Aerobic Degradation of Alkanes

Aerobic alkane degraders activate alkane molecules using O2 as a reactant. The alkane-activating monooxygenase overcomes the low reactivity of the hydrocarbon by producing reactive oxygen species. Oxidation of methane leads to formation of

Carbon Assimilation

Carbon Assimilation

Serine pathway Ribulose-P pathway

Serine pathway Ribulose-P pathway

Methane Oxidation

Fig. 17.2 Aerobic pathways of methane oxidation (after Rojo 2009)

methanol which is subsequently transformed to formaldehyde and then to formic acid (Fig. 17.2). This compound either gets converted to CO2 or assimilated for biosynthesis of other organic compounds either by the ribulose monophosphate pathway or by the serine pathway depending upon the organism (Lieberman and Rosenzweig 2004). The complete degradation of hydrocarbons mainly occurs under aerobic conditions (Riser-Robert 1998). This process involves several steps as illustrated in Fig. 17.3: (1) Accessibility of chemicals to microbes having degradation ability. Since hydrocarbons are insoluble in water, their degradation essentially requires biosurfactants which are produced by bacteria. (2) Activation and incorporation of oxygen is the vital reaction catalysed by oxygenase and peroxidase. (3) Peripheral degradation pathways which convert hydrocarbons into intermediates of the tricarboxylic acid cycle (TCA) and (4) Biosynthesis of cell biomass from the central precursor metabolites i.e. acetyl-CoA, succinate and pyruvate, sugars are required for various biosynthesis and gluconeogenesis for growth.

Degradation of n-alkanes is initiated by the oxidation of a terminal methyl group to render a primary alcohol, which gets further oxidized to the corresponding aldehyde, and finally converted into a fatty acid. Fatty acids are conjugated to CoA and further processed by b-oxidation to generate acetyl-CoA (Wentzel et al. 2007) (Fig. 17.4). However, in some cases, both ends of the alkane molecule are oxidized through m-hydroxylation of fatty acids at the terminal methyl group (m position), rendering an m-hydroxy fatty acid that is further converted into a dicarboxylic acid and processed by b oxidation (Coon 2005). Sub-terminal oxidation of n-alkanes has also been reported (Kotani et al. 2007). The product generated a secondary alcohol which is converted to the corresponding ketone, and then oxidized by a Baeyer-Villiger monooxygenase to render an ester. The ester is hydrolysed by an esterase, generating an alcohol and a fatty acid. Both terminal and sub-terminal oxidation can co-exist in some microorganisms.

Fig. 17.3 Process of microbial aerobic degradation of hydrocarbons associated with growth process (after Fritsche and Hofrichter 2000)

Some strains of Pseudomonas are able to utilize alkanes as the sole carbon and energy source (Stanier et al. 1966). The initial pathway of alkane oxidation is the following:

This pathway has been established by simultaneous adaptation experiments (Heringa et al. 1961) and chromatographic analysis of the products of alkane oxidation (Thijsse and van der Linden 1963). Acinetobacter spp. can split a hydrocarbon at the number of ten position, forming hydroxyl acids. The initial steps appear to involve terminal attack to form carboxylic acid, sub-terminal dehydrogenation at the number ten position to form an unsaturated acid, and splitting of carbon chain to form a hydroxyl acid and alcohol. Highly branched isoprenoid alkanes, such as Pristane, have been found to undergo ro-oxidation with the formation of dicarboxylic acids as the major degradative pathway.

Fig. 17.4 Aerobic pathways of n-alkane degradation (after Fritsche and Hofrichter 2000)

Intermediary metabolism

Fig. 17.4 Aerobic pathways of n-alkane degradation (after Fritsche and Hofrichter 2000)

Methyl branching increases the resistance of hydrocarbons to microbial attack. Methyl branching at b-oxidation requires an additional strategy, such as a-oxidation, ro-oxidation or ß alkyl group removal (Atlas 1981). Acremonium spp. oxidize ethane to ethanol by NADPH dependent monooxygenase, which is subsequently oxidized to acetaldehyde and acetic acid. Acetate, thus formed, is assimilated into cellular carbon via reverse tricarboxylic acid cycle and glyoxalate bypass. Similarly, a number of propane and butane utilizers have been reported that are also capable of growth on long chain alkanes, such as n-dodecane and n-hexadecane.

Long chain hydrocarbons (C10-C18) can be used rapidly by many high G + C Gram-positive bacteria, but only a few bacteria can oxidize C2-C8 hydrocarbons. Degradation of n-alkanes requires activation of the inert substrates by molecular oxygen with the help of oxygenases by three possible ways that are associated with membranes:

1. Monooxygenase attacks at the end producing alkan-1-ol:

2. Dioxygenase attack produces hydroperoxides, which are reduced to yield also alkan-1-ol:

R-CH3 + O2 ! R-CH2OOH + NAD(P)H + H+ ! R-CH2OH + NAD(P)++ H2O

3. Rarely, subterminal oxidation at C2 by monooxygenase yields secondary alcohols.

Brevibacterium erythrogenes can use 2-methylundecane as substrate for growth by a combination of m- and b-oxidation. Arthrobacter sp. has been reported to metabolize squalene (C30-multiple, methyl branched compound) to geranylace-tone, which is accumulated in the medium as it cannot be further metabolized. Similarly, Corynebacterium sp. and B. erythrogenes have been shown to degrade pristane (2,6,10,14-tetramethyl pentadecane) involving m-oxidation, followed by b-oxidation, yielding propionyl-CoA and acetyl-CoA units alternately.

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