Enrichment of Anaerobic Alkane Degraders

Activated sludge (50 ml) and anaerobic digestion sludge (100 ml) from a full-scale wastewater treatment plant (Luggage Point, Brisbane, Australia) were used as inoculum for the ethane and n-butane (hereafter butane) bioreactor enrichment. This choice of inoculum was based on previous successful enrichment of anaerobic propane degradation bacteria from this source and the small quantities of ethane and butane detected in anaerobic digestion systems [36 (link)]. The incubations with ethane or butane as a sole carbon source were set up in a lab bioreactor with a volume of 1.12 and 2.3 l, respectively. An anoxic mineral medium [16 (link)] of 0.67 and 1.69 l was initially added to the ethane and butane reactor (~1:4.5 and 1:11.3 of sludge to medium ratios, respectively), leaving a headspace of 0.3 and 0.46 l, respectively. The ethane/butane reactors were periodically flushed with pure ethane/butane gas (99.99%, Coregas, Australia) to maintain the ethane/butane partial pressure in the headspace between 0.9 and 1.2 atm. A concentrated stock solution (80 g NO₃⁻N l⁻¹) was manually pulse-fed to the reactors to replenish NO₃⁻ to 20–30 mg N l⁻¹. The bioreactors were continuously mixed using a magnetic stirrer (IKA, Labtek, Australia) at 650 rpm and operated in a thermostatic chamber (35 ± 1°C). Every 1–4 months, the stirrers were stopped for 24 h to allow biomass to settle, and the supernatant of 0.2–0.8 l was then replaced with fresh medium. The pH was manually adjusted to 6.8–7.5 using a 1 M anoxic HCl solution. Liquid samples (0.4–0.6 ml each) were collected periodically (2–5 samples per week) and filtered immediately using a 0.22 μm membrane filter (polyethersulfone filter, Millex, USA) for the analysis of NO₃⁻, NO₂⁻, and NH₄⁺. A gas sample (100 μl) from the headspace was withdrawn regularly (three to five times per week) using a gas-tight syringe (1710 SLSYR, Hamilton) for the determination of C₂H₆ and N₂.