Innovative approaches to understand complex microbial communities for eco-engineering the degradation of herbicides in stressed agricultural soils

Bioaugmentation protocols to improve herbicide removal in soils have been implemented. The bioaugmentation protocol serves to assess the performance of the specialized catabolic strain both in the herbicide removal rate and in the strain survival. To assess the effect of bioaugmentation on soil bacterial population the bioaugmentation protocol can be combined with culture independent tools and approaches also developed within the framework of this project. Bioaugmentation protocol can be applied for strains catabolizing either s-triazine herbicides such as atrazine or simazine, or chlorinated phenoxyalkanoic herbicides such as 2,4-dichlorophenoxyacetate. Use of soil microcosm studies as a previous step before field studies is recommended. Procedures to test abiotic factor effects in both herbicide removal rates and strain survival, such as lixiviation and soil sterilization through irradiation, are also considered. Some soil management procedures such as use of plants, use of nitrogen source, or addition of antimicrobial compounds are also addressed. Although tested in three types of soils, and five different catabolic strains, the use of these protocols can be extended to other soils ad catabolic strains.

We have developed MetaRouter, an application intended for laboratories working in biodegradation and bioremediation which need to maintain and consult public and private data, linked internally and with external databases, and to extract new information from it. Among the data-mining features, a program is included for locating bio-degradative pathways for chemical compounds according to a given set of constraints and requirements. The integration of biodegradation information with the corresponding protein and genome data provides a suitable framework for studying the global properties of the bioremediation network. The system can be accessed and administrated through a web. MetaRouter is a system for maintaining heterogeneous information related with bioremediation and biodegradation in a framework that allows its updating, query, modification and mining. The core of the system is a relational database where the information on chemical compounds, reactions, enzymes and organisms is stored in an integrated framework. MetaRouter allows not only to interactively consult the database but also to formulate new questions with associated programs that run on top of the database.

To more rapidly analyse catabolic gene diversity, but also diversity of expressed catabolic genes, molecular fingerprinting methods are necessary. We therefore established reliable PCR-SSCP (single stranded chain polymorphism) methods to sort catabolic genes (see Junca et al., 2003). New methods were needed to analyze the diversity of catabolic key genes involved in the degradation of phenoxyalkanoate herbicides. Two kinds of genes are known to encode enzymes for 2,4-D transformation, termed tfdA encoding 2,4-D/a-ketoglutarate dioxygenase and cad encoding 2,4-D monooxygenases. Different primer combinations and phosphorylated derivatives were evaluated for tfdA genes to obtain fragment mixtures comprising only fragments of the target genes. Best results were obtained for primers termed tfdAa, which were capable to amplify both tfdA and tfdAa-genes. Separation allowed distinguishing between highly similar gene variants, and successfully separated tfdA and tfdAa gene variants. Separation conditions were also evaluated for cad genes using fragments. The current method allows separating successfully different groups of cad genes, however, singling base differences cannot yet be evaluated. The current methods are of special use for evaluating the catabolic potential and diversity for phenoxyalkanoate degradation in diverse soil systems. Bacterial community responses were assessed through T-RFLP (See Sanchez et al. 2004). To do so, bacterial community DNA was digested with MspI or HhaI restriction enzymes and the corresponding T-RFLP signals were plotted as relative abundance vs fragment length to obtain richness (number of bacteria or ribotypes in the sample) and diversity values that informed on the bacterial community structure. Two additional outputs from these T-RFLP plots could be obtained. First, comparisons of the bacterial community structures under different conditions (presence/absence of herbicides or plants, time of incubation, type of soil, etc) were carried out, and were, usually, very informative of the effect of specific conditions on bacterial community structures (see below). Statistical significance of such comparisons was assessed by NMDS, PCA, and ANOSIM analyses. Second, taxonomic assignations, based on public databases such as the Ribosomal Database Project, were also possible. This allowed distinction of the effect of different conditions at the level of phyla and some classes. The robustness of comparisons of soil bacteria taxonomic profiles obtained in this way was assessed by PCA, ANOSIM, and analysis of similarity of percentages (SIMPER).

Microorganisms capable of detoxifying or mineralizing organic pollutants are not ubiquitous at contaminated sites. A collection of diverse species of microorganisms capable of degrading atrazine and simazine, as well as 2,4-dichlorophenoxyacetate and 2,4-dichlorobutyrate have been isolated. Those microorganisms can be used to support biodegradation by bio-augmentation at contaminated sites. They are used to extend knowledge on degradative genes in different genera of microorganisms. Two culture collections are available at UTFSM and UBA, respectively.

Metabolic processes, like the whole metabolism have to function within the context of the host and be tightly regulated, so detrimental energy fluxes are avoided in order to not compromise host-fitness and survival. These mechanisms need to link the biodegradation pathway to host physiology by several bridges. We have designed and used custom DNA arrays to be able of studying as a whole, the links between the cell physiology and a given degradative pathway. We choose genes, which report the status of the cell; sigma factors, heat shock related chaperones, iron-regulated genes, sulfur regulated or nitrogen response elements etc. In one of the arrays, we printed such genes of Pseudomonas along with the well-characterized genes coding for the TOL pathway, which is composed by four transcriptional units. The array of both stress genes and TOL genes was employed to see how the cells behave in the presence of different effector molecules of the pathway and the response of the pathway on mRNA levels. The second designed array is based on the genome of the 2,4-dichlorophenoxyacetate degrader Cupriavidus necator JMP134. The available complete genome sequence allowed performing the metabolic reconstruction of the aromatic catabolism of this bacterium. This strain is able to use more than 60 aromatic compounds as a sole carbon and energy source, including halo-, and nitro- aromatics, C6-C2, and C6-C3 compounds. About 300 catabolic genes have been identified, encoding the peripheral reactions and the twelve main ring cleavage routes that the in vivo studies predict. We developed a catabolic/physiological 50-mer oligonucleotide microarray containing 348 key probe sequences, including most of the genes involved in catabolic pathways for aromatic compounds and key genes involved in global regulation. A third designed system is a phylogeny-based DNA array for application to the analysis of microbial population diversity and dynamics in soils and other complex ecosystems. The microbial phylogeny-based DNA array system is dependent upon the sequence differences contained within hyper-variable regions of the rRNA genes. More than 2000 bacteria were evaluated for determining the best regions of the 16S rRNA genes to be used for differentiation and identification. These regions are targeted for PCR-amplification to generate the polynucleotide products to be used as probes (as opposed to using oligonucleotide probes) of approximately 250-300 nucleotides in length. A major advantage of the proposed strategy is the potential for generating the reference DNA or probes, from microbial isolates, from cloned DNA or from total PCR-amplified community DNA/RN, without prior sequence knowledge or dependence upon evolving sequence databases. PCR-products or genomic DNAs used as reference DNAs are spotted and total community DNA polynucleotide probes are generated by PCR for hybridisation. The level of resolution for such hybridisation probes is approximately 5% sequence difference, corresponding to genus and sub-genus level differentiation. Through a combination of probes with progressively higher resolution levels, the identities of the individual members of a given microbial community can be analysed.

Extraction and analysis of atrazine and 2,4-D in soils with high content of humic compounds Due to the widespread implementation of herbicides in the modern farming practices, it became necessary to develop analytical techniques for monitoring the presence and fate of these compounds and its metabolites in the environment in a fast and non-expensive way. That knowledge is an irreplaceable tool to assess the impact of agrochemicals on the different ecosystems and improve the management strategies for their application, preserving in that way the environment. UBA group has improved processes to extract and analyze chlorinated herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, as well as some of their derivatives and metabolites in soils with high amounts of organic matter (especially humic compounds). In this way, a new prospective emerge for analysis and monitoring of those xenobiotics in the highly complex matrix of humic soils, without increasing the costs or the technological requirements. In countries with agro-exporting schemes, where herbicides are widely used, this kind of improvement acquires a central relevance, especially when there is a limited economical support for technology. That is why, in the future, this simple and low cost technology could be an available tool for people dedicated to organic farming practices and agricultural biochemical analyses, for monitoring bioremediation protocols, and even for Health and Environmental Control organisms. The improved methods allow assessing environmental herbicides levels without any lost in accuracy or sensibility. Perkin Elmer and UTFSM group has improved processes to extract and analyze chlorinated herbicides such as simazine and atrazine in soil samples.

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