Aerobic granular systems for biological treatment of industrial wastewateroperation and characterization of microbial populations

Abstract

The new regulations and policies, both at a State and European Community level, are the more and more exigent regarding the concentrations of pollutants in the generated effluents at industrial and urban level. This fact, together with the reduction of the available surfaces for their management, is promoting the development of new more effective and compact systems and technologies for wastewater treatment where the simultaneous organic matter and nutrients removal, like nitrogen and phosphorus, can be performed. The use of sequencing batch reactors (SBR) with aerobic granular biomass applied for the biological wastewater treatment in aerobic conditions is presented as an alternative to the conventional activated sludge processes. These reactors are operated in sequential cycles that comprise filling, reaction, settling and withdrawal phases that are characterised by the short length of the filling and settling ones aiming to the development of biomass in the shape of granules in aerobic conditions. These systems allow the treatment of higher loading rates than the activated sludge systems because higher biomass concentrations can be achieved. On the other hand, the excellent settling properties of aerobic granules and their low biomass productivity allow doing without the big and expensive secondary settlers and moreover smaller footprint is required for its implantation. Another advantage of these granular systems is that organic matter and nitrogen removal can be simultaneously carried out in the same unit by means of simultaneous nitrification and denitrification processes (SND). In this way not only the improvement of the quality of the generated effluents but also a reduction of the implementation and sludge management costs are attained. First studies focused on aerobic granulation were developed at laboratory scale reactors using synthetic media in order to better understand all the variables that affected the granulation process. In this way the settling time, the hydrodynamic shear and stress forces, the type of substrate, the organic matter (COD) and nitrogen concentrations, the dissolved oxygen concentration in the bulk liquid or the presence of possible inhibitory substances were identified to be among the key factors affecting the operation of these systems. The knowledge of the optimal operational conditions for the formation of granules allowed the reduction of the start-up time of these systems. From here, the lines of research in this field have been diversified during time into more specific subjects that go from the study of the effect of the stress conditions, the mass transfer limitations or the study of the characteristics of the granules after long periods of storage. In most of the cases the works have been carried out using a synthetic medium. With the aim of evaluating the applicability of this technology to treat effluents from different industrial sectors studies treating wastewater in reactors, at laboratory scale and later at pilot plant scale reactors, have been performed. This information is indispensable in order to carry out the scale up of these systems to industrial level, since this is the last purpose of the design of new technologies. Taking the existent knowledge as starting point (Chapter 1), in the present thesis the start up and the performance of aerobic granular reactors employing two different types of wastewater were studied. Special attention was paid to organic matter and nitrogen removal together with the physical properties of the generated granules. The studied effluents were obtained from a fish canning industry (Chapter 3) and from a pig farm (Chapters 5 and 6) since these are two types of effluents generated as a result of economic activities of great importance in Galicia. The common characteristics of these two effluents are the large volume of wastewater to be treated and the high contained organic matter and nitrogen concentrations. When working with biological systems it is very important not only to consider them as black boxes but also to pay attention to the organisms involved in the different processes. Molecular biology techniques like the DGGE (denaturing gradient gel electrophoresis), clone libraries and FISH (fluorescent in situ hibrydization) allow identifying and quantifying the microorganisms, as well as to follow their populations dynamics during the operation of the biological systems. These techniques of molecular biology were used to identify the main microbial populations that carried out the biological processes in the used reactors (Chapters 4, 7 and 8). The main contents of each one of the chapters of the present thesis and the achieved objectives are detailed in the following sections: In Chapter 1, a bibliographic review of the different actors involved in wastewater treatment processes is presented. First, the reasons that drive to the need of the establishment of wastewater treatment systems are also indicated. Later a brief description is provided of the biological processes for organic matter and nitrogen removal from wastewater, including aspects as stoichiometry, kinetic parameters or the inhibitory effect of certain compounds. Information related to aerobic granular systems and its state of the art, including studies performed up to date treating industrial or urban wastewater at laboratory and pilot plant scale, is presented. Finally, the main bacterial populations involved in the nitrogen removal biological processes (ammonia oxidizing, nitrite oxidizing, anammox and denitrifying bacteria) are indicated, providing with a brief description of the most relevant aspects related to their activity. In Chapter 2, the material and methods, used in the different experiments performed along the following chapters, are described. Either conventional parameters used for the characterisation of the wastewater liquid fraction (organic matter, nitrogenous compounds, pH, dissolved oxygen and carbon compounds), or those used for the characterisation of the granular biomass from its physical properties and biological activities to the present microbial populations. On one hand parameters like granules density and sludge volume index were determined and techniques based on the observation under a stereomicroscope for the estimation of particles average diameter and size distribution were used. On the other hand, molecular biology techniques used for the identification of the different bacterial populations based on the DNA amplification, isolation and sequencing are described. The primers used for the amplification of the 16S ribosomal RNA and the amoA gene, the way of use of the DGGE technique and the procedure for the cloning libraries construction, to obtain the separation of the different fragments of DNA, are specified. Furthermore the used protocol for the application of the FISH technique together with a detailed classification of all the probes that have been applied during the distinct chapters of this thesis are described. In this chapter the procedure for the calculation of some parameters with the aim of evaluating the operation of the utilized reactors is also included. In Chapter 3, the start up and performance of a granular aerobic SBR reactor treating the wastewater from a fish canning industry was studied. In the Community of Galicia these industries, especially those situated in coastal zones, are characterised by the low space availability for the installation of a conventional system for wastewater treatment, the variability in the composition and the presence of potentially inhibitory substances like chlorides in the produced effluent. Due to their advantages, the use of granular systems would be a viable alternative for the treatment of these effluents. In this chapter a SBR reactor with aerobic granular biomass was operated at scale laboratory (1.5 L) to treat the effluent from a fish canning industry located in the Ria of Vigo. This effluent presented a high concentration of chlorides due to the use of sea water in the industrial process which could affect the biological activity of the treatment system. Therefore, the start up of the process was performed by applying different dilutions with tap water. Although the time needed to achieve complete granulation was longer than that previously reported in systems using a synthetic medium, after 75 days of operation, the granules were stable. At this moment the obtained granules had a diameter of 3.2 mm, a sludge volumetric index of 30 mL/g TSS and a solids concentration of 12 g VSS/L. The applied dilution factor to the feeding was progressively diminished and the system could treat simultaneously an organic loading rate of 1.46 kg COD/(m3 d) and a nitrogen load rate of 0.18 kg N/(m3 d) with removal efficiencies of 95% and 40%, respectively. The evolution of the concentration profiles of organic matter and nitrogen during the operational cycles from distinct operational stages was evaluated. Finally, by means of respirometric batch assays, a close relation between the nitrifying activity of the granules and their diameters was proved. Therefore, the larger the diameter of the granules the lower the nitrifying specific activity of the biomass, due to mass transfer limitations. In Chapter 4, the characterisation and monitoring of the main bacterial populations present in the biomass of the granular reactor was carried out by means of the use of molecular techniques. The DGGE technique showed the evolution of the microbial populations from the start up to the end of the operation of the reactor. The evaluation was performed according to the presence or absence of characteristic bands. The sequencing of the most representative bands allowed identifying the genus Bacteroidetes and Thauera as the representatives of the first stages of operation, and Thiothrix and Chloroflexi as the representatives of the last stages. The construction of the clone library allowed the identification of the genus Nitrosomonas as the main ammonia oxidizing bacteria, and also other members of the heterotrophic population, that belonged to the family Comamonadaceae and to different genera of the Rhodocyclaceae family. Two different genera of filamentous bacteria, Chloroflexi and Thiothrix, were also identified in the aerobic granules. They could act like a skeleton in the granule formation process. The application of the FISH technique to cryosectioned granules allowed to visualise the spatial distribution of the ammonia oxidizing and Thiothrix genera bacteria. In Chapter 5, the start up and performance of a granular reactor to treat the liquid fraction of the slurry generated in a swine farm located in Santiago de Compostela was studied. Swine slurry is characterised by its high organic matter and nitrogen concentration because of the faecal material and the urine of the animals. The application of these effluents on the crop lands as a fertilizer is an alternative for its management but is not a valid solution when the extension of available land to spread them is limited. It is necessary to consider questions like the over fertilization and that the current legislation marks the maximum value of application in 170 kg N/ha/year, and this makes a treatment of slurries prior to be spread necessary. Nowadays, the anaerobic digestion is being boosted as an alternative for its valorisation because of the energetic use of the produced biogas, but it has the disadvantage of its low efficiency regarding the nitrogen removal. The use of aerobic granular systems would allow the simultaneous nitrogen and organic matter removal. Furthermore their compact design and easy operation would make them appropriate for its installation in the farms. A SBR reactor with a volume of 1.5 L, fed with the liquid fraction of swine slurry diluted with tap water, was used in order to evaluate the suitability of the granular systems. Aerobic granules were observed after 15 days of operation and were stable during the whole reactor operation. As an example, on the day 140 of operation, the solids concentration inside the reactor was of 13 g TSS/L and the aerobic granules had an average diameter of 5 mm, a sludge volumetric index of 32 mL/g TSS and a density around 55 g VSS/Lgranule. The stability of the system while the applied loading rate increased was studied. When 4.4 kg COD/(m3 d) and 0.83 kg N/(m3 d) were applied, the system removed 87% of the organic matter and 70% of nitrogen simultaneously. In order to treat the swine slurry without previous dilution the volumetric exchange ratio was reduced, but this promoted the excessive growth of dispersed biomass with worse settling properties than the granular biomass. However it was checked that the system continued with a similar efficiency in terms of organic matter removal when loads up to 7.0 kg COD/(m3 d) were applied whereas the elimination of nitrogen was reduced, making necessary the application of a post-treatment for nitrogen removal. In Chapter 6, the feasibility of the application of the CANON (Completely Autotrophic Nitrogen Removal Over Nitrite) process to treat the effluent generated in the aerobic granular reactor fed with swine slurry was studied. This wastewater, with a similar composition to those generated in anaerobic digesters, was characterised by a low organic matter concentration and high nitrogen content (low COD/N ratio). Two groups of autotrophic bacteria, ammonia oxidizing and anammox bacteria, coexist in the CANON systems, so the former oxidise part of the ammonium to nitrite and the later combine both to produce nitrogen gas. Even though the anammox process has been previously used with effluents from anaerobic digesters treating swine slurry, the present work is one of the first in which the CANON process has been used as a post-treatment of swine slurry operated at low temperature. A SBR reactor with a volume of 1.5 L was used. It was characterized by the use of pulsed air to maintain the oxygen concentration in the bulk liquid at the values accurate for the ammonia oxidizing and anammox bacteria and also favour the mixing and the mass transfer in the bulk liquid. In this case, the capacity of the system to treat the effluent produced in the aerobic granular reactor was evaluated, with an applied nitrogen loading rate of 1.25 kg N/(m3 d), with a removal efficiency of 75%. In this chapter a comparative study of the applicability of the CANON system with other processes that have been posed in the last years for nitrogen removal from livestock effluents is performed. In Chapter 7, an insight of the physical properties and microbial populations characterization of the biomass from the CANON reactor is carried out. The coexistence between the heterotrophic bacteria, which could develop because of the presence of organic matter and the microaerobic conditions, and the autotrophic bacteria (anammox and ammonia oxidizing bacteria) is intended to be evaluated. The biomass from this reactor was distributed in two fractions: one fraction in the form of granules with an average feret diameter of 2.6 mm and with an intense reddish colour, because of the presence of anammox bacteria, and another one in the form of dispersed biomass, mainly composed by ammonia oxidizing and heterotrophic bacteria. By means of the realisation of a clone library and the application of the DGGE technique, members of the phylum Bacteroidetes, the family Comamonadaceae and the genus Zoogloea were identified as members of the heterotrophic bacteria population. The use of specific primers for the identification of the anammox population revealed the presence of bacteria that belonged to the genus Candidatus Brocadia anammoximans and Candidatus Brocadia fulgida. Finally, ammonia oxidizing bacteria were identified to belong to the genus Nitrosomonas. The use of the FISH technique allowed an estimation of the abundance of the main organisms involved in the CANON process, with a relative abundance of 35% of ammonia oxidizing bacteria and 30% of anammox. The presence of heterotrophic bacteria in anammox systems could be responsible for the events of competition for the common substrates like the nitrite for denitrification. However, thanks to the operational conditions of this reactor the destabilization of the system was not observed since the competition would be established for the oxygen with the ammonia oxidizing bacteria. This would be the reason why these organisms are forced to grow in suspension. Finally, Chapter 8 was focused on the identification, by means of the FISH technique, of filamentous organisms that were observed in repeated occasions when the analysis of the microbial populations present in the aerobic granules of different reactors was performed. The characterisation of the filamentous organisms is of great interest since they are responsible of multiple episodes of bulking and foaming in wastewater treatment systems based on activated sludge. In the case of aerobic granules, it is thought that they can have a structural function and form part of the skeleton of the granule, but, its presence can cause settleability problems and even cause the breakage of the granules. In this chapter three granular reactors treating three different types of wastewater were analysed: the effluent from a fish canning industry (R1), the effluent from a plant processing marine products (R2) and a synthetic medium supplemented with coagulant and floculant (R3). The use of the FISH technique allowed identifying and visualising the following filamentous organisms: Thiothrix and Chloroflexi in the reactor R1, Meganema perideroedes in the reactor R2 and Chloroflexi and Sphaerotilus natans in the reactor R3. The operational conditions of each one of the reactors and also the possible causes and factors that can promote the excessive development of these organisms in wastewater treatment systems were described.