Microcosms setup.
Both experiments were performed in 158 mL serum bottles containing 50 mL tailings (SS g/L), 50 mL methanogenic media, and 0.4 mM polycyclic aromatic hydrocarbons (PAHs). Bottles were capped with butyl rubber stoppers and crimped with aluminum caps to maintain methanogenic environment, then incubated without shaking under 21°C room temperature in darkness. The tailings were collected from 10.9 meters below water surface of the Base Mine Lake operated by Syncrude Canada Ltd-Research. Methanogenic media was prepared to provide indigenous microbes with macronutrients, micronutrients and vitamins for growth rate improvement (Fedorak et al., 1984, and Holowenko et al., 2000). Naphthalene, Phenanthrene, and Pyrene were spiked to microcosms. All the PAHs used in this study exists in the form of crystalline particles under room temperature, thus toluene was used to dissolve PAHs before spiking. Treatments. Inoculated treatments were the microcosms inoculated by PAHs and active inoculums. Solvent controls were set to compare and remove the methane production from methanogenic degradation of solvent (toluene). Sterile controls were prepared to eliminate any degradations that are not due to bioactivities. Methylcyclohexane was used as the internal standard for the measurements of toluene concentration. Background controls were used to monitor baseline methane production from tailings. PAH Soxhlet Extraction. All microcosms were gently shaken before taking samples with syringes. A 5 g slurry sample and 2.5 g anhydrous sodium sulphate Fig. 3 A microcosm
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were placed in a soxhlet thimble, with dichloromethane as extractant. The process continued ~24 hours before the extracts were cleaned with chromatography columns to remove non-polar compounds. Dichloromethane was used to moisturize the column and remove the potential contaminants before cleaning. The cleaned extracts were reduced to 1.5 mL through evaporation and analyzed with Gas Chromatography-Mass Spectrometry (GC-MS).
The 1 mM of Naphthalene, Phenanthrene, and Pyrene were injected into the Amend and Sterile Control microcosms on the Day 0 of the experiment. Microcosms were kept in darkness for 24 hours after spiking to guarantee the comprehensive distribution of PAHs into tailings. Chemical Analysis. Toluene and methane were taken out from microcosm headspace with syringes before being manually injected into GC-MS for analysis. Samples were measured by a TRACE™ 1300 Series GC System connected to an ISQ™ Single Quadruple MS and a TriPlus™ RSH SMART Autosampler. Stoichiometric Analysis. The maximum production of methane from hydrocarbons degradation in this study was calculated from the following equation: CnHaOb + (n-a/4-b/2)H2O = (n/2-a/8+b/4)CO2 + (n/2+a/8-b/4)CH4 (Equation 1) Determination of Microbial Communities. Samples were taken periodically from each microcosm for molecular analysis. A FastDNA™ SPIN Kit was used for extracting DNA from samples, and metagenomic sequencing was used for taxa identification and characterization. Fig. 4 The Methanogenic Media
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Disclaimer: All the contents presented in this website were produced by Henian Guo as an assignment for RENR 580 at the University of Alberta and should NOT be interpreted outside of the scope of this assignment. Parts of the findings were not from the real experiment.