Insights on the recovery, characterization and valorization of Extracellular Polymeric substances (EPS) from granular sludge applied in innovative wastewater treatment systems

The urge for more sustainable wastewater treatment solutions based on the well-established concept of “circular economy” is progressively paving the way towards new resource recovery-oriented strategies. A paradigm shift has been driven conceiving wastewater treatment plants (WWTPs) as collection points of resources (e.g., nutrient, water, energy, etc.): their redesign from treatment facilities into water resource recovery factories (WRRFs) is considered able to provide multiple opportunities to contribute to a more circular economy-based water sector. The resource recovery potential is particularly emphasized in the case of granular sludge (GS)-based technologies that were recognized as viable alternative to conventional activated sludge (CAS) systems for a wide range of biological wastewater treatment processes. Biogranulation consists in forcing microrganisms to form granules (i.e., self-aggregated biofilms without the presence of carrier materials) rather than flocs: the compact granular form endows excellent settleability, easier solid/liquid separation, and enhanced biomass retention. As in conventional biofilms, in granules microorganisms are embedded in a matrix of highly hydrated Extracellular Polymeric Substances (EPS) secreted by bacterial consortia during cell metabolism. The complex and diverse biopolymeric matrix mainly consists of proteins (PN), polysaccharides (PS), uronic acids, lipids, nucleic acids, humic-like substances, etc. EPS contribute to the initial aggregation of microbial cells and are mainly associated with the structural integrity, rheological behaviour, physic-chemical properties, and functional stability of granules. Hence, EPS exert multiple functions within the granular biofilm such as protection, nutrient source, maintenance of a stable structure, and organic substance sorption. The recovery and conversion of EPS into bio-based commodities is considered an appealing route to enhance the economics and sustainability of wastewater treatment according to a circular economy pattern in waste sludge management. Thanks to their versatile properties, GS-derived EPS can be valorized in multiple industrial/environmental solutions alternatively to synthetic polymers (e.g., coating/sizing agents in paper and textile industry, cement curing, biosorption, etc.), thus contributing to a less fossil-fuel dependent manufacturing sector. The development of EPS recovery-oriented solutions is currently hampered by many bottlenecks which can be mainly identified in a still incomplete understanding of various fundamental aspects in terms of both EPS composition/properties and production regulation. Further research effort is therefore demanded to progress towards the sustainable EPS recovery and conversion into value-added biomaterials able to generate a change in the critical status of waste sludge management in WWTPs. In this perspective, the present thesis mainly aimed to give insights on the recovery, characterization, and valorization of Extracellular Polymeric Substance (EPS) from waste granular sludge of different origin. Particular emphasis was dedicated on potential approaches to move towards the production of EPS-based biomaterials to be valorized in environment-related applications. Particularly, two types of GS were investigated (aerobic granular sludge, AGS, and anammox granular sludge, AmxGS) and distinct resource recovery-oriented scenarios were addressed depending on the nature of the studied microbial aggregates. Various solutions to engineer the most attractive features of these waste-derived biopolymers were hence proposed and all the evaluation criteria and methodologies were consequently adapted: hydrogel-based materials with great potential in sustainable agronomic practises (AGS-recovered EPS) and biosorbent media for the treatment of heavy metal-contaminated wastewaters (AmxGS-recovered EPS). More detailed, the thesis structure, methodologies and main findings can be summarized as follows. Chapter 1 introduces the general background of the thesis. The urge for more sustainable wastewater treatment solutions based on the concepts of “resource recovery” and “circular economy” was emphasized. The attractiveness of GS-based technologies as viable alternative to conventional activated sludge systems was presented: particularly, their potential in terms of EPS production/recovery was pointed out. The main bottlenecks limiting the large-scale implementation of EPS-based biomaterials have been comprehensively discussed. Finally, the outlines and main goals of the thesis are presented. Chapter 2 proposes a comprehensive analysis of the hydrogel-forming ability and resulting post-gelling mechanical properties of structural extracellular polymeric substances (sEPS) extracted from aerobic granular sludge (AGS). The gelling protocols in presence of divalent metal cations (e.g., Ca2+) were adapted with the aim to minimize the polymer consumption and optimize the hydrogel geometry for the analytical investigation. The high-complexity and diversity of AGS-derived sEPS was addressed by evaluating the overall process of hydrogel-formation in comparison with well-known biopolymers (i.e., alginate and k-/ι-carrageenan). The post-gelling mechanical behaviour was evaluated under both compression and shear stress conditions via rheometry. Particularly, sensitive parameters were extrapolated from the observed mechanical profiles (e.g., Young’s modulus, E, storage modulus, G’, loss modulus, G’’, complex viscosity, η*) and correlated with the applied gelling conditions to gain insights on the main drivers of the hydrogel-formation processes. Based on the results emerged from the mechanical characterization, the minimum sEPS (weight) concentration enabling the formation of an extended cross-linked polymeric network was recognized in the range of 2.5 – 5 wt% (for sEPS concentrations lower than 2.5 wt% only weakly interconnected polymeric clusters were probably present). The higher polymer and (ionic) cross-linker concentrations needed for the sEPS hydrogel-formation with respect to the studied reference polymers gave hence a proof-of-principle of the greater complexity and diversity of the sEPS matrix (likely involving also compounds not really contributing to the gelling processes and resulting post-gelling mechanics). Distinct mechanical responses to consecutive compression-decompression cycles were observed among the studied biopolymer-based hydrogels. Particularly, sEPS and ι-carrageenan hydrogels behaved similarly under mechanical stresses: their linear elastic behaviour was preserved along the subsequent loading-unloading cycles, but lower levels of stiffness were achieved compared to alginate and k-carrageenan-based systems. For all the studied biopolymers, the post-gelling stiffness varied significantly depending on the applied hydrogel-forming conditions, even if the overall mechanical response remained almost unchanged: E increased upon increasing the polymer and (ionic) cross-linker concentration and varied based on the (divalent) metal cation used as cross-linking agent. The oscillatory shear measurements confirmed that sEPS were able to form hydrogels with solid-like mechanical properties. From an applicative point of view, the feasibility of forming sEPS-based hydrogels with mechanical properties comparable to other biopolymer-based systems currently applied for commercial purposes was presented, thus suggesting potential resource recovery-oriented solutions able to progress towards a less fossil fuel-dependent manufacturing sector. Chapter 3 offers a consistent approach to engineer the hydrogel-forming properties of AGS-derived sEPS based on the high qualitative standards imposed by the agronomic sector. Particularly, the influence of various chemicals in the extraction and gelling processes on the quantity/quality of the extractable EPS macromolecules was pointed out, emphasizing the importance to adapt the methodologies on the research objectives. With this regard, extraction and gelling protocols widely discussed in literature were adapted providing chemical reagents containing no sodium or chlorine which are considered phytotoxic in large quantities: K2CO3 or (NH4)2CO3/HNO3/KOH (extraction) and Ca(NO3)2∙H2O or Ca(C2H5COO)2 (cross-linking). The quality/quantity of the extractable EPS macromolecules as well as their overall hydrogel-forming ability did not appear strongly influenced by the distinct chemicals applied. Conversely, more significant differences were observed in terms of compositional analysis (e.g., macronutrients, Na, Cl, heavy metals, etc.). Overall, the obtained sEPS and derived biomaterials (e.g., hydrogels) were consistent with the current environmental legislation in matter of soil improvers and fertilizing products, resulting within the maximum limits imposed in terms of heavy metals (values related to Cr(VI) to be investigated). The great potential of the obtained sEPS hydrogels in agronomy-oriented solutions was emphasized evaluating their swelling ability and nutrient release capacity. Particularly, the behaviour of sEPS-based hydrogels as superabsorbent polymers (SAPs) able to sorb and hold high quantities of water (up to 16 g H2O per g hydrogel as dry matter) was suggested. Moreover, a preliminary proof-of-principle of the potential application of sEPS hydrogels as carrier systems for nutrient loading and release was given. The biodegradability assessment was preliminarily carried out by adapting respirometric techniques (single- and multiple-OUR experiments): conclusions in terms of organic matter biodegradability were drawn based on the partitioning of the sample Chemical Oxygen Demand (COD) in soluble biodegradable, particulate biodegradable, soluble inert and particulate inert fractions. It has been found that sEPS and derived hydrogels can be utilized as substrate from the microbial communities inhabiting the activated sludge, but their biodegradation was influenced by the chemicals applied in the extraction and gelling processes. Moreover, it has been observed that the readily biodegradable carbonaceous fraction decreased upon hydrogel-formation: the establishment of an extended 3D polymeric network in which the sEPS macromolecules were more confined likely resulted in a decreased substrate accessibility, thus requiring further hydrolytic reactions before their microbial utilization. Finally, guidelines to progress towards new resource recovery-oriented solutions in agriculture exploiting the versatile properties of AGS-derived sEPS were proposed. Chapter 4 deals with the feasibility of exploiting EPS extracted from waste anammox granular sludge (AmxGS) in high-performance and cost-effective technologies for the treatment of heavy metal-contaminated wastewater. With this regard, the metal-binding ability of the recovered EPS was addressed in comparison to that of pristine granules in single- and multi-metal biosorption studies mainly to shed light on the biosorption mechanisms of extracted and non-extracted EPS in native biomass. Metal-binding capacities equivalent or higher than conventional and/or unconventional sorbent media were found both for extracted EPS and pristine anammox granules, but distinct biosorption pathways were suggested. These differences could be due to three main reasons: (i) multiple mechanisms participating in the heavy metal biosorption by native granules, in addition to the heavy metal uptake by EPS; (ii) EPS chemical modifications induced by the extraction method applied; (iii) different polymer chain mobility and binding site availability of extracted EPS in aqueous dispersions and non-extracted EPS in pristine granules. Mechanistic hypothesis on metal biosorption were suggested: as emerged from a detail molecular-level analysis carried out combining various analytical techniques, a multifaceted mechanism based on a combination of electrostatic interaction, ion exchange, complexation and precipitation has been proposed. Particularly, it has been found that AmxGS-extracted EPS behaved like flocculant/complexing agents in presence of high-concentrated heavy metal-contaminated aqueous systems, leading to the spontaneous precipitation of EPS-metal composite aggregate. Finally, a proof-of-principle of the potential application of the extracted EPS in composite sorbent media (e.g., with activated carbon) able to target single and multiple heavy metals simultaneously from contaminated aqueous systems was given, thus suggesting encouraging strategies to enhance the industrial applicability of AmxGS-derived EPS. Chapter 5 draws the general conclusions and outlooks of the thesis, thus suggesting the next challenges to be addressed in follow up research. The outstanding potential of both AGS- and AmxGS-derived EPS in environment-related applications has been emphasized, thus shedding light on recourse recovery-oriented solutions able to progress towards a more circular economy-based water sector.