Inuence of Variations in Wastewater on Simultaneous Nutrient Removal in a Pre-anoxic Selector Attached Full-scale Sewage Treating SBR

In addition to many other well-documented factors, local conditions are rudimentary conditions of sharp change observed in wastewater characteristics from place to place. An optimized and exible treatment strategy is required to handle these uctuations and variations in quality. Pre-anoxic selector-equipped sequencing batch reactors (SBR) perform eciently in removing COD, BOD 5 , TSS, NH 4+ -N, TN, and Fecal Coliforms. The monitoring of 3-MLD Full-scale SBR installed at IIT, Roorkee, drew attention to the processes involving simultaneous nitrication and denitrication (SND) and biological phosphorous removal (BPR) undergoing with the variations in inuent wastewater, particularly the readily biodegradable COD (rbCOD), and their effects on micro-biota. Regular monitoring of all the units for a period of six months revealed that the overall average treatment/ removal eciencies were >94% COD (17.9±7.7 mg/L in euent), >95% BOD 5 (5.9±2.2 mg/L in euent), >95% TSS (9.3±2.1 mg/L in euent), >96% NH 4 -N (0.7±0.5 mg/L in euent), >86% TKN (4.2±2.9 mg/L in euent), >69% TN (9.7±3.0 mg/L in euent), >31% Ortho-PO 4 -P (1.8±0.7 mg/L in euent) and >42% TP (3.6±1.8 mg/L in euent) and achieved <50 MPN/ 100 mL fecal coliform in the nal euent after disinfection. Anoxic tri-sectional selector and an aeration tank constituted one SBR followed by the other availed 76± 9% SND at rbCOD/TCOD of 0.12±0.04, rbCOD/sCOD of 0.33±0.10, sCOD/TCOD of 0.35±0.06, and COD/TN of ~13. The study claries the degree of variations in key factors included in design guidelines for laying out an optimized treatment system for COD, Nitrogen, and Phosphorus removal in the Indian scenario. Evaluation of Pre-anoxic selector-equipped full-scale SBR based STP was performed. of Bio-selector improved the The rbCOD concentration signicantly affects the denitrication and TP removal.


Introduction
A comprehensive data of total organic matter present in the wastewater can be achieved by characterizing total COD (TCOD) into its various fractions. Additionally, the major characteristics of wastewater can be studied based on COD fractionations following ATV-A.131, 2000 guidelines, and subsequent modi cations (Płuciennik-Koropczuk and Myszograj 2019). The TCOD of wastewater, segregated in fractions, can be calculated as the sum of readily biodegradable COD (rbCOD) (metabolism), non-biodegradable soluble COD (nbsCOD) (observed in the treated e uent), biodegradable particulate COD (bpCOD) (i.e., slowly biodegradable COD (sbCOD)) (adsorption, hydrolysis, and metabolism) and non-biodegradable particulate COD (nbpCOD) (regarded in the sludge production) as g O 2 m -3 (Choi et al. 2017; Płuciennik-Koropczuk and Myszograj 2019). The general rbCOD/ TCOD in South African wastewater's raw sewage: 0.08 to 0.25 (Rossle and Pretorius 2001). The substantial the amount of rbCOD, the faster the nitrate reduction rate (Metcalf and Eddy, Inc. 2003). It has been investigated in different studies that carbon to nitrogen ratio (C/N) is an essential factor in biologically removing the nutrients (N and P) from domestic wastewaters, however readily biodegradable content in the TCOD also in particular directs the nutrient removal e ciency (Khursheed et al. 2018). Denitrifying bacteria requires an optimum carbon source for succeeding in excellent denitri cation, and therefore they have to contend with further heterotrophs. Lesser C/N ratio in the in uent effects in a rapid carbon discrepancy and consequences in unstable simultaneous nitri cation and denitri cation (SND) (Zhao et al. 2008; Phanwilai et al. 2020).
Enhanced biological phosphorus removal (EBPR) governs the prominent characteristic of uptake of organic matter and release of phosphorus in anaerobic states, and uptake of excess phosphorus under subsequent aerobic conditions. High phosphorus is accumulated in the sludge by phosphate accumulating organisms (PAOs). Polyphosphates are reduced to supply adenosine triphosphate (ATP) obligatory for the formation of Poly-β-hydroxybutyrate (PHB), and the degradation of polyphosphates is achieved by the discharge of ortho PO 4 -P, Mg, Ca and K. (Toerien et al. 1990; Davis 2013). The rbCOD concentration in the in uent predicts more accurately the performance of biological nutrient removal, consists of complex soluble COD that can be fermented to volatile fatty acids (VFA); therefore initial rbCOD to TP ratio is a better indication of the EBPR's process and performance than the total COD to P ratio (Barnard et al. 2017). Hence, in uent parameters like C/N, BOD 5 /COD, rbCOD/ TCOD (/or rbCOD/ sCOD), and rbCOD/TP play an essential role in enhancing the SND and BPR as observed in the study.
Barely limited literature is available based on investigating the in uence of wastewater characteristics on the nutrient removal process's e ciency in SBRs, so novel ndings in the present study may prove useful for further researches in this eld. Even the effect of an anoxic selector on the SND and EBPR process in SBR has not been explored to date. Moreover, the present study investigates the importance of anoxic bioselectors in improving sludge properties. As the main features concerning the activated sludge microbial diversity are the possible substrate composition of the incoming sewage and the ongoing signi cant operational variations in the treatment plant (Mielczarek 2012), therefore, this study aims to understand the pre-anoxic selector's effect and observe the in uence of wastewater characteristics on Nitri cation, Denitri cation, and Biological Phosphorus removal in SBR. Concomitantly, the sludge biomass and wastewater microbiome were considered as critically important.

Materials And Methods
This 3-MLD SBR has been set up in close vicinity to the residential area near the IIT, Roorkee campus, Uttarakhand (India). The important features of this institutional STP are the deodorization system's additional odor control for sump well, pre-treatment units, and advanced tertiary treatment facility (Fibre Disc ltration and UV disinfection) ( Figure 1). The onsite monitoring of various parameters was performed in the bio-selectors and aeration tanks of the 3 MLD SBR Plant.

Physicochemical parameters' analysis
Onsite monitoring of dissolved oxygen (DO), pH, oxidation-reduction potential (ORP), and SV 30 are executed regularly in the bio-selectors and aeration tanks of the 3 MLD SBR Plant. To determine the DO, temperature, and pH in the aeration tanks and selectors, a portable DO meter (Hach 110Q multimeter, Hach, USA) and pH meter (HQ11d pH Meter, Hach) was used. ORP was measured by the convenient ORP meter (HQ11d ORP Meter, Hach). Complete performance evaluation of the plants in terms of COD (total COD (TCOD) and soluble COD (sCOD)), Ultimate BOD (UBOD), BOD 5   The mass of total nitrogen in the dissipated sludge (Kg/d) was computed by the product of daily sludge wasted in L/d (q wasted), MLSS of the wasted sludge, and a fraction (%) of total nitrogen contained in the sludge wasted. The fraction was around 1.6% to 2.7% in the sludge.   Table 2 gives us an estimated range of different COD fractions in reported studies.  The overall nitri cation of ammonia was 96.7 ± 2.6%; total nitrogen removal was 69.1 ± 11.5%. Figure S3 (Supplementary Material) illustrates the total nitrogen, ammonia, and nitrates in in uent and e uent.
During the sampling period, the plant runs e ciently with total nitrogen in e uent achieved ≤ 10 mg/L.
The average TN in the e uent was 9.7 ± 3.0 mg/L. Higher values in the e uent that crossed the Indian standards (Recent Noti ed E uent standards of National Green Tribunal (NGT) (2019)) are observed at COD: TN range of 12-14 and 18-20 ( Figure 3). However, at COD: TN ratios <11, the TN in the e uent was <10 mg/L. According to Randall (1992), entire denitri cation can be attained at a TCOD/ TKN ratio of 7, which is also observed in the present study. Generally, at least a value of 9 is obligatory for accomplishing biological nutrient removal (Goronszy 1992). Isaacs and Henze (1994) proposed that 1.5 to 2.5 g COD/ g P is utilized for the removal of phosphates while the COD: TN ratio for denitri cation varies from 3.5 to 4.5 g COD/ g N (Pochana and Keller 1999), which is near to the hypothetical requirement supporting denitri cation with no COD loss during aerobic processes. A characteristic variation in the C/N ratio in the plant can be seen in Figure 3. Figure 3 represents that in the wastewater, the optimum COD: TN Ratio at which excellent TN Removal was obtained at ratios between 7 and 11. The range contained eight values of TN Removal in which best removal ~83% was observed at COD: TN of 9.

Relationship with rbCOD (Readily Biodegradable COD) and Simultaneous Nitri cation and Denitri cation undergoing in the plant
Denitri ers are recognized to struggle in search of carbon supply amid other heterotrophs; a lesser C/N ratio in the incoming wastewater outcomes in a quick carbon shortage, originating unstabilized SND (Zhao et al. 2008). SND in the SBR plant was 76 ± 9%, where average ammonia from the in uent wastewater was removed from ~21.8 mg/L to ~0.7 mg/L in the e uent. At the same time, absolute nitrate observed in the in uent and e uent was ~0.9 mg/L and ~5.6 mg/L, respectively. Detailed analysis of 3 MLD SBR, IIT Roorkee, was performed to analyze the relationship between rbCOD% and SND% ( Figure 4).

Effect of rbCOD: TN and BOD 5 : TKN on the denitri cation rate and the TN removal
To carry out the denitri cation process during biological treatment, the presence of readily biodegradable organic carbon is an indispensable factor (Randall 1992). Under anaerobic/ anoxic conditions, denitri cation capacity is evaluated by the requirement of available carbon source of nitrates, which is managed by the readily biodegradable fraction of COD (Tas et al. 2009). Operation data from 3 MLD SBR showed the effects of the in uent rbCOD: TN ratios on the e uent Nitrate concentrations operating in SND mode ( Figure S4), and the relationship showed a decreasing linear trend. It can be observed that higher rbCOD speci cally is needed to achieve denitri cation and which strongly in uences SND performance (Pochana and Keller 1999;Jimenez et al. 2010). Higher rbCOD: TN ratio above 2.0 showed higher SND (> 80%).
The BOD 5 : TKN ratio also shows signi cant effects on denitri cation rates and TN removal in the plant ( Figure S4). At a higher BOD 5 : TKN ratio above 6.0, more than 80% denitri cation was achieved, and when the BOD 5 : TKN fraction dropped below 2, lesser denitri cation was attained (Jimenez et al. 2010).
Similarly, at higher values of soluble BOD 5 to TKN ratio (sBOD 5 : TKN) > 3.0, better and consistent TN removal was observed in the plant, and e uent TN reached the stabilized results of 8.1 ± 2.2 mg/L while lower values of sBOD 5 : TKN < 1.6 showed higher TN in e uent 11.1 ± 3.9 mg/L ( Figure S4, Supplementary Material).

Total Nitrogen Balance
In the SBR, a typical total nitrogen (TN) balance has been observed ( gure 5). The destination of the incoming TN ~ 74.6 Kg/d in the inlet is followed by its expedition in three ways

Relationship with Readily Biodegradable COD and Biological Phosphorus Removal undergoing in the plant
The fraction of rbCOD to TP is an improved implication of the biological phosphorus removal process performance besides the total COD: TP ratio suggested in classic models. In the EBPR process, the soluble readily biodegradable fraction of COD gets fermented to VFA in the anaerobic zone (Majed and Gu 2019). The examined stoichiometry requisite of carbon for an elemental quantity of phosphorus to be removed has subsisted in the range of 10 to 20 mg rbCOD/ mg P eliminated (Barnard et al. 2017).
Elevated rbCOD/P ratios, i.e., 40 to 50 mg rbCOD/ mg P, have been perceived to be related to GAOcontrolled diversity, and smaller ratios < 10 to 20 mg-rbCOD/mg P have been related with PAO ledcommunity (Broughton et al. 2008). In 3 MLD SBR plant mg rbCOD/ mg TP ratio was on an average 9.6 ± 4.8 and mg COD/ mg TP and mg BOD 5 / mg TP ratio were observed as 81. 8 Figure 7 exhibits the effect of rbCOD/TCOD (%) and rbCOD/sCOD (%) on the total phosphorus and orthophosphate removal of the plant.
Zone-wise, PO 4 -P removal is illustrated in gure S6 (Supplementary Material). The release of phosphate is observed as 26.4% in the anoxic selectors, and then a reduction in the aeration tanks was observed as 52.9%, which shows uptake of 26.5%. Return Activated Sludge (RAS) in nitrifying processes planned to eliminate ammonia includes considerable nitrate concentrations that are not suited to two-stage (anoxicaerobic) EBPR systems. In the following circumstances, prerequisites must be taken care of for denitrifying the return solids to circumvent negotiating the anaerobic zone's integrity, which might be ful lled by having one or more anoxic phases (Minnesota Pollution Control Agency 2006). Other than the requirements of rbCOD: TP ratio, the necessary conditions of EBPR are VFA to TP ratio should be more than 7, and pH supposed to be between 8.0 to 8.5 and 7.0 to 7.5, for anaerobic and aerobic zones respectively, for e cient EBPR process (Mino et al. 1998). ORP in the anaerobic, anoxic, and aerobic zones should cover the range of -100 mV to -200 mV, -50 mV to +50 mV, and +100 to +300 mV, respectively (Burkhardt 2012). Acid formation from the fermentation of rbCOD occurs at an ORP of -100 to -250 mV (Goronszy et al. 1992(Goronszy et al. , 1996. Even the sludge's phosphorous content should be reasonably more signi cant than the stoichiometric value. The phosphorous content in the sludge was 1.95 ± 0.80% of MLSS in the plant. In the anaerobic zone, VFAs are stored inside the bacterial cell. PAOs use PHAs & PHBs in the aerobic process during a lack of exogenous substrates sequestering soluble phosphorus as poly-phosphates (known as P uptake). This uptake is greater than the P released in anaerobic processes since substantial additional energy is generated by aerobic oxidation of the accumulated carbon compounds than used to conserve them in an anaerobic environment (Oehmen et al. 2007). Anaerobic long-covered sewer lines contain a high amount of VFAs and compensate for the need for a complete anaerobic chamber before SBR basins. Wastewaters that are more septic, from collection systems in warm climates and minimal slope, will contain a high concentration of VFAs (Broughton et al. 2008). But, if fermentation could not happen in the collection system, it must take place in the anaerobic region so that EBPR can work sound.
The hydraulic retention times of the anaerobic zone must ful ll the limit between 0.5 to 2.0 hours (Burkhardt 2012). However, RAS's falling in the anoxic chamber of selectors dampens plants' productivity in removing TP biologically. If the anoxic selectors could not reach the particular requirement, su cient formation of VFAs might not occur. VFAs' sources were observed inadequate for proper conditions of PAOs' growth and effective EBPR in the 3 MLD SBR plant. Though some fermentation of rbCOD occurred at an ORP of -90 ± -24 mV in the third compartment of anoxic selectors of the SBR plant, and EBPR occurred as ~18%.

Storage products (PHBs) for SND and EBPR
The prospectives for PHB to supply electrons for an e cient SND process can be observed in SBR plants.
The non-rapid degradation characteristics of PHB clari es that it is a deserving active substrate for the SND process (Third et al. 2003;Miao et al. 2015). Internally stored PHBs are removed much slowly than the soluble substrate and therefore can be employed as an electron donor for denitri cation when exogenous carbon sources are not present (Table S4, Supplementary Material) (Third et al. 2003). The capacity of heterotrophs to quickly sequester the soluble substrate and conserve it as a slowly biodegradable polymer signi es expedient chances in preserving reducing power for SND. PHBs are found su cient as granules within a lamentous sludge or inside the large ocs governing SND.
Granular formation of biomass, producing better-quality supernatant-biomass separation and high concentrations of mixed liquor suspended solids (MLSS), and its capability to achieve more excellent loading rates have been monitored in several anaerobic processes. However, the mechanism of this phenomenon remains to be incomprehensible (Morgenroth et al. 1997). As discussed earlier in the phosphorus removal; Polyphosphate Accumulating Organisms (PAOs) uptake organic substrate, and PHB formation occurs by sequestering rbCOD, PO 4 -P is released, and exogenous BOD is consumed in anaerobic condition. PHA, glycogen, and poly-P are the storage products for PAOs. PHAs are 0.2 to 0.5 µm sized granules, which are present in the cytoplasm of the cell enclosed by a lm (membrane). Frequent PHA preserved by bacteria is Poly-β-hydroxybutyrate (PHB), a lipid-resembling polymer of 3hydroxybutyrate. However, there are some poly-P collecting bacteria (e.g., M. phosphovorus), which do not accumulate PHA but preserve trehalose, poly-P, and glycogen (Sathasivan 2009).
The literature suggests that PHB formation has a signi cant role during the processes of SND (a potential substrate for denitri cation) and EBPR (a potential substrate for excess phosphorus uptake by PAOs in the aerobic phase). In

Microbial characteristics and identi cation
This study substantiated the prospect of attaining granular sludge in an anaerobic/aerobic sequencing batch reactor with a complete SND and biological phosphorus removal performance. Operational litheness of the SBR (capability to lessen settling time, initial reactor volume, etc.) played a pivotal role in promoting compact granular biomass formation and maintenance. After staining the samples for PHB, microscopic observations revealed that the biomass consisted of a microbial community diverse in terms of morphology, physiology, and anaerobic PHB storage. The biomass assessment concerning the microscopic features of the EBPR phenomenon, SND, and phylogenetic identi cation of the microbial populations should be performed simultaneously to put adjacent to the function of microorganisms and identity observed in EBPR systems (Dulekgurgen et al. 2003).
Several protozoa species are identi ed in the sludge samples (Table S5,

Pathogens removal (Total Coliforms and Fecal Coliforms control)
The disinfection of the nally treated e uent was carried out by Ultraviolet radiations and chlorine dosing together. The in uent and e uent Total Coliforms were 3600000 ± 80 MPN/ 100 mL and 5400 ± 10 MPN/ 100 mL, respectively resulted in 3 log removal. Fecal coliforms also reduced to 35 ± 9 MPN/ 100 mL from 160000 ± 13 MPN/ 100 mL (~ 4 log removal). Fecal coliforms in the nal e uent completely satis ed the latest NGT standards.
3.12. The overall effect of qualitative and quantitative variations on plant performance The ratio of in uent BOD 5 to COD impacts nutrient removal performance. BOD 5 : total COD signi es the biodegradable carbon content in wastewater (biochemical oxygen demand) from the whole organic matter (chemical oxygen demand) in wastewater, which is quite imperative for e cient nutrient removal in wastewater treatment plants. Therefore, the evaluation of wastewater propensity for biological treatment is widely revolving around BOD 5 / COD (Gajewska et al. 2015). An increasing linear trend was observed in BOD 5 to total COD ratio, and TN and TP removal in the SBR plant has been observed in the plant ( Figure S7, Supplementary material).
The temperature varied from 10 C to 30 C during the study. The average MLSS and MLVSS in the aeration tanks were 7189 and 3087 mg/L (Aeration tank 1) and 7518 and 3740 mg/L (Aeration tank 2). Average in uent NH 4 -N decreased from 22 ± 5.8 mg/L to 0.7 ± 0.5 mg/L (96.7% removal), and TN removal was 69%. DO concentration varied from 0 mg/L to 2.48 mg/L during aeration and 0.02-0.22 mg/L during settling/ decanting. ORP uctuates between -66 mV to -114 mV in the anoxic selector compartments and reaches ~140 mV in the peak hours during the aeration phase in the SBR. It ultimately attained ≤ 50 mV during the settling and decanting phases. Table S1 in the Supplementary material demonstrates the following parameters. The DO and ORP pro les with the COD and nutrient removal can be observed in Figure 9. Figure S8 (Supplementary Material) illustrates the pro les in the compartments of the anoxic selector. The third compartment of the selector ORP reduces to < -90 mV, contributing to 39.7% denitri cation. When RAS (15 -30 minutes contact time) from aeration tanks goes to these selectors, the microorganisms meet a greater amount of substrate and limited DO concentration in the anoxic selector, natural selection of foremost oc-formers occurs. Large ocs produce, which strengthens the SND e ciency of the plant.

Conclusion
This study demonstrates the impact of pre-anoxic selector in the variation of wastewater and its in uence on the treatment e ciency of the SBR plant. The plant was e cient in treating the simultaneous organic matter, suspended solids, nutrients, and pathogens in wastewater. An authentic relationship of rbCOD was observed with SND, and total phosphorus removal and some enhanced uptake of phosphorus were observed. ORP control, rbCOD: TP ratio, upcoming VFA from the sewer lines are the concrete parameters for providing characteristic enhanced biological phosphorus removal. SBR has multi-compartment anoxic bio-selectors that effectively make bigger ocs in sizes and encourage SND to occur delightfully in the aeration tanks. The soluble organics in the raw sewage are sequestered as intracellular compounds in the biomass utilized for SND. Biological phosphorus removal took place under managed cyclic aeration sequences in SBR in the aeration zone, informing good sludge devoid of bulking and foaming. Bioselector improved the sludge morphology, and the prevalence of protozoa indicated excellent sludge formation. Besides, some denitri cation also occurred in the selectors. Stored substrates are visible using Sudan Black B staining, also con rming superior SND in the plants. Finally, disinfecting the e uent by UV and chlorination intensi ed the quality of discharged e uent and made it satisfactory for Indian e uent discharge standards. Further assessment of microbial ecology present in the full-scale plant's activated sludge could open novel wastewater treatment dimensions.

Declarations Acknowledgment
The rst author is thankful to the Indian Institute of Technology, Roorkee, for Ph.D. assistantship during the study and monitoring and researching the 3 MLD SBR plant, authorized by IIT Roorkee. The authors are also thankful to the operator and workers inside the plant who cooperated during regular sampling and monitoring of the plant.