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Assessing the diversity and metabolism of oxalotrophic bacteria

Author Daniel BRAVO
Director of thesis Pilar Junier
Co-director of thesis
Summary of thesis

In soils, low molecular weight organic acids are usually associated with microbial decay of organic matter and/or root secretions in the rhizosphere. Oxalic acids (H2C2O4) are produced by plants and fungi and deposed in soil like wewellite crystals (CaC2O4) and are able to interact with rhizosphere bacterial communities to be precipitated in calcite or calcium carbonate form (CaCO3); in this sense the oxalate is a organic acid used like only carbon and/or energy source by a limited number of soil bacterial species that are involved in the mineralization of oxalate. This process is called the oxalate-carbonate pathway and the microorganisms that would be able to transform the oxalate in carbonate are called oxalotrophic. In the bacteria, there must to be several metabolic ways to assimilate the oxalate and must of the studies are performed in serine and glycolate pathways. However, there are not studies about the diversity of oxalotrophic bacteria in tropical soil ecosystems and the possible metabolic diversity in implied in oxalate-carbonate cycle. For this reason this study are focus to elucidate the diversity of oxalotrophic bacteria and the metabolic diversity involved in that process. In this way new oxalotrophic bacteria were isolated from three different types of tree three tropical soils. At the moment 160 isolates were obtained in three different oxalogenic systems in Bolivia, India and Cameroon. 32 strains were isolated from Bolivian soil 88 strains from Indian soil and 40 strains from Cameroon. All the strains were isolated in solid Schlegel medium supplemented with calcium-oxalate like only carbon source. The growing of the strains was evaluated in Schlegel liquid media supplemented with calcium and potassium oxalate separately. And for isothermal microcalorimetric studies the cultures were cultured in Angle's media with potassium oxalate as carbon source. Enrichment cultures of soil samples from every tropical soil were performed as well. The DNA was extracted from 6 soil samples from Bolivia (one profile and a negative control) 30 soil samples from India (two soil profiles) and 15 soil samples from Cameroon. Positive test was obtained of PCR amplification for all soil samples using the 16S rRNA gene. It was also tested the efficiency of a frc gene. frc gene is a specific molecular marker for oxalotrophic bacteria which present a big and a small fragment of amplification and codify for the enzyme formylCoA transferase. All strains from Bolivia amplified the small fragment of frc gene. In the other hand Indian's strains are irregulars in the amplification of the gene with the marker used. Due to inconsistencies in the characterization of oxalotrophic bacteria by this target, it was propose to characterize the oxalotrophic activty by isothermal microcalorimetry (IMC). Measurement of the metabolic activity by IMC, was performed using two strains isolated from Bolivia which corespond at Streptomyces sp. and was named as BV1m3 and BV1m4, and 6 oxalotrophic bacteria reported before and used as reference in the oxalotrophic enthalpy. It was found high oxalotrophic activity for Pseudomonas oxalaticus, Cupriavidus necator, Methylobacterium extorquens and Streptomyces violaceoruber with 90, 40 and 55 microJulies per second respectively. After phisiological description, most of the strains isolated in all tropical soils are slow-growing actinobacteria, and at the moment the kinetics of growing in cultures with nutrient broth are ready for Bolivian isolates. Sequenciation stage is going on for all strains using the 16srRNA gene amplifications. Some experiments was performed with DNA extracts of soil samples using the PCR products of 16srRNA gene and frc gene to be analyzed by DGGE. The optimization of the technique as well the ladder design with oxalotrophic strains are in process.

Administrative delay for the defence
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