with only soil and used lubricating oil. Also, an additional control (comprising of autoclaved soil and
0.5% (w/w) NaN3) in vessel A was set up to observe the degradation of lubricating oil in the soil due to
non-biological factors. The moisture content was adjusted to 60% by the addition of water, and the
content was tilled for aeration three times a week throughout the experimental period. The plastic vessels
were incubated at room temperature (30 ± 2q
C) in triplicates.
2.3. Determination of total petroleum hydrocarbon
Anhydrous sodium sulphate (Na2SO4) was added to soil samples to absorb soil moisture. Hydrocarbon
content of the soil samples was determined by suspending 10g of soil sample in 20ml of toluene (AnaLar
grade) in a 250ml flask. After shaking for 60 minutes on an orbital shaker (model N-Biotek-101M), at
200 RPM, the liquid phase of the extract was measured at 420nm using DR/4000 Spectrophotometer. The
total petroleum hydrocarbon (TPH) in soil was estimated using the standard curve derived from fresh
used engine oil diluted with toluene. TPH data was fitted to first-order kinetics model [20] with,