Microbial degradation of the multiply branched alkane 2,6,10,15,19, 23-hexamethyltetracosane (Squalane) by Mycobacterium fortuitum and Mycobacterium ratisbonense

Abstract

Among several bacterial species belonging to the general Gordonia, Mycobacterium, Micromonospora, Pseudomonas, and Rhodococcus, only two mycobacterial isolates, Mycobacterium fortuitum strain NF4 and the new isolate Mycobacterium ratisbonense strain SD4, which was isolated from a sewage treatment plant, were capable of utilizing the multiply branched hydrocarbon squalane (2,6,10,15,19, 23-hexamethyltetracosane) and its analogous unsaturated hydrocarbon squalene as the sole carbon source for growth. Detailed degradation studies and high-pressure liquid chromatography analysis showed a clear decrease of the concentrations of squalane and squalene during biomass increase. These results were supported by resting-cell experiments using strain SD4 and squalane or squalene as the substrate. The degradation of acyclic isoprenoids and alkanes as well as of acids derived from these compounds was also investigated. Inhibition of squalane and squalene degradation by acrylic acid indicated the possible involvement of beta-oxidation in the degradation route. To our knowledge, this is the first report demonstrating the biodegradation of squalane by using defined axenic cultures.

FIG. 1

Growth of M. ratisbonense strain SD4 and M. fortuitum strain NF4 on squalane and squalene. Cells of strain SD4 (A and B) and strain NF4 (C) were grown in 30 ml of MSM in 250-ml Erlenmeyer flasks containing 0.5% (vol/vol) squalane (A and C) or 0.5% (vol/vol) squalene (B) as the sole carbon source. The cultures were inoculated from washed cells obtained from a Luria-Bertani medium preculture, and incubation was done at 30°C on a rotary shaker (120 rpm). At the indicated time intervals, samples were taken from the flasks and the indicated parameters were measured. Symbols: ⧫, living-cell count; ○, total protein content.

FIG. 2

Degradation of squalane and squalene by M. ratisbonense strain SD4. At the indicated time intervals, the culture supernatant was extracted and treated as described in Materials and Methods. The disappearance of squalane or squalene was estimated by HPLC analysis. The data recorded represent the mean value from three different cultivation experiments.

FIG. 3

Putative pathway for the degradation of squalane in Mycobacterium spp. The initial dioic acid intermediate is oxidized via β-oxidation reactions analogous to those postulated for the pristane degradation pathway. For further degradation, the β-methyl group is replaced by a carbonyl group, as found in the citronellol pathway.