Assessing soil microbial populations responding to crude-oil amendment at different temperatures using phylogenetic, functional gene (alkB) and physiological analyses

The effect of temperature as a determinant for selecting microbial populations associated with alkane-degradation was examined in crude oil-amended soil microcosms. After a 30-day incubation, >95% of n-alkane components in the crude-oil were depleted and approximately 40 and 60% of added [ ¹⁴C] hexadecane was converted to ¹⁴CO₂ at 4-10 and 25°C, respectively. Concomitant with crude-oil depletion, 16S rRNA gene sequence analysis revealed the emergence of a prominent Rhodococcus-like 16S rRNA sequence at all temperatures and a prominent Pseudomonas-like sequence at 4 and 10°C. The diversity of alkane hydroxylase genes (alkB) associated with the amendments was examined using group-specific alkB-PCR primers targeting phylogenetically distinct groups of alkane-degrading bacteria and subsequent cloning, denaturing gradient gel electrophoresis and sequencing analyses. Diverse Rhodococcus-alkB genes were detected at all temperatures, while a single prominent Pseudomonas-alkB genotype was detected only at lower temperatures. Two isolates obtained from the microcosms were shown to have 16S rRNA and alkB genes identical to those observed and were used to examine growth as a function of temperature. The Pseudomonas isolate exhibited a substantially higher growth rate at 4 and 10°C than the Rhodococcus isolate, consistent with the inference that differences in adaptation to low temperature explain the observed shift in populations. High resolution analysis of alkB genes enabled the differentiation of distinct alkane-degrading populations responding to crude-oil amendment from other closely related, well-studied strains with different temperature adaptations.