petrochemical industrial waste: bioremediation techniques ... · the petrochemical industry is one...

International Journal of Advancements in Research & Technology, Volume 2, Issue 7, July-2013 211 ISSN 2278-7763

Copyright © 2013 SciResPub. IJOART

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: [email protected]

ABSTRACT

The petrochemical industry is one such major source of hazardous waste, produced during

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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mailto:[email protected]



1. 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

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:

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]

amp: melting point;bbp: boiling point; c Sol: aqueous solubility. d log Kp: logarithm of theoctanol:water partitioning coefficient.

PAH No. of rings

mpa (oC) bpb(oC) Solc(mg l-1) log Kpd Vapour pressure

(torr at 20(oC)

Phenanthrene

3 101 340 1.29 4.46 6.8 x10-4

Anthracene 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 536 0.062 7.66 1.0 x 10-10

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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].

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 Soil Assessment

< 200 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

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

dehydrogenase to yield a dihydroxylated derivative [41].Further oxidation of the cis-

dihydrodiols 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 Organisms References

Naphthalene

Acinetobacter calcoaceticus, Alcaligenes

denitrificans, Mycobacterium sp., Pseudomonas

sp., P. putida, P. fluorescens, Sp.

paucimobilis,Brevundimonasvesicularis,

Burkholderiacepacia, Comamonastestosteroni,

Rhodococcus sp., Corynebacteriumrenale,

Moraxella sp.,Streptomyces sp., B. cereus, P.

marginalis, P. stutzeri, P. saccharophila,

Neptunomonasnaphthovorans, Cycloclasticus sp.

Ryu et al. (1989), Weissenfels et al. (1990, 1991), Kelly et al. (1991), Dunn

and Gunsalus (1973), Davies and Evans (1964), Foght and Westlake (1988),

Jeerey et al. (1975), Mueller et al. (1990a), Kuhm et al. (1991), Walter et al.

(1991), Dua and Meera (1981), Tagger et al. (1990), Garcia-Valdes et al.

(1988), Trower et al. (1988), Grund et al. (1992), Barnsley (1975a);

Barnsley (1983a), Yang et al. (1994), Burd and Ward (1996), Allen et al.

(1997), Stringfellow and Aitken (1995), Filonov et al. (1999), Hedlund et al.

(1999), Geiselbrecht et al. (1998), Foght and Westlake (1996), Goyal and

Zylstra (1996)

Acenaphthene

Beijernickia sp., P. putida, P.

fluorescens,Bu.cepacia, Pseudomonas sp.,

Cycloclasticus sp., Neptunomonasnaphthovorans,

Alcaligeneseutrophus,

Alcaligenesparadoxus

Chapman (1979), Schocken and Gibson (1984), Ellis et al. (1991),

Geiselbrecht et al. (1998), Hedlund et al. (1999), Selifonov et al. (1993)

Phenanthrene

Aeromonas sp., A. faecalis, A. denitrificans,

Arthrobacter polychromogenes, Beijernickia sp.,

Micrococcus sp., Mycobacterium sp., P. putida,

Sp. paucimobilis, Rhodococcus sp., Vibrio sp.,

Nocardia sp., Flavobacterium sp., Streptomyces

sp., S. griseus, Acinetobacter sp., P. aeruginosa,

P. stutzeri, P. saccharophila, Stenotrophomonas

maltophilia, Cycloclasticus sp., P. uorescens,

Acinetobacter calcoaceticus,

Acidovorax dela®eldii, Gordona sp.,

Sphingomonas sp., Comamonas

testosteroni, Cycloclasticus pugetii, Sp.

yanoikuyae, Agrobacterium sp.,

Bacillus sp., Burkholderia sp., Sphingomonas sp.,

Pseudomonas sp., Rhodotorula glutinis,

Nocardioides sp., Flavobacterium gondwanense,

Halomonas meridiana

Kiyohara et al. (1976, 1982, 1990), Weissenfels et al. (1990, 1991), Keuth

and Rehm (1991), Jerina et al. (1976), Colla et al. (1959), West et al. (1984),

Kiyohara and Nagao (1978), Heitkamp and Cerniglia (1988), Guerin and

Jones (1988a, 1989), Treccani et al. (1954), Evans et al. (1965), Foght and

Westlake (1988), Mueller et al. (1990b), Sutherland et al. (1990), Ghosh and

Mishra (1983), Savino and Lollini (1977), Trower et al. (1988), Barnsley

(1983b), Yang et al. (1994), Kohler et al. (1994), Stringfellow and Aitken

(1995), Boonchan (1998), Juhasz (1998), Geiselbrecht et al. (1998), Foght

and Westlake (1996),

Kastner et al. (1998), Lal and Khanna (1996), Shuttleworth and Cerniglia

(1996), Mahro et al. (1995), Goyal and Zylstra (1996), Dyksterhouse et al.

(1995), Allen et al. (1999), Aitken et al. (1998), Romero et al. (1998),

Iwabuchi et al. (1998), Churchill et al. (1999),

Juhasz (1991)

Anthracene

Beijernickia sp., Mycobacterium sp., P. putida,

Sp. paucimobilis, Bu.

cepacia, Rhodococcus sp., Flavobacterium sp.,

Arthrobacter sp., P. marginalis, Cycloclasticus

sp., P. fluorescens, Sp. yanoikuyae,

Acinetobactercalcoaceticus, Gordona sp.,

Sphingomonas sp.,

Comamonastestosteroni,Cycloclasticuspugetii

Colla et al. (1959), Akhtar et al. (1975), Jerina et al. (1976), Evans et al.

(1965), Ellis et al. (1991), Weissenfels et al. (1991), Foght and Westlake

(1988), Walter et al. (1991), Mueller et al. (1990a), Savino and Lollini

(1977), Tongpim and Pickard (1996), Burd and Ward (1996), Geiselbrecht

et al. (1998), Foght and Westlake (1996), Kim et

al. (1997), Lal and Khanna (1996), Mahro et al. (1995), Goyal and Zylstra

(1996), Dyksterhouse et al. (1995), Allen et al. (1999)

Fluoranthene

A. denitrificans, Mycobacterium sp., P. putida, Sp.

paucimobilis, Bu.

cepacia, Rhodococcus sp., Pseudomonas sp.,

Stenotrophomonas

maltophilia, Acinetobactercalcoaceticus,

Acidovoraxdelafieldii, Gordona

sp., Sphingomonas sp., P. saccharophilia,

Pasteurella sp.

Kelly and Cerniglia (1991), Walter et al. (1991), Weissenfels et al.

(1991), Foght and Westlake (1988), Barnsley (1975b), Mueller et al.

(1990a, 1990b), Ye et al. (1996), Kelly et al. (1993), Boonchan (1998),

Juhasz (1998), Lal and Khanna (1996), Shuttleworth and Cerniglia (1996),

Mahro et al. (1995), Willumsen and Karlson (1998), Sepic et al. (1998),

Willumsen et al. (1998), Churchill et al. (1999), Chen and Aitken (1999)

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Pyrene

A. denitrificans, Mycobacterium sp., Rhodococcus

sp., Sp. paucimobilis,

Stenotrophomonasmaltophilia,

Acinetobactercalcoaceticus, Gordona sp.,

Sphingomonas sp., P. putida, Bu cepacia, P.

saccharophilia

Heitkamp et al. (1988a), Walter et al. (1991), Weissenfels et al. (1991),

Grosser et al. (1991), Schneider et al. (1996), Ye et al. (1996),

Boonchan (1998), Juhasz (1998), Kastner et al. (1998), Lal and Khanna

(1996), Mahro et al. (1995), McNally et al. (1999), Jimenez and Bartha

(1996), Churchill et al. (1999), Juhasz et al. (1997), Chen and Aitken (1999)

Chrysene

Rhodococcus sp., P. marginalis, Sp. paucimobilis,

Stenotrophomonas

maltophilia, Acinetobacter calcoaceticus,

Agrobacterium sp., Bacillus sp.,

Burkholderia sp., Sphingomonas sp.,

Pseudomonas sp., P. saccharophilia

Walter et al. (1991), Burd and Ward (1996), Ye et al. (1996), Boonchan

(1998), Lal and Khanna (1996), Aitken et al. (1998), Chen and Aitken

(1999)

Benz[a]

anthracene

A. denitrificans, Beijernickia sp., P. putida, Sp.

paucimobilis,

Stenotrophomonasmaltophilia, Agrobacterium

sp., Bacillus sp.,

Burkholderia sp., Sphingomonas sp.,

Pseudomonas sp., P. saccharophilia

Gibson et al. (1975), Maha€ey et al. (1988), Weissenfels et al. (1991),

Schneider et al. (1996), Ye et al. (1996), Boonchan (1998), Juhasz (1998),

Aitken et al. (1998), Chen and Aitken (1999)

Dibenz[a,h]

anthracene

Sp. paucimobilis, Stenotrophomonasmaltophilia Ye et al. (1996), Boonchan (1998), Juhasz (1998)

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,o-

demethylation, 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 2-

naphthoic 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-

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-2-

napthoic acid (III), which could be hydrated to generate hydroxydecahydro-2-naphthoic acid

(V) and subsequently oxidizedto oxodecahydro-2-naphthoic acid (VI). Decahydro-2-

naphthoic acid (IV) is probably a dead-end metabolite. Athiolytic ring cleavage and a

subsequent oxidation can generatethe tentatively identified C11H16O4-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) C11H16O4-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 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 –CH2OH which is further converted to –CHO and –COOH, resulting

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-methyl-

succinyl- 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 trans-

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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 CO2[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

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

Naphthalene

Absidaglauca, Aspergillusniger, Basidiobolusranarum, Candida utilis,

Choanephoracampincta, Circinella sp., Clavicepspaspali, Cokeromyces

poitrassi, Conidiobolusgonimodes, C. bainieri, C. elegans, C. japonica,

Emericellopsis sp., Epicoccumnigrum, Gilbertellapersicaria,

Gliocladium sp., Helicostylumpiriforme, Hyphochytriumcatenoides,

Linderinapennispora, Mucorhiemalis, Neurosporacrassa,

Panaeoluscambodginensis, Panaeolus subbalteatus,

Penicilliumchrysogenum, Pestalotia sp., Phlyctochytrium

reinboldtae, Phycomyesblakesleeanus, Phytophthoracinnamomi,

Psilocybe cubensis, Psilocybestrictipes, Psilocybestuntzii,

Psilocybesubaeruginascens,

Rhizophlyctisharderi, Rhizophlyctisrosea, Rhizopusoryzae, Rhizopus

stolonifer, S. cervisiae, Saprolegniaparasitica, Smittiumculicis, Smittium

culisetae, Smittiumsimulii, Sordaria ®micola,

Syncephalastrumracemosum,

Thamnidiumanomalum, Zygorhynchusmoelleri

Cerniglia and Gibson (1977), Cerniglia

et al. (1978, 1982a),

Smith and Rosazza (1974), Cerniglia

and Crow (1981), Ferris etal. (1973)

Acenaphthene

C. elegans, T, versicolor

Pothuluri et al. (1992a), Johannes et al.

(1998)

Phenanthrene

C. elegans, P. chrysosporium, P. laevis, Pleurotusostreatus, T. versicolor,

Bjerkanderaadjusta, Pleurotusostreatus, Cylindrocladium simplex,

Monosporiumolivaceum, Curvularialunata, Curvulariatuberculata,

Laetiporus

sulphureus, Daedaelaquercina, Flamulinavelutipes, marasmiellus sp.,

Penicullium sp., Kuehneromycesmutabilis, Laetiporussulphureus,

Agrocybe aegerita, Aspergillusniger, Syncephalastrumracemosum

Cerniglia and Yang (1984), Cerniglia et

al. (1989), Morgan et al. (1991),

Sutherland et al. (1991), Bumpus

(1989), Hammel et al. (1992), Bezalel et

al. (1996), Brodkorb and Legge (1992),

Schutzendubel et al. (1999), Lisowska

and Dlugonski (1999), Bogan and

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.

laevis, Ramaria sp., R. solani, Trametesversicolor, Pleurotusostreatus,

Cylindrocladium simplex, Monosporiumolivaceum, Curvularialunata,

Curvulariatuberculata, Cryphonectriap arasitica,

Ceriporiopsissubvermispora, Oxysporus sp., Cladosporiumherbarum,

Drechsleraspicifera, Verticillium

lecanii, Fusariummoniliforme, Rhizopusarrizus, Coriolopsispolyzona,

Laetiporussulphureus, Daedaelaquercina, Flamulinavelutipes,

marasmiellus sp., Penicullium sp.

Cerniglia (1982), Cerniglia and Yang

(1984), Hammel et al.

(1991), Sutherland et al. (1992), Field et

al. (1992), Collins et al.

(1996), Schutzendubel et al. (1999),

Lisowska and Dlugonski (1999),

Krivobok et al. (1998), Andersson and

Henrysson (1996), Johannes et al.

(1996), Bogan et al. (1996), Bogan and

Lamar (1996), Vyas et al. (1994), Sack

and Gunther (1993)

Fluoranthene

C. elegans, C. blackesleeana, C. echinulata, Bjerkanderaadjusta,

Pleurotus ostreatus, Sporormiellaaustralis, Cryptococcus albidus,

Cicinoboluscesatii, Pestalotiapalmarum, Beauveria alba,

Aspergillusterreus, Mortierella ramanniana, Rhizopusarrhizus,

Pothuluri et al. (1990, 1992b),

Schutzendubel et al. (1999), Salicis

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.

glabrum, P.ostreatus, Syncephalastrumracemosum, Bjerkanderaadjusta,

Pleurotus sp., Dichomitussqualens, Flammulinavelutipe,

Trammetesversicolor, Kuehneromycesmutabilis, Laetiporussulphureus,

Agrocybeaegerita

Cerniglia et al. (1986), Hammel et al.

(1986), Launen et al.(1995), Bezelel et

al. (1996), Schutzendubel et al. (1999),

Martens & Zadrazil (1998), Sack et al.

(1997b), Wunder et al. (1997), Sack and

Fritsche (1997), Lang et al. (1996),

Stanley et al.(1999), Boonchan (1998)

Chrysene

P. janthinellum, Syncephalastrumracemosus, Penicillium sp.

Cerniglia et al. (1980a), Andersson and

Henrysson (1996), Bogan

and Lamar (1996), Stanley et al. (1999),

Boonchan (1998)

Benz[a]

anthracene

C. elegans, Trametesversicolor, P. laevis, P. janthinellum

Kiehlmann et al. (1996), Stanley et al.

(1999), Boonchan (1998)

Dibenz[a,h]anth

racene

Trametesversicolor, P. janthinellum Andersson and Henrysson (1996),

Stanley et al. (1999), Boonchan (1998)

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

Naphthalene

Oscillatoria sp., Microcoleuschthonoplastes, Nostoc sp.,

Anabaena sp., Agmenellumquadruplicatum, Coccochloriselabens,

Aphanocapsa sp., Chlorella sorokiniana, Chlorella autotrophica,

Dunaliellatertiolecta, Chlamydomonasangulosa, Ulvafasciata,

Cylindrotheca

sp., Amphora sp., Nitzschia sp., Navicula sp.,

Porphyridiumcruentum

Cerniglia et al. (1979, 1980d,

1980b, 1982b), Narro et al.

(1992a)

Phenanthrene

Oscillatoria sp., Agmenellumquadruplicatum

Narro et al. (1992b)

3.7Biodegradation by Yeast

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 land-

treatment 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].

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 pre-

treatment.

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

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 CO2 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

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

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 sulfate-

reducing [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 (CH4,

NO2 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:

(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

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. cepacea-

Penicillium 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-

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

MICRO-BLAZE

MC

Verde Environmental, Inc.,

Houston, TX

OIL SPILL EATER II NA/EA

Oil Spill Eater International,

Corporation, Dallas, TX

OPPENHEIMER

FORMULA

MC

Oppenheimer Biotechnology,

Inc., Austin, TX

PRISTINE SEA II MC

Marine Systems, Baton Rouge,

LA

SYSTEM E.T. 20. MC

Quantum Environmental

Technologies, Inc(QET), La

Jolla, CA

VB591TMWATER,

VB997TMSOIL,

AND BINUTRIX

NA

BioNutraTech, Inc.,

Houston,TX

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

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-molecular-

weight polycyclic aromatic hydrocarbons other than naphthalene.

Zhou. et.al [116] successfully cloned a bio-degradative gene encoding catechol 2,3-

dioxygenase (C23O) from Pseudomonas sp. CGMCC2953, isolated from oil-polluted soil,

into the plasmid pK4 derived from pRK415 with a broad host range. The apparent

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 plasmids and their

hosts.[117]

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

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

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.

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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