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Note added. Determination of Biodegradation Analytical methods for oil compound analyses

Since oil consists of thousands of different compounds (Marshall and Rogers 2003) measurements of individual compounds is a challenge.  Bulk oil biodegradation may be determined by traditional gravimetric analyses (e.g. Horowitz and Atlas 1977), while broader groups of oil components (saturates, aromatics, resins and asphaltenes = SARA) may be determined by Iatroscan thin-layer chromatography with flame ionization detection (TLC-FID; Stevens 2004).  Using this method, crude oil components are determined according to their polarity.  The saturate fraction consists of nonpolar material including linear, branched, and cyclic saturated hydrocarbons (paraffins). Aromatics, which contain one or more aromatic rings, are slightly more polarizable.  The remaining two fractions, resins and asphaltenes, have polar substituents.  Additional bulk oil analytical methods include Fourier Transform Infrared (FTIR) spectroscopy and Nuclear Magnetic Resonance (NMR) spectroscopy.  FTIR is an absorption technique that uses infrared (IR) electromagnetic radiation to examine the identity of chemical bonds within the substance of interest.  As microbial degradation of the oil is expected to result in the addition of oxygen atoms into the structure of oil compounds this method may be a method for measuring bulk changes in composition, although the resolution and sensitivity is poor compared to other methods.  NMR is a nondestructive technique that is well-suited for identifying and quantifying different hydrocarbon classes and can provide information on the relative content of aliphatic, olefinic, and aromatic components.  Studies have shown that NMR spectra in conjunction with multivariate statistical analysis can be correlated to a number of physicochemical properties and standard distillation cut yields (Molina et al. 2007).  Mass spectrometry (MS) has become one of the most important detection principles in modern analytical chemistry.  The principle behind MS is that molecules can be identified through their molecular weight and fragmentation patterns.  MS is very often connected to a separation step, usually gas (GC) or liquid (LC) chromatography.  These methods may be used to identify and quantify targeted oil compounds or for fingerprinting of complex chemical mixtures.  To separate between different oil compound groups gas chromatographic analyses (GC-FID and GC-MS) are the standards today, but these methods favor detection of nonpolar compounds.  The common use of these methods therefore limits our knowledge of oil biodegradation, mainly to some compound groups, such as the C10-C40 saturates, cyclic saturates (decalines), BTEX, phenols, 2-6 ring PAHs, and a variety of biomarkers.  LC-MS analyses may therefore be an important supplement to the gas chromatographic analyses for more polar compound groups.  In addition, biodegradation studies of compounds like naphthenic acids have been of interest in specific areas like Canada.  Several high-resolution instruments, like time-of-flight mass spectrometers (ToF-MS) coupled to GCxGC systems (GCxGC-ToF-MS)(e.g. Tran et al. 2010) and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (e.g. Hughey et al. 2008) provide powerful techniques for the analytical separation of complex mixtures combined with methods for characterizing the resolved compounds. Minor components hidden in the large background can be detected by these instruments, and both resolution and sensitivity allow for searching of spectra from very narrow peaks.  For instance FT-ICR MS can separate masses of <0.002 Dalton of compounds that contain heteroatoms such as N, O, S and other elements, identifying oil compounds by mass and molecular formula at high resolution. Experimental apparatus

Advances in microbial sampling capabilities, in particular sampling of the ocean in drilling areas, came with advances in drilling technology.  The Ocean Drilling Project (ODP) and subsequent Integrated Deep Ocean Drilling Program (IODP 2003-2013) and planned International Ocean Discovery Program (IODP 2013-2023) provide a framework for these activities (Edwards et al. 2012).  Each of the named programs includes or will include a sampling component for microbial ecology research.  In particular, the 2003 IODP included extensive evaluations of seafloor and sub-seafloor microbial communities (Cyranoski 2003).  In conjunction with new microbiological techniques, these samples provided new perspectives on deep ocean microbial community composition and function (D'Hondt et al. 2004; Schippers et al. 2005; Inagaki et al. 2006; Biddle et al. 2008; Kobayashi et al. 2008; Forschner et al. 2009; Lomstein et al. 2012).  Understanding native populations in Arctic drilling fields requires sampling such as has been carried out in these programs.

Much of the sampling that is associated with drilling activities focuses on the deep subsea floor while water column and sediment samples can be collected with remote samplers or can also  be collected by autonomous underwater vehicles (AUVs).  The Chemosynthetic Ecosystem Science (ChEss) project of the Census of Marine Life (2002-2010) was one such project that generated a substantial amount of new information about marine microbial communities.  Much of the success of the ChEss project was attributed to the development of improved deep-ocean AUVs (German et al. 2011) that allowed systematic exploration of previously understudied areas, including cold seeps.  Modern AUVs are capable of rapid deployment and operation at a range of depths.  They have been effectively deployed to sample in response to events such as the Deepwater Horizon spill of 2010 (Camilli et al. 2010).  These vehicles contribute to the ability to observe natural processes and conduct in situ experiments, particularly at depth in harsh marine environments.

Another strategy is to employ microbial observatories in marine environments that incorporate real-time sensors, time-lapse cameras, and other experimental devices.  These observatories, along with autonomous and cabled sensors, allow direct measurement of microbial processes in the deep ocean.  In particular, beginning some 20 years ago, circulation obviation retrofit kits (CORKs) came into use to study connectivity of hydraulics and biogeochemistry at the interface of the ocean bottom and open water (; Cowen et al. 2003) rather than relying on extrapolation from controlled laboratory experiments (e.g. Bartlett 2002; Tapilatu et al. 2010) or inference from population composition (Simonato et al. 2006) as is more commonly done.  Another type of observatory, the Microbial Methane Observatory for Seafloor Analysis (MIMOSA), is an autosampler that collects and archives microbial material for later recovery and analysis.  Two of these devices recently were deployed in the Gulf of Mexico to evaluate petroleum seeps and spills as they affect microbial population structure (Balinski 2012).  This type of observatory may be useful to implement in situ experiments to monitor biodegradation rates and processes and further advance knowledge of petroleum hydrocarbon degradation processes in the deep ocean environment.

Finally, another tool that is directly relevant to petroleum biodegradation and carbon utilization is the so-called “bug trap,” in which hydrophobic beads or woven matrix is dosed with petroleum hydrocarbons to evaluate in situ degradation potential and analyzed to characterize degrading community composition.  Because petroleum-degrading microorganisms can be chemotaxic to suitable substrates, these experimental devices can be used to attract and study degraders in the laboratory (Brakstad and Bonaunet 2006) and in situ experiments (Raloff 2010; DeAngelis et al. 2011). Biodegradation data processing

In standard laboratory studies, oil degradation is usually determined by comparison of depletion in normal seawater or cultures to depletion in sterile (killed) controls. In this way processes like evaporation, wall effects, dissolution of compounds from the oil phase etc. may be accounted for and separated from the biodegradation process. However, in field and meso-/large-scale studies biodegradation is determined by normalization of degradable compounds to less degradable (recalcitrant) compounds. Common compounds for this internal normalization are pentacyclic triterpane biomarkers (e.g.C3017α(H),21β(H)-hopane) and the isoprenoids pristane and phytane (Prince et al. 1994; Douglas et al. 1996; Page et al. 1996). The isoprenoids have proven to be biodegradable themselves, although at slower rates than their corresponding n-alkanes (e.g. Douglas et al. 1996). Hopanes also have limitations if used to determine biodegradation of compounds with low boiling points, since it may be difficult to separate biodegradation from evaporation. In addition, determination of biodegradation as ratios between biodegradable and more persistent compounds has also been suggested using other compounds, like 2-methylphenanthrene/1-methylphenanthrene, and C3-phenanthrene/C3-dibenzothiophene (Fedorak and Westlake 1981; Christensen and Larsen 1993; Wang et al. 1998; Lamberts et al. 2008).