The important role of amino acids in spore germination was established by Hills (1949) and Stewart and Halvorson (1953). Their studies, however, were limited to the aerobic forms because the anaerobes could not be made to produce sufficient quantities of spores in media within a specified time. Using an autoclaved 1 per cent solution of glucose phosphate, Wynne et al. (1954) germinated anaerobic spores in 1 hr, but rapid germination in a medium of well-defined nutrients has not been reported. The investigation reported here had three phases: (1) the development of media and methods for growing and harvesting anaerobic spores, (2) the determination of the optimum conditions and minimal nutritional requirements for their rapid germination, and (3) a study of the effect of inhibitors on germination. MATERIALS AND METHODS Organisms. Cultures of Clostridium butylicum strain 6014, Clostridium roseum strain 6012 (both of the American Type Culture Collection), and Clostridium acetobutylicum were tested for their ability to sporulate in various media and under different conditions. Stock cultures maintained on brain heart infusion medium (Difco) were used to inoculate 150 ml of clear sporulation medium in 6 oz bottles. The sporulation medium (TS-5) giving optimum spore production contained trypticase (BBL), 1.5 per cent; glucose, 0.2 per cent; sodium 1 This research was supported in part by Swift and Company, Office of Naval Research, and taken in part from a dissertation presented by the senior author in partial fulfillment of the requirements for the Ph.D.
Summary A microbial-enhanced waterflooding field project is being conducted in the Mink Unit of the Delaware-Childers field in Nowata County, OK. A microbial formulation was injected into four injection wells and has been followed by periodic injections of molasses as nutrient. Laboratory and field procedures that were used to design the test are described. Improvements in both oil production rate and WOR's have been observed. Introduction A microbial-enhanced waterflood field experiment was initiated during Oct. 1986 by the U.S. DOE, the Natl. Inst. for Petroleum and Energy Research (NIPER), Microbial Systems Corp., and Injectech Inc. The purpose of the project was to conduct and document a microbial-enhanced waterflooding field test using procedures that could be implemented easily by an independent petroleum producer. Laboratory studies were conducted to design a microbial formulation for the field and to conduct a field pilot test of microbial-enhanced waterflooding method. Field Data. The Mink Unit (Sec. 36, Twp. 27N, Rge. 16E) is located in the Delaware-Childers field in Nowata County, OK (see Fig. 1), and is producing oil from the Bartlesville sandstone formation. The Mink Unit covers a 60-acre [24-ha] area with 21 injection wells and 15 producing wells drilled on a 5-acre [2-ha] spacing. Only one of the producing wells is being pumped. Well completions are open hole. Table 1 lists average reservoir properties. The Mink Unit contains the Sallie and Candy Mink leases. Net pay thickness in the Mink Unit decreases from about 40 feet to less than 10 ft [12 to less than 3 m] in a northeasterly direction from the southwest comer of the unit. The original oil in place is estimated from historical oil production records to be 1,666,000 bbl [265 × 103 m3], of which 341,000 bbl [54 × 103 m3] had been produced as of the end of 1986. The remaining 1,325,000 bbl [211 X 103 m3] of oil in place in the 2,900 acre-ft [3.58 × 106 m3] of net pay yield an average oil saturation of about 40 bbl/acre-ft [0.6 m3/m3] (30%). The annual oil production rate from the Mink Unit has remained relatively constant since 1982. The stable oil production rate should provide a relatively reliable basis upon which to judge the effects of the microbial treatment. The pilot site for the project was four adjacent inverted five-spot patterns within the Mink Unit (see Fig. 2). The pilot site covers an area of 17 8 acres [7.2 ha] and a net pay volume of 516 acre-ft [636 × 103 m3]. The pilot area has four injection and eight production wells. In addition, two off-pattern wells (Wells C-BP-2 and S-AP-4) were also monitored as part of the test. All possible efforts were made to ensure that no changes in operating conditions or procedures were made during the pilot test. No workovers were performed during the test, and the normal procedure of backflushing all injection wells each week continued. Baseline Monitoring. Field sampling was initiated in Nov. 1986 and continued to March 17, 1987. The data obtained included total oil production (Mink Unit), total water production (Mink Unit), injection-well pressures and fluid rates from each well, and WOR's from each production well. Samples of produced fluids were collected each week and analyzed. Total dissolved solids (TDS); pH; trace mineral and ion analysis; microbial populations, including sulfate-reducing bacteria; and oil viscosity were determined for each sample. Table 2 gives the data from these baseline studies. The TDS of the produced water from each producer and the injection plant water remained constant to within +/-0.01 %. The pH of the samples remained between 6.4 and 7.0, and trace mineral and ion analyses indicated no marked changes in concentrations of sodium, calcium, magnesium, strontium, barium, carbonate, hydroxide, and phosphate ions. The microbial counts from the producing wells were consistent throughout the monitoring period (Table 3). The counts were very low, ranging from 0 to 100 colony-forming units per milliliter of water in the producing wells. Sulfate-reducing bacteria were consistently present at low levels in the tank battery water and intermittently present in the plant injection water. There were sporadic occurrences of sulfate-reducing bacteria in the produced waters. The field data during the baseline monitoring period remained consistent. Fig. 3 shows the oil production for the Mink Unit site. Single-well injection tests were performed in February 1987 to establish certain parameters before injection of the microbial system was initiated in the Mink site. An off-pattern injection well (Well S-DW-1; see Fig. 2) was injected with 100 L of microbial formulation (about 1 X 108 cells/mL; NIPER Bac 1) and shut in for 12 days. The well was backflushed and samples were collected every 10 to 15 minutes until microorganisms and molasses were detected. The injection rates and pressures after the shut-in period were normal, indicating that no plugging had occurred. All the injected microorganisms were detected in the backflush samples in high numbers, indicating that the microbes were still growing after 12 days of incubation under reservoir conditions.
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