Final Report Summary - BIOTOOL (Biological procedures for diagnosing the status and predicting evolution of polluted environments)
The BIOTOOL project aimed to assess, evaluate and predict natural attenuation processes in order to implement natural attenuation as a principal soil and groundwater remediation strategy in Europe. Therefore, innovative monitoring tools, focusing on biological processes, were developed and assessed on environmental samples and sites. Moreover, protein biomarkers were identified as descriptors of soil and groundwater conditions and biological attenuation. Finally, the project established tree core analyses as an innovative method for the rapid monitoring of subsurface contamination and biodegradation, through exploitation of the qualitative relationship between soil, groundwater and plant contamination.
BIOTOOL specific objectives were to:
1. establish the correlation between soil and groundwater contamination and plant contamination;
2. design and utilise deoxyribonucleic acid (DNA) technology to examine the catabolic potential of any given particulate sample;
3. access and analyse the soil and groundwater meta-proteome as a biomarker;
4. employ lipid biomarkers as general prediction instruments of stress and toxicity on soil and groundwater organisms;
5. elucidate the roles of natural and chemical stress and plant and microbe interactions on the metabolic activities of soil and groundwater microbial catalysts;
6. validate the robustness of the developed diagnostic instruments and utilise the proposed technology for assessment of contaminated sites.
The selected experimental sites were polluted with chlorinated or petroleum hydrocarbons and were characterised in detail regarding their geological and hydrogeological characteristics. Their evolution was closely monitored throughout the project by application of adequate techniques. The transport of indicator chemicals in trees was monitored through a mathematical model which described uptake, translocation and volatilisation of chemicals. The model was verified using laboratory and field data. The model was also adapted to simulate herbs and grass; nevertheless this type of vegetation was not suitable for indicators' monitoring. The correlation between trees and subsurface pollution was verified, but was exploitable for pollutants' identification and not for determination of their subsurface concentration. The method was highly successful in monitoring chloroethene in shallow groundwater aquifers, thus relevant guidelines were developed.
Analysis of different experimental systems resulted in the conclusion that fatty acids formed an excellent toxicity tool for laboratory experiments; however, they represented an urgent response system and could not be utilised in real bioremediation plants where long term adaptation mechanisms were evolving. The phospholipid fatty acids (PLFA) profile appeared to be a more reliable technique in the field; hence, a method for rapid extraction of lipids from soil samples was developed.
In addition, DNA array technology was used to decipher the interplay between expression of catabolic genes and stress caused on a model soil bacterium. Extensive literature review resulted in the selection and evaluation of eleven gene families which catalysed key steps of aerobic and anaerobic catabolic pathways. The proposed method increased available knowledge on the functional potential of microbial communities and helped to identify shifts in the gene structure that allowed for the deduction of the evolutionary fitness of the genes under changing environmental conditions. The use of genomic libraries enabled to retrieve genes from natural bacterial communities without cultivation and was combined with a generic approach which translated biotransformations lacking easily observable phenotypes into traits that could be selected.
The complexity and dynamics of natural ecosystems imposed the investigation of the interactions between microbial communities and the environment in bioremediation and natural attenuation scenarios. Thus, BIOTOOL utilised state-of-the-art proteomic technologies to locate protein biomarkers, which were descriptive of the soil status and predictive of its evolution. The potential of camel antibody technology for production of large numbers of antibodies which recognised specifically given catabolic enzymes was pinpointed, though a soil protein extraction procedure able to reveal the biodegradative landscape was yet to be established.
A bioinformatics system was also developed, in order to organise and maintain heterogeneous information related to biodegradation. In addition, a machine learning approach was proposed in order to identify the degradation potential of new compounds based on their chemical descriptors. The system could assist the implementation of recent international regulations on the use of new chemicals.
BIOTOOL resulted in numerous exploitable outcomes. Protocols for the catabolic genes fingerprinting processes, which could be utilised to define degradation in sites of interest, were available. In addition, the tree core sampling approach was highly recommended for monitoring chloroethene in shallow groundwater aquifers and relevant knowledge was broadly disseminated. Moreover, lipids could be utilised for rapid monitoring of microbial communities and protocols were developed for that purpose. The designed DNA extraction kit was at commercial exploitation stage, as well as the proposed biodegradation prediction server. Finally, genetic tools were constructed to allow for the identification of enzymes catalysing novel reactions.
BIOTOOL specific objectives were to:
1. establish the correlation between soil and groundwater contamination and plant contamination;
2. design and utilise deoxyribonucleic acid (DNA) technology to examine the catabolic potential of any given particulate sample;
3. access and analyse the soil and groundwater meta-proteome as a biomarker;
4. employ lipid biomarkers as general prediction instruments of stress and toxicity on soil and groundwater organisms;
5. elucidate the roles of natural and chemical stress and plant and microbe interactions on the metabolic activities of soil and groundwater microbial catalysts;
6. validate the robustness of the developed diagnostic instruments and utilise the proposed technology for assessment of contaminated sites.
The selected experimental sites were polluted with chlorinated or petroleum hydrocarbons and were characterised in detail regarding their geological and hydrogeological characteristics. Their evolution was closely monitored throughout the project by application of adequate techniques. The transport of indicator chemicals in trees was monitored through a mathematical model which described uptake, translocation and volatilisation of chemicals. The model was verified using laboratory and field data. The model was also adapted to simulate herbs and grass; nevertheless this type of vegetation was not suitable for indicators' monitoring. The correlation between trees and subsurface pollution was verified, but was exploitable for pollutants' identification and not for determination of their subsurface concentration. The method was highly successful in monitoring chloroethene in shallow groundwater aquifers, thus relevant guidelines were developed.
Analysis of different experimental systems resulted in the conclusion that fatty acids formed an excellent toxicity tool for laboratory experiments; however, they represented an urgent response system and could not be utilised in real bioremediation plants where long term adaptation mechanisms were evolving. The phospholipid fatty acids (PLFA) profile appeared to be a more reliable technique in the field; hence, a method for rapid extraction of lipids from soil samples was developed.
In addition, DNA array technology was used to decipher the interplay between expression of catabolic genes and stress caused on a model soil bacterium. Extensive literature review resulted in the selection and evaluation of eleven gene families which catalysed key steps of aerobic and anaerobic catabolic pathways. The proposed method increased available knowledge on the functional potential of microbial communities and helped to identify shifts in the gene structure that allowed for the deduction of the evolutionary fitness of the genes under changing environmental conditions. The use of genomic libraries enabled to retrieve genes from natural bacterial communities without cultivation and was combined with a generic approach which translated biotransformations lacking easily observable phenotypes into traits that could be selected.
The complexity and dynamics of natural ecosystems imposed the investigation of the interactions between microbial communities and the environment in bioremediation and natural attenuation scenarios. Thus, BIOTOOL utilised state-of-the-art proteomic technologies to locate protein biomarkers, which were descriptive of the soil status and predictive of its evolution. The potential of camel antibody technology for production of large numbers of antibodies which recognised specifically given catabolic enzymes was pinpointed, though a soil protein extraction procedure able to reveal the biodegradative landscape was yet to be established.
A bioinformatics system was also developed, in order to organise and maintain heterogeneous information related to biodegradation. In addition, a machine learning approach was proposed in order to identify the degradation potential of new compounds based on their chemical descriptors. The system could assist the implementation of recent international regulations on the use of new chemicals.
BIOTOOL resulted in numerous exploitable outcomes. Protocols for the catabolic genes fingerprinting processes, which could be utilised to define degradation in sites of interest, were available. In addition, the tree core sampling approach was highly recommended for monitoring chloroethene in shallow groundwater aquifers and relevant knowledge was broadly disseminated. Moreover, lipids could be utilised for rapid monitoring of microbial communities and protocols were developed for that purpose. The designed DNA extraction kit was at commercial exploitation stage, as well as the proposed biodegradation prediction server. Finally, genetic tools were constructed to allow for the identification of enzymes catalysing novel reactions.
