MICROBIOLOGICAL AND MOLECULAR BIOLOGICAL METHODS FOR MONITORING MICROBIAL INOCULANTS AND THEIR EFFECTS IN THE SOIL ENVIRONMENT

As the use of biotechnology products, such as genetically modified mic roorganisms (GMMs), in the environment might bring about undesirable e cological effects, it is important that the environmental fate of inoc ulant organisms, as well as any effects of their release, are assessed . Ideally, pilot studies in microcosms or small mesocosms are thus per formed prior to a larger-scale (commercial) application, and the resul ts of these studies serve to guide further environmental use of the GM M. In these pilot studies, the methods employed to assess the environm ental fate and effects of the GMM will have to be fine-tuned and optim ized so as to assess these phenomena in an optimal way. This review ex amines the methods that are currently available for the assessment of the environmental fate of genetically modified and unmodified microorg anisms, as well as the impact following their release. The emphasis wi ll be on monitoring of these phenomena in soil, as a paradigm of asses sments in complex environmental matrices. Detection methods based on c ultivation, which generally rely on the use of added or intrinsic mark ers, serve to assess the fate of the culturable fractions of released microorganisms. The specificity of this detection can be enhanced by u sing a combination of cultivation-based and immunology- and/or DNA-bas ed assessments. Furthermore. specific immunofluorescence or in situ hy bridization techniques are suitable to quantify populations of GMMs at the level of microscopy-detectable cells, even though fluorescent in situ hybridization is still plagued by sensitivity problems in oligotr ophic environments. Detection methods based on nucleic acids (DNA or R NA) extracted from the environment offer the possibility to monitor th e fate of the heterologous genes released, including that following a horizontal gene transfer. Moreover, these methods can also provide a p icture of the dynamics of the total numbers of microbial cells release d. For a sound assessment of the biosafety of environmental releases, a polyphasic approach to environmental monitoring is recommended, as i n most analyses information will be needed on the fate of culturable a nd nonculturable cells, as well as on that of the specific gene sequen ces released. Finally, the use of impact analysis via an array of diff erent methods is briefly reviewed, and the merits of novel approaches to assessments of community structure via molecular means (PCR/DGGE, A RDRA or T-RFLP) and metabolic profiling via Biolog are discussed as me thods specifically aimed at the detection of shifts in community struc ture and function (metabolic complement analysis). (C) 1998 Elsevier S cience B.V.