Análisis de la biodiversidad microbiana asiociada a la desnitrificación autótrofa en lodo granular

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 01-07-2009Nitrate is a common water contaminant, its removal is important to prevent a wide array of publichealth and environmental impacts. The most common strategy to face this problem is the heterotrophic denitrification. Problems of this process can be avoided using inorganic substances as electron donors. Chemolithotrophic denitrification with reduced sulfur compounds can attain the simultaneous removal of N and S contamination in a single-phase system, offering a great biotechnological potential. In spite of this, little is know about the microbes implicated in this kind of reactors. The present work aimed to study microbial populations involved in sulfur-based autotrophic denitrifying reactors, their adjustments to different operational conditions, including the impacts of bioaugmentation. For that, granular sludge from UASB denitrifying reactors under auto-, mixo- and heterotrophic conditions were studied with molecular techniques such as 16S rRNA cloning, DGGE or CARD-FISH as well as SEM in combination with classical techniques for bacterial isolation. The design of specific probes targeting on autotrophic denitrifying organisms was conclusive to the work. The structure of anaerobic granules suffered a great degradation under chemolithoautotrophic conditions due to the cellular lysis inside them that, together with sulfur deposition increased the sludge buoyancy. Molecular techniques showed a huge biodiversity change when shifting from methanogenic to denitrifying conditions. Methanogenic inoculum was dominated by Actinobacteria (15.6%), Alfa-Proteobacteria (16.3%), and Methanosaeta concilii. Filotipes recovered were mainly related with fermentative organisms from anaerobic digesters. Denitrifying sludge was clearly dominated by Thiobacillus sp. and members of Gamma, Beta and Alfa-Proteobacteria whereas Archaea and other groups almost disappeared. The main responsibles for autotrophic denitrification under these conditions were Thiobacillus denitrificans and Thiobacillus thiophilus. Nevertheless, bioaugmentation with T. denitrificans and S. denitrificans of UASB reactors inoculated with granular sludge do not improve their operation. Some filotipes were also close to denitrifying heterotrophs and fermentative microorganism. A heterotrophic network was established fedded by the organic matter released from the endogenous biomass decay in combination with influent inputs. DGGE band profiles suggest that denitrifying microbial communities under the same nutritional conditions were stable thorough time. In addition, several denitrifying microorganisms were isolated. Among them one strain could represent a new specie. The range of the autotrophic denitrification study was extended by including arsenite as electron donor, to obtain insights into the types of bacteria responsible for the occurrence of anoxic As(III) oxidation. Data recovered from different enrichment cultures suggested that both Azoarcus genus and members of Comamonadaceae family are the main As(III) oxidizers in the microbial communities of the cases studied