Pseudomonas aureofaciens in soil: survival and recovery efficiency

Efficient methods for the recovery of genetically engineered organisms (GEM) added to soil are critical if the safety of potential releases is to be evaluated and the actual release is to be monitored. Pseudomonas aureofaciens strain 3732 RN-L11 (lacZY) was added to 10 g sieved soil microcosms and incubated for 5 and 28 days. Various diluents, shaking methods, and settling of soil were examined to determine the optimum method for recovery of the GEM from the soil. Of the diluents examined, 0.1% agar gave significantly lower numbers than distilled water, 1.0% sodium metaphosphate, 1% peptone, and phosphate-buffered water. After 5 days of incubation, shaking for 10 min with glass beads and shaking for 30 min without glass beads resulted in the highest recovery of the GEM from soil, while sonification resulted in the lowest recovery. After 28 days of incubation, sonification produced significantly lower numbers than any of the other treatments. The addition of 1% CaCl2 to enhance settling significantly increased recovery efficiency. Although the use of CaCl2 in distilled water and shaking for 10 min was an effective method for recovering P. aureofaciens from a Maryland soil, when the same extraction procedure was compared with a standard technique (dd H2O, shaking for 10 min) for eight divergent soils, neither extraction method was consistently better than the other. Statistical analysis of the data showed the need for log transformation of the raw data. Four microcosm and two plate replicates for each dilution provided the greatest ability to detect differences between treatment means while maximizing experimental efficiency.