Enhanced bioremediation of soil containing 2,4-dinitrotoluene by a genetically modified Sinorhizobium meliloti

The symbiotic nitrogen-fixing soil bacterium, Sinorhizobium meliloti, is well known for its ability to interact with the leguminous plant Medicago sativa L. It has, however, not been reported that this species possesses the capability to degrade toxic nitroaromatic compounds, such as 2,4-dinitrotoluene (DNT) which is commonly associated with the degradation of the explosive trinitrotoluene (TNT). In this study, the pJS1 DNT-biodegradative plasmid was genetically transferred to S. meliloti strain USDA 1936, which was confirmed by plasmid profile analysis. Several standard analytical and chemical tests including high performance liquid chromatography (HPLC), nitrite (NO₂) release assays, rhizosphere population and plant greenhouse studies were conducted to test the ability of S. meliloti to degrade 2,4-DNT. The possible presence of 2,4-DNT remaining in the treated soil was tested, and no 2,4-DNT had been absorbed by the soil. The pJS1-carrying recombinant strain DHK1 produced ‘ARC’ alfalfa plants that were almost 2-fold higher in shoot dry weight than that produced by the parent strain on soil containing 0.14 mM 2,4-DNT. The transconjugant strain DHK1 reduced significantly one-third more 2,4-DNT in both 0.14 and 0.28 mM contaminated soil, and in 0.55 mM contaminated soil it degraded 94% of the 2,4-DNT present. In liquid cultures, however, only about 4% reduction in 2,4-DNT concentrations was obtained in 10 days. We interpret the results as clearly establishing that genetic modification was successfully used, for the first time, to improve the capability of the symbiotic nitrogen-fixing soil bacterium S. meliloti DHK1 to bioremediate in situ 2,4-DNT-contaminated soil in the presence of alfalfa plants.     

The role of laboratory, glasshouse and field scale experiments in understanding the interactions between genetically modified crops and soil ecosystems: A review of the ECOGEN project

The interactions of genetically modified (GM) crops with soil species and ecosystems is complex, requiring both specific and broad spectrum assessments. In the ECOGEN project we undertook experiments at three scales of increasing complexity, using Bt maize expressing the Cry1Ab protein from Bacillus thuringiensis as an example. Test species were selected for laboratory-scale experiments to represent taxonomic groups that we could also monitor at glasshouse and field scales (e.g., nematodes, protozoa, micro-arthropods, earthworms, and snails). In the laboratory, single species were exposed to purified Cry1Ab protein or to Bt maize leaf powder incorporated into simplified diets under controlled conditions. In the glasshouse, multiple test species and soil microbial communities taken from ECOGEN's field sites were exposed to Bt maize plants growing under glasshouse or mesocosm conditions. In the field, evaluations were conducted on our selected indicator groups over multiple sites and growing seasons. Field evaluation included assessment of effects due to the local environment, crop type, seasonal variation and conventional crop management practice (tillage and pesticide use), which cannot be assessed in the glasshouse. No direct effects of Cry1Ab protein or Bt leaf residues were detected on our laboratory test organisms, but some significant effects were detected in the glasshouse. Total nematode and protozoan numbers increased in field soil under Bt maize relative to conventional maize, whilst microbial community structure and activity were unaffected. Field results for the abundance of nematodes and protozoa showed some negative effects of Bt maize, thus contradicting the glasshouse results. However, these negative results were specific to particular field sites and sampling times and therefore were transient. Taking the overall variation found in maize ecosystems at different sites into account, any negative effects of Bt maize at field scale were judged to be indirect and no greater than the impacts of crop type, tillage and pesticide use. Although the ECOGEN results were not predictive between the three experimental scales, we propose that they have value when used with feedback loops between the scales. This holistic approach can used to address questions raised by results from any level of experimentation and also for putting GM crop risk:benefit into context with current agricultural practices in regionally differing agro-ecosystems.