Crude oil bioremediation by alcanivorax borkumensis

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Alcanivorax borkumensis is a recognized alkane-degrading organism that has the potential to be useful for crude oil spill bioremediation.

A. borkumensis Overview

Physiology and Phylogeny

A. borkumensis is a gram-negative, rod shaped, aerobic, catalase negative and oxidase positive species (1). A. borkumensis is halotolerant, which is necessary to survive in ocean salinity; optimal NaCl concentration for growth in culture is between 3-10% (1). It can use linear and branched alkanes as a primary fuel source, but cannot use aromatic alkanes for this purpose (1,2). This is an important factor in determining A. borkumensis’ role in bioremediation as discussed in section 5.2. It is classified as a γ-proteobacterium based on 16S rRNA sequencing (1). The γ-proteobacterium class includes another known alkane degrading genus, Marinobacter (1).

Distribution

A. borkumensis is not typically observed in the microbial communities of uncontaminated water (4). However, it can make up 70-90% of the composition of microbial communities after contamination with crude oil, which is indicative of a competitive advantage (4). It is important to note that alkane degradation is not unique to A. borkumensis and thus, the ability to degrade alkanes alone does not explain why it is the dominant species in crude oil contaminated areas (5). It is suspected that the reason for its dominance lies in the ability to degrade branched alkanes (2).

Composition of Crude Oil

A total of three million tons of crude oil enter the ocean each year as a result of human activity and more natural mechanisms such as seepage(6). Crude oil in the ecosystem is viewed as a chronic pollutant that is toxic to most forms of life (6). Crude oil is challenging to clean up as it has over 17000 distinct compounds (3). Chief among them are saturated hydrocarbons, aromatic hydrocarbons, resins and asphaltenes (3).

Crude oil can be removed from the environment through evaporation, photooxidation and microbial bioremediation (1). After a spill, the typical approach is to use booms, skimmers and adsorbents to remove as much oil from the water as possible; however, the effectiveness of this approach is very limited (7).

A) Shows the composition of crude before (above) and shortly after (below) biodegradation begins. B) shows the preferential degradation of shorter hydrocarbons by the microbial communities present in oil contaminated sea water. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Microbiology Head I, Jones MD, Roling W. “Marine microorganisms make a meal of oil.” (2006)

Structure and Function of alk operon

The two major alkane hydroxylases, (alkane 1-monooxygenases), are encoded by alkB1 and alkB2, each are part of separate operons (9). The presence of these genes allows A. borkumensis to degrade alkanes (9). An upstream locus, alkS, regulates the alkB1 operon (10). The operon includes alkH - an aldehyde dehydrogenase, alkJ - an alcohol dehydrogenase and alkG – a rubredoxin reductase, which reduces the electron shuttling protein rubredoxin (9,11). The alkane hydroxylases alkB1 and alkB2 preferentially degrade alkanes that are 5-12 and 8-16 carbons in length, respectively (2). This is seen in Figure 1B, which shows the preferential degradation of specific length hydrocarbons.

The genome lacks a cyclohexane monooxygenase gene (9). As a result, A. borkumensis cannot utilize aromatic alkanes as a primary fuel source (9). It is suspected that alkB1 or alkB2 are able to cometabolize cycloalkanes via oxygenase activity (9). This permits A. borkumensis to break down cycloalkanes only when there are aliphatic alkanes present, since the expression of the necessary genes is induced by the presence of aliphatic alkanes.

A. borkumensis likely has another mechanism of alkane degradation as alkB1 and alkB2 knockouts do not completely lose alkane degradation capacity (12). This is suspected to be due to the presence of three putative cytochrome P450 proteins that can break down aliphatic alkanes (12).

Mechanism of Unbranched Alkane Degradation

Emulsification

A. borkumensis must first ensure that crude oil is bioavailable. Crude oil floating on the surface of water must be emulsified before it can be metabolized (13). This is achieved through the production of biosurfactant. Biosurfactants act as emulsifiers to mediate the interaction between the bacteria, water and oil (14). By increasing the solubility of the crude oil, the rate of biodegradation also increases (14).

Formation of alcohol from alkane

NADH reduces rubredoxin reductase, which then reduces rubredoxin. Rubredoxin shuttles electrons to the alkane hydroxylase (15). The alkane hydroxylase uses dioxygen to oxidize the alkane with one molecule of oxygen, while reducing the other molecule of oxygen to water (10). This yields an alcohol.

There is a series of reactions involving rubredoxin and rubredoxin reductase that lead to the formation of an alkyl alcohol from an alkane with NADH as the reductant

Formation of a fatty acid

The alcohol is oxidized by an alcohol dehydrogenase to yield an aldehyde (9). The aldehyde is subsequently oxidized to yield a fatty acid via an aldehyde dehydrogenase (9). The fatty acid that is produced proceeds through β-oxidation to yield acetyl CoA (16).

The steps of beta oxidation to yield acetyl CoA from a fatty acid

The mechanism of branched alkane degradation in A. borkumensis is not fully understood but it is suspected to involve simultaneous and α and β-oxidation (16).

Applications and Limiting Factors

The use of fertilizer to increase the activity of A. borkumensis has become an accepted technique for crude oil spill clean ups (17). The process of bioremediation is limited by nutrient availability (discussed below) and fertilizer helps overcome this (18). This has been shown to increase the rate of biodegradation two to three fold (7). The Exxon Valdez spill in Prince William Sound, Alaska is an early example of the large-scale applications of A. borkumensis alkane degradation as a method of bioremediation (7).

Nutrient availability

Low availability of nitrogen and phosphorous in crude oil is a major limiting factor for bioremediation (1). The application of fertilizer to increase available nitrogen and phosphorous has been demonstrated as an effective strategy to increase alkane degradation, allowing A. borkumensis to grow beyond the typical carrying capacity of the environment (7).

Metabolic Constraints

A. borkumensis can only degrade select alkanes in crude oil (3). These include linear and branched alkanes in addition to the cometabolism of aromatic alkanes (9). It has been observed that fertilizer application may increase the rate at which the end point is reached without changing the actual end point in terms of components of crude oil that remain (3). Although alkanes constitute a large portion of crude oil, after A. borkumensis bioremediation, non-degraded compounds will persist.

Bioavailability

Despite the production of biosurfactant, as the population of A. borkumensis grows, the surface area of the interface between oil and water becomes a limiting factor. Limited emulsification has been implicated in causing the transition from the exponential to a linear growth phase (19). The addition of biosurfactant producing bacteria is being explored as a way to increase the rate of crude oil bioremediation by prolonging exponential growth, resulting in higher density A. borkumensis populations and more rapid alkane degradation (18). Acinteobacter radioresistens is a candidate that can potentially be used for this purpose (19).

Temperature

Water temperature is a limiting factor for the rate of alkane degradation (17). In the case of the Exxon Valdez spill, fertilizer application could only occur during the summer months when the water temperature was warm enough to facilitate relatively rapid alkane degradation by A. borkumensis (18). Temperature limits the window of time during which fertilizer application can be considered a viable option (18).

References

(1) Yakimov M, Golyshin P, Lang S, Moore E, Abraham W, Lunsdorf H, Timmis K. “Alcanivorax borkumensis gen. nov., sp. Nov., a new hydrocarbon-degrading and surfactant-producing marine bacterium.” International Journal of Systems and Evolutionary Microbiology. 1998. 2:339-48. DOI: 10.1099/00207713-48-2-339

(2) Hara A, Syutsubo K, Harayama S. “Alcanivorax which prevails in oil-contaminated seawater exhibits broad substrate specificity for alkane degradation.” Environmental Microbiology. 2003. 5(9): 746-753. DOI: 10.1046/j.1468-2920.2003.00468.x

(3) Head I, Jones MD, Roling W. “Marine microorganisms make a meal of oil.” Nature Reviews Microbiology. 2006. 4:173-182. DOI: 10.1038/nrmicro1348

(4) Roling WFM, Milner M, Jones MD, Fratepietro F, Swannell RPJ, Daniel R, Head I. “Bacteria; Community Dynamics and Hydrocarbon Degradation during a Field-Scale Evaluation of Bioremediation on a Mudflat Beach Contaminated with Buried Oil.” Applied Environmental Microbiology. 2004. 70(5):2603-2613. DOI: 10.1128/AEM.70.5.2603-2613.

(5) Floodgate GD. “Some environmental aspects of marine hydrocarbon bacteriology.” Aquatic Microbiology. Ecol. 1995. 9:3-11.

(6) Golyshin P, Martins Dos Santos VAP, Kaiser O, Ferrer M, Sabriova Y, Lunsdorf H, Chemikova TN, Golyshina O, Yakimov MM, Puhler A, Timmis K. “Genome sequence completed of Alcanivorax borkumensis, a hydrocardon-degrading bacterium that plays a global role in oil removal from marine systems.” Journal of Biotechnology. 2003. 106(2-3): 215-220. DOI: 10.1016/j.jbiotec.2003.07.013

(7) Prince R. “Petroleum Spill Bioremediation in Marine Environments.” Critical Reviews in Microbiology. 1993. 19(4): 217-242. DOI: 10.3109/10408419309113530

(8) Kasai Y, Kishira H, Sasaki T, Syutsubo K, Watanabe K, Harayama S. “Predominant growth of Alcanivorax strains in oil-contaminated and nutrient supplemented seas water.” Environmental Microbiology. 2002. 4(3): 141-147. DOI: 10.1046/j.1462-2920.2002.00275

(9) Sabirova JS, Ferrer M, Regenhardt D, Timmis K, Golyshin P. “Proteomic Insights into Metabolic Adaptations in Alcanivorax borkumensis induced by Alkane Utilization.” Journal of Bacteriology. 2006. 188(11): 3762-3773. DOI: 10.1128/JB.00072-06

(10) van Beilen J, Marin M, Smits T, Rothlisberger M, Franchini A, Witholt B, Rojo R. “Characterization of two alkane hydroxylase genes from the marine hydrocabonoclastic bacterium Alcanivorax borkumensis.” Environmental Microbiology. 2004. 6(3), 264-273. DOI: 10.1111/j.1462-2920.2004.00567.x

(11) Teimoori A, Ahmadian S, Mdadkar-Sobhani A, Bambai B. “Reubredoxin reductase from Alcanivorax borkumensis: expression and characterization.” Biotechnology Progress. 2001. 27)5):1383-1389. DOI: 10.1002/btpr.653

(12) Miri M, Bambai B, Tabandeh F, Sadeghizadeh M, Kamali N. “Production of a recombinant alkane hydroxylase (AlkB2) from Alcanivorax borkumensis.” Biotechology letters. 2010. 32(4): 497-502. DOI: 10.1007/s10529-009-0177-0

(13) Abraham WR, Meyer H, Yakimov M. “Novel glycine containing glucolipids from the alkane using bacterium Alcanivorax borkumensis.” Lipids and Lipid Metabolism. 1998. 1:57-62. DOI: 10.1016/S0005-2760(98)00058-7

(14) Banat I, Franzetti A, Gandolfi I, Bistetti G, Martinotti M, Fracchia L, Smyth T, Marchant R. “Microbial biosurfactant production, applications and future potenital.” Applied Microbiology and Biotechnology. 2010. 87:427-444. DOI: 10.1007/s00253-010-2589-0.

(15) van Beilen J, Neunschwander M, Smits T, Roth C, Balada S, Witholt B. “Rubredoxins involved in alkane oxidation.” Journal of Bacteriology. 2002. 184(6): 1722-1732. DOI: 10.1128/JB.184.6.1722-1732.2002

(16) Dutta TK, Harayama S. “Biodegredation of n-alkylcycloalkanes and n-alkylbenzenes via New Pathways in Alcanivorax sp. Strain MBIC 4326”. Applied Environmental Microbiology. 2001. 67(4): 1970-1974. DOI: 10.1128/AEM.67.4.1970-1974.2001

(17) Nikolopoulou M, Eickenbusch P, Pasadakis N, Venieri D, Kalogerakis N. “Microcosm evaluation of autochthonous bioaugmentation to combat marine oil spills.” New Biotechnology. 2013. 30(6):734-742. DOI: 10.1016/j.nbt.2013.06.005

(18) Pritchard PH, Mueller JG, Rogers JC, Kremer FV, Glaser JA. “Oil spill bioremediation: experiences, lessons and results from the Exxon Valdez oil spill in Alaska.” Biodegradation. 1992. 3:315-335.

(19) Ron E, Rosenberg E. ”Biosurfactants and oil bioremediation.” Current Opinion in Biotechnology. 2002. 13(3):249-252. DOI: 10.1016/S0958-1669(02)00316-6