Post-Treatment of Kennel Wastewater With Different Aeration Strategies

The objective of this study was to evaluate the effect of the introduction of a complementary aerobic treatment composed of a submerged aerated biological lter (SABF) with a secondary clarier (SC), followed by horizontal subsurface ow constructed wetlands (CWs), after anaerobic units, on the ability to remove pollutants in different aeration phases (Ph1, Ph2, and Ph3) at the euent treatment station of the Parque Francisco de Assis (PFA) dog shelter. Ph1 and Ph2 had 7 and 5 hours of daily aeration, respectively, and Ph3 had intermittent aeration every 2 hours. The phases were monitored regarding the removal eciency of organic matter, solids, nutrients (N, P), coliforms, and detection of Giardia and Cryptosporidium. It was found that post-treatment provided greater removal eciencies and that the aeration strategy of Ph3 showed mean eciencies of 71% for COD removal and 77% for BOD removal, being similar or statistically higher, even with less biodegradable euent, than those of Ph1 and Ph2. The SABF and SC removed N by nitrication and denitrication, leaving a total Kjeldahl nitrogen (TKN) concentration in the euent of 18 mg L −1 . The CW showed potential for simultaneous nitrication and denitrication (SND), in addition to solid ltration. The system did not satisfactorily remove thermotolerant coliforms (ThermC) (1 ± 0 log). PCR suggested the presence of the pathogens Giardia and Cryptosporidium in all post-treatment units in Ph1 and Ph2.


Introduction
The lack of or ine cient treatment of wastewater before it is returned to the environment may result in reduced levels of dissolved oxygen (DO) and eutrophication of the aquatic environment, in addition to contamination with microplastics, endocrine disruptors, heavy metals, and pathogenic organisms, causing environmental, economic, and aesthetic losses (Gao et  In Brazil, a large number of wastewater treatment plants (WWTPs) still apply only primary and secondary anaerobic treatment stages, which is insu cient for reducing the concentration of nutrients, such as nitrogen (N) and other compounds, before discharge into a waterbody. Systems that combine anaerobic and aerobic treatment with potential for nutrient removal are an alternative for improving wastewater treatment (Chernicharo et al. 2015;Brasil 2017).
Furthermore, to achieve biological N removal, it is necessary to alternate aerobic and anoxic conditions so that nitri cation and denitri cation and/or absorption of N inorganic forms occur (Zoppas et   The WWTP is composed of two parallel systems of preliminary and primary/secondary anaerobic treatments that receive different amounts of wastewater because the WWTP was built on uneven terrain. One of these systems, which receive the largest volume of wastewater, performs preliminary treatment by grating and primary treatment in a 4-m 3 clari er. Next, the wastewater is directed to a primary/secondary treatment unit consisting of an ST-ANF unit with 10 m 3 of capacity. The other treatment system, which receives the lowest volume of wastewater, is composed of grating followed by an ST-ANF with 5 m 3 of capacity, without a clari er (

Aeration phases and system monitoring
The physical, chemical, and microbiological variables were monitored at all treatment stages by collecting e uent at the following points ( Figure 1): P1A -grating of the larger ST-ANF unit, P1B -grating of the smaller ST-ANF unit, P2A -after the larger ST-ANF unit, P2B -after the smaller ST-ANF unit, P3 -e uent pumping tank of the A and B ST-ANF units (in ow into the SABF), P4 -out ow from the SABF, P5 -out ow from the last SC, and P6 -out ow from the last CW.
Monitoring was performed during periods with different SABF aeration times, with an oxygen application rate of 0.28 g L -1 h -1 O 2 .

Physical, chemical, and microbiological analyses
For the physical and chemical analyses, simple samplings of the wastewater were performed weekly at all monitoring points, using a previously cleaned polyethylene ask, for the duration of each phase. The removal e ciencies in each treatment unit and in the system as a whole were calculated from the weekly results.
The data were subjected to Tukey's and t test at a 5% signi cance level to assess whether the means were signi cantly different from each other. The test was performed using STATISTICA ® software.

DNA extraction and molecular analysis by PCR for the detection of Giardia and Cryptosporidium
The last samples from the SABF, SC, and CWs (P4, P5, and P6) from aeration phases 1 and 2 (obtaining a microbiota more adapted to the condition of each phase) were tested for the protozoans Giardia and Cryptosporidium by polymerase chain reaction (PCR). The samples underwent basic sedimentation and sludge separation procedures. The sludge was pipetted (2 mL to 5 mL), stored in Eppendorf tubes, and sent to the Laboratory of Water and Sewage Microbiology of the Department of Sanitary and Environmental Engineering of Federal University of Minas Gerais (Universidade Federal de Minas Gerais (UFMG)).
Genomic DNA was extracted from 0.5-g aliquots of all samples using the FastDNA ® SPIN Kit for Soil (MP Biomedicals) according to the manufacturer's instructions. Aliquots of 3.0 μL of DNA extracted from the samples were subjected to electrophoresis in a 1.2% agarose gel (80 volts, 40 minutes) to determine the result, followed by quanti cation in a Nanodrop 1000 spectrophotometer (Thermo Scienti c).
For PCR, primers P241F-P241R for Giardia and P702F-P702R for Cryptosporidium were used at a concentration of 30 pmol µL -1 . A premix (Phoneutria, Brazil) containing standard buffer, nucleotides, and Taq polymerase was used in the PCR. The annealing temperature followed the speci cations of each primer. The presence and size of the ampli ed fragments were visualized on a 1% agarose gel with a 2-μL aliquot of the PCR product. Negative controls containing only the PCR reagent and ultrapure water were used for quality control of the reactions.

Results And Discussion
The monitoring results are shown in Figure 2 and are discussed below by the treatment stage (A and B anaerobic systems and aerobic post-treatment) and the system as a whole.

A and B anaerobic systems
The mean pH values of the e uent in owing into the two anaerobic treatment systems (Figure 2a, b, and c) were higher than the typical value (7.0) of domestic sewage (von Sperling 2017a), than that found by Silva and Souza (2011) in the studied ST-ANF (7.3), and than the optimal pH range for the development of methanogenic archaea (6.6 -7.4) (Chernicharo 2007). Therefore, the system may not be performing well in terms of microbiological degradation, requiring pH control interventions.
In B system, however, the pH remained within the range reported by von Sperling (2017a), 6.7 to 8.0, while in A system, the mean pH was above the upper limit. This may be due to the presence of some chemical substances at higher concentrations in the e uent generated by the washing of the stalls, which is directed to P1A. The system in question covers more animals, in addition to stalls sheltering dogs that are undergoing treatment and receiving medication or that underwent surgical procedures. In addition to this hypothesis, the mean COD concentrations (Figure 2d, e, and f) found in system A, in the three phases, were higher than those in B system. In both systems, the mean COD concentration was higher than the mean observed by Souza  The mean BOD/COD ratio of the e uent in owing into the system, calculated from the data presented in Figure 2d, The mean organic matter (BOD and COD) removal e ciencies in A and B systems were not satisfactory, as the removal expected for these treatment units, 80 to 85% for BOD and 70 to 80% for COD, was not achieved (von Sperling 2017a) and the minimum e ciency required by law for e uent discharge into waterbodies, 85% for BOD and 75% for COD (Minas Gerais 2008), was not met ( Table 2). The concentrations at the end of the anaerobic treatment were also higher than the maximum concentrations allowed for discharge   The removal e ciencies of A system were higher than those of B system, not only for organic matter but also for solids and nutrients. As shown in Figure 2, A system has a primary clari er before the ST-ANF unit, which helps to increase the e ciencies through the retention of solids, including organic compounds and nutrients (nitrogen and phosphorus) bound to the solids.
It is known the nutrient removal of anaerobic treatment units is limited (Chernicharo 2007 Therefore, the e ciencies found in the anaerobic treatment of the PFA-WWTP were low, and there were no signi cant differences (p>0.05) between the nutrient concentrations of the inlet and outlet of either system. within what is considered satisfactory (Figure 2v, w, and x), although there were no signi cant differences between the inlet and outlet.
In addition, the geometric mean at the outlet of the anaerobic treatments at P3 was still high, 2x10 6 MPN 100 mL -1 .

Aerobic post-treatment
As it was necessary to increase the e ciency of the PFA-WWTP, the facultative lagoons were replaced by an SABF, a clari er, and CWs, and the contribution of the reactors is discussed below. The mean pH values (Figure 2a, b, and c) at P3 to P6 remained within the range appropriate for the activity of nitrifying microorganisms, 6.0 to 8.0, and decreased throughout the treatment, indicating nitri cation since H + ions are released in the involved reactions (von Sperling 2012; Zoppas et al. 2016).
Nitri cation was higher at P4 (aerobic treatment by SABF) than in the other post-treatment units (Figure 3a and b), as expected, with higher nitrite and nitrate concentrations due to the air blowing and aerobic conditions, which are favorable to the activity of nitrifying bacteria (nitrosation and nitration).
Using the Tukey test, it was observed that only for the variable BOD there was a signi cant difference in the in uent concentration in the different phases. The BOD concentration in phase 3 was signi cantly lower than in phase 2. Then, indicating that differences in post-treatment performance can be explained to the aeration conditions.
The nitrosation ( Figure 3a) and nitration (Figure 3b) in the SABF were signi cantly higher in Ph3 (intermittent aeration and higher O 2 supply), with higher mean concentrations of nitrite (0.05 mg L -1 ) and nitrate (3 mg L -1 ) than Ph1 (p<0.02 for nitrite and nitrate) and Ph2 (p<0.001 for nitrite; p<0.02 for nitrate). The results are consistent with the nitrifying bacteria count performed by Souza et al. (2020), an evaluation that showed that the highest density of nitrifying bacteria was found in this phase.
The increase in the concentration of NO 2 and NO 3 resulted in a higher concentration at the SABF outlet in Ph3, reducing the removal e ciency, which could explain the fact that there was no signi cant difference in the removal of nitrites and nitrates in Phase 3, as reported in Table 3.   In Ph3, there was also greater accumulation of sludge in the SC reactor (Figure 2), making the environment more anaerobic, which favored greater adaptation of heterotrophic bacteria, reducing nitrate to nitrogen gas (N 2 ). Under these conditions, denitrifying bacteria may become predominant due to a higher growth rate and cell yield (7. In phases Ph1 and Ph2, the sludge accumulation was lower because periodic cleaning of the SC was performed every 7 months, occurring once at the beginning of the monitoring, again during Ph2, and coinciding with the end of the study; thus, Ph3 did not include a cleaning intervention. Another factor that may have contributed to the sludge accumulation in the SC in Ph3 was the greater oxygen supply. Aerobic treatments are commonly characterized by large sludge generation (von Sperling 2017a; Jordão and Pessoa 2017), as observed in P4 (Figure 2m, n, and o); therefore, a increased O 2 availability contributed to greater organic degradation and sludge generation.
In Ph3, the SS concentration in the SC was 119 mg L -1 , higher values than in other phases, however, the e ciencies of TS (dissolved and suspended) and TP retention were only numerically greater than in phases 1 and 2 (Table 3). It is known that TP removal is related to TS retention, through the incorporation of the nutrient in the microbial biomass and, consequently, in the removed sludge as , and a Pall ring), a solid retention chamber after aerobic treatment was essential. According to these authors, due to the presence of the sludge retention chamber, immediately after the bio lter, it was possible to achieve a mean SS removal e ciency of 94% and a COD of 72%. In addition to increasing the solids removal, the clari er is important for increasing the service life of the CWs (Matos et al. 2018).
Higher organic matter degradation e ciency was observed, considering the COD removal in the SABF during Ph3 (statistically compared to phase 1), a period with greater applied aeration rate (Table 3) Thus, there was a greater percent reduction in the BOD concentrations than the COD concentrations by biological processes. It is noteworthy that, although the SABF a uent was less biodegradable in Phase 3 compared to Phase 2, with BOD concentration signi cantly lower, without the same occurring with COD, there was no signi cant difference in organic matter removal e ciencies. This condition indicates the importance of oxygen supply to increase the ability to remove organic matter.
The organic matter (BOD) removal by the SABF was complemented by the SC, which showed good e ciency; therefore, the BOD concentration at the outlet of the SC was 59 mg L -1 in Ph2 and 53 mg L -1 in Ph3, complying with the legal limit (60 mg L -1 ), which was not the case for COD, except in Ph3. In the phase with the longest aeration time, the CW was able to reduce the COD concentration to acceptable values for discharge into the watercourse (180 mg L -1 ) (Minas Gerais 2008), with an outlet concentration of 166 mg L -1 .
As described in other studies (Lizama et  Therefore, there were conditions for SND to occur in the CWs, as observed by the nitration and denitri cation e ciency data ( Table 3). The co-occurrence of aerobic, anoxic, and anaerobic conditions within a CW can increase the nitri cation and denitri cation activity The CW in Ph2 removed 1 log ThermC, as the SABF followed by the SC in Ph3, that showed better conditions for ThermC removal, which may be due to oxidation or competition with other microorganisms. However, the sludge dragged to the CW and, consequently, the accumulation of microorganisms may have reduced the inactivation conditions (as it could have happened on the CW in phase 3), considering that cultivated CWs operated with lower organic loads usually have a greater capacity for coliform removal due to the release of antimicrobial substances by plants (Avelar et al. 2014;Brix et al. 1989).
Regarding the detection performed by the PCR technique, the presence of the protozoan genera Giardia and Cryptosporidium was con rmed in the e uent samples in Ph1 and Ph2 at all analyzed stages (Table 4), con rming that the post-treatment was not able to remove pathogenic microorganisms. 3.3 Evaluation of the PFA-WWTP as a whole Analysis of the PFA-WWTP treatment system as a whole (Table 5) shows that Ph3, in addition to presenting satisfactory organic matter concentrations at the WWTP outlet (P6) (Figure 3f and i), showed higher removal e ciency than that established by law for COD (70%) and BOD (75%) (Minas Gerais 2008) ( Table 5). In the other phases, the removal e ciency reached the standards established by law only for BOD. Therefore, the addition of more aeration time, with intermittent supply into the bio lter, contributed to N removal and COD stabilization, which was high in the e uent studied, as in the study by Hasan et al. (2014). The removal of SS was satisfactory in all phases, despite the accumulation and dragging of the material in the SC and CW in Ph3, due to the greater sludge generation in the SABF with the greater oxygen supply. Possibly for this reason, the removal e ciency of TKN was low in this phase, while P removal did not differ statistically (Tables 3 and 5).
However, the concentration of N (TKN and NT) at the WWTP outlet (P6) in Ph3 (Figures 2r and 3c) was close to the legal limit. This shows that the treatment system has the capacity to provide e cient N removal, and this aeration strategy can be adopted, as observed by Pelissari et al. (2017;. However, a shorter interval between SC cleanings should be adopted to take advantage of the potential of the operating condition. The accumulation and dragging of sludge in Ph3 may also have been factors that reduced the e ciency of ThermC inactivation because the system showed good removal of microorganisms in Ph1, beginning after the SCs were cleaned, and in Ph2, in which cleaning was performed through monitoring (Table 3)

Conclusion
The anaerobic treatment composed of the ST-ANF units was not su cient for compliance with the legislation for the discharge of the treated wastewater from PFA into the local watercourse, especially regarding the variables COD and N. This justi es the use of subsequent post-treatment steps.
Post-treatment of the wastewater from PFA with an SABF, an SC, and CWs increased the treatment e ciencies, and the operation of the SABF with intermittent aeration every 2 hours provided the best results in terms of terms of nitri cation/denitri cation and organic matter removal (even being less biodegradable). Under this condition, in Ph3, the e uent COD was removed, and nitri cation rate was greater.
In general, the SC acted in the retention of solids and phosphorus incorporated in the sludge and in denitri cation. The SABF + SC combination was responsible for the removal of N in the PFA-WWTP, while the CWs exerted a polishing function, removing the remaining COD and N by SND, and acted in ltration, retaining solids, and dragging P and N from the SC.
The implementation of mechanisms for discharging the accumulated sludge in the SCs at intervals of up to 6 months is recommended to ensure greater nutrient and pathogenic microorganism removal e ciencies. The amount to be discarded should be better evaluated to prevent reduction of the denitri cation capacity of the SC due to anoxic conditions in the presence of the sludge.
The quanti cation of Giardia cysts and Cryptosporidium oocysts at the inlet and outlet of the system is recommended to correlate the density of these microorganisms to the high presence of coliforms. Methods for improving the quality of the treatment system, such as adding a disinfection phase and considering, in addition to the removal of organic compounds, the removal of pathogenic microorganisms, can also be considered. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. This paper originates from the PhD work of the rst and corresponding author.

d) Competing interests
The authors declare that they have no competing interests.