Ecological pre-release risk assessment of two genetically engineered, bioluminescent Rhizobium meliloti strains in soil column model systems

In order to identify potential ecological risks associated with the environmental release of two Rhizobium meliloti strains, genetically engineered with the firefly-derived luciferase gene (luc), a pre-release greenhouse investigation was conducted. The upper 4 cm of soil columns (30 cm diameter; 65 cm depth), which were filled according to the horizons of an agricultural field (loamy sand), were inoculated with seeds of Medicago sativa (alfalfa) and R. meliloti cells at approximately 5x10⁶ cells-g^-1 soil. Four treatments were tested: inoculation with a non-engineered wild type strain (2011), strain L33 (luc⁺), strain L1(luc⁺, recA^-) and non-inoculated controls. The fate of the engineered strains was followed by two methods: (1) selective cultivation and subsequent detection of bioluminescent colonies and (2) PCR detection of the luc gene in DNA, directly extracted from soil. Strain R. meliloti L33 declined to 9.0x10⁴ cfu · g^-1 soil within 24 weeks and to 2.8x10³ cfu · g^-1 soil within 85 weeks in the upper 25 cm of the soil columns. Decline rates for R. meliloti L1 were not significantly different. Vertical distribution analysis of the recombinant cells after 37 weeks revealed that in three of four columns tested, the majority of cells (>98%) remained above 10 cm soil depth and no recombinant cells occurred below 20 cm depth. However, in one column all horizons below 20 cm were colonized with 2.2x10⁴ to 6.8x10⁴ cfu g^-1 soil. Ecological monitoring parameters included organic substance, total nitrogen, ammonium and nitrate, microbial biomass, culturable bacteria on four different growth media and the immediate utilization of 95 carbon sources (BiologGN) by soil-extracted microbial consortia. None of the parameters was specifically affected by the genetically engineered cells.