# A Novel Approach to Enhance Crude Oil Recovery Ratio Using Selected Bacterial Species
## 2 Materials and Methods
### Sand-Packed Column Experiment  
Five core samples and four sand packs were collected from the Sirte Basin and formation type (Dolomite), Concession 11 (Alghni Field, northeast of Libya), sand-packed model (4 cm in diameter; and 30 cm in length) were prepared with different grain size. The oil sample was heated to 65 \({}^{\circ}\)C for 960 h. K = 200 psi net confining stress, Q = 100 psi Helium. The dolomite sandstone core plugs were drilled in horizontal and vertical directions using a diamond core bit with water as the bit coolant and lubricant. The plug sample dolomite sandstone cores (absolute permeability 230 \(\times\) 10\({}^{-2}\)\(\mu\)m\({}^{2}\)) were extracted of hydrocarbons using toluene, leached of salt using methanol, and oven dried at 80 \({}^{\circ}\)C. A silver nitrate solution was used to ensure the removal of salts present in the pore spaces. The clean, dry samples were subjected to various analyses to determine permeability, porosity, and grain density values, and were saturated with filtered, sterilized formation water for 12 h in a desicator under vacuum. The formation water was collected from one of the Libyan oil fields. The experimental procedures and equipment specifications were tested according to API RP 40 (recommended practice for core analysis).  
A model of an oil-saturated dolomite formation column was used to mimic the reservoir condition. The crashed dolomite cores saturated with Bouri oil were placed in the packed column apparatus and heated in the oven at 50 \({}^{\circ}\)C, then were saturated with 5-6 pore volume formation water until no further oil was produced. The pore volume (PV) was calculated as the difference in the wet and dry weights of the core. The residual oil was calculated by measuring the amount of oil produced from water flooding. Then, 5-6 PV heavy crude oil was injected until no more water was produced (i.e., it reached the irreducible water saturation, (Swr)). The initial oil saturation of the cores was calculated volumetrically from the amount of injected oil and produced water. Further extra oil recovery was determined by injected 5-6 PV of the bacterial culture as a tertiary recovery.  
The 24 h-grown isolates in Luria Bertani broth (LB) served as the inoculum for the packed column experiment, (OD600 = 0.764; 32.8 \(\times\) 10\({}^{7}\) CFU/mL for _Nocardiia cylari-georgica_, OD600 = 1.88; 21.8 \(\times\) 10\({}^{7}\) CFU/mL for _Bacillus_ species, and OD600 = 1.734; 47.3 \(\times\) 10\({}^{7}\) CFU/mL for _Pseudomonas putida_). The bacterial inoculum was injected into the core and the column model was incubated for 12 weeks at 55 \({}^{\circ}\)C. A control experiment was performed without injection of the isolates at the same conditions. Core columns were incubated under anaerobic conditions and simulated reservoir conditions (pressure, temperature 55 \({}^{\circ}\)C, and flow rate).  
Samples were taken to measure the viscosity reduction and oil recovery. The residual crude heavy oil in the core was measured from the volume of oil produced. The extra recovered oil was collected and measured using graduated tubes, and then analyzed by GC-FID for determining the biotransformation of heavy crude oil. The pH of the aqueous phase in the packed column was also measured before and during the incubation period.