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

Understanding soil organic carbon dynamics at the landscape scale: hotspot mapping

<p>The recombinant Caulobacters were co-incubated with HIV pseudotype virus SVPB13 and TZM-bl cells to demonstrate inhibition of infection. TZM-bl cells were also incubated alone, with virus, or with virus and neutralizing monoclonal antibody. Pseudotype virus infection was measured by ELISA for β-galactosidase. Significant levels of inhibition of infection were observed (denoted by asterisks) between both Cc-MIP1a and the Cc-CTRL (<0.001) and Cc-CD4 and Cc-CTRL (<0.01). HIV infections are presented as a percentage of the untreated control infections using the same pseudotype virus, SVPB13 and with the background for uninfected cells subtracted out. Each separate experiment was performed with 3 assay wells per condition. Data represent mean + standard error of the mean (s.e.m) from 4 separate experiments.</p

Incubation of both recombinant Caulobacters with pseudotype HIV-1 shows combinatorial effects and heightened inhibition of infection against the clade B viruses.

<p>The recombinant Caulobacters were combined in equal amounts and co-incubated with one of six different HIV pseudotype viruses and TZM-bl cells to demonstrate inhibition of infection. TZM-bl cells were also incubated alone, with virus, or with virus and neutralizing monoclonal antibody. Pseudotype virus infection was measured by ELISA for β-galactosidase. Significant levels of inhibition of infection were observed between both Cc-MIP1a/Cc-CD4 and the Cc-CTRL (<0.01 for all six pseudotype viruses). HIV infections are presented as a percentage of the untreated control infections using the same pseudotype virus and with the background for uninfected cells subtracted out. Each separate experiment was performed with 3 assay wells per condition. Data represent mean + s.e.m from 3–4 separate experiments.</p

MIP1α surface display on Caulobacter.

<p>A. SDS-PAGE of normalized low pH extraction of S-layer protein (RsaA) from <i>C. crescentus</i> JS 4022. Lane 1- RsaA obtained from Cc-CTRL. Lane 2 - RsaA obtained from Cc-MIP1a. B. Fluorescence microscopy using anti-MIP1α polyclonal antibody and an FITC-labeled secondary.</p

Heat inactivation of the recombinant Caulobacters retains inhibition of infection for pseudotype HIV-1 subtype B virus clone SVPB13.

<p>The recombinant Caulobacters were heat inactivated and co-incubated with HIV pseudotype virus SVPB13 and TZM-bl cells to demonstrate inhibition of infection. TZM-bl cells were also incubated alone, with virus, or with virus and neutralizing monoclonal antibody. Pseudotype virus infection was measured by ELISA for β-galactosidase. Significant levels of inhibition of infection were observed (denoted by asterisks) between HIC Cc-MIP1a and Live Cc-MIP1a (<0.005), HIC Cc-CTRL and Live Cc-CTRL (<0.001), and HIC Cc-CD4 and Live Cc-CD4 (<0.001). HIV infections are presented as a percentage of the untreated control infections using the same pseudotype virus, SVPB13 and with the background for uninfected cells subtracted out. Each separate experiment was performed with 3 assay wells per condition. Data represent mean + s.e.m from 3 separate experiments.</p

Display of CD4 domain 1 on Caulobacter.

<p>A. SDS-PAGE of normalized low pH extraction of S-layer protein (RsaA) from <i>C. crescentus</i> JS 4022. Lane 1- RsaA obtained from Cc-CTRL. Lane 2- RsaA obtained from Cc-CD4. Asterisks indicate the RsaA proteins. B. Fluorescence microscopy using anti-CD4 polyclonal antibody and an Alexa488-labeled secondary.</p