Aerobic Granulation in Wastewater Treatment

CHAPTER 4

Aerobic Granulation in Wastewater Treatment: A General Overview Sumit Sharma1, Saurabh Jyoti Sarma1, Joo-Hwa Tay2 1

Department of Biotechnology, School of Engineering and Applied Sciences, Bennett University, Greater Noida, India; 2Department of Civil Engineering, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada

1. Introduction: Aerobic Granules The cellecell interaction in a microorganism is a capability of aggregation to make them self-immobilized. These self-immobilized aggregates, mostly formed in a spherical shape, are named as granules (Khan et al., 2013). The rate of oxygen flow and other physical parameters results in the aerobic granules development. The granule formed in the absence of oxygen categories as anaerobic granules. Aerobic granules have become the most promising tool in the wastewater treatment technologies. The treatment efficiency depends on the growth of the microorganism and settling properties. Bulk sludge with higher filamentous microorganisms causes the difficulty to settle in the clarifiers because cells naturally have the dispersion characteristic, not aggregation (Etterer, 2006). The applied adhesive force and shearing form aerobic granules which have characteristics features like more settling property (Lochmatter, 2008), high retention time (Liu et al., 2016), and high activity (Bindhu and Madhu, 2013). There are various uses of the aerobic granules in treatment process such as biodegradation of dye, Bisphenol A, fluorinated compounds, antibiotics, herbicides, pesticides, phenols, volatile organic compounds, etc. (Sarma et al., 2016). Recently, aerobic granulation is being used in paper and pulp wastewater treatment (Vashi et al., 2017). In this chapter, various aspects of the aerobic granulation are discussed such as granular characteristics, mechanism of granules formation, microbial diversity, reactors used for granulation, process parameters, and the role and application of aerobic granules in wastewater treatment.

2. Physiochemical Properties and Granule Formation The ability of microorganisms to aggregate resultsin the information of granule like structure. One cell interacts with another cell through the release of a special kind of Microbial Wastewater Treatment. https://doi.org/10.1016/B978-0-12-816809-7.00004-X Copyright © 2019 Elsevier Inc. All rights reserved.

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58 Chapter 4 extracellular material called as extrapolymeric substances (EPS). These substances are made up of polysaccharides and stick together to join the cells and make the complex microbial granule. One single granule can have millions of copies of a cell type. These granules are very compact and densely aggregated with each other; therefore they have good settling property and resistivity for a long duration. When this aggregation occurs between the same species, then it is called auto-aggregation, while in the case of distinct cells, it is named as coaggregation.

2.1 Physio-Chemical Characteristics 2.1.1 Structure and Morphology The aerobic granules morphologically are spherically shaped (700e1900 mm in size) (Zhou et al., 2016), highly dense, and compact structures. The granular density is measured in glucose-fed mature granule at 1.048 g/l and settled at a velocity of 2.0 cm/s (Etterer and Wilderer, 2001). Tay et al. reported the diameter size of 2.4 mm, settling velocity 35 m/h, biomass density 41.1 g/L, physical strength 98%, hydrophobicity 68%. These granules can survive longer time at low temperature, i.e., up to four months at 4
C (Tay et al., 2002). When granules are settled down in the reactor, they look like a huge biomass with small beads clumped together. Due to the EPS and shear forces, the bacterial cells move close to each other in a centric manner so that an inner substratum is formed at which these cells adhere. Sarma et al. proposed that the structure is divided into three zones (Fig. 4.1) (Sarma et al., 2017). The central zone is made up of dead cell biomass covered by an anoxic anaerobic contact area of the growing bacterial biomass. The outer layer consists of fully active bacterial cell stacked together which is around 600  50 mm (Tohet al., 2003) in thickness and directly in contact with the aerobic environment in the reactor. This structure is generated when the granule is in the fully matured stage.

Figure 4.1 Structure of aerobic granule.

Aerobic Granulation in Wastewater Treatment: A General Overview 59 2.1.2 Hydrophobicity The environmental changes allow the modifications in the cells and to release the vesicles of the outer membrane. This increases the cell’s hydrophobicity and enhance the biofilm formation. In Pseudomonas putida DOT-T1E, the high osmotic stress of the salts releases the cell outer membrane vesicles within 10 min and increases the cell hydrophobicity (Baumgarten et al., 2012). When there is a stress condition, the self-aggregation starts with the decreases in cell-free energy due to the increase in hydrophobicity. Sometimes, by applying multiple starvation phases between the growth increases the cell hydrophobicity. It was suggested that microbes change their structural properties and come close to each other and interact more during starvation condition (Liu et al., 2004a) and this aggregation complete the granule formation. Adav et al. describe the cellecell aggregates can be used for the treatment of waste containing phenol, nitrogen, phosphorus, etc. There is positive as well as negative aspects of cell interaction like attachment and detachment of the cell under biotic and abiotic conditions (Adav et al., 2008). It affects the biofilm to distort or accumulates more for the nonremoval or removal of pollutants, respectively (Krasowska and Sigler, 2014). This approach helps in identification of the granular stability in an unsuitable environmental condition (Liu et al., 2009). 2.1.3 Extracellular Polymeric Substances Extracellular polymeric substances (EPS) are the mixture of polymeric units of different macromolecules like carbohydrates, proteins, lipids, and nucleic acids (Liu and Fang, 2002); 75%e89% of EPS accounts for the polysaccharides and proteins present in it (Tsuneda et al., 2003a). These are secreted outside the cell, means every polymeric substance is not attached directly with the outer membrane. EPS give structural integrity to biofilm and is the main component of biofilm formation. A gel-like three-dimensional hydrated matrix encloses the cell within it called as EPS (Wingender et al., 1999). EPS has a functional relationship with the microbial aggregates like mass transfer, the surface charge against the negative charge of sludge, flocculation ability, its role in settling ability of microbial cell aggregates (Sheng et al., 2010). Generally, these substances produced during the exponential phase and regulate the integrity of the granules by acting as an energy source when there is a starvation condition. There are various functions contributes by EPS which includes cells adherence, aggregation, the formation of biofilm, biofilm stabilization, protection of cell from biocides or another toxic environment, biosorption of organic compounds, nutrient accumulation, and water retention (Laspidou et al., 2002). EPS overall provides a stable, highly dense, microbial community placed in close proximity to each other. EPS is segmented into two parts: bound EPS and soluble EPS (Fig. 4.2).

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Figure 4.2 Tightly bounded and loosely bounded EPS on the outer surface of the cell.

Bound EPS is made up of condensed gels, polymers, and organic components while soluble EPS consists of slimes, macromolecules, and colloids. Soluble EPS are loosely bounded with week interaction and can easily dissolve in the large amount of solution (Su et al., 2013). Bound EPS is reverse of soluble EPS. They bound tightly with the cell wall and make a visible compact layer outside the boundary of the cell (Ramesh et al., 2007). EPS is a source of a natural ligand for the binding of charged particles and absorbed them and provide better functioning of microbes in wastewater treatment (Alaba et al., 2018).

2.2 Development of Granules The surface hydrophobicity and van der Waal forces allow the cell to adhere together. The cellular signaling and environmental conditions stimulate the cells to release extracellular polymeric substances. The hydrodynamic shearing through the air and water flow and the interactions of various ions with EPS lead to the granulation of the microbial colonies shown in Fig. 4.3 (shodhganga.inflibnet.ac.in/bitstream/10603/12869/11/11_chapter% 203.pdf). These microbial granules come close together to form a multi-granule complex which is further separated into free granules when the oxygen supply diminishes (Liu et al., 2008). The resulted granules come out from the complex due to shearing forces formed in between the granules. These aerobic granules can easily settle down and can be roughly used multiple time for wastewater treatment.

Aerobic Granulation in Wastewater Treatment: A General Overview 61

Figure 4.3 Microbial cell aggregation and granule formation (A) Free microbial cells, (B) Cell-cell interaction, (C) Aggregation, (D) Aerobic granulation, (E) Multi-granule complex, (F) Complex lysis, (G) Free granules.

2.2.1 Mechanism of Aerobic Granules Formation Liu proposed the mechanism of the aerobic granule formation (Liu and Tay, 2002). The mechanism is divided into four phases. Phase 1: Cellecell contact by physical movement. Various forces involved in the physical interaction of cells are hydrodynamic, gravity, thermodynamics, Brownian movement, and diffusion force along with the natural mobility of the cell (flagella and cilia). The hydrodynamic forces allow the microbial cell to interact due to their surface charge interaction. The cells make clumps and setting of the cells increases as biomass increases and the gravitational force allows the whole aggregate to settle fast and remain intact in the compact formation. Phase 2: Stable contact between cells by attractive forces. Attractive forces categorized as physical forces, chemical forces, and biochemical forces. (i) Physical forces comprise of surface free energy, hydrophobicity, surface tension, cross-linking bridges, van der Waal interactions, and attraction by opposite charges. As described above in Section 2.1.2, cell hydrophobicity plays a major role in the granule formation. A decrease in the free energy reversibly impacts the increment of the hydrophobicity which further enhances cells aggregation.

62 Chapter 4 (ii) Chemical forces include ionic pairing, hydrogen bonding, triple ion formation, and bridge formation between particulates. These interactions form the ionic bond in between the charged molecules and strengthen the complexities of the granule. (iii) Biochemical forces imply on the fusion of the membranes and dehydration of the surface of the cell. Protein translocation helps in the dehydration of the cell surface (Toe et al., 2000). When cell membrane fuses with each other, there is an ion channel transport starts dehydration of the cell surface occurs due to the change in the solute to solvent concentration in the medium. Phase 3: Maturation by forces generated by microbes. Metabolic changes, production of EPS and higher cellular biomass increase the cellular structure to become more compact and matured. EPS enhances the surface charge between the cell and hydrophobicity, which leads to the tight bonding of the microbial cells to adhere more due to tight bounded EPS of the microbes. Phase 4: Stability and shape. The hydrodynamic shear forces give the three-dimensional shape to the granule. Shear stimulates the polysaccharides production and allows the more stability to the granule structure (Tay et al., 2001). The fully stable granules become like a tight ball which contains millions of microbial cell stick together. these cells are highly active and more stable to act on different kinds of wastewater. Overall mechanism given by Liu is started with the physical movement of the cell to contact each other through hydrodynamic, gravity and thermodynamic forces. Then cells aggregate by EPS released by cells followed by study state condition formed by hydrodynamic forces. This results in a highly organized three-dimensional microbial structure. Multicell coordination and quorum sensing are also some contributing factors help in the development of aerobic granules (Sarma et al., 2017).

2.3 Bio-Film Formation and Flocculation For an effective bioprocess treatment of wastewater, arobust bio-film formation plays an important role. Quorum sensing is a well-studied mechanism for the biofilm formation. The social behavior occurs in between cells through signaling molecules. These signaling molecules are called as autoinducers (Miller et al., 2001). Biofilm is formed through the whole granulation process from initial attachment to the final stage of maturation. Initially, cell attaches to the substratum and then adheres through the release of some extracellular polymeric substances (Parsek et al., 2005). The positively charged coagulants(iron and ammonium salts) are added to destabilize the negative charge

Aerobic Granulation in Wastewater Treatment: A General Overview 63 particulates and contaminants. Flocculation is the process of mixing and colliding of the particles, separates them and then again aggregates into large precipitates. The mixing parameters like speed, intensity and time affect the rate of flocculation (https:// www.mrwa.com/WaterWorksMnl/Chapter%2012% 20Coagulation.pdf). The proper attention on mixing to time ratio is required to prevent the flow from shearing apart. The gentle mixing increases the size of the micro floc and suspended particles easily visible called as pin floc. The pin flocs continue to bound together to form the macro flocs, which gives optimum strength and increases the sedimentation power.

3. Microbial Community Microbial community of the aerobic granules is diversified into ammonia oxidizing (AO) bacteria (Tsuneda et al., 2003b), nitrifying (Liu et al., 2004b) denitrifying (Jang et al., 2003), phosphate removal bacteria (Wachtmeister et al., 1997; Bond et al., 1995), sulfur removing bacteria (Lens et al., 1995), and heavy metal removing bacteria (Liu et al., 2003). These bacteria are present in the aerobic and anaerobic zone of the granule and effectively work with the good porosity for the treatment of the compound present in the wastewater. A list of microbes with their function is illustrated in Table 4.1.

Table 4.1: Different kind of microorganisms involved in aerobic granulation treatment. Type of Microorganism Ammonium oxidizing and nitrifying bacteria Denitrifying bacteria Polyphosphateaccumulating bacteria

Organism Name Nitromonas spp., Nitrobacter sp., Nitrospira sp. Comamonas, Nitromonas, Alcaligenes, Rhodocyclus Thauera, Nitosospira, Accumulibacter, Rholocyclus Candidatus, Accumulibacter, Tetrasphaera

Sulfur removing bacteria

Thiobacillus, Sulfurimonas, Arcobacter Desulfobulbus, Desulfobacter, Desulfomicrobium, Desulfosarcina, Desulfovibrio

Function

References

Oxidized ammonia into nitrite and nitrate

Kim and Seo (2006).

Reduction of nitrate/ nitrite to free nitrogen Phosphates accumulation to polyphosphates by absorbing orthophosphates Phosphates accumulation to polyphosphates by absorbing orthophosphates Oxidising of sulfur into sulfate

(Adav et al., 2010a) Zhang et al. (2011)

Reduction of sulfate to sulfur derivatives

Gu ¨nther et al. (2009)

Yang et al. (2016). Hao et al. (2013).

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3.1 Ammonium Oxidizing, Nitrifying and Denitrifying Bacteria The oxidation of the ammonia into nitrite or nitrate is accomplished by ammoniaoxidizing bacteria found in sludge. In the mature granule, diversification of these nitrifying bacteria is as filamentous shaped on the outer surface and cocci shaped in inner side (Shi et al., 2009). These bacteria simultaneously remove the carbon and nitrogen in the reactor. The Fluorescence in situ hybridization (FISH) analysis reveals the presence of 16.2%e18.3% of Nitrobacter and 69.4%e70.8% of AO bacteria in the aerobic granule (Shi et al., 2009). The nitrification rate for nitrogen removal was obtained in aerobic fluidized bed reactor with ammonia removal rate of 1.5 kg/m3/day by Nitromonas-like bacteria found in the outer region of granules (Tsuneda et al., 2003b). In a sequencing batch airlift reactor, Nitromonas spp. (Nsm156), Nitrobacter (Nit3) and Nitrospira (Ntspa662) were detected after 100 days of operation (Kim and Seo, 2006). The slowgrowing nitrifying bacteria improve the granule stability (Liu et al., 2004c). The granules which have slow growth rate are highly stable with high specific gravity and hydrophobicity and made them good settler and dense structure. This is done by maintaining the N/COD ratio in the medium. Adav et al. studied the denitrifying microbial community in aerobic sludge and found the diversity with the genera of b-proteobacteria includes Comamonas, Nitromonas, Alcaligenes, and Rhodocycluswith a 100e700 mg/L nitrite to nitrogen conversion (Adav et al., 2010a).

3.2 Polyphosphate-Accumulating Organisms The denitrifying polyphosphate accumulating microorganisms effectively use in the limitation of the oxygen because these are present in the anoxic zone and coexist with the nitrifying bacteria in the oxic zone. A synthetic wastewater was treated in sequencing batch reactor, and after 30 days of process a granule size of 500 mm was formed and the nitrogen and phosphate comes out form outlet was less than 1 mg from NH4eN (60 mg/ L), PO4eP (10 mg/L) in the reaction mixture (Kishida et al., 2006). Zhang et al. found rod-shaped filamentous bacteria in initial granulation then matured with the coccoid bacteria and more diversity included when maturation completed. The microorganism such as Thauera, Nitosospira, Accumulibacter, Rholocyclus, etc. was investigated in the mature granule (Zhang et al., 2011). Flow cytometric analysis by using bright green fluorescence of tetracycline antibiotic formed a divalent complex with the phosphorylated granules was identified the microorganisms such as Candidatus, Accumulibacter, and Tetrasphaera (Gu¨nther et al., 2009).

3.3 Sulfur and Metal Removing Bacteria Sulfur-oxidizing bacteria (SOB) and sulfur-reducing bacteria (SRB) are mostly found in the aerobic granules. These bacteria remove the sulfur as well as denitrifying the

Aerobic Granulation in Wastewater Treatment: A General Overview 65 wastewater. Through 16S rRNA analysis done by Yang et al. found the autotrophic bacterial genera named as Thiobacillus, Sulfurimonas, and Arcobacter (Yang et al., 2016). Sulfate-reducing up-flow sludge bed (SRUSB) reactor granules were studied for the identification of sulfur reducing bacteria. FISH and pyrosequencing of 16S rRNA of microbial community diversified the genera of SRB as Desulfobulbus, Desulfobacter, Desulfomicrobium, Desulfosarcina, and Desulfovibrio was found in the SRUSB reactor (Hao et al., 2013). Various metal removal studies were done for the wastewater treatment by aerobic granules bio-sorption mechanism. Aerobic granules can effectively remove metals like nickel (Ni2þ) (Xu at al., 2006), zinc (Zn2) (Liu et al., 2002), and other metals. The biosorption capacity of the granules for Cd2þ, Cu2þ, and Zn2þ metals found 172.7, 59.6 and 164.5 mg/g of granular biomass and confirmed that granules are very effective bio-sorbents in removing metals from the wastewater (Liu et al., 2003). The EPS are good biosorbing elements for heavy metals. Due to polyanionic nature of EPS, they form a complex with the metal ions through electrostatic interactions and subsequently precipitate into the polymeric mass to detoxify the heavy metals from the wastewater (Pal and Paul, 2008). Liu and Xu studied the mechanism of metal removal again and found the ionexchange mechanism was followed highly than only bio-sorption. The bio-sorption of Ni2þ was ion exchanged by the release of Ca2þ ions. 1 meq of Ni2þ is exchanged by 0.68 meq of Ca2þ. It was also investigated that an increase in temperature increases the biosorption from 25 to 55
C and the sorption is endothermic in nature and it can diffuse in the aerobic granule uniformly (Liu and Xu, 2007).

4. Types of Bioreactors Formation of highly compact aerobic granules depends on the design of the reactor and its operational parameters. There are various kinds of bioreactors evaluated for successful formation of aerobic granules. Sequencing batch reactor, Membrane reactor and integrated reactor are best suited and preferred mostly for aerobic granule formation.

4.1 Sequencing Batch Reactor Sequencing batch reactor (SBR) is the stable and time-based system. In this system, the fill to draw all the process occurs sequentially. It is an advanced method against activated sludge treatment approach and has the ability to remove phosphates and nitrogenous compounds much higher. SBR is a single tank in which fill and draw process does not require secondary clarifier as shown in Fig. 4.4. The tank can be rectangular or cylindrical in shape and filled with the sample for a period of time then left it to settle and treated water is extracted out from the tank.

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Figure 4.4 The activated sludge process and sequencing batch reactor (SBR) process.

4.1.1 Sequencing batch reactor (SBR) operation cycle The SBR process cycle completed in five steps: (1) Influent Fill, (2) React/Mixing, (3) Settle/Solid Seperation, (4) Decant/Draw, and (5) Idle or stable (Vigneswaran et al., 2009). It is a single tank cycle with steps continues one by one at several intervals of time. Aeration is also provided by jet aerator or diffusers and mixing are done by the agitator. Fig. 4.5 describes the whole cycle and process is proposed by Liu and Tay in 2002 given in the following sections in detail. 4.1.1.1 Fill

This is the first step of the cycle, in which influent is added into the reactor. Influent contains nutrient for the aerobic sludge microbes. The influent is filled and mixed until it reaches the top level and after the complete fill, influent valve turned off, and continue the batch process. Type of fill and mixing with aeration depends on the objective of the treatment such as Static Fill, Mixed Fill, or Aerated Fill is described further. i. Static Fill Static means no aeration and no mixing of the influent. This is used for the steady phase of treatment plant which does not require nitrification or denitrification at the initial stage. So that power consumption is low, but substrate concentration is high at the end of fill. ii. Mixed Fill In this type of fill, no aeration but mixing is there. The influent mixed with the microbial biomass. Because air is not provided, the anaerobic condition arises and starts denitrification of the influent and biomass release the phosphorous. There is energy input of mechanical mixer and reduction in BOD also observed.

Aerobic Granulation in Wastewater Treatment: A General Overview 67

Figure 4.5 Sequencing batch reactor (SBR) process cycle.

iii. Aerated Fill The aerated type of fill consist aeration and mixing both. The influent mixed with air which leads to the nitrification and organic removal. For de-nitrification, turn the switchoff the air supply and anaerobic condition generated. This oxic and anoxic condition allows nitri and de-nitrification both in the same tank.The measured dissolved oxygen level must be maintained below the 0.2 mg/L. This type of fill reduces the cycle time. 4.1.1.2 React

In the react phase, there is no further addition of the wastewater. The aeration and mixing increase the biological treatment and starts decreasing the organic loading continuously. Similar to fill, if air is provided with high dissolve oxygen is present there, then it is called an “aerated react,” and if no air, then it is as “mixed react.” Time is spaced in the cycle according to the treatment like for organics removal 15 min, for nitrification 4 h, and for denitrification long no air time and less aeration time. 4.1.1.3 Settle

The SBR also act as clarifier where the settle phase allows the solids/biomass to settle and there is no air flow, no mechanical mixing. The activated sludge settled in the form of floc mass also called as sludge blanket. The settling time must be very low so that it reduces the total cycle time and also does not allow to draw off some sludge as in case of activated

68 Chapter 4 sludge second clarification. It separates the clear liquid from the solid which can easily drain out without allowing the solid to come out. Aerobic granules have very low settling time and very compact shape so that they can easily separate the treated liquid from itself and completely settle down very fast. 4.1.1.4 Draw

This is the process of decanting the effluent from the reactor. To decant simply a pipe on outlet can be fitted above the level of the settled biomass to the minimum low volume so that the entire effluent can come out. This can be done by a pump or valve works to open and close the flow through gravity. Another type of decanter is a floating decanter, which maintains the nonremoval of solid by placing the inlet near below the water surface and helps in flexibility to draw and fill. The time suggested for the decanting process is 5%e30% of total process time otherwise sludge starts coming up and out. 4.1.1.5 Idle

The idle is the condition where granular sludge is in completely stable condition. During the process, some solid increases the net solid in the reactor so that some small amount sludge is removed to maintain the constant aerobic sludge volume in the reactor and the sludge pumped out from bottom named as waste sludge. This is the final step of the cycle and then the cycle begins again. 4.1.2 Aerobic Granulation in Sequencing Batch Reactor (SBR) The aerobic granulation in the SBR is the best-suited mechanism for maximum separation of effluent and settling of the granular sludge in a short period of time. Bunn et al. observe the aerobic granule formation in SBR at short hydraulic retention time and high shear force (Bunn et al., 1999). Height to column diameter ratio (H/D) also affects the granulation formation. Higher the H/D shows the high quality of settling for a shorter period of time. Bunn et al. developed the granules in a SBR airlift type with acetate COD 2.5 kg/m3d loading in 1 week (Bunn et al., 2002). The compact and dense granules selected based on the settling time, slow setting are flocs and filamentous while fast settling is compact granules with 2.5 mm in size, setting rate >10 m/h and 60 g volatile suspended solids (VSS)/l density (Bunn et al., 2002). The dairy wastewater was treated with aerobic granular sludge in SBR at a retention time 15e30 min with 90% COD, 80% N and 67% P removal efficiency (Schwarzenbeck et al., 2005). Another study revealed the degradation of p-nitrophenol (PNP) with a 0.6 kg/m3/day with glucose and found the complete removal of p-nitrophenol (Yi et al., 2006). Maximum degradation of PNP achieved up to 19.3 mg/g of VSS. These granules also found the ability to degrade other

Aerobic Granulation in Wastewater Treatment: A General Overview 69 phenolics like hydroquinone, dichlorophenol, or catechols. The low-strength municipal wastewater was treated with SBR aerobic granules resulted in 90% COD removal and 95% nitrogen removal after 300 days (Ni et al., 2009). Similarly, aerobic SBR process is well established and studied for various kinds of wastewater.

4.2 Membrane Bioreactor The treatment using membrane is the basic operation of the membrane bioreactor (MBR). There is a physical separation of biomass filtration through the membrane and the quality of the effluent water increase. The MBR is configured by two kinds of systems: Extractive and diffusive (Judd, S., 2008). Extractive means to extract the specific compound by a membrane, while in diffusive, the membrane is used for maximum utilization of the gas diffuses on the surface and the bio-film attaches to the membrane get the direct air into it. Leob and Sourirajanduring the 1960s used first time the cellulose acetate membrane for reverse osmosis in wastewater treatment process. Since then, membranes are studied for better performance in treatment. Membranes are classified based on the material, filtration, module, surface and module status. On the basis of material, organic or ceramic membranes are used while according to type, microfilter or ultrafilter are used. Similarly, plate, frame, tubular, hollow fiber for module type, innereouter skin for filtration surface and static or dynamic membranes for module status are preferred accordingly. Submerged hollow fiber membrane with module outer skin does not work as well as compared to ceramic microfilters. Membrane bioreactor with aerobic granule shows an effective treatment process with more than 80% removal of compounds. The simultaneous nitrification and denitrification were observed in combining aerobic granular sludge with membrane bioreactor. There is 84.7%e91.9% and 85.4%e99.7% removal of total organic carbon and ammonia nitrogen was observed in MBR, where initial concentration was 56.8e132.6 mg/L and 28.1e38.4 mg/L, respectively (Wang et al., 2008). The membrane filtration in aerobic granule treatment is a posttreatment advancement. A baffled membrane separation reactor was operated with organic loading of COD 15 kg/m3d observed the membrane fouling with an 84% increase in the EPS in the broth medium which affects the membrane efficiency (Thanh et al., 2008). To reduce the membrane fouling and the pressure of the organic load on the reactor, powder activated carbon (PAC) was used, and 5 g/l of PAC shows the best results with 0.8 kPa/day rise in pressure as compared to 2.4 kPa/day rise in without PAC membrane reactor (Aun et al., 2006). It is summarized that MBR is an efficient technology with aerobic granulation for wastewater treatment and recycling of the biomass. To reduce the membrane fouling, PAC-MBR (Aun et al., 2006), porous carrier MBR (Jun et al., 2003) or SBR (Zhang et al., 2006) can be used.

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4.3 Integrated Bioreactor The integrated bioreactor approach is the combining of the two bioreactors like anaerobic and aerobic reactors. Bioreactor sometimes called integrated when the membrane bioreactor is combined with the sequencing batch reactor. There are four kinds of integrated bioreactors are categorized as (1) reactors integrated with separated aerobic and anaerobic zone by physical mean; (2) reactors integrated without separated aerobic and anaerobic zone by physical mean; (3) SBR with temporally separated zones; and (4) combined cultures with limited oxygen supply (Chan et al., 2009). Single SBR is mostly preferred to be used than integrated bioreactor to minimize the operating cost and duration of the process.

5. Operational Parameters The efficiency of the reactor for treatment depends on the parameters of the reactor process. The optimized value of the operational parameter is considered best for the process to be done in the bioreactor. There are various parameters described in detail as follows.

5.1 Hydraulic Retention Time Hydraulic retention time is the time to provide the hydraulic condition. A short retention period is recommended for the aerobic specific growth. Pan et al. investigated the impact of HRT on the stability of aerobic granules and found that, at very short one hour HRT biomass washout, while at very long 24 h HRT, the volumetric exchange is low and resulted as bio-flocs formed in granule. When the retention time is in between 2 and 12 h, stable aerobic granules formed (Pan et al., 2004). Hydraulic retention time at 5.8 h was also observed in treating textile wastewater through aerobic granulator sludge (Muda et al., 2011).

5.2 Specific Oxygen Utilization Rate The dissolved oxygen actively valued in the maintenance of the aerobic granules and their structure. The higher the superficial up-flow velocity higher is the stability of the granules formation. Tay et al. found a compact granular structure with up-flow velocity at 1.2 cm/s or higher (Tay et al., 2001). Similarly, Sturm and Irvine also observed the disintegration of the granules into flocs below the up-flow velocity of less than 1.0 cm/s and dissolved oxygen below 5 g/L (Sturm and Irvine, 2008). Benn et al. also confirmed the granule formation in 7 days at air velocity of 1.33 cm/s (Buen et al., 2002).

Aerobic Granulation in Wastewater Treatment: A General Overview 71

5.3 Organic Loading Rate The main base of the effective aerobic granulation treatment is variability of the organic loads. The variation in the organic loading will result in the taking long time to achieve stable granule formation (Awang et al., 2017). A moderately high loading rate is suitable for the aerobic granules stability. Tay et al. tested four sequential aerobic sludge blanket reactors with loading 1,2,4,8 kg COD/m3 d and found that best granules formed at 4 kg COD/m3 d but not in lower or higher than that. In case of very high loading, granules become unstable after a certain period of time and then washed out (Tay et al., 2004). There is a transformation of the microbial population in the species richness according to the loading rate. Very less diverse species are found in a reactor having higher loading and higher diversity on lower organic loading reactor (Li et al., 2008). The physical characteristics of the granules also change at low and high loading rate. The loose fluffy structure with dominant filamentous bacteria at low organic loading exhibits while irregular, depressed cervices with folds appeared when loading rate is higher (Moy et al., 2002). Adavet al. reported the study in SBR with 9e21.3 kg COD/m3 day and 94%e96% removal was observed up to 19.5 kg COD/m3day but granule disintegrates above this loading at 21.3 kg COD/m3 day (Adav et al., 2010b). The granules lose their capacity to aggregates and to release EPS effectively. The potential causes of the granules disintegration at higher organic loading was proposed due to the limitation of mass transfer (Zheng et al., 2006), high filamentous growth (Liu et al., 2006) and presence of a different kind of gamma-proteobacteria, proteobacteria, and bacteroidetes (Adav et al., 2010b).

5.4 Temperature and pH The optimum temperature for the reaction is of mesophilic range and the treatment efficiency increases when temperature increases to 35
C (Liu et al., 2006). The stable and compact granules with small size 0.24 mm and high effective nitrification were investigated at mesophilic temperature 35
C (Cui et al., 2014). Ab Halim et al. did the wastewater treatment experiment in SBR at 30, 40 and 50
C and found that the efficiency for the removal of COD, ammonia, and phosphate was 98.17%, 94.45%, and 72.46%, respectively at 50
C (Ab Halim et al., 2015). This means that high temperature can also be used for the treatment process. The stable pH generally closes to neutral pH 7 good for the activity of cells. Methanogens are mostly affected than acidogens with pH change. The methanogens have the pH stability at a range of 6.3e7.8 (Tiwari et al., 2006). Biosorption of metals by aerobic granule was greatly affected at low pH. The surface charge is characterized by zeta potential of biogranule and inversely proportional to the pH. Nickel absorption at high pH favors and low the zeta potential. The cell surface binding sites are available due to less number of Hþ ions and open up more functional groups for

72 Chapter 4 absorption. However, at low pH (w2), the Hþ ions are more in number on the surface and restrict the absorption of the metal ions (Xu et al., 2006).

5.5 Hydrodynamic Shear Forces The shear force is directly proportional to the superficial velocity of the air flow. At high up-flow velocity, the shear forces are high and disintegrate the granule and wash out from reactor due to lack of ability to form granule (Tiwari et al., 2006). At high velocity, 1e1.5 m/h creates high shear force and breaks the granules while at low velocity 0.25e0.5 m/h granules start accumulating and increase in size and become denser for fast settling (Kosaric et al., 1990). The granule is matured after the step of nucleation in which nuclei initially starts to form flocs. The nucleation at high shear force rate 12.42/s is faster than the lower shear rate of 0.04/s. The mechanism behind this is the secretion of high EPS with high shearing but theoverproduction of EPS leads to the nuclei to weaken cause disruption and wash out (Wu et al., 2009).

5.6 Growth Rate and Mass Transfer The growth rate gradually changes due to various parameters retention time (long), substrate concentration (low), dissolved oxygen (low), nutrients (low) and a shift in temperature. Mostly, the granulation shifted to higher filamentous growth and causes the less stable structure of granule (Liu et al., 2006). So that controlling the growth rate is a major recommendation for the proper functioning of granules. Yeng et al. researched on the growth rate kinetics in SBR on different conditions and found the direct relation to high organic load (9 kg COD/m3 d) and inverse relation to the high shear force (3.6 cm/s air flow) and high N/COD ratio (0.3) (Yeng et al., 2004). The kinetics of the aerobic granule depends on the size of granule and mass transfer of substrate. An effectiveness factor (h) define the reaction rate with and without the diffusion resistance as shown in Eq. (4.1) and Thiele modulus for reaction rate is based on diffusivity shown in Eq. (4.2) (Liu et al., 2005), proposed by Liu and Tay. Rate with diffusion resistance (4.1) Rate without diffusion resistance h is at equilibrium when the granular radius is lower than 350 mm where mass transfer diffusion is limited and starts decreasing when radius increasing above 350 mm where mass transfer dominated the diffusion. Thiele modulus (4) is an entity to identify the reaction rate with diffusion rate obtained by mass balance and can be calculated by Eq. (4.2) !1=2 X0 $mm (4.2) 4¼R YX=S $S0 $Ds ðhÞ ¼

Aerobic Granulation in Wastewater Treatment: A General Overview 73 Where R is the radius of the granule, mm is maximum growth rate, X0 is biomass concentration, S0 is initial substrate concentration, Ds is substrate diffusivity, and YX/S is yield of biomass. The reaction rate is higher with low diffusion rate is at higher value of 4.a.

5.7 Sludge Volume Index Sludge volume index is defined as “the activated sludge volume of 1 gram of sludge settled in 30 min.” It is the standard measurement of the activated sludge physical characteristics (Dick et al., 1969). Sludge volume index (SVI) is directly proportional to the settling velocity of the sludge flux curve which is developed for the solid flux containing solids with respect to zone settling velocity against solids concentration (Bye et al., 1998). Bye et al. suggested that the SVI can be divided into diluted sludge volume index (DSVI) and stirred specific volume index (SSVI). There is more than 250 mL volume of sludge is required for DSVI, below 250 mL settling volume is the same. So that diluted sludge volume is used and the index is measured as DSVI. SSVI differs from standard SVI in the size of the settling column as used by Bye et al. in a stirred reactor with diameter 100 mm and height 500 mm with 4-L working volume (Bye et al., 1998). In a case report, it was found that the aerobic granular sludge settle ability is very good when sludge volume index range was in between 80 and 100 mL/g of sludge (Dangcong et al., 1999).

6. Role of Aerobic Granules in Wastewater Treatment 6.1 Organic Removal The higher amount of the dissolved oxygen enhances the organic removal rate. More than 90% organic carbon removal efficiency was investigated in SBR with high DO concentration (7e8 mg/L) (Di Bella and Torregrossa, 2013). Sarma et al. proposed the mechanism of the carbon removal in the form of organic and inorganic carbon. Organic carbon is in the form of biomass and methane, while inorganic carbon such as carbonate, carbonic acid, and carbon dioxide. Calcium carbonate is main inorganic found in the aerobic granule (Sarma and Tay, 2018). The phosphates are accumulated in the form of polyphosphates by absorbing orthophosphates (calcium and magnesium phosphates) in the absence of organic carbons (Sarma and Tay, 2018). The slow-growing organism can contribute to the organic removal at low oxygen concentration. The heterotrophic organism of the granule showed the 100% COD removal with 94% phosphate removal efficiency (De Kreuk et al., 2005).

74 Chapter 4

6.2 Nitrification and Denitrification Nitrogen is present in the wastewater in the form of ammonium, nitrate, nitrite and free ammonia. These all are toxic to aquatic life. The biological treatment method is used to remove nitrogen forms through nitri- and denitrification process (http://lequia.udg.edu/ sanitas-itn/wp-content/uploads/2013/10/Deliverable-1.10_Guideline_Design_Granular_ Sludge_Reactor.pdf). The nitrification occurs with the oxidation of the ammonium into nitrite and then nitrite to nitrate aerobically as shown in Eqs. (4.3) and (4.4). NH4 þ þ 1:5 O2 /NO2  þ H2 O þ 2Hþ NO2  þ 0:5 O2 /NO3 

(4.3) (4.4)

Denitrification is the process of reduction of nitrate/nitrite to nitrogen by microbes in anaerobic condition through enzyme nitrate, nitrite, nitric oxide, and nitrous reductases, respectively (Mellor et al., 1992; Adav et al., 2010a) as shown in Eq. (4.5) in the conversion process. Heterotrophic microbes use nitrate/nitrite in the form of an inorganic compound as an electron donor and convert them into free nitrogen. NO3  /NO2  /NO/N2 O/N2

(4.5)

Shi et al. studied that the partial nitrification was observed by AOB (ammonia oxidizing bacteria) about 64% through aerobic granulation in SBR for 60 days operation (Shi et al., 2011). An 8 L SBR was setup to remove nitrogen by aerobic granule having 1.7 mm diameter, 1.035 specific gravity, 62 g VSS/l, ZSV (zone settling velocity) 51 m/h and SVI 22 mL/g resulted in more than 97% nitrogen removal efficiency where nitri- and denitrification simultaneously occurs (Cassidy and Belia, 2005).

6.3 Heavy Metal Biosorption Various treatment strategies were used for the removal of heavy metal but the bio-sorption of heavy metal by aerobic granular EPS system is best suited. There are diverse functional groups like hydroxyl, carboxyl, amino, and a sulfate group leads to the role of bio-sorption (Arief et al., 2008). The mechanism behind the metal absorption was estimated as the presence of the EPS, ion exchange, and chemical precipitation. Xu and Liu found the release of the Ca2þ, Mg2þ, and Kþ in replacement of Cd2þ, Cu2þ and Ni2þ through aerobic granulation in SBR (Xu and Liu, 2008). The Sips equation derived by Liu described the change in state of adsorbate with a decrease in effective free energy and increase in metal absorption (Tsuneda et al., 2003a; Liu and Liu, 2008). Morel and Hering expressed the free energy with the bio-sorption to favor the increase in adsorption resistance and a decrease in the driving force and increase in overall free energy can be calculated by Eq. (4.6) (Morel and Hering, 1993).

Aerobic Granulation in Wastewater Treatment: A General Overview 75 0

DG ¼ DG0 þ RT ln

Resistance Driving force

(4.6)

0

Where DG is overall free energy, DG0 is the effective free energy, T is temperature and R is ideal gas constant. The biosorption study for various metal was researched by Hawari and Mulligan for metal ion exchange with microbial aggregate treated with Ca2þ ions used as a cation exchanger and found with higher binding capacity of qmax 255, 60, 55, and 26 mg/g for Pb2þ, Cd2þ, Cu2þ, and Ni2þ ions respectively (Hawari and Mulligan, 2006). In some cases anionic carboxyl group was found in EPS of the granule for the absorption of Cu2þ (Sun et al., 2011) and the polyethylene grafted aerobic granules found with the 71.239 mg/g and 348.125 mg/g of Cu2þ and Cr4þ ions (Wang et al., 2010). So that, it was found that the aerobic granules bio-sorption with ion exchange mechanism is a convenient way for removing heavy metals from the wastewater.

6.4 Biodegradation Biodegradation studies were mostly found for the phenolic compounds. p-nitrophenol was degraded upto 19.3 mg/g of VSS (Yi et al., 2006), pyridine degradation 73.0 mg/g of VSS (Adav et al., 2007), and Azo dye blue 59 upto 5 g/L. Azo dye degradation was accomplished by azo reductase and cytochrome P-450 of aerobic granular sludge containing a, b, and Yeproteobacteria (Kolekar et al., 2012); p-cresol was degraded in SBR to 800 mg/L with 88% efficiency of removal and degradation rate was 0.96 g/g VVS day (Basheer and Farooqi, 2012). The Congo red dye biodegradation was also observed in SBR with a removal rate of 93% for congo red and 91% of efficiency for removal of COD content. The leftover concentration was 3.4 mg/L and 43.3 mg/L for congo red and COD, respectively (Ma et al., 2014). Sharma et al. describe the degradation of various toxic compounds such as dye (eriochrome black T, congo red, azo dye, acid red 18, acid orange and reactive blue 59), antibiotics (trimethoprim and sulphamethoxazole), phenols and butyl alcohols (Sarma et al., 2016).

7. Application of Aerobic Granulation Technology Aerobic granulation technology is the advancement in the convenient activated sludge methodology. It is a robust technique, which can be used for the significant removal of the nutrient and toxic compounds from the wastewater. There are various applications suggested by Oh, J.H. are given below (http://home.eng.iastate.edu/wtge/ce421-521/Jin% 20Hwan%20Oh. pdf) and their corresponding role is described in detail in the previous section 3.6. (a) Organic and inorganic carbon removal (methane, carbonate, carbonic acid, carbon dioxide) (Sarma and Tay, 2018). (b) Nitrogen removal (ammonia, nitrate, nitrite to nitrogen) (Shi et al., 2011).

76 Chapter 4 (c) (d) (e) (f) (g) (h)

Phosphorus removal (orthophosphates) Phenolics removal (nitrophenols) Heavy metal removal (Pb2þ, Cd2þ, Cu2þ, and Ni2þ absorption). Dyestuff removal (azo dye, pyridine) High and low strength domestic wastewater treatment (COD value) Municipal, Dairy, and Industrial wastewater treatment.

8. Summary The aerobic granulation is the most widely used technology for the treatment of wastewater. The highly compact and dense structure of granules formed by the cell to cell interaction mechanism and aggregation by extrapolymeric substances to form biofilm to finally bioaccumulation in a form of granule makes it resistant to use it for a long duration of time. The excellent physiochemical characteristics of the granules such as small diameter, high settling velocity, high density, high physical strength, high hydrophobicity, high growth rate, high temperature tolerance and require short retention time, and high reusability are the major advantage over the activated sludge process to be use it for the effective wastewater treatment. The sequential batch reactor is best for the aerobic granules formation and releases a negligible amount of wash out in the effluent. Various parameters like organic load, retention time, airflow velocity, mass transfer balance, sludge volume index, and the H/D ratio optimization greatly enhance the settle ability and complexity of the granule in the reactor and also improve the treatment process. Microorganisms found in the granular sludge have a microbial diversity of ammoniaoxidizing, nitrifying, denitrifying, phosphate accumulating, sulfur-oxidizing and sulfur-reducing bacteria present in the anoxic and oxic zone of the aerobic granules. These microbes are used for the organic carbon, nitrogen, phosphorus, and sulfur removal and convert them into nontoxic compounds which are used by microbes for their growth by increasing in biomass and granular size. It is concluded that the aerobic granulation technology is the most suitable, reliable and cost-effective technology for treating a wide range of wastewater.

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