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==What is the mechanism of hydrocarbon biodegradation?==

==What is the mechanism of hydrocarbon biodegradation?==

There are two main pathways for hydrocarbon biodegradation. The first occurs in the presence of oxygen (aerobic). The second process can occur in the absence of oxygen (anaerobic) The aerobic mechanism is highlighted in [[:File:GeoWikiWriteOff2021-Aljezen-Figure2.jpg|Figure 2]].

There are two main pathways for hydrocarbon biodegradation. The first occurs in the presence of oxygen (aerobic). The second process can occur in the absence of oxygen (anaerobic) The aerobic mechanism is highlighted in [[:File:GeoWikiWriteOff2021-Aljezen-Figure2.png|Figure 2]].

[[File:GeoWikiWriteOff2021-Aljezen-Figure2.jpg|framed|center|{{figure number|2}} shows the mechanism to biodegrade hydrocarbons with the presence of oxygen.<ref name="6Das" />]]

[[File:GeoWikiWriteOff2021-Aljezen-Figure2.png|framed|center|{{figure number|2}} shows the mechanism to biodegrade hydrocarbons with the presence of oxygen.<ref name="6Das" />]]

The normal alkane (C₁-C₈ ''n''-alkane) in [[:File:GeoWikiWriteOff2021-Aljezen-Figure2.jpg|Figure 2]] will react with oxygen with the help of monooxygenase enzyme produced by living organisms (e.g. bacteria), and converts the normal alkane to an alcohol and oxidize iron from Fe²⁺ to Fe³⁺. Under aerobic conditions, simple aromatics such as benzene, xylene and toluene can be degraded.  Typically, this requires 3mg/L of dissolved oxygen to degrade 1 mg/L of these aromatics (i.e. 3:1 ratio).  If the dissolved oxygen content is lower than 3:1 then the biodegradation rate is slower.

The normal alkane (C₁-C₈ ''n''-alkane) in [[:File:GeoWikiWriteOff2021-Aljezen-Figure2.png|Figure 2]] will react with oxygen with the help of monooxygenase enzyme produced by living organisms (e.g. bacteria), and converts the normal alkane to an alcohol and oxidize iron from Fe²⁺ to Fe³⁺. Under aerobic conditions, simple aromatics such as benzene, xylene and toluene can be degraded.  Typically, this requires 3mg/L of dissolved oxygen to degrade 1 mg/L of these aromatics (i.e. 3:1 ratio).  If the dissolved oxygen content is lower than 3:1 then the biodegradation rate is slower.

Compared to aerobic degradation, anaerobic degradation is considered to proceed much slower This is because anaerobic pathways require more energy, i.e. they are energetically unfavorable. There are three different pathways that anaerobic microbes can utilize to biodegrade hydrocarbons. All three pathways require inserting an oxidizing group into the molecule, which makes it more active and thus easier to transform to microbial-consumable products such as fatty acids. The first pathway is called fumarate addition and this pathway is used by bacteria to activate C3 to C20 alkanes and alkyl-substituted aromatics such as xylene and toluene. The mechanism proceeds via addition to the double bond by terminal or pre-terminal alkyl group from the alkanes or alkyl-substituted aromatics then followed by the removal of a carbon dioxide molecule ([[:File:GeoWikiWriteOff2021-Aljezen-Figure3.jpg|Figure 3]]).<ref name="7Fuchsetal">Fuchs, G., M/ Boll, and J. Heider, 2011, [https://www.nature.com/articles/nrmicro2652 Microbial degradation of aromatic compounds—from one strategy to four]: Nature Reviews Microbiology, v. 9, p.803–816.</ref><ref name="8Bianetal">Bian, X. Y., S, M. Mbadinga, Y. F. Liu, S. Z. Yang, J. F. Liu, R. Q. Ye, J. D. Gu, and B. Z. Mu, 2015, [https://www.nature.com/articles/srep09801 Insights into the anaerobic biodegradation pathway of n-alkanes in oil reservoirs by detection of signature metabolites]: Scientific Reports, v. 5. article no. 9801.</ref>  

Compared to aerobic degradation, anaerobic degradation is considered to proceed much slower This is because anaerobic pathways require more energy, i.e. they are energetically unfavorable. There are three different pathways that anaerobic microbes can utilize to biodegrade hydrocarbons. All three pathways require inserting an oxidizing group into the molecule, which makes it more active and thus easier to transform to microbial-consumable products such as fatty acids. The first pathway is called fumarate addition and this pathway is used by bacteria to activate C3 to C20 alkanes and alkyl-substituted aromatics such as xylene and toluene. The mechanism proceeds via addition to the double bond by terminal or pre-terminal alkyl group from the alkanes or alkyl-substituted aromatics then followed by the removal of a carbon dioxide molecule ([[:File:GeoWikiWriteOff2021-Aljezen-Figure3.png|Figure 3]]).<ref name="7Fuchsetal">Fuchs, G., M/ Boll, and J. Heider, 2011, [https://www.nature.com/articles/nrmicro2652 Microbial degradation of aromatic compounds—from one strategy to four]: Nature Reviews Microbiology, v. 9, p.803–816.</ref><ref name="8Bianetal">Bian, X. Y., S, M. Mbadinga, Y. F. Liu, S. Z. Yang, J. F. Liu, R. Q. Ye, J. D. Gu, and B. Z. Mu, 2015, [https://www.nature.com/articles/srep09801 Insights into the anaerobic biodegradation pathway of n-alkanes in oil reservoirs by detection of signature metabolites]: Scientific Reports, v. 5. article no. 9801.</ref>  

[[File:GeoWikiWriteOff2021-Aljezen-Figure3.jpg|framed|{{figure number|3}}shows the mechanism to activate hydrocarbons using Fumarate addition strategy.]]

[[File:GeoWikiWriteOff2021-Aljezen-Figure3.png|framed|{{figure number|3}}shows the mechanism to activate hydrocarbons using Fumarate addition strategy.]]

Oxygen-independent hydroxylation is another pathway that selected microbes can utilize to activate hydrocarbons in the absence of oxygen. This is illustrated below using ethylbenzene as an example (Figure 4). The process will start with the attack of a hydroxyl group (-OH) to the closest carbon atom to the ring with the help of ethylbenzene dehydrogenase enzyme, forming (S)-1-phenylethanol. Then a molecule of carbon dioxide will react with (S)-1-phenylethanol with the help of (S)-1-phenylethanol dehydrogenase enzyme to form benzoylacetate. The last two steps of the process involve reaction with a thiol group that contains co-enzyme A (CoA). The first thiol attack will convert benzoylacetate to benzoylacetyl-CoA. The second thiol attack will cleave the benzoylacetyl-CoA to benzoyl-CoA and acetyl-CoA ([[:File:GeoWikiWriteOff2021-Aljezen-Figure4.jpg|Figure 4]]).<ref name="7Fuchsetal" /><ref name="9Heider">Heider, J., 2007, [https://www.sciencedirect.com/science/article/abs/pii/S1367593107000269?via%3Dihub Adding handles to unhandy substrates: Anaerobic hydrocarbon activation mechanisms]: Current Opinion in Chemical Biology, v. 11, no. 2, p. 188-194.</ref><ref name="10Bolletal">Boll, M., C. Loffler, B. E. Morris, and J. W. Kung, 2014, [https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.12328 Anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl-coenzyme A esters: Organisms, strategies and key enzymes]: Environmental Microbiology, v. 6, no. 3, p. 612-627.</ref><ref name="11Callaghan">Callaghan, A. V., 2013, [https://www.frontiersin.org/articles/10.3389/fmicb.2013.00089/full Enzymes involved in the anaerobic oxidation of ''n''-alkanes: From methane to long-chain paraffins]: Frontiers in Microbiology, v. 4, article no. 89, 4 p.</ref>

Oxygen-independent hydroxylation is another pathway that selected microbes can utilize to activate hydrocarbons in the absence of oxygen. This is illustrated below using ethylbenzene as an example (Figure 4). The process will start with the attack of a hydroxyl group (-OH) to the closest carbon atom to the ring with the help of ethylbenzene dehydrogenase enzyme, forming (S)-1-phenylethanol. Then a molecule of carbon dioxide will react with (S)-1-phenylethanol with the help of (S)-1-phenylethanol dehydrogenase enzyme to form benzoylacetate. The last two steps of the process involve reaction with a thiol group that contains co-enzyme A (CoA). The first thiol attack will convert benzoylacetate to benzoylacetyl-CoA. The second thiol attack will cleave the benzoylacetyl-CoA to benzoyl-CoA and acetyl-CoA ([[:File:GeoWikiWriteOff2021-Aljezen-Figure4.png|Figure 4]]).<ref name="7Fuchsetal" /><ref name="9Heider">Heider, J., 2007, [https://www.sciencedirect.com/science/article/abs/pii/S1367593107000269?via%3Dihub Adding handles to unhandy substrates: Anaerobic hydrocarbon activation mechanisms]: Current Opinion in Chemical Biology, v. 11, no. 2, p. 188-194.</ref><ref name="10Bolletal">Boll, M., C. Loffler, B. E. Morris, and J. W. Kung, 2014, [https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.12328 Anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl-coenzyme A esters: Organisms, strategies and key enzymes]: Environmental Microbiology, v. 6, no. 3, p. 612-627.</ref><ref name="11Callaghan">Callaghan, A. V., 2013, [https://www.frontiersin.org/articles/10.3389/fmicb.2013.00089/full Enzymes involved in the anaerobic oxidation of ''n''-alkanes: From methane to long-chain paraffins]: Frontiers in Microbiology, v. 4, article no. 89, 4 p.</ref>

[[File:GeoWikiWriteOff2021-Aljezen-Figure4.jpg|framed|{{Figure number|4}}shows the mechanism to activate hydrocarbons using Oxygen-Independent Hydroxylation strategy.]]

[[File:GeoWikiWriteOff2021-Aljezen-Figure4.png|framed|{{Figure number|4}}shows the mechanism to activate hydrocarbons using Oxygen-Independent Hydroxylation strategy.]]

The third pathway is carboxylation. Carboxylation of non-branched alkyl chains the need of the presence of an alkyl group attached to it (e.g. benzene and naphthalene). The exact carboxylation reaction mechanism is still debated, but it is agreed that carbon dioxide is attached directly to the aromatic or aliphatic hydrocarbon via a carboxylase enzyme, requiring the presence of iron and nitrate reducing conditions.<ref name="12Gluecketal">Glueck, S. M., Gümüs, W. M. F. Fabian, and K. Faber, 2010, [https://pubs.rsc.org/en/content/articlelanding/2010/cs/b807875k Biocatalytic carboxylation]: Chemical Society Reviews, v. 39, no. 1, p. 313-328.</ref>

The third pathway is carboxylation. Carboxylation of non-branched alkyl chains the need of the presence of an alkyl group attached to it (e.g. benzene and naphthalene). The exact carboxylation reaction mechanism is still debated, but it is agreed that carbon dioxide is attached directly to the aromatic or aliphatic hydrocarbon via a carboxylase enzyme, requiring the presence of iron and nitrate reducing conditions.<ref name="12Gluecketal">Glueck, S. M., Gümüs, W. M. F. Fabian, and K. Faber, 2010, [https://pubs.rsc.org/en/content/articlelanding/2010/cs/b807875k Biocatalytic carboxylation]: Chemical Society Reviews, v. 39, no. 1, p. 313-328.</ref>

==How biodegradation of hydrocarbons is identified?==

==How biodegradation of hydrocarbons is identified?==

Crude oils that has been biodegraded typically has a lower API gravity, high sulfur content (%S) and vanadium & nickel contents than corresponding non-biodegraded crude oils.<ref name="13Moldowanetal">Moldowan, M. J., K. E. Peters, and C. C. Walters, 2007, Biodegradation parameters, ''in'' Biomarker guide: Volume 2, biomarkers and isotopes in petroleum systems and earth history (Second, Vol. 2, pp. 645–705). essay, Cambridge University Press.</ref>. Gas Chromatography (GC) analysis of crude oils is commonly used to identify and assess hydrocarbon biodegradation. GC utilizes chromatography to firstly separate individual hydrocarbon components within crude oil mixtures and a detector (either flame ionization or mass spectrometer) to measure the amount of the separated hydrocarbons which are as relative intensities thus allowing comparison between different peak intensities (e.g. low peak intensity will indicate low amounts and high peak intensity will indicate high amounts).  In general, microbes consume the simplest hydrocarbons first, i.e. normal alkanes starting from C4 then C5 then C6 and then of increasing chain length. At more advanced levels of biodegradation, branched hydrocarbons (i.e. alkyled) are biodegraded. The general sequence of hydrocarbon biodegradation, by compound class is shown in [[:File:GeoWikiWriteOff2021-Aljezen-Figure5.jpg|Figure 5]].  

Crude oils that has been biodegraded typically has a lower API gravity, high sulfur content (%S) and vanadium & nickel contents than corresponding non-biodegraded crude oils.<ref name="13Moldowanetal">Moldowan, M. J., K. E. Peters, and C. C. Walters, 2007, Biodegradation parameters, ''in'' Biomarker guide: Volume 2, biomarkers and isotopes in petroleum systems and earth history (Second, Vol. 2, pp. 645–705). essay, Cambridge University Press.</ref>. Gas Chromatography (GC) analysis of crude oils is commonly used to identify and assess hydrocarbon biodegradation. GC utilizes chromatography to firstly separate individual hydrocarbon components within crude oil mixtures and a detector (either flame ionization or mass spectrometer) to measure the amount of the separated hydrocarbons which are as relative intensities thus allowing comparison between different peak intensities (e.g. low peak intensity will indicate low amounts and high peak intensity will indicate high amounts).  In general, microbes consume the simplest hydrocarbons first, i.e. normal alkanes starting from C4 then C5 then C6 and then of increasing chain length. At more advanced levels of biodegradation, branched hydrocarbons (i.e. alkyled) are biodegraded. The general sequence of hydrocarbon biodegradation, by compound class is shown in [[:File:GeoWikiWriteOff2021-Aljezen-Figure5.png|Figure 5]].  

[[File:GeoWikiWriteOff2021-Aljezen-Figure5.jpg|framed|{{Figure number|5}}shows the PM scale for biodegradation. The shaded area at the end of the bars represent the qualitative extent of the partial removal of thecompound within a class.<ref name="15Larteretal">Larter, S., H. Huang, J. Adams, B. Bennett, and L. R. Snowden, 2012, [https://www.sciencedirect.com/science/article/abs/pii/S0146638012000083 A practical biodegradation scale for use in reservoir geochemical studies of biodegraded oils]: Organic Geochemistry, v. 45, p. 66-76.</ref>]]

[[File:GeoWikiWriteOff2021-Aljezen-Figure5.png|framed|{{Figure number|5}}shows the PM scale for biodegradation. The shaded area at the end of the bars represent the qualitative extent of the partial removal of thecompound within a class.<ref name="15Larteretal">Larter, S., H. Huang, J. Adams, B. Bennett, and L. R. Snowden, 2012, [https://www.sciencedirect.com/science/article/abs/pii/S0146638012000083 A practical biodegradation scale for use in reservoir geochemical studies of biodegraded oils]: Organic Geochemistry, v. 45, p. 66-76.</ref>]]

==Biodegradation scales==

==Biodegradation scales==

In order to assess the level of crude oil biodegradation, a scale has been developed by Peters and Moldowan.<ref name="13Moldowanetal" /> The scale is called the PM scale. The PM scale ranges from 1 to 10, with 10 to be the most altered i.e. most biodegraded. This scale is efficient and can be evaluated in conjunction with observation of the corresponding gas chromatography traces of crude oil samples. This scale illustrates very clearly that microbes favor consumption of simple hydrocarbons starting from n-alkanes and proceed to heavy aromatics at higher levels of biodegradation ([[:File:GeoWikiWriteOff2021-Aljezen-Figure5.jpg|Figure 5]]).

In order to assess the level of crude oil biodegradation, a scale has been developed by Peters and Moldowan.<ref name="13Moldowanetal" /> The scale is called the PM scale. The PM scale ranges from 1 to 10, with 10 to be the most altered i.e. most biodegraded. This scale is efficient and can be evaluated in conjunction with observation of the corresponding gas chromatography traces of crude oil samples. This scale illustrates very clearly that microbes favor consumption of simple hydrocarbons starting from n-alkanes and proceed to heavy aromatics at higher levels of biodegradation ([[:File:GeoWikiWriteOff2021-Aljezen-Figure5.png|Figure 5]]).

Other authors have proposed their own biodegradation scales, e.g. Another biodegradation scale is Wenger et al. although that scale only describes the oil based on very slight, slight, moderate, heavy and severe biodegradation levels. The scale is based on the presence or absence of specific compound classes ([[:File:GeoWikiWriteOff2021-Aljezen-Figure6.jpg|Figure 6]]). The main problem with these scales is that, there are insufficient changes observable in chemical classes at very high biodegradation levels (heavy to severe) thus biodegradation between PM 5-8 on the PM scale and heavy to severe on Wenger et al. scale.

Other authors have proposed their own biodegradation scales, e.g. Another biodegradation scale is Wenger et al. although that scale only describes the oil based on very slight, slight, moderate, heavy and severe biodegradation levels. The scale is based on the presence or absence of specific compound classes ([[:File:GeoWikiWriteOff2021-Aljezen-Figure6.png|Figure 6]]). The main problem with these scales is that, there are insufficient changes observable in chemical classes at very high biodegradation levels (heavy to severe) thus biodegradation between PM 5-8 on the PM scale and heavy to severe on Wenger et al. scale.

[[File:GeoWikiWriteOff2021-Aljezen-Figure6.jpg|framed|Figure 6 shows the Wenger 2002 scale with the corresponding PM scale and the key compounds altered or removed. <ref name="15Larteretal" />]]

[[File:GeoWikiWriteOff2021-Aljezen-Figure6.png|framed|{{Figure number|6}}shows the Wenger 2002 scale with the corresponding PM scale and the key compounds altered or removed. <ref name="15Larteretal" />]]

==What are the effects of biodegradation on crude oil economically?==

==What are the effects of biodegradation on crude oil economically?==