CONTRATADOS
GIULIETTI Ana Maria
congresos y reuniones científicas
Título:
Analysis of the influence of plant species and rhizosphere organisms on herbicide metabolism
Autor/es:
CUADRADO,V.; MERINI, L.J.; FLOCCO, C.G; GIULIETTI A M
Lugar:
Gotenburgo
Reunión:
Workshop; 6th ACCESS meeting; 2005
Resumen:
The primary objective of ACCESS has been to generate and establish the knowledge base for the eco-engineering of soil ecosystems polluted with herbicides, atrazine, 2,4-D, and their derivatives. This was proposed to be achieved by 1) the detailed physical, chemical and microbiological characterization of herbicide contaminated agricultural soils; 2) the design and optimization of molecular methods to study changes in gene expression of the responses to stress in microbial isolates and communities, the changes in diversity, structure and catabolic performance of complex microbial communities; 3) extension of the knowledge base of microbial capabilities for degrading the target compounds and of the influences on degradation through community interactions; 4) evaluation, with newly developed tools and methods, of the factors that positively affect biodegradation by microbial communities in microcosms of increasing complexity, including plant-microbe interactions and 5) design of knowledge based farming practices to more actively reduce pollution caused by organochlorine herbicides. Two experimental areas located in Chile and Argentina and treated with triazine or chlorophenoxy herbicides were chosen based on the applied agricultural practice. Monitoring tools were optimized with a particular emphasis on molecular biology diagnostics. DNA extraction procedures were adapted to the soil types under study and a new RNA extraction method for agricultural soils rich in humic acids was developed. Catabolic gene profiling methods were developed to assess their diversity under changing environmental conditions. To establish the bottlenecks that hinder expression of herbicide-degrading genes in strains in soil environments, two dedicated DNA arrays were developed and used for examining the stress caused by substrate addition on the general physiological status of P. putida and C. necator. We assessed how cells respond to potential carbon sources which, simultaneously, are noxious compounds for cell survival, how expression of catabolic genes is influenced by the simultaneous exposure of the cells to nutritional and abiotic stresses and how bacteria assign a share of the transcriptional machinery to meet either nutritional demands or to endure environmental stresses. Our experiments show that in P. putida a mixture of possible toxic growth substrates is sensed, primarily, as an environmental threat and not as a potential nutrient. The delay in catabolic gene expression reflects a genuine hierarchy of transcriptional responses and shows that cells cope first with environmental stress and only then do they activate a metabolic program for dealing with the nutrients-to-be. The advanced molecular diagnostic tools, in combination with accurate analytic determinations were applied to analyze different experimental systems, ranging from pure cultures to consortia, soil microcosms, including plant microcosms and field studies and used to evaluate the effects of environmental stress factors and specifically herbicides on microbial community composition and degradative performance. New isolates degrading the target herbicides were obtained. Specificall,y simazine degrading isolates were shown to differ in their regulation pattern from previously described strains, making them promising candidates for bioaugmentation. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by catabolic gene and expression analyses, which showed a high natural attenuation potential in most of the soils analyzed. The high natural attenuation potential was validated by field studies, wherein rapid degradation of herbicides was observed due to the relatively high abundance of herbicide degraders and catabolic genes involved in herbicide metabolism. However, the soils differed significantly in their resistance to higher loads of herbicides, as well as speed of adaptation and metabolic flux and microcosm experiments indicated significant shifts in catabolic gene abundance, composition and expression during metabolism of the herbicides. In the case of 2,4-D degradation, both the monooxygenolytic pathway and the dioxygenolytic pathway responded to herbicide stress and new subfamilies of genes belonging to both classes were identified in the soils, evidencing the broad bacterial diversity and flexibility. In the case of simazine degradation, the archetype atz genes were of major importance. However, different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by catabolic gene and expression analyses, which showed a high natural attenuation potential in most of the soils analyzed. The high natural attenuation potential was validated by field studies, wherein rapid degradation of herbicides was observed due to the relatively high abundance of herbicide degraders and catabolic genes involved in herbicide metabolism. However, the soils differed significantly in their resistance to higher loads of herbicides, as well as speed of adaptation and metabolic flux and microcosm experiments indicated significant shifts in catabolic gene abundance, composition and expression during metabolism of the herbicides. In the case of 2,4-D degradation, both the monooxygenolytic pathway and the dioxygenolytic pathway responded to herbicide stress and new subfamilies of genes belonging to both classes were identified in the soils, evidencing the broad bacterial diversity and flexibility. In the case of simazine degradation, the archetype atz genes were of major importance. However, different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by catabolic gene and expression analyses, which showed a high natural attenuation potential in most of the soils analyzed. The high natural attenuation potential was validated by field studies, wherein rapid degradation of herbicides was observed due to the relatively high abundance of herbicide degraders and catabolic genes involved in herbicide metabolism. However, the soils differed significantly in their resistance to higher loads of herbicides, as well as speed of adaptation and metabolic flux and microcosm experiments indicated significant shifts in catabolic gene abundance, composition and expression during metabolism of the herbicides. In the case of 2,4-D degradation, both the monooxygenolytic pathway and the dioxygenolytic pathway responded to herbicide stress and new subfamilies of genes belonging to both classes were identified in the soils, evidencing the broad bacterial diversity and flexibility. In the case of simazine degradation, the archetype atz genes were of major importance. However, different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. growth substrates is sensed, primarily, as an environmental threat and not as a potential nutrient. The delay in catabolic gene expression reflects a genuine hierarchy of transcriptional responses and shows that cells cope first with environmental stress and only then do they activate a metabolic program for dealing with the nutrients-to-be. The advanced molecular diagnostic tools, in combination with accurate analytic determinations were applied to analyze different experimental systems, ranging from pure cultures to consortia, soil microcosms, including plant microcosms and field studies and used to evaluate the effects of environmental stress factors and specifically herbicides on microbial community composition and degradative performance. New isolates degrading the target herbicides were obtained. Specificall,y simazine degrading isolates were shown to differ in their regulation pattern from previously described strains, making them promising candidates for bioaugmentation. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by catabolic gene and expression analyses, which showed a high natural attenuation potential in most of the soils analyzed. The high natural attenuation potential was validated by field studies, wherein rapid degradation of herbicides was observed due to the relatively high abundance of herbicide degraders and catabolic genes involved in herbicide metabolism. However, the soils differed significantly in their resistance to higher loads of herbicides, as well as speed of adaptation and metabolic flux and microcosm experiments indicated significant shifts in catabolic gene abundance, composition and expression during metabolism of the herbicides. In the case of 2,4-D degradation, both the monooxygenolytic pathway and the dioxygenolytic pathway responded to herbicide stress and new subfamilies of genes belonging to both classes were identified in the soils, evidencing the broad bacterial diversity and flexibility. In the case of simazine degradation, the archetype atz genes were of major importance. However, different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by catabolic gene and expression analyses, which showed a high natural attenuation potential in most of the soils analyzed. The high natural attenuation potential was validated by field studies, wherein rapid degradation of herbicides was observed due to the relatively high abundance of herbicide degraders and catabolic genes involved in herbicide metabolism. However, the soils differed significantly in their resistance to higher loads of herbicides, as well as speed of adaptation and metabolic flux and microcosm experiments indicated significant shifts in catabolic gene abundance, composition and expression during metabolism of the herbicides. In the case of 2,4-D degradation, both the monooxygenolytic pathway and the dioxygenolytic pathway responded to herbicide stress and new subfamilies of genes belonging to both classes were identified in the soils, evidencing the broad bacterial diversity and flexibility. In the case of simazine degradation, the archetype atz genes were of major importance. However, different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by catabolic gene and expression analyses, which showed a high natural attenuation potential in most of the soils analyzed. The high natural attenuation potential was validated by field studies, wherein rapid degradation of herbicides was observed due to the relatively high abundance of herbicide degraders and catabolic genes involved in herbicide metabolism. However, the soils differed significantly in their resistance to higher loads of herbicides, as well as speed of adaptation and metabolic flux and microcosm experiments indicated significant shifts in catabolic gene abundance, composition and expression during metabolism of the herbicides. In the case of 2,4-D degradation, both the monooxygenolytic pathway and the dioxygenolytic pathway responded to herbicide stress and new subfamilies of genes belonging to both classes were identified in the soils, evidencing the broad bacterial diversity and flexibility. In the case of simazine degradation, the archetype atz genes were of major importance. However, different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. are noxious compounds for cell survival, how expression of catabolic genes is influenced by the simultaneous exposure of the cells to nutritional and abiotic stresses and how bacteria assign a share of the transcriptional machinery to meet either nutritional demands or to endure environmental stresses. Our experiments show that in P. putida a mixture of possible toxic growth substrates is sensed, primarily, as an environmental threat and not as a potential nutrient. The delay in catabolic gene expression reflects a genuine hierarchy of transcriptional responses and shows that cells cope first with environmental stress and only then do they activate a metabolic program for dealing with the nutrients-to-be. The advanced molecular diagnostic tools, in combination with accurate analytic determinations were applied to analyze different experimental systems, ranging from pure cultures to consortia, soil microcosms, including plant microcosms and field studies and used to evaluate the effects of environmental stress factors and specifically herbicides on microbial community composition and degradative performance. New isolates degrading the target herbicides were obtained. Specificall,y simazine degrading isolates were shown to differ in their regulation pattern from previously described strains, making them promising candidates for bioaugmentation. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by catabolic gene and expression analyses, which showed a high natural attenuation potential in most of the soils analyzed. The high natural attenuation potential was validated by field studies, wherein rapid degradation of herbicides was observed due to the relatively high abundance of herbicide degraders and catabolic genes involved in herbicide metabolism. However, the soils differed significantly in their resistance to higher loads of herbicides, as well as speed of adaptation and metabolic flux and microcosm experiments indicated significant shifts in catabolic gene abundance, composition and expression during metabolism of the herbicides. In the case of 2,4-D degradation, both the monooxygenolytic pathway and the dioxygenolytic pathway responded to herbicide stress and new subfamilies of genes belonging to both classes were identified in the soils, evidencing the broad bacterial diversity and flexibility. In the case of simazine degradation, the archetype atz genes were of major importance. However, different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by catabolic gene and expression analyses, which showed a high natural attenuation potential in most of the soils analyzed. The high natural attenuation potential was validated by field studies, wherein rapid degradation of herbicides was observed due to the relatively high abundance of herbicide degraders and catabolic genes involved in herbicide metabolism. However, the soils differed significantly in their resistance to higher loads of herbicides, as well as speed of adaptation and metabolic flux and microcosm experiments indicated significant shifts in catabolic gene abundance, composition and expression during metabolism of the herbicides. In the case of 2,4-D degradation, both the monooxygenolytic pathway and the dioxygenolytic pathway responded to herbicide stress and new subfamilies of genes belonging to both classes were identified in the soils, evidencing the broad bacterial diversity and flexibility. In the case of simazine degradation, the archetype atz genes were of major importance. However, different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by catabolic gene and expression analyses, which showed a high natural attenuation potential in most of the soils analyzed. The high natural attenuation potential was validated by field studies, wherein rapid degradation of herbicides was observed due to the relatively high abundance of herbicide degraders and catabolic genes involved in herbicide metabolism. However, the soils differed significantly in their resistance to higher loads of herbicides, as well as speed of adaptation and metabolic flux and microcosm experiments indicated significant shifts in catabolic gene abundance, composition and expression during metabolism of the herbicides. In the case of 2,4-D degradation, both the monooxygenolytic pathway and the dioxygenolytic pathway responded to herbicide stress and new subfamilies of genes belonging to both classes were identified in the soils, evidencing the broad bacterial diversity and flexibility. In the case of simazine degradation, the archetype atz genes were of major importance. However, different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. growth substrates is sensed, primarily, as an environmental threat and not as a potential nutrient. The delay in catabolic gene expression reflects a genuine hierarchy of transcriptional responses and shows that cells cope first with environmental stress and only then do they activate a metabolic program for dealing with the nutrients-to-be. The advanced molecular diagnostic tools, in combination with accurate analytic determinations were applied to analyze different experimental systems, ranging from pure cultures to consortia, soil microcosms, including plant microcosms and field studies and used to evaluate the effects of environmental stress factors and specifically herbicides on microbial community composition and degradative performance. New isolates degrading the target herbicides were obtained. Specificall,y simazine degrading isolates were shown to differ in their regulation pattern from previously described strains, making them promising candidates for bioaugmentation. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by catabolic gene and expression analyses, which showed a high natural attenuation potential in most of the soils analyzed. The high natural attenuation potential was validated by field studies, wherein rapid degradation of herbicides was observed due to the relatively high abundance of herbicide degraders and catabolic genes involved in herbicide metabolism. However, the soils differed significantly in their resistance to higher loads of herbicides, as well as speed of adaptation and metabolic flux and microcosm experiments indicated significant shifts in catabolic gene abundance, composition and expression during metabolism of the herbicides. In the case of 2,4-D degradation, both the monooxygenolytic pathway and the dioxygenolytic pathway responded to herbicide stress and new subfamilies of genes belonging to both classes were identified in the soils, evidencing the broad bacterial diversity and flexibility. In the case of simazine degradation, the archetype atz genes were of major importance. However, different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. different regulatory regimes in different bacteria are responsible for adaptability. The influence of plants was generally of minor importance, however, the herbicides were rapidly degraded in a reasonable time and in a safe way. Bioaugmentation studies were carried out at different scales and usually the natural attenuation potential was increased only slightly. One major reason for the failure of bioaugmentation could be attributed to specific grazing, which can be rapidly followed by molecular tools. Our results also indicate that a detailed knowledge of the regulatory net of bacteria is a prerequisite for successful bioaugmentation, as is the case specifically for organisms degrading simazine and related nitrogen containing herbicides. Overall, the project developed a variety of new molecular tools and a multifaceted approach over different scales of complexity generated a knowledge base which can be used for the ecoengineering of sites polluted with atrazine, 2,4-D, and their derivatives. Soil microcosms of different complexity were shown to be useful tools for analyzing the degradation of herbicides, the effectiveness of bioaugmentation procedures, the catabolic potential of microbial communities and the changes in bacterial communities after herbicide exposure. Community complexity was evaluated at three levels, i.e., through cloned sequence libraries, DNA fingerprinting methods as well as statistical analysis of T-RFLP fingerprints. Whereas the first methods gave indications of the presence of various bacteria with relatively distant relationships to described bacteria and possibly currently unknown bacterial species with unknown metabolisms, the second set of methods allowed gross analyses of differences in community composition and indicated a surprisingly high abundance of Archaea, whereas statistical evaluation of fingerprints enabled a general evaluation of the effects of different stress situations on the overall community structure. More detailed insights in the soils analyzed at different scales were obtained by ca