2.2. Microcosm experiment design

Soil microcosms were established using 700 g of either soil A and B in sterile 1 L, wide-mouth amber glass jars. Four different microcosm conditions for each soil were established and tested in triplicate (Table 1). Soil grinding was done using mortars and pestles made from hard chemical-porcelain ware. The mortars had a lip and were glazed on the outside. The pestles were glazed to the grinding surface. Soil aliquots of about 70 g were ground for about 15 min in the mortar with the pestle to pass the soil through a 42.5 μm sieve. The ground soil aliquots were then combined for additional sample preparation. Nutrients were added in the form of ammonium nitrate and potassium orthophosphate to obtain a C:N:P ratio of 100:1:0.1. The hydrocarbon-degrading inoculum was composed of three bacterial isolates supplied by Remedios Limited (Aberdeen). The bacterial isolates were isolated from an attenuated enrichment culture from No.6 oil impacted soil (Alzahrany, 2011). Two bacterial isolates were related to Pseudomonas sp. (100% match) and one to Klebsiella sp. (99% match). Inoculum was grown using a minimal medium supplemented with diesel (equivalent to 5.5 mg C l⁻¹) as carbon source. The cell concentration added to each microcosm was such as to give 5 × 10⁷ CFU g⁻¹ soil. For each amendment, a few woodchips were added to 10 ml of Bushnell-Haas broth supplemented with 1 g l⁻¹ salicylic acid and 1% ethanol, adjusted to pH 7. The mixture was placed in an orbital shaker at 150 rpm in the dark at 20 °C and left overnight, after which 1 ml was added to 100 ml of fresh medium and grown to a stationary phase (about 24 h, checked by optical density readings at 600 nm). The cell number at stationary phase was 10⁸ cells ml⁻¹. The inoculum solution was then added to the soils at 0.01 ml g⁻¹ dry wt soil to achieve 10⁶ cells g⁻¹ dry wt soil. The moisture content of each microcosm was adjusted to 80% of the soil’s water holding capacity using deionised water. The microcosms were incubated in the dark at 15 °C. High humidity was maintained using damp cotton wool and moisture checked periodically. Each microcosm was mixed weekly and capped loosely to allow oxygen transfer. Soil from each microcosm was sampled at 0, 7, 14, 28, 56, and 112 days for subsequent microorganism respiration monitoring and hydrocarbon analysis.

Microcosm experiment setup.