Diseño, caracterización y aplicación de microelectrodos para el estudio de biopelículas sulfuroxidantes

Abstract

This thesis is focused on the development of microsensors specially designed for biofilms monitoring and on the application of these devices to study and characterize different biofilms.
 Using micro-electromechanical systems (MEMS) technology, an amperometric microsensor for DO monitoring, which improves the performance of the available microsensors, has been designed, constructed and validated. The multi-electrode design of these device, allows to obtain simultaneous information about dissolved oxygen (DO) concentration at 8 different depths through a biofilm, with an excellent DO sensitivity and a high spatial resolution (<50 µm). The high robustness exhibited by the microsensor has been exploited for the monitoring of biotrickling filters, obtaining instantaneous information of DO concentration inside biofilms. 
 Heterotrophic biofilms monitoring has served to quantify mass transport rates within biofilms. These results have been used to increase the knowledge of mechanisms involved in these processes and to establish the relationship between mass transport mechanisms, hydrodynamic conditions and biofilm structure. The information obtained in these studies highlighted how important is to include biofilms heterogeneity in mass transport description, and has been used in the development of two correlations for the estimation of mass transport rate within biofilms.
 Biofilms monitoring has been also used in the development of specific kinetic models to replace those developed in suspension cultures, used conventionally to describe biofilms activity. From DO, H2S and pH recorded profiles was possible to calibrate kinetic models, which accurately described the activity of heterotrophic and sulfuroxidizing biofilms. Microsensors measurements proved to be an excellent tool to validate biofiltration models and to more accurately predict their behaviour from the characterization of mass transport and biokinetic. Results obtained during the simulations revealed that using microsensors measurements within biofilms, as central elements of bioprocess, increase the thoroughness and the ability to accurately predict the behavior of biofilms.
 The developed methodology allowed to design a novel MEMS microsensor, based on DO microsensor, integrating into a single device DO and pH measurement. The design and construction of the firts prototype was modified to include a second microelectrodes array to potentiometric pH detection, and some improvements. The reduction of microsensors dimensions and the protection of microelectrodes reduced biofilm perturbations during needle insertion and increased sensor response stability. These improvements place the microsensor as a powerful tool, and as an alternative to conventional monitoring systems, allowing obtaining simultaneous spatial and temporal information within low thickness biofilms. Esta tesis se centra en el desarrollo de nuevos microsensores diseñados específicamente para la monitorización de biopelículas y en su aplicación para el estudio y caracterización de biopelículas de diferente origen (heterótrofas y autótrofas). Utilizando tecnología de sistemas micro-electromecánicos se ha diseñado, fabricado y validado un microsensor amperométrico para la detección del oxígeno disuelto (OD), que mejora las prestaciones de los microsensores disponibles. El diseño multi-electrodo de este dispositivo permite obtener información simultánea de la concentración de OD en 8 puntos diferentes a lo largo de una biopelículas, con una excelente sensibilidad para la detección del OD y una elevada resolución espacial (<50 µm). La elevada robustez que presenta este tipo de dispositivos ha sido aprovechada para monitorizar biofiltros percoladores, obteniendo información a tiempo real de la concentración de OD en el interior de las biopelículas. La monitorización de las biopelículas heterótrofas ha servido para cuantificar en su interior las velocidades de transporte de materia. Estos resultados han sido utilizados para incrementar el conocimiento sobre los mecanismos que intervienen en estos procesos y para establecer la relación entre estos mecanismos, las condiciones hidrodinámicas del sistema y la estructura de la biopelícula. La información obtenida en estos estudios ha demostrado la importancia de incluir la heterogeneidad de las biopelículas en la descripción del transporte de materia, y ha sido utilizada para desarrollar dos correlaciones para la estimación de las velocidades de transporte de materia en el interior de las biopelículas estudiadas. La monitorización de biopelículas también se ha utilizado para desarrollar modelos cinéticos específicos para sustituir los modelos desarrollados en cultivos en suspensión, utilizados tradicionalmente para describir la actividad de biopelículas. A partir de la adquisición de perfiles de OD, H2S y pH en el interior de biopelículas ha sido posible calibrar modelos cinéticos que describen con precisión la actividad de microorganismos en el interior de biopelículas heterótrofas y sulfuroxidantes. Las medidas obtenidas con microsensores demostraron ser una excelente herramienta para validar los modelos de biofiltración y predecir con mayor precisión su comportamiento a partir de la caracterización del transporte de materia y la biocinética. Los resultados obtenidos en la simulación demostraron que la utilización de medidas directas en el interior de biopelículas, como elementos centrales del proceso biotecnológico, aumenta la rigurosidad de los modelos de biofiltración. La metodología desarrollada permitió diseñar un nuevo microsensor MEMS, basado en el de OD, que incorporó en un solo dispositivo la medida de OD y pH. El diseño y la fabricación del primer prototipo se modificaron para incluir una segunda matriz de electrodos para la detección potenciométrica del pH y para incorporar mejoras. La reducción de las dimensiones del microsensor y la protección de los electrodos hizo menos invasiva su medida y aumentó la estabilidad de su respuesta. Estas mejoras sitúan el microsensor como una herramienta de gran potencial, alternativa a los sistemas de monitorización convencionales, que permite obtener información simultánea espacial y temporal de la concentración de OD y pH en el interior de biopelículas con bajo grosor

Computational fluid dynamics techniques for fixed-bed biofilm systems modeling : numerical simulations and experimental characterization

“…In the fluids which are flowing over the biofilm, the transport of substrates is governed mainly by advection, having the diffusion a more participative role in the concentration boundary layer (CBL) (Wäsche et al, 2002;Lewandowski and Beyenal, 2007;Guimerà et al, 2016). This implies that the biochemical processes and biofilm dynamics can be affected by biosystem fluid conditions, which may even cause problems in biofilm growth limited by the amount of nutrient available and/or limitations in bioreactors efficiency operated for pollutants removal (Iliuta and Larachi, 2004;Liotta et al, 2014).…”

“…Microsensors can be used for measurements of oxygen, sulphide, hydrogen, chlorine, hydrogen peroxide, ammonia, nitrate, sulfide and pH among others (Lewandowski and Beyenal, 2007;Guimerà, 2016). They have also been used to characterize mass-transport dynamics and nutrients biodegradation processes inside the biofilms (Lewandowski et al, 1993;Zhang and Bishop, 1994;Horn and Hempel, 1995;Guimerà et al, 2015Guimerà et al, , 2016.…”

“…These measurements were related to the volatile suspended solids (VSS) concentration according to the correlation described by Guimerà and coworkers (Guimerà et al, 2016). Further details about the recorded profiles and the applied procedure can be found in Guimerà (2016).…”

“…DO microsensors measurements were performed in order to characterize the biological activity within the biofilm under both endogenous and substrate consumption conditions and to study biofilm heterogeneous character, as it has been reported in previous works (Guimerà et al, 2015). Several oxygen concentration profiles were measured at six different regions along the bioreactor during both endogenous and substrate consumption conditions (Guimerà, 2016). Using the methodology proposed by Wäsche et al (2002), the characterization of these profiles allowed to distinguish three phases (see Figure 4.1): the liquid phase, in which DO concentration can be assumed to be constant; the boundary layer (liquid-biofilm interface), in which DO mass transfer from liquid to biofilm occurs; and the biofilm, in which DO is diffused and consumed by the microbial activity.…”

“…Glucose, fed in excess to avoid biomass growth limitation, was used as the sole carbon source. During biofilm growth, flow rate and residence time were adjusted to approximately 1 m h −1 and 12 h. Further details about reactor setup and operation can be found in Guimerà et al (2016). After heterotrophic biofilm growth, the well-fixed samples were carefully removed from the FPB and transferred into the rheometer plate, following the indications of Wloka et al (2005).…”