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[[wikipedia: Hydrocarbon | Hydrocarbons]] are major constituents of crude oil and petroleum. They can be biodegraded by naturally-occurring microorganisms in freshwater and marine environments under a variety of aerobic and anaerobic conditions. The ability of microorganisms - bacteria, archaea, fungi, or algae - to break down hydrocarbons is the basis for natural and enhanced bioremediation. To promote biodegradation, amendments such as nitrogen and phosphorous fertilizer are often added to stimulate microbial growth and metabolism. Oxygen, nitrate, or sulfates are sometimes added as electron acceptors to enhance biodegradation rates. The addition of hydrocarbon-degrading microbial cultures to contaminated sites might also be considered, although the practice of bioaugmentation is uncommon for hydrocarbons owing to the natural abundance of hydrocarbon-degrading microbes. Aside from excavation, various forms of bioremediation from simple tilling of soils and landfarming to in situ biostimulation are common forms of remediation for hydrocarbon contamination. Compared to other technologies, bioremediation is lower cost, environmentally-friendly, and often non-invasive<ref name= "Atlas2005">Atlas, R.M. and Philp, J. (eds.), 2005. Bioremediation: Applied microbial solutions for real-world environmental cleanup, 366 pgs. American Association for Microbiology, Washington. [http://www.asmscience.org/content/book/10.1128/9781555817596 DOI: 10.1128/9781555817596]</ref>. However, the time needed for full site cleanup can be longer and less predictable than for other methods, and successful outcomes may not be guaranteed due to variability in site conditions. Nonetheless, it is the most common approach for remediation of hydrocarbon-contaminated environments.
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<onlyinclude>[[wikipedia: Hydrocarbon | Hydrocarbons]] are major constituents of crude oil and petroleum. They can be biodegraded by naturally-occurring microorganisms in freshwater and marine environments under a variety of aerobic and anaerobic conditions. The ability of microorganisms - bacteria, archaea, fungi, or algae - to break down hydrocarbons is the basis for natural and enhanced bioremediation. To promote biodegradation, amendments such as nitrogen and phosphorous fertilizer are often added to stimulate microbial growth and metabolism. </onlyinclude>Oxygen, nitrate, or sulfates are sometimes added as electron acceptors to enhance biodegradation rates. The addition of hydrocarbon-degrading microbial cultures to contaminated sites might also be considered, although the practice of bioaugmentation is uncommon for hydrocarbons owing to the natural abundance of hydrocarbon-degrading microbes. Aside from excavation, various forms of bioremediation from simple tilling of soils and landfarming to in situ biostimulation are common forms of remediation for hydrocarbon contamination. Compared to other technologies, bioremediation is lower cost, environmentally-friendly, and often non-invasive<ref name="Atlas2005">Atlas, R.M. and Philp, J. (eds.), 2005. Bioremediation: Applied microbial solutions for real-world environmental cleanup, 366 pgs. American Association for Microbiology, Washington. [http://www.asmscience.org/content/book/10.1128/9781555817596 DOI: 10.1128/9781555817596]</ref>. However, the time needed for full site cleanup can be longer and less predictable than for other methods, and successful outcomes may not be guaranteed due to variability in site conditions. Nonetheless, it is the most common approach for remediation of hydrocarbon-contaminated environments.
 
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'''Related Article(s)''':
 
'''Related Article(s)''':
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*[[Bioremediation - Anaerobic]]
 
*[[Bioremediation - Anaerobic]]
 
*[[Chemical Oxidation (In Situ - ISCO)]]
 
*[[Chemical Oxidation (In Situ - ISCO)]]
 
*[[Chemical Reduction (In Situ - ISCR)]]
 
*[[Chemical Reduction (In Situ - ISCR)]]
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*[[Landfarming]]
 
*[[Polycyclic Aromatic Hydrocarbons (PAHs)]]
 
*[[Polycyclic Aromatic Hydrocarbons (PAHs)]]
  
  
'''CONTRIBUTOR(S):''' [[Elisse Magnuson]] and [[Dr. Elizabeth Edwards]]
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'''Contributor(s):''' [[Elisse Magnuson]] and [[Dr. Elizabeth Edwards]]
  
  
 
'''Key Resource(s)''':  
 
'''Key Resource(s)''':  
*[https://doi.org/10.1007/s12010-015-1603-5 A Comprehensive Review of Aliphatic Biodegradation - Hydrocarbons by Bacteria]<ref name= "Abbasian2015">Abbasian, F., Lockington, R., Mallavarapu, M. and Naidu, R., 2015. A comprehensive review of aliphatic hydrocarbon biodegradation by bacteria. Applied Biochemistry and Biotechnology, 176(3), 670-699. [https://doi.org/10.1007/s12010-015-1603-5 doi: 10.1007/s12010-015-1603-5]</ref>
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*[https://doi.org/10.1159/000443997 Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment]<ref name= "Rabus2016">Rabus, R., Boll, M., Heider, J., Meckenstock, R.U., Buckel, W., Einsle, O., Ermler, U., Golding, B.T., Gunsalus, R.P., Kroneck, P.M. and Krüger, M., 2016. Anaerobic microbial degradation of hydrocarbons: from enzymatic reactions to the environment. Journal of Molecular Microbiology and Biotechnology, 26(1-3), 5-28. [https://doi.org/10.1159/000443997 doi:10.1159/000443997]</ref>
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*[https://doi.org/10.1007/s12010-015-1603-5 A Comprehensive Review of Aliphatic Biodegradation - Hydrocarbons by Bacteria]<ref name="Abbasian2015">Abbasian, F., Lockington, R., Mallavarapu, M. and Naidu, R., 2015. A comprehensive review of aliphatic hydrocarbon biodegradation by bacteria. Applied Biochemistry and Biotechnology, 176(3), 670-699. [https://doi.org/10.1007/s12010-015-1603-5 doi: 10.1007/s12010-015-1603-5]</ref>
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*[https://doi.org/10.1159/000443997 Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment]<ref name="Rabus2016">Rabus, R., Boll, M., Heider, J., Meckenstock, R.U., Buckel, W., Einsle, O., Ermler, U., Golding, B.T., Gunsalus, R.P., Kroneck, P.M. and Krüger, M., 2016. Anaerobic microbial degradation of hydrocarbons: from enzymatic reactions to the environment. Journal of Molecular Microbiology and Biotechnology, 26(1-3), 5-28. [https://doi.org/10.1159/000443997 doi:10.1159/000443997]</ref>
  
 
==Introduction==
 
==Introduction==
[[wikipedia: Hydrocarbon | Hydrocarbons]] are compounds composed entirely of carbon and hydrogen. Aromatic hydrocarbons, alkanes, alkenes, cycloalkanes, alkynes, and combinations of these compounds comprise different types of hydrocarbons. Complex mixtures of hydrocarbons occur naturally in crude oil and gasoline. Most can be used as substrates in metabolism by bacteria, archaea, fungi, and algae<ref name= "Abbasian2015"/><ref name= "Rabus2016"/>. While fungi and algae degrade hydrocarbons aerobically, bacteria and archaea are capable of both aerobic and [[Bioremediation - Anaerobic | anaerobic degradation]]<ref>Weelink, S.A., van Eekert, M.H. and Stams, A.J., 2010. Degradation of BTEX by anaerobic bacteria: physiology and application. Reviews in Environmental Science and Bio/Technology, 9(4), 359-385. [https://doi.org/10.1007/s11157-010-9219-2 doi: 10.1007/s11157-010-9219-2]</ref><ref>Widdel, F., Knittel, K. and Galushko, A., 2010. Anaerobic hydrocarbon-degrading microorganisms: an overview. In Handbook of Hydrocarbon and Lipid Microbiology, pgs. 1997-2021. Springer Berlin Heidelberg. [https://doi.org/10.1007/978-3-540-77587-4_146 doi: 10.1007/978-3-540-77587-4_146]</ref><ref name="Haritash2009">Haritash, A.K. and Kaushik, C.P., 2009. Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): A review. Journal of Hazardous Materials, 169(1), 1-15. [http://dx.doi.org/10.1016/j.jhazmat.2009.03.137 http://dx.doi.org/10.1016/j.jhazmat.2009.03.137]</ref><ref name="Xue2015">Xue, J., Yu, Y., Bai, Y., Wang, L. and Wu, Y., 2015. Marine oil-degrading microorganisms and biodegradation process of petroleum hydrocarbon in marine environments: a review. Current Microbiology, 71(2), 220-228. [https://doi.org/10.1007/s00284-015-0825-7 doi: 10.1007/s00284-015-0825-7]</ref>. Owing to the relatively reduced nature of most hydrocarbons, they are typically the energy source or electron donors for microbial metabolism, and their oxidation must be coupled to the reduction of a suitable electron acceptor. Aerobic degradation occurs with molecular oxygen (O<sub>2</sub>) as both a reactant to oxidize the substrate and an electron acceptor for microbial respiration. In contrast, anaerobic degradation uses different biotransformation pathways that don’t depend on oxygen, coupled to microbial respiration of a variety of electron acceptors. Under anaerobic conditions, biodegradation often results from the stepwise concerted action of many different microbes in a process called [[wikipedia: Syntrophy | syntrophy]].  
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[[wikipedia: Hydrocarbon | Hydrocarbons]] are compounds composed entirely of carbon and hydrogen. Aromatic hydrocarbons, alkanes, alkenes, cycloalkanes, alkynes, and combinations of these compounds comprise different types of hydrocarbons. Complex mixtures of hydrocarbons occur naturally in crude oil and gasoline. Most can be used as substrates in metabolism by bacteria, archaea, fungi, and algae<ref name="Abbasian2015" /><ref name="Rabus2016" />. While fungi and algae degrade hydrocarbons aerobically, bacteria and archaea are capable of both aerobic and [[Bioremediation - Anaerobic | anaerobic degradation]]<ref>Weelink, S.A., van Eekert, M.H. and Stams, A.J., 2010. Degradation of BTEX by anaerobic bacteria: physiology and application. Reviews in Environmental Science and Bio/Technology, 9(4), 359-385. [https://doi.org/10.1007/s11157-010-9219-2 doi: 10.1007/s11157-010-9219-2]</ref><ref>Widdel, F., Knittel, K. and Galushko, A., 2010. Anaerobic hydrocarbon-degrading microorganisms: an overview. In Handbook of Hydrocarbon and Lipid Microbiology, pgs. 1997-2021. Springer Berlin Heidelberg. [https://doi.org/10.1007/978-3-540-77587-4_146 doi: 10.1007/978-3-540-77587-4_146]</ref><ref name="Haritash2009">Haritash, A.K. and Kaushik, C.P., 2009. Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): A review. Journal of Hazardous Materials, 169(1), 1-15. http://dx.doi.org/10.1016/j.jhazmat.2009.03.137</ref><ref name="Xue2015">Xue, J., Yu, Y., Bai, Y., Wang, L. and Wu, Y., 2015. Marine oil-degrading microorganisms and biodegradation process of petroleum hydrocarbon in marine environments: a review. Current Microbiology, 71(2), 220-228. [https://doi.org/10.1007/s00284-015-0825-7 doi: 10.1007/s00284-015-0825-7]</ref>. Owing to the relatively reduced nature of most hydrocarbons, they are typically the energy source or electron donors for microbial metabolism, and their oxidation must be coupled to the reduction of a suitable electron acceptor. Aerobic degradation occurs with molecular oxygen (O<sub>2</sub>) as both a reactant to oxidize the substrate and an electron acceptor for microbial respiration. In contrast, anaerobic degradation uses different biotransformation pathways that don’t depend on oxygen, coupled to microbial respiration of a variety of electron acceptors. Under anaerobic conditions, biodegradation often results from the stepwise concerted action of many different microbes in a process called [[wikipedia: Syntrophy | syntrophy]].  
  
Biodegradation occurs naturally because hydrocarbons have always been present in the environment, released from seeps and reservoirs through various geologic processes. Natural attenuation of hydrocarbon contamination can be monitored by a number of analytical methods, including gas chromatography and infrared spectroscopy<ref>Agency for Toxic Substances and Disease Registry, 1999. Toxicological profile for total petroleum hydrocarbons (TPH). [[Media:ATSDR-1999-Tox_profile_for_TPH.pdf|Report pdf]]</ref>. Absolute proof of degradation versus dilution or other non-degradative processes can be obtained using compound [[Compound Specific Isotope Analysis (CSIA) |specific isotope analysis (CSIA)]], where an enrichment in the natural abundance of heavier isotopes of carbon and hydrogen in the parent hydrocarbons confirms biotransformation. Often biostimulation - the addition of limiting nutrients - may be desired in sites where attenuation occurs slowly or not at all.
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Biodegradation occurs naturally because hydrocarbons have always been present in the environment, released from seeps and reservoirs through various geologic processes. Natural attenuation of hydrocarbon contamination can be monitored by a number of analytical methods, including gas chromatography and infrared spectroscopy<ref>Agency for Toxic Substances and Disease Registry, 1999. Toxicological profile for total petroleum hydrocarbons (TPH). [//www.enviro.wiki/images/0/08/ATSDR-1999-Tox_profile_for_TPH.pdf Report pdf]</ref>. Absolute proof of degradation versus dilution or other non-degradative processes can be obtained using compound [[Compound Specific Isotope Analysis (CSIA) |specific isotope analysis (CSIA)]], where an enrichment in the natural abundance of heavier isotopes of carbon and hydrogen in the parent hydrocarbons confirms biotransformation. Often biostimulation - the addition of limiting nutrients - may be desired in sites where attenuation occurs slowly or not at all.
[[File:Edwards Article 1-figure 1.PNG|400 px|thumbnail|left|Figure 1. Components of hydrocarbon biodegradation. Understanding and facilitating biodegradation at a contaminated site requires knowledge of the environmental conditions, compound properties, and microorganisms present (adapted after Sutherson, 1999)<ref name= "Suthersan1999"/>.]]
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[[File:Edwards Article 1-figure 1.PNG|400 px|thumbnail|left|Figure 1. Components of hydrocarbon biodegradation. Understanding and facilitating biodegradation at a contaminated site requires knowledge of the environmental conditions, compound properties, and microorganisms present (adapted after Sutherson, 1999)<ref name="Suthersan1999" />.]]
  
Many bioremediation efforts involve injection and circulation of oxygen to subsurface environments in order to increase the availability of oxygen as both reactant and electron acceptor. Common methods include using a vacuum pump to promote air circulation<ref name= "NRC1993">National Research Council, 1993. In Situ Bioremediation – When does it work? Committee on In Situ Bioremediation, Water Science and Technology Board, Commission on Engineering and Technical Systems, National Academy Press, Washington D.C. [https://doi.org/10.17226/2131 doi: 10.17226/2131]</ref>, bioventing or biosparging that add air to the subsurface<ref>US Environmental Protection Agency, 1994. How to evaluate alternative cleanup technologies for underground storage tank sites. [[Media:USEPA-1994-How_to_evalutate_alternative_cleanup_tech_for_UST_Sites.pdf|Report pdf]]</ref>, water circulation aboveground to add oxygen<ref name= "NRC1993"/>, and the introduction of substances that release oxygen like peroxides or ozone. Remediation may also involve introduction of bacterial strains (bioaugmentation) as well as nutrients to stimulate hydrocarbon degradation. This strategy is most often used at sites with mid-weight or heavier petroleum products, as lighter compounds volatilize<ref name= "Contsitecleanup2016">'''Contaminated Site Clean-Up Information, 2016. Bioremediation'''</ref>. Related commercial products include microbial culture, enzyme, and nutrient additives. These products may be less viable in the field than in laboratory settings due to varying site conditions such that field testing is necessary to determine their effectiveness<ref name= "das2011">Das, N. and Chandran, P., 2011. Microbial degradation of petroleum hydrocarbon contaminants: an overview. Biotechnology Research International, 941810, 13 pgs. [https://doi.org/10.4061/2011/941810 doi: 10.4061/2011/941810]</ref>. As the biochemical processes of anaerobic hydrocarbon biotransformation have become more clearly established, bioremediation efforts have shifted towards adding nitrate or sulfate as electron acceptors instead of oxygen because these electron acceptors are much more soluble than oxygen and easier to deliver, particularly for in situ applications<ref>Cunningham, J.A., Hopkins, G.D., Lebron, C.A. and Reinhard, M., 2000. Enhanced anaerobic bioremediation of groundwater contaminated by fuel hydrocarbons at Seal Beach, California. Biodegradation, 11(2-3), 159-170. [https://doi.org/10.1023/a:1011167709913 doi: 10.1023/A:1011167709913]</ref>.  
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Many bioremediation efforts involve injection and circulation of oxygen to subsurface environments in order to increase the availability of oxygen as both reactant and electron acceptor. Common methods include using a vacuum pump to promote air circulation<ref name="NRC1993">National Research Council, 1993. In Situ Bioremediation – When does it work? Committee on In Situ Bioremediation, Water Science and Technology Board, Commission on Engineering and Technical Systems, National Academy Press, Washington D.C. [https://doi.org/10.17226/2131 doi: 10.17226/2131]</ref>, bioventing or biosparging that add air to the subsurface<ref>US Environmental Protection Agency, 1994. How to evaluate alternative cleanup technologies for underground storage tank sites. [//www.enviro.wiki/images/7/74/USEPA-1994-How_to_evalutate_alternative_cleanup_tech_for_UST_Sites.pdf Report pdf]</ref>, water circulation aboveground to add oxygen<ref name="NRC1993" />, and the introduction of substances that release oxygen like peroxides or ozone. Remediation may also involve introduction of bacterial strains (bioaugmentation) as well as nutrients to stimulate hydrocarbon degradation. This strategy is most often used at sites with mid-weight or heavier petroleum products, as lighter compounds volatilize<ref name="Contsitecleanup2016">US EPA, CLU-IN, Bioremediation. [https://clu-in.org/techfocus/default.focus/sec/Bioremediation/cat/Overview/ Website]</ref>. Related commercial products include microbial culture, enzyme, and nutrient additives. These products may be less viable in the field than in laboratory settings due to varying site conditions such that field testing is necessary to determine their effectiveness<ref name="das2011">Das, N. and Chandran, P., 2011. Microbial degradation of petroleum hydrocarbon contaminants: an overview. Biotechnology Research International, 941810, 13 pgs. [https://doi.org/10.4061/2011/941810 doi: 10.4061/2011/941810]</ref>. As the biochemical processes of anaerobic hydrocarbon biotransformation have become more clearly established, bioremediation efforts have shifted towards adding nitrate or sulfate as electron acceptors instead of oxygen because these electron acceptors are much more soluble than oxygen and easier to deliver, particularly for in situ applications<ref>Cunningham, J.A., Hopkins, G.D., Lebron, C.A. and Reinhard, M., 2000. Enhanced anaerobic bioremediation of groundwater contaminated by fuel hydrocarbons at Seal Beach, California. Biodegradation, 11(2-3), 159-170. [https://doi.org/10.1023/a:1011167709913 doi: 10.1023/A:1011167709913]</ref>.  
  
Site conditions affect the occurrence and rate of biodegradation in contaminated environments (Fig. 1). Nitrogen, phosphorus, and iron are important nutrients for bacteria and may become limiting in certain environments, particularly in seawater and freshwater wetlands which can be nutrient deficient. Addition of inorganic nutrients can bolster biodegradation rates. However, high levels of nitrogen, phosphorus, and potassium may inhibit biodegradation, particularly of [[Polycyclic Aromatic Hydrocarbons (PAHs) | aromatics]]<ref name= "das2011"/>. Nitrogen at reported levels of 200-4000 mg N/kg soil have been shown to reduce hydrocarbon loss, while lower levels stimulated degradation. This may depend on site-specific conditions such as soil type and contaminants<ref>Braddock, J.F., Ruth, M.L., Catterall, P.H., Walworth, J.L. and McCarthy, K.A., 1997. Enhancement and inhibition of microbial activity in hydrocarbon-contaminated arctic soils: implications for nutrient-amended bioremediation. Environmental Science & Technology, 31(7), 2078-2084. [https://doi.org/10.1021/es960904d doi: 10.1021/es960904d]</ref><ref>Chaineau, C.H., Rougeux, G., Yepremian, C. and Oudot, J., 2005. Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil. Soil Biology and Biochemistry, 37(8), 1490-1497. [http://dx.doi.org/10.1016/j.soilbio.2005.01.012 doi: 10.1016/j.soilbio.2005.01.012]</ref>. In addition, the pH of a site affects the solubility and availability of nutrients, and must be in the tolerant range for the responsible microorganisms, generally between pH 6-8<ref name= "Contsitecleanup2016"/><ref name= "Suthersan1999">Suthersan, S.S. 1999. Soil vapor extraction. In: Remediation Engineering Design Concepts. CRC Press, S.S. Suthersan, ed., Boca Raton, Florida. [https://www.crcpress.com/Remediation-Engineering-Design-Concepts/Suthersan/p/book/9781566701372 ISBN: 978-1-5667-0137-2]</ref>. The presence of organic matter may increase persistence of contaminants due to contaminant partitioning into the organic phase, reducing their bioavailability to microorganisms<ref name= "Chikere2011">Chikere, C.B., Okpokwasili, G.C. and Chikere, B.O., 2011. Monitoring of microbial hydrocarbon remediation in the soil. 3 Biotech, 1(3), 117-138. [https://doi.org/10.1007/s13205-011-0014-8 doi: 10.1007/s13205-011-0014-8]</ref>.  
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Site conditions affect the occurrence and rate of biodegradation in contaminated environments (Fig. 1). Nitrogen, phosphorus, and iron are important nutrients for bacteria and may become limiting in certain environments, particularly in seawater and freshwater wetlands which can be nutrient deficient. Addition of inorganic nutrients can bolster biodegradation rates. However, high levels of nitrogen, phosphorus, and potassium may inhibit biodegradation, particularly of [[Polycyclic Aromatic Hydrocarbons (PAHs) | aromatics]]<ref name="das2011" />. Nitrogen at reported levels of 200-4000 mg N/kg soil have been shown to reduce hydrocarbon loss, while lower levels stimulated degradation. This may depend on site-specific conditions such as soil type and contaminants<ref>Braddock, J.F., Ruth, M.L., Catterall, P.H., Walworth, J.L. and McCarthy, K.A., 1997. Enhancement and inhibition of microbial activity in hydrocarbon-contaminated arctic soils: implications for nutrient-amended bioremediation. Environmental Science & Technology, 31(7), 2078-2084. [https://doi.org/10.1021/es960904d doi: 10.1021/es960904d]</ref><ref>Chaineau, C.H., Rougeux, G., Yepremian, C. and Oudot, J., 2005. Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil. Soil Biology and Biochemistry, 37(8), 1490-1497. [http://dx.doi.org/10.1016/j.soilbio.2005.01.012 doi: 10.1016/j.soilbio.2005.01.012]</ref>. In addition, the pH of a site affects the solubility and availability of nutrients, and must be in the tolerant range for the responsible microorganisms, generally between pH 6-8<ref name="Contsitecleanup2016" /><ref name="Suthersan1999">Suthersan, S.S. 1999. Soil vapor extraction. In: Remediation Engineering Design Concepts. CRC Press, S.S. Suthersan, ed., Boca Raton, Florida. [https://www.crcpress.com/Remediation-Engineering-Design-Concepts/Suthersan/p/book/9781566701372 ISBN: 978-1-5667-0137-2]</ref>. The presence of organic matter may increase persistence of contaminants due to contaminant partitioning into the organic phase, reducing their bioavailability to microorganisms<ref name="Chikere2011">Chikere, C.B., Okpokwasili, G.C. and Chikere, B.O., 2011. Monitoring of microbial hydrocarbon remediation in the soil. 3 Biotech, 1(3), 117-138. [https://doi.org/10.1007/s13205-011-0014-8 doi: 10.1007/s13205-011-0014-8]</ref>.  
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<onlyinclude>
  
 
==Aerobic Degradation==
 
==Aerobic Degradation==
Hydrocarbons are readily degraded under aerobic conditions. Bacteria, fungi, and algae are all capable of aerobic hydrocarbon degradation<ref name="Haritash2009"/>. In general, [[wikipedia: Alkene | alkenes]] (hydrocarbons containing double bonds) and short-chain [[wikipedia: Alkane | alkanes]] (hydrocarbons containing only single bonds) are the most easily degraded, followed by branched alkanes (alkanes with side chains) and then aromatics (hydrocarbons in a stable ring structure)<ref name="Xue2015"/>. However, degradation rates vary based on environmental parameters and decrease as hydrocarbon complexity increases. Reported degradation rates vary considerably because hydrocarbon composition depends on the source of the petroleum and age of the spill. For example, compound degradation varies from 5% to 30% in 28 days, while up to 100% degradation occurrs with nitrogen addition<ref>Röling, W.F., Milner, M.G., Jones, D.M., Lee, K., Daniel, F., Swannell, R.J. and Head, I.M., 2002. Robust hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced oil spill bioremediation. Applied and Environmental Microbiology, 68(11), 5537-5548. [https://doi.org/10.1128/aem.68.11.5537-5548.2002 doi: 10.1128/AEM.68.11.5537-5548.2002]</ref>. Degradation rates by fungal species reportedly range from ~ 30-100% degradation over 28 days or less<ref>Zafra, G. and Cortés-Espinosa, D.V., 2015. Biodegradation of polycyclic aromatic hydrocarbons by Trichoderma species: a mini review. Environmental Science and Pollution Research, 22(24), 19426-19433. [https://doi.org/10.1007/s11356-015-5602-4 doi: 10.1007/s11356-015-5602-4]</ref>. As discussed above, the primary rate-limiting factor in aerobic biodegradation is delivery of oxygen. Oxygen availability is dependent on the ability of oxygen to move or diffuse through the site environment as well as on the uptake rate by microorganisms. Addition of oxygen can increase degradation rates several orders of magnitude over naturally occurring rates<ref name= "Contsitecleanup2016"/>.  
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Hydrocarbons are readily degraded under aerobic conditions. Bacteria, fungi, and algae are all capable of aerobic hydrocarbon degradation</onlyinclude><ref name="Haritash2009" /><onlyinclude>. In general, [[wikipedia: Alkene | alkenes]] (hydrocarbons containing double bonds) and short-chain [[wikipedia: Alkane | alkanes]] (hydrocarbons containing only single bonds) are the most easily degraded, followed by branched alkanes (alkanes with side chains) and then aromatics (hydrocarbons in a stable ring structure)</onlyinclude><ref name="Xue2015" /><onlyinclude>.</onlyinclude> However, degradation rates vary based on environmental parameters and decrease as hydrocarbon complexity increases. Reported degradation rates vary considerably because hydrocarbon composition depends on the source of the petroleum and age of the spill. For example, compound degradation varies from 5% to 30% in 28 days, while up to 100% degradation occurrs with nitrogen addition<ref>Röling, W.F., Milner, M.G., Jones, D.M., Lee, K., Daniel, F., Swannell, R.J. and Head, I.M., 2002. Robust hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced oil spill bioremediation. Applied and Environmental Microbiology, 68(11), 5537-5548. [https://doi.org/10.1128/aem.68.11.5537-5548.2002 doi: 10.1128/AEM.68.11.5537-5548.2002]</ref>. Degradation rates by fungal species reportedly range from ~ 30-100% degradation over 28 days or less<ref>Zafra, G. and Cortés-Espinosa, D.V., 2015. Biodegradation of polycyclic aromatic hydrocarbons by Trichoderma species: a mini review. Environmental Science and Pollution Research, 22(24), 19426-19433. [https://doi.org/10.1007/s11356-015-5602-4 doi: 10.1007/s11356-015-5602-4]</ref>. As discussed above, the primary rate-limiting factor in aerobic biodegradation is delivery of oxygen. Oxygen availability is dependent on the ability of oxygen to move or diffuse through the site environment as well as on the uptake rate by microorganisms. Addition of oxygen can increase degradation rates several orders of magnitude over naturally occurring rates<ref name="Contsitecleanup2016" />.
  
 
===Alkanes and Alkenes===
 
===Alkanes and Alkenes===
Alkanes containing 14 carbons or fewer are prone to volatilization, while alkanes containing more carbons are less volatile<ref name= "Chikere2011"/>. Regardless, alkanes and alkenes, with the exception of cyclic alkanes (alkanes in a ring structure), are the most readily degraded hydrocarbons with reported degradation of alkanes containing up to 44 carbons<ref name= "Abbasian2015"/><ref name= "Chikere2011"/>. Both alkanes and alkenes are degraded with addition of molecular oxygen. Oxygen availability and the initial step of degradation are rate-limiting<ref name= "Atlas2005"/>. In the initial steps of aerobic alkane degradation, enzymes called [[wikipedia: Oxygenase | oxygenases]] add molecular oxygen to the hydrocarbon molecules, forming alcohols that are further oxidized to fatty acids that are subsequently metabolized to acetyl-CoA and finally to CO<sub>2</sub> and H<sub>2</sub>O. Many mono- and di-oxygenases and other oxidases have a wide substrate range and act readily on a variety of hydrocarbons<ref name= "Abbasian2015"/>.
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Alkanes containing 14 carbons or fewer are prone to volatilization, while alkanes containing more carbons are less volatile<ref name="Chikere2011" />. Regardless, alkanes and alkenes, with the exception of cyclic alkanes (alkanes in a ring structure), are the most readily degraded hydrocarbons with reported degradation of alkanes containing up to 44 carbons<ref name="Abbasian2015" /><ref name="Chikere2011" />. Both alkanes and alkenes are degraded with addition of molecular oxygen. Oxygen availability and the initial step of degradation are rate-limiting<ref name="Atlas2005" />. In the initial steps of aerobic alkane degradation, enzymes called [[wikipedia: Oxygenase | oxygenases]] add molecular oxygen to the hydrocarbon molecules, forming alcohols that are further oxidized to fatty acids that are subsequently metabolized to acetyl-CoA and finally to CO<sub>2</sub> and H<sub>2</sub>O. Many mono- and di-oxygenases and other oxidases have a wide substrate range and act readily on a variety of hydrocarbons<ref name="Abbasian2015" />.
  
 
===Aromatic Hydrocarbons===
 
===Aromatic Hydrocarbons===
Aromatic hydrocarbons are generally more difficult to degrade than shorter alkanes and alkenes due to their greater toxicity, yet they are readily degraded aerobically by many bacteria and fungi (see also [[Polycyclic Aromatic Hydrocarbons (PAHs)]]). Degradability decreases with increasing number of rings and increased molecular size, due to increased hydrophobicity and sorption capacity<ref name= "Chikere2011"/>. The median primary degradation rate of benzene, toluene, ethylbenzene, and xylene (BTEX compounds) ranges from 0.05-0.2 day<sup>-1</sup><ref name="Lawrence2006">Lawrence, S.J., 2006. Description, properties, and degradation of selected volatile organic compounds detected in ground water--A Review of Selected Literature No. 2006-1338. [[Media:Lawrence-2006-Description_properties_degradation_of_VOCs.pdf|Report pdf]]</ref>. Oxidation of BTEX requires 3.1 mg/L dissolved oxygen (DO) to degrade 1 mg/L of BTEX. When DO is below 2 mg/L, biodegradation slows<ref name="Lawrence2006"/>. The general pathway for degradation of aromatic compounds begins with the addition of O<sub>2</sub> by mono and di-oxygenases<ref>Baboshin, M.A. and Golovleva, L.A., 2012. Aerobic bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) and its kinetic aspects. Microbiology, 81(6), 639-650. [https://doi.org/10.1134/s0026261712060021 doi: 10.1134/S0026261712060021]</ref>. This yields key intermediate products such as benzyl alcohol, phenol or catechol, protocatechuate, and gentisate<ref name="Fuchs2011">Fuchs, G., Boll, M. and Heider, J., 2011. Microbial degradation of aromatic compounds-from one strategy to four. Nature Reviews Microbiology, 9(11), 803-816. [https://doi.org/10.1038/nrmicro2652 doi: 10.1038/nrmicro2652]</ref>. These intermediates then undergo ring cleavage, also by a variety of oxygenases, resulting in carboxylic acids<ref>Vaillancourt, F.H., Bolin, J.T. and Eltis, L.D., 2006. The ins and outs of ring-cleaving dioxygenases. Critical Reviews in Biochemistry and Molecular Biology, 41(4), 241-267. [https://doi.org/10.1080/10409230600817422 doi: 10.1080/10409230600817422]</ref><ref name="Fuchs2011"/>. Degradation then continues to acetyl-CoA and succinyl-CoA<ref name="Fuchs2011"/> that enter into central metabolism. Fungal degradation occurs by non-specific extracellular oxidizing enzymes that form radical intermediates, although many reactions are similar to those found in bacteria<ref name= "Haritash2009"/>.  
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Aromatic hydrocarbons are generally more difficult to degrade than shorter alkanes and alkenes due to their greater toxicity, yet they are readily degraded aerobically by many bacteria and fungi (see also [[Polycyclic Aromatic Hydrocarbons (PAHs)]]). Degradability decreases with increasing number of rings and increased molecular size, due to increased hydrophobicity and sorption capacity<ref name="Chikere2011" />. The median primary degradation rate of benzene, toluene, ethylbenzene, and xylene (BTEX compounds) ranges from 0.05-0.2 day<sup>-1</sup><ref name="Lawrence2006">Lawrence, S.J., 2006. Description, properties, and degradation of selected volatile organic compounds detected in ground water--A Review of Selected Literature No. 2006-1338. [//www.enviro.wiki/images/5/5f/Lawrence-2006-Description_properties_degradation_of_VOCs.pdf Report pdf]</ref>. Oxidation of BTEX requires 3.1 mg/L dissolved oxygen (DO) to degrade 1 mg/L of BTEX. When DO is below 2 mg/L, biodegradation slows<ref name="Lawrence2006" />. The general pathway for degradation of aromatic compounds begins with the addition of O<sub>2</sub> by mono and di-oxygenases<ref>Baboshin, M.A. and Golovleva, L.A., 2012. Aerobic bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) and its kinetic aspects. Microbiology, 81(6), 639-650. [https://doi.org/10.1134/s0026261712060021 doi: 10.1134/S0026261712060021]</ref>. This yields key intermediate products such as benzyl alcohol, phenol or catechol, protocatechuate, and gentisate<ref name="Fuchs2011">Fuchs, G., Boll, M. and Heider, J., 2011. Microbial degradation of aromatic compounds-from one strategy to four. Nature Reviews Microbiology, 9(11), 803-816. [https://doi.org/10.1038/nrmicro2652 doi: 10.1038/nrmicro2652]</ref>. These intermediates then undergo ring cleavage, also by a variety of oxygenases, resulting in carboxylic acids<ref>Vaillancourt, F.H., Bolin, J.T. and Eltis, L.D., 2006. The ins and outs of ring-cleaving dioxygenases. Critical Reviews in Biochemistry and Molecular Biology, 41(4), 241-267. [https://doi.org/10.1080/10409230600817422 doi: 10.1080/10409230600817422]</ref><ref name="Fuchs2011" />. Degradation then continues to acetyl-CoA and succinyl-CoA<ref name="Fuchs2011" /> that enter into central metabolism. Fungal degradation occurs by non-specific extracellular oxidizing enzymes that form radical intermediates, although many reactions are similar to those found in bacteria<ref name="Haritash2009" />.  
 
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<onlyinclude>
 
==Anaerobic Degradation==
 
==Anaerobic Degradation==
[[File:Edwards Article 1-figure 2.PNG|600px|thumbnail|right|Figure 2. Comparison of the longitudinal redox zonation concept (A) and the plume fringe concept (B). Both concepts describe the spatial distribution of electron acceptors and respiration processes in a hydrocarbon contaminant plume. (B) Iron(III) reduction, manganese(IV) reduction, and methanogenesis may occur simultaneously in the core of the contaminant plume (reproduced with permission from &copy; 2015 American Chemical Society<ref>Meckenstock, R.U., Elsner, M., Griebler, C., Lueders, T., Stumpp, C., Aamand, J., Agathos, S.N., Albrechtsen, H.J., Bastiaens, L., Bjerg, P.L. and Boon, N., '''More Authors Here''', 2015. Biodegradation: updating the concepts of control for microbial cleanup in contaminated aquifers. Environmental Science & Technology, 49(12), 7073-7081. [https://doi.org/10.1021/acs.est.5b00715 doi: 10.1021/acs.est.5b00715]</ref>).]]
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</onlyinclude>
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[[File:Edwards Article 1-figure 2.PNG|600px|thumbnail|right|Figure 2. Comparison of the longitudinal redox zonation concept (A) and the plume fringe concept (B). Both concepts describe the spatial distribution of electron acceptors and respiration processes in a hydrocarbon contaminant plume. (B) Iron(III) reduction, manganese(IV) reduction, and methanogenesis may occur simultaneously in the core of the contaminant plume (reproduced with permission from &copy; 2015 American Chemical Society<ref>Meckenstock, R.U., Elsner, M., Griebler, C., Lueders, T., Stumpp, C., Aamand, J., Agathos, S.N., Albrechtsen, H.J., Bastiaens, L., Bjerg, P.L., Boon, N., Dejonghe, W., Huang, W.E., Schmidt, S.I., Smolders, E., Sørensen, S.R., Springael, D, and van Breukelen, B.M., 2015. Biodegradation: Updating the Concepts of Control for Microbial Cleanup in Contaminated Aquifers. Environmental Science and Technology, 49(12), pp. 7073-7081. [https://doi.org/10.1021/acs.est.5b00715 doi: 10.1021/acs.est.5b00715]</ref>).]]
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Hydrocarbon degradation under anaerobic conditions is often slower compared to aerobic degradation, due to less favorable reaction energetics with alternate electron acceptors. Despite this limitation, both facultative and obligately anaerobic bacteria and archaea are known to degrade hydrocarbons without oxygen. Such microorgansims develop readily at hydrocarbon-impacted sites owing to rapid consumption of oxygen, and therefore anaerobic processes significantly impact the fate of hydrocarbons in the environment. </onlyinclude>Initial steps in anoxic hydrocarbon degradation that involve adding an oxidized functional group to activate the molecule are typically rate-limiting. Doubling times of anaerobic hydrocarbon degraders range from days to months<ref>Meckenstock, R.U. and Mouttaki, H., 2011. Anaerobic degradation of non-substituted aromatic hydrocarbons. Current Opinion in Biotechnology, 22(3), 406-414. [https://doi.org/10.1016/j.copbio.2011.02.009 doi: 10.1016/j.copbio.2011.02.009]</ref>. Despite slow growth rates, complete degradation of many different types of hydrocarbons occurs in the absence of oxygen. For example, degradation of [[Polycyclic Aromatic Hydrocarbons (PAHs) | polycyclic aromatic hydrocarbons]] (compounds composed of multiple aromatic rings) has been reported to occur within 90 days, while benzene degradation occurred over a timescale of 120 weeks<ref name="Meckenstock2016">Meckenstock, R.U., Boll, M., Mouttaki, H., Koelschbach, J.S., Cunha Tarouco, P., Weyrauch, P., Dong, X. and Himmelberg, A.M., 2016. Anaerobic degradation of benzene and polycyclic aromatic hydrocarbons. Journal of Molecular Microbiology and Biotechnology, 26(1-3), 92-118. [https://doi.org/10.1159/000441358 doi: 10.1159/000441358]</ref>. Under methanogenic conditions, linear alkanes have reportedly been degraded in under 200 days<ref>Jiménez, N., Richnow, H.H., Vogt, C., Treude, T. and Krueger, M., 2016. Methanogenic hydrocarbon degradation: evidence from field and laboratory studies. Journal of Molecular Microbiology and Biotechnology, 26(1-3), 227-242. [https://doi.org/10.1159/000441679 doi: 10.1159/000441679]</ref>.
  
Hydrocarbon degradation under anaerobic conditions is often slower compared to aerobic degradation, due to less favorable reaction energetics with alternate electron acceptors. Despite this limitation, both facultative and obligately anaerobic bacteria and archaea are known to degrade hydrocarbons without oxygen. Such microorgansims develop readily at hydrocarbon-impacted sites owing to rapid consumption of oxygen, and therefore anaerobic processes significantly impact the fate of hydrocarbons in the environment. Initial steps in anoxic hydrocarbon degradation that involve adding an oxidized functional group to activate the molecule are typically rate-limiting. Doubling times of anaerobic hydrocarbon degraders range from days to months<ref>Meckenstock, R.U. and Mouttaki, H., 2011. Anaerobic degradation of non-substituted aromatic hydrocarbons. Current Opinion in Biotechnology, 22(3), 406-414. [https://doi.org/10.1016/j.copbio.2011.02.009 doi: 10.1016/j.copbio.2011.02.009]</ref>. Despite slow growth rates, complete degradation of many different types of hydrocarbons occurs in the absence of oxygen. For example, degradation of [[Polycyclic Aromatic Hydrocarbons (PAHs) | polycyclic aromatic hydrocarbons]] (compounds composed of multiple aromatic rings) has been reported to occur within 90 days, while benzene degradation occurred over a timescale of 120 weeks<ref name= "Meckenstock2016">Meckenstock, R.U., Boll, M., Mouttaki, H., Koelschbach, J.S., Cunha Tarouco, P., Weyrauch, P., Dong, X. and Himmelberg, A.M., 2016. Anaerobic degradation of benzene and polycyclic aromatic hydrocarbons. Journal of Molecular Microbiology and Biotechnology, 26(1-3), 92-118. [https://doi.org/10.1159/000441358 doi: 10.1159/000441358]</ref>. Under methanogenic conditions, linear alkanes have reportedly been degraded in under 200 days<ref>Jiménez, N., Richnow, H.H., Vogt, C., Treude, T. and Krueger, M., 2016. Methanogenic hydrocarbon degradation: evidence from field and laboratory studies. Journal of Molecular Microbiology and Biotechnology, 26(1-3), 227-242. [https://doi.org/10.1159/000441679 doi: 10.1159/000441679]</ref>.  
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Anaerobic microbes use terminal electron acceptors other than oxygen in respiration, including compounds such as nitrate, sulfate, carbon dioxide, oxidized metals, or even certain organic compounds<ref name="Contsitecleanup2016" />. At a contaminated site, microbes tend to use electron acceptors sequentially as a function of decreasing reduction potential in the order of oxygen, nitrate, ferric iron, sulfate, and H<sub>2</sub> (Fig. 2)<ref name="Abbasian2015" />. In a few cases, specific species of denitrifying or sulfate reducing microorganisms have been shown to metabolise certain hydrocarbons completely to CO<sub>2</sub> and water. However, anaerobic degradation of hydrocarbons more often occurs via syntrophy, where the degradation of a substrate by one microbe is dependent on the activity of another microbe responsible for keeping intermediate products such as formate and H<sub>2</sub> at low concentrations. Low product concentrations drive otherwise thermodynamically unfavorable reactions. Syntrophy is more common under anaerobic conditions because the use of oxygen as a terminal electron acceptor is more energetically favorable<ref>Morris, B.E., Henneberger, R., Huber, H. and Moissl-Eichinger, C., 2013. Microbial syntrophy: interaction for the common good. FEMS Microbiology Reviews, 37(3), 384-406. [https://doi.org/10.1111/1574-6976.12019 doi: 10.1111/1574-6976.12019]</ref>. Syntrophic processes are absolutely necessary for complete degradation to methane and carbon dioxide, since methanogens (archaea that produce methane) are only able to metabolize simple substrates like acetate and hydrogen. Multiple syntrophic relationships may be present in any given environment, based on available substrates and conditions (Fig. 3). In methanogenic environments, where all other electron acceptors are used up, primary degraders such as ''Peptococcaceae'' and ''Clostridium'' degrade hydrocarbons to intermediates like H<sub>2</sub> and acetate, which are consumed by methanogens<ref name="Gieg2014">Gieg, L.M., Fowler, S.J. and Berdugo-Clavijo, C., 2014. Syntrophic biodegradation of hydrocarbon contaminants. Current Opinion in Biotechnology, 27, 21-29. [https://doi.org/10.1016/j.copbio.2013.09.002 doi: 10.1016/j.copbio.2013.09.002]</ref>.  
  
Anaerobic microbes use terminal electron acceptors other than oxygen in respiration, including compounds such as nitrate, sulfate, carbon dioxide, oxidized metals, or even certain organic compounds<ref name= "Contsitecleanup2016"/>. At a contaminated site, microbes tend to use electron acceptors sequentially as a function of decreasing reduction potential in the order of oxygen, nitrate, ferric iron, sulfate, and H<sub>2</sub> (Fig. 2)<ref name= "Abbasian2015"/>. In a few cases, specific species of denitrifying or sulfate reducing microorganisms have been shown to metabolise certain hydrocarbons completely to CO<sub>2</sub> and water. However, anaerobic degradation of hydrocarbons more often occurs via syntrophy, where the degradation of a substrate by one microbe is dependent on the activity of another microbe responsible for keeping intermediate products such as formate and H<sub>2</sub> at low concentrations. Low product concentrations drive otherwise thermodynamically unfavorable reactions. Syntrophy is more common under anaerobic conditions because the use of oxygen as a terminal electron acceptor is more energetically favorable<ref>Morris, B.E., Henneberger, R., Huber, H. and Moissl-Eichinger, C., 2013. Microbial syntrophy: interaction for the common good. FEMS Microbiology Reviews, 37(3), 384-406. [https://doi.org/10.1111/1574-6976.12019 doi: 10.1111/1574-6976.12019]</ref>. Syntrophic processes are absolutely necessary for complete degradation to methane and carbon dioxide, since methanogens (archaea that produce methane) are only able to metabolize simple substrates like acetate and hydrogen. Multiple syntrophic relationships may be present in any given environment, based on available substrates and conditions (Fig. 3). In methanogenic environments, where all other electron acceptors are used up, primary degraders such as ''Peptococcaceae'' and ''Clostridium'' degrade hydrocarbons to intermediates like H<sub>2</sub> and acetate, which are consumed by methanogens<ref name= "Gieg2014">Gieg, L.M., Fowler, S.J. and Berdugo-Clavijo, C., 2014. Syntrophic biodegradation of hydrocarbon contaminants. Current Opinion in Biotechnology, 27, 21-29. [https://doi.org/10.1016/j.copbio.2013.09.002 doi: 10.1016/j.copbio.2013.09.002]</ref>.
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[[File:Edwards Article 1-figure 3.PNG|550px|thumbnail|left|Figure 3. Conceptual model for syntrophic anaerobic degradation of benzene and alkylbenzenes. Acetate and H<sub>2</sub> are consumed in reactions 1, 2, and 3, keeping the fermentation reaction energetically favorable. When external electron acceptors (e.g., nitrate, iron, or sulphate) are no longer available, methanogens consume acetate and hydrogen (adapted from<ref name="Gieg2014" />).]]
 
 
[[File:Edwards Article 1-figure 3.PNG|550px|thumbnail|center|Figure 3. Conceptual model for syntrophic anaerobic degradation of benzene and alkylbenzenes. Acetate and H<sub>2</sub> are consumed in reactions 1, 2, and 3, keeping the fermentation reaction energetically favorable. When external electron acceptors (e.g., nitrate, iron, or sulphate) are no longer available, methanogens consume acetate and hydrogen (adapted from<ref name= "Gieg2014"/>).]]
 
  
 
Anaerobic microbes use diverse strategies to activate hydrocarbons without requiring molecular oxygen (O<sub>2</sub>). Each strategy is discussed separately below, and is applicable to both aliphatic and aromatic compounds. The general strategy is to insert a more oxidized group into the molecule to make it more reactive to further transformation to more common intermediates (typically fatty acids and other carboxylic acids) that can enter central metabolic pathways. Most aromatic compounds are activated and funnelled towards the central anaerobic intermediate, benzoate, or more accurately, its coenzyme A (CoA) thioester derivative, benzoyl-CoA.
 
Anaerobic microbes use diverse strategies to activate hydrocarbons without requiring molecular oxygen (O<sub>2</sub>). Each strategy is discussed separately below, and is applicable to both aliphatic and aromatic compounds. The general strategy is to insert a more oxidized group into the molecule to make it more reactive to further transformation to more common intermediates (typically fatty acids and other carboxylic acids) that can enter central metabolic pathways. Most aromatic compounds are activated and funnelled towards the central anaerobic intermediate, benzoate, or more accurately, its coenzyme A (CoA) thioester derivative, benzoyl-CoA.
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===Strategies For Activating Hydrocarbons Without Oxygen===
 
===Strategies For Activating Hydrocarbons Without Oxygen===
 
'''1. Fumarate Addition'''<br />
 
'''1. Fumarate Addition'''<br />
Bacteria use fumarate addition to activate alkanes from 3-20 carbons as well as alkyl-substituted aromatics like toluene, xylenes, or methylnaphthalene<ref name= "Abbasian2015"/>. Fumarate is a common cellular metabolite that contains two carboxylic acid groups and a double bond. The terminal or subterminal methyl group of alkanes or of alkyl benzenes adds across the double bond of fumarate. For alkanes, this reaction is catalyzed by alkylsuccinate synthase and results in intermediates such as 2-(1-methylalkyl)succinates or 2-alkylsuccinates. These compounds are further degraded via carbon rearrangement, decarboxylation, and β-oxidation<ref>Bian, X.Y., Mbadinga, S.M., Liu, Y.F., Yang, S.Z., Liu, J.F., Ye, R.Q., Gu, J.D. and Mu, B.Z., 2015. Insights into the anaerobic biodegradation pathway of n-alkanes in oil reservoirs by detection of signature metabolites. Scientific Reports, 5. [https://doi.org/10.1038/srep09801 doi: 10.1038/srep09801]</ref>. Aromatic hydrocarbons degraded by fumarate addition include toluene, xylene, and 2-methylnaphthalene<ref name="Fuchs2011"/>. This process is catalyzed by the enzyme benzylsuccinate synthase. Many anaerobic bacteria, including pure cultures of sulfate- and nitrate-reducing bacteria, convert toluene readily to (R)-benzylsuccinate through fumarate addition<ref name= "Abbasian2015"/>.  
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Bacteria use fumarate addition to activate alkanes from 3-20 carbons as well as alkyl-substituted aromatics like toluene, xylenes, or methylnaphthalene<ref name="Abbasian2015" />. Fumarate is a common cellular metabolite that contains two carboxylic acid groups and a double bond. The terminal or subterminal methyl group of alkanes or of alkyl benzenes adds across the double bond of fumarate. For alkanes, this reaction is catalyzed by alkylsuccinate synthase and results in intermediates such as 2-(1-methylalkyl)succinates or 2-alkylsuccinates. These compounds are further degraded via carbon rearrangement, decarboxylation, and β-oxidation<ref>Bian, X.Y., Mbadinga, S.M., Liu, Y.F., Yang, S.Z., Liu, J.F., Ye, R.Q., Gu, J.D. and Mu, B.Z., 2015. Insights into the anaerobic biodegradation pathway of n-alkanes in oil reservoirs by detection of signature metabolites. Scientific Reports, 5. [https://doi.org/10.1038/srep09801 doi: 10.1038/srep09801]</ref>. Aromatic hydrocarbons degraded by fumarate addition include toluene, xylene, and 2-methylnaphthalene<ref name="Fuchs2011" />. This process is catalyzed by the enzyme benzylsuccinate synthase. Many anaerobic bacteria, including pure cultures of sulfate- and nitrate-reducing bacteria, convert toluene readily to (R)-benzylsuccinate through fumarate addition<ref name="Abbasian2015" />.  
  
 
'''2. Oxygen-Independent Hydroxylation'''<br />
 
'''2. Oxygen-Independent Hydroxylation'''<br />
Oxygen-independent hydroxylation can denitrify bacteria metabolizing ethylbenzene<ref name= "Heider2007">Heider, J., 2007. Adding handles to unhandy substrates: anaerobic hydrocarbon activation mechanisms. Current Opinion in Chemical Biology, 11(2), 188–194. [http://doi.org/10.1016/j.cbpa.2007.02.027 doi: 10.1016/j.cbpa.2007.02.027]</ref><ref name="Fuchs2011"/>. In this pathway, a hydroxyl (-OH) group is added to the C1 carbon (carbon atom closest to the aromatic ring) on the side chain by ethylbenzene dehydrogenase to form S-1-phenylethanol, followed by oxidation to acetophenone and transformation to benzoyl-CoA and acetyl-CoA<ref name= "Heider2007"/>. The first step is catalyzed by a molybdenum cofactor-containing hydroxylase<ref name= "Heider2007"/><ref name="Boll2014">Boll, M., Löffler, C., Morris, B.E. and Kung, J.W., 2014. Anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl‐coenzyme A esters: organisms, strategies and key enzymes. Environmental Microbiology, 16(3), 612-627. [https://doi.org/10.1111/1462-2920.12328 doi: 10.1111/1462-2920.12328]</ref>. Enzymes called flavocytochrome c hydrolases have also been implicated in the oxidation of alkyl side chains on aromatic rings to their corresponding alcohols<ref name= "Boll2014"/>. A similar mechanism of hydroxylation has been proposed to act on the subterminal carbon of alkanes producing an alcohol functionality that is then more susceptible to further oxidation<ref>Callaghan, A.V., 2013. Enzymes involved in the anaerobic oxidation of n-alkanes: from methane to long-chain paraffins. Frontiers in Microbiology, 4, 89. [https://doi.org/10.3389/fmicb.2013.00089 doi:10.3389/fmicb.2013.00089]</ref>.
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Oxygen-independent hydroxylation can denitrify bacteria metabolizing ethylbenzene<ref name="Heider2007">Heider, J., 2007. Adding handles to unhandy substrates: anaerobic hydrocarbon activation mechanisms. Current Opinion in Chemical Biology, 11(2), 188–194. [http://doi.org/10.1016/j.cbpa.2007.02.027 doi: 10.1016/j.cbpa.2007.02.027]</ref><ref name="Fuchs2011" />. In this pathway, a hydroxyl (-OH) group is added to the C1 carbon (carbon atom closest to the aromatic ring) on the side chain by ethylbenzene dehydrogenase to form S-1-phenylethanol, followed by oxidation to acetophenone and transformation to benzoyl-CoA and acetyl-CoA<ref name="Heider2007" />. The first step is catalyzed by a molybdenum cofactor-containing hydroxylase<ref name="Heider2007" /><ref name="Boll2014">Boll, M., Löffler, C., Morris, B.E. and Kung, J.W., 2014. Anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl‐coenzyme A esters: organisms, strategies and key enzymes. Environmental Microbiology, 16(3), 612-627. [https://doi.org/10.1111/1462-2920.12328 doi: 10.1111/1462-2920.12328]</ref>. Enzymes called flavocytochrome c hydrolases have also been implicated in the oxidation of alkyl side chains on aromatic rings to their corresponding alcohols<ref name="Boll2014" />. A similar mechanism of hydroxylation has been proposed to act on the subterminal carbon of alkanes producing an alcohol functionality that is then more susceptible to further oxidation<ref>Callaghan, A.V., 2013. Enzymes involved in the anaerobic oxidation of n-alkanes: from methane to long-chain paraffins. Frontiers in Microbiology, 4, 89. [https://doi.org/10.3389/fmicb.2013.00089 doi:10.3389/fmicb.2013.00089]</ref>.
  
 
'''3. Carboxylation'''<br />
 
'''3. Carboxylation'''<br />
All mechanisms described to date only apply to hydrocarbons with alkyl groups, and not to unsubstituted aromatic hydrocarbons like benzene or naphthalene. In fact, anaerobic degradation of benzene is much slower than that of toluene, or xylenes, and may not occur at all sites<ref name="Lawrence2006"/>. One mechanism for naphthalene degradation and postulated for anaerobic benzene degradation is [[wikipedia: Carboxylation | carboxylation]]. In this process, CO<sub>2</sub> is added directly to aliphatic and aromatic hydrocarbons<ref name= "Abbasian2015"/>. The process is thought to be somewhat analogous to the mechanism of anaerobic phenol (=hydroxybenzene) degradation where phenol is first activated using energy from ATP to phosphophenol prior to carboxylation to para-hydroxybenzoate. Enzymes called carboxylases catalyze the reaction that adds a carboxyl (-COOH) group to their substrate, although the mechanism is still under investigation. Activation of benzene is thought to occur by carboxylation to benzoate under iron- and nitrate-reducing conditions<ref name= "Meckenstock2016"/>. Benzene ring activation is difficult due to the stability of the ring and the correspondingly high dissociation energy. While no biochemical evidence for benzene carboxylation yet exists, gene expression and proteomic studies correlate expression of the putative anaerobic benzene carboxylase (called AbcDA) with benzene degradation<ref>Luo, F., Gitiafroz, R., Devine, C.E., Gong, Y., Hug, L.A., Raskin, L. and Edwards, E.A., 2014. Metatranscriptome of an anaerobic benzene-degrading, nitrate-reducing enrichment culture reveals involvement of carboxylation in benzene ring activation. Applied and Environmental Microbiology, 80(14), 4095-4107. [https://doi.org/10.1128/aem.00717-14 doi:10.1128/AEM.00717-14]</ref><ref>Abu Laban, N., Selesi, D., Rattei, T., Tischler, P. and Meckenstock, R.U., 2010. Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron‐reducing enrichment culture. Environmental Microbiology, 12(10), 2783-2796. [http://dx.doi.org/10.1111/j.1462-2920.2010.02248.x doi:10.1111/j.1462-2920.2010.02248.x]</ref>. Naphthalene carboxylation has been demonstrated in crude cell extracts<ref>Mouttaki, H., Johannes, J. and Meckenstock, R.U., 2012. Identification of naphthalene carboxylase as a prototype for the anaerobic activation of non‐substituted aromatic hydrocarbons. Environmental Microbiology, 14(10), 2770-2774. [http://doi.org/10.1111/j.1462-2920.2012.02768.x doi: 10.1111/j.1462-2920.2012.02768.x]</ref>. The active sites of aromatic ring carboxylases are thought to be similar to the UbiD family of carboxylases<ref name= "Boll2014"/> involved in general aromatic metabolism.  
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All mechanisms described to date only apply to hydrocarbons with alkyl groups, and not to unsubstituted aromatic hydrocarbons like benzene or naphthalene. In fact, anaerobic degradation of benzene is much slower than that of toluene, or xylenes, and may not occur at all sites<ref name="Lawrence2006" />. One mechanism for naphthalene degradation and postulated for anaerobic benzene degradation is [[wikipedia: Carboxylation | carboxylation]]. In this process, CO<sub>2</sub> is added directly to aliphatic and aromatic hydrocarbons<ref name="Abbasian2015" />. The process is thought to be somewhat analogous to the mechanism of anaerobic phenol (=hydroxybenzene) degradation where phenol is first activated using energy from ATP to phosphophenol prior to carboxylation to para-hydroxybenzoate. Enzymes called carboxylases catalyze the reaction that adds a carboxyl (-COOH) group to their substrate, although the mechanism is still under investigation. Activation of benzene is thought to occur by carboxylation to benzoate under iron- and nitrate-reducing conditions<ref name="Meckenstock2016" />. Benzene ring activation is difficult due to the stability of the ring and the correspondingly high dissociation energy. While no biochemical evidence for benzene carboxylation yet exists, gene expression and proteomic studies correlate expression of the putative anaerobic benzene carboxylase (called AbcDA) with benzene degradation<ref>Luo, F., Gitiafroz, R., Devine, C.E., Gong, Y., Hug, L.A., Raskin, L. and Edwards, E.A., 2014. Metatranscriptome of an anaerobic benzene-degrading, nitrate-reducing enrichment culture reveals involvement of carboxylation in benzene ring activation. Applied and Environmental Microbiology, 80(14), 4095-4107. [https://doi.org/10.1128/aem.00717-14 doi:10.1128/AEM.00717-14]</ref><ref>Abu Laban, N., Selesi, D., Rattei, T., Tischler, P. and Meckenstock, R.U., 2010. Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron‐reducing enrichment culture. Environmental Microbiology, 12(10), 2783-2796. [http://dx.doi.org/10.1111/j.1462-2920.2010.02248.x doi:10.1111/j.1462-2920.2010.02248.x]</ref>. Naphthalene carboxylation has been demonstrated in crude cell extracts<ref>Mouttaki, H., Johannes, J. and Meckenstock, R.U., 2012. Identification of naphthalene carboxylase as a prototype for the anaerobic activation of non‐substituted aromatic hydrocarbons. Environmental Microbiology, 14(10), 2770-2774. [http://doi.org/10.1111/j.1462-2920.2012.02768.x doi: 10.1111/j.1462-2920.2012.02768.x]</ref>. The active sites of aromatic ring carboxylases are thought to be similar to the UbiD family of carboxylases<ref name="Boll2014" /> involved in general aromatic metabolism.
  
 
==Implications for Remediation==
 
==Implications for Remediation==
Hydrocarbon remediation occurs more quickly under aerobic conditions than anaerobic. The primary concern in in situ aerobic remediation is oxygen delivery and mixing, which may be achieved by a number of previously-established methods including landfarming, sparging, groundwater recirculation, and peroxide addition. However, aerobic remediation is not feasible in all environments, particularly low permeability soils<ref name= "Contsitecleanup2016"/>. In these cases, anaerobic bioremediation may be preferred. Important considerations for biodegradation include the nature of the suite of hydrocarbons present (light or heavy hydrocarbons), bioavailability, microorganism community composition, nutrient availability, soil permeability, and pH. Biostimulation through addition of nutrients such as nitrogen, phosphorus, and iron is often helpful. Bioaugmentation by addition of actively degrading microbial cultures and nutrients may accelerate biotransformation of particularly recalcitrant hydrocarbons like benzene and [[Polycyclic Aromatic Hydrocarbons (PAHs) | polycyclic aromatic hydrocarbons (PAHs)]], but site conditions must first be assessed to predict and ensure their effectiveness. Knowing the degradation pathways and responsible organisms is useful to (a) assess potential for natural and enhanced remediation and (b) track biodegradation at a site by monitoring functional genes biomarkers and degradation intermediates.  
+
Hydrocarbon remediation occurs more quickly under aerobic conditions than anaerobic. The primary concern in in situ aerobic remediation is oxygen delivery and mixing, which may be achieved by a number of previously-established methods including landfarming, sparging, groundwater recirculation, and peroxide addition. However, aerobic remediation is not feasible in all environments, particularly low permeability soils<ref name="Contsitecleanup2016" />. In these cases, anaerobic bioremediation may be preferred. Important considerations for biodegradation include the nature of the suite of hydrocarbons present (light or heavy hydrocarbons), bioavailability, microorganism community composition, nutrient availability, soil permeability, and pH. Biostimulation through addition of nutrients such as nitrogen, phosphorus, and iron is often helpful. Bioaugmentation by addition of actively degrading microbial cultures and nutrients may accelerate biotransformation of particularly recalcitrant hydrocarbons like benzene and [[Polycyclic Aromatic Hydrocarbons (PAHs) | polycyclic aromatic hydrocarbons (PAHs)]], but site conditions must first be assessed to predict and ensure their effectiveness. Knowing the degradation pathways and responsible organisms is useful to (a) assess potential for natural and enhanced remediation and (b) track biodegradation at a site by monitoring functional genes biomarkers and degradation intermediates.  
  
 
==References==
 
==References==
  
<references/>
+
<references />
  
 
==See Also==
 
==See Also==
*[https://www.serdp-estcp.org/Program-Areas/Environmental-Restoration/Contaminated-Groundwater/ER-199522 Enhanced Bioremediation - Anaerobic of Fuel-Contaminated Groundwater]
+
 
 +
*[https://www.serdp-estcp.org/Program-Areas/Environmental-Restoration/Contaminated-Groundwater/ER-199522 Enhanced Bioremediation - Anaerobic of Fuel-Contaminated Groundwater]
 
*[https://clu-in.org/techfocus/default.focus/sec/Bioremediation/cat/Overview/ EPA Contaminated Site Clean-Up Information: Bioremendiation Overview]
 
*[https://clu-in.org/techfocus/default.focus/sec/Bioremediation/cat/Overview/ EPA Contaminated Site Clean-Up Information: Bioremendiation Overview]

Latest revision as of 21:19, 1 April 2024

Hydrocarbons are major constituents of crude oil and petroleum. They can be biodegraded by naturally-occurring microorganisms in freshwater and marine environments under a variety of aerobic and anaerobic conditions. The ability of microorganisms - bacteria, archaea, fungi, or algae - to break down hydrocarbons is the basis for natural and enhanced bioremediation. To promote biodegradation, amendments such as nitrogen and phosphorous fertilizer are often added to stimulate microbial growth and metabolism. Oxygen, nitrate, or sulfates are sometimes added as electron acceptors to enhance biodegradation rates. The addition of hydrocarbon-degrading microbial cultures to contaminated sites might also be considered, although the practice of bioaugmentation is uncommon for hydrocarbons owing to the natural abundance of hydrocarbon-degrading microbes. Aside from excavation, various forms of bioremediation from simple tilling of soils and landfarming to in situ biostimulation are common forms of remediation for hydrocarbon contamination. Compared to other technologies, bioremediation is lower cost, environmentally-friendly, and often non-invasive[1]. However, the time needed for full site cleanup can be longer and less predictable than for other methods, and successful outcomes may not be guaranteed due to variability in site conditions. Nonetheless, it is the most common approach for remediation of hydrocarbon-contaminated environments.

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Contributor(s): Elisse Magnuson and Dr. Elizabeth Edwards


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Introduction

Hydrocarbons are compounds composed entirely of carbon and hydrogen. Aromatic hydrocarbons, alkanes, alkenes, cycloalkanes, alkynes, and combinations of these compounds comprise different types of hydrocarbons. Complex mixtures of hydrocarbons occur naturally in crude oil and gasoline. Most can be used as substrates in metabolism by bacteria, archaea, fungi, and algae[2][3]. While fungi and algae degrade hydrocarbons aerobically, bacteria and archaea are capable of both aerobic and anaerobic degradation[4][5][6][7]. Owing to the relatively reduced nature of most hydrocarbons, they are typically the energy source or electron donors for microbial metabolism, and their oxidation must be coupled to the reduction of a suitable electron acceptor. Aerobic degradation occurs with molecular oxygen (O2) as both a reactant to oxidize the substrate and an electron acceptor for microbial respiration. In contrast, anaerobic degradation uses different biotransformation pathways that don’t depend on oxygen, coupled to microbial respiration of a variety of electron acceptors. Under anaerobic conditions, biodegradation often results from the stepwise concerted action of many different microbes in a process called syntrophy.

Biodegradation occurs naturally because hydrocarbons have always been present in the environment, released from seeps and reservoirs through various geologic processes. Natural attenuation of hydrocarbon contamination can be monitored by a number of analytical methods, including gas chromatography and infrared spectroscopy[8]. Absolute proof of degradation versus dilution or other non-degradative processes can be obtained using compound specific isotope analysis (CSIA), where an enrichment in the natural abundance of heavier isotopes of carbon and hydrogen in the parent hydrocarbons confirms biotransformation. Often biostimulation - the addition of limiting nutrients - may be desired in sites where attenuation occurs slowly or not at all.

Figure 1. Components of hydrocarbon biodegradation. Understanding and facilitating biodegradation at a contaminated site requires knowledge of the environmental conditions, compound properties, and microorganisms present (adapted after Sutherson, 1999)[9].

Many bioremediation efforts involve injection and circulation of oxygen to subsurface environments in order to increase the availability of oxygen as both reactant and electron acceptor. Common methods include using a vacuum pump to promote air circulation[10], bioventing or biosparging that add air to the subsurface[11], water circulation aboveground to add oxygen[10], and the introduction of substances that release oxygen like peroxides or ozone. Remediation may also involve introduction of bacterial strains (bioaugmentation) as well as nutrients to stimulate hydrocarbon degradation. This strategy is most often used at sites with mid-weight or heavier petroleum products, as lighter compounds volatilize[12]. Related commercial products include microbial culture, enzyme, and nutrient additives. These products may be less viable in the field than in laboratory settings due to varying site conditions such that field testing is necessary to determine their effectiveness[13]. As the biochemical processes of anaerobic hydrocarbon biotransformation have become more clearly established, bioremediation efforts have shifted towards adding nitrate or sulfate as electron acceptors instead of oxygen because these electron acceptors are much more soluble than oxygen and easier to deliver, particularly for in situ applications[14].

Site conditions affect the occurrence and rate of biodegradation in contaminated environments (Fig. 1). Nitrogen, phosphorus, and iron are important nutrients for bacteria and may become limiting in certain environments, particularly in seawater and freshwater wetlands which can be nutrient deficient. Addition of inorganic nutrients can bolster biodegradation rates. However, high levels of nitrogen, phosphorus, and potassium may inhibit biodegradation, particularly of aromatics[13]. Nitrogen at reported levels of 200-4000 mg N/kg soil have been shown to reduce hydrocarbon loss, while lower levels stimulated degradation. This may depend on site-specific conditions such as soil type and contaminants[15][16]. In addition, the pH of a site affects the solubility and availability of nutrients, and must be in the tolerant range for the responsible microorganisms, generally between pH 6-8[12][9]. The presence of organic matter may increase persistence of contaminants due to contaminant partitioning into the organic phase, reducing their bioavailability to microorganisms[17].


Aerobic Degradation

Hydrocarbons are readily degraded under aerobic conditions. Bacteria, fungi, and algae are all capable of aerobic hydrocarbon degradation[6]. In general, alkenes (hydrocarbons containing double bonds) and short-chain alkanes (hydrocarbons containing only single bonds) are the most easily degraded, followed by branched alkanes (alkanes with side chains) and then aromatics (hydrocarbons in a stable ring structure)[7]. However, degradation rates vary based on environmental parameters and decrease as hydrocarbon complexity increases. Reported degradation rates vary considerably because hydrocarbon composition depends on the source of the petroleum and age of the spill. For example, compound degradation varies from 5% to 30% in 28 days, while up to 100% degradation occurrs with nitrogen addition[18]. Degradation rates by fungal species reportedly range from ~ 30-100% degradation over 28 days or less[19]. As discussed above, the primary rate-limiting factor in aerobic biodegradation is delivery of oxygen. Oxygen availability is dependent on the ability of oxygen to move or diffuse through the site environment as well as on the uptake rate by microorganisms. Addition of oxygen can increase degradation rates several orders of magnitude over naturally occurring rates[12].

Alkanes and Alkenes

Alkanes containing 14 carbons or fewer are prone to volatilization, while alkanes containing more carbons are less volatile[17]. Regardless, alkanes and alkenes, with the exception of cyclic alkanes (alkanes in a ring structure), are the most readily degraded hydrocarbons with reported degradation of alkanes containing up to 44 carbons[2][17]. Both alkanes and alkenes are degraded with addition of molecular oxygen. Oxygen availability and the initial step of degradation are rate-limiting[1]. In the initial steps of aerobic alkane degradation, enzymes called oxygenases add molecular oxygen to the hydrocarbon molecules, forming alcohols that are further oxidized to fatty acids that are subsequently metabolized to acetyl-CoA and finally to CO2 and H2O. Many mono- and di-oxygenases and other oxidases have a wide substrate range and act readily on a variety of hydrocarbons[2].

Aromatic Hydrocarbons

Aromatic hydrocarbons are generally more difficult to degrade than shorter alkanes and alkenes due to their greater toxicity, yet they are readily degraded aerobically by many bacteria and fungi (see also Polycyclic Aromatic Hydrocarbons (PAHs)). Degradability decreases with increasing number of rings and increased molecular size, due to increased hydrophobicity and sorption capacity[17]. The median primary degradation rate of benzene, toluene, ethylbenzene, and xylene (BTEX compounds) ranges from 0.05-0.2 day-1[20]. Oxidation of BTEX requires 3.1 mg/L dissolved oxygen (DO) to degrade 1 mg/L of BTEX. When DO is below 2 mg/L, biodegradation slows[20]. The general pathway for degradation of aromatic compounds begins with the addition of O2 by mono and di-oxygenases[21]. This yields key intermediate products such as benzyl alcohol, phenol or catechol, protocatechuate, and gentisate[22]. These intermediates then undergo ring cleavage, also by a variety of oxygenases, resulting in carboxylic acids[23][22]. Degradation then continues to acetyl-CoA and succinyl-CoA[22] that enter into central metabolism. Fungal degradation occurs by non-specific extracellular oxidizing enzymes that form radical intermediates, although many reactions are similar to those found in bacteria[6].

Anaerobic Degradation

Figure 2. Comparison of the longitudinal redox zonation concept (A) and the plume fringe concept (B). Both concepts describe the spatial distribution of electron acceptors and respiration processes in a hydrocarbon contaminant plume. (B) Iron(III) reduction, manganese(IV) reduction, and methanogenesis may occur simultaneously in the core of the contaminant plume (reproduced with permission from © 2015 American Chemical Society[24]).

Hydrocarbon degradation under anaerobic conditions is often slower compared to aerobic degradation, due to less favorable reaction energetics with alternate electron acceptors. Despite this limitation, both facultative and obligately anaerobic bacteria and archaea are known to degrade hydrocarbons without oxygen. Such microorgansims develop readily at hydrocarbon-impacted sites owing to rapid consumption of oxygen, and therefore anaerobic processes significantly impact the fate of hydrocarbons in the environment. Initial steps in anoxic hydrocarbon degradation that involve adding an oxidized functional group to activate the molecule are typically rate-limiting. Doubling times of anaerobic hydrocarbon degraders range from days to months[25]. Despite slow growth rates, complete degradation of many different types of hydrocarbons occurs in the absence of oxygen. For example, degradation of polycyclic aromatic hydrocarbons (compounds composed of multiple aromatic rings) has been reported to occur within 90 days, while benzene degradation occurred over a timescale of 120 weeks[26]. Under methanogenic conditions, linear alkanes have reportedly been degraded in under 200 days[27].

Anaerobic microbes use terminal electron acceptors other than oxygen in respiration, including compounds such as nitrate, sulfate, carbon dioxide, oxidized metals, or even certain organic compounds[12]. At a contaminated site, microbes tend to use electron acceptors sequentially as a function of decreasing reduction potential in the order of oxygen, nitrate, ferric iron, sulfate, and H2 (Fig. 2)[2]. In a few cases, specific species of denitrifying or sulfate reducing microorganisms have been shown to metabolise certain hydrocarbons completely to CO2 and water. However, anaerobic degradation of hydrocarbons more often occurs via syntrophy, where the degradation of a substrate by one microbe is dependent on the activity of another microbe responsible for keeping intermediate products such as formate and H2 at low concentrations. Low product concentrations drive otherwise thermodynamically unfavorable reactions. Syntrophy is more common under anaerobic conditions because the use of oxygen as a terminal electron acceptor is more energetically favorable[28]. Syntrophic processes are absolutely necessary for complete degradation to methane and carbon dioxide, since methanogens (archaea that produce methane) are only able to metabolize simple substrates like acetate and hydrogen. Multiple syntrophic relationships may be present in any given environment, based on available substrates and conditions (Fig. 3). In methanogenic environments, where all other electron acceptors are used up, primary degraders such as Peptococcaceae and Clostridium degrade hydrocarbons to intermediates like H2 and acetate, which are consumed by methanogens[29].

Figure 3. Conceptual model for syntrophic anaerobic degradation of benzene and alkylbenzenes. Acetate and H2 are consumed in reactions 1, 2, and 3, keeping the fermentation reaction energetically favorable. When external electron acceptors (e.g., nitrate, iron, or sulphate) are no longer available, methanogens consume acetate and hydrogen (adapted from[29]).

Anaerobic microbes use diverse strategies to activate hydrocarbons without requiring molecular oxygen (O2). Each strategy is discussed separately below, and is applicable to both aliphatic and aromatic compounds. The general strategy is to insert a more oxidized group into the molecule to make it more reactive to further transformation to more common intermediates (typically fatty acids and other carboxylic acids) that can enter central metabolic pathways. Most aromatic compounds are activated and funnelled towards the central anaerobic intermediate, benzoate, or more accurately, its coenzyme A (CoA) thioester derivative, benzoyl-CoA.

Strategies For Activating Hydrocarbons Without Oxygen

1. Fumarate Addition
Bacteria use fumarate addition to activate alkanes from 3-20 carbons as well as alkyl-substituted aromatics like toluene, xylenes, or methylnaphthalene[2]. Fumarate is a common cellular metabolite that contains two carboxylic acid groups and a double bond. The terminal or subterminal methyl group of alkanes or of alkyl benzenes adds across the double bond of fumarate. For alkanes, this reaction is catalyzed by alkylsuccinate synthase and results in intermediates such as 2-(1-methylalkyl)succinates or 2-alkylsuccinates. These compounds are further degraded via carbon rearrangement, decarboxylation, and β-oxidation[30]. Aromatic hydrocarbons degraded by fumarate addition include toluene, xylene, and 2-methylnaphthalene[22]. This process is catalyzed by the enzyme benzylsuccinate synthase. Many anaerobic bacteria, including pure cultures of sulfate- and nitrate-reducing bacteria, convert toluene readily to (R)-benzylsuccinate through fumarate addition[2].

2. Oxygen-Independent Hydroxylation
Oxygen-independent hydroxylation can denitrify bacteria metabolizing ethylbenzene[31][22]. In this pathway, a hydroxyl (-OH) group is added to the C1 carbon (carbon atom closest to the aromatic ring) on the side chain by ethylbenzene dehydrogenase to form S-1-phenylethanol, followed by oxidation to acetophenone and transformation to benzoyl-CoA and acetyl-CoA[31]. The first step is catalyzed by a molybdenum cofactor-containing hydroxylase[31][32]. Enzymes called flavocytochrome c hydrolases have also been implicated in the oxidation of alkyl side chains on aromatic rings to their corresponding alcohols[32]. A similar mechanism of hydroxylation has been proposed to act on the subterminal carbon of alkanes producing an alcohol functionality that is then more susceptible to further oxidation[33].

3. Carboxylation
All mechanisms described to date only apply to hydrocarbons with alkyl groups, and not to unsubstituted aromatic hydrocarbons like benzene or naphthalene. In fact, anaerobic degradation of benzene is much slower than that of toluene, or xylenes, and may not occur at all sites[20]. One mechanism for naphthalene degradation and postulated for anaerobic benzene degradation is carboxylation. In this process, CO2 is added directly to aliphatic and aromatic hydrocarbons[2]. The process is thought to be somewhat analogous to the mechanism of anaerobic phenol (=hydroxybenzene) degradation where phenol is first activated using energy from ATP to phosphophenol prior to carboxylation to para-hydroxybenzoate. Enzymes called carboxylases catalyze the reaction that adds a carboxyl (-COOH) group to their substrate, although the mechanism is still under investigation. Activation of benzene is thought to occur by carboxylation to benzoate under iron- and nitrate-reducing conditions[26]. Benzene ring activation is difficult due to the stability of the ring and the correspondingly high dissociation energy. While no biochemical evidence for benzene carboxylation yet exists, gene expression and proteomic studies correlate expression of the putative anaerobic benzene carboxylase (called AbcDA) with benzene degradation[34][35]. Naphthalene carboxylation has been demonstrated in crude cell extracts[36]. The active sites of aromatic ring carboxylases are thought to be similar to the UbiD family of carboxylases[32] involved in general aromatic metabolism.

Implications for Remediation

Hydrocarbon remediation occurs more quickly under aerobic conditions than anaerobic. The primary concern in in situ aerobic remediation is oxygen delivery and mixing, which may be achieved by a number of previously-established methods including landfarming, sparging, groundwater recirculation, and peroxide addition. However, aerobic remediation is not feasible in all environments, particularly low permeability soils[12]. In these cases, anaerobic bioremediation may be preferred. Important considerations for biodegradation include the nature of the suite of hydrocarbons present (light or heavy hydrocarbons), bioavailability, microorganism community composition, nutrient availability, soil permeability, and pH. Biostimulation through addition of nutrients such as nitrogen, phosphorus, and iron is often helpful. Bioaugmentation by addition of actively degrading microbial cultures and nutrients may accelerate biotransformation of particularly recalcitrant hydrocarbons like benzene and polycyclic aromatic hydrocarbons (PAHs), but site conditions must first be assessed to predict and ensure their effectiveness. Knowing the degradation pathways and responsible organisms is useful to (a) assess potential for natural and enhanced remediation and (b) track biodegradation at a site by monitoring functional genes biomarkers and degradation intermediates.

References

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See Also