Biological study of extractive membrane biofilm reactor systems for the treatment of groundwater contaminated by fuel oxygenates
- Autores: Isabel María Guisado Requena
- Directores de la Tesis: Clementina Pozo Llorente (dir. tes.), Jesús Juan González López (codir. tes.)
- Lectura: En la Universidad de Granada ( España ) en 2015
- Idioma: español
- Tribunal Calificador de la Tesis: Concepción Calvo (presid.), M. J. Belen Juarez Jimenez (secret.), Ramiro Vilchez Vargas (voc.), Magdalena Constantí Garriga (voc.), Massimiliano Fenice (voc.)
- Programa de doctorado: Programa Oficial de Doctorado en Biología Fundamental y de Sistemas
- Materias:
- Enlaces
- Tesis en acceso abierto en: DIGIBUG
- Resumen
The application of new technologies for the biological treatment of groundwater contaminated with methyl tert-butyl ether (MTBE, an oxygenate compound present in the formulation of fuel) is an instrument of crucial importance in the search to optimise the quality of treated water. The aim of this approach is, on the one hand, to protect the environment and, on the other, to make the water thus treated fit for human consumption and agricultural purposes. In recent years, a new approach has been proposed, one that is highly important in the field of bioremediation, based on the use of fixed biofilms attached to semipermeable membranes within bioreactors. From a biological standpoint, incorporating fixed biofilm processes benefits extractive membrane bioreactor (EMBR) systems in various ways, such as the increased activity and greater resistance of the biomass to xenobiotics. An extractive membrane biofilm reactor (EMBFR) is presented, as a novel configuration of EMBR technology and a viable alternative for the treatment of groundwater contaminated with fuel oxygenates. EMBFR makes it possible to control the growth conditions of microorganisms and thus ensure the efficient degradation of pollutant compounds, by forming an active biofilm on the semipermeable membranes, regardless of the conditions in the effluent to be treated.
This Doctoral Thesis is concerned with the Biological study of extractive membrane biofilm reactor systems for the treatment of groundwater contaminated by fuel oxygenates.
In the initial phase, we examined various environmental samples and isolated and selected 20 bacterial strains capable of biotransforming MTBE. Most of the bacterial isolates (60%) belonged to the phyla Firmicutes and Actinobacteria. The other bacterial strains corresponded to the phylum Proteobacteria.
Taking into account the results obtained for the growth and degradation of MTBE and aromatic compounds (benzene, toluene, ethylbenzene and xylene ¿ BTEX) in the media examined, together with the data generated from the search for genes involved in the degradation of ethers, five bacterial strains were selected for further study: Rhodococcus ruber A5, Rhodococcus ruber EE1, Rhodococcus ruber EE6, Agrobacterium sp. MS2 and Paenibacillus sp. SH7.
The ability of these strains to become attached as a biofilm on semipermeable tubular membranes, the components of the future EMBFR, and the production of exopolysaccharide (EPS) by these strains in culture media to which MTBE had been added, was assessed in the laboratory. For this purpose, laboratory-scale batch systems were designed, with semipermeable tubular membranes that were inoculated with the above-mentioned strains. The acute toxicity exhibited by the bacterial cultures was then evaluated by the Microtox® bioassay.
The bacterial strains Agrobacterium sp. MS2, Paenibacillus sp. SH7 and Rhodococcus ruber EE6 were found to have appropriate characteristics regarding pollutant degradation, biofilm formation and ecotoxicity (EC50) for use as selective inocula (either alone or in consortium).
To evaluate the efficiency of EMBFR technology for the elimination of MTBE from samples of water contaminated with this compound, laboratory scale bioreactors were designed and built, and then selectively inoculated with the three strains mentioned above. The inoculated bioreactors were operated for 28 days (7 days in recirculation and 3 weeks continuously), under three different hydraulic retention times (1, 6 and 12 h). Various parameters, such as pH, temperature, dissolved oxygen and bacterial growth in suspension (measured as the optical density) in the biomedium of each system, were measured daily, together with the concentrations of oxygenate present in the input to the systems (untreated influent), in the biomedium and in the treated effluent, using gas chromatography-mass spectrometry. After each assay, the formation of biofilm on the semipermeable tubular membranes was assessed by scanning electron microscopy field emission and by the ecotoxicity present in the biomedium and in the effluent, according to the Microtox® bioassay.
In the development of this Thesis, we also performed a taxonomic study of a bacterial species belonging to the genus Paenibacillus (Paenibacillus sp. SH7), which was isolated and selected for the above assays on the basis of its ability to use MTBE as the sole source of carbon and energy. The results of the morphological, physiological, chemotaxonomic and phylogenetic studies conducted, together with those of DNA-DNA hybridisation, confirmed that this strain is a new species of the genus Paenibacillus, which is proposed as a type strain, to be designated Paenibacillus oxygenati nov.
This bacterial strain and Agrobacterium tumefaciens MS2 were the two microorganisms selected because, although they degraded significant quantities of MTBE, a gene search did not reflect the presence of any known genes encoding for enzymes involved in the degradation of this xenobiotic compound (monooxygenase alkane and/or cytochrome P-450).
The results obtained and presented in this Thesis highlight the major importance of new complementary methods ¿ physiological, genetic and ecotoxicological ¿ for the isolation and selection of bacteria capable of degrading xenobiotics (such as MTBE), for subsequent use in processes of bioremediation. The results also show that the selection of bacterial strains as inocula for application in different technologies should be determined not only by their ability to degrade a pollutant, but also by other factors, such as the capability of bacterial strains to adhere to supports and to become attached as a biofilm, and the level of acute toxicity achieved by their degradation activities. Finally, extractive membrane biofilm reactor technology is shown to be an efficient alternative (under certain operating conditions) for the bioremediation of water contaminated with semi-volatile compounds (such as MTBE), overcoming the problems encountered by other technologies such as low bacterial density, or the volatilisation of the oxygenate from the aqueous phase.
