Degradation of alkanes is a widespread phenomenon in nature, and numerous microorganisms, both prokaryotic and eukaryotic, capable of utilizing these substrates as a carbon and energy source have been isolated and characterized. In this review, we summarize recent advances in the understanding of bacterial metabolism of long-chain n-alkanes. Bacterial strategies for accessing these highly hydrophobic substrates are presented, along with systems for their enzymatic degradation and conversion into products of potential industrial value. We further summarize the current knowledge on the regulation of bacterial long-chain n-alkane metabolism and survey progress in understanding bacterial pathways for utilization of n-alkanes under anaerobic conditions.

Bacterial metabolism of long-chain n-alkanes.

Acinetobacter calcoaceticus BD413 accumulates wax esters and triacylglycerol under conditions of mineral nutrient limitation. Nitrosoguanidine-induced mutants of strain BD413 were isolated that failed to accumulate wax esters under nitrogen-limited growth conditions. One of the mutants, Wow15 (without wax), accumulated wax when grown in the presence of cis-11-hexadecenal and hexadecanol but not hexadecane or hexadecanoic acid. This suggested that the mutation may have inactivated a gene encoding either an acyl-acyl carrier protein or acyl-coenzyme A (CoA) reductase. The Wow15 mutant was complemented with a cosmid genomic library prepared from wild-type A. calcoaceticus BD413. The complementary region was localized to a single gene (acr1) encoding a protein of 32,468 Da that is 44% identical over a region of 264 amino acids to a product of unknown function encoded by an open reading frame associated with mycolic acid synthesis in Mycobacterium tuberculosis H37Ra. Extracts of Escherichia coli cells expressing the acr1 gene catalyzed the reduction of acyl-CoA to the corresponding fatty aldehyde, indicating that the gene encodes a novel fatty acyl-CoA reductase.

Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzyme A reductase.

Biogenic formation of methane from coal is of great interest as an underexploited source of clean energy. The goal of some coal bed producers is to extend coal bed methane productivity and to utilize hydrocarbon wastes such as coal slurry to generate new methane. However, the process and factors controlling the process, and thus ways to stimulate it, are poorly understood. Subbituminous coal from a nonproductive well in south Texas was stimulated to produce methane in microcosms when the native population was supplemented with nutrients (biostimulation) or when nutrients and a consortium of bacteria and methanogens enriched from wetland sediment were added (bioaugmentation). The native population enriched by nutrient addition included Pseudomonas spp., Veillonellaceae, and Methanosarcina barkeri. The bioaugmented microcosm generated methane more rapidly and to a higher concentration than the biostimulated microcosm. Dissolved organics, including long-chain fatty acids, single-ring aromatics, and long-chain alkanes accumulated in the first 39 days of the bioaugmented microcosm and were then degraded, accompanied by generation of methane. The bioaugmented microcosm was dominated by Geobacter sp., and most of the methane generation was associated with growth of Methanosaeta concilii. The ability of the bioaugmentation culture to produce methane from coal intermediates was confirmed in incubations of culture with representative organic compounds. This study indicates that methane production could be stimulated at the nonproductive field site and that low microbial biomass may be limiting in situ methane generation. In addition, the microcosm study suggests that the pathway for generating methane from coal involves complex microbial partnerships.

Stimulation of methane generation from nonproductive coal by addition of nutrients or a microbial consortium.

The microbial degradation of petrochemicals has attracted much interest due to its potential for the development of bioremediation processes for oil spill environments, as well as for the production of fine and moderately priced chemicals. Acinetobacter spp. are known to utilize long-chain n-alkanes and to accumulate intracellular wax esters (8), which are enveloped by a single membrane (25) and serve as cell reserves. The composition of intracellular wax esters as to carbon chain length or the degree of unsaturation can be controlled by altering the growth substrate or temperature (5, 16, 17). Therefore, Acinetobacter spp. are expected to have great potential for industrial utilization.

Recently, the hydroxylation of n-alkanes, involving rubredoxin and rubredoxin reductase, has been found to be indispensable for n-alkane degradation by Acinetobacter sp. strain ADP1 (9, 19). From these facts, together with this evidence of the existence of a membrane-bound aldehyde dehydrogenase (2, 12, 27), the main pathway of n-alkane oxidation to acyl coenzyme A (acyl-CoA) via carboxylic acid is assumed to proceed through the membrane-bound enzymes in these Acinetobacter strains. This pathway is analogous to that confirmed in the alkBAC operon of Pseudomonas oleovorans (29), although the genetic organization of the alk genes is different from that in Acinetobacter spp. (9, 19). On the other hand, a variety of enzymes that catalyze the oxidation of n-alkanes and related compounds have also been found in soluble fractions of cell extracts of Acinetobacter spp. (1, 11, 7, 30), but their physiological significance in n-alkane metabolism has not been confirmed.

In this study, we found a long-chain aldehyde dehydrogenase, Ald1, in a soluble fraction of Acinetobacter sp. strain M-1 cells. In order to determine the physiological role of Ald1 in Acinetobacter sp. strain M-1, we cloned the Ald1-encoding gene, ald1, and expressed and characterized the recombinant enzyme. From these results together with those of investigation of an ald1 disruptant strain, Ald1 was confirmed to play a significant role in n-alkane utilization, especially in intracellular wax ester synthesis.

Long-Chain Aldehyde Dehydrogenase That Participates in n-Alkane Utilization and Wax Ester Synthesis in Acinetobacter sp. Strain M-1

http://link.springer.com/content/pdf/10.1007/978-3-540-77587-4_59.pdf

Enzymes for Aerobic Degradation of Alkanes

Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD+-dependent enzyme and two NADP+-dependent enzymes. One of the NADP+-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent K
m value of 5.2 μM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD+-dependent enzyme, an NADP+-dependent enzyme and one which was nucleotide independent. The NAD+-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP+-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent K
m value of 0.2 μM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Wurster's Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent K
m of 0.36 mM for decanal.

F. M. Dickinson

Papers

Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N.