Full-Scale Application of One-Stage Simultaneous Nitrification and Denitrification Coupled with Anammox Process for Treating Collagen Casing Wastewater

The ammonia nitrogen (NH4+-N) concentration in the effluent released from the secondary sedimentation tank of the original collagen enteric coating wastewater treatment process considerably exceeded the Chinese effluent discharge standard. Therefore, a one-stage simultaneous nitrification and denitrification coupled with the anaerobic ammonia oxidation (SNDA) process was designed to terminally treat collagen enteric coating wastewater containing low COD/NH4+-N (C/N). The entire process start-up and NH4+-N loading (NLR) domestication phase was completed within two months. During the NLR domestication, the NH4+-N removal rate was more than 90% and its effluent concentration was less than 15 mg/L, guaranteeing that the NH4+-N in the subsequent effluent was within the standard value. The results of microbial diversity show that Acinetobacter, Bacillus, and other heterotrophic nitrification–aerobic denitrification bacteria, and anammox ammonia oxidation bacteria were the main functional bacteria at the genus level, exhibiting high denitrification performance. The one-stage SNDA process effectively and stably removed nitrogen; the treated sewage satisfied the national comprehensive wastewater discharge standard (GB8978-1996), effectively saving 30–40% of the floor area and reducing 67.6% of the additionally added alkali, wherein the system’s denitrifying bacteria compensated for some alkali consumed during the nitrification reaction.


Introduction
Guangxi Wuzhou Shenguan protein-casing factory specializes in casing production using cow leather as a raw material, and the acid hydrolysis method to prepare and extract collagen. The wastewater produced during casing processing contains copious amounts of animal protein, oil, and suspended solids. The ammonia nitrogen (NH 4 + -N) concentration in the effluent, after the original activated sludge treatment process, exceeded the Chinese effluent discharge standard. Its direct entry into the water body reduces the dissolved oxygen (DO) concentration, which directly affects the survival of aquatic organisms and causes water eutrophication [1]. Increased ammonia pollution from the food processing industry has heightened the need for nitrogen abatement strategies. In the traditional biological denitrification process, oxygen and carbon source requirements differ between aerobic nitrifying and anaerobic denitrifying bacteria, requiring the establishment and operation of separate aerobic and anaerobic reactors, resulting in complex processes and higher costs [2,3]. Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria have the advantages of both traditional nitrifying and denitrifying bacteria, and can perform

Project Introduction
The one-stage SNDA process flow chart is shown in Figure 1, and contains an inlet pump, sludge circulating pump, aerobic tank, advection sedimentation tank, NH 4 + -N online monitoring, pH and DO online monitoring, lye tank and pump. The aerobic tank size is 40 m × 2.7 m × 2.45 m, with an effective volume of 216 m 3 . The sedimentation tank size is 40 m × 1.68 m × 2.45 m, with an effective volume of 131 m 3 . After being treated in the aerobic tank, the effluent from the secondary sedimentation tank flowed through the porous water distribution plate and baffle plate, and entered the advection sedimentation tank. After separating the mud water, the supernatant was discharged over the triangular weir. The sludge collected into the sludge bucket by the mud scraper was returned to the inlet of the aerobic tank via a circulating pump. An aeration device was placed at the bottom of the aerobic tank, and an NH 4 + -N online monitor, and pH and DO online monitor were installed at the end of the aerobic tank. The NH 4 + -N online monitor was used for operating and stopping the inlet pump, and regulating the NH 4 + -N concentration in the outlet water. When the NH 4 + -N value in the mixed liquid was lower than the lower limit of the NH 4 + -N online setting value, the inlet pump was run; when the NH 4 + -N value in the mixed liquid was higher than the upper limit of the NH 4 + -N online setting value, the inlet pump stopped. The alkali liquor pipe was evenly arranged along the body of the aerobic tank with 12 alkali liquor adding valves, and each alkali liquor valve was adjusted to ensure that the pH of the entire aerobic tank was maintained in the appropriate range. The pH online analyzer was used for operating and stopping the alkali pump. When the pH value in the mixed solution of the aerobic tank was lower than the lower limit of the pH online setting value, the alkali pump was run; when the pH value in the mixed solution of the aerobic tank was higher than the upper limit of the pH online setting value, the alkali pump stopped. One-stage SNDA records the changes in pH, DO, and NH 4 + -N content in real-time and regulates them according to the actual situation. During the process, the changes in pH, DO, and NH 4 + -N content were recorded in real-time and adjusted according to the actual situation.
operating and stopping the inlet pump, and regulating the NH4 + -N concentration in the outlet water. When the NH4 + -N value in the mixed liquid was lower than the lower limit of the NH4 + -N online setting value, the inlet pump was run; when the NH4 + -N value in the mixed liquid was higher than the upper limit of the NH4 + -N online setting value, the inlet pump stopped. The alkali liquor pipe was evenly arranged along the body of the aerobic tank with 12 alkali liquor adding valves, and each alkali liquor valve was adjusted to ensure that the pH of the entire aerobic tank was maintained in the appropriate range. The pH online analyzer was used for operating and stopping the alkali pump. When the pH value in the mixed solution of the aerobic tank was lower than the lower limit of the pH online setting value, the alkali pump was run; when the pH value in the mixed solution of the aerobic tank was higher than the upper limit of the pH online setting value, the alkali pump stopped. One-stage SNDA records the changes in pH, DO, and NH4 + -N content in real-time and regulates them according to the actual situation. During the process, the changes in pH, DO, and NH4 + -N content were recorded in real-time and adjusted according to the actual situation.

Inoculated Sludge and Wastewater
The secondary sedimentation tank effluent of a sausage processing plant was the wastewater used in this study. Influenced by the original wastewater treatment process, the NH4 + -N concentration in the secondary sedimentation tank effluent considerably exceeded the standard, necessitating its retreatment. The wastewater quality and effluent treatment design is presented in Table 1. The process start-up and operation were mainly divided into two stages; the specific operating conditions are shown in Table 2. In the initial stage of the start-up, the lower and upper limits of pH value online were set to 7.3 and 7.6, respectively; the lower and upper limits of NH4 + -N online were set to 6 and 9 mg/L, respectively; and the flow of the inlet pump was set at 50 m 3 /h. During NLR adjustment, the lower and upper limits of pH value online were set to 7.2 and 7.4, respectively, by adding alkali (NaOH), while the lower and upper limits of NH4 + -N online were increased accordingly. The DO concentration was controlled to be below 1.5 mg/L, and the inlet pump was adjusted to 25 m 3 /h to prolong the inlet time. The treatment mode varied from intermittent water inflow to continuous water inflow.

Inoculated Sludge and Wastewater
The secondary sedimentation tank effluent of a sausage processing plant was the wastewater used in this study. Influenced by the original wastewater treatment process, the NH 4 + -N concentration in the secondary sedimentation tank effluent considerably exceeded the standard, necessitating its retreatment. The wastewater quality and effluent treatment design is presented in Table 1. The process start-up and operation were mainly divided into two stages; the specific operating conditions are shown in Table 2. In the initial stage of the start-up, the lower and upper limits of pH value online were set to 7.3 and 7.6, respectively; the lower and upper limits of NH 4 + -N online were set to 6 and 9 mg/L, respectively; and the flow of the inlet pump was set at 50 m 3 /h. During NLR adjustment, the lower and upper limits of pH value online were set to 7.2 and 7.4, respectively, by adding alkali (NaOH), while the lower and upper limits of NH 4 + -N online were increased accordingly. The DO concentration was controlled to be below 1.5 mg/L, and the inlet pump was adjusted to 25 m 3 /h to prolong the inlet time. The treatment mode varied from intermittent water inflow to continuous water inflow. The sludge to be inoculated was procured from the CASS tank of a sewage treatment plant. Activated sludge was added during the early stages of aerobic tank start-up, with MLSS of 2000~3000 mg/L. After one week of start-up and operation, 1% laboratory cultured AnAOB-activated carbon filler was added, as shown in Figure 2. The sludge to be inoculated was procured from the CASS tank of a sewage treatment plant. Activated sludge was added during the early stages of aerobic tank start-up, with MLSS of 2000~3000 mg/L. After one week of start-up and operation, 1% laboratory cultured AnAOB-activated carbon filler was added, as shown in Figure 2.

Analytical Method
The amount of nitrous nitrogen was determined using N-(1-naphthyl)-ethylenediamine spectrophotometry [20], and COD was determined using the rapid digestion method. The pH and DO were determined by the online monitoring system, and NH4 + -N was determined online using the German WTW ammolyt plus 700 iq. During the entire experiment, the NH4 + -N content, pH, and DO concentration in the effluent were monitored in real-time to monitor the reactor performance. For the convenience of calculation, all units of nitrogen and COD concentrations are in mg/L. The removal efficiency (%) of NH4 + -N and COD is calculated using Formula (1) [21]: where C(inf) and C(eff) are the concentration of NH4 + -N and COD in the influent and effluent, respectively. OriginPro 2022 software (origin lab, Northampton, MA, USA) and SPSS 23.0 software were used for data analysis. The Pearson correlation test was

Analytical Method
The amount of nitrous nitrogen was determined using N-(1-naphthyl)-ethylenediamine spectrophotometry [20], and COD was determined using the rapid digestion method. The pH and DO were determined by the online monitoring system, and NH 4 + -N was determined online using the German WTW ammolyt plus 700 iq. During the entire experiment, the NH 4 + -N content, pH, and DO concentration in the effluent were monitored in real-time to monitor the reactor performance. For the convenience of calculation, all units of nitrogen and COD concentrations are in mg/L. The removal efficiency (%) of NH 4 + -N and COD is calculated using Formula (1) [21]: where C(inf ) and C(eff ) are the concentration of NH 4 + -N and COD in the influent and effluent, respectively. OriginPro 2022 software (origin lab, Northampton, MA, USA) and SPSS 23.0 software were used for data analysis. The Pearson correlation test was performed to determine the correlation between the values of certain parameters. The results were considered to significantly differ at p < 0.05.

Microbial Diversity Analysis
To more intuitively understand the changes in the microbial community structure and diversity, high-throughput sequencing technology, which is a molecular biology technology, was used to determine the microbial community structure in the sludge samples before and after the reactor start-up. The sludge was sampled thrice after the sludge domestication stage (T1, T2, and T3), and was transported to sangon Bioengineering (Shanghai) Co., Ltd., (Shanghai, China) for performing high-throughput sequencing analysis to analyze the changes in microbial diversity and identify the dominant strains. The analysis method performed was according to that described by Liu et al. [7].

Start-Up Period
It can be seen from Figure 3a that only nitrification and denitrification sludge was added to the aerobic tank during the first 7 days. This initial stage of the start-up contained less biomass, and SV30 was maintained at approximately 16.3%, with low nitrification and denitrification rates. Deng and Yang et al. found that acidic (pH < 5) or alkaline (pH > 10) conditions inhibit the growth and reproduction of HN-AD bacteria [22,23], and weak alkaline conditions are more conducive to their metabolism [24]. Therefore, the lower and upper limits of the pH online were set at 7.3 and 7.6, respectively, and the flow of the inlet pump was 50 m 3 /h. The lower and upper limits of NH 4 + -N online were set at 6 and 9 mg/L, respectively, by shortening the inlet time and prolonging the treatment time, and a low-NH 4 + -N load (NLR) was maintained in the aerobic tank for the growth of HN-AD bacteria. It can be seen from Figure 3b that at the initial stage of the start-up, the influent of NH 4 + -N was approximately 130 mg/L, the NLR was stable at approximately 0.18 kg-N m −3 d −1 , the effluent NH 4 + -N concentration was less than 10 mg/L, and the NRE was maintained at more than 90%. During this phase, a stable denitrification performance was observed, indicating that the HN-AD bacteria in the aerobic tank rapidly adapted to the surrounding environment and assisted in NH 4 + -N removal. Therefore, the activated carbon filler attached to AnAOB was added on the eighth day, and the NLR gradually increased to 0.366 kg-N m −3 d −1 . The added AnAOB competed with HN-AD for NH 4 + -N substrate. After several days of acclimation, NRE gradually increased and stabilized at more than 90%, indicating the successful establishment and start of the denitrification process. The DO concentration in the system was maintained below 1.5 mg/L; a lower DO concentration is conducive to the formation of an anoxic microenvironment in the activated sludge floc, which helps coordinate the anaerobic ammonia oxidation and aerobic denitrification processes, further strengthening the denitrification effect.
analyze the changes in microbial diversity and identify the dominant strains. The analysis method performed was according to that described by Liu et al. [7].

Start-Up Period
It can be seen from Figure 3a that only nitrification and denitrification sludge was added to the aerobic tank during the first 7 days. This initial stage of the start-up contained less biomass, and SV30 was maintained at approximately 16.3%, with low nitrification and denitrification rates. Deng and Yang et al. found that acidic (pH < 5) or alkaline (pH > 10) conditions inhibit the growth and reproduction of HN-AD bacteria [22,23], and weak alkaline conditions are more conducive to their metabolism [24]. Therefore, the lower and upper limits of the pH online were set at 7.3 and 7.6, respectively, and the flow of the inlet pump was 50 m 3 /h. The lower and upper limits of NH4 + -N online were set at 6 and 9 mg/L, respectively, by shortening the inlet time and prolonging the treatment time, and a low-NH4 + -N load (NLR) was maintained in the aerobic tank for the growth of HN-AD bacteria. It can be seen from Figure 3b that at the initial stage of the start-up, the influent of NH4 + -N was approximately 130 mg/L, the NLR was stable at approximately 0.18 kg-N m −3 d −1 , the effluent NH4 + -N concentration was less than 10 mg/L, and the NRE was maintained at more than 90%. During this phase, a stable denitrification performance was observed, indicating that the HN-AD bacteria in the aerobic tank rapidly adapted to the surrounding environment and assisted in NH4 + -N removal. Therefore, the activated carbon filler attached to AnAOB was added on the eighth day, and the NLR gradually increased to 0.366 kg-N m −3 d −1 . The added AnAOB competed with HN-AD for NH4 + -N substrate. After several days of acclimation, NRE gradually increased and stabilized at more than 90%, indicating the successful establishment and start of the denitrification process. The DO concentration in the system was maintained below 1.5 mg/L; a lower DO concentration is conducive to the formation of an anoxic microenvironment in the activated sludge floc, which helps coordinate the anaerobic ammonia oxidation and aerobic denitrification processes, further strengthening the denitrification effect.

NLR Domestication Stage
After the start-up period, the one-stage SNDA system entered a stable operation period for a total of 37 days. During this period, the lower and upper limits of pH value online were set to 7.2 and 7.4, respectively; the lower and upper limits of NH 4 + -N online were set to 11 and 12 mg/L, respectively; and the inlet pump flow was adjusted to 25 m 3 /h. The inlet time was prolonged and the treatment time was shortened to improve the NLR in the aerobic tank. In Figure 4b, the influent COD changed in the range of 74~318.46 mg/L, with large fluctuations, which reflected the fluctuating characteristics of the effluent from the original secondary sedimentation tank; however, the effluent COD was maintained at approximately 60 mg/L, meeting China's sewage discharge standards, which also demonstrates the ability of the fully mixed aeration system in resisting the impact load. Wang et al. also suggested that a complete mixed aeration system can provide a suitable growth environment for heterotrophic microorganisms including aerobic denitrifying bacteria, and most aerobic denitrifying bacteria have the capacity for both heteroxic nitrification and the metabolic degradation of organic matter [24]. During reactor operation, controlling the growth environment and operating conditions specific to the nitrifying bacteria, HN-AD bacteria, and AnAOB continuously eliminated several miscellaneous bacteria, which considerably reduced the number of microbial communities and the biomass, and reduced the SV30 from 70% to approximately 36.20%. Figure 4b shows that at higher influent COD concentration, the COD removal rate is relatively large, and that this decreases when the influent COD concentration is low. Carbon is the structural unit and a source of energy for microorganisms, whereas nitrogen can promote the synthesis of amino acids, proteins, and nucleic acids. Therefore, carbon and nitrogen are crucial for the growth and function of microbial cells [25]. Due to the low C/N in wastewater, aerobic heterotrophic bacteria that use organic matter as a nutrient substrate have insufficient nutrients available when the organic matter concentration is low, which renders the removal effect of COD unstable.
with large fluctuations, which reflected the fluctuating characteristics of the effluent from the original secondary sedimentation tank; however, the effluent COD was maintained at approximately 60 mg/L, meeting China's sewage discharge standards, which also demonstrates the ability of the fully mixed aeration system in resisting the impact load. Wang et al. also suggested that a complete mixed aeration system can provide a suitable growth environment for heterotrophic microorganisms including aerobic denitrifying bacteria, and most aerobic denitrifying bacteria have the capacity for both heteroxic nitrification and the metabolic degradation of organic matter [24]. During reactor operation, controlling the growth environment and operating conditions specific to the nitrifying bacteria, HN-AD bacteria, and AnAOB continuously eliminated several miscellaneous bacteria, which considerably reduced the number of microbial communities and the biomass, and reduced the SV30 from 70% to approximately 36.20%. Figure 4b shows that at higher influent COD concentration, the COD removal rate is relatively large, and that this decreases when the influent COD concentration is low. Carbon is the structural unit and a source of energy for microorganisms, whereas nitrogen can promote the synthesis of amino acids, proteins, and nucleic acids. Therefore, carbon and nitrogen are crucial for the growth and function of microbial cells [25]. Due to the low C/N in wastewater, aerobic heterotrophic bacteria that use organic matter as a nutrient substrate have insufficient nutrients available when the organic matter concentration is low, which renders the removal effect of COD unstable. As observed in Figure 4d, NLR gradually decreased from day 14 to day 20 and reached the lowest value of 0.127 kg-N m −3 d −1 on day 20. The influent during this period contained more white sediment and a higher COD concentration. After the influent returned to normal, NLR gradually increased. On the 26th day, the NLR increased and was maintained at 0.32 kg-N m −3 d −1 . HN-AD bacteria adapted to the surrounding environment, exhibiting increased activity, which gradually stabilized the denitrification rate. With the increase in influent concentration, the lower and upper limits of NH 4 + -N online were adjusted to 7 and 12 mg/L, respectively, on the 42nd day. The NLR gradually improved and accelerated the nitrogen removal efficiency in the aerobic tank. Furthermore, the upper limit of NH 4 + -N online was adjusted to 7~15 mg/L on the 48th day, which increased the NLR to ensure continuous influent supply on the 50th day. NLR is strongly positively correlated with NRE (p = 0.006 < 0.05); hence, improving the NLR is conducive to efficient and stable denitrification.
It can be seen from Figure 4a,d that HN-AD bacteria and AnAOB continued to grow and reproduce with NH 4 + -N as the substrate, and the NLR continued to increase from 0.127 kg-N m −3 d −1 to 0.501 kg-N m −3 d −1 . The NRE was more than 94%, and the NH 4 + -N concentration in the effluent was less than 15 mg/L, indicating that the subsequent effluent did not exceed the standard limit for ammonia and nitrogen. Heterotrophic nitrifying and aerobic denitrifying bacteria can oxidize NH 4 + -N to NO 2 − -N or NO 3 − -N, and denitrify these products to N 2 O or N 2 [26]. NO 2 − -N accumulation is often observed during the aerobic denitrification process [26]. Li et al. found that although HN-AD can achieve NO 3 − -N and NO 2 − -N denitrification, it cannot eliminate NO 2 − -N accumulation, which is consistent with the application results of this study [27]. However, the continuously high NH 4 + -N content in the influent inhibited NOB, and did not significantly change the effluent NO 3 − -N and NO 2 − -N contents, indicating that the increase in the NLR did not significantly affect the HN-AD bacteria and AnAOB in the system, and that the system was relatively stable. The uneven distribution of aeration devices easily produces a large proportion of localized anoxic microenvironment. Therefore, in this system, simultaneous nitrification and denitrification coupled with anaerobic ammonia oxidation can be effectively achieved by adjusting the NLR, DO, and pH. Compared with the traditional denitrification method, the one-stage SNDA process has the advantages of achieving nitrification, denitrification, and anaerobic ammonia oxidation simultaneously in one system, avoiding acidification of the nitrification system and balancing multifunctional microbial mixing culture, etc. It not only solves the problem of insufficient carbon source in the wastewater to be treated, but also effectively saves floor space and reduces the amount of alkali dosing in the system. Synchronous nitrification-denitrification not only shortens the reaction time, but also reduces the consumption of organic carbon sources and simplifies the process, and HN-AD bacteria have higher growth rate, resistance to environmental stresses such as salinity, heavy metal ions and antibiotics than traditional autotrophic nitrifying bacteria, and are suitable for a wide range of wastewater treatments [25]. The anaerobic ammonia oxidation process does not require an additional carbon source and saves 60% in terms of energy consumption, but the process start-up time is longer and the enrichment of anaerobic ammonia-oxidizing bacteria is difficult [28]. Table 3 lists some of the practical applications of simultaneous nitrification-denitrification and anaerobic ammonia oxidation processes, from which it can be seen that the anaerobic ammonia oxidation process and simultaneous nitrification-denitrification process are commonly used for the denitrification of wastewater with a low carbon to nitrogen ratio and a high concentration of ammonia nitrogen, such as piggery wastewater, waste leachate wastewater, and coking industrial wastewater. Therefore, the one-stage SNDA process couples the simultaneous nitrification-denitrification and anaerobic ammonia oxidation processes for denitrification, in which heterotrophic denitrifying bacteria can also lift the inhibition of the anaerobic ammonia oxidation process by organic matter and dissolved oxygen, so the one-stage SNDA process in this study has a good application prospect in treating high-ammonia-nitrogen wastewater.

Microbial Analysis
To determine the microbial community structure of one-stage SNDA process after startup and nitrogen load acclimation, the sludge samples (T1, T2, and T3) collected after the load test were analyzed. It can be seen from Figure 5 that the sludge samples T1, T2, and T3 mainly include five bacteria phyla: Proteobacteria, Firmicutes, Verrucomicrobia, Ignavibacteriae, and Armatimonadetes, among which Proteobacteria are the dominant phylum at the microbial level in the sludge [36]. In the activated sludge ecosystem, the stability of the system does not contribute to the change in microbial diversity; it mainly depends on the growth and change in functional bacteria [37]. Due to the changes in the inoculated sludge environment, some microorganisms die and collapse due to maladjustment. The COD breakdown in the cell provides a small amount of carbon source for denitrifying bacteria, and helps in the multiplication of HN-AD in the system. It can be seen from Table 4 that the rich microbial diversity in the sludge system after load acclimation establishes an efficient denitrification process. Acinetobacter, Sporosarcina, and Planococcaceae are the most abundant genera, of which HN-AD bacteria-related genera (such as Acinetobacter and Bacillus) are absolutely dominant, accounting for more than 70% of the total. Anaerobic ammonia oxidation and aerobic denitrification promote and compete with each other in the one-stage reaction system. Although both accept NH 4 − -N as the reaction substrate, AnAOB find it difficult to compete with HN-AD bacteria in terms of population number. Moreover, NO 2 − -N produced by aerobic denitrification can also provide electronic receptors for anaerobic ammonia oxidation. They are efficient and synergistic, thereby ensuring the efficient and stable denitrification performance of the system.

Engineering Benefit
The one-stage SNDA process reduces the reactor volume by 30-40% and saves the capital construction cost. The total investment for the entire study mainly comprised civil engineering cost, total equipment cost, and others (Figure 6a), with a total cost of approximately 1.62 million yuan. Since the sewage treatment process adopts PLC automatic control operation, and the technicians of the original sewage treatment plant are responsible for the operation, the labor cost is ignored. Therefore, the operating cost mainly consisted of electricity, chemicals, repair and maintenance, sludge disposal, and other expenses (Figure 6b), totaling at RMB 0.449 per m 3 (USD 0.07/m 3 , exchange rate USD 1 = RMB 6.36), based on 600 m 3 of wastewater treated per day. Electricity was mainly generated by pumps and blowers, consuming approximately 20 kwh per day. Considering the price of RMB 0.7/kW·h (large industrial electricity), the electric charge was RMB 0.196 per m 3 of wastewater treatment (USD 0.02/m 3 , exchange rate USD 1 = RMB 6.36), accounting for 44% of the total operating cost. The cost for chemical agents was largest for NaOH, with a daily requirement of 150 kg. Therefore, a chemical agent fee of RMB 0.089 is required for each m 3 of wastewater treatment (USD 0.014/m 3 , exchange rate of USD 1 = RMB 6.36). The repairs and maintenance costs are calculated at 1.65% of the total construction cost, based on a conventional fixed asset formation rate of 80%, so the repair and maintenance cost is RMB 0.097 per m 3 (USD 0.015/m 3 , exchange rate of USD 1 = RMB 6.36). Sludge disposal cost is RMB 0.046 per m 3 , accounting for 10% of the total operating cost. Other costs include sewage treatment for flushing various structures, pool surface, sludge treatment

Engineering Benefit
The one-stage SNDA process reduces the reactor volume by 30-40% and saves the capital construction cost. The total investment for the entire study mainly comprised civil engineering cost, total equipment cost, and others (Figure 6a), with a total cost of approximately 1.62 million yuan. Since the sewage treatment process adopts PLC automatic control operation, and the technicians of the original sewage treatment plant are responsible for the operation, the labor cost is ignored. Therefore, the operating cost mainly consisted of electricity, chemicals, repair and maintenance, sludge disposal, and other expenses (Figure 6b), totaling at RMB 0.449 per m 3 (USD 0.07/m 3 , exchange rate USD 1 = RMB 6.36), based on 600 m 3 of wastewater treated per day. Electricity was mainly generated by pumps and blowers, consuming approximately 20 kwh per day. Considering the price of RMB 0.7/kW·h (large industrial electricity), the electric charge was RMB 0.196 per m 3 of wastewater treatment (USD 0.02/m 3 , exchange rate USD 1 = RMB 6.36), accounting for 44% of the total operating cost. The cost for chemical agents was largest for NaOH, with a daily requirement of 150 kg. Therefore, a chemical agent fee of RMB 0.089 is required for each m 3 of wastewater treatment (USD 0.014/m 3 , exchange rate of USD 1 = RMB 6.36). The repairs and maintenance costs are calculated at 1.65% of the total construction cost, based on a conventional fixed asset formation rate of 80%, so the repair and maintenance cost is RMB 0.097 per m 3 (USD 0.015/m 3 , exchange rate of USD 1 = RMB 6.36). Sludge disposal cost is RMB 0.046 per m 3 , accounting for 10% of the total operating cost. Other costs include sewage treatment for flushing various structures, pool surface, sludge treatment equipment water consumption, sewage, sludge test fees, greening fees, etc., totaling RMB 0.021 per m 3 (USD 0.003/m 3 , exchange rate of USD 1 = RMB 6.36). Compared with the traditional nitrification and denitrification process, the electricity cost was reduced by 20%. The alkali produced during denitrification in the one-stage SNDA process can reduce the additional dosage of alkali for nitrification, which reduces the reagent cost by approximately 67.6%. Therefore, the one-stage SNDA process can replace the expensive nitrification-denitrification process. 0.021 per m 3 (USD 0.003/m 3 , exchange rate of USD 1 = RMB 6.36). Compared with the traditional nitrification and denitrification process, the electricity cost was reduced by 20%. The alkali produced during denitrification in the one-stage SNDA process can reduce the additional dosage of alkali for nitrification, which reduces the reagent cost by approximately 67.6%. Therefore, the one-stage SNDA process can replace the expensive nitrification-denitrification process.

Conclusions
The one-stage SNDA process effectively treated the effluent from the secondary sedimentation tank of the collagen casing plant containing low C/N. The COD removal rate varied with the change in influent COD concentration, but the effluent COD was maintained at approximately 60 mg/L, which satisfied China's sewage discharge standard. The engineering application results indicate that by adjusting the NLR, continuous water inflow was achieved on the 50th day. The average TN removal rate was approximately 80%, and the NRE reached more than 94%, indicating that NH4 + -N in the subsequent effluent did not exceed the standard. The stability of the system mainly depended on the growth and change in functional bacteria; the main functional bacteria in the nitrogen load acclimation stage were Acinetobacter, Bacillus, Armatimonadetes_gp5, and Candidatus Kuenrnia. The symbiosis of these dominant bacteria contributed to the simultaneous and efficient removal of NH4 + -N and COD. In addition, combined with the online application of NH4 + -N, pH, and DO monitoring, automatic control of the system was achieved, which simplified the operation and management of sewage treatment, thereby saving approximately 43% of the operation cost compared with that of the traditional nitrification and denitrification process. Moreover, the system reduced the volume of the traditional reactor by 30%, thereby decreasing the capital construction cost.

Conclusions
The one-stage SNDA process effectively treated the effluent from the secondary sedimentation tank of the collagen casing plant containing low C/N. The COD removal rate varied with the change in influent COD concentration, but the effluent COD was maintained at approximately 60 mg/L, which satisfied China's sewage discharge standard. The engineering application results indicate that by adjusting the NLR, continuous water inflow was achieved on the 50th day. The average TN removal rate was approximately 80%, and the NRE reached more than 94%, indicating that NH 4 + -N in the subsequent effluent did not exceed the standard. The stability of the system mainly depended on the growth and change in functional bacteria; the main functional bacteria in the nitrogen load acclimation stage were Acinetobacter, Bacillus, Armatimonadetes_gp5, and Candidatus Kuenrnia. The symbiosis of these dominant bacteria contributed to the simultaneous and efficient removal of NH 4 + -N and COD. In addition, combined with the online application of NH 4 + -N, pH, and DO monitoring, automatic control of the system was achieved, which simplified the operation and management of sewage treatment, thereby saving approximately 43% of the operation cost compared with that of the traditional nitrification and denitrification process. Moreover, the system reduced the volume of the traditional reactor by 30%, thereby decreasing the capital construction cost.