Biodegradation - Hydrocarbons

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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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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.


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See Also