Ferrero P. P. MICROBIAL COMMUNITY ANALYSIS, INFLUENCE OF REACTOR HYBRIDATION AND EFFECT OF THE PROPORTION OF GLYCOL ETHERS/ETHANOL MIXTURES IN EGSB REACTORS TESIS DOCTORAL PROGRAMA DE DOCTORADO EN INGENIERÍA QUÍMICA, AMBIENTAL Y DE PROCESOS Autor: Pablo Ferrero Aguar Directores: THE PROPORTION OF GLYCOL GLYCOL THE PROPORTION OF ETHERS/ETHANOL MIXTURES IN EGSB REACTORS MICROBIAL COMMUNITY ANALYSIS, HYBRIDATIONINFLUENCE OF REACTOR OF AND EFFECT D. Vicente Martínez Soria D. Josep Manuel Peñarrocha Oltra 2018 Departament d’Enginyeria Química MICROBIAL COMMUNITY ANALYSIS, INFLUENCE OF REACTOR HYBRIDATION AND EFFECT OF THE PROPORTION OF GLYCOL ETHERS/ETHANOL MIXTURES IN EGSB REACTORS Programa de Doctorado en Ingeniería Química, Ambiental y de Procesos Memoria que, para optar al Título de Doctor por la Universitat de València, presenta PABLO FERRERO AGUAR Valencia, octubre de 2018 Agradecimientos En primer lugar, me gustaría agradecer a mis directores de tesis Vicente Martínez y Josep Manuel Peñarrocha por haberme ofrecido la posibilidad de realizar incorporarme este grupo de investigación, y su dedicación y esfuerzo en la realización de esta tesis doctoral, su realización no hubiera sido posible sin su apoyo. También ampliar mi agradecimiento a Carmen Gabaldón por la confianza depositada en mi, y al resto de miembros del grupo de investigación (Paula Marzal, Javier Álvarez y Marta Izquierdo) ya que también gracias a su ayuda y sus consejos se ha podido desarrollar este trabajo. También me gustaría agradecer a Luc Malhautier y todo el equipo del Laboratoire du Génie de l’Environment Industriel por la acogida durante la realización de la estancia y sus enseñanzas sobre las técnicas de identificación microbiana. Tampoco quiero dejar pasar la oportunidad de agradecer a Manuel Toledo los buenos momentos que pasamos en Alès. Gracias a mis compañeros de laboratorio, Pau, Maria y Carlos, por haberme acogido tan bien desde el principio, los buenos consejos y estar siempre con una sonrisa. A Dani y a Nadine por las risas y buenos momentos compartidos. A Keisy, Lidia y Alejo, por hacer el día a día más ameno y divertido, sin vosotros no hubiera sido lo mismo. A las nuevas incorporaciones del grupo, Miguel y Helena, desearos lo mejor en esta nueva etapa que comenzáis. Gracias a mi familia, y en especial a mis padres, por ser el pilar básico de todo, por estar siempre, por su apoyo y confianza incondicional. Sin ellos nada de esto hubiera sido posible. A mis abuelos porque esto también es gracias a vosotros. Por ultimo agradecer al Ministerio de Ciencia, Innovación y Universidades por la beca concedida (CTM2014-54517-R) para realizar esta tesis. Summary Volatile organic compound (VOC) emissions can cause different problems in the public health and in the environment, acting as a primary pollutant and allowing the formation of secondary pollutants as tropospheric ozone. Due to these problems VOC emissions are regulated in many countries, such as USA and the European Union, in this case by the Council Directive 2010/75/EU. Because of the use of solvents in its productive process, flexographic industry is one of the major contributors to the emissions of these compounds, and biological techniques have been considered as one of the best available technologies for the treatment of VOC emissions in this industrial sector. Among these processes, a new technology the anaerobic bioscrubber is emerging as a feasible technology (patent number WO2015114436A1). In this process, VOC are transferred from the gas phase (air emission) to the liquid phase (water) and then transformed into biogas in an anaerobic expanded granular sludge bed (EGSB) reactor. So, the VOC emissions can be converted into bioenergy. However, the use of EGSB reactors for this process has some inherent barriers as the lack of information about the anaerobic degradation of some of the compounds typically used in the flexographic industry or the loss of biomass in the effluent due to the use of a high up flow liquid velocity. For this last aspect, an alternative configuration to the EGSB reactors should be studied in order to avoid the biomass leakage. In this regard, an anaerobic hybrid reactor configuration, which consists in the installation of a filter of polypropylene rings inside the gas- liquid-solid separator in the upper zone of the reactor, seems to be a good alternative to improve the biomass retention capacity of the EGSB reactors. Furthermore, the flexographic industry uses synthetic organic solvents as glycol ethers, such as 1-ethoxy-2-propanol and/or 1-methoxy-2-propanol, but the anaerobic biodegradation of mixtures of these compounds remain unknown yet. In addition, in the literature there is also barely information available about the possible negative or toxic effect of these glycol ethers on the microbial population responsible of the biotransformation of VOC emission into biogas. In this regard, different molecular techniques, as denaturing gradient gel electrophoresis (DGGE), quantitative polymerase chain reaction (qPCR) and high throughput sequencing technologies, are available to characterize the microbial community and to analyse the microbial evolution in biological systems, such as anaerobic reactors, which can be helpful to check these negative or toxic effects by the presence of some of these solvents. In this context, the main objectives of this PhD thesis are: i) to evaluate an alternative reactor to the EGSB reactor to improve the biomass retention capacity and the performance of a reactor treating glycol ethers and ethanol mixtures; ii) to study the pathways for the anaerobic degradation of glycol ethers as 1-ethoxy-2- propanol (E2P) and 1-methoxy-2-propanol (M2P) used in the flexographic industry and the possible impact on the microbial community; iii) to evaluate the effect of the ethanol / glycol ethers ratio in an EGSB reactor treating mixtures of these compounds; and iv) to compare the performance and microbial communities from laboratory scale reactors and an industrial prototype reactor. An alternative configuration based on the modification of the EGSB with a filter of polypropylene rings, called anaerobic hybrid reactor (AHR), has been compared with the conventional EGSB (control reactor). Both reactors were operated at the same conditions and the experiment was divided in seven stages (from S-I to S-VII). First (S-I), the organic loading rate (OLR) was increased step by step up to 45 kg chemical oxygen demand (COD) m-3 d-1 using a readily biodegradable substrate such as ethanol, then E2P was introduced (S-II and S-III), resulting in a binary mixture of ethanol and E2P, and the total OLR was maintained around 45 kg COD m-3d-1. After that, M2P was also introduced as a new substrate in the reactor feed (ternary mixture of ethanol, E2P and M2P) maintaining the same total OLR (S-IV). Later on, the feeding was switched off to simulate a long-term shutdown of production process, which typically occurs in the printing facilities, and so to check the influence of a long starvation period (S-V). Then, reactors were restarted again by increasing the OLR, step by step with the ternary mixture, and finally, in the last stage the proportion of M2P was slightly increased (S-VI and S-VII). Proportion of glycol ethers in this experiment was always lower than 30% in weight. Results showed a high performance of both reactors with global removal efficiencies (RE) higher than 92% even treating OLR of 54 kg COD m-3 d-1 and RE only decreased below 90% in both reactors during the first days after the feeding of E2P, indicating that biomass was not adapted to this solvent and an adaptation period was needed to be able to metabolize it. The adaptation period was also observed in the evolution of RE of E2P, as only around 20% RE was achieved in both reactors when this compound was introduced, but after 40 - 50 days the RE of E2P increased to 80% and maintained during all the experimental period. Regarding M2P, RE was almost complete (100%) immediately after its introduction in the reactor feed and no adaptation period was needed, which would suggest that both glycol ethers have the same mechanism of degradation. Furthermore, the removal of M2P was practically complete along all the experimental period, even after the increased in its proportion in the feed (S-VII). In addition, during the first days of exposure to the glycol ethers, some intermediate products of their degradation (methanol, acetone and isopropanol) were detected and identified, allowing the clarification of the anaerobic degradation pathways of these compounds. Regarding the biomass retention capacity, the accumulated solids in the effluent (563.2 g in the EGSB reactor and only 293.7 g in the AHR) showed that the AHR had a higher biomass retention capacity than the EGSB reactor. However, the filter installed in the AHR was finally clogged and the rings of the filter had to be replaced by new polypropylene rings to maintain its higher biomass retention capacity. Despite the higher biomass concentration in the AHR, both reactors performed similarly. These results, included in the Chapter 4 of this PhD thesis, have been published in the Journal of Environmental Management (Ferrero, P., San-Valero, P., Gabaldón, C., Martínez-Soria, V., Penya-roja, J.M., 2018. Anaerobic degradation of glycol ether-ethanol mixtures using EGSB and hybrid reactors: Performance comparison and ether cleavage pathway. J. Environ. Manage. 213, 159–167). The dynamics of the microbial community of both reactors was also analysed using different molecular tools, such as denaturing gradient gel electrophoresis (DGGE), quantitative polymerase chain reaction (qPCR) and high throughput sequencing technologies. These analyses revealed an important impact in the microbial populations caused by the introduction of both glycol ethers (E2P and M2P). DGGE technique showed an evolution in the bacterial community from the beginning to the end of the experiment.