Environ Eng Res > Volume 28(3); 2023 > Article
Desireddy, Madhavan, and Sabumon: Development and long-term operation of aerobic granular system for simultaneous removal of COD, nitrogen, and phosphorous in a conical SBR

### Abstract

This study evaluates the performance of a simple sequencing batch reactor (SBR) with conical geometric configuration in terms of aerobic granulation and simultaneous removal of COD, nitrogen, and phosphorous from synthetic wastewater. The reactor was operated for 328 days in 3 different phases. Stable granules measuring around 0.9±0.3 mm with good settling properties were formed in phase III of operation. Optimum removals of COD (90%), NH4+-N (91%), total nitrogen (87%), and PO43−-P (83%) were achieved in phase III, while the influent concentrations were COD (640±32 mg/L), NH4+-N (53±2.5 mg/L), and PO43−-P (9±0.6 mg/L). Mixed liquor suspended solids increased from 0.26 g/L to 2.3 g/L while sludge volume index (SVI30) decreased from 380 mL/g to 65 mL/g during start-up to end of the study, respectively. The conical geometry induces an effective velocity gradient along with aeration in the reactor for better flocculation of biomass which has a good impact on the formation of stable and resistant aerobic granules. This system in conical SBR is advantageous as it attains simultaneous nutrient removal (CNP) with effective biomass retention without automatic process control. This treatment system has the potential to employ in low volume wastewater treatment in many decentralized applications.

### 1. Introduction

Organic carbon, nitrogen, and phosphorus are the main sources of pollution from domestic, industrial, and agricultural sectors. When present in excess in natural water bodies they stimulate the growth of algal blooms and promote eutrophication which eventually threatens the safety of aquatic species and human health [1, 2]. Hence, there is a need to treat the nutrient-rich wastewaters before discharging into water bodies. Traditional biological treatment processes employ diverse microbes under various environmental conditions to remove organic matter, nitrogen, and phosphorous from wastewaters.
Recently, aerobic granular sludge technology is being regarded as an alternative to conventional activated sludge process owing to its eco-friendly and economic benefits. Aerobic granules are compact structures of diverse microorganisms that do not require any carrier [3]. Compared to activated sludge flocs, the aerobic granules hold several advantages like (i) efficient settling properties and short settling time, (ii) low SVI values, (iii) high biomass concentration, (iv) diverse microbial structures, (v) tolerates high shock loading rates, (vi) prevalence of aerobic, anoxic, and anaerobic zones in each granule, and (vii) efficient treatment [4, 5]. Hence, this technique is being extensively employed in treating several types of wastewaters comprising organic matters, nitrogenous species, and phosphorous.
The stratified layers in the granules allow the growth of aerobic autotrophs and heterotrophs in the outer layer where abundant oxygen levels are available. In the below layer, oxygen levels are comparatively less which provides a suitable environment for the growth of facultative anaerobes. In the core of the granules complete anoxic or anaerobic conditions prevail which predominates the growth of anaerobes [6]. Thus, each granule provides aerobic, anoxic, and anaerobic conditions which allows the co-existence of autotrophs and heterotrophs and favors a rich bacterial diversity which helps in concurrent removal of organics, and nutrients (N, P) [7]. Mainly, heterotrophs involve in the removal of organic matter and phosphorus accumulating organisms (PAOs) helps in phosphate removal and also plays a major role in denitrification process [8]. These organisms utilize internally stored compounds as carbon source. In feast phase, organic matter gets stored in the cells as polyhydroxyalkanoates (PHA), while, in starvation phase PHAs are utilized for microbial growth [9]. Unlike PAOs, another group of microorganisms called as denitrifying PAOs (dPAOs) can utilize NO2-N or NO3-N as electron acceptors instead of oxygen and these dPAOs are more prominent in granular sludge [10]. Overall, the oxygen gradient in the layered structures of granules helps in providing the required conditions for nitrifiers, denitrifiers, and dPAOs leading to effective nutrient removal [9].
Formation of aerobic granules is a complex process and involves several steps. Though the mechanism of exact granule formation is not known, several researchers hypothesized the probable mechanism which generally includes the interaction among cells via various biological or physicochemical forces. The granulation process includes: (i) aggregate formation among microbes by hydrodynamic, diffusional, gravitational, or thermodynamic forces; (ii) increase in stability of initial aggregates via physicochemical or biological forces like vander walls attraction, surface tension, hydrophobic interactions, ionic/inter-particle bonds, fusion of cell membranes, cell receptors attractions, dehydration of cell surfaces, (iii) generation of extracellular polymers which help in maturation of granules, (iv) transformation in to three dimensional granules in steady state with smooth surfaces and organized structure [11, 12].
To obtain stable aerobic granules, several operational strategies as described below are followed: (i) settling time (low settling time is a main selection pressure on the biomass and helps in washing out of flocculent biomass from the reactor) [13], (ii) type of inoculum (hydrophobic bacterial cells as inoculum helps in rapid formation of granules) [14], (iii) substrates composition and concentration (low substrate and high microbial concentration lead to excess growth of filamentous bacteria) [15], (iv) feed strategies (feast famine system supports the co-habitation of microbes in accumulation of PHA and helps to select microbes with low growth rate) [16], (v) aeration and DO (aeration rate and up-flow air velocities lead to hydrodynamic shear forces which enhances the EPS generation and thus improves the structural integrity of granules) [17], (vi) pH and temperature [12].
In the various reactor system configurations reported, the development of aerobic granules was found to be appropriate in sequencing batch reactors (SBRs). All the stages of reactor operation involved in conventional activated sludge process occur in a single unit in SBR and hence, there is no requirement for a clarifier which saves the footprint [18]. The high biomass retention lets a rise in the volumetric exchange amounts. Also, the operational parameters can be easily manipulated in SBRs to achieve desired results [19].
Granular sludge technology is being successfully employed in the treatment of domestic and industrial wastewaters contaminated with C, N, and P. A lab scale SBR was operated to treat synthetic wastewater containing higher concentrations of P by aerobic granular sludge technology. Other than glucose, two other volatile fatty acids named butyrate and valerate were used as additional C source and their effect on granulation process was studied. It was found that presence of butyrate enhanced the enrichment of PAOs and removal efficiencies of 95% of COD, 75% of P, and 99% of N were achieved [20]. Desireddy and Sabumon [21] developed a stable aerobic granular system to simultaneously remove C, N, and P under alternating aerobic and anoxic environments in an automated sequential batch airlift reactor. Strategies like varying dissolved oxygen concentration and feed cycle times were applied to achieve stable and dense aerobic granules of size 1.3 to 8 mm. Effective removals of 90% of N, 90% of COD, and 83% of P were attained. In another study long term operation of aerobic granular sludge system treating real wastewaters with low C/N ratio (3.8 ± 1.6) and influent COD of 133 ± 12 mg/L was carried out. Long SRT (sludge retention time) of 61 d, lead to less biomass yield per COD removed, and effective removals of COD (84%), N (71%), and P (96%) were achieved [22]. Nevertheless, this technology faces certain disadvantages like requirement of pre/post treatments in the presence of higher concentration of suspended solids, high start-up time required for the formation of granules, and disintegration of aerobic granules affecting the process performance [23, 24].
This work attempts to develop stable aerobic granules to achieve simultaneous nutrient removal in a lab-scale SBR where imhoff cone is used as the reactor and such studies are not reported where a conical geometry of the reactor for promoting aerobic granulation. Various studies reported the disintegration and washout of aerobic granules after few days of operation leading to process failure [25]. In this context, the granules were developed in an imhoff cone and maintained for a long period of operation of 328 days with zero sludge discharge. Hydraulic selection pressures by short settling time, low hydraulic retention time, and high organic loading rate were reported as important factors in granule formation, shape, and stability [26]. Compared to the planar bottom of cylinder, the tip of the cone consists of boundless set of eddies even at a very slow motion which gives rise to swirl of fluid at the edges of the reactor and flows upstream. These eddies provide homogeneous mixing and hence uniform growth conditions prevail [27]. It also provides uniform hydrodynamic shear stress which helps in granulation. Generally, conical shaped hydro cyclones are used as sludge separators to intensify the deammonification process [28]. However, conical shaped reactors were not used as reactors to culture aerobic granules. To compare the benefits of conical geometric configuration in the formation of granules, a cylindrical reactor of the same volumetric capacity was operated as control with similar operating conditions. Extracellular Polymeric Substances (EPS) produced were also quantified during the operation. Kinetic studies were carried out in the best phase III of reactor operation to understand the removal rates of COD, NH4+-N, and PO43−-P. The results presented in this work would help to promote design and operate low-cost decentralized wastewater treatment unit to handle low volume wastewater in a conical geometry by cultivating aerobic granules.

### 2.1. Mineral Media and Seed Biomass

The mineral media composition used in this study is as given in Table 1 and is intended to represent a typical COD, NH4+-N, and PO43−-P composition of domestic wastewaters. The seed inoculum was procured from an aeration tank treating domestic wastewater. It consisted of activated sludge flocs with an MLSS (Mixed Liquor Suspended Solids) of 0.26 g/L and SVI30 (Sludge Volume Index) of 380 mL/g.

### 2.2. Experimental Procedure

SBR studies were carried out for 328 days using two reactors with 1 L capacity each. An imhoff cone (BOROSIL) was used as the experimental reactor, while a measuring cylinder (BOROSIL) was used as a control reactor. During start-up, both the reactors were added with 1 L of mineral media inoculated with seed sludge. The initial MLSS was 0.26 g/L. An air pump was used to provide aeration and the diffuser stone was placed at the bottom portion of the reactors.
The SBRs were operated at room temperature (30 ± 2 ºC) in four phases by varying the COD and cycle time. In the start-up phase and phase III of reactor operation, each reactor was fed once a day (24 h intervals), and in phase I and phase II reactor was fed twice a day (unequal cycle times of 9 h and 15 h). The SBRs were operated as fill and draw method comprising four stages during every cycle which consist of feeding mineral media (15 min), reaction (8 h/14 h/23 h; varied in each phase), settle (30 min), and decant (15 min). The operating parameters during various phases of reactor operation are given in Table. 2. The samples after each cycle were quantified for dissolved oxygen (DO), pH, NH4+-N, NO2-N, NO3-N, PO43−, and COD every day, while MLSS, EPS (carbohydrates and proteins content), and SVI were analyzed once every week.

### 2.3. Kinetics of Nutrient Removal

Kinetic studies of COD, NH4+-N, and PO43−-P removals were carried out in steady state phase III of reactor operation. The reactor was operated similarly, and samples were collected at every 2 h intervals from 0 h to 8 h and one sample after 24 h. All the samples were quantified for NH4+-N, NO2-N, NO3-N, COD, PO43−-P, and pH. The order and rate coefficients were determined graphically using integrated rate laws. The differential rate law for zero-order reaction is:
##### (1)
$Rate=k[S]0=k$
The integrated rate law is:
##### (2)
$[S]=-kt+[S]0$
Where,
• [S]0 is the initial substrate concentration

• [S] is the concentration at time ‘t’

• k is the rate constant

The plot of [S] versus t is a straight line with slope of –k and intercept of [S]0.
The differential rate law for first-order reaction is:
##### (3)
$Rate=-d[s]dt=k[S]$
The integrated rate law is:
##### (4)
$ln[S]=-kt+ln[S]0$
The plot of ln[S] versus t is a straight line with slope of –k and intercept of ln[S]0.
The differential rate law for second-order reaction is:
##### (5)
$Rate=k[A]2$
The integrated rate law is:
##### (6)
$1[S]=kt+1[S]0$
The plot of 1/[S] versus t is a straight line with slope of k and intercept of 1/[S]0.

### 2.4. Analytical Techniques

All the physico-chemical analyses were conducted as per the protocols of standard methods for the examination of water and wastewater [29]. The instruments used to measure DO and pH were DO meter (YSI 55 model, USA) and pH meter (Mettler Toledo-Five Easy Plus) respectively. The nitrogenous species (NH4+-N, NO2-N, and NO3-N) were analyzed using Ion Chromatography (IC) (883 Basic IC Plus, Metrohm) with incorporated MagicNet program to process the data. NH4+-N analysis was performed using cationic column (Metrosep C4-150) and eluent comprising 1.7 mM of HNO3, 0.7 mM diplocolinic acid, and 100 ml/L of acetonitrile as organic carbon suppressor. NO2-N, NO3-N, and PO43−-P analyses were performed with an anionic column (Metrosep A Supp5-250) and eluent comprising of Na2CO3 (0.3487 g/L) and NaHCO3 (0.084 g/L), and 100 ml/L of acetonitrile as organic carbon suppressor. Closed reflux dichromate digestion method was used to analyze COD using WTW-CR-3200 digester, (Germany). Microbial growth was measured by OD550 using a UV-Vis spectrophotometer (Evolution 300, Thermo Scientific). Later, the MLSS was quantified with a standard calibration curve that was established by relating MLSS with corresponding OD550.
EPS extraction was done by ethanol extraction technique. To begin with, the sludge samples were centrifuged at 20,000 rpm for 20 min at 4 ºC. To the obtained supernatant three measurements of ice-cold ethanol were supplemented and incubation was done at 4 ºC for 12 h. Later the EPS as precipitates were separated by centrifugation of the incubated samples at 4000 rpm for 15 min. The proteins and carbohydrates contents in EPS were quantified by the Lowry’s technique (BSA as standard) [30] and the phenol sulfuric acid technique (with glucose as standard) [31], respectively.

### 3.1. Performance of Conical SBR

The performance profile of conical SBR is shown in Fig. 1. The percentage nutrient removals at various phases of reactor operation are shown in Table 3 and the values of SVI30, MLSS, and MLVSS are given in Table S1. In the start-up phase effective nitrification was observed with an average NH4+-N removal of 78%, while the denitrification (42% of total nitrogen (TN) removal) was not effective due to higher DO levels in the reactor. Ineffective PO43−-P (43%) removal in the start-up phase could also be due to high DO levels. Also, the MLSS was very less and sludge lysis of un-acclimated biomass could be attributed to inefficient removals. This is evident from the decreased MLSS from 0.26 g/L at start-up to 0.14 g/L by day 14 of the reactor operation. DO in the reactor during start-up phase was in the range of 3.21 mg/L to 4.26 mg/L. In such DO levels, aerobic digestion of MLSS is possible on long starvation and the obtained results support aerobic digestion of biomass.
In phase I, the reactor was fed twice a day with cycle times of 9 h and 15 h respectively. Though the cycle time was decreased the NH4+-N and the TN removals remained almost stable which could be attributed to the acclimated biomass and gradual increase in MLSS (Fig. 2) due to the availability of abundant nutrients. A slight increase in COD and PO43−-P removals was also attained. The efficiency of the SBR in terms of nutrient removal was higher at a cycle time of 15 h compared to 9 h (Table 3).
In phase II, two feed cycles per day was maintained as such, while the average influent COD was increased from 470 to 640 mg/L. The COD/N ratio increased from 9 in previous phases to 12 in this phase. The aeration was decreased to enhance denitrification and PO43−-P removal and the corresponding DO levels were in the range of 0.98 mg/L and 1.87 mg/L. Slight increase in removals of NH4+-N and PO43−-P was observed compared to previous phases. The increased organic loading rate did not affect the nitrification efficiency of the system. An increase in denitrification was also observed giving a TN removal of 64% which could be due to availability of COD and utilization of DO due to higher substrate concentration. TN removal efficiency increased at high COD/N ratio as a drastic decrease is seen in the effluent NO2-N and NO3-N concentrations. Similar results were obtained by Zaman et al. [32], where at a COD/N ratio of 7, 69% of TN was removed, while at COD/N ratio of 10 it increased to 86%. The COD/N ratio and DO levels show a direct effect on TN and PO43−-P removal. At a DO of 1 mg/L and COD/N ratio of 4, only 65% of TN and 37% of PO43−-P removals were achieved by Wang et al. [33], whereas the removal efficiencies increased at high COD/N ratio. The COD removal percentage was slightly less in this phase which could be attributed to increased influent concentrations. The increase in PO43−-P removal efficiency could be due to the decrease in DO levels and also less effluent NO2-N and NO3-N concentrations. Denitrifiers dominate the PAOs and dPAOs in utilizing COD in the presence of high NO2-N and NO3-N levels. Also, high DO levels are not favorable for the enrichment of dPAOs as the genes responsible for nitrite reductase cannot be expressed at high DO. In such studies with high DO, phosphorus removal by dPAOs was not emphasized [34]. Several studies related to aerobic granules and simultaneous nutrient removal were carried out at DO levels of 2 mg/L to 5 mg/L and COD/N ratio of 7 to 11 [32]. By the end of this phase, few small granules were formed in the SBR which would lead to development of denitrifiers and PAOs in the granule interior thereby leading to effective TN and PO43−-P removal. However, complete granulation and mature granules were not obtained even in phase II of operation which could be due to overgrowth of filamentous biomass on the outer layers of flocs at high influent COD concentrations and low DO.
In phase III the reactor was fed only once a day with a cycle time of 24 h. In this phase complete granulation was seen after day 236 with stable granules in the size of 0.9 ± 0.3 mm. Less DO levels would decrease the diffusion depth of oxygen in the granules leading to small aerobic zones and large anoxic zones which impact nitrification and denitrification [35]. Phase III was operated for a very long time and no decrease in the removal efficiencies was noticed. After stable granule formation, the MLSS in the SBR stabilized. Effective average removals of COD (90%), NH4+-N (91%), TN (87%), and PO43−-P (83%) were achieved in this phase. Generally, removal of PO43−-P is efficient at low sludge age. However, in this reactor, though the sludge age was high, no effect on PO43−-P removal was noticed which could be due to the formation of precipitates of phosphorous in the inside of granules. The MLVSS was less during the initial few days of phase III (0.23 g/L). However, by the end of phase III the MLVSS increased to only 0.52 g/L though the SVI decreased which confirms that a part of PO43−-P is removed by precipitation. The presence of Ca2+ and Mg2+ in the mineral media helps in the precipitation and this is enhanced by the presence of PAOs. Formation of precipitates is also beneficial to the system as the granules get densified and settle easily eliminating the need for clarifiers [36].

### 3.2. Development of Aerobic Granules

The reactor was inoculated with activated sludge flocs with an SVI30 of 380 mL/g and an MLSS of 0.26 g/L. As the MLSS was very less, a settling time of 30 min was maintained to avoid the washout of biomass. However, before inoculating, the seed sludge was washed thrice with mineral media with a settling time of 10 min to allow the washout of low settling biomass. The physical observation of the biomass by the end of start-up phase did not show the formation of granules and bigger flocs were still dominant in the reactor. A slight increase in the SVI30 to 440 mL/g was observed by the end of start-up phase which could be due to the sludge lysis during acclimation period. During start-up and phase I of reactor operation the DO levels in the reactor were ranging between 3.21 mg/L and 4.26 mg/L. By the end of phase I of operation, the size of activated sludge flocs decreased and smaller flocs with good settling abilities were formed. SVI30 also decreased greatly from the inoculation to day 53. In phase II, the aeration was decreased and the DO levels in the system were in the range of 0.98 mg/L to 1.87 mg/L. As the influent substrate concentration was increased, during the initial few days of phase II slight filamentous growth was observed on the exterior of the flocs. Slight increase in the SVI30 was also noticed compared to phase I of operation. At incomplete organic substrate uptake, aerobic degradation of organic matter occurs at the surface of flocs or granules leading to the development of fluffy granules and similar results were reported [37].
In phase III of operation, the cycle time was again increased to 24 h. After stable operation from day 88 without varying the operating parameters, from day 126 onwards formation of granules was noted. By day 236 mature granules of size 0.9 ± 0.3 mm were developed without any filamentous growth. The size was measured as reported by Desireddy and Sabumon [21]. To find the size of granules homogenous sample of 500 mL was collected and passed through a series of sieves with various mesh sizes (1.5 mm, 1.2 mm, 1 mm, 800 μm, and 500 μm). The corresponding MLSS and MLVSS in the reactor were 2.15 g/L and 0.52 g/L (24%), respectively. The MLSS and MLVSS on the first day of phase III was 1.76 g/L and 0.23 g/L (13%), respectively. The percentage of inorganic content increased while the SVI30 and SVI15 decreased, which indicates the formation of phosphorous precipitates in the core of granules after long-term operation. Decreased DO levels would have helped in the generation of layers in the granule with nitrifiers, denitrifiers, and PAOs on the outer layers and dPAOs in the interiors of granules. The reactor was operated for 240 days in phase III. By the end of reactor operation (day 328) also the granules were not disintegrated even at a very high SRT of 328 days: considering negligible biomass in the decanted effluent in each cycle. Slight fluctuations in the granules size ranging between 0.6 mm and 1.2 mm was observed in phase III. However, the granules remained intact and stable which could be due to their smaller size. The conical geometric configuration and induced shear force by low rate of aeration would have helped in maintaining the granule size. If the granules were bigger, they easily get breakdown into smaller granules and again regrow. Sometimes due to endogenous respiration, the granules get destabilized leading to smaller fractions. By the end of phase III, the SVI30 decreased to 65 mL/g while SVI5 was 67 mL/g giving an SVI5/SVI30 ratio of 1.03 indicating good settling nature of the developed granules.
There are fluctuations in the EPS during the study period (Fig. 2). EPS during start-up was high which could be due to the sludge lysis and enhanced aerobic digestion at high DO on prolonged starvation period. From phase III of operation gradual increase in the EPS was noticed which helps in the formation of granules. Also, it is observed that there were not many fluctuations in EPS after 200 days of operation till the end of the study, which could have helped in maintaining stable granules in the reactor.

### 3.3. Comparison with Control SBR

A measuring cylinder of 1 L capacity was used as a control reactor operated at the same operational conditions as shown in Table 2. The average percentage removals of COD, NH4+-N, TN, and PO43−-P in various phases of reactor operation are given in Table S2, and the profile of SBR is shown in Fig. 3 and Fig. 4. Up to phase I of operation, the nutrient removal efficiencies in control reactor were almost similar to that of conical SBR while no improvement in COD, TN, and PO43−-P removals occurred. The SVI30 and SVI5 are not comparable with that of conical SBR. This could be due to the non-uniform aeration in the measuring cylinder which led to settling of some amount of biomass at the bottom corners of the control SBR. There was no proper shear force and velocity gradient to help in the enhanced formation of aerobic granules. Hence effective granulation was not seen in the control reactor. However, the nitrification efficiency throughout the study was similar to that of conical SBR, while the TN removal efficiency is not satisfactory. The ineffective COD, TN, and PO43−-P removals could be due to the existence of non-granular biomass in the reactor. The MLSS in the conical SBR on the last day of reactor operation was 2.31 g/L, while it was 1.96 g/L in the control reactor. As there were no proper granule formation and the settling was not effective, there would be a higher chance for the escape of biomass from the reactor during decant phase. Also, the MLSS measured occasionally (results not shown) confirms that the effluents from conical SBR are clear with negligible solids compared to the control reactor. The EPS on day 328 in conical SBR and control SBR were 124 mg/g/VSS and 98 mg/g/VSS, respectively. The microscopic images (4 times magnification using phase contrast microscope; Zeiss-Primo star) of biomass drawn on day 150 from the conical SBR compared with that of control SBR is shown in the supplementary section (Fig. S1). From the images, it is evident that conical SBR is able to hold denser and granular biomass compared to the control reactor. Fig. S2 (supplementary) shows the schematic of SBRs with the highlights of the results obtained. Based on these results, it can be confirmed that the SBR with conical configuration is beneficial in terms of granule formation and simultaneous removal of COD, NH4+-N, and PO43−-P cost-effectively without needing any costly instrumentation control. Also, the results obtained on the long-term operation are helpful in further designing and piloting conical configurations of SBR for cultivating aerobic granules in low DO concentrations for simultaneous removal of COD, nitrogen, and phosphorous.

### 3.4. Kinetics of Nutrient Removal

Kinetics data of COD, NH4+-N, NO2-N, NO3-N, pH, and PO43−-P are plotted in Fig. 5. Results show a gradual decrease in NH4+-N up to 8 h with slight accumulation of NO2-N and NO3 N whereas slight increase in NH4+-N and high accumulation of NO2-N and NO3-N levels were observed at 24 h. Similar trends were observed in the removals of COD. This indicates the lysis of biomass after depletion of nutrients. Immediately after adding the feed, homogenous sample was collected and measured for COD. While the feed contained 640 mg/L of COD, it swiftly decreased to 313 mg/L at 0 h. The high concentration of COD leads to penetration of COD into the interior of granules swiftly and ultimately could be stored as PHB by heterotrophic microbes inside the granules. At 0 h slight increase in PO43−-P (10.2 mg/L) was also noticed which could have been released due to assimilation of carbon during the feeding stage. However, the phosphorous released to COD used ratio (0.004) is negligible compared to the typical ratios (0.3) of anaerobic periods as reported in the literature [38]. Up to 8 h, no much accumulation of NO2-N and NO3-N was seen even though effective nitrification occurred. Also, a gradual decrease in COD and PO43−-P removals occurred which implies the occurrence of nitrification, denitrification, and denitrifying P removal simultaneously. After majority of the PO43−-P is removed by 8 h, at 24 h increase in accumulation of NO2-N and NO3-N was seen which signifies the presence of dPAOs in the reactor. The DO was in the range of 0.9 mg/L to 1.5 mg/L during the kinetic studies. The presence of heterotrophic nitrification and aerobic denitrifcation (HNAD) species is also possible in the SBR under these operating conditions, which have unique ability to denitrify in both aerobic and anoxic environments [39]. The reactor might be dominant in nitrifiers compared to denitrifiers due to the prevalence of continuous aerobic conditions. However, the gradual decrease in COD and PO43−-P removal shows that the granules contained mixed microbial consortium that involves in SNDPR. Slight decrease in pH was observed at 4 h which could be due to aerobic nitrification, however, the pH again increased at 8 h and 24 h indicating simultaneous denitrification. The pH was in the range of 6.6 to 7.2 during kinetic studies. Filipe et al. [40] reported that at a pH higher than 7.25, denitrifying glycogen accumulating organisms (dGAOs) dominate dPAOs. Hence in this system dPAOs would be the dominant organisms that are favorable, as dPAOs are known to reduce NO2-N and NO3-N more effectively than dGAOs. Also, literature shows that dPAOs prevail effectively at lower DO concentrations compared to dGAOs due to the high oxygen affinity of PAOs. Carvalheira et al. [41] reported that phosphorous uptake rate of PAOs decreased by 20% while that of GAOs decreased by 77% when DO was decreased from 8 mg/L to 0.6 mg/L. Hence the low DO and pH values could have led to the favoritism of PAOs or dPAOs in this system. However, the composition of mixed culture was not done in this study by advanced microbiological identification techniques, due to lack of facility.
NH4+-N and PO43−-P removals followed zero-order kinetics with a rate constant of k = 6.22 M/h (R2 = 0.98), and k = 1.03 M/h (R2 = 0.98), respectively. The kinetics of COD removal was fitted well with first-order kinetic model with k = 0.28/h and R2 = 0.98. Though the pH was not controlled in the reactor, it was in the neutral range all through the study and hence there was no need for an external supply of alkalinity. The obtained results indicate the possibility of SNDPR in a single unit. Also, the kinetics study reveals that for effective and optimal treatment, the cycle time required is 8 h. Therefore, it is possible to operate conical SBR in 8 h cycle time yielding effective SNDPR.

### 4. Conclusion

This study reveals the effectiveness of a SBR with conical geometric configuration in development of aerobic granules for simultaneous nutrient removal via SNDPR. Low DO values are beneficial in simultaneous C, N, P removal in comparison with high DO values due to presence of large anoxic core in the granules. The mature and stable granules of size 0.9 ± 0.3 mm formed in phase III of operation were maintained for long term without any disintegration. The stable operating conditions and aeration in the SBR helped in effective granulation in conical SBR which was not the case in cylindrical SBR. Optimum average removal efficiencies of COD (90%), NH4+-N (91%), TN (87%), and PO43−-P (83%) were achieved in phase III of operation in conical SBR. This system holds several advantages like (i) decreased footprint, (ii) non-requirement of alkalinity, (iii) less energy input, and (iv) zero sludge discharge on prolonged starvation period in a cycle. While this study has shown a favorable approach for long-term maintenance of aerobic granules to assist in SNDPR, microbial population dynamic studies would further help in understanding the process and shall be taken up as next phase of research preferably in a pilot scale.

### Acknowledgement

We gratefully acknowledge Department of Science and Technology (DST), Government of India, for supporting this work through the research grant DST/TM/WIC/WTI/2K17/82(G2).

### Notes

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author Contributions

Dr. Swathi Desireddy (Senior Research Fellow) conducted experiments, investigation, formal analysis, validation, and writing (original draft). Ms. Sneha Madhavan (Engineer, B.Tech) assisted in conducting experiments. Dr. Sabumon P.C. (Professor) involved in conceptualization, data curation, funding acquisition, project administration, supervision, and writing (review & editing).

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##### Fig. 1
Performance profile of Conical SBR (a) COD profile (b) NH4+-N, TN profile (c) NO2-N, NO3-N profile (d) PO43−-P profile (e) DO profile (f) pH profile.
##### Fig. 2
Profile of EPS content and MLSS in conical SBR.
##### Fig. 3
Performance profile of control SBR (a) COD profile (b) NH4+-N, TN profile (c) NO2-N, NO3-N profile (d) PO43−-P profile (e) DO profile (f) pH profile.
##### Fig. 4
Profile of EPS content and MLSS in control SBR.
##### Fig. 5
Kinetics of (a) COD, PO43−-P removal, and pH (b) NH4+-N, NO2-N, and NO3N.
##### Table 1
Mineral Media Composition
Macro nutrients Concentration (mg/L)
Urea 183.48
NH4Cl 12.75
CH3COONa 79.37
Peptone 17.41
MgHPO4.3H2O 29.02
KH2PO4 23.4
FeSO4.7H2O 5.8
Starch 122
Milk powder 116.19
Yeast 52.24
Soy oil 29.02

Trace elements solution (1ml/L) (g/L)

ZnCl2 0.28
NiCl2.6H2O 0.51
AlSO4.16H2O 0.913
MnSO4.2H2O 0.38
H3BO3 0.4
Al2(MoO4)3 0.5
##### Table 2
Operational Parameters of SBRs
Phase Days of operation (days) Influent (mg/L) COD/N ratio Cycle time (h)
NH4+-N COD PO43−-P
Start-up 1 to 14 53 ± 2.5 470 ± 16 9 ± 0.6 9 24
I 15 to 53 53 ± 2.5 470 ± 16 9 ± 0.6 9 9, 15
II 54 to 84 53 ± 2.5 640 ± 32 9 ± 0.6 12 9, 15
III 85 to 328 53 ± 2.5 640 ± 32 9 ± 0.6 12 24
##### Table 3
Average Percentage Removals During Various Phases of Conical SBR Operation
Phase % Removals

COD NH4+-N TN PO43−-P
Start-up 66 ± 18 78 ± 20 42 ± 20 43 ± 11

I 76 ± 7# 78 ± 8# 55 ± 12# 61 ± 7#
75 ± 7* 73 ± 11* 40 ± 23* 47 ± 4*

II 70 ± 14# 82 ± 10# 64 ± 14# 73 ± 4#
64 ± 11* 64 ± 13* 57 ± 14* 67 ± 3*

III 90 ± 5 91 ± 7 87 ± 7 83 ± 3

* Cycle time – 9 h;

# Cycle time – 15 h

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