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Metabolic Pathways for Degradation of Aromatic Hydrocarbons by Bacteria
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DOI: 10.1007/978-3-319-23573-8_5
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105© Springer International Publishing Switzerland 2016
P. de Voogt (ed.), Reviews of Environmental Contamination and Toxicology
Volume 237, Reviews of Environmental Contamination and Toxicology 237,
DOI 10.1007/978-3-319-23573-8_5
Metabolic Pathways for Degradation
of Aromatic Hydrocarbons by Bacteria
Guillermo Ladino-Orjuela , Eleni Gomes , Roberto da Silva ,
Christopher Salt , and John R. Parsons
G. Ladino-Orjuela (*) • E. Gomes • R. da Silva
Laboratory of Biochemistry and Applied Microbiology, Institute of Biosciences ,
Letters and Exact Sciences (IBILCE) – São Paulo State University (Unesp) ,
Rua Cristóvão Colombo , 2265, São José do Rio Preto , São Paulo 15013-000 , Brazil
e-mail:
ambos.ong@gmail.com; eleni@ibilce.unesp.br; dasilva@ibilce.unesp.br
C. Salt • J. R. Parsons
Institute for Biodiversity and Ecosystem Dynamics (IBED) , Universiteit Van Amsterdam ,
P.O. Box 94248 , Amsterdam 1090 GE , The Netherlands
e-mail:
christopher.salt@student.uva.nl; J.R.Parsons@uva.nl
Contents
1 Introduction ....................................................................................................................... 105
2 Biodegradation of Aromatic Compounds ......................................................................... 107
2.1 Aromatic Hydrocarbon Biodegradation Under Aerobic Conditions ....................... 108
2.2 Aromatic Hydrocarbon Biodegradation Under Anaerobic Conditions.................... 112
3 Practical Applications of Knowledge About Metabolic Pathways ................................... 116
4 Summary ........................................................................................................................... 116
References ............................................................................................................................... 117
1 Introduction
The aromatic compounds present in the environment are from natural sources and
anthropogenic activities. The chemical characteristic of these compounds is the
presence of one benzene ring (monoaromatic hydrocarbon—MAHs) or more than
one fused rings (polyaromatic hydrocarbon—PAHs) (Favre and Powell 2013 ).
The ring provides structural and chemical stabilities due to a symmetric π-electron
system and therefore recalcitrance of these compounds (Vogt et al. 2011 ). In accor-
dance with Molecular Orbital Theory, in a molecule of benzene the p electrons on
106
each carbon atom are delocalized and contribute to the development of the so-called
π system. An electrostatic potential map of benzene (Fig. 1 ) shows that the electrons
in the π-system are evenly distributed around the ring (Bruice 2004 ).
Aromatic hydrocarbons are classifi ed as biological and non-biological com-
pounds. Biological aromatic hydrocarbons are produced by plants and by microor-
ganisms. In plants, this is mainly through the shikimic acid pathway (Ghosh et al.
2012 ) and microorganisms mainly via the malonic acid pathway (Zhan 2009 ).
Capsaicin, estradiol, caffeine, theobromine, gallic acid, aromatic aminoacids (tyro-
sine, phenylalanine, and tryptophan), salicylic acid and the monolignols (p- coumaryl,
coniferyl, and sinapyl) of lignin are the most well-known.
The monolignols are synthesized in plants via the shikimic acid pathway and are
the most important natural aromatic compounds since they appear in large quanti-
ties in the environment as lignin and humic acids. p-coumaryl is a minor component
of grass and forage type lignins, and coniferyl is the predominant lignin monomer
found in softwoods (hence the name). Both coniferyl and sinapyl are the building
blocks of hardwood lignin (Li and Chapple 2010 ).
The main sources of non-biological aromatics hydrocarbons are the effl uents
from fuel, chemical, plastic, explosive, ink, metal, pharmaceutical, and electric
industries among others (Table 1 ). These compounds are called xenobiotics in func-
tion of their non-biological origin and their bioaccumulation, toxicity and carcino-
genic action are well documented (USEPA 2005 ).
The physic-chemical properties of aromatic hydrocarbons have environmental
signifi cance because they determine fate in soil, water and atmosphere. For instance,
adsorption on soils or sediments, due to hydrophobicity, is a major factor in their
transportation and eventual degradation (Karickhoff 1981 ). The soil organic carbon-
water partitioning coeffi cient (K
OC
), that refl ects the ratio between the quantity of
the compound absorbed in the soil (normalized to organic carbon content) and the
concentration in water has been used in predicting the mobility and bioavailability
of organic soil contaminants. Low K
OC
values correlate to more mobile organic
chemicals and higher bioavailability (Wilczyńska-Piliszek et al. 2012 ) (Table 1 ).
The United States Environmental Protection Agency (USEPA 2005 ) published in
1986 guidelines to characterize the human carcinogenic potential of agents according
to the Weight of Evidence (WoE). The characterization was done by a six-category
Fig. 1 Electron delocalization of π-electron system in benzene ring. ( a ) Shows the sigma-bonding
framework of benzene. ( b ) Shows the p orbitals which form the delocalized π-bonding system
in benzene. ( c ) Shape of the π-electron clouds above and below the plane of the ring in benzene.
( d ) Electrostatic potential map of benzene (Bruice
2004 )
G. Ladino-Orjuela et al.
107
alphanumeric classifi cation system (A, B1, B2, C, D and E). Group A includes human
carcinogenic agents and Group E is for substances with evidence of non-carcinoge-
nicity. The approach outlined in USEPA’s guidelines for carcinogen risk assessment
(USEPA 2005 ) considers all scientifi c information in determining whether, and under
what conditions, an agent may cause cancer in humans and provides a narrative
approach to characterize carcinogenicity rather than categories (Table 1 ).
Among the techniques for the study of metabolic pathways of aromatic hydro-
carbon degradation by bacteria, the molecular biology technique Stable Isotope
Probing (SIP) is particularly interesting because it allows for detailed metabolic and
taxonomic analysis. SIP involves the incorporation of heavy isotopes (
13
C,
15
N or
18
O) into newly synthesized nucleic acids allowing the metabolic capacity of culti-
vated or uncultivated microorganisms to be linked to taxonomic identity (Aanderud
and Lennon 2011 ; Abu Laban et al. 2015 ; Cupples 2011 ; Rettedal and Brözel 2015 ;
Taubert et al. 2012 ; Zhang et al. 2012b ). Briefl y, in the nucleic acid-based SIP with
carbon, light
12
C nucleic acid and heavy
13
C nucleic acid (DNA or RNA) are sepa-
rated through ultracentrifugation and then characterized by denaturing gradient gel
electrophoresis (DGGE) or terminal restriction fragment length polymorphism
(T-RFLP) and sequencing of 16S rRNA gene. These results are used for genomic
and/or metagenomic analysis (Cupples 2011 ; Kim et al. 2014 ; Kleinsteuber et al.
2012 ; Zhang et al. 2012b ). The protein-based SIP relies on the detection and quan-
tifi cation of the peptides that incorporate heavy isotope
13
C or
15
N for proteomic
and/or metaproteomic analysis using high-resolution mass spectrometry
(Kleinsteuber et al. 2012 ; Taubert et al. 2012 ).
2 Biodegradation of Aromatic Compounds
The hydrophobicity and chemical stability of aromatic hydrocarbons, described
above, give negligible biological activity to these molecules. Therefore, to break them
down, in either aerobic or anaerobic conditions, bacteria need to destabilize the ben-
zene ring through reversible and irreversible chemical modifi cations (Díaz et al. 2013 ).
Table 1 Aromatic compounds from industrial activities
Compound Formula WoE
*
K
oc
CASRN
Aniline C
6
H
5
NH
2
B2 0.96 62-53-3
Phenol C
6
H
6
O D 1.24 108-95-2
Benzene C
6
H
6
A 1.82 71-43-2
2,4,6 Trinitrotoluene C
6
H
2
CH
3
(NO
2
)
3
C 2.48 118-96-7
Bisphenol A C
15
H
16
O
2
N.A. 2.74 80-05-7
Tetrachlorobenzene 1,2,3,4 C
6
H
2
Cl
4
N.A. 4.60 634-66-2
Benzo-a-pyrene C
20
H
12
B2 5.98 50-32-8
*
WoE weight of evidence approach, N.A. not applicable, K
oc
sorption coeffi cient (log L/Kg),
CASRN chemical abstract service registry numbers
Metabolic Pathways for Degradation of Aromatic Hydrocarbons by Bacteria
108
In both aerobic and anaerobic pathways of aromatic hydrocarbon biodegradation
a Terminal Electron Acceptor (TEA) is required. TEA determines the energy bal-
ance and the metabolic reaction used by microorganisms (Table 2 ) (Philipp and
Schink 2012 ; Schink et al. 2000 ). However, studies with microcosms and stable
isotope probing (SIP) have shown that, in environments dominated by a particular
TEA, the dominant bacterial strain was not specialized to degrade the aromatic
hydrocarbon being evaluated (Kleinsteuber et al. 2012 ; Pilloni et al. 2011 ). These
results suggest that aromatic-degrading strains are specialized and the dominant
bacterial strains are generalists and are able to use compounds other than aromatic
hydrocarbons as carbon and energy sources (Staats et al. 2011 ).
The aerobic and anaerobic processes of aromatic hydrocarbon biodegradation
have been divided (see also below) into upper pathways, which go from the original
aromatic compound to so-called central intermediates, and lower pathways, which
go from the ring cleavage of intermediates down to molecules for biomass (Cafaro
et al. 2004 ; Carmona et al. 2009 ).
2.1 Aromatic Hydrocarbon Biodegradation Under Aerobic
Conditions
In nature, oxygen is the most common and strongest oxidizing agent found (DeLaune
and Reddy 2005 ). In this sense, bacteria will fi rstly use oxygen as the TEA to
degrade aromatic hydrocarbons.
In aerobic conditions, the fi rst step of upper pathways is an oxidation catalyzed
by monooxygenases (hydroxylases) or by dioxygenases (Huijbers et al. 2014 ;
Parales and Resnick 2004 ).
The monooxygenases catalyze the cleavage of the oxygen-oxygen bond of O
2
,
inserting one oxygen atom into the aromatic ring while the other is reduced to H
2
O
(Fig. 2 ). These enzymes are classifi ed in eight groups according to their structure,
sequence, type of reaction catalyzed and type of electron donor. The group A–B
Table 2 TEAs (terminal electron acceptors) used by bacteria for aromatic hydrocarbon degradation
Anaerobic Aerobic
Environmental
condition
Highly
reduced Reduced
Moderately
reduced
Oxidized Redox condition
CO
2
SO
4
2−
Fe(III) Mn(IV) NO
3
−
O
2
Terminal Electron
Acceptor (TEA)
CH
4
HS
−
Fe(II) Mn(II) NO
2
H
2
O Products
Anaerobic
Facultative
Aerobic Microbial metabolism
−300 −200 −100 0 +100 +200 +300 +400 +500 +600 +700 E° (mV)
Downward arrow indicates products of TEAs reduction. Dashed arrow indicates sequential order
of TEAs preferences from higher redox potential (E° = +) to lower redox potential (E° = −)
(DeLaune and Reddy
2005 ; Gibson and Harwood C 2002 )
G. Ladino-Orjuela et al.
