase, HA hydratasealdolase, ALD aldehyde
dehydrogenase, HX hydroxylase,
PAH-RHD PAH ringhydroxylating dioxygenase, DC
decarboxylase, RHD ringhydroxylating dioxygenase, BDO
biphenyl dioxygenase, XM xylene
monooxygenase, HL hydrolase

Appl Microbiol Biotechnol

Methylocystis, 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 possess
a [2Fe-2S] catalytic center and utilize FAD and NADH as
cofactors and oxidize C1C8 chain-length alkanes, halogenated alkanes, alkenes, and cycloalkanes. The pMMOs have a
mono- or di-copper center and hydroxylate only C1C5 substrates such as alkanes, halogenated alkanes, and alkenes (van
Beilen and Funhoff 2007).
Cytochrome P450 comprises several oxidase systems in
Bacteria, Archaea, and Eukarya. Soluble cytochromes P450
involved in alkane oxidation have been described in
Proteobacteria and Actinobacteria (van Beilen et al. 2006).
Seven Rhodococcus erythropolis strains possess two cytochromes 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 in
sulfate-reducing Deltaproteobacteria. In Desulfococcus
oleovorans Hxd3, n-alkanes are activated by carboxylation
at C-3 position and elimination of the two adjacent terminal
carbons to yield a carboxylic acid that contains one carbon
less than the initial n-alkane (Fig. 1) (So et al. 2003). Hxd3
enzymes involved in anaerobic alkane catabolic pathways
have not been identified. Desulfatibacillum alkenivorans
AK-01 is able to grow on C13C18 chain-length n-alkanes,
1-hexadecene, and 1-pentadecene. Alkane activation is
achieved via conjugation with fumarate at C-2 position by
the alkyl succinate synthase (Ass) to yield (1-methylalkyl)succinate. After carbon rearrangement and decarboxylation, a methylated fatty acid that is two carbons larger than
the original n-alkane is formed (Callaghan et al. 2006). The
resulting fatty acid is funneled into -oxidation pathway
(Fig. 1). Although the genome of Desulfatibacillum
alkenivorans strain AK-01 has been sequenced, mechanisms
for (1-methyl-alkyl)succinate carbon rearrangement and fumarate regeneration remains poorly known (Callaghan et al.
2012).

Bacterial aromatic hydrocarbon degradation

Aerobic microbial metabolism for aromatic hydrocarbons
such as biphenyl, naphthalene, phenanthrene, and pyrene
(Fig. 2) involves an initial dioxygenation reaction by a ringhydroxylating dioxygenase (RHD) (Kim et al. 2007; Pagnout
et al. 2007; Peng et al. 2008; Seo et al. 2012). In contrast, the
xylene/toluene pathway from P. putida mt-2 (P. putida
KT2440 (pWW0)) starts with a monooxygenation of a methyl
group (Fig. 2). PAH-RHD catalytic sites share a Rieske [2Fe2S] non-heme iron catalytic center and are less diverse than
alkane 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 aromatic ring can undergo fission in meta- or ortho-position
(Peng et al. 2008). After ring cleavage, aromatic hydrocarbons
with at least two rings give rise to different carboxy-aromatic
metabolic intermediates, which are further transformed into
intermediates that enter central metabolism, including catechol, salicylate, gentisate, homogentisate, and protocatechuate
central pathways (Chain et al. 2006; Mndez et al. 2011;
Romero-Silva et al. 2013). Eventually, activation of the original aromatic substrate generates molecules with even higher
toxicity, leading to dead-end metabolic pathways (Cmara
et al. 2004; Pieper and Seeger 2008).
Benzene, toluene, ethylbenzene, and xylenes (BTEX) are
main components of the volatile organic fraction from crude
oil. For toluene degradation, five aerobic pathways and one
anaerobic route have been reported (Shinoda et al. 2004).
P. putida mt-2 oxidizes toluene at the methyl group by the
xylene monooxygenase into benzaldehyde (Fig. 2). P. putida
F1 oxidizes toluene by the toluene dioxygenase TodC1C2BA
into cis-toluene dihydrodiol. Burkholderia cepacia G4,
Ralstonia pickettii PKO1, and Pseudomonas mendocina
KR1 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 extensively
studied. The enzymes for toluene (m-xylene, p-xylene) degradation are encoded by the xyl genes from pWW0 plasmid
from P. putida mt-2 (Domnguez-Cuevas et al. 2006). The
upper operon xylUWCMABN encodes the enzymes for the
oxidation of the methyl group of toluene, m-xylene, and pxylene into the corresponding (alkyl)benzoate. Oxidation is
followed by the dioxygenation of the (alkyl)benzoate and the
meta-cleavage of the resulting (methyl)catechol, which is
funneled into the Krebs cycle. This second set of reactions is
catalyzed by the meta TOL gene products from the
xylXYZLTEGFJQKIH gene cluster. Positive transcriptional
regulators XylR and XylS orchestrate the coordinated expression of the upper and lower pathways. The xylE gene encoding
catechol 2,3-dioxygenase enzyme of the toluene/xylene degradation pathway has been detected in novel hydrocarbondegrading Acinetobacter, Kocuria, and Pseudomonas strains
isolated from a crude oil-contaminated soil (Mndez et al.
2010).
The naphthalene catabolic pathway encoded by the nah
genes from the NAH7 plasmid in P. putida G7 shares notorious resemblances with xyl catabolic route. The upper catabolic
operon nahAaAbAcAdBFCED encodes enzymes for the conversion of naphthalene into salicylate. Enzymes encoded by
the lower operon nahGTHINLOMKJ transform salicylate via
meta-cleavage into pyruvate and acetaldehyde. The

Appl Microbiol Biotechnol

naphthalene dioxygenase NahAaAbAcAd from P. putida G7

incorporates two hydroxyl groups in cis-position. The nah
genes-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. Bacteria able to use a wide
range of compounds for growth should have an advantage
over bacteria able to use only few compounds. In evolution,
this feature is favored and is more likely to be maintained in
time (Copley 2000). The low substrate specificity allows
RHDs to oxidize related compounds usually found together
in nature, but with different conversion rates. Mycobacterium
vanbaalenii PYR-1 possesses the NidAB and NidA3B3 enzymes involved in the mono- and dioxygenation of toluene,
m-xylene, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, and the
non-hydrocarbons carbazole and dibenzothiophene (Kweon
et al. 2010). NidAB showed higher conversion rates toward
pyrene, whereas NidA3B3 presents higher conversion rates
for phenanthrene and fluoranthene. Their low substrate specificity correlates with larger binding pocket sizes in the active
site (Kweon et al. 2010).
Biphenyl is present naturally in crude oil and is of special
concern because it is the starting molecule for the synthesis of
polychlorobiphenyls (PCBs). Biphenyl and PCB degradation
by enzymes encoded by the bph genes occurs in
Pseudomonas, Burkholderia, Sphingomonas,
Achromobacter, and Rhodococcus strains (Chain et al. 2006;
Pieper and Seeger 2008; Seeger et al. 1997). Biphenyl-2,3dioxygenase (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 from
mono- to hexachlorobiphenyls (Seeger et al. 1999; Seeger and
Pieper 2009). Biphenyls with fluoro, bromo, nitro, and hydroxy substituents, dibenzofuran, dibenzodioxin, and natural
and synthetic isoflavonoids are also accepted as substrates by
the LB400 BDO (Seeger et al. 2001, 2003; Overwin et al.
2012). A wide array of dioxygenases and hydroxylasesencoding genes from strain LB400 accounts for its unusual
catabolic versatility toward aromatic compounds (Mndez
et al. 2011; Romero-Silva et al. 2013). In addition to the
relaxed substrate range, protein derivatives generated by combination of gene segments (Cmara et al. 2007) or random
mutagenesis (Zielinski et al. 2006) can give rise to enzymes
that oxidize novel substrates, which could be useful for bioremediation and biotransformation processes. Improved PCB
bioremediation was observed with the genetically modified
Cupriavidus necator JMS34 in which the bph locus of
B. xenovorans LB400 that encodes the PCB-degradation pathways was incorporated (Saavedra et al. 2010).
An aromatic hydrocarbon can be a carbon source as well as
a toxic signal for bacteria. In response to hydrocarbons,

P. putida mt-2 induces a solvent extrusion system and exhibits

a tolerance response. Efflux pumps and several stressresponse related proteins are up-regulated in presence of both
toluene and the non-substrate o-xylene (Domnguez-Cuevas
et al. 2006). In B. xenovorans LB400, stress responses due to
the presence of toxic aromatic compounds such as biphenyl
and PCBs include the induction of general stress and oxidative
stress proteins (Agull et al. 2007). Bacteria possess sophisticated mechanisms to counteract aromatic toxicity, inducing
genes involved in stress response and degradation. Bacterial
isolates from crude oil-polluted environments showed increased catalase and peroxidase activities. Enzymatic defenses
were crucial for the degradation of pollutants but also to
counteract reactive oxygen species (Bukov et al. 2010).
Induction of components of the OxyR regulon was observed
during toluene degradation in P. putida KT2440 (pWW0)
(Domnguez-Cuevas et al. 2006). Antioxidant enzymes from
the OxyR regulon such as alkylhydroperoxide reductase and
catalase possess a key role in cellular response to oxidative
damage. Bacterial adaptation to toxic compounds comprises
changes at genome and physiological levels (Agull 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 the
degree and age of the spill. The aim of bioremediation is to
overcome the limiting factors that slow down biodegradation
rates. Bioremediation can be accomplished either by in situ or
ex situ treatments. During in situ applications, the pollution is
treated at the site. The ex situ technologies involve the transport 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 hydrocarbonpolluted soils and sediments
Remediation strategy

Treating site

Cost (US $/m3)a

Biological
Biostimulation
Bioaugmentation
Bioventing
Biopiles
Composting
Landfarming
Physicochemical

In situ
In situ
In situ
Ex situ
Ex situ
Ex situ

30100
30100
79970
130260
630757
3070

In situ
Ex situ

4051,485
44252

Vapor extraction
Thermal desorption
a

Data from the US Federal Remediation Technologies Roundtable (2014).

Additional costs for laboratory and pilot scale experiments are not included

Appl Microbiol Biotechnol

Strategy selection depends on rate-limiting factors and the

pollutants chemical nature. For petroleum hydrocarbons, four
well-known scenarios arise: (1) The excess of carbon source
due to hydrocarbon input results in limitation of other nutrients. Nitrogen and phosphorus amendments can be used to
restore balance and thus increase biodegradation rates; (2)
insufficient oxygen availability decreases biodegradation
rates. Air injection or simple stirring can overcome oxygen
limitation during aerobic hydrocarbon degradation; (3) low
bioavailability of hydrocarbons. Addition of surfactants can
improve solubility and thus bioavailability of hydrocarbons.
The application of biosurfactants (i.e., surfactants produced by
microorganisms or plants) is an environmentally friendly alternative, as they are non-toxic and biodegradable; and (4)
non-efficient catabolic machinery from native microbial communities. An input of hydrocarbon-degrading microorganisms
as pure culture or microbial consortium can enhance degradation rates.
Bioremediation costs are commonly lower than physicochemical treatments and depend largely on the amounts of soil
to remediate the degree and depth of pollution and the soil
type (Table 2). In general, costs per soil volume increase when
the site is small, the pollution is deep, and the soil particles are
small, i.e., clay/silt soil is more difficult to remediate than
sandy soils (US Federal Remediation Technologies
Roundtable 2014).
Main in situ remediation strategies for hydrocarbonpolluted soils are biostimulation, bioaugmentation, and
bioventing. Biostimulation involves the enhancement of
native microorganisms metabolism by management of environmental factors and nutrients. Bioaugmentation is
achieved by the addition of native or exogenous
hydrocarbon-degrading microorganisms, when native microbial 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 to
enhance aerobic metabolism.
The most common ex situ bioremediation technologies for
oil-polluted soil treatments are biopiles, composting, and
landfarming. The aim is to speed up hydrocarbon degradation
by adding low-cost nutrients and oxygen. Biopiles and windrow composting involve the mixing of polluted soil with
organic material as a bulking agent. This mixture promotes
microbial activity by improving soil texture, aeration, and
moisture maintenance (Jrgensen et al. 2000). Organic matter
can be also a substrate for microbial growth or even a microbial source (Prez-Armendriz et al. 2004). Main difference
between both strategies is the aeration methodology.
Composts are aerated by turning the soil/bulking agent mixture periodically with a modified windrow turner, whereas in
biopiles a pipe network delivers air. Biopiles and windrows
have been successfully used for the remediation of a wide
range of contaminants (Namkoong et al. 2002; Van Gestel

et al. 2003). Landfarming is based on the controlled spreading

of organic waste on the soil surface to allow native microorganisms to aerobically degrade pollutants. Main features of
these strategies are summarized in Table 3.
A key aspect to take into account for designing and scaling
up a bioremediation technique is the technology cost. Organic
material from industrial residues allows to scale up applications with low-associated costs. Moreover, it has the ecological
advantage of minimizing organic industrial waste. Some illustrative examples include orange peel addition, which resulted
as an effective strategy to enhance hydrocarbon degradation. In
a pilot scale with high total petroleum hydrocarbon (TPH)
concentration (58,000 mg kg1), higher degradation rates correlated with increasing amounts of peel added (Roldn-Martn
et al. 2006). The addition of coffee beans on composting
indicated that higher degradation rates were achieved with
lower amounts of coffee beans (Roldn-Martn et al. 2007).
Increasing coffee proportions may lower pH to a range that is
detrimental for hydrocarbon-degrading microorganisms.
Besides being used as carbon source, fruit residues can counteract oxidative stress during degradation of aromatic compounds. Improved degradation rates by B. xenovorans
LB400 was obtained with the addition of berry extract to
PCB-contaminated soils (Ponce et al. 2011). Organic material
that possesses antioxidant properties can increase bacterial
tolerance to the toxicity of aromatics and the oxidative stress
during their degradation (Ponce et al. 2011).
Sugarcane bagasse addition improved microbial oil degradation in polluted soils. Increased degradation rates were observed
with both bagasse and soil microorganisms, compared with
either bagasse or soil microorganisms. Thus, biostimulation
and bioaugmentation approaches can be contributing to biodegradation (Prez-Armendriz et al. 2004). For bioaugmentation,
bagasse can also be used as a low-cost non-toxic carbon source
for microbial growth. Bacteria and Fungi grown in bagasse
degraded phenanthrene in soil, probably due to preadaptation
to use aromatic compounds present in lignocellulosic residues.
As a rich nutrient source, bagasse allows to exploit synergic
interactions 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 4.4
and >73 % (Chvez-Gmez 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 using
lignocellulose residues for inoculum growth instead of conventional and usually more expensive carbon sources.

Lessons from an emblematic oil spill

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

Appl Microbiol Biotechnol

Table 3 Main characteristics of bioremediation processes for petroleum-contaminated soils
Bioremediation strategy

Main features

Main remarks

Bioaugmentation
Isolated strain
Microbial consortium
Biostimulation
Fertilizers
(Bio)surfactants
Bioventing
Biopiles/composting

Addition of efficient pollutant-removing microbes

Degradation of single molecules or simple mixtures
Degradation of complex pollutant mixtures
Management of environmental factors
Restoration of nutrient balance, C/N/P ratio optimization
Enhancement of pollutant bioavailability
Increase of oxygen availability
Mixing with bulking agents

Introduced strains can be inhibited by co-pollutants or native

microorganisms

Landfarming

Land tilling. Can be combined with amendments

Fertilizer addition enhanced crude oil degradation by the indigenous microorganisms. Speeding up biodegradation gave rise
to hydrocarbon losses in field as high as 1.2 %/day.
Nonetheless, after the more readily degradable components
were depleted, degradation rates decreased even when fertilizer
was reapplied (Atlas and Hazen 2011). The oleophilic fertilizer
Inipol EAP22 (Elf Aquitaine, France) and the slow-release
fertilizer Customblen 28-8-0 (Sierra Chemicals, West
Sacramento, CA, USA) were selected, based on retention time
in shorelines, bioavailability, and lack of toxicity. More than
48 tons of nitrogen-containing fertilizers was applied between
1989 and 1991. Sediment analyses carried out during 1989
indicated that 2530 % of hydrocarbons on shorelines were
removed within the first days to weeks. By 1992, only 1.3 % of
the shoreline remained polluted (Atlas and Hazen 2011; Bragg
et al. 1994). Population dynamics of oil-degrading bacteria
gave some insights into microbial response. Oil-degrading
bacteria represented about 110 % of the heterotrophic bacterial
community (15103 cells mL1) just after the oil spill in 1989.
However, late that year, oil-degrading bacteria reached 1105
cells mL1, becoming about 40 % of the heterotrophic community in oiled shoreline pore waters (Atlas and Hazen 2011).
After massive fertilizer amendments during summer 1990, oildegrading bacteria returned to background levels. This bacterial
shift and decline has been described for crude oil-degrading
bacteria afterwards (Yakimov et al. 2007).
Monitoring biodegradation after the EVOS accident left
lessons about the importance of nutrients balance for bioremediation. Appropriate nitrogen-containing fertilizer amounts
showed to be critical. Oxygen, phosphorous, hydrocarbon bioavailability, and oil age were less important (Bragg et al. 1994).

Microbial community dynamics during hydrocarbon

biodegradation
Culture of hydrocarbon-degrading bacteria and heterotrophic
bacteria is the classical methodology for assessing microbial

Optimal C/N/P ratio and pollutant bioavailability have to be

determined
Adequate pipe network design
Optimal bulking agent/soil ratio and aeration regime have to
be determined
Large soil surface is required

community changes. However, less that 1 % of environmental

bacteria are cultivable. Culture-independent techniques have
opened gateways for a deeper knowledge of microbial communities, revealing new metabolic routes and phylogenetic
groups.
Culture-independent methods provide molecular snapshots
of the microorganisms present in a specific space at a given
time. Nucleic acids can be analyzed by clone libraries or
community fingerprinting of PCR-amplified fragments of a
functional (e.g., catabolic) gene or a genetic marker, usually
the small subunit ribosomal RNA gene (Altimira et al. 2012;
Hernndez et al. 2011; Morgante et al. 2010; Smalla et al.
2007). Fluorescence in situ hybridization is a powerful technique for assessing active microbes (Morgante et al. 2010). In
the last decade, massive parallel sequencing technologies have
experienced an exponential development. Preferred platforms
for metagenomic surveys are pyrosequencing (i.e., 454 from
Roche), Illumina, and IonTorrent (Loman et al. 2012).
Metagenomic assembly allows the reconstruction of
metagenomes and metabolic networks directly from environment without PCR bias inherent to universal primers
(Narasingarao et al. 2012). Amplicon-based approaches are
used to assess phylogenetic relationships and changes among
different environments based on a genetic marker. Ecological
studies with millions of sequences per sample can be achieved
at an unprecedented depth (Caporaso et al. 2011).
Changes in microbial communities and its diversity influenced by hydrocarbon pollution and bioremediation treatments have been reported using 16S rRNA genes.
Bioaugmentation and biostimulation strategies using nitrogen,
phosphorous, or biosurfactants allow highlighting microbial
community dynamics. Changes in Proteobacteria,
Actinobacteria, Firmicutes, and Fungi were highly treatment
dependent (Grace Liu et al. 2011; Morgante et al. 2010). Main
changes in microbial community occur during the first days of
treatment (Rling et al. 2002).
Mono- and dioxygenase-encoding genes became a commonly used molecular marker in hydrocarbon biodegradation

Appl Microbiol Biotechnol

surveys due to their key role in degradation, wide phylogenetic distribution, and high sequence divergence (Iwai et al.
2011; Wang et al. 2010). These genes usually become
enriched after hydrocarbon input. Different RHD gene shifts
were observed in response to diverse PAHs. After spiking the
same soil with naphthalene, phenanthrene, or pyrene, nahAcrelated genes from Pseudomonas became enriched in
naphthalene-polluted soil (N Chadhain et al. 2006). During
phenanthrene degradation, abundance, diversity, and phylogenetic identity of enriched RHD genes were soil-type dependent (Ding et al. 2010). Similar results were observed in Arctic
soils after long-term (i.e., 1 year) diesel pollution. Levels of
RHD genes changed significantly in two soil-types during
bioremediation. Additionally, 16S rRNA, alkane
monooxygenase, and nitrate reductase encoding genes
showed different dynamics at both soils (Yergeau et al.
2009). Similar shifts have been reported in seawater, where
alkB gene copy number increased up to 100-fold in less than
1 week after pollution (Sei et al. 2003). Genes encoding
enzymes catalyzing downstream reactions seem to behave in
a similar way as RHD genes. For example, levels of catechol
2,3-dioxygenase xylE gene from (methyl)toluene degradation
pathway correlate with degradation rates in hydrocarbon polluted soils. A positive correlation between hydrocarbon degradation rate and functional alkB, xylE and nahAc genes
abundance 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 revealed
some interesting trends. At low temperatures, nutrient amendments with nitrogen and phosphorous achieved significant
degradation rates. In the northern Alert site, 58 % degradation
was achieved just 1 month after nutrient amendment, and 91 %
degradation was observed after 1 year. In the southern Eureka
site, 47 % of diesel was degraded after 1 month, but the
degradation increased up to only 52 % after 1 year (Yergeau
et al. 2009). These dissimilar results can be explained by
differences in microbial communities at both sites. RHD genes
from Gram-negative bacteria were higher in Alert site, whereas
RHD from Gram-positive strains were higher in Eureka site.
Probably Gram-negative strains responded more efficiently to
hydrocarbon pollution. In a previous survey on Arctic and
Antarctic soils, alkB genes from Rhodococcus genus were
dominant in pristine and hydrocarbon-contaminated soils,
whereas alkM genes from Acinetobacter genus were less abundant. However, alkB genes from Pseudomonas showed the
main 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 in
cold soils. Metagenomic assembly of Alert site microbial
communities showed that Gammaproteobacteria were predominant at higher degradation rates. After 1 year, abundance
of these taxa as well as degradation rates decreased. After

1 month, alkane hydroxylase and catechol 2,3-dioxygenase

genes from Gammaproteobacteria and cytochrome P450
genes from Actinobacteria were enriched, whereas
protocatechuate 3,4-dioxygenase genes from both taxa codominated (Yergeau et al. 2012). Other surveys carried out in
Antarctic soils support the role of Gammaproteobacteria in
hydrocarbon degradation, especially Pseudomonas strains
(Ruberto et al. 2003).
Hydrocarbon degradation at high altitude Alpian cold
soils showed that NPK fertilization achieved higher diesel degradation (Margesin and Schinner 2001). Higher degradation occurred at first 78 days during summer.
Nonetheless, degradation was less effective during the second summer even after a second nutrient amendment
(Margesin and Schinner 2001). During the first year, pH
decreased ~0.5, whereas after the second year, only a ~0.1
pH reduction was observed. Higher diesel decay was observed after the first 3 months. Both acidification and diesel
aging may have contributed to lower degradation efficiency
at the second year. Gamma- and Betaproteobacteria are
present at higher abundances in polluted soils (Labb
et al. 2007). Additionally, levels of alkB, xylE, and ndoB
genes from Pseudomonas, as well as alkM genes from
Acinetobacter, were higher in polluted soils than in pristine
sites (Margesin et al. 2003). These results suggest that
Proteobacteria, and particularly Gammaproteobacteria, are
the 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 temperatures during summer, the reduced bioavailability is maybe
an overlooked factor contributing to lower degradation rates
in winter. Stable isotope probing (SIP) and metagenomic
analyses also indicated that Proteobacteria dominated in
two long-term aromatic-contaminated soils after addition
of biphenyl, naphthalene, or benzoate (Uhlik et al. 2012).
The ecology of a special group of ubiquitous marine
hydrocarbon-degrading bacteria offers a model of microbial
dynamics after oils spills. Obligate hydrocarbonoclastic bacteria (OHCB) are an unusual ecological group that plays a key
role in biodegradation of petroleum hydrocarbons in marine
environments. OHCB are able to grow on a reduced spectrum
of hydrocarbons. In non-polluted marine environments,
OHCB are found in low numbers, but after an oil pollution
event, members of this group undergo a bloom (Atlas and
Philp 2005; Yakimov et al. 2004). OHCB growth is favored in
hydrocarbon-polluted seawater because they can breakdown
substrates that are useless for most bacteria (Yakimov et al.
2007). The model OHCB Alcanivorax borkumensis SK2 is
able to degrade n-alkanes of chain-length up to C32, longchain isoprenoids, phytane, pristine, and alkyl-aromatic hydrocarbons and to synthesize biosurfactants and
exopolysaccharides probably involved in biofilm formation

Appl Microbiol Biotechnol

(Schneiker et al. 2006). SK2 genome revealed multiple systems for hydrocarbon degradation pathways, i.e., two alkane
hydroxylase systems, AlkB1 and AlkB2, and three P450
cytochromes. The absence in strain SK2 of genes belonging
to glucose breakdown pathways highlights its amazing metabolic specialization (Schneiker et al. 2006).

Management of environmental conditions such as dissolved

oxygen, pH, temperature, nutrient availability, and water content will increase the bioremediation. The knowledge of the
system will be the best advisor for choosing the adequate
bioremediation strategy. Improved bioremediation strategies
are required for an efficient removal of hydrocarbons from
increasing polluted environments.

Concluding remarks and perspectives

Acknowledgments The authors gratefully acknowledge Conicyt PhD

(SF, VM), Mecesup FMS0710 PhD (PA, SF), and Fulbright (SF) fellowships. MS acknowledges financial support of FONDECYT (1110992 and
1070507) (http://www.fondecyt.cl), Conicyt-BMBF, Center for Nanotechnology and Systems Biology (http://www.usm.cl), and USM
(131342, 131109, 130948) (http://www.usm.cl) grants. The funders had
no role in study design, data collection and analyses, decision to publish,
or preparation of the manuscript.

Hydrocarbon breakdown in nature seems to be dominated by

aerobic processes. Hydroxylation is the main aerobic strategy
for hydrocarbon activation. High diversity of mono- and
dioxygenases for alkane hydroxylation has been reported.
Aromatic hydrocarbons are oxidized by RHDs, which are less
diverse than alkane oxygenases. Hydrocarbon degradation
pathways expand the microbial metabolic versatility and the
carbon source range for growth. In alkane degradation, successive oxidations produce carboxylic acids that can be degraded by the -oxidation pathway. In PAH degradation,
metabolic intermediates are channeled into central aromatic
routes such as catechol, gentisate, and protocatechuate
pathways.
In nature, hydrocarbons are mainly present as complex
mixtures. Relaxed substrate range enables a given enzyme to
transform various structurally related compounds. In addition,
same bacterial strains harbor several enzymes, which overlap
in substrate range or are expressed in different conditions.
This expands the substrate range and provides an additional
level of robustness and versatility to the system. A conserved
panorama making sense with evolution theory has been observed: Bacterial shifts occur anytime the selective pressure
comes, where bacteria capable to tolerate and use hydrocarbon
as carbon source will be selected. Bioremediation can also
impact microbial communities, favoring the growth of selected phylogenetic groups.
The genetic and biochemical basis of bacterial degradation
of aliphatic and aromatic hydrocarbons has been elucidated in
model bacteria. The role of biosurfactants on microbial degradation of hydrocarbons has been highlighted, pushing further studies to reveal the structure and the biosynthetic pathways of novel surfactants.
Culture-dependent methods indicate that three phyla degrade successfully hydrocarbons: Proteobacteria,
Actinobacteria and Firmicutes. Interestingly, cultureindependent environmental surveys tend to confirm that mainly these bacterial groups respond to oil spills. The characterization of the microbial communities involved in hydrocarbon
removal by modern approaches such as next-generation sequencing techniques, PCR, and community fingerprinting or
clone libraries, FISH, qPCR, and SIP provides critical knowledge on hydrocarbon degradation. Bioremediation is usually
case-specific; however, some general rules can be pointed out.

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