High speed centrifuge

Approximate 4–5 L mixed oil/water liquid from the ASP-flooded production wells and 2–3 L from the water-flooded production wells were centrifuged at 4°C and 12000 × g for 20 min in a high-speed centrifuge (Beckman, United States) to pellet microbial cells. Total genomic DNA was extracted by a bead shaker treatment combined with AxyPrep^TM Genomic DNA Miniprep Kit (United States) as previously described (Gao et al., 2016 (link)).

GC cell-derived exosomes were extracted by differential centrifugation. All centrifugation steps were performed at 4°C, and other steps were performed on ice. In short, cell debris and dead cells were removed following centrifugation at 500 g for 10 min and at 2,000 g for 20 min in a cryogenic centrifuge (Eppendorf, Germany). The supernatant was centrifuged at 110,000 g for 45 min in a high-speed centrifuge (Beckman, USA) to remove cell debris, large cells and extracellular vesicles. The supernatant was filtered using a 0.22 μm filter. Next, the exosomes were isolated at 110,000 g for 90 min in an ultracentrifuge (Beckman, USA). The collected pellets were resuspended in phosphate buffered saline (PBS) and repeatedly centrifuged once. Finally, the pellets were resuspended in 50–100 μL PBS and stored at −80°C for subsequent use. The exosome markers TSG101, CD63 and Alix were measured by Western blot analysis.

Samples were collected between July 2011 and September 2012 from the wellheads of the production wells in each reservoir block. Sterilized plastic bottles (15 L) were completely filled, then immediately capped and sealed to avoid contamination and oxygen intrusion. The residual oil in each sample was firstly removed by heating the sample to 60 °C for 15 min and conducting phase separation in sterilized separatory funnels. Microbial cells were then collected from the 5 L water sample by centrifugation (12,000 × g) at 4 °C for 15 min, in a high-speed centrifuge (Beckman, USA). Total genomic DNA was extracted from the cell deposits using methods previously described15 (link). The detailed process is described in the Supplementary text.

The EE of RLX/NRG NLCs was estimated by separation of un-entrapped RLX and NRG from RLX/NRG NLCs using the centrifugation process as described in [44 (link)]: 2 mL of the NLCs loaded with drugs were centrifuged for 40 min at 15,000 rpm using a high-speed centrifuge (Beckman Coulter India Pvt. Ltd., Mumbai, India). Subsequently, separation of the supernatant layer was carefully followed by proper methanol dilution and filtration through a syringe membrane filter (0.45 μm). Then, the filtrated solution was subjected to drug estimation for free RLX and NRG spectrophotometrically (Shimadzu, Japan). The EE was computed using the following equation: $$\mathrm{Entrapment} \mathrm{efficiency} \left(\mathrm{EE}\%\right)=\frac{ {\left[\mathrm{D}\right]}_{ \left[\mathrm{Total}\right]}-{\left[\mathrm{D}\right]}_{\left[\mathrm{Free}\right]} }{{\left[\mathrm{D}\right]}_{\left[\mathrm{Total}\right]}}\times 100$$
where [D]_Total is the initial weight of the RLX and NRG added to the formulation and [D]_Free is the weight of free unloaded RLX and NRG detected in the supernatant layer. Therefore, we consider that all the RLX and NRG not present in the supernatant layer were successfully loaded in the lipid matrix of the NLCs [45 (link)].

Crude bacteriophage lysates (~5×10¹⁰ PFU/mL) were filter-sterilized using a 0.22-µm membrane (Millipore, Waltham, MA, USA) and then pelleted at 25,000g for 1 h at 4°C using a high-speed centrifuge (Beckman Coulter, Palo Alto, CA, USA). The bacteriophage pellet was resuspended in 150 μl of SM-buffer supplemented with CaCl₂ (5 mM) after washing twice in a neutral solution of ammonium acetate (0.1 M). Bacteriophage particles were deposited onto a carbon-coated Formvar film on copper grids and stained with 20 μl of 2% potassium phosphotungstate (pH 7.2). After dye removal with filter paper, bacteriophage particles were examined under a transmission electron microscope (TECNAI 12; FEI, Hillsboro, OR, USA) at 120 kEv. Images were collected and analyzed using Digital Micrograph™ (Gatan, Pleasanton, CA, USA). Taxonomic assignments were made according to the classification scheme for bacteriophages developed by Ackermann and Berthiaume (Berthiaume and Ackermann, 1977 (link)) and the International Committee on the Taxonomy of Viruses.

Production mixed oil/water samples were collected directly from each wellhead of P140, P141 and P40 in July 2015, March 2016 and December 2016, while samples were collected from the zone near the downhole of the production wells P140 and P141 in July 2015 and March 2016 by field personnel from PetroChina. The samples from the downhole of the injection wells were obtained by water backflow. A total of 13 samples were collected. To avoid contamination and oxygen intrusion, the samples were used to fill 10 L sterilised plastic bottles, which were immediately sealed with screw caps. The bottles were then transported to the laboratory as soon as possible for further analysis. After the oil and water phases were separated, 5 L of each water sample was centrifuged at 4°C for 15 min at 10,000 × g in a high‐speed centrifuge (Beckman, Pasadena, CA) to collect microbial cells, and 100 mL of water was used for chemical analysis. Total genomic DNA was extracted from the cell deposits using methods previously described (Gao et al., 2015 ).