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Petrochemical Industrial Waste: Bioremediation Techniques
An Overview
Sheetal Koul and MH Fulekar*
Department of Life Sciences, University of Mumbai, Santacruz (E), Mumbai- 400 098, India
*
School of Environment and Sustainable development, Central University of Gujarat, Gandhinagar –
482030, India
*
Email: mhfulekar@yahoo.com
ABSTRACT
The petrochemical industry is one such major source of hazardous waste, produced during
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manufacture of Petrochemical products. These wastes are often released in the environment.
Petrochemical Solid waste is generally associated with more hazardous constituents and
accordingly carries a higher level of public health and environmental risk potential. In the
present paper the petrochemical waste compound in particular Polycyclic Aromatic
Hyrocarbons (PAH) were described as persistent pollutant in soil-water environment. The
microbial sources which have been found and reported for PAH compound degradation have
been cited with examples viz: potential species of Bacteria, species of Fungal and
actinomycetes have been described for microbial degradation. The factors influencing
microbial degradation including the influence of GMO’s on bioremediation have been cited
in the paper. This would serve as a bioremediation technique for microbial degradation of
petrochemical waste.
Keywords:
Bioremediation, Polycyclic Aromatic Hydrocarbons’ (PAH), Petrochemical
Solid waste, GMO’s
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212
Introduction:
The persistence of a pollutant in the environment is influenced by the nature of the
contaminant, the amount of the contaminant present and the interplay between chemical,
geological, physical and biological characteristics of the contaminated site. In the present era,
petroleum hydrocarbon contamination is considered a major widespread environmental
problem distributed in atmosphere, terrestrial soil, marine waters and sediments [1].
Anthropogenic activities which include improper management and disposal of oil sludge
waste results in leaks and accidental spills during the exploration, production, refining,
transport and storage of petroleum products. Polycyclic aromatic hydrocarbons (PAHs) are
one class of toxic environmental pollutants and perhaps the first recognized environmental
carcinogens that have accumulated in the environment whether accidentally or due to human
activities [2].
Polycyclic aromatic Hydrocarbons (PAHs) are fused ring hydrocarbon compounds
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that are highly recalcitrant under normal conditions due to their structural complexity, strong
molecular bonds, low volatility and aqueous solubility and high affinity for soil material and
particulate matter [3]. Overtime, they accumulate in the surrounding soil sediments and
ground water causing extensive damage to animal tissues due to their carcinogenic,
mutagenic and potentially immune toxicant properties. [4], [5]. Although in the natural
environments they are readily degraded by indigenous microbial communities, these
processes are very time consuming. Various physical and chemical applications like
mechanical burying, evaporation, dispersion and washing are currently employed to
remediate the problems caused by PAHs pollution. However, these forms of treatments are
either expensive or can lead to incomplete decomposition of contaminants [6].
The process of bioremediation, defined as the use of microorganisms to detoxify or
remove pollutants owing to their diverse metabolic capabilities is an evolving method for the
removal and degradation of many environmental pollutants including the products of
petroleum industry [7]. In addition, bioremediation technology is believed to be non-invasive
and relatively cost effective [8]. Bioremediation by natural populations of microorganisms
represents one of the primary mechanisms by which petroleum and other hydrocarbon
pollutants can be removed from the environment [9] and is cheaper than other remediation
technologies [10].The success of PAH degradation and bioremediation depends on one’s
ability to establish and maintain conditions that favour enhanced oil biodegradation rates in
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the contaminated environment which include the employment of correct population of
microorganism [3] with the appropriate metabolic capabilities [6] and ensuring adequate
concentration
of
nutrients,
oxygen
and
optimal
pH
and
temperature
[11],[12],[13],[14],[15].The characterization of novel catalytic mechanisms, physiological
adaptations of microbes, biochemical mechanisms involved in hydrocarbons accession,
uptake and the application of genetically engineered and enhanced microbes for
bioremediation are the recent advances employed in the removal of persistent organic
pollutants like PAHs.
Therefore, the intent of this review is to update information on microbial degradation
of Polycyclic Aromatic Hydrocarbons towards the better understanding in bioremediation
challenges.
2. Polycyclic Aromatic Hydrocarbons (PAHs)
2.1 Physical and chemical properties:
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Polycyclic Aromatic Hydrocarbons or PAHs as they are fondly called are chemical
compounds made up of two or more fused benzene rings [3] and some “Penta-cyclic
Moieties” in linear, angular, and/or cluster arrangements [16].
Many PAHs contain a “bay-region” and a “K-region”. The bays-and K-region
epoxides, which can be formed metabolically, are highly reactive both chemically and
biologically. Phenanthrene is the simplest aromatic hydrocarbon which contains these
regions. The bay-region of phenanthrene is a sterically hindered area between carbon atom 4
and 5 and the K-region is the 9, 10 double bond [17] According to the Schmidt-Pullman,
electronic theory K-region epoxides should be more Carcinogenic than the parent
hydrocarbon.[17].
PAHs generally accumulate in the environment because they are thermodynamically
stable compounds, due to their large negative resonance energies; they have low aqueous
solubility’s and they absorb to soil particles [16].Generally solubility decreases and
hydrophobicity increases with an increase in number of fused benzene rings also volatility
decreases with an increasing number of fused rings [18] High molecular weight [HMW]
PAHs (four or more rings) sorb strongly to soils and sediments and are more resistant to
microbial degradation. Because of solid state, high molecular weight and hydrophobicity
expressed as its log P value between 3 and 5, PAHs are very toxic to whole cells [19].
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Figure1. PAHs representatives and their chemical structures [17]
Table 1. Structure and physical-chemical properties of some three-, four-, five- and six-ring polycyclic aromatic
hydrocarbons . [20]
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No. of
rings
mpa (oC)
bpb(oC)
Solc(mg l-1)
log K p d
Vapour pressure
(torr at 20(oC)
3
101
340
1.29
4.46
6.8 x10-4
3
216
340
4.45
0.07
2.0 x 10-4
Fluoranthene
4
111
250
0.26
5.33
6.0 x 10-6
Benz[a ]anthracene
4
158
400
0.014
5.61
5.0 x 10-9
Pyrene
4
149
360
0.14
5.32
6.8 x 10-7
Chrysene
4
255
488
0.002
5.61
6.3 x 10-7
Benzo[a ]pyrene
5
179
496
0.0038
6.04
5.0 x 10-7
Dibenz[a,h]anthracene
5
262
524
0.0005
5.97
1.0 x 10-10
Benzo[g,h,i]perylene
6
222
-
0.0003
7.23
1.0 x 10-10
Indeno[1,2,3-c,d ]pyrene
6
6163
0.062
7.66
1.0 x 10-10
PAH
Phenanthrene
Anthracene
536
a
mp: melting point;bbp: boiling point; c Sol: aqueous solubility.
d
log K p : logarithm of theoctanol:water partitioning coefficient.
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2.2 Production, Occurrence and Toxicity of PAHs
The major source of PAHs is from the incomplete combustion of organic material
(Guerin & Jones., 1988). PAHs are formed naturally during thermal geologic production and
during burning of vegetation in forest and bush fires [21].In Industrial countries, anthrogenic
combustion activities are a principal source of PAHs in soils where they arise from
atmospheric deposition [22]. PAHs have been detected in a wide variety of environmental
samples including air [23],soil [22], sediments [24],water, oils, tars and foodstuff [25],
[26].Oil leakage from storage tank bottoms, oil-water separators, and drilling operations etc.
has led to an increase in soil PAH concentration over the last few decades [17] PAHs are also
a major constituent of Creosote (approximately 85% PAH by weight) and anthracene oil,
which are commonly used pesticides for wood treatment. [27].These contaminated soils vary
in hydrocarbon composition.
Table2. Characteristics of a typical PAH-contaminated soil [28].
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PAH-Contaminated areas pose a health risk to humans [29]. One-, two and three-ring
compounds are acutely toxic [20]. Low Molecular weight PAH pollutants exert toxic,
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mutagenic, carcinogenic and potential endocrine-disrupting properties [29],while higher
molecular weight PAHs are considered to be Genotoxic [30].
Table 3.standard limiting PAH content (µg/kg) in the soil surface layer [31]
Total PAH Content
Pollution Class
< 200
Soil Assessment
0
Unpolluted (natural content)
200-600
I
Unpolluted (natural content)
600-1000
II
slightly polluted
1000-5000
III
Polluted
5000-10000
IV
Heavily Polluted
>10000
V
Very heavily polluted
3. Biodegradation of PAHs- Bioremediation Strategies
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Biodegradation of petroleum hydrocarbons particularly PAHs is a complex process.
Petroleum hydrocarbon compound bind to soil components, and they are difficult to degrade
[32]. The natural presence of PAHs in the environment allows many microorganisms to adapt
to the use and exploitation of these naturally occurring potential growth substrates. Thus
many bacterial, fungal and algal strains have been shown to degrade a wide variety of PAHs
containing from three to five aromatic rings [27].
3.1General Aspects of PAH-Degradation:
The fate of PAHs in the soil very much depends on the physical characteristics of the
PAH constituents, like molecular size and the topology of the compound [33] For low
molecular weight (LMW) PAHs, removal through evaporation is the first line of elimination
[3].
Generally the increase in molecular size and angularity of the PAH compound results
in a concomitant increase in hydrophobicity and electrochemical stability and due to their
lipophilic nature, PAH have potential for bio-magnification through tropic transfers
[33].However, with prolonged exposure to soil particles, bioavailability is greatly reduced
and biodegradation rate become slower, therefore in order to enhance the biodegradation
process, bioavailability of PAHs in soil needs to be increased [34].The indigenous organisms
have only limited capacity to degrade all the fractions of hydrocarbons present, hence, a
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consortia consisting of outside microbes isolated from the contaminated sites and the
indigenous microorganisms can degrade the constituents and have a higher tolerance to
toxicity [35]. The efficiency range for biodegradation of hydrocarbons by bacteria, yeast and
fungi is reported to be 6%-82% [36] for soil fungi, 0.13%-50% [37],[38] for soil bacteria and
0.003%-100% [39] [40] for marine bacteria.
3.2Bacterial Degradation
Bacteria are the most active agents in petroleum degradation and they work as
primary degraders of spilled oil in environment [40],[39],Bacteria initially oxidise aromatic
hydrocarbons to cis-dihydrodiols [41],[42], [43] by incorporating both atoms of molecular
oxygen, catalysed by a dioxygenase into the aromatic ring to produce a cis-dihydrodiol [44].
The dioxygenase that catalyses these initial reactions is a multicomponent enzyme
system. The initial ring oxidation is usually the rate-limiting step in the biodegradation
reaction of PAH [45] Cis-dihydrodiols are re-aromatised through a cis-dihydrodiol
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dehydrogenase to yield a dihydroxylated derivative [41].Further oxidation of the cisdihydrodiols leads to the formation of catechols [46] via a NAD+ dependent dehydrogenation
reaction. The important step in catabolism of PAHs is ring fission by dioxygenase enzymes
that cleave the aromatic ring to give aliphatic intermediates [47] Catechol can be oxidised via
two pathways, the Ortho-pathway involves cleavage of the bond between carbon atoms with
a hydroxyl group. Ring cleavage results in the production of succinic, fumaric, pyruvic and
acetic acids and aldehydes, all of which are utilised by micro-organisms for the synthesis of
cellular constituents and energy [48] A by-product of these reactions is the production of
carbon dioxide and water.
Once the initial hydroxylated aromatic ring of the PAH is degraded to pyruvic acid
and carbon dioxide, the second ring is then attached in the same manner. High molecular
weight PAH’s such as benzo(a)pyrene (BaP) are only degraded with difficulty. However
degradation has been observed via a co-oxidation or co-metabolism mechanism through less
recalcitrant compound.
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Figure 2: Microbial metabolism of the aromatic ring by ortho or meta cleavage [45]
A great diversity of bacterial genera have been repeatedly isolated mainly from the
soil that are capable of degrading the low molecular weight PAHs like naphthalene,
acenapthanes and phenantherne, these are usually gram-negative bacteria, most of them
belong to the genus Pseudomonas. The biodegradative pathways have also been reported in
bacteria from the genera Mycobacterium ,Corynebacterium, Acromonas, Rhodococcusand
Bacillus [49], [50].
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Table 4: Polycyclic aromatic hydrocarbons oxidised by different species of bacteria [45]
PAH
Naphthalene
Organisms
References
Acinetobacter calcoaceticus, Alcaligenes
Ryu et al. (1989), Weissenfels et al. (1990, 1991), Kelly et al. (1991), Dunn
denitrificans, Mycobacterium sp., Pseudomonas
and Gunsalus (1973), Davies and Evans (1964), Foght and Westlake (1988),
sp., P. putida, P. fluorescens, Sp.
Jeerey et al. (1975), Mueller et al. (1990a), Kuhm et al. (1991), Walter et al.
paucimobilis,Brevundimonasvesicularis,
(1991), Dua and Meera (1981), Tagger et al. (1990), Garcia-Valdes et al.
Burkholderiacepacia, Comamonastestosteroni,
(1988), Trower et al. (1988), Grund et al. (1992), Barnsley (1975a);
Rhodococcus sp., Corynebacteriumrenale,
Barnsley (1983a), Yang et al. (1994), Burd and Ward (1996), Allen et al.
Moraxella sp.,Streptomyces sp., B. cereus, P.
(1997), Stringfellow and Aitken (1995), Filonov et al. (1999), Hedlund et al.
marginalis, P. stutzeri, P. saccharophila,
(1999), Geiselbrecht et al. (1998), Foght and Westlake (1996), Goyal and
Neptunomonasnaphthovorans, Cycloclasticus sp.
Beijernickia
sp.,
fluorescens,Bu.cepacia,
Acenaphthene
P.
putida,
P.
Pseudomonas
sp.,
Cycloclasticus sp., Neptunomonasnaphthovorans,
Zylstra (1996)
Chapman (1979), Schocken and Gibson (1984), Ellis et al. (1991),
Geiselbrecht et al. (1998), Hedlund et al. (1999), Selifonov et al. (1993)
Alcaligeneseutrophus,
Alcaligenesparadoxus
Phenanthrene
Aeromonas sp., A. faecalis, A. denitrificans,
Kiyohara et al. (1976, 1982, 1990), Weissenfels et al. (1990, 1991), Keuth
Arthrobacter polychromogenes, Beijernickia sp.,
and Rehm (1991), Jerina et al. (1976), Colla et al. (1959), West et al. (1984),
Micrococcus sp., Mycobacterium sp., P. putida,
Kiyohara and Nagao (1978), Heitkamp and Cerniglia (1988), Guerin and
Sp. paucimobilis, Rhodococcus sp., Vibrio sp.,
Jones (1988a, 1989), Treccani et al. (1954), Evans et al. (1965), Foght and
Nocardia sp., Flavobacterium sp., Streptomyces
Westlake (1988), Mueller et al. (1990b), Sutherland et al. (1990), Ghosh and
sp., S. griseus, Acinetobacter sp., P. aeruginosa,
Mishra (1983), Savino and Lollini (1977), Trower et al. (1988), Barnsley
P. stutzeri, P. saccharophila, Stenotrophomonas
(1983b), Yang et al. (1994), Kohler et al. (1994), Stringfellow and Aitken
maltophilia, Cycloclasticus sp., P. uorescens,
(1995), Boonchan (1998), Juhasz (1998), Geiselbrecht et al. (1998), Foght
Acinetobacter calcoaceticus,
and Westlake (1996),
Acidovorax dela®eldii, Gordona sp.,
Kastner et al. (1998), Lal and Khanna (1996), Shuttleworth and Cerniglia
Sphingomonas sp., Comamonas
(1996), Mahro et al. (1995), Goyal and Zylstra (1996), Dyksterhouse et al.
testosteroni, Cycloclasticus pugetii, Sp.
(1995), Allen et al. (1999), Aitken et al. (1998), Romero et al. (1998),
yanoikuyae, Agrobacterium sp.,
Iwabuchi et al. (1998), Churchill et al. (1999),
Bacillus sp., Burkholderia sp., Sphingomonas sp.,
Juhasz (1991)
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Pseudomonas sp., Rhodotorula glutinis,
Nocardioides sp., Flavobacterium gondwanense,
Halomonas meridiana
Anthracene
Beijernickia sp., Mycobacterium sp., P. putida,
Colla et al. (1959), Akhtar et al. (1975), Jerina et al. (1976), Evans et al.
Sp. paucimobilis, Bu.
(1965), Ellis et al. (1991), Weissenfels et al. (1991), Foght and Westlake
cepacia, Rhodococcus sp., Flavobacterium sp.,
(1988), Walter et al. (1991), Mueller et al. (1990a), Savino and Lollini
Arthrobacter sp., P. marginalis, Cycloclasticus
(1977), Tongpim and Pickard (1996), Burd and Ward (1996), Geiselbrecht
sp., P. fluorescens, Sp. yanoikuyae,
et al. (1998), Foght and Westlake (1996), Kim et
Acinetobactercalcoaceticus, Gordona sp.,
al. (1997), Lal and Khanna (1996), Mahro et al. (1995), Goyal and Zylstra
Sphingomonas sp.,
(1996), Dyksterhouse et al. (1995), Allen et al. (1999)
Comamonastestosteroni,Cycloclasticuspugetii
Fluoranthene
A. denitrificans, Mycobacterium sp., P. putida, Sp.
Kelly and Cerniglia (1991), Walter et al. (1991), Weissenfels et al.
paucimobilis, Bu.
(1991), Foght and Westlake (1988), Barnsley (1975b), Mueller et al.
cepacia, Rhodococcus sp., Pseudomonas sp.,
(1990a, 1990b), Ye et al. (1996), Kelly et al. (1993), Boonchan (1998),
Stenotrophomonas
Juhasz (1998), Lal and Khanna (1996), Shuttleworth and Cerniglia (1996),
maltophilia, Acinetobactercalcoaceticus,
Mahro et al. (1995), Willumsen and Karlson (1998), Sepic et al. (1998),
Acidovoraxdelafieldii, Gordona
Willumsen et al. (1998), Churchill et al. (1999), Chen and Aitken (1999)
sp., Sphingomonas sp., P. saccharophilia,
Pasteurella sp.
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Pyrene
220
A. denitrificans, Mycobacterium sp., Rhodococcus
Heitkamp et al. (1988a), Walter et al. (1991), Weissenfels et al. (1991),
sp., Sp. paucimobilis,
Grosser et al. (1991), Schneider et al. (1996), Ye et al. (1996),
Stenotrophomonasmaltophilia,
Boonchan (1998), Juhasz (1998), Kastner et al. (1998), Lal and Khanna
Acinetobactercalcoaceticus, Gordona sp.,
(1996), Mahro et al. (1995), McNally et al. (1999), Jimenez and Bartha
Sphingomonas sp., P. putida, Bu cepacia, P.
(1996), Churchill et al. (1999), Juhasz et al. (1997), Chen and Aitken (1999)
saccharophilia
Chrysene
Rhodococcus sp., P. marginalis, Sp. paucimobilis,
Walter et al. (1991), Burd and Ward (1996), Ye et al. (1996), Boonchan
Stenotrophomonas
(1998), Lal and Khanna (1996), Aitken et al. (1998), Chen and Aitken
maltophilia, Acinetobacter calcoaceticus,
(1999)
Agrobacterium sp., Bacillus sp.,
Burkholderia sp., Sphingomonas sp.,
Pseudomonas sp., P. saccharophilia
Benz[a]
A. denitrificans, Beijernickia sp., P. putida, Sp.
Gibson et al. (1975), Maha€ey et al. (1988), Weissenfels et al. (1991),
anthracene
paucimobilis,
Schneider et al. (1996), Ye et al. (1996), Boonchan (1998), Juhasz (1998),
Stenotrophomonasmaltophilia, Agrobacterium
Aitken et al. (1998), Chen and Aitken (1999)
sp., Bacillus sp.,
Burkholderia sp., Sphingomonas sp.,
Pseudomonas sp., P. saccharophilia
Dibenz[a,h]
Sp. paucimobilis, Stenotrophomonasmaltophilia
Ye et al. (1996), Boonchan (1998), Juhasz (1998)
anthracene
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3.3 Anaerobic PAH Degradation:
Rapid depletion of dissolved oxygen during PAH degradation results in decrease of redox
potential which eventually favours the growth of denitrifying, sulphate reducing and even
methanogenic microbial population [51], [52] proposed links of bacterial genera like
Bacillus, Rhodococcus and Herbaspirillum with anaerobic anthracene degradation using
16srRNA analysis under methanogenic conditions in an aquifer.[53] suggested that the
archaeal members most closely affiliated with Methanosaeta and Methanocellues and
bacterial members most closely realted to “Clostridiaceae” play an important role in
methanogenic metabolism of substituted.
There has been tremendous interest in understanding the fate of PAHs in ground
water subsurface environments, that are largely micro-aerobic or anaerobic [51]. In anaerobic
conditions like that of aquifers the degradation of PAH compounds depend on the availability
of suitable nutrients and soil microorganisms which can degrade the compounds through
utilization of electron acceptors other than oxygen.[54]. Microbial transformation of aromatic
compounds under denitrifying, sulphate-reducing and methanogenic conditions, however is
fundamentally different from degradation under aerobic condition [51]
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Kartikeyan A & Bandari A [51] summarized the suggested pathways of anaerobic bacterial
degradation
involving
enzymes
that
include
CoA
ligase,
oxido-reductases
and
decarboxylases. The possible peripheral metabolic reactions occurring during the anaerobic
transformation process are carboxylation, reductive dehydroxylation, reductive deamination,
reductive dehydroxylation, oxidation of carboxymethyl groups, methyl oxidation,odemethylation, trans- hydroxylation and decarboxylation. [55] identified carboxylation
reaction as a prototype for the initial activation of anaerobic degradation of naphthalene.
Meckenstock., etal [56], studied the degradation of naphthalene by sulphate-reducing culture
isolated from a freshwater source. The authors suggested a stepwise reduction of the aromatic
ring system before ring cleavage. The first step is the carboxylation of the aromatic ring to 2naphthoic acid, which may activate the aromatic ring prior to hydrolysis. Stepwise reduction
of 2-naphthoic acid via a series of hydrogenation reactions results in decaclin-2-carboxylic
acid which is subsequently converted to decahydro-2-naphthoic acid [57]
In
a
similar
study by
[58],
anaerobic
degradation
of
naphthalene,
2-
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methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a
sulfate-reducing enrichment culture obtained from a contaminated aquifer (fig) .It was
proposed that the naphthalene (compound A) or2-methylnaphthalene (B) is activated in
peripheral, upper degradationpathways to generate 2-naphthoic acid (I), naphthoic acid isthen
reduced to 5,6,7,8-tetrahydro-2-naphthoic acid (II), whichis also the entry of anaerobic
tetralin (C) degradation into thepathway. A further hydrogenation may lead to octahydro-2napthoic acid (III), which could be hydrated to generate hydroxydecahydro-2-naphthoic acid
(V) and subsequently oxidizedto oxodecahydro-2-naphthoic acid (VI). Decahydro-2naphthoic acid (IV) is probably a dead-end metabolite. Athiolytic ring cleavage and a
subsequent oxidation can generatethe tentatively identified C 11 H 16 O 4 -diacid (VII).
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Fig3: Proposed reductive 2-naphthoic acid pathway of anaerobic PAH degradation. (I) 2-naphthoic acid, (II)
5,6,7,8-tetrahydro-2-naphthoic acid, (III) hydroxydecahydro-2-naphthoic acid, (IV) b-oxo-decahydro-2
naphthoic acid, (V) C 11 H16 O 4 -diacid, (VI) 2- carboxycyclohexylacetic acid. Compounds III and IV are putative
intermediates. [58]
3.4 Alkyl Substituted Poly Aromatic Hydrocarbons:
The ring structure of PAH compounds are generally substituted with alkyl groups
having one to four saturated carbon atoms, producing different structural isomers and
homologs for each poly aromatic hydrocarbon family. Abundance of alkyl substituted PAHs
such as alkyl naphthalenes, alkyl phenanthrenes and alkyl dibenzothiophenes are found in
fossil fuels, crude oil, petroleum and petroleum derived products [59].Alkylated PAH’s are
found to be more abundant, less water soluble and persistent than their respective “Parent”
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PAH
compound
[60].
Alkyl
PAHs
like
1-Methylanthracene
223
and
7,
12-
Diethylbenzo[a]anthracene are among the 16 Priority pollutant PAHs designated by US-EPA.
Within an aromatic series, acute toxicity increases with increasing alkyl substitution on the
aromatic nucleus [60].
Since the presence of alkyl branch inhibits the proper orientation and accessibility of PAHs to
dioxygenase enzyme resulting the involvement of more diverse enzymes. These include
oxidation of methyl group to alcohol, aldehyde, or carboxylic acid, decarboxylation,
demethylation, and dioxygenation [59].
Mahajan et al [61], suggested the possibility of occurrence of multiple pathways in the
degradation of 1 and 2 methyl naphthalene. Enzyme activity study of Pseudomonas putida
CSV86, growing on 1&2 methyl naphthalene in one of the proposed pathway suggests the
oxidation of the aromatic ring adjacent to the one bearing the methyl moiety ,leading to the
formation of methyl-salicylates and methyl catechols. In the other pathway the methyl side
chain is hydroxylated to –CH 2 OH which is further converted to –CHO and –COOH, resulting
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in the formation of napthoic acid as the end product.
Annweiler et al, [60] investigated the anaerobic degradation of 2-methyl naphthalene
by a sulphate reducing enrichment culture. The findings proposed the addition of fumarate to
the methyl group of 2-methylnapthalene as the first activation step by naphthyl-2-methyl
succinate synthase, Napthyl-2-methyl-succinic acid is activated by a succinyl CoA dependent
CoA transferase and subsequent oxidation to yield napthyl-2 methylene succinyl CoA. A
sequence of reactions proceeds via beta oxidation and leads to the 2-napthoic acid CoA ester
intermediate.(Fig 4).
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FIG 4.. Proposed scheme of the upper pathway of anaerobic 2-methylnaphthalene (1) degradation to the central
intermediate 2-naphthoic acid ( 2), fumaric acid; (3), naphthyl-2-methyl-succinic acid;(4), naphthyl-2-methylsuccinyl- CoA; (5), naphthyl-2-methylene-succinyl-CoA;(6), naphthyl-2-hydroxymethyl-succinylCoA; (7),
naphthyl-2-oxomethyl-succinyl-CoA. Asterisk marked compounds were identified as free acids.
3.5Fungal Degradation
Some fungi have the enzymatic apparatus to degrade a wide variety of structurally
diverse PAHs via pathways that are generally similar to those used by mammalian enzyme
systems [16]. Many fungi oxidize PAHs via a cytochrome P-450 monooxygenase by
incorporating one atom of the oxygen molecule into the PAH to form an arene oxide and the
other atom into water. Most arene oxides are unstable and can undergo either enzymatic
hydration via epoxide hydrolase to form trans-dihydrodiols or nonenzymatic rearrangement
to form phenols, which can be conjugated with sulfate, glucose, xylose, or glucuronic acid.
Whereas a diverse group of non ligninolytic fungi is able to oxidize PAHs to transCopyright © 2013 SciResPub.
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dihydrodiols, phenols, tetralones, quinones, dihydrodiol epoxides, and various conjugate of
the hydroxylated intermediates, only a few have the ability to degrade PAHs to CO 2 [16].
Fungal genera, namely, Amorphoteca, Neosartorya, Talaromyces, and Graphiumwere
isolated from petroleum contaminated soil and proved to be the potential organisms for
hydrocarbon degradation [62] .A group of terrestrial fungi, namely, Aspergillus,
Cephalosporium, and Pencillium which were also found to be the potential degrader of crude
oil hydrocarbons [63]. The yeast species, namely, Candida lipolytica, Rhodotorula
mucilaginosa, Geotrichum sp, and Trichosporon mucoides isolated from contaminated water
were noted to degrade petroleum compounds [64].
White-rot fungi have remarkable potential to degrade PAHs. Degradation of substrate
is carried out through the action of extracellular enzymes, the best characterized of which are
laccase, lignin peroxidases and manganese peroxidases. The specificity of these enzymes is
low, so the substrate spectrum is broad. Other advantage of this non-specific mode of enzyme
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action is that it does not require preconditioning to individual pollutants and avoids the
uptake of potentially toxic substances [65]. Furthermore, because the induction of the
degradative enzymes is independent of the presence of the pollutants, the fungi can degrade
pollutants at extremely low concentrations. White-rot fungus, Phanerochaete chrysosporium,
has been reported to mineralise phenanthrene, fluorene, fluoranthene, anthracene and pyrene
in nutrient-limited cultures [66]. Degradation of BaP to carbon dioxide and water has also
been reported [67].
However, since a huge amount of fungal inoculum is required for bioremediation, it
makes this technique costly also achieving homologous distribution of mycelium into the soil
remains a setback for employment of fungal strains for bioremediation [16].
.
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Table 5: Polycyclic aromatic hydrocarbons oxidised by different species of fungi
PAH
Organisms
References
Absidaglauca, Aspergillusniger, Basidiobolusranarum, Candida utilis,
Choanephoracampincta, Circinella sp., Clavicepspaspali, Cokeromyces
poitrassi, Conidiobolusgonimodes, C. bainieri, C. elegans, C. japonica,
Naphthalene
Emericellopsis sp., Epicoccumnigrum, Gilbertellapersicaria,
Gliocladium sp., Helicostylumpiriforme, Hyphochytriumcatenoides,
Linderinapennispora, Mucorhiemalis, Neurosporacrassa,
Cerniglia and Gibson (1977), Cerniglia
Panaeoluscambodginensis, Panaeolus subbalteatus,
et al. (1978, 1982a),
Penicilliumchrysogenum, Pestalotia sp., Phlyctochytrium
Smith and Rosazza (1974), Cerniglia
reinboldtae, Phycomyesblakesleeanus, Phytophthoracinnamomi,
and Crow (1981), Ferris etal. (1973)
Psilocybe cubensis, Psilocybestrictipes, Psilocybestuntzii,
Psilocybesubaeruginascens,
Rhizophlyctisharderi, Rhizophlyctisrosea, Rhizopusoryzae, Rhizopus
stolonifer, S. cervisiae, Saprolegniaparasitica, Smittiumculicis, Smittium
culisetae, Smittiumsimulii, Sordaria ®micola,
Syncephalastrumracemosum,
Thamnidiumanomalum, Zygorhynchusmoelleri
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Pothuluri et al. (1992a), Johannes et al.
Acenaphthene
C. elegans, T, versicolor
(1998)
Cerniglia and Yang (1984), Cerniglia et
Phenanthrene
C. elegans, P. chrysosporium, P. laevis, Pleurotusostreatus, T. versicolor,
al. (1989), Morgan et al. (1991),
Bjerkanderaadjusta, Pleurotusostreatus, Cylindrocladium simplex,
Sutherland et al. (1991), Bumpus
Monosporiumolivaceum, Curvularialunata, Curvulariatuberculata,
(1989), Hammel et al. (1992), Bezalel et
Laetiporus
al. (1996), Brodkorb and Legge (1992),
sulphureus, Daedaelaquercina, Flamulinavelutipes, marasmiellus sp.,
Schutzendubel et al. (1999), Lisowska
Penicullium sp., Kuehneromycesmutabilis, Laetiporussulphureus,
and Dlugonski (1999), Bogan and
Agrocybe aegerita, Aspergillusniger, Syncephalastrumracemosum
Lamar (1996), Sack & Gunther (1993),
Sack et al. (1997a), Collins and Dobson
(1996), Casillas et al. (1996), Sutherland
et al. (1993)
Anthracene
Bjerkandera sp., Bjerkanderaadjusta, C. elegans, P. chrysosporium, P.
Cerniglia (1982), Cerniglia and Yang
laevis, Ramaria sp., R. solani, Trametesversicolor, Pleurotusostreatus,
(1984), Hammel et al.
Cylindrocladium simplex, Monosporiumolivaceum, Curvularialunata,
(1991), Sutherland et al. (1992), Field et
Curvulariatuberculata, Cryphonectriap arasitica,
al. (1992), Collins et al.
Ceriporiopsissubvermispora, Oxysporus sp., Cladosporiumherbarum,
(1996), Schutzendubel et al. (1999),
Drechsleraspicifera, Verticillium
Lisowska and Dlugonski (1999),
lecanii, Fusariummoniliforme, Rhizopusarrizus, Coriolopsispolyzona,
Krivobok et al. (1998), Andersson and
Laetiporussulphureus, Daedaelaquercina, Flamulinavelutipes,
Henrysson (1996), Johannes et al.
marasmiellus sp., Penicullium sp.
(1996), Bogan et al. (1996), Bogan and
Lamar (1996), Vyas et al. (1994), Sack
and Gunther (1993)
C. elegans, C. blackesleeana, C. echinulata, Bjerkanderaadjusta,
Fluoranthene
Pleurotus ostreatus, Sporormiellaaustralis, Cryptococcus albidus,
Pothuluri et al. (1990, 1992b),
Cicinoboluscesatii, Pestalotiapalmarum, Beauveria alba,
Schutzendubel et al. (1999), Salicis
Aspergillusterreus, Mortierella ramanniana, Rhizopusarrhizus,
et al. (1999), Sack and Gunther (1993)
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Laetiporussulphureus, Daedaelaquercina,
Flamulinavelutipes, marasmiellus sp., Penicullium sp.
Pyrene
C. elegans, P. chrysosporium, Penicillium sp., P. janthinellum, P.
Cerniglia et al. (1986), Hammel et al.
glabrum, P.ostreatus, Syncephalastrumracemosum, Bjerkanderaadjusta,
(1986), Launen et al.(1995), Bezelel et
Pleurotus sp., Dichomitussqualens, Flammulinavelutipe,
al. (1996), Schutzendubel et al. (1999),
Trammetesversicolor, Kuehneromycesmutabilis, Laetiporussulphureus,
Martens & Zadrazil (1998), Sack et al.
Agrocybeaegerita
(1997b), Wunder et al. (1997), Sack and
Fritsche (1997), Lang et al. (1996),
Stanley et al.(1999), Boonchan (1998)
Cerniglia et al. (1980a), Andersson and
Chrysene
P. janthinellum, Syncephalastrumracemosus, Penicillium sp.
Henrysson (1996), Bogan
and Lamar (1996), Stanley et al. (1999),
Boonchan (1998)
Kiehlmann et al. (1996), Stanley et al.
Benz[a]
anthracene
C. elegans, Trametesversicolor, P. laevis, P. janthinellum
(1999), Boonchan (1998)
Dibenz[a,h]anth
Trametesversicolor, P. janthinellum
Andersson and Henrysson (1996),
Stanley et al. (1999), Boonchan (1998)
racene
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3.6` Degradation of PAH compounds by Algae and Cyanobacteria.
Algae and cyanobacteria have also been shown to oxidise PAH [2]. Eukaryotic algae
and cyanobacteria (blue-green algae) oxidize PAHs under photoautotrophic conditions to
form hydroxylated intermediates. Cyanobacteria oxidize naphthalene and phenanthrene to
metabolites that are similar to those formed by mammals and fungi. In contrast, the green
alga Selenastrum capricornutum oxidizes benzo[a]pyrene to isomeric cis-dihydrodiols similar
to bacterial metabolites [68].
Walker et al [69] isolated an alga, Protothecazopfi which was capable of utilizing
crude oil and a mixed hydrocarbon substrate and exhibited extensive degradation of n-alkanes
and iso-alkanes as well as aromatic hydrocarbons.
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Table 6:Polycyclic aromatic hydrocarbons oxidised by different species of cyanobacteria and algae [45]
PAH
Organism
Reference
Oscillatoria sp., Microcoleuschthonoplastes, Nostoc sp.,
Anabaena sp., Agmenellumquadruplicatum, Coccochloriselabens,
Cerniglia et al. (1979, 1980d,
1980b, 1982b), Narro et al.
Naphthalene
Aphanocapsa sp., Chlorella sorokiniana, Chlorella autotrophica,
(1992a)
Dunaliellatertiolecta, Chlamydomonasangulosa, Ulvafasciata,
Cylindrotheca
sp., Amphora sp., Nitzschia sp., Navicula sp.,
Porphyridiumcruentum
Phenanthrene
Oscillatoria sp., Agmenellumquadruplicatum
Narro et al. (1992b)
3.7Biodegradation by Yeast
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Studies on yeast able to use various petroleum components as sole carbon source, showed
that their biodegradability decreases from n-alkanes >branched alkanes>low molecular
weight aromatic hydrocarbons >cycloalkanes>high molecular weight aromatic and polar
compounds. Yeasts, however cannot grow on polycyclic aromatic hydrocarbons (PAH) but
are able to cooxidizebiphenyl, naphtalene and benzopyrene using the mono-oxigenase
cytochrome P450 pathway induced by the presence of n-alkanes. Studies on fungi and yeast
(Candida,Rhodotorula, Trichosporon) communities from aquatic environments polluted with
PAH, especially phenantherene, revealed high degradation rates for Trichosporon
penicillatum [70].
3.8 Field Scale Application of Biodegradation of PAHs
The bioremediation of PAH contaminated sites include a wide range of remediation
strategies like land farming, slurry-phase bioreactors, land-treatment systems, enhanced
washing of PAHs from soil using surfactants, combination of advanced chemical
oxidation with bioremediation by thermal pre-treatment etc.
An engineered land treatment system was operated for the treatment of soils
containing wood-preserving chemicals at a creosote wood-treatment site. The landtreatment unit contained polluted soil which was nutrient amended, tilling weekly and
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moisture was applied periodically; pH was maintained at 6.5-7.5. The decrease in total
PAH concentration was seen during active bioremediation period from 21%-82%.The
treatment period s ranged from 2months to 12 months. The end point was observed at
1000mg/Kg [16].
Pilot study of slurry phase removal of 10,000 tons of creosote-contaminated soil from
the southern wood preserving superfund site in Canton, Missippi [71] was performed by
the American Company OHM remediation services Corporation. Four slurry phase
bioreactors were employed each with volume of 680m3. The soil was sieved to less than
200 mesh and Aeration of the slurry was provided by diffusers and blower. The initial
total PAH concentration ranged from 8000 to 1500 mg/kg, the concentration of
carcinogenic PAH was from 1000 to 2500 mg/kg. The majority of the PAH
biodegradation occurring in the initial 5-10 days of treatment. The treatment goal was
easily achieved after 10days residence time in a bioreactor [71].
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4. FACTORS AFFECTING PAH DEGRADATION
A number of physical, chemical, biological or environmental factors may influence the rate
and extent of PAH degradation [72]
4.1Bioavailability (Use of Biosurfactant):
The composition and inherent biodegradability of the petroleum hydrocarbon
pollutant is the first and foremost important consideration when the suitability of a
remediation approach is to be assessed [73]. Bioavailability could be the rate determining
factor for the degradation of PAH. Decrease in the mineralisation of high molecular weight
PAHs attributed to the association of PAHs to soil organic matter [74],[75] which results in
the reduction of the rate and extent of PAH degradation due to the slowing of PAH
desorption from soil organic matter into the soil aqueous phase [76].
According to Leahy & Colwell [10], biosurfactants are important agents in the
effective uptake of PAHs by bacteria and fungi. The formation of emulsions in the presence
of biosurfactants is reported to be in 96% of hydrocarbon metabolizing freshwater bacteria
[77]. Additives and bulking agents enhance the overall hydrocarbon catabolism [78] .The use
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of surfactants like SDS, TritonX-102, Brij 35, Marlipal 013/90 and Genapol X150 increases
the concentration of hydrophobic compounds in the water phase by solubilisation or
emulsifycation [79].
The increase of biodegradation rate has been observed in studies in which organic
solvents were used to improve PAH-bioavailability to bacteria [80]. Soils pre-treated with
solvents like acetone and ethanol showed faster biodegradation rate than soils without pretreatment.
Chemotactic migration of mobile bacterial cells towards or away from the elevated
level of attractant chemical compounds has also been found to play an important role in the
biodegradation of organic compounds.
Calvo ortega et al [81] Chemotaxis of PAH degrading microorganisms present in
polluted rhizosphere soils inferred that the chemotactic attraction towards PAHs increase
their bioavailability and consequently the biodegradation rate of PAH.
4.2 pH
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pH is an important factor for the degradation activity of introduced microorganisms in
the soil or water systems. PAH mineralization is favoured by near neutral pH values [82].
Small pH shifts have dramatic effects on the degradation of low concentrations of PAH’s in
oligotrophic aquatic environments [83]. However fungi are known to be more tolerant to
acidic conditions. Biodegradation study of PAH’s in extremely acidic environments in the
presence of acidophilic microorganisms by [84] reported that the indigenous microorganisms
oxidized about 50% of the supplied naphthalene to CO 2 and water within 24 weeks, while the
extent of mineralization of phenenthrene and anthracene was only 10-20%. It was suggested
that initial fungal attacks on the hydrocarbons may have produced intermediates that were
available for further degradation by bacteria [82].
4.3Temperature
Temperature plays a significant role in controlling the natural and the extent of
microbial hydrocarbon metabolism, which is of special significance for in-situ
bioremediation. [82]. Temperature also affects the bioavailability and solubility of
hydrocarbons [85] possessing direct effect on the physical nature and chemical composition
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of the PAHs constituents [46]. At low temperature PAHs tend to be more viscous with less
microbial growth and propagation [86]. Studies show increased temperature made PAH’s
more bioavailable to microorganisms because the increased temperature made the PAH’s
more soluble [57]. In a field study done by [87].on the biodegradation of dispersed crude oil
in cold and icy seawater (-1.8 to5.50 C), half-life times of PAHs ranged from 1.5-1.7 days
(naphthalene) to 2.4-7.5 days(phenanthrene) under favourable conditions, i.e. at temperature
above 0ºC and with effective chemical dispersion.
The highest degradation rates that generally occur are in the range of 30-40 ºC in soil
environments and 15-20oC in marine environment [88],[89], [90] isolated microorganisms
which are able to convert naphthalene, phenanthrene and antheracene under thermophilic
conditions. Bacillus thermoleovorans thermophilic bacteria degraded naphthalene at 60oC
showing significantly different metabolites and different metabolic pathway known for
mesophilic bacteria. For mesophilic bacteria at temperature above optimal, enzymatic
activities are inhibited as protein denatures [10].
4.4Nutrients
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Apart from degradable carbon source in the form of PAH compounds,
microorganisms require mineral nutrients such as nitrogen (N), phosphate (P) and potassium
(K) for cellular metabolism and growth. In contaminated sites, where organic carbon levels
are often high due to the nature of the pollutant, available nutrients like Nitrogen,
phosphorous and in some cases Iron (Fe)[89] rapidly deplete during microbial metabolism
hence become limiting factor and greatly affect the microbial degradation of hydrocarbons
[72] e.g in marine environment where low levels of Nitrogen and Phosphorous are found
[86]. Therefore it is common practice to supplement contaminated land with nutrients,
generally nitrogen and phosphates to stimulate the in situ microbial community and therefore
enhance bioremediation. Fungi are able to effectively recycle nutrients (specifically nitrogen).
In fact, the high molecular weight PAH-oxidising ligninolytic enzymes of the white-rot fungi
are produced under nutrient deficient (often low nitrogen) conditions. Excessive nutrient
concentrations can also inhibit the biodegradation activity [91] negative effects of high N, P,
K levels on aromatic hydrocarbon degradation have also been reported [92],[93], [94].
4.5Salinity:
There is an inverse relationship between salinity and solubility of PAHs, with the
increase in salinity there was an increase in the sorption of aromatic hydrocarbon as seen in
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pyrene in various sediment types, was due to the salting-out effects occurring in both the
solution and solid (sediment organic matter) phases. However hyper salinity results in the
decreased microbial growth but an unidentified halophytic archaeon was found to degrade
PAH’s (acenaphtene, phenanthrene, anthracene; 500mg/l. Four bacterial strains, belonging to
the genera Micrococcus, Pseudomonas and Alcaligenes and tolerating 7.5% w/v NaCl, could
grow on 0.1% naphthalene and anthracene. [95]
Rhykerd et al [96] Showed inhibitory effect of artificial salinity on mineralization of
Petroleum oil (50g/ Kg soil),soils fertilized with inorganic N and P and emended with NaCl (
0.4,1.2 and 2% w/w) . Highest salt concentration after 80 days at 25º C had considerably
inhibited the mineralization of petrochemical compounds. Thus the removal of salt from PAH
contaminated soils may reduce the time required for bioremediation, however some
indigenous microorganisms are expected to be salt adapted.
4.6 Oxygen
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The amount of available oxygen will determine whether the system is aerobic or
anaerobic. PAHs degradation occurs primarily under aerobic condition, however in anaerobic
environment such as aquifers and marine sediments anaerobic biodegradation has also been
reported [97] with negligible rate and were initially limited to halogenated aromatic
compounds like halobenzoates chlorophenols etc only [98], [99].Though it is now well
established that bioremediation of organic contaminants such as PAHs can proceed under
both aerobic and anaerobic conditions. During aerobic PAH metabolism, oxygen is integral to
the action of mono- and dioxygenase enzymes in the initial oxidation of the aromatic ring. In
the absence of molecular oxygen, alternative electron acceptors such as nitrate, ferrous iron
and sulphate are necessary to oxidise these aromatic compounds, with recent research clearly
demonstrating that PAH degradation will occur under both denitrifying [100] and sulfatereducing [97] anaerobic conditions. Promotion of anaerobic bioremediation however has
several drawbacks, for not all environments contain an active population of anaerobic PAH
degraders. Also, under anaerobic conditions when electron acceptors like nitrate, ferric iron
and sulphate are reduced, this results in the release of excess of phosphorous and ferrous iron,
both of which are toxic to the environment. In addition, release of greenhouse gases (CH 4 ,
NO 2 etc) and increase in pH has also been observed during anaerobic degradation of PAH.
[57].
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To increase the oxygen amount in the soil it is possible to till or sparge air. In some
cases, hydrogen peroxide or magnesium peroxide can be introduced in the environment.
4.7Genetic Enhancement:
A bacterium needs the appropriate catabolic gene in order to be a degrader of a
compound these catabolic genes can be either chromosomal or plasmid borne. The metabolic
pathways for compounds such as naphthalene, salicylate, camphor, octane, Xylene and
toluene have been shown to be encoded on plasmid in Pseudomonas spp.by [101]
Applications for genetically engineered microorganisms (GEM) in bioremediation
have received a great deal of attention, but have largely been confined to the laboratory
environment. This has been due to regulatory risk assessment concerns and to a large extent
the uncertainty of their practical impact and delivery under field conditions. There are at least
four principal approaches to GEM development for bioremediation application. These
include:
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(1) modification of enzyme specificity and affinity,
(2) pathway construction and regulation,
3) bioprocess development, monitoring, and control, and
(4) bioaffinity bioreporter sensor applications for chemical sensing, toxicity reduction, and
end point analysis. [102]
Collective metabolism by mixed culture of microorganism may result in enhanced PAH
utilization as compared to single bacterial strain degradation by indigenous community of
bacteria.
5. Current Approaches to Improve PAHs Degradation
5.1 Bioaugementation
Bioaugementation is an in situ treatment method involving the addition of
microorganisms indigenous or exogenous to the contaminated sites. The addition of
exogenous microorganisms with PAH degrading capabilities overcome the catabolic
limitations of the indigenous microflora towards PAH [2], [90] and [103] proposed that
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bioaugementation is especially important for sites containing high PAH concentrations, site
which contain a significant proportion of high molecular weight PAHs and for recently
polluted soils which do not have an adapted microbial population. Bioaugementation study
demonstrated by [2] showed significant decrease in the concentration of all PAH compounds
including Benzo(a) pyrene at bench scale trials for the clean-up of petroleum contaminated
soil using a mixed bacterial culture isolated from MGP site [104] and had previously shown
the ability to degrade three-,four-,five- and seven-ring PAH compounds. Some factors
however limit the use of added microbial cultures in land which include die-off of the
laboratory strains, and the inability of the inocula to compete with the indigenous microflora
to develop and sustain useful population levels.
5.2 Bacterial-Fungal Co-cultures
Degradation of PAH is by bacteria is often limited by the incapability of the
organisms to hydroxylate the compound, and the inability of high molecular weight PAH
compounds like BaP to pass through bacterial cell wall [105]. Since fungi have the ability to
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produce extracellular enzymes (lignin-degrading enzymes)the initial transformation of PAH
by fungi followed by bacterial degradation of polar metabolites could lead to an effective
strategy for PAH degradation[2].
Gomez et.al [106]performed a study consisting of sixteen co-cultures composed of
four bacteria and four fungi grown on sugarcane bagasse pith were tested for phenanthrene
degradation in soil. The four bacteria were identified as Pseudomonas aeruginose, Ralstonia
pickettii, Pseudomonas sp. and Pseudomonas cepacea. The four fungi were identified as:
Penicillium sp., Trichoderma viride, Alternaria tenuis and Aspergillus terrus that were
previously isolated from different hydrocarbon-contaminated soils. Fungi had a statistically
significant positive (0.0001<p) effect on phenanthrene removal, that ranged from 35% to
50% and bacteria removed the compound by an order of 20%.Co-cultures B. cepaceaPenicillium sp., R. pickettii-Penicillium sp., and P. aeruginose-Penicillium sp. exhibited
synergism for phenanthrene removal, reaching 72.84 ± 3.85%, 73.61 ± 6.38% and 69.47 ±
4.91%; in 18 days, respectively.
5.3 Application of Immobilized Cells
Immobilized cells have been used and studied for the bioremediation of numerous
toxic chemicals. Immobilization not only simplifies separation and recovery of immobilized
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cells but also makes the application reusable which reduces the overall cost [107] used free
suspension and immobilized Pseudomonas sp. to degrade petrol in an aqueous system. The
study indicated that immobilization resulted in a combination of increased contact between
cell and hydrocarbon droplets and enhanced level of rhamnolipids production.
Immobilization can be done in batch mode as well as continuous mode. Packed bed reactors
are commonly used in continuous mode to degrade hydrocarbons. It can be concluded that
immobilization of cells is a promising application in the bioremediation of hydrocarbon
contaminated site.
5.4 Commercially Available Bioremediation Agents
Microbiological cultures, enzyme additives, or nutrient additives that significantly
increase the rate of biodegradation to mitigate the effects of the discharge were defied as
bioremediation agents by U.S.EPA [108]. Bioremediation agents are classified as bio-
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augmentation agents and bio-stimulation agents based on the two main approaches to oil spill
bioremediation. The U.S. EPA compiled a list of 15 bioremediation agents [100, 101] as a
part of the National Oil and Hazardous Substances Pollution Contingency Plan (NCP)
Product Schedule, which was required by the Clean Water Act, the Oil Pollution Act of
1990(Table 7) Studies showed that bioremediation products may be effective in the
laboratory but significantly less so in the field. However, However, due to the limitations of
common fertilizers (e.g., being rapidly washed out due to tide and wave action), several
organic nutrient products, such as oleophilic nutrient products, have recently been evaluated
and marketed as bioremediation agents.
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Table 7: Bioremediation agents in NCP product schedule [16]
Name or Trademark
Product Type
Manufacture
BET BIOPETRO
MC
BioEnviro Tech, Tomball, TX
BILGEPRO
NA
International Environmental
Products, LLC, Conshohocken,
PA.
INIPOL EAP 22
NA
Societe, CECA S.A., France
LAND AND SEA
NA
Land and Sea Restoration LLC,
San Antonio
RESTORATION
MC
MICRO-BLAZE
Verde Environmental, Inc.,
Houston, TX
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OIL SPILL EATER II
NA/EA
Oil Spill Eater International,
Corporation, Dallas, TX
OPPENHEIMER
FORMULA
PRISTINE SEA II
MC
Oppenheimer Biotechnology,
Inc., Austin, TX
MC
Marine Systems, Baton Rouge,
LA
SYSTEM E.T. 20.
MC
Quantum Environmental
Technologies, Inc(QET), La
Jolla, CA
VB591TMWATER,
NA
VB997TMSOIL,
BioNutraTech, Inc.,
Houston,TX
AND BINUTRIX
WMI-2000
MC
WMI International, Inc
Abbreviations of product type:
MC: Microbial Culture,EA: Enzyme Additive, NA: Nutrient Additive
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5.5 Phytoremediation
Phytoremediation is an emerging technology that uses plants to manage a wide variety
of environmental pollution problems, including the cleanup of soils and groundwater
contaminated with hydrocarbons and other hazardous substances. The different mechanisms,
namely, hydraulic control, phytovolatilization, rhizoremediation, and phytotransformation.
could be utilized for the remediation of a wide variety of contaminants. Advantages of using
phytoremediation
include
cost-effectiveness,
aesthetic
advantages,
and
long-term
applicability.
5.6 Genetically Modified Bacteria and Use of Plasmids
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Studies on PAH metabolism are entering a new era with the application of genetically
engineered microorganisms (GEM) s for bioremediation processes. Modified microorganisms
have shown potential for bioremediation of many chemical contaminants. However,
ecological and environmental concerns and regulatory constraints are major obstacles for
testing GEM in the field. The use of genetically engineered bacteria was applied to
bioremediation process monitoring, strain monitoring stress response, end-point analysis and
toxicity assessment. A bacterium needs the appropriate catabolic genes in order to be a
degrader of a compound. Many of the genes involved in the degradation of PAHs are often
located on plasmids [109]. Plasmids that carry structural genes that code for the degradation
of many naturally occurring organic compounds and xenobiotics, are referred to as
degradative or catabolic plasmids. A plasmid may encode a complete degradative pathway or
partial degradative step. Some other plasmids code for enzymes that have specificity for
several substrates. For example, the genes encoding the upper and lower pathways of
naphthalene in the NAH plasmids of several pseudomonads have broad specificities, allowing
the host to grow on several two and three -ring PAHs, as sole carbon and energy sources
[110].
Dunn &Gunsalus [111] reported for the first time, the involvement of plasmids in the
degradation of PAHs. All the genes involved in the degradation of naphthalene by
Pseudomonas putida PpG7 are now known to be plasmid borne and transmissible [112],[113]
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demonstrated that the genes encoding the enzymes of the first 11 steps of the naphthalene
oxidation pathway are located on the NAH7 plasmid. In plasmid NAH7, the naphthalene
catabolic genes are organized into two operons; nah and sal. The naphthalene oxidation genes
are organized in two operons. The first operon includes genes nahABCDEF, coding for the
conversion of naphthalene to salicylate, and the second operon includes genes nahGHIJK,
coding for the oxidation of salicylate via the catechol meta-cleavage pathway.
The naphthalene dioxygenase enzyme encoded by the NAH7 genes is known today to
be a highly versatile enzyme system, encoding a wide range of reactions [114], [115],[102]
produced the first report which provides direct biochemical evidence that the naphthalene
plasmid degradative enzyme system is involved in the degradation of higher-molecularweight polycyclic aromatic hydrocarbons other than naphthalene.
Zhou. et.al [116] successfully cloned a bio-degradative gene encoding catechol 2,3dioxygenase (C23O) from Pseudomonas sp. CGMCC2953, isolated from oil-polluted soil,
into the plasmid pK4 derived from pRK415 with a broad host range. The apparent
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phenanthrene biodegradation parameters of the recombinant microorganism (Pseudomonas
sp. CGMCC2953-pK) were determined and compared with those of the wild type. As the key
enzyme of phenanthrene degradation, C23O, could be expressed constitutively in the
recombinant strain, Pseudomonas sp. CGMCC2953-pK showed an increased ability to
degrade phenanthrene. The excessive production of C23O in Pseudomonas sp.
CGMCC2953-pK could serve as an effective approach to construct genetically engineered
microorganisms for the bioremediation of environmental contaminations
The combination of microbiological and ecological knowledge, biochemical
mechanisms and field engineering designs are essential elements for successful in situ
bioremediation using genetically modified bacteria.
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Table
8:
Selected
PAH
degrading
bacterial
239
plasmids
and
their
hosts.[117]
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Table 9: Genes borne on the NAH7 plasmid and enzymes encoded by them.
[117]
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Protoplast fusion is another practically useful technology which has significant
engineering applications in generating microbial strains with specific properties for
environmental bioremediation. [118] constructed a high-efficiency phenanthrene-degrading
bacterial fusant strain F14 protoplast fusion between Sphingomonas sp. GY2B (GenBank
DQ139343) and Pseudomonas sp. GP3A (GenBank EU233280). Results showed within 24
hours Phenanthrene could be almost completely degraded by the fusant strain F14, which was
much quicker than GY2B and GP3A.The fusant strain F14 had a wider range of temperature
(25-30 °C) and pH value (6.5-9.0) than GY2B did. Phenanthrene was metabolized through a
pathway having less accumulation of potentially toxic metabolites than GY2B. The results
demonstrated the feasibility of accelerating the phenanthrene degradation, enhancing the
adaptability of bacteria, and accumulating less potentially toxic metabolite(s) by using the
protoplast fusion technology.
5.7Bioinformatic Approach: PAHbase
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Bioinformatics based analysis and prediction is playing a pivotal role in
understanding and capturing the in-depth knowledge of biological molecules particularly with
reference to proteomics and genomics.[119]. Bioinformatics technology has been developed
to identify and analyse various components of cells such as gene and protein functions,
interactions, metabolic and regulatory pathways. Bioinformatics analysis will facilitate and
quicken the analysis of bioremediation processes [120].
There is constantly increasing need for new ways of comparing multiple sets of data
and information related to the occurrence and potential of PAH degrading bacteria [121]. Due
to the generation of huge number of sequences and information from the fast and user
friendly implementations of bioinformatics and in order to access and use the information
about PAH degrading organisms details from the research papers were extracted, analysed
and presented in form of a precise informative database: PAHbase: reflecting the diversity
and functional analysis of PAHs degrading bacteria
PAHbase (URL: www.pahbase.in.) is a freely available functional database of
Polycyclic Aromatic Hydrocarbons (PAHs) degrading bacteria. The database consists of
relevant information obtained from scientific literature and databases (i.e. NCBI, DDBJ and
EMBL.). The database provides a comprehensible representation of PAH degrading bacteria
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with reference to its occurrence, extremophilic nature (Halophilic/Thermophilic/Mesophilic),
taxonomy and phylogenetic relatedness with nearby species, and stress adaptation, preferred
PAH source of utilization as carbon source, biodegradative ability, media used in laboratory
physical, chemical and environmental conditions provided for degradation, metabolic
pathways , enzymes involved in degradation, genetic basis of degradation, gene/s involved
and gene location,16S ribosomal gene sequence and references.
5.8Bacterial Biosensors
Biosensors are analytical tools, which use the biological specificity in sensing the
target molecule. Many studies demonstrate the design and application of molecular
biosensors for use in bioremediation.[122]. The genetic information, located on a plasmid
vector, is inserted into a bacterial strain so that the engineered fusion replicates along with the
cell’s normal DNA. Biosensor systems include a wide range of integrated devices that
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employ enzymes, antibodies, tissues, or living microbes as the biological recognition
element. Bacterial biosensors uniquely measure the interaction of specific compounds
through highly sensitive bio-recognition processes and offer great sensitivity and selectivity
for the detection and quantification of target compounds. Whole-cell biosensors, constructed
by fusing a reporter gene to a promoter element induced by the target compound, offer the
ability to characterize, identify, quantify, and determine the biodegradabilty of specific
contaminants present in a complex mixture without pre treatment of the environmental
samples [72].
The presence of toxic compounds and the potential associated ecological risks can be
determined by using bacterial biosensor and toxicity tests. Several biosensors have been
developed for the detection of many petrochemical waste compounds including PAH.
[123],[124]constructed a biosensor for detecting the toxicity of PAHs in contaminated
soilswith an immobilized recombinant bioluminescent bacterium, GC2 (lac::luxCDABE),
which constitutively produces bioluminescence. The monitoring of phenanthrene toxicity was
achieved through measurement of the decrease in bioluminescence when a sample extracted
with the rhamnolipid biosurfactant was injected into a mini-bioreactor. This system was
proposed to be used as an in situ system to detect the toxicity of hydrophobic contaminants in
soils and for the performance evaluation of PAH degradation in soils.
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6) Conclusion and Future Prospects.
The increasing incidents of oil spills and production of PAHs demand the degradation
of complex hydrocarbons. The complex hydrocarbons which are otherwise harmful to soil
and aquatic flora and fauna, must be degraded to simpler nontoxic compounds. A better
understanding of the mechanisms of biodegradation has a high ecological significance that
depends on the indigenous microorganisms to transform or mineralize the organic
contaminants and the potential benefits of using genetically modified bacteria. Although
bioremediation is generally regarded as an economical remediation option for the clean-up of
PAH-Contaminated soil, the successful application of this technology is restricted to low
molecular weight PAHs, however PAHs containing five or more fused benzene rings, such as
BaP, is limited. Hence there is an urgent need to address this issue and to direct future
research on expanding our knowledge on the practical application of co-metabolic process,
bioaugementation, application of GEMs etc.
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Petroleum microbiology research is advancing on many fronts, spurred on most
recently by new knowledge of cellular structure and function gained through molecular and
protein engineering techniques combined with more conventional microbial methods.
Improved systems for biodegradation of PAH compounds are being commercialized with
positive economic and environmental advantages.
Systems biology is an integrated research approach to study complex biological
systems. Modern tools of genomics, transcriptomics, proteomics, metabolomics, phenomics
and lipidomics have been applied to investigate systems biology of microbial communities in
a myriad of environments .Currently, user friendly bioinformatics tools including “omics”
tools
(Qiime-qiime.sourceforge.net
and
Phylotrac-www.phylotrac.org/)
provide
comprehensive database of all available genomics, proteomics and metabolomics information
from bioremediation research for scientists to exchange information leading to generation of
judicious predictive models and strategies for successful implementation of bioremediation
applications in future [125].
Ground breaking research is being done to engineer new biocatalysts and biosensors
for achieving complete mineralization of high molecular weight PAH compounds Future
research exploiting molecular techniques and metagenomic studies are expected to explore
and harness microbial potential for effective Bioremediation.
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