A) process through gas-mixing strategy: System evaluation and microbial analysis

Journal Pre-proofs Efficient Partial-Denitrification/Anammox (PD/A) process through gas-mixing strategy: System evaluation and microbial analysis Rui Du, Shenbin Cao, Xiangchen Li, Jincheng Wang, Yongzhen Peng PII: DOI: Reference:

S0960-8524(19)31904-2 https://doi.org/10.1016/j.biortech.2019.122675 BITE 122675

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

23 October 2019 19 December 2019 21 December 2019

Please cite this article as: Du, R., Cao, S., Li, X., Wang, J., Peng, Y., Efficient Partial-Denitrification/Anammox (PD/A) process through gas-mixing strategy: System evaluation and microbial analysis, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122675

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Efficient Partial-Denitrification/Anammox (PD/A) process through gas-mixing strategy: System evaluation and microbial analysis

Rui Du a, Shenbin Cao b, Xiangchen Li a, Jincheng Wang a, Yongzhen Peng a *

a National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Engineering Research Center of Beijing, Beijing University of Technology, Beijing 100124, China b State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China

*Corresponding author: [email protected]

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Abstract Partial denitrification (PD, NO3--N → NO2--N) provides a promising opportunity for anammox application in wastewater nitrogen removal. In this study, a continuous-flow PD/Anammox (PD/A) process operated with a novel gas mixing was reported in an up-flow anaerobic bed reactor. A high nitrogen removal rate of 2.42 kgN/(m3∙d) was achieved at a relatively short hydraulic retention time (HRT) of 0.5 h with both influent NH4+-N and NO3--N of 30 mg/L. Sludge floatation was eliminated by mixing with gas of the reactor due to an efficiently improved mass transfer. Further optimization of gas flowrates at high NLR could avoid the overproduction of tight-bound extracellular polymeric substances (TB-EPS) and benefit sludge stability. Functional microorganisms of PD and anammox were effectively retained, and Zoogloea affecting sludge settleability kept increasing throughout the operation. This study clearly demonstrated the effectiveness of gas mixing strategy for a high-rate continuous-flow PD/A process with stable nitrogen removal performance.

Keywords: Partial-denitrification; anammox; gas mixing; sludge flotation; upflow anaerobic bed reactor (UASB)

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1. Introduction The nitrate (NO3--N) residue is a critical issue in current wastewater treatment plants (WWTPs) due to the carbon-limitation of municipal wastewater. Biological denitrification is the most common method for nitrogen removal from wastewater (McCarty, 2018). However, it requires massive organic carbon source serving as electron donor, accompanying with huge quantity of waste sludge production due to 0.3~0.5 g dry biomass/g COD removed (Gao et al., 2017; Liu et al., 2018). As an efficient solution, anaerobic ammonium oxidation (anammox) process is economically and environmentally acceptable for the 60% saving in aeration and 100% saving in organic carbon demand in nitrogen removal (Eq.1). In this process, the anammox bacteria converts NH4+-N and NO2--N to N2 directly under anoxic condition (Gao et al., 2018; Kartal et al., 2010). However, it cannot be utilized for NO3-N removal directly due to its specific metabolic characteristics. Concurrently, in view of increasing stringent discharge limitation, developing a high-efficiency nitrogen removal technology has become a highly urgent issue in WWTPs (Winkler & Straka, 2019; Zhang et al., 2018). NH4+-N + 1.32 NO2--N →N2 + 0.26 NO3--N

(Eq.1)

The partial-denitrification (NO3--N→NO2--N, PD) process (Eq.2), in which the NO3--N is reduced with NO2--N as end products, has attracted increasing attention (Cao et al., 2019b; Du et al., 2019d). The nitrate-to-nitrite transforming ratio (NTR) in PD has been reported to reach a high level of about 80% in a lab-scale sequencing batch reactor (SBR) (Cao et al., 2019a; Cao et al., 2013). Moreover, this high NO2--N accumulation maintained stably in a continuous-flow reactor (Cao et al., 2016; Li et al., 2018). The excellent stability of PD enabled us to develop the novel integrations of PD and anammox for simultaneous NO3--N and NH4+-N removal. In the PD coupling with anammox process, the NO3--N will be reduced to NO2--N and removed with NH4+-N as electron donor by anammox

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bacteria (Eq.3). Moreover, there is 11% nitrogen production as NO3--N in anammox process (Eq.1), for which reason the maximum nitrogen removal efficiency will be 89% on theory. This NO3--N could be reduced to NO2--N in PD process and removed by anammox bacteria. Therefore, the nitrogen removal efficiency could be significantly improved to achieve as high as 100% in the PD/A process. Furthermore, this new bioprocess allows for up to 100% aeration energy saving and 60% organic carbon reduction, offering a promising alternative for a cost-efficient treatment of NH4+-N and NO3-N wastewaters (Cao et al., 2019a; Du et al., 2019a). NO3--N + 2e (COD) → NO2--N

(Eq.2)

NH4+-N + 1.06 NO3--N + 2.12e (COD) → N2

(Eq.3)

This emerging approach has been successfully demonstrated in multiple lab-scale bioreactors. The removal efficiencies of NH4+-N and NO3--N in an SBR-type PD/A process reached 91.4% and 95.4%, respectively, with both influent NH4+-N and NO3--N of 50 mg/L using acetate as electron donor (Du et al., 2017). However, a considered limitation of this system was the relatively long hydraulic retention time (HRT) (12~16 h), which resulted in the low nitrogen removal rates (NRRs) that could not meet the requirement of high-flowrate wastewater treatment. Thereby, the feasibility of a continuous-flow PD/A process in an up-flow anaerobic sludge bed (UASB) reactor was explored, and results showed that the TN removal efficiency reached 89.1% with influent NH4+-N and NO3--N both of 30 mg/L at HRT of 2.0 h (Du et al., 2019b). However, a serious sludge floatation was found in a PD/A UASB reactor, causing a drastic decrease in activities of the anammox bacteria, followed by the rapidly deteriorated nitrogen removal performance (Du et al., 2019b). In fact, such a problem is always observed in the up-flow reactor, which still needs an efficient solution especially for the low gas production process (Campos et al.,

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2017; Wang et al., 2018). This would be related to the relatively slow rate of gas release, and thus the gas bubbles trapped inside granule sludge forming bubble-sludge complexes and leading to the sludge floatation. Even though some mitigation measures have been proposed, i.e. breaking up granules and chemicals addition, enhancing reactor mixing is technically accepted as it does not change the aggregate structure and requires no additional chemical dosage (Li et al., 2014; Winkler et al., 2018). Furthermore, the mass transfer of the PD/A reactor can be efficiently improved by hydrodynamic mixing with adjusted recirculation ratio (Du et al., 2019b). However, NRR was still much lower (0.64 kg N/(m3d)) than values in the reported anammox processes (Meng et al., 2014; Tang et al., 2011). It still faced the risk of performance disturbance with further increasing nitrogen loads. Effectively controllable strategies are extremely needed to achieve high-efficiency nitrogen removal in continuously feeding PD/A process. With the above in mind, a novel strategy of gas mixing was explored in this study. The overall nitrogen removal behavior, physicochemical property of biomass aggregates, and characteristics of extracellular polymeric substances (EPS) production were systematically analyzed under the liquid and gas mixing modes. Microbial community evolution was revealed to in-depth understand the functional bacteria interactions and the mechanism of nitrogen transformation influenced by mixing strategy in the continuously feeding PD/A process. 2. Materials and methods 2.1 Reactor design and operation In this study, a UASB reactor with a total volume of 2.0 L was used, comprising an effective volume of 1.6 L and 0.4 L of headspace as shown in Fig.1. It had an inner diameter of 50mm and height of 800 mm. The reactor was equipped with a gas-liquid-solid separator. The synthetic influent

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was transferred to the reactor from the bottom by a peristaltic pump. Effluent or gas in the reactor were recirculated to provide continuous mixing of the liquor in different operational phases. During phase L (day 1~25), liquid recirculation was conducted from a flow point at 5 cm below the effluent port to the influent port at bottom of reactor by a separated peristaltic pump. When the gas mixing strategy was employed, the gas outlet set at the top of reactor was connected to the influent port with the help of a recirculation pump. Since the feeding NH4+-N and NO3--N were converted to nitrogen gas in PD/A process, the major content in gas phase was assumed to be nitrogen gas. The reactor was operated under room temperature without heating during the whole operation. Fig.1 During phase L (day 1-25), the liquid recirculation was conducted for enhancing the substrates and sludge mixing with the liquid recirculating flowrates (fL) of 4.74 L/h. The HRT was 1.5h. The mixing strategy was shifted to gas mixing from phase G-1 to G-6 during the operational day 26 to 229. The HRT was stepwise reduced from 1.5 h (G-1) to 1.0 h (G-2), 0.75 h (G-3), and 0.5 h (G4~6). The gas flowrate (fG) was adjusted to ensure the sludge fluidization in the reactor. Considered that there would be increasing gas entrapped inside the sludge under higher NLR and lead to the undesirable sludge floatation, increasing gas flowrate was applied to improve the mixing performance and prompt the gas release. The fG increased from 0.42 L/h (G-1 and G-2) to 0.45 L/h (G-3), 0.63 L/h (G-4), 0.72 L/h (G-5) to 0.83 L/h (G-6) in accordance with the step-wise decrease in HRT. 2.2 Wastewater and inoculation Synthetic wastewater containing NH4+-N and NO3--N was fed to the PD/A reactor. The concentration of both NH4+-N and NO3--N maintained constantly at 30 mg/L during the entire

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operation. The NH4+-N and NO3--N were made of NH4Cl and NaNO3. The other composition of influent was according to the previous study (Du et al., 2019b). The sodium acetate served as organic carbon source for NO3--N conversion to NO2--N in PD process, and it was introduced separately using a peristaltic pump. The COD/NO3--N parameter kept at 3.4 from phase L to phase G-2 according to the previous study (Du et al., 2019b), and it was adjusted according to the performance of NO3--N and NH4+-N removal. The COD/NO3--N was reduced to 3.0 in phase G-3 ~ G-6 due to the improved mass transfer under gas mixing condition. The inoculum was driven from a stable operated PD/A reactor as described in our previous study (Du et al., 2019b). The original reactor was operated with the liquid recirculation. A nitrogen removal rate of 0.72 kgN/(m3∙d) was achieved for treating the NO3--N and NH4+-N contained wastewater during stable operation. 2.3 Ex-situ specific activities of PD and anammox bacteria The ex-situ activities of PD and anammox bacteria were evaluated regularly by batch tests during the entire operation. The sludge was washed to remove the residual substrates and filled to the wide neck glass vessels for the measurement after being taken from the reactor. The specific NH4+-N removal rates, NO3--N reduction rates and NO2--N accumulation rates were measured in each test according to variation in the nitrogen concentration with reaction duration. The batch tests were conducted under the same condition in all the experiments. The detailed description was according to the previous study (Du et al., 2019b). 2.4 EPS extraction and fluorescence spectra EPS were extracted from the sludge samples by a modified heat extraction method (Du et al., 2019b). The compositions of loosely bound (LB-EPS) and tightly bound (TB-EPS) were analyzed

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for the sludge aggregations. The protein (PN) in the EPS was measured with bovine serum albumin as the standard (Lowry et al., 1951). Polysaccharides (PS) were determined by the anthrone method with glucose as the standard (Loewus, 1952). Fluorescence spectra of three-dimensional Excitation-Emission Matrix (3D-EEM) were obtained in duplicate (Sheng & Yu, 2006), using a LS-55 spectrometer (Perkin Elmer, USA). The EPS samples were diluted with pure water (Milli-Q, Millipore Co. Ltd, USA) to avoid the signals exceeding the maximum limit. The dilution ratio was determined by measurement at successive dilution ratios (Jacquin et al., 2017). The excitation (Ex) and emission (Em) wavelengths ranged from 200 nm to 400 nm at 10 nm intervals, and 250 nm to 550 nm at 5-nm intervals, respectively. The scanning speed was set at 1200 nm/min, with the excitation and emission slit bandwidths fixed at 10 nm. Milli-Q water was regarded as blanks and was analyzed under the same conditions as the samples. All the data used for plotting were deducted from those of the blank samples. The 3D-EEM data were processed using the software Origin 9.5. 2.5 Analytical method The effluent samples of PD/A reactor were collected on a daily basis. The nitrogen concentration including NO3--N, NO2--N and NH4+-N were analyzed by Lachat QuickChem 8000 flow injection analyzer (Lachat Instrument, Milwaukee, WI). The concentration of COD was analyzed by a COD quick-analysis apparatus (Lian-Hua Tech Co., Ltd., 5B-1, China). The temperature was measured by a detection sensor (WTW Company, WTW 340i, Germany). Sludge samples were regularly taken from the top, middle and bottom of the PD/A reactor, and were mixed thoroughly for analyzing the physical, chemical and biological properties of the sludge aggregations. The particle size distribution was examined by a series of screens with the uniform

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diameters of 0.02mm, 0.50mm, 1.00mm, 1.50mm, 2.00mm, 2.5mm, 3.30mm. The size distribution was calculated as weight percentages at different diameter range. Microbial community of the PD/A sludge was analyzed by high-throughput 16S rRNA sequencing of bacteria. The samples were taken from the reactor on day 61, 114, and 219 during the long-term operation. DNA extraction was in accordance with the previous study (Du et al., 2019a). The PCR amplification was conducted with forward primer 338F and reverse primer 806R. The amplified DNA sequencing was performed by MiSeq platform. Results of sequencing information were analyzed according to the previous study. 3. Result and Discussion 3.1 Performance of PD/A process with elevating nitrogen loading rates 3.1.1 Operation with hydrodynamic mixing The PD/A reactor was operated continuously for 229 days under increasing nitrogen loading rate (NLR) with decreasing HRT. Two mixing strategies were applied during the entire experiment. The overall nitrogen removal performance was showed in Fig.2 During the initial operation (phase L, day 1-25) (Fig.2a), the liquid recirculation was applied with a constant fL of 4.74 L/h to provide appropriate liquid mixing based on our previous study (Du et al., 2019b). After inoculation, satisfactory nitrogen removal performance was observed with effluent NH4+-N, NO2--N and NO3--N concentration below 4 mg/L (Fig.2b). This was comparable with previous study on continuously feeding PD/A process at HRT of 2.0 h (Du et al., 2019b). It indicated that the anammox bacteria could be competitive for substrate NO2--N over denitrifiers in this reactor. However, effluent NH4+-N and NO3--N began to accumulate even the temperature increased gradually and averaged 17.3 ºC during this phase.

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Meanwhile, serious sludge floatation occurred at this stage along with the biomass stacking at the top of the reactor. Considering the elevating NLR of reactor, which had been operated at HRT of 2.0 h and NLR of 0.72 kgN/(m3∙d) as describe in previous study (Du et al., 2019b), the sludge floatation was mainly attributed to the increasing nitrogen gas entrapment inside the sludge under the insufficient shear force (Li et al., 2014; Song et al., 2017). In this scenario, the nitrogen removal performance deteriorated with the average NH4+-N and NO3--N removal efficiencies of 82.9% and 81.8% in phase L, respectively. Additionally, the decreasing gas production in turns caused a worse sludge mixing performance, which was a significant issue that should be addressed in a continuously feeding PD/A process under the increasing NLR. Fig.2 3.1.2 Optimization with gas mixing strategy Starting from day 26, gas mixing was performed instead of liquid recirculation to achieve adequate mixing. During phase G-1 (day 26-37), fG was set at 0.42 L/h to ensure the sludge fluidization. Sludge floatation disappeared rapidly once the gas mixing strategy was employed. Moreover, a remarkable decrease in effluent nitrogen concentration was observed with NH4+-N, NO2--N and NO3--N effluent concentration of 4.0 mg/L, 2.4 mg/L, and 3.61 mg/L, corresponding to the average removal efficiencies of NH4+-N and NO3--N was 86.7% and 80.0%, respectively (Fig.2b). When the HRT was further reduced to 1.0 h, the NH4+-N and NO3--N removal efficiency increased to a higher level of 93.5% and 85.6%, with no sludge floatation observed. It clearly suggested that the increasing NLR had little impact on the TN removal performance of PD/A process with gas mixing strategy. This was different from the operation with liquid recirculation as mentioned above. Moreover, it maintained at a desirable level at further lowering HRT to 0.75 h

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(phase G-3, day 62-98), with the effluent NH4+-N, NO2--N and NO3--N concentration below 2.0 mg/L. This resulted in a much higher TN removal efficiency of 93.2% with the NH4+-N and NO3--N removal of 95.4% and 91.0%, respectively (Table 1). Compared to the liquid recirculation stage, at which the TN removal decreased to 64.3% on day 25, a significantly enhanced nitrogen removal capability was achieved by the gas mixing strategy. Table 1 In the last phase, the shortest HRT of 0.5 h was applied in PD/A reactor. However, the stable performance was disrupted, and sludge floatation happened again in phase G-4 (day 99-114), causing a high accumulation of NH4+-N and NO3--N in effluent, correspondingly the NH4+-N, NO3--N and TN removal efficiency declined to 78.4%, 71.7% and 75.1%, respectively (Table 1). To enhance the mixing effect, fG increased from 0.64 to 0.72 L/h, but the limited improvement of nitrogen removal was observed. Subsequently, fG was further increased to 0.83 L/h in phase G-7 (day 166-229). Consequently, sludge floatation was effectively resolved and did not occur until the end of the operation. During this stage, the effluent NH4+-N, NO2--N and NO3--N concentration rapidly decreased to below 4.0 mg/L, corresponding to the removal efficiency of NH4+-N, NO3--N and TN increasing to 90.3%, 77.5% and 83.9%, respectively. According to the best of our knowledge, nitrogen removal in the present study of a continuousflow PD/A system appeared to be relatively stable and high-efficiency, especially for treating the low-strength NH4+-N and NO3--N wastewater. Compared to the liquid mixing mode, the gas mixing facilitated a more robust and efficient performance under shorter HRT. Previous study also reported that the sludge flotation potential could be reduced by pneumatic mixing (Wang et al., 2017). In the present study, the excellent NH4+-N and NO3--N removal along with the enhanced sludge fluidization

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in reactor clearly demonstrated that the upflow PD/A bioreactor with gas mixing was a viable and efficient technology for practical application. 3.2 Enhancing activities of PD and anammox bacteria by gas-mixing strategy 3.2.1 Effect of mixing mode on nitrogen conversion capability The above results illustrated that optimization by gas mixing strategy significantly promoted the nitrogen removal capacity of continuous-flow PD/A process. The NRR increased from 0.79 to 2.42 kgN/(m3∙d) with the HRT shortening from 1.5 h to 0.5 h, corresponding to increasing NLR from 0.96 to 2.88 kgN/(m3∙d) (Fig.2c). This was most likely related to the enhanced sludge fluctuation and the associated mass transfer (Wang et al., 2018). The gas mixing strategy enabled vigorous mixing and dispersed sludge in dead zones and released the gas bubbles trapped inside the granules (Wang et al., 2017). In comparison, the major drawback of liquid recirculation was related to a limited intensity of mixing provided by rising gas bubbles, especially for the low gas produced process. In this case, the gas bubbles were trapped and encapsulated in clogged sludge thus causing flotation. Compared to the liquid recirculation, the gas recirculation could produce a much higher shear rate that enhance the gas bubbles trapped in the sludge to release effectively (Wang et al., 2018). On the other hand, the mixing mode played an important role in the substrate competition between different functional bacteria in the single-stage PD/A system. Supported by the mass balance results shown in Fig.2d, anammox sustained as the main process governing the nitrogen removal, while the heterotrophic denitrification played only a minor role. However, the different contributions of the anammox process should be noted with the change of mixing strategy. Under the liquid recirculation mode, anammox contributed to 86.5±5.3% of TN removal, and it increased to a higher level of 92.3±2.8% by applying the novel gas mixing strategy. This was most likely attributed

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to the even substrate distribution and sufficient mass transfer, preventing the anammox bacteria from being substrates-limited. Consequently, the possible competition for available NO2--N and living space between the low-yield anammox bacteria and heterotrophic denitrifying microorganism could be minimized. Therefore, it clearly confirmed the significant effect of mixing strategy on the stability of continuous-flow PD/A process. 3.2.3 Effect of gas mixing strategy on biomass activities of PD and anammox bacteria To understand the long-term effect of mixing mode on the PD/A process, batch tests were conducted to determine the ex-situ activities of PD and anammox bacteria. With the liquid mixing mode, the specific NO3--N reduction rates (rNO3) of PD process were initially at a low level of 11.65 mgN/(g VSS∙h), corresponding to the specific NO2--N accumulation rates (rNO2) of 10.58 mgN/(g VSS∙h) (Fig.3). This was much lower than the reported values (rNO3 of 50.6 and rNO2 of 48.5 mgN/(g VSS∙h)) at a comparatively lower NLR of 0.72 kg N/(m3d) (Du et al., 2019b). This was most probably owing to the decay of bacteria cells at the long-term lack of available substrate under insufficient sludge fluidization. Notably, significantly improved PD and anammox activities were observed when the gas mixing strategy was applied. The rNO3 and rNO2 reached 47.96 and 44.43 mgN/(g VSS∙h), respectively, as a response to a relatively short HRT of 0.5 h. Furthermore, the increasing gas flowrates facilitated an extra enhancement in the rNO3 and rNO2, which achieved 72.52 and 59.97 mgN/(g VSS∙h) at the end phase. It should be noted that the NTR sustained at high level of 87.8% to 97.1% through the entire operation. This was even higher than the previously reported value in an UASB reactor for sole PD process (81.4% at stable operation) (Cao et al., 2016). It strongly suggested the importance of sufficient mixing for NO2--N production activities in the continuous

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flow reactor. In addition to the PD activities, gas mixing was demonstrated to significantly promote the nitrogen removal capability of anammox bacteria. With liquid mixing, the specific ammonia consumption rates (rNH4) decreased from 1.35 to 1.04 mgN/(g VSS∙h) possibly due to the substratelimited condition (Fig.3). In comparison, applying gas recirculation resulted in a significantly increasing rNH4 to 5.34 mgN/(g VSS∙h). This was most likely resulted from the enhanced mass transfer from the external liquid environment to cells inside of bacteria. Fig. 2 It was noted that the TN removal performance maintained at a stable level despite the temperature decreasing from 28.8 ℃ to 11.2 ℃. This was markedly different from the previous study where the anammox activities were negatively impacted by decreased temperature, especially below 20oC (Dosta et al., 2008; Lotti et al., 2015). This was closely related to the granular sludge structure discussed in the following sections, which played a protective role for the microorganism against the adverse environment. These results suggested that gas mixing was an effective strategy in terms of performance stability and sufficient bacterial activities. 3.4 Morphology and EPS analysis of sludge aggregate in high-rates PD/A system 3.4.1 Morphological characteristic and size distribution The characteristics of mature PD/A granules developed in the UASB reactor was investigated. It could be seen that the inoculated sludge was relatively regular in shape, dark red-color and spherical particles with a smooth surface. In comparison, the morphological character of sludge appeared a significant difference after the 175-days cultivation. At the end of operation, most granules maintained an elliptical shape with different sizes. However, the granules had a darker color than the

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inoculum, which was mainly due to the cell decay of anammox bacteria as mentioned above. The size distribution showed that more than 90% of granules were over 0.5 mm. Furthermore, granules having larger diameters from 2.0 mm to 2.5 mm accounted for 19.7 %, whereas the granular size in the inoculum was mainly displayed from 0.2 mm to 0.5 mm with a percentage of 53.4%. It indicated that the granule continued to develop and grew to a larger size during 229-days cultivation. Moreover, the VSS/SS ratio gradually decreased with the increased diameter, indicating that there would be more inorganic fraction in the bigger aggregates. The largely homogeneous diameter distribution of granules was attributed to the even substrate distribution. Granular structure was assumed to offer an external advantage for sludge stability since the formation of granules could not only benefit for achieving effective biomass retention but also increase the resistance of bacteria to adverse environmental conditions. This was in accordance with the previous study that an increasing settleability of granular sludge favored a higher effluent quality (Corsino et al., 2018). Results in the present study indicated that the mixing condition had an important effect on biomass aggregation and granules stability, and further study is necessary for the characterization of the PD/A granular sludge. 3.4.2 EPS stratification and chemical composition The EPS exhibited a key effect on the sludge properties and bacteria consortia aggregation. It has been regarded that bacteria could respond to stressful culture conditions by regulating their energy metabolism, which significantly affected the biological nitrogen removal performance (Sheng et al., 2010). In this study, the joint influence of increasing NLR and optimal mixing on the EPS stratification and chemical composition in PD/A system was investigated. Results suggested that the mixing mode had an important influence on the EPS production. With the liquid mixing mode, the total EPS content at HRT of 1.5 h appeared to be lower than the

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reported value at HRT of 2.0 h (Du et al., 2019b), followed by the occurrence of sludge floatation (Fig.4a). It was mainly attributed to the decreased biomass activities under insufficient mass transfer. Significantly, the EPS amount had a sharp increase from 73.40 (phase G-2) to 148.03 mg/g VSS (phase G-3) by employing a gas mixing strategy but decreased to 109.58 mg/g VSS when the sludge floatation happened again in phase G-4. Subsequently, the total content of EPS rose to a much higher level of 185.91 mg/g VSS after the optimization of gas flowrates. Previous study reported that a higher EPS content would benefit high granule stability (Sheng et al., 2010), which was in accordance with the results in the present study. Similar trend was observed for the TB-EPS concentrations, which increased from 47.70 mg/g VSS to 103.93 mg/g VSS. Furthermore, the TBEPS concentrate was higher than the LB-EPS, except for that in phase G-4, when the sludge floatation happened again under an insufficient mixing condition. This indicated that the TB-EPS had an important role in the sludge stability in a continuous PD/A system. Fig.4 Moreover, it should also be noted that the PD/A performance had close relation with the portion of EPS in different stratification. The ratio of TB-EPS and LB-EPS (TB/LB) exhibited a remarkable distinction with changing mixing mode. A high TB/LB ratio of 2.19 was observed during the liquid recirculation stage, in which the sludge floatation and a deteriorated reactor performance was observed. It was likely to be caused by the thick TB-EPS layer, leading to a poor transfer of gas and substrate inside the sludge aggregation. It was supported by the previous study that excessive EPS could lead to poor flocculation (Li & Yang, 2007). With the optimization of gas mixing strategy, the TB/LB ratio decreased gradually to 0.92, followed by an improved nitrogen removal performance. Further increasing of NLR resulted in a slight increase of TB/LB ratio to 1.34 at the stable operation,

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which was also lower than that in the liquid recirculating phase. This would be mainly attributed to the effect of higher shear force on avoiding the overproduction of TB-EPS by the increasing gas flowrates. Taken together, the sludge stability was closely related to both TB-EPS abundance and the TB /LB ratio. Deep understanding of the mechanism of structural EPS influencing the sludge stability is necessary for further study of the novel PD/A systems. In addition, PN and PS contents were measured in different EPS stratification (Fig.4b and 4c). It showed that the concentration of PN was much higher than PS in both of LB-EPS and TB-EPS during the entire operation (Fig.4b and 4c). The PN production was significantly enhanced at phase G-3 after using the gas mixing strategy. However, a sharp decrease of PN concentration in TB-EPS was observed at phase G-4 when the sludge stability deteriorated due to a sudden increase of NLR. With the optimization of increasing fG, PN was found to increase again from 46.42 mg/g VSS to 91.83 mg/g VSS under the same NLR of 2.88 kg N/(m3∙d). This strongly confirmed the important role of PN contents in EPS on the stability of PD/A aggregation, especially in the TB layer. It to some extent agreed with the present assumption that excessively low PN/PS also had a negative effect on the sludge stability due to the decay of aggregation capability. Notably, the PN/PS ratio in LB-EPS and TB-EPS of the PD/A sludge were as high as 9.2 and 8.7, respectively (Fig.4c), which were much higher than the reported value of the anammox culture. Previous study reported the PN/PS about 2.7 in the anammox aggregates (Hou et al., 2015). A lower PN/PS ratio of 0.51 was reported in granules of anammox UASB (Ni et al., 2010). In contrast, a relatively high contribution of PN in EPS was found in the single PD granule reactor without combination with anammox, in which the PN/PS was over 7.0 (Cao et al., 2019c). It strongly implied that the heterotrophic bacteria related to oxidization of organic carbon and accumulation of NO2--N played an important role in the

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EPS secretion of the PD/A aggregates. In addition, increased EPS content was assumed to protect biomass from the negative influence of the low temperature and to improve the resistance to increasing NLR. This also well explained the excellent nitrogen conversion activities during the entire operation. 3.4.2 Fluorescence spectra of LB-EPS and TB-EPS Three-dimensional EEM analysis has been regarded as a promising method for fluorescent characteristics of microbial EPS (Jacquin et al., 2017). Fig.5 presented the 3D-EEM of the different stratification of EPS. The fluorescent peaks of protein-like substrates were observed in all EPS samples. Specifically, two peaks in all TB-EPS were referred to tryptophan-like (Ex/Em= 250-280/330-360 nm) and the tyrosine-like protein (Ex/Em= 200-250/330-360 nm) (Fig.5b, 5d, and 5f). Moreover, the peak locations were not substantially influenced by nitrogen loading. These were different from the LBEPS. Two protein-like peaks (peak A, B) were observed in the LB-EPS fraction of phase G-2 (Fig.5a). However, there was no obvious tryptophan-like peak (peak E) and the location of tyrosine-like substrates (peak F) shifted in phase G-4 (Fig.5c), following by the occurrence of sludge flotation. Both of two typical peaks (peak I, J) were observed again following by the gas flowrates adjustment in phase G-6 (Fig.5e). This indicated that the mixing strategy had more influence on LB-EPS than TB-EPS in PD/A aggregates, typically on the tryptophan-like composition. Fig.5 Prior to this, the tryptophan was assumed to trigger the aggregation process and stabilize the amyloidal structures of the EPS (Biancalana et al., 2015). Significant tryptophan production was observed in the EPS of the aerobic granules enriching the aerobic ammonia-oxidizing bacteria for enhancing the aggregation (Lin et al., 2018). In this study, the PD/A granular sludge was expected to

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secret high tryptophan to strengthen the stable and compact structure under increasing NLR in the continuously feeding reactor. Moreover, a higher intensity was observed for the tyrosine than tryptophan-like substance, which was associated with the key amino acid composition that played an important role in the aggregation of protein. These results suggested that the tryptophan and tyrosinelike substances had a significant effect on the resistance to the increasing nitrogen and organic stress. The persistent secretion of these functional EPS composition was expected to be the main reason for the increased stability of the continuous PD/A reaction by gas-mixing strategy. Moreover, the humic acid-like peak (peak E) was observed in the LB-EPS fraction of phase G-4, which was mainly caused by the cell lysis and decay in the reactor. It disappeared at the end period of operation, attributing to better mass transfer under the optimized gas mixing condition. Taken together, this study indicated that the gas mixing strategy offered considerable benefits to maintain the suitable matric structure and sufficient mass transfer in the continuous PD/A reactor. 3.5 Effect of gas mixing strategy on the microbial community of PD/A biomass 3.5.1 Bacterial diversity in continuously feeding PD/A system A total of 122,095 reads were obtained through quality filtering across three samples during the operational period with gas mixing. The high-quality sequences were clustered into 847 OTUs at a threshold of 97% sequence similarity. The microbial diversity increased from 4.63 (sample U1-1, day 61) to 5.11 (sample U1-2, day 114), but declined to 4.62 (sample U1-3, day 219). It suggested that both NLR and mixing intensity could affect the bacterial diversity in continuously feeding PD/A system. High NLR increased the microbial diversity as many opportunistic bacteria could grow on available nitrogen and organic substrates. Under the same NLR, a lower bacterial diversity was observed corresponding to a higher gas flow rate, which possibly led to the washout of some flocculent

19

biomass and sludge granulation under the higher hydraulic shear force. This was supported by previous results that the bacteria diversity in granular anammox sludge was much lower than the flocs (Guo et al., 2016). The microbial community displayed a remarkable variation with the nitrogen loads and gas flowrates at the phylum level (Fig.6a). Proteobacteria, Chloroflexi, Acidobacteria, and Planctomycetes were the most dominant phylum in the reactor. The relative abundance of Proteobacteria significantly increased from 18.21% (day 61) to 68.18% (day 219), whereas that of Chloroflexi and Acidobacteria decreased dramatically from 46.94% to 5.66% and from 9.46% to 1.42%, respectively. This was consistent with previous results in a PD/A SBR that dominated with the Proteobacteria phylum (Du et al., 2017). Chloroflexi was associated with the sludge bulking in the wastewater treatment process due to the high coverage of filamentous-shaped bacterium (Guo & Zhang, 2012). The higher abundance of Chloroflexi on day 61 would be due to the insufficient mixing under the low gas flowrates as well as a high COD/NO3--N in this period, which favored the growth of filamentous bacteria. Similarly, Acidobacteria, Ignavibacteriae, and Actinobacteria consisting a large group of heterotrophic bacteria showed decreasing trends. Planctomycetes known to include the anammox species showed an increasing trend. Fig. 6 3.5.2 Microbial community dynamic of functional bacteria under gas mixing condition Taxonomic analysis of genera classification showed a high-level enrichment of the functional bacteria (Fig.6b). A pronounced increase in abundance of Thauera genera was found from 0.15% (day 61) to 14.7 % (day 219). The members of Thauera were previously assumed to be capable of considerable NO2--N generation during NO3--N reduction. This was likely attributed to the adequate

20

substrate distribution and sufficient mass transfer in the gas-mixing system, where PD bacteria could compete over the bacteria capable of full denitrification for the available electron donor. Consequently, PD activities in the reactor were improved with a constantly high NTR level. The structure of anammox bacteria in this study showed many distinctions compared with previous studies. Three genera of anammox bacteria including Ca. Brocadia, Ca. Kuenenia and Ca. Jettenia were detected in all three samples (Fig.6b), while only one or two (Ca. Brocadia or Ca. Kuenenia) genera were identified in the other PD/A systems (Du et al., 2019c; Wang et al., 2019). With the increasing NLR, a remarkable increase in Ca. Brocadia from 0.90% (day 61) to 3.55% (day 219) was observed, which was much higher than the reported value in a PD/A SBR (generally below 2.0%) (Du et al., 2017; Du et al., 2019a). This would be related to the sludge granulation in this study, which enhanced the enrichment of slow-growth anammox bacteria within matrix structure. In comparison, genus Ca. Kuenenia of 0.85% was identified; however, it decreased to 0.09% at the end phase. Similar trend was observed in genus Ca. Jettenia. It impacted that, Ca. Kuenenia and Ca. Jettenia was less competitive than Ca. Brocadia under a high NLR in the single-stage PD/A system, which could be related to the lower growth rates of the former species, especially at the presence of organic matter (Oshiki et al., 2011). Moreover, Anaerolineaceae-related genera belonging to heterotrophic bacteria dramatically reduced from 34.49% to 3.80%. Meanwhile, the relative abundance of Denitratisoma also declined during the operation, which was capable of reducing NO3--N to N2 using organic carbon source. Similar trend was observed for the Ignavibacterium as the filamentous bacterium capable of denitrification, indicating the selective effect of granular aggregation on denitrifying bacteria. On the contrary, genus Zoogloea increased from 0.10% to 5.09%, which was well known to have a crucial

21

effect on the synthesis of EPS and forming sludge aggregation (An et al., 2016). This would facilitate biomass granulation and increase the stability of PD/A system. Venn diagrams illustrated that 258 OTUs were shared (Fig.6c), among which the key genus responsible for PD and anammox were identified with high percentages of 8.11% (Thauera) and 2.17% (Ca. Brocadia), respectively (Fig.6d). It suggested that the functional bacteria of PD/A process coexisted stably in the continuously feeding reactor and facilitated a relatively stable performance at increasing NLR. 4. Conclusion A stable and high-rate PD/A process in continuous-flow reactor was developed for treatment of low-strength NH4+-N and NO3--N wastewater. The effectiveness of gas mixing strategy was demonstrated by the improved mass transfer and bacterial activities, thus achieving the desirable effluent qualities at a relatively short HRT. Optimization of gas mixing effectively prevented the sludge floatation and affected the EPS production in the granular aggregates. The functional bacteria of PD and anammox coexisted stably under optimal mixing conditions, resulting in a relatively stable performance at increasing NLR.

Acknowledgments This research was supported by National Postdoctoral Program for Innovative Talents (BX20180019); China Postdoctoral Science Foundation (2018M641139); Beijing Postdoctoral Research Foundation; Basic Research Foundation of Beijing University of Technology (005000546319557).

E-supplementary data of this work can be found in the online version of the paper.

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Table and Figures List Table 1 Nitrogen removal rates and effluent quality under different operational condition. Fig.1 Apparatus for single-stage PD/A process with different mixing conditions: (a) liquid recirculation and (b) gas mixing strategy. Fig.2 Operational conditions and nitrogen removal performance of continuous-flow PD/A system with different mixing strategies: (a) HRT, COD/NO3--N and gas flowrate, (b) concentration of influent TN, effluent NH4+-N, NO2--N and NO3--N, and temperature variation, (c) NLR, NRR and TN removal efficiency, (d) Anammox, denitrification and NO2--N consumed by anammox pathway. Fig.3 Effect of gas mixing on the ex-situ activities of PD and anammox bacteria. Fig.4 Characterization of EPS production in PD/A aggregates during long-term operation: (a) total concentration of EPS and ratio of TB-EPS/LB-EPS; concentration of PN, PS and PN/PS ratios in (b) loosely bound layer, and (c) tightly bound layer. Fig.5 Influence of gas mixing on EEM profiles of LB-EPS and TB-EPS at different NLR: (a) LBEPS and (b) TB-EPS on day 35 (phase G-2); (c) LB-EPS and (d) TB-EPS on day 103 (phase G-4); (e) LB-EPS and (f) TB-EPS on day 218 (phase G-6). Fig. 6 Microbial community of continuously feeding PD/A system under gas mixing at different conditions: Taxonomic analysis on (a) phylum and (b) genus classification; (c) Venn analysis of different samples; (d) shared genus among all the samples.

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Table 1 Nitrogen removal rates and effluent quality under different operational condition. fLb Mixing mode

Phase

Days

a

HRT

VL

or

(h)

(L/h)

fGc

Gas

(mg/L)

NO3--N

(L/h) Liquid

Effluent concentration

COD/ ratio

NH4+-N

NO2--N

NO3--N

NLR

NRR

(kgN/(m3·d))

TN removal efficiency (%)

L

1~25

1.5

1.04

4.74

3.4

5.14±2.54

2.52±1.47

2.94±2.52

0.96

0.79±0.09

82.3±8.9

G-1

26~37

1.5

1.04

0.42

3.4

4.00±1.92

2.40±1.17

3.61±1.54

0.96

0.80±0.05

83.1±5.5

G-2

38~61

1.0

1.56

0.42

3.4

1.97±2.58

2.06±1.12

2.28±0.99

1.44

1.29±0.09

89.5±6.0

G-3

62~98

0.75

2.08

0.45

3

1.38±1.68

1.55±1.04

1.16±0.94

1.92

1.79±0.07

93.2±3.8

G-4

99~114

0.5

3.12

0.63

3

6.48±3.89

2.87±1.32

6.24±2.73

2.88

2.16±0.28

75.1±9.6

G-5

115~165

0.5

3.12

0.72

3

5.87±2.34

3.09±1.39

5.94±2.66

2.88

2.17±0.2

75.2±6.8

G-6

166~229

0.5

3.12

0.83

3

2.91±2.19

3.15±1.37

3.61±1.47

2.88

2.42±0.19

83.9±6.8

a: Upflow velocity of wastewater in the reactor. b: Calculated flowrate of recirculated liquid at the liquid mixing stage. c: Calculated flowrate of gas inside the reactor at the gas mixing stage.

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Gas phase

Organic carbon

(a) Liquid recirculation

COD COD

(b) Gas mixing

Effluent

NH4+ -

NO3

Influent

Pump

Fig.1 Apparatus for single-stage PD/A process with different mixing conditions: (a) liquid recirculation and (b) gas mixing strategy.

30

0.4

0

Nitrogen concentration (mg/L)

120 Time (d)

200

0.0 240

32

40

24

20

16

0 0

40 NLR

80

120 Time (d)

NRR

160

200

TN removal efficiency

8 240 100

4

80

3

60

2

40

1

20

0

0

40

Anammox

80

120 Time (d)

Denitrification

100

Contribution to TN by different pathway (%)

160

60

5

NLR and NRR (kgN/(m3·d))

80

Inf.TN Eff.NH4+-N Eff.NO2--N Temperature Eff.NO3 -N

(b)

(c)

40

160

200

Gas flowrates (L/h)

0.8

1 0

(d)

Gas flowrates

HRT C/N

2

1.2

0 240

NO2--N consumed by anammox

60

80

45

60

30

40

15

20 0 0

40

80

120 Time (d)

Temperature (oC)

HRT (h) and C/N

3

Gas mixing

TN removal efficiency (%)

Liquid mixing

160

200

0 240

NO2--N consumed by anammox (mg/L)

(a)

Fig.2 Operational conditions and nitrogen removal performance of continuous-flow PD/A system with different mixing strategies: (a) HRT, COD/NO3--N and gas flowrate, (b) concentration of influent TN, effluent NH4+-N, NO2--N and NO3--N, and temperature variation, (c) NLR, NRR and TN removal efficiency, (d) Anammox, denitrification and NO2--N consumed by anammox pathway.

31

Gas mixing

2.0

60

1.5 rNO3 rNO2 rNH4

40

0

HRT

L

G-1

80 60

1.0

NTR

20

100

0.5

G-2

G-3 G-4 Phase

G-5

G-6

0.0

40

NTR (%)

80

HRT (h)

Specific biomass activity (mgN/(gVSS·h)

Liquid mixing

20 0

Fig. 3 Effect of gas mixing on the ex-situ activities of PD and anammox bacteria.

32

Liquid mixing

2.0

Flotation

150 100

1.0

50

0.5 0.0

L

G-2

G-3 G-4 Phase

LB-PN LB-PS

80

G-5

G-6

0 12

LB-PN/PS

9

60 40

6

20

3

0

L

G-2

(c) 100

G-4 G-3 Phase

G-5

G-6

60

9

TB-PN/PS

6

40

3

20 0

0 12

TB-PN TB-PS

80

L

G-2

G-4 G-3 Phase

PN/PS ratio

Concentration (mg/gVSS)

200

1.5

(b) 100

Concentration (mg/gVSS)

Total EPS TB-EPS/LB-EPS

G-5

G-6

PN/PS ratio

TB-EPS/LB-EPS ratio

Flotation

2.5

Total EPS (mg/gVSS)

Gas mixing

(a) 3.0

0

Fig.4 Characterization of EPS production in PD/A aggregates during long-term operation: (a) total concentration of EPS and ratio of TB-EPS/LB-EPS; concentration of PN, PS and PN/PS ratios in (b) LB layer, and (c) TB layer.

33

400

850.0 708.3

Excitation (nm)

(a)

350

566.7

300

A

425.0 283.3

250

B

850.0

(b)

350 300 250

425.0

C

283.3

C D

141.7

200 250 300 350 400 450 500 550

0.000

200 250 300 350 400 450 500 550

400

850.0

400

Emission (nm)

(c)

350

E

708.3 566.7

300

425.0 283.3

F

250

850.0

(d)

350 300

G

425.0 283.3

250

H

200 250 300 350 400 450 500 550

400

850.0

400

Emission (nm)

708.3

Exitation (nm)

Exitation (nm)

141.7

0.000

566.7

I

300

425.0 283.3

250

J

200 250 300 350 400 450 500 550

708.3 566.7

200 250 300 350 400 450 500 550

(e)

0.000

Emission (nm)

141.7

350

708.3 566.7

141.7

Exitation (nm)

Exitation (nm)

Excitation (nm)

400

0.000

Emission (nm) 850.0

(f)

350

708.3 566.7

300

K

250

L

141.7

425.0 283.3 141.7

200 250 300 350 400 450 500 550

0.000

Emission (nm)

0.000

Emission (nm)

Fig.5 Influence of gas mixing on EEM profiles of LB-EPS and TB-EPS at different NLR: (a) LBEPS and (b) TB-EPS on day 35 (phase G-2); (c) LB-EPS and (d) TB-EPS on day 103 (phase G-4); (e) LB-EPS and (f) TB-EPS on day 218 (phase G-6).

34

Fig.6 Microbial community of continuously feeding PD/A system under gas mixing at different conditions: Taxonomic analysis on (a) phylum and (b) genus classification; (c) Venn analysis of different samples; (d) shared genus among all the samples.

CRediT author statement

Rui Du: Conceptualization, Formal analysis, Writing- Original draft preparation, Funding acquisition. Shenbin Cao: Methodology, Writing- Reviewing and Editing.

35

Xiangchen Li: Data Curation. Jincheng Wang: Resources. Yongzhen Peng: Supervision.

Highlights

 High-efficiency continuous PD/A process was achieved by a novel gas mixing strategy.  Excellent effluent quality was obtained with short HRT of 0.5 h at temperature of 11.2 ºC.  Gas mixing significantly promoted nitrite production and anammox activities of PD/A.  Sludge floatation was avoided due to enhanced mass transfer and key EPS secretion.  Functional bacteria enrichment in PD/A granules was demonstrated under gas mixing. Declaration of interests ☒ 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.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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