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INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 7, 2011
© Copyright 2010 All rights reserved Integrated Publishing Association
Research article ISSN 0976 – 4402
Received on March, 2011 Published on April 2011 1420
Crude oil PAH constitution, degradation pathway and associated bioremediation microflora: an overview
Kumar Arun 1 , Munjal Ashok 1 , Sawhney Rajesh 2 1 Department of Bioscience and Biotechnology, Banasthali Vidyapith, Banasthali, Rajasthan
(India)304022 2 Department of Microbiology, Bhojia Institute of Life Sciences, Budh, Baddi. Distt.
Solan,Himachal Pradesh (India)173205 [email protected]
ABSTRACT
Crude oil, a dark sticky liquid, is a complex mixture of varying molecular weight which is used for the preparation of petroleum products. Crude oil contains more than 30 parent polyaromatic hydrocarbons (PAHs). The U.S.EPA has designated 16 PAH compounds (naphthalene, acenaphthylene, acenaphthene, fluorene, phenenthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a, h]anthracene, benzo[g, h, i]perylene, and indeno[1,2,3cd]pyrene) as priority pollutants. PAHs are one of the most widespread organic pollutants and potentially health hazard. Besides other environmental components, they are also found in foods (cereals, oils, fats, vegetables, cooked meat). They are carcinogenic , mutagenic , and teratogenic . Thus, key focus is to eliminate these hazardous pollutants from the environment. The present review highlights the presence of various PAHs in the crude oil, key metabolic pathway for the degradation and the associated microbial degraders. The current approach to bioremediation uses various bacterial and fungal genera under aerobic or anaerobic conditions to directly target the specific PAH. However, there is need to explore newer approaches to design an efficient, effective and ecofriendly bioremediation tool. The dearomatization of crude oil might be a useful comprehensive approach and one shot solution to multiple PAH population.
Keywords: Crude oil, PAHs, Bioremediation, Phytoremediation, Rhizoremediation
1 Introduction
Crude oil is a complex mixture of varying molecular weight hydrocarbons and other organic compounds found beneath the earth's surface. It is a dark sticky fluid naturallyoccurring in certain rock formations. Crude oil contains carbon and hydrogen, with or without non metallic elements such as oxygen and sulfur. It is highly flammable and generates energy. Its derivative i.e. natural gas, is an excellent fuel. The term "Petroleum" has been used as a synonym to crude oil. This term was first used in the treatise “De Natura Fossilium” published in 1546 by the German mineralogist Georg Bauer (BauerGeorg et al., 1955).
1.1 Origin, constitution and use
Crude oil is the product of heating of ancient organic materials over geological period. It is formed from pyrolysis of hydrocarbon, in a variety of reactions, mostly endothermic at high
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temperature and/or pressure. Crude oil reserves were formed from the preserved remains of prehistoric zooplankton and algae , which had settled to a sea or lake bottom in large quantities under anoxic conditions. On the other hand, the remains of prehistoric terrestrial plants led to form coal. During the formation of crude oil, diagenesis followed catagenesis. The studies documented that over a period, the organic matter mixed with the mud and got buried under heavy layers of sediments resulting in generation of high levels of heat and pressure (diagenesis). This process transformed the organic matter into a waxy material known as kerogen, followed by its further conversion to liquid and gaseous hydrocarbons ( catagenesis). The change from kerogen to natural gas through oil is a temperature dependent event. Sometimes the oil formed at extreme depths migrates and is entrapped at shallower depths. eg. Athabasca oil sands.
The crude oil is a heterogeneous entity, composed of hydrocarbon chains of varied lengths. It contains hundreds of different hydrocarbon compounds such as paraffins , naphthenes , aromatics as well as organic sulfur compounds, organic nitrogen compounds and oxygen containing hydrocarbons (phenols). Crude oils generally lack in olefins (Gary et al., 1984). The most common distillations of petroleum are fuels. Fuels generally include, ethane and other shortchain alkanes , diesel fuel (petrodiesel), fuel oils , gasoline (petrol), jet fuel , kerosene, liquefied petroleum gas (LPG). The following table1 depicts various fuels with their use.
Table 1: Different distillations of Petroleum (Fuels) and their use.
S. No. Fuel/ Derivatives Uses 1 Alkenes (Olefins) Manufacture of plastics or other compounds 2 Lubricants Synthesis of light machine oils, motor oils and
greases, as viscosity stabilizers 3 Wax Used in the packaging of frozen foods 4 Petroleum coke
(asphalt) Used in carbon products or as solid fuel, Paraffin wax , Aromatic petrochemicals as precursors in other chemical synthesis.
5 Paraffin wax & aromatic petrochemicals
As precursor in chemical production
The different fractions of the crude oil, produced exhibit boiling point ranges, instead of a single boiling point eg. a crude oil fractionator produces an overhead fraction called "naphtha ". This fraction becomes a gasoline component after it is further processed through a catalytic hydrodesulfurizer and a catalytic reformer into molecules having higher octane rating value (Nelson, 1958; and Gary et al., 1984).
1.2 Variety of PAHs in crude oil
PAHs, commonly termed as polyaromatic hydrocarbons or polynuclear aromatic hydrocarbons, are chemical compounds that consist of fused aromatic rings and do not contain heteroatoms or carry substituents (Fetzer, 2000). The natural crude oil contains significant amounts of polycyclic aromatic hydrocarbons (PAHs) that arise from chemical conversion of natural product molecules, like steroids, to aromatic hydrocarbons. PAHs are
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also found in processed fossil fuels, tar and various edible oils (Glenn, 1995). It is described that the distributions of PAHs with respect to the relative amounts of individual PAHs and that of the isomers produced, determine the type of combustion and acts as the indicators of the burning history.
The simplest PAHs are phenanthrene and anthracene (International Union on Pure and Applied Chemistry (IUPAC). Benzene and naphthalene have been formally excluded from the list of PAHs. However, they are chemically related to PAHs and referred to as monoaromatic or diaromatics.
The literature documents that the number of aromatic rings determine the type of PAHs. The number in PAH may vary from 4 to 7, with 5 or 6 ringed PAH being more common. PAHs composed only of sixmembered rings are called alternant PAHs. Certain alternant PAHs, lacking in complete benzene ring, are called "benzenoid" PAHs. The figure1 and table2 enlists different PAHs constituents of crude oil.
PAHs are classified as small and large depending on the presence of number of rings. The “small” PAHs contain up to six fused aromatic rings where as “large” PAHs contain more than six aromatic rings.
PAHs have characteristic UV absorbance spectra with many bands each unique for each ring structure. Thus, each isomer has a different UV absorbance spectrum (200nm400nm). This helps in the identification of PAHs. Most of the PAHs are also fluorescent. The extended pi electron electronic structures of PAHs lead to these spectra, as well as to certain large PAHs also exhibiting semiconducting and other behaviors.
Polycyclic aromatic hydrocarbons are lipophilic . The larger compounds are less water soluble and less volatile . These properties gives PAHs, it’s a place in the environment, primarily in soil , sediment and oily substances. However, they are also a component of concern in particulate matter suspended in air.
PAHs, the aromatic compounds, exhibit varying degree of aromaticity for each ring segment. Clar's rule, given by Erich Clar in 1964 explains that benzenelike moieties are the most important for the characterization of the properties of PAHs (Kim et al., 2003). The degree of aromacity determines its level of reactivity.
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Figure 1: Radial depiction showing parent polyaromatic hydrocarbons present in crude oil.
Ova Pya
Hec
Hep
Trp
Cor
Rub
Hex
Hep
Tpl
Pec
Pen
Per
Per
Pic
Ple Npc Chr Pyr
Tpl
Aca
Acp
Flt
Ant
Phr
Phe
Flu
Ach
sIn
aIn
Bip
Hep
Azu
Nap Ind Pen
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Table 2: Parent Polyaromatic hydrocarbons present in crude oil.
S.N. Radial Depiction for PAH
PAH Name PAH structure Molecular formula
1. Pen Pentalene C8H6
2. Ind Indene C9H8
3. Nap Naphthalene C10H8
4. Azu Azulene C10H8
5. Hep Heptalene C12H10
6. Bip Biphenylene C12H8
7. aIn asIndacene C12H8
8. sIn sIndacene C12H8
9. Can Acenaphthylene C12H8
10. Flu Fluorene C13H10
11. Phe Phenalene C13H10
12. Phr Phenanthrene C14H10
13. Ant Anthracene C14H10
14. Flt Fluoranthene C16H10
15. Acp Acephenanthrylene C16H10
16. Aca Aceanthrylene C16H10
17. Tpl Triphenylene C18H12
18. Pyr Pyrene C16H10
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19. Chr Chrysene C18H12
20. Npc Naphthacene C18H12
21. Ple Pleiadene C18H12
22. Per Perylene C20H12
23. Pic Picene C22H14
24. Pen Pentaphene C22H14
25. Pec Pentacene C22H14
26. Tpl Tetraphenylene C24H16
27. Hep Hexaphene C26H16
28. Hex Hexacene C26H16
29. Rub Rubicene C26H14
30. Cor Coronene C24H12
31. Trp Trinaphthylene C30H18
32. Hep Heptaphene C30H18
33. Hec Heptacene C30H18
34. Pya Pyranthrene C30H16
35. Ova Ovalene C32H14
The United States Environmental Protection Agency (USEPA) has designated 16 PAHs compounds as priority pollutants (Table3). They are naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a, h]anthracene, benzo[g, h, i]perylene, and indeno[1,2,3cd]pyrene. These priority PAHs are generally targeted for measurement in environmental samples.
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Table 3: The U.S. EPA has designated 16 PAH compounds.
Naphthalene Acenaphthylene
Acenaphthene Phenanthrene
Anthracene Benz[a,h] anthracene Benz[a] anthracene
Chrysene
Pyrene
Benzo[a]pyrene Indeno[1,2,3cd] pyrene Benzo[g, h, i]
perylene
Fluorene Fluoranthene Benzo[k] fluoranthene Benzo[b]
fluoranthene
2. PAHs and Human health
PAHs are one of the most widespread organic pollutants and potentially health hazard. In addition to their presence in fossil fuels they are also formed by incomplete combustion of carboncontaining fuels such as wood, coal, diesel, fat, tobacco, or incense. They have been identified as carcinogenic, mutagenic, and teratogenic. PAHs are also found in foods. Studies have shown that most food intake of PAHs comes from cereals, oils and fats. Smaller intakes come from vegetables and cooked meats (Larsson et al., 1983; and Agency for toxic substances and disease registry 1996, European Commission, 2002). The toxicity of PAHs is dependent on its structure and the isomers may exhibit variable toxicity. Benzo[a]pyrene, is the first chemical carcinogen to be discovered. It is one of the constituent found in cigarette smoke . The EPA has classified seven PAH compounds as probable human carcinogens: benz[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, chrysene, dibenz[a,h]anthracene, and indeno[1,2,3cd]pyrene. Besides these, Benzo[j]fluoranthene, benzo[ghi] perylene , coronene , and ovalene are known for carcinogenic , mutagenic and teratogenic properties (Luch, 2005).
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3. Bioremoval strategies for PAHs
Microorganisms degrade PAHs either via metabolism or cometabolism. Cometabolism is especially relevant for the degradation of mixtures of PAHs. Both aerobic and anaerobic metabolism exist for PAH degradation. However aerobic pathways, their kinetics and enzymatic and genetic regulation is well documented. The present focus is on aerobic metabolism of PAHs The metabolic pathways, the degradation kinetics and the enzymatic and genetic regulation are well understood (Wirtz et al., 1981; Digiovanni, 1992; Goyal and Zylstra, 1997).
The literature cites four types of aromatic metabolism (Fuchs, 2008):
a) Aerobic Metabolism
b) Hybrid type aerobic metabolism
c) Reductive aromatic metabolism
d) Reductive metabolism in anaerobes
The flow chart exhibits the aerobic metabolic pathway of degradation for anthracene, as a model compound (Fig 2).
The aerobic aromatic metabolism is characterized by the extensive use of molecular oxygen as cosubstrate for oxygenases that introduce hydroxyl groups and cleave the aromatic ring. The aerobic PAH catabolism is mediated by the enzymatic activity of dioxygenase/monooxygenase. It incorporates atoms of molecular oxygen into the aromatic nucleus and as a result aromatic ring is oxidized (Digiovanni, 1992; Auger et al., 1995; Goyal et al., 1997,). On the basis of the substituents on the original molecule, two hydroxyl groups may be positioned either ortho (catechol and protocatechuate) or para to each other (gentisate and homogentisate). The cisdihydrodiols that are formed in this reaction are further oxidized to the aromatic dihydroxy compounds (catechols). These compounds are further oxidized through the ortho or meta cleavage pathways (Denome et al., 1993; Baboshin et al 2008).
Finally, the reactions culminate into synthesis of the precursors of TCA cycle (tricarboxylic acid) intermediates. The degradation of all PAHs is carried out by this common scheme. However, its known that the number of aromatic rings govern the kinetic efficiency of the pathway and the type of reaction intermediates produced.
Hybrid type aerobic metabolism is used by facultative aerobes eg. aerobic metabolism of benzoate, phenylacetate, and anthranilate. This pathways uses coenzyme A thioesters of the substrates and do not require oxygen for ring cleavage. An oxygenase/reductase leads to dearomatization of the ring.
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Anthracene
Naphthalene 1, 2dioxygenase
Cis1, 2Dihydroanthracene1, 2diol
Cis1, 2dihydrodihydroxynaphthalene dehydrogenase
Anthracene1, 2diol
Anthracene1,2diol1,2 dioxygenase
Anthracene1, 2diol 1, 2dioxygenase
3[(Z)2carboxyvinyl]2naphthoate
4(2hydroxynaph3yl)2oxobut3enoate
4(2hydroxynaph3yl)2oxobut3enoate hydratasealdolase
6, 7Benzocoumarin 3Hydroxy2naphthoate
3hydroxy2naphthoate hydroxylase
2, 3Dihydroxynaphthalene
Phthalate
Figure 2: Aerobic oxidation of polyaromatic hydrocarbon (model compound anthracene).
In the presence of oxygen, facultative aerobes and phototrophs use a reductive aromatic metabolism. The reduction of the aromatic ring of benzoylcoenzyme A is catalyzed by
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benzoylcoenzyme A reductase. This reduction is led by the hydrolysis of 2 ATP molecules. It has been documented that a little characterized benzoylcoenzyme A reductase operates in strict anaerobe as they can not afford the costly ATPdependent ring reduction (Georg, 2008). Both fungi and bacteria are involved in biodegradation of PAHs (Table 4 & 5).
Table 4: Bacterial genera involved in PAHs degradation
Bacterial species strain PAHs References Achromobacter sp. NCW Carbazole Guo et al., 2008 Alcaligenes denitrificans Fluoranthene Weissenfels et al., 1990 Arthrobacter sp. F101 Fluorene Casellas et al., 1997 Arthrobacter sp. P11 Phenanthrene, Carbazole,
Dibenzothiophene Seo et al., 2006
Arthrobacter sulphureus RKJ4 Phenanthrene Samanta et al., 1999 Acidovorax delafieldii P41 Phenanthrene Samanta et al., 1999 Bacillus cereus P21 Pyrene Kazunga et al., 2000 Bacillus subtilis BMT4i (MTCC9447)
Benzo[a]pyrene Lily et al., 2009
Brevibacterium sp.HL4 Phenanthrene Samanta et al., 1999 Burkholderia sp.S3702, RP007, 2A12TNFYE5, BS3770
Phenanthrene Kang et al., 2003, Balashova et al., 1999, Laurie et al., 1999
Burkholderia sp. C3 Phenanthrene Seo et al., 2006 Burkholderia cepacia BU3 Phenanthrene
Pyrene, Naphthalene
Kim et al., 2003
Burkholderia xenovorans LB400
Benzoate, Biphenyl Denef et al., 2005
Chryseobacterium sp. NCY Carbazole Guo et al., 2008 Cycloclasticus sp. P1 Pyrene Wang et al., 2008 Geobacillus sp. Napthalene, Phenanthrene,
Fluorene Bubians et al., 2007
Geobacillus stearothermophilus “AAP7919”
Anthracene Kumar et al., 2011
Janibacter sp. YY1 Phenanthrene, Fluorene, Anthracene, Dibenzofuran, Dibenzopdioxin, Dibenzothiophene
Yamazoe et al., 2004
Marinobacter NCE312 Naphthalene Hedlund et al., 2001 Mycobacterium sp.PYR, Benzo[a]pyrene Cheung et al., 2001,
Grosser et al., 1991 Mycobacterium sp. JS14 Fluoranthene Lee et al., 2007 Mycobacterium sp. 6PY1, KR2, AP1
Pyrene Rehmann et al., 1998, Vila et al., 2001, Krivobok et al., 2003
Mycobacterium sp. RJGII135 Benzo[a]pyrene, Schneider et al., 1996
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Benz[a]anthracene
Pyrene Mycobacterium sp.PYR1, LB501T
Pyrene, Phenanthrene, Fluoranthene, Anthracene
Mody et al., 2001, Kelley et al., 1993, Sepic et al., 1998, Ramirez et al., 2001,
Van et al., 2003
Mycobacterium sp. CH1, BG1, BB1, KR20
Pyrene, Phenanthrene, Fluorene Boldrin et al., 1993,
Rehmann et al., 2001 Mycobacterium flavescens Pyrene, Fluoranthene DeanRoss et al., 2002,
DeanRoss et al., 1996 Mycobacterium vanbaalenii PYR1
Phenanthrene
Pyrene, Dimethylbenz[a]anthracene
Kim et al., 2005,
Moody et al., 2003
Mycobacterium sp. KMS Pyrene Miller et al., 2004 Nocardioides aromaticivorans IC177
Carbazole Inoue et al., 2006
Pasteurella sp. IFA Fluoranthene Sepic 1999 Polaromonas naphthalenivorans CJ2
Naphthalene Pumphrey et al., 2007
Pseudomonas sp. C18, PP2, DLCP11
Phenanthrene, Naphthalene Denome et al., 1993,
Prabhu et al., 2003 Pseudomonas sp. BT1d 3hydroxy2
formylbenzothiophene Bressler et al., 2001
Pseudomonas sp. HH69 Dibenzofuran Fortnagel et al., 1990 Pseudomonas sp. CA10 Chlorinated dibenzopdioxin,
Carbazole Habe et al., 2001
Pseudomonas sp. NCIB 98164 Fluorene, Dibenzofuran, Dibenzothiophene
Resnick et al., 1996
Pseudomonas sp. F274 Fluorene Grifoll et al., 1994 Pseudomonas paucimobilis Phenanthrene Weissenfels et al., 1990 Pseudomonas vesicularis OUS82
Fluorene Weissenfels et al., 1990
Pseudomonas putida P16, BS3701, BS3750, BS590P, BS202P1
Phenanthrene, Naphthalene Kiyohara et al., 1994, Balashova et al., 1999
Pseudomonas fluorescens BS3760
Phenanthrene, Benz[a]anthracene, Chrysene
Balashova et al., 1999
Pseudomonas stutzeri P15 Pyrene Kazunga et al., 2000
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Pseudomonas saccharophilia Pyrene Kazunga et al., 2000 Pseudomonas aeruginosa Phenanthrene Romero et al., 1998 Ralstonia sp. SBUG 290, U2 Naphthalene, Dibenzofuran Becher et al., 2000,
Zhou et al., 2002 Rhodanobacter sp. BPC1 Benzo[a]pyrene Kanaly et al., 2002 Rhodococcus sp. Pyrene, Fluoranthene DeanRoss et al., 2002,
Walter et al., 1991 Rhodococcus sp. WUK2R Benzothiophene,
Naphthothiophene Kirimura et al., 2002
Rhodococcus erythropolis I19 Alkylated dibenzothiophene Folsom et al., 1999 Rhodococcus erythropolisD1 Dibenzothiophene Matsubara et al., 2001 Staphylococcus sp. PN/Y Phenanthrene Mallick et al., 2007 Stenotrophomonas maltophilia VUN 10,010
Benzo[a]pyrene
Pyrene, Fluoranthene
Boonchan et al., 1998
Stenotrophomonas maltophilia VUN 10,003
Pyrene, Fluoranthene, Benz[a]anthracene
Juhasz et al., 2000
Sphingomonas yanoikuyae R1 Pyrene Kazunga et al., 2000 Sphingomonas yanoikuyae JAR02
Benzo[a]pyrene Rentz et al., 2008
Sphingomonas sp.P2, LB126 Phenanthrene, Fluoranthene, Fluorene, Anthracene
Pinyakong et al., 2003, Van et al., 2003, Pinyakong et al., 2000
Sphingomonas sp. Dibenzofuran, Carbazole, Dibenzothiophene
Gai et al., 2007
Sphingomonas paucimobilis EPA505
Phenanthrene, Fluoranthene, Anthracene, Naphthalene
Story et al., 2001,
Mueller et al., 1990 Sphingomonas wittichii RW1 Chlorinated dibenzopdioxin Nam et al., 2006 Sphingomonas sp. KS14 Phenanthrene, Naphthalene Cho et al., 2001 Terrabacter sp.DBF63 Fluorene, Dibenzofuran,
Chlorinated dibenzopdioxin, Chlorinated dibenzothophene
Habe et al., 2004, Habe et al., 2001, Habe et al., 2002
Xanthamonas sp. Benzo[a]pyrene
Pyrene, Carbazole
Grosser et al., 1991
White rot fungi often prepare aromatic compounds for ring cleavage by first converting them to quinones. The initial oxidation of anthracene (to 9,10anthraquinone), benzo[a]pyrene ( Haemmerli, et al., 1986 ) and several other PAHs is catalyzed by lignin peroxidases from Phanerochaete chrysporium, Bjerkandera sp. strain BOS55 (Field, J.A. et al., Enzyme and Micro. Tech. 18:300308, 1996) and other white rot fungi. Manganese peroxidases, another family of lignin degrading peroxidases produced by white rot fungi, can also oxidize anthracene (Eibes et al., 1986). Laccases, coppercontaining enzymes that are also involved in lignin degradation by Trametes versicolor, have also been shown to oxidize anthracene ( Collins et al., 1986 ). Not all white rot fungi produce laccases. P. chrysosporium can completely mineralize anthracene. It cleaves 9,10anthraquinone to phthalate and, here
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proposed, catechol, though obenzoquinone or aliphatic compounds are also possible ( Hammel et al., 1991).
Table 5: Fungal genera capable of degrading PAHs.
Name of Fungus PAH Reference Phanerochaete chrysporium Anthracene Field et al.,1996 Bjerkandera sp. strain BOS55
Anthracene Field, et al.,1996
Trametes versicolor Anthracene Collins et al., 1986 Cunninghamella elegansoxidizes
Anthracene Cernigilia, 1997
P. chrysosporium Anthracene Hammel et al., 1991 Aspergillus flavus Benzo[a]pyrene Romero et al., 2010 Paecilomyces farinosus Benzo[a]pyrene Romero et al., 2010
Different technologies such as biostimulation, bioaugmentation, bioaccumulation, biosorption, phytoremediation and rhizoremediation are the key focus of present bioremediation strategies.
4. Conclusion
Crude oil contains variety of PAHs, which are known pollutants and potential health hazards. Besides other approaches, dearomatization of crude oil might be a direct hit to target and curb the PAH pollution. Voluminous researches have evolved different bioremediation tools in the form of efficient bacteria and fungi as potential degraders. The metabolism involved in degradation pathways is also well understood. The present day developments and newer approaches primarily focus to target the specific PAHs. However, development of precise, effective and composite technology to treat the complex mixtures is still a matter of concern.
Acknowledgement
We are thankful to Professor Aditya Shastri for kindly extending “Banasthali Centre for Education and Research in Basic Science” sanctioned under CURIE (Consolidation of University Research for Innovation and Excellence in Women University) program of department gratefully acknowledged. The authors are indebted to Bhojia Charitable Trust for Science Research and Social Welfare for providing adequate facilities to prepare this manuscript.
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