Long-Term Incubation Reveals Methanogenic Biodegradation of C₅ and C₆ <i>iso</i>-Alkanes in Oil Sands Tailings

<i>iso</i>-Alkanes are major components of petroleum and have been considered recalcitrant to biodegradation under methanogenic conditions. However, indigenous microbes in oil sands tailings ponds exposed to solvents rich in 2-methylbutane, 2-methylpentane, 3-methylpentane, <i>n</i>-pentane, and <i>n</i>-hexane produce methane in situ. We incubated defined mixtures of <i>iso-</i> or <i>n-</i>alkanes with mature fine tailings from two tailings ponds of different ages historically exposed to different solvents: one, ∼10 years old, receiving C₅–C₆ paraffins and the other, ∼35 years old, receiving naphtha. A lengthy incubation (>6 years) revealed <i>iso-</i>alkane biodegradation after lag phases of 900–1800 and ∼280 days, respectively, before the onset of methanogenesis, although lag phases were shorter with <i>n</i>-alkanes (∼650–1675 and ∼170 days, respectively). 2-Methylpentane and both <i>n</i>-alkanes were completely depleted during ∼2400 days of incubation, whereas 2-methylbutane and 3-methylpentane were partially depleted only during active degradation of 2-methylpentane, suggesting co-metabolism. In both cases, pyrotag sequencing of 16S rRNA genes showed codominance of Peptococcaceae with acetoclastic (Methanosaeta) and hydrogenotrophic (Methanoregula and Methanolinea) methanogens. These observations are important for predicting long-term greenhouse-gas emissions from oil sands tailings ponds and extend the known range of hydrocarbons susceptible to methanogenic biodegradation in petroleum-impacted anaerobic environments