New Advances in Aerobic Granular Sludge Technology Using Continuous Flow Reactors: Engineering and Microbiological Aspects

water Review New Advances in Aerobic Granular Sludge Technology Using Continuous Flow Reactors: Engineering and Microbiological Aspects Aurora Rosa-Masegosa 1,2, Barbara Muñoz-Palazon 1,2,* , Alejandro Gonzalez-Martinez 1,2, Massimiliano Fenice 3, Susanna Gorrasi 3and Jesus Gonzalez-Lopez 1,2   Citation: Rosa-Masegosa, A.; Muñoz-Palazon, B.; Gonzalez-Martinez, A.; Fenice, M.; Gorrasi, S.; Gonzalez-Lopez, J. New Advances in Aerobic Granular Sludge Technology Using Continuous Flow Reactors: Engineering and Microbiological Aspects. Water 2021, 13, 1792. https://doi.org/10.3390/ w13131792 Academic Editor: Hongyu Ren Received: 26 May 2021 Accepted: 25 June 2021 Published: 29 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Faculty of Pharmacy, Campus de Cartuja, University of Granada, s/n, 18071 Granada, Spain; [email protected] (A.R.-M.); [email protected] (A.G.-M.); [email protected] (J.G.-L.) 2Department of Microbiology, Institute of Water Research, University of Granada, C/Ramón y Cajal, 4, 18071 Granada, Spain 3Dipartimento di Ecologia e Biologia, Universitàdegli Studi della Tuscia, Largo Universitàsnc, 01100 Viterbo, Italy; [email protected] (M.F.); [email protected] (S.G.) *Correspondence: [email protected] Abstract: Aerobic granular sludge (AGS) comprises an aggregation of microbial cells in a tridimensional matrix, which is able to remove carbon, nitrogen and phosphorous as well as other pollutants in a single bioreactor under the same operational conditions. During the past decades, the feasibility of implementing AGS in wastewater treatment plants (WWTPs) for treating sewage using fundamentally sequential batch reactors (SBRs) has been studied. However, granular sludge technology using SBRs has several disadvantages. For instance, it can present certain drawbacks for the treatment of high flow rates; furthermore, the quantity of retained biomass is limited by volume exchange. Therefore, the development of continuous flow reactors (CFRs) has come to be regarded as a more competitive option. This is why numerous investigations have been undertaken in recent years in search of different designs of CFR systems that would enable the effective treatment of urban and industrial wastewater, keeping the stability of granular biomass. However, despite these efforts, satisfactory results have yet to be achieved. Consequently, it remains necessary to carry out new technical approaches that would provide more effective and efficient AGS-CFR systems. In particular, it is imperative to develop continuous flow granular systems that can both retain granular biomass and efficiently treat wastewater, obviously with low construction, maintenance and exploitation cost. In this review, we collect the most recent information on different technological approaches aimed at establishing AGS-CFR systems, making possible their upscaling to real plant conditions. We discuss the advantages and disadvantages of these proposals and suggest future trends in the application of aerobic granular systems. Accordingly, we analyze the most significant technical and biological implications of this innovative technology. Keywords: aerobic granular sludge; continuous flow reactor; granular stability; microbial community; reactor design 1. Introduction Aerobic granular sludge (AGS) technology is today often regarded as a promising option for biological wastewater treatment because it has demonstrated excellent physicochemical performance at the pilot scale; even the AGS full-scale has gained an optimum position in the area of treatment of urban and industrial sewage [ 1 ]. Granular sludge was first discovered in upflow sludge blanket anaerobic systems in the 1980s [ 2 ]. Later, at the end of the 1990s, it achieved the growth of AGS without any supporting carrier operated in sequential batch reactors (SBR) [ 3 , 4 ]. Currently, aerobic granular sludge technology remains challenging to implement for treating sewage water, several authors reporting Water 2021,13, 1792. https://doi.org/10.3390/w13131792 https://www.mdpi.com/journal/water Water 2021,13, 1792 2 of 20 the instability of granular biomass for long-term operations [ 5 , 6 ]. Nevertheless, several mainstream full-scale systems are being substituted by AGS technology worldwide by Nereda ® for treating urban and even industrial wastewaters [ 7 – 10 ]. Undoubtedly, AGS technology has numerous advantages in comparison with the most common technology implemented: conventional activated sludge (CAS). Conventional technology needs longer hydraulic and sludge retention times, more time to treat and recycle sludge and a larger surface area for implementation. For these reasons, AGS has become increasingly popular among the scientific and engineering communities. AGS technology has been employed for treating highly toxic and recalcitrant compounds in wastewater at full scale, including particulate matter, nitrogen, phosphorous, pharmaceutical compounds, phenols, nuclear waste and heavy metals [ 11 – 14 ]. However, it is important to mention that most recently applications are being focused on the denitrification of groundwater contaminated with pesticides and nitrates, implemented at full scale and thereby providing biosafety and high-quality drinking water [ 15 ]. Thus, nowadays the technology continues innovate in the water biological treatment area [16]. Granules are defined as compact and dense spherical biofilms with a solid structure, conformed by aggregates of prokaryotic and eukaryotic microorganisms embedded in extracellular polymeric substances, constituting a tridimensional matrix without any supporting material and encouraged by self-immobilization [ 17 ]. The excellent properties of granular biomass encouraged the high settleability, and consequently, the technology is able to remove organic matter and nutrients in a compact design reactor. Moreover, the microbial relationships found in the granular sludge encourage several metabolic pathways to degrade pollutants in syntrophic association, such as reported by Kasina et al. [ 18 ] These factors promote a technology with a low footprint, a small surface in need of implementation and low energy costs [16]. 2. Characterization of Aerobic Granular Sludge Biomass 2.1. Physicochemical Characterization (a) Regular shape Granular sludge is defined as a biofilm with a high degree of sphericity and a smooth surface, owing to the hydrodynamic pressure exerted by the circular and continuous motion within the bioreactor. Viruses, bacteria, archaea, protozoa, fungi and metazoan microorganisms, among others, are joined through the excretion of extracellular polymeric substances, which act as glue among cells. The spherical conformation is produced without the presence of any supporting material for promoting growth, as seen in other biofilm technologies [19,20]. (b) Excellent settleability Settleability is a significant advantage of AGS relative to CAS. In wastewater treatment plants (WWTPs) with CAS systems, it is essential to separate the liquid phase from the solid fraction. In this sense, CAS systems require a larger land surface for building a WWTP due to floccules’ lack of settleability. However, the incomparable granular decanting ability allows for easy and fast solid-liquid separation because the granules are in different phases. This fact is driven by the negative cell surface charge of granules and the hydrophobic compounds they produce, which are mainly constituted by proteins [ 21 ]; the feast–famine periods produced changes on cell surface charges that intensified the better settleability. This settling capacity allows granules to reach a decanting velocity of up to 138 m h −1 , as reported by Muñoz-Palazon et al. [ 17 ], whereas floccules of CAS show velocities below 10 m h −1 [ 11 ]. Such impressive settleability allows operations to be conducted in shorter time periods, ranging from 2 to 10 min. The recurrence of this action enables the selection of the most compact granules, while fluffy and filamentous flocs are washed out [ 22 ] in sequential batch reactors. The separation of solid–liquid phases takes place in the same reactor; hence, secondary clarification after biological treatment is not required. All these Water 2021,13, 1792 3 of 20 characteristics reduce both the surface required for implementation and the investment for the build, as also given by the vertical design of the technology. (c) Dense structure Dense and compact granules are selected due to their excellent settleability, as mentioned in the previous paragraph. However, this structure is also important for conferring one of the most important characteristics and advantages of granular sludge: the dense layers promote the existence of several microniches, driven by mass transfer behaviors from the outer layers to the internal layers. This mass transfer encourages the limitation of oxygen in the environment as well as the nutrients consumed in the external layers; their metabolites are thus transported to the interlayer and the nuclei. These physical properties bestow the coexistence of aerobic, anoxic and anaerobic organisms in a single granule (Figure 1). Therefore, the outermost layer is in contact with a higher dissolved oxygen concentration in the medium, which is where the heterotrophic and nitrifying organisms that carry out the oxidation process of raw water are located. Subsequently, polyphosphateaccumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) compete in the interlayers for the substrate, where oxygen is presented in small concentrations or is even absent. Finally, denitrifying microorganisms, which need anaerobic conditions, are located in the core of the granules in order to carry out the denitrification process in the absence of oxygen. In this way, the granular structure enables the removal of nutrients and organic matter within the same reactor, whereas modifications of CAS are required to remove phosphate and nitrogen [12]. Water 2021, 13, x FOR PEER REVIEW 3 of 21 same reactor; hence, secondary clarification after biological treatment is not required. All these characteristics reduce both the surface required for implementation and the investment for the build, as also given by the vertical design of the technology. (c) Dense structure Dense and compact granules are selected due to their excellent settleability, as mentioned in the previous paragraph. However, this structure is also important for conferring one of the most important characteristics and advantages of granular sludge: the dense layers promote the existence of several microniches, driven by mass transfer behaviors from the outer layers to the internal layers. This mass transfer encourages the limitation of oxygen in the environment as well as the nutrients consumed in the external layers; their metabolites are thus transported to the interlayer and the nuclei. These physical properties bestow the coexistence of aerobic, anoxic and anaerobic organisms in a single granule (Figure 1). Therefore, the outermost layer is in contact with a higher dissolved oxygen concentration in the medium, which is where the heterotrophic and nitrifying organisms that carry out the oxidation process of raw water are located. Subsequently, polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) compete in the interlayers for the substrate, where oxygen is presented in small concentrations or is even absent. Finally, denitrifying microorganisms, which need anaerobic conditions, are located in the core of the granules in order to carry out the denitrification process in the absence of oxygen. In this way, the granular structure enables the removal of nutrients and organic matter within the same reactor, whereas modifications of CAS are required to remove phosphate and nitrogen [12]. Figure 1. Schematic diagram of niches located in aerobic granular sludge biomass. (d) Removal of organic matter and nutrients within the reactor As mentioned in Subsection c, granules are able to remove organic matter and nutrients such as phosphorous and nitrogen under the same operational conditions. Nitrogen is removed via several steps. First, ammonium is oxidized to nitrite and subsequently to nitrate under aerobic redox conditions. Then, nitrite and nitrate are reduced by denitrifying microorganisms, which transforms them into nitric oxide, nitrous oxide and finally dinitrogen gas [12,16,23]. The degradation product of one microorganism can serve Figure 1. Schematic diagram of niches located in aerobic granular sludge biomass. (d) Removal of organic matter and nutrients within the reactor As mentioned in Subsection c, granules are able to remove organic matter and nutrients such as phosphorous and nitrogen under the same operational conditions. Nitrogen is removed via several steps. First, ammonium is oxidized to nitrite and subsequently to nitrate under aerobic redox conditions. Then, nitrite and nitrate are reduced by denitrifying microorganisms, which transforms them into nitric oxide, nitrous oxide and finally dinitrogen gas [ 12 , 16 , 23 ]. The degradation product of one microorganism can serve as the substrate for another microorganism, facilitating the complete degradation of pollutants. Water 2021,13, 1792 4 of 20 In the enhanced biological phosphorus removal (EBPR) process, PAOs can store polyphosphate through the release of phosphorous in the anaerobic phase and then the ‘feast uptake’ of phosphorus in the aerobic stage. Both PAOs and extracellular polymeric substances can accumulate phosphorous and act as a reservoir in the biomass [ 24 , 25 ]. Moreover, granules can eliminate organic matter, nitrogen, phosphorous and more; they are even able to biotransform, biodegrade, bioadsorb and remove priority and emerging pollutants as highly recalcitrant substances from the wastewater of industries such as tanneries, textiles, pharmaceuticals and the chemical and mining sectors [1]. (e) Tolerance to a high organic loading ratio and high biomass retention The high biomass retention and concentration reached in the system allow AGS to withstand high organic loads, treating more than 15 kg m −3 d −1 [ 26 , 27 ]. AGS has been reported to be highly resistant to a wide range of organic loading rates (OLRs). The organic loading ratio and the substrate determine the kinetic behavior and morphology of granules; likewise, the granulation process is dependent on the load [ 26 , 27 ]. Due to the high specific surface and biomass retention capacity of granules, the technology can handle high organic loading ratios in comparison with conventional activated sludge [ 27 ]. A significant substrate concentration can promote increased microbial activity and a greater mean diameter [ 13 ]. Some studies have defined OLR as a key operational parameter for the design of WWTPs [28,29]. In addition, AGS has a high biomass retention capacity, making it possible to treat large amounts of inflow in a smaller bioreactor relative to other technologies such as CAS because, as mentioned before, the surface in contact with raw water is greater [ 11 ]. Some research has reported a concentration of mixed liquor suspended solids (MLSS) close to 10 g L−1[6], while in CAS technology this usually ranges from 2.5 to 4.5 g L−1[30]. (f) High toxicity resistance Biological processes are poorly effective for biodegraded toxic substances due to their recalcitrant nature. However, several studies have demonstrated AGS’s resistance to the toxicity of several chemicals due to the microbial activities and high levels of biomass retention provided by the layered structure [ 1 , 11 , 14 ]. Bioaggregation is a means of protecting single cells from environmental stresses and toxic compounds. Furthermore, significant levels of microbial diversity can affect various organisms differently, causing syntrophic relationships of cohabitants within granules. Redox conditions and diffusional resistance towards external molecules and then high tolerance to toxicity make granules different from the flocs found in CAS or membrane bioreactors (MBRs) [31]. 2.2. Advantages over Conventional Activated Sludge The use of AGS has emerged as a feasible alternative technology to traditional flocbased CAS for the second step of wastewater treatment. It has been demonstrated at the laboratory, pilot and full scale that granular sludge has remarkable advantages relative to CAS systems (Table 1). All of the characteristics of granular biomass described previously render this technology a promising system for biological wastewater treatment, whereas activated sludge is typified by slow-settling flocculent biomass, demanding a large secondary clarifier, a lower MLSS concentration and large amounts of system energy [ 32 ]. Furthermore, AGS systems reduce the footprint due to the lower hydraulic retention time. Given these advantages, CAS infrastructure may be replaced through the implementation of AGS. Indeed, AGS requires a 25–50% smaller area for implementation, 23–40% lower electricity costs and 20–25% lower operational costs. CAS systems are able to remove pathogen microorganisms. Floc biomass is an ecosystem where predation and biomass adsorption promote inactivation or destroy pathogens. The main way to predation is carried out by protozoan microorganisms, which have a high capacity to consume particulate matter; in this sense, pathogen bacteria can be ingested by eukaryotic organisms [ 13 ]. Alternatively, viral removal can be promoted by physical procedures, mainly by biomass adsorption, because viruses are adhered to the surface and Water 2021,13, 1792 5 of 20 subsequently microbial inactivation occurs [ 33 ]. Despite efforts to increase knowledge about the removal of pathogens, few studies have concluded that AGS is able to remove F-specific RNA bacteriophages such as E. coli, Enterococci and thermotolerant coliforms as efficiently as CAS [ 34 ]. However, there is a clear need for further studies to determine if the removal mechanisms in the two systems are comparable. This issue is promoted by the complex ecosystem found in the granular sludge able to establish several relationships between organisms. Moreover, the high abundance of extracellular polymeric substances (EPS) could act with success mechanisms to bioadsorb viral particles, which have an effect on the microbial dynamics. Table 1. Comparison of AGS and CAS. Parameters CAS AGS Settling ability <10 m h−115 to 140 m h−1 Size <0.2 mm 0.2 to 50 mm Redox Aerobic Aerobic, anoxic and anaerobic Compactness Absence High EPS production Low High Resistance to toxicity Low High Another important aspect of AGS is its resistance against toxic compounds, meanwhile AGS could treat high load shocks; in terms of organic matter, nutrients or chemical compounds, biomass concentration of CAS is deeply affected by these kinds of influents. Actually, CAS technology is a treatment not specifically designed for the removal of emergent contaminants. Then, the discharge of effluent from this kind of WWTP is a focus point for the release of emergent and priority contaminants such as pharmaceutical compounds, which reach natural water bodies [35]. 3. Granular Formation Mechanisms and Technological Characterization Most of the studies pointed out that the ideal conditions for the aggregation, compactness and stability of the granules are mainly given by the pressure exerted in SBR systems due to the hydrodynamic shear force and slow-sludge discharge. [ 11 – 13 , 36 , 37 ]. Nevertheless, this batch operation is almost incompatible with most WWTPs worldwide due to the nature of continuous flow. Some essential parameters to obtain granules are hydrodynamic shear force, the feast–famine period, circular and continuous motion and the segregation of extracellular polymeric substances. In fact, granular formation is usually described in terms of the four stages involved. First is cell-to-cell attachment, stimulated by the physical motion caused by the contact among cells, which encourages movement within the reactor. Some of these physical forces are hydrodynamics, diffusion, gravity, cell mobility and thermodynamic forces such as Brownian movement, as reported by Sarma et al. [ 38 ]. The second stage is based on the attachment of cells and the formation of microaggregates. The forces acting on granulation can be physical, chemical and biochemical. The consensus indicates that cellular surface dehydration, cellular membrane fusion, the formation of ion pairs or an interparticle bridge, Van der Waals forces or opposite charge attraction are the strongest. In this sense, it is important to note that hydrophobicity is indirectly related to the excess Gibbs energy of the cell surface; when this is low, cell-to-cell contact is encouraged. The third stage is enhanced attachment by EPS production, whereby the essential mechanism involved is quorum sensing (QS), whose signals contribute to increased polysaccharide content, promoting strong aggregates due to their role as glue. Higher QS activity during the previous granulation stages is linked with the greater production of gel-forming EPS [ 39 ]. By contrast, the opposite process of quorum quenching (QQ) has a relevant influence on granulation because with higher QQ activity, granules trend to be fluffier and floccular [ 40 ]. The last stage comprises granular conformation and maturation by hydrodynamic shear Water 2021,13, 1792 6 of 20 forces, allowing mass transfer behavior in order to facilitate diverse metabolic pathways to obtain energy. 3.1. Extracellular Polymeric Substance Segregation The role of EPS is to assist cell adhesion, which promotes the initial stages of granulation. Usually, the EPS are constituted by lipids, carbohydrates, proteins and polysaccharides. Some authors have noticed that they can act as a bridged union between cell and particulate matter [ 11 ] (Figure 2). Proteins and humic compounds are the most representative substances, followed by polysaccharides. These substances can be degraded during the starvation phase, which is one of the two periods of the feast–famine regime, in which there is a shortage of carbon source. That degradation during starvation stage causes an increase of the cell surface hydrophobicity and the promotion of the stability of granules. Although the enzymatic hydrolysis of proteins, lipids and α -polysaccharides has little effect on the structure of granules, the hydrolysis of β -polysaccharides causes granules to break and disintegrate [ 41 ]. Actually, some minerals could be bonded to the internal layers through EPS or fatty acids [ 18 ]. The efficient capacity of bioadsorption of AGS can be given by the EPS content [ 42 ]. It also protects single microorganism cells from shock loads and toxic compounds such as antibiotics or phenolic compounds. Reino et al. [ 43 ] have reported that EPS support the survival of a microbial consortium under adverse environmental conditions. Water 2021, 13, x FOR PEER REVIEW 6 of 21 content, promoting strong aggregates due to their role as glue. Higher QS activity during the previous granulation stages is linked with the greater production of gel-forming EPS [39]. By contrast, the opposite process of quorum quenching (QQ) has a relevant influence on granulation because with higher QQ activity, granules trend to be fluffier and floccular [40]. The last stage comprises granular conformation and maturation by hydrodynamic shear forces, allowing mass transfer behavior in order to facilitate diverse metabolic pathways to obtain energy. 3.1. Extracellular Polymeric Substance Segregation The role of EPS is to assist cell adhesion, which promotes the initial stages of granulation. Usually, the EPS are constituted by lipids, carbohydrates, proteins and polysaccharides. Some authors have noticed that they can act as a bridged union between cell and particulate matter [11] (Figure 2). Proteins and humic compounds are the most representative substances, followed by polysaccharides. These substances can be degraded during the starvation phase, which is one of the two periods of the feast–famine regime, in which there is a shortage of carbon source. That degradation during starvation stage causes an increase of the cell surface hydrophobicity and the promotion of the stability of granules. Although the enzymatic hydrolysis of proteins, lipids and α-polysaccharides has little effect on the structure of granules, the hydrolysis of β-polysaccharides causes granules to break and disintegrate [41]. Actually, some minerals could be bonded to the internal layers through EPS or fatty acids [18]. The efficient capacity of bioadsorption of AGS can be given by the EPS content [42]. It also protects single microorganism cells from shock loads and toxic compounds such as antibiotics or phenolic compounds. Reino et al. [43] have reported that EPS support the survival of a microbial consortium under adverse environmental conditions. Figure 2. Segregation of EPS found in the core of the granules treating wastewater. Many efforts have been made to determine EPS in AGS due to the difficulties reported by the extraction of substrates from the complex matrix in comparison with activated sludge [41]. It has been discovered that the high content of alginate polysaccharides provides the hydrophobicity and elastic assembly of granules, generating great interest in recovering the alginate contained in granular waste for food industries [44] because it acts as a stabilizing and gelling agent used in bakeries, canneries and drink industries Moreover, alginate extends shelf life and minimizes changes during the storage and handling of food [45]. Another interesting substance is named Granulan, an EPS exclusively found in granules that treat high concentrations of N and P from wastewaters, although it is unclear whether it can be found in other systems [46]. Both Granulan and alginate are soluble under mild alkaline conditions, attributed to their easy isolation and characterization, although completely opposite to the extraction of other EPS from granules [46]. Figure 2. Segregation of EPS found in the core of the granules treating wastewater. Many efforts have been made to determine EPS in AGS due to the difficulties reported by the extraction of substrates from the complex matrix in comparison with activated sludge [ 41 ]. It has been discovered that the high content of alginate polysaccharides provides the hydrophobicity and elastic assembly of granules, generating great interest in recovering the alginate contained in granular waste for food industries [ 44 ] because it acts as a stabilizing and gelling agent used in bakeries, canneries and drink industries Moreover, alginate extends shelf life and minimizes changes during the storage and handling of food [ 45 ]. Another interesting substance is named Granulan, an EPS exclusively found in granules that treat high concentrations of N and P from wastewaters, although it is unclear whether it can be found in other systems [ 46 ]. Both Granulan and alginate are soluble under mild alkaline conditions, attributed to their easy isolation and characterization, although completely opposite to the extraction of other EPS from granules [46]. 3.2. Technological Operation AGS technology is operated in sequencial batch reactors (SBRs), in four stages: feeding, aeration, settling and draining. Granular cultivation and operation in a steady state is thoroughly understood for achieving dense and compact biomass as well as impressive physicochemical performance in sequential batch cycles. For example, it is well-known that a longer reaction time promotes feast–famine phases, and consequently, the starvation stage Water 2021,13, 1792 7 of 20 encourages the density of granular sludge. Furthermore, the pressure exerted by shorter settling times makes a strong selection of rapidly decanting granules, while slow-decanting flocs are washed out. Both of the previously described examples are essential for obtaining a stable granule structure for long-term operations. However, the lack of knowledge about aerobic granular sludge rectors operated in continuous flow reactors (AGS-CFR) makes diffuse the reactor design and the mechanisms to filamentous microorganisms washed-out. In general, it is referred to as the optimum operation condition for getting stable granules using activated sludge as inoculum or to maintain the compact structure of mature granules. The loss of stability for long-term operations has been reported in SBR [ 47 ] to a greater extent than in CFRs [ 48 ]. In recent years, many efforts have been made to develop AGS technology in CFRs because control and operations in WWTPs at the full scale are easier, and the current infrastructure could be re-used [ 49 ]. In this respect, interesting advances have been achieved. Several authors have designed engineering models for the successful cultivation of granules using a range of strategies, and the number of scientific papers about AGS operated in continuous flow is continuing to increase. Perhaps one of the biggest problems facing the development of a continuous flow design for AGS is the impact of the outgrowth of filamentous bacteria in the granules because an uncontrolled proliferation of such slow-decanting microorganisms could instigate the granules’ loss of stability and breakage. 4. Aspects of AGS in Continuous-Flow Reactors 4.1. Design Reactors and Engineering Aspects In general terms, AGS systems are built on a cylindrical column with a variable height/diameter (H/D) ratio and are operated in sequential batch cycles. The cycles in batch mode firstly consist in an aeration stage, which is used for the degradation of organic matter, nutrients and pollutants. Then, the aeration is stopped, and the granular biomass is allowed to settle at the bottom of the reactor. This parameter is essential because biomass selection pressure depends on settle time. The wash-out of biomass is subsequently linked with the discarded effluent. Finally, the reactor is re-filled with raw water. However, this operational mode presents certain disadvantages, such as the relatively low volume of treated water compared with other technologies that operate continuously. Therefore, from a technical point of view, the development of AGS working in continuous flow may represent an advantage over sequential systems. In recent years, different designs of potentially useful granular CFR systems have been tested, in each case in an attempt to attain an optimal system that would enable a reactor to keep the granules in a steady-state, preventing the proliferation of slow-growing microorganisms. These microorganisms are responsible for the instability of granular biomass as a consequence of the outgrowth of filamentous organisms. Despite the different approaches carried out, we can affirm that for the moment, none of the experiences reported at the laboratory scale have been able to be transferred to the real scale, sometimes due to the complexity of the design or the lack of requirements for implementation. In this context, a large number of CFRs are being tested at the laboratory scale using different strategies, for example, bubble columns, serial multiple chambers, clarifiers after or within the bioreactors, or submerged membranes. Some designs are even hybrid reactors, combining aspects of SBR and CFR. A brief description of these designs is presented below. (a) Bubble columns with baffles Chen et al. [ 48 ] reported an upflow sludge bed reactor (USB) connected to an aeration column as a continuous flow granular system (Figure 3). The effluent from the USB was flown to the aeration tank and water and granular biomass returned to the USB to ensure a high level of biomass retention. In order to avoid the loss of biomass, a solid–liquid–gas separator was set to the top of the USB reactor. A similar separator has been employed by other authors [ 50 , 51 ]. Thus, Zhou et al. [ 50 ] operated a continuous flow airlift fluidized bed reactor (CAFB) that consisted in two columns, one inside the other, with a bubble burst at the top (Figure 3). The granules flew up through the internal tube and fell down by Water 2021,13, 1792 8 of 20 the annular tube that rounds the internal cylinder. To acclimate the sludge, the reactor operated in batch mode for 2 days and was then configured in continuous mode. In the same way, Wan et al. [ 51 ] designed a continuous-flow aerobic granular reactor (CFAGR) composed of a cylindrical reactor containing a three-phase separator and a two-decker stabilizer to avoid the loss of biomass. Kishida et al. [ 52 , 53 ] also designed a reactor with a similar baffle that operated in an aerobic upflow fluidized bed (AUFB) in continuous flow. This reactor consisted of a column with a gas–solid separator (located in the upper part of the reactor) and a lamella dividing the aeration zone from the effluent output zone (Figure 3). Water 2021, 13, x FOR PEER REVIEW 8 of 21 (a) Bubble columns with baffles Chen et al. [48] reported an upflow sludge bed reactor (USB) connected to an aeration column as a continuous flow granular system (Figure 3). The effluent from the USB was flown to the aeration tank and water and granular biomass returned to the USB to ensure a high level of biomass retention. In order to avoid the loss of biomass, a solid– liquid–gas separator was set to the top of the USB reactor. A similar separator has been employed by other authors [50,51]. Thus, Zhou et al. [50] operated a continuous flow airlift fluidized bed reactor (CAFB) that consisted in two columns, one inside the other, with a bubble burst at the top (Figure 3). The granules flew up through the internal tube and fell down by the annular tube that rounds the internal cylinder. To acclimate the sludge, the reactor operated in batch mode for 2 days and was then configured in continuous mode. In the same way, Wan et al. [51] designed a continuous-flow aerobic granular reactor (CFAGR) composed of a cylindrical reactor containing a three-phase separator and a two-decker stabilizer to avoid the loss of biomass. Kishida et al. [52,53] also designed a reactor with a similar baffle that operated in an aerobic upflow fluidized bed (AUFB) in continuous flow. This reactor consisted of a column with a gas–solid separator (located in the upper part of the reactor) and a lamella dividing the aeration zone from the effluent output zone (Figure 3). Figure 3. Diagram of AGS operated in a continuous flow reactor using bubble columns with baffles adapted from ( A ) Chen et al. [48], (B) Zhou et al. [50] and (C) Kishida et al. [53]. (b) Serial multiple chambers A different mechanism to avoid the wash-out of granular biomass was operated using serial multiple chambers to retain the granules, such as the bioreactor designed by Li et al. [ 54 ]. The length of the reactor was divided by baffles to create 10 chambers in order to increase the H/D of the full reactor. Each chamber was connected with the chambers placed next to it. There was an aerator in all the cells. The direction of the influent flow Water 2021,13, 1792 9 of 20 changed every 2 h. On the opposite sections of the extremes, there was an agitator, but only one of them was working at the same time: the agitator located in the chamber of the influent input. Following this idea, the aerator of the last section was disconnected to create a settling zone at the bottom, where the granules were accumulated to avoid their wash-out. This configuration improved the feast–famine mechanism (Figure 4). Water 2021, 13, x FOR PEER REVIEW 9 of 21 Figure 3. Diagram of AGS operated in a continuous flow reactor using bubble columns with baffles adapted from (A) Chen et al. [48], (B) Zhou et al. [50] and (C) Kishida et al. [53]. (b) Serial multiple chambers A different mechanism to avoid the wash-out of granular biomass was operated using serial multiple chambers to retain the granules, such as the bioreactor designed by Li et al. [54]. The length of the reactor was divided by baffles to create 10 chambers in order to increase the H/D of the full reactor. Each chamber was connected with the chambers placed next to it. There was an aerator in all the cells. The direction of the influent flow changed every 2 h. On the opposite sections of the extremes, there was an agitator, but only one of them was working at the same time: the agitator located in the chamber of the influent input. Following this idea, the aerator of the last section was disconnected to create a settling zone at the bottom, where the granules were accumulated to avoid their wash-out. This configuration improved the feast–famine mechanism (Figure 4). Figure 4. Diagram of a continuous flow reactor using a serial multiple chamber, adapted from Li et al. [54]. Figure 4. Diagram of a continuous flow reactor using a serial multiple chamber, adapted from Li et al. [54]. (c) Use of clarifiers Several studies have presented CFR designs connected with clarifiers, where granules could be retained. Long et al. [ 55 ] reported a CFR system with a double column cyclic aerobic granular reactor (DCCAGR), which consisted in two equal bioreactors composed by a column and a settling tank connected by an inclined tube (Figure 5). Both reactors were able to separate the solid, liquid and gas phases. The settling tank had a removable baffle, allowing the selection pressure to be modified. Moreover, both reactors functioned alternatively as the first and the second reactor, using the effluent of the first as the influent of the second one. Another example of a CFR system combined with clarifiers was reported by Chen et al. [ 56 ]. In this case, the CFR system was built with a cylindrical column with an H/D ratio of 1, composed by a mixer and an aerator. Moreover, the system incorporated a clarifier tank, where granules were accumulated and returned to the aeration tank Water 2021,13, 1792 16 of 20 the treatment of saline wastewater, suggesting that this technology is especially useful for the treatment of industrial effluents with extreme characteristics that are difficult to treat by other conventional biological systems. However, a lack of knowledge about the capacity and efficiency to remove recalcitrant compounds by granular sludge in continuous flow remains an issue. Many efforts should be made to develop a deeper understanding of the initial granulation process, stability in the absence of pressure selection, the chemical behavior of EPS and the relationship among cells from layered niches. AGS-CFR systems are especially beneficial for treating chemicals, as they are maintained at lower concentrations than in SBRs [83]. It could be argued that CFR technology is experimentally mature enough to make a leap to the real scale in the near future and should be applied to mainstream wastewater treatment, given by the continuous flow nature of the WWTP already implemented worldwide. Furthermore, interest in the comprehensive operation of this mode should be increased. Some knowledge previously well described in conventional SBR systems could be used for the improvement of CFR systems. Author Contributions: A.R.-M.—conceptualization, investigation, writing—original draft preparation; B.M.-P.—conceptualization, investigation, writing—original draft preparation; A.G.-M.— supervision, writing—review and editing; S.G.—validation, writing—review and editing; M.F.— supervision, writing—review and editing; J.G.-L.—funding acquisition, project administration, supervision, writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing not applicable. Acknowledgments: The authors would like to acknowledge the support given by the Institute of Water Research of the University of Granada, Spain and Universitádegli Studi della Tuscia, Italy. Conflicts of Interest: The authors declare that there are no conflict of interest in this work. Abbreviations ACFR advanced continuous flow reactor AGR aerobic granular sludge AOB ammonia oxidizing-bacteria AUFB aerobic upflow fluidized bed CAFB continuous flow airlift fluidized bed CAS conventional activated sludge CFAGR continuous-flow aerobic granular reactor CFR continuous flow reactor CFR-TST continuous-flow reactor with two-zone sedimentation tank CGSFDMBR continuous-flow granular self-forming dynamic membrane bioreactor COD chemical oxygen demand CSTR completely stirred tank reactor D diameter DCCAGR double column cyclic aerobic granular reactor DMBR dynamic membrane bioreactor DO dissolved oxygen EBPR enhanced biological phosphorus removal EPS extracellular polymeric substances GAO glycogen-accumulating organism H height HRT hydraulic retention time Water 2021,13, 1792 17 of 20 MBR membrane bioreactor MLSS mixed liquor suspended solids NOB nitrite oxidizing bacteria OLR organic loading rate PAO polyphosphate-accumulating organism QS quorum sensing QQ quorum quenching RNA ribonucleic acid SBAR sequencing batch airlift reactor SBR sequential batch reactor USB upflow sludge bed WWTP wastewater treatment plant References 1. 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