Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp

Abstract

The psychorotrophic Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5 degrees C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C10 to C21 alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5 degrees C. Mineralization of hexadecane at 5 degrees C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction-gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis the A gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25 degrees C.

FIG. 1

Mineralization of [1-¹⁴C]dodecane by the psychrotroph Rhodococcus sp. strain Q15 and the mesophile P. oleovorans at 10, 20, and 30°C. Mineralization by Rhodococcus sp. strain Q15 and P. oleovorans was determined in 20 ml of MSM supplemented with 50 mg of YE/liter, as described in Materials and Methods. Each point represents the mean from duplicate cultures.

FIG. 2

Mineralization of [1-¹⁴C]dodecane, [1-¹⁴C]hexadecane, [14,15-¹⁴C]octacosane, or [16,17-¹⁴C]dotriacontane by Rhodococcus sp. strain Q15 at 5°C, determined as described in the legend to Fig. 1. Each point represents the mean from duplicate cultures.

FIG. 3

GC profiles of diesel oil extracted from the aqueous phase of MSM medium after 28 days of incubation at 5°C with and without inoculation with Rhodococcus sp. strain Q15. (A) Abiotic control (uninoculated); (B) inoculated with Q15. IS, injection standard; ES, extraction standard; C10 to C21, n-alkanes (numbers designate the number of C atoms); B₁ to B₅, branched alkanes; C, substituted cyclohexane; Na, naphthalene; N₁ to N₄, substituted naphthalenes; Pr, pristane; Ph, phytane. The alkane, naphthalene, phytane, and pristane peaks were identified by comparison of their retention times and mass spectra with authentic standards. The branched alkanes, substituted naphthalenes, and substituted cyclohexane were tentatively identified by mass spectra database comparisons following GC-MS analysis.

FIG. 4

Degradation of specific diesel fuel components by Rhodococcus sp. strain Q15 at 5°C in MSM containing 0.1% (vol/vol) diesel fuel after 14 days of growth in MSM alone (solid bars) or supplemented with 10 ppm of YE (hatched bars) and after 28 days of growth in MSM alone (shaded bars) or supplemented with 10 ppm of YE (open bars). Abbreviations are described in the legend to Fig. 3 and correspond to the peaks shown in Fig. 3. Each point represents the mean from duplicate cultures.

FIG. 5

GC profile of intermediate metabolites of hexadecane degradation by Rhodococcus sp. strain Q15. Q15 was grown in MSM supplemented with 50 ppm of hexadecane and 50 ppm of YE at 23°C. Metabolic intermediates were extracted from the culture supernatant by SPME and analyzed by GC-MS. (A) Intermediates extracted at time zero; (B) intermediates extracted after 24 h of incubation.

FIG. 6

Utilization of dodecane and hexadecane and their potential metabolic intermediates by Rhodococcus sp. strain Q15 at 23°C. Q15 was grown in MSM supplemented with 100 ppm of the indicated n-alkane or metabolic intermediate as the sole carbon and energy source. Bacterial growth was monitored by CO₂ respiration. Each point represents the mean number of micromoles of CO₂ evolved from triplicate samples, with the error bars representing the standard deviations of the means. The control values represent the background CO₂ respired from MSM inoculated with Rhodococcus sp. strain Q15 but lacking a carbon source.

FIG. 7

Detection of plasmids in parental Rhodococcus sp. strain Q15 and the plasmid-cured strains Q15 H and Q15 NP by plasmid and Southern analyses. Total DNA (chromosomal and plasmid DNAs) from the three strains was isolated and examined by agarose gel electrophoresis. (A) Agarose gel electrophoresis (0.7%) showing the presence of the ∼90- and ∼115-kb plasmids in Q15, the ∼90-kb plasmid in Q15 H, and the lack of both plasmids in Q15 NP. (B) Southern hybridization analysis of chromosomal and plasmid DNAs shown in panel A transferred to a nylon membrane and probed with a DNA probe constructed from an ∼4-kb EcoRI fragment derived from the Q15 90-kb plasmid. Lanes 1, lambda (HindIII) ladder; lanes 2, Rhodococcus sp. strain Q15; lanes 3, Q15 H; lanes 4, Q15 NP.

FIG. 8

The effect of bioaugmentation with Rhodococcus sp. strain Q15 (10⁸ CFU/g of soil) on mineralization of [1-¹⁴C]hexadecane at 5°C in pristine and hydrocarbon-contaminated soil microcosms. Each point represents the mean cumulative mineralization (percent CO₂) from triplicate soil microcosms, with the error bars representing standard deviations of the means. Contam., contaminated.