A strategy of high-efficient nitrogen removal by an ammonia-oxidizing bacterium consortium

Accepted Manuscript A strategy of high-efficient nitrogen removal by an ammonia-oxidizing bacterium consortium Xiaolong Yang, Lihua Liu, Shoubing Wang PII: DOI: Reference:

S0960-8524(18)31718-8 https://doi.org/10.1016/j.biortech.2018.12.057 BITE 20814

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

20 October 2018 9 December 2018 16 December 2018

Please cite this article as: Yang, X., Liu, L., Wang, S., A strategy of high-efficient nitrogen removal by an ammoniaoxidizing bacterium consortium, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech. 2018.12.057

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A strategy of high-efficient nitrogen removal by an ammonia-oxidizing bacterium consortium Xiaolong Yang a, Lihua Liu b, Shoubing Wang a,* a

Department of Environmental Science and Engineering, Fudan University, 2005 Songhu

road, Shanghai 200433, PR China b

Maths & Physics College, Jinggangshan University, 28 Xueyuan road, Ji’an 343009, PR

China

* Corresponding author: Prof. Shoubing Wang, Fax: +86-21-65642297, E-mail: [email protected] Department of Environmental Science and Engineering, Fudan University, 2005 Songhu road, Shanghai 200433, PR China

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Abstract An ammonia-oxidizing bacterium consortium showed approximately 100% removal of NH4+-N with an initial concentration of 262.28  8.21 mg·L-1 within 10 days, and only 16.54  0.52% of NH4+-N was converted to NO2--N in this study. The consortium removed ammonium by heterotrophic nitrification and aerobic denitrification (HNAD) without N2O emission. The activity of AOB was not affected by low concentrations of FA or FNA, but completely inhibited by 0.04 mg HNO2·L-1. In a bioaugmentation treatment of eutrophic wastewater using the consortium, the removal efficiency of NH4+-N reached 90.85 ± 0.8% and 77.88 ± 1.86% at initial concentrations of 1.80 ± 0.04 mg·L-1 and 40.31 ± 0.57 mg·L-1, respectively, and the dissolved oxygen level had a significant impact on the consortium activity. No significant changes in the bacterial community structure were observed after the consortium addition, and local functional bacteria were enriched by aeration and contributed to ammonium nitrogen removal with AOB.

Keywords Ammonia-oxidizing

bacterium

consortium;

ammoxidation

characteristics;

bioaugmentation; eutrophic water; bacterial community structure

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1. Introduction Nitrogen enters natural water bodies through surface runoff and atmospheric deposition due to rapid urbanization, the application of large doses of nitrogen fertilizers (notably, the chemically reduced forms) and the intensification of livestock farming in the past few decades (Wu et al., 2017; Janssen et al., 2017; Jani et al., 2018). Ammonium is the most basic form of nitrogen, and pollution with it has become a prominent and intractable water environmental problem in China (Miao et al., 2017). Meanwhile, ammonium is also favored by non-nitrogen-fixing cyanobacteria such as Microcystis and Oscillatoria, which can cause massive algal blooms in lakes and rivers (Monchamp et al., 2014; Gardner et al., 2017). Currently, ammonium nitrogen has exceeded chemical oxygen demand (COD) as the primary index affecting surface water quality in China (Miao et al., 2017). A water quality assessment of 41 national aquatic germplasm resource conservation areas in 2015 revealed that only 1.6% of the water bodies meet the national ammonium standard, while the regions not meeting the standard were 3.72 million hectares in area (MEE of China, 2017). Meanwhile, discharge of excess ammonium to water bodies results in ammonia (NH3) autointoxication of fishes by dynamic exchange between NH4+ and NH3, even at a very low ammonia level (0.2 mg·L-1) (Zhang et al., 2018). Therefore, the removal of ammonium nitrogen is of great urgency for protecting surface water and drinking water supplies. Ammonium oxidation, as a crucial process within biological nitrogen transformation, is mostly accomplished by chemolithoautotrophic bacteria. Ammonium is first oxidized to nitrite by ammonia-oxidizing bacteria (AOB), such as Nitrosomonas and Nitrosococcus. Then, nitrite is oxidized to nitrate by nitrite-oxidizing bacteria (NOB), such as Nitrobacter

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and Nitrospira (Daims et al., 2016; Pelissari et al., 2018). In the nitrogen-polluted water bodies, however, the process occurs at a low level due to insufficient organisms and feedback inhibition of the bacterial cell by the intermediate product nitrite (NO2-) (Bai et al., 2012). Bioaugmentation can enhance pollutant removal by the inoculation of exogenous degrading bacteria and has been widely used (Liu et al., 2017; Figdore et al., 2018). Nevertheless, the difficulty of isolating and maintaining pure cultures of AOB has restricted their application to nitrogen removal. It is easier to obtain a bacterial consortium than a pure strain. Many studies have reported the enrichment of AOB by nitrifying activated sludge systems, such as sequencing batch reactors (SBRs) (Vadivelu et al., 2007; Zou et al., 2014; Zhang et al., 2015; Yuan et al., 2016); however, they have mainly focused on the susceptibility of AOB to the environment and changes in population structure, and the isolation and application of these populations were ignored. AOB and NOB populations typically coexist within bacterial clusters in the biogeochemical cycle of nitrogen due to the close spatial co-aggregation, the tight interaction between them are often observed in nitrifying biofilms and activated sludge flocs (Daims et al., 2016; Pelissari et al., 2018). Metabolites, signalling molecules, genetic material and defensive compounds are interchanged by short diffusion pathways, which achieves the maximization of substrate use (Flemming et al., 2016). For instance, Zou et al. (2014) demonstrated that the coupling of an enriched autotrophic nitrifying consortium and a heterotrophic denitrifying consortium in SBRs greatly enhanced nitrogen removal and reduced carbon supply. The prolonged purification effect and minor impact on the bacterial

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community have been observed in wastewater treatment by inoculating with a bacterial consortium (Festa et al., 2016). Hence, to develop a bioaugmentation measure using ammonia-oxidizing bacterium consortium for enhancing in-situ biological nitrification and the understanding interaction between AOB populations and local bacteria are important. The objective of this study was to investigate the performance of ammonia-oxidizing bacterium consortium on nitrogen removal and validate its effectiveness in bioaugmentation treatment for eutrophic water. A batch of tests were performed to evaluate the ammoxidation performance of AOB consortium and the effect of FA, FNA and carbon sources on ammoxidation activity. Bioaugmentation test was conducted by a laboratory-simulated water system with intermittent aeration, and microbial community structures and functional microorganisms were analyzed to explore the impact of the consortium on the bacterial community. These findings will provide a new way of thinking about removing nitrogen from water bodies by bioaugmentation strategies and facilitate researches on AOB. 2. Materials and methods 2.1. Cultivation of an ammonia-oxidizing bacterium consortium and its morphological characteristics A consortium of AOB was isolated from landfill leachate through persistent domestication. The activation and cultivation of this consortium were performed in a 250 mL Erlenmeyer flask containing 100 mL of modified ammonia-oxidizing bacterium medium at 30 C with shaking (160 rpm). Ammonia-oxidizing bacterium consortiums harvested in the mid-log growth phase were used for morphological analysis with scanning electron microscopy (SEM, TESCAN VEGA3) (Jung et al. 2011). 5

2.2. Ammoxidation characteristics of an ammonia-oxidizing bacterium consortium An activated ammonia-oxidizing bacterium consortium was inoculated into 300 mL of ammonia-oxidizing bacterium medium (5% v/v), which was then incubated in a 500 mL Erlenmeyer flask for 10 days at 30 °C with shaking (160 rpm). The culture was sampled every day to examine the removal of ammonium, the production of oxidation products (NO2--N, NO3--N) and the consumption of nitrogen for bacterial cell growth. In the incubation, the levels of the ammonia monooxygenase catalytic subunit A (amoA), hydroxylamine oxidase (hao), nitrite reductase (nirK) and nitric oxide reductase (norB) genes were determined by RT-qPCR. RNA was extracted from the cultures at 2, 4 and 6 days using TRIzol Reagent (Invitrogen, USA) and treated with RNase-free DNase I. Quantified RNA was used to synthesize cDNA using FastKing-RT SuperMix (Tiangen, China). RT-qPCR was performed in a volume of 20 μL containing 1 μL cDNA template, 0.3 μM of each forward and reverse primer and 10 μL SuperReal PreMix Plus (SYBR Green) (Tiangen, China) in a Roche LightCycler® 480 system using published primers (Kapoor et al., 2015). All samples were analyzed in triplicate. Standard curves were created with 10-fold dilution series of plasmids containing the sequences for each of the targeted genes. The threshold cycle (Ct) values obtained for each gene were compared with the standard curve to determine their copy numbers (Kapoor et al., 2016). 2.3. Influence of carbon sources on the removal of ammonium nitrogen Organic carbon (OC) with 1 g·L-1 sodium succinate and inorganic carbon (IC) with 1 g·L1

sodium bicarbonate were used as carbon sources of ammonia-oxidizing bacterium medium

to cultivate ammonia-oxidizing bacterium consortiums. In these media, CaCO3 was replaced with 1.0 g·L-1 CaCl2. The initial concentration of ammonium nitrogen was 261.09 6

 1.03 mg·L-1. The inoculation percentage and culture conditions were as described above. The cultures were sampled to measure the concentrations of NH4+-N, NO2--N, NO3--N, intracellular nitrogen and total nitrogen (TN) every day. Each test was conducted in triplicate. 2.4. Influence of FA and FNA on the removal of ammonium nitrogen Ammonia-oxidizing bacterium media containing various initial concentrations of NH4+N (50, 100, 200, 300, 400, 500, 600, 800, and 1000 mg·L-1) were used to test the inhibition of ammoxidation by free ammonia (FA). Inhibition by free nitrous acid (FNA) was tested in ammonia-oxidizing bacterium medium with various concentrations of NO2--N (0, 5, 10, 20, 40, 60, 80, 160, and 320 mg·L-1) and 260 mg·L-1 ammonium. The pH of the medium was 8.0 for the FA test and 7.2 for the FNA test, where the effects of FA inhibition would be nonsignificant (Sun et al., 2013; Kinh et al., 2017). The temperature of incubation was fixed at 30 °C. The changes in NH4+-N, NO2--N and pH in media were analyzed in the test. Each test was performed in triplicate. 2.5. Bioaugmentation treatment for eutrophic water with an ammonia-oxidizing bacterium consortium An experimental system was constructed to simulate natural water environments in the laboratory. In this device, 20 liter plastic buckets were used as reactors, and LED light tubes running 12 h every day served as “sunlight”. An aerator was used to supply dissolved oxygen (DO) in the experimental group and operated for 3 h every morning and evening. The temperature was maintained at 30 °C during the daytime and 22 °C at night with the air conditions in the laboratory.

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Eutrophic water collected from landscape ponds was used in the experiment, and the characteristics of the water are listed in Table 1. A total of 18 L of water was poured into each bucket of the above experimental device. A 50 mL bacterial suspension of the ammonia-oxidizing bacterium consortium was added into each bucket of the experimental group, and the treatment was performed for 10 days according to the above operation conditions. A group without an inoculation was used as the control. Water samples were collected every day, their pH value, concentrations of NH4+-N, NO2--N, NO3--N, TN, and total phosphorus (TP), and chemical oxygen demand (CODcr) were determined. To further investigate the performance of the ammonia-oxidizing bacterium consortium for the treatment of high-ammonium water. The eutrophic wastewater with 40 mg·L-1 NH4+N was used for test and the characteristics of water are listed in Table 1. To simulate a natural water body, surface sediment from a landscape water body was taken and mixed fully with landscape water adjusted as above and then placed into a bucket. The mixed liquor suspended solid (MLSS) concentration was 3.5 ± 0.62 g·L-1. After standing for 3 days, the culture of ammonia-oxidizing bacterium consortium was transferred into experimental bucket and cultivated for one month. The bucket without the consortium addition was used as the control. The operating conditions were the same as above. Each treatment was performed in triplicate. Every two days, the pH value of the water and the levels of NH4+-N, NO2--N, NO3--N, TN, CODcr, TP and DO were determined. 2.6. Analysis methods The concentrations of NH4+-N, NO2--N, NO3--N, TN, TP and CODcr were measured in accordance with standard methods (APHA, 2005). The pH was determined by a pH meter (Mettler Toledo, FE28, Germany). The DO concentration in water was determined by a DO 8

meter (INESA, JPB607, China). N2O was determined using a Trace GC Ultra gas chromatograph (Thermo Finnigan, USA) equipped with a thermal conductivity detector and packed with a PoraPlot Q capillary column. The data were all expressed as the mean ± standard deviation from triplicate experiments, and graphical analyses were performed using SigmaPlot software. 2.7. Illumina sequencing of 16S rRNA after bioaugmentation using the ammonia-oxidizing bacterium consortium After 30 days of cultivation, mixtures of sediment and overlying water were collected to assess shifts of microbial community structure. The V3-V4 region of the 16S rRNA gene was amplified with the primers 338f 5'ACTCCTACGGGAGGCAGCA-3' and 806r 5'GGACTACHVGGGTWTCTAAT-3', where the adapter sequences and barcode sequences had been combined. PCR reactions were performed in 50 μL volumes with Q5 HighFidelity DNA Polymerase system (New England Biolabs, USA). All PCR products were pooled and purified with the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA). Purified amplicons were quantified using the HS-DNA Quant-it™ dsDNA HS Assay Kit (Invitrogen, USA) and sequenced on an Illumina Hiseq 2500 platform. The effective sequences were clustered into operational taxonomic units (OTUs) with 97% sequence similarities in QIIME v1.8.0. All raw reads were submitted to the NCBI with the accession number SRP136570. 3. Results and discussion 3.1. Growth and morphological characteristics of the ammonia-oxidizing bacterium consortium

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After the AOB had been cultivated in medium for 7 days, the organisms and calcium carbonate powder combined to form a floc. As revealed by SEM analysis, the AOB were mainly composed of rod-shaped and filamentous bacteria, and these microorganisms attached to and grew on the surface of the calcium carbonate powder, intertwining with each other to form a solid mass. This result was consistent with the adhering growth characteristics of known AOB. Katsuyuki and Sadao (1983) reported that the growth of AOB and ammonium oxidation were hastened with the addition of calcium carbonate to soil because it created a favorable environment for enzyme activity. Thus, calcium carbonate not only was used to adjust the alkalinity of the medium to maintain an appropriate pH but also provided an ideal habitat for AOB. 3.2. Ammoxidation performance of the ammonia-oxidizing bacterium consortium The ammonia-oxidizing bacterium consortium had a perfect performance in the removal of ammonium nitrogen (Fig. 1a). The removal efficiency was as high as 99.6  0.2% with an initial NH4+-N concentration of 262.28  8.21 mg·L-1 after 10 days of cultivation; of this NH4+-N, 13.84  0.44% was changed to biomass, and 32.0  1.01% was converted to ammoxidation products (NO2--N and NO3--N). The net removal efficiency of nitrogen reached 53.92  1.05%. However, comparatively little attention has been focused on the obtainment and application of ammonia-oxidizing bacterium consortiums. Nakano et al. (2008) constructed a consortium comprising AOB, and denitrifying bacteria removed roughly 90% of NH4+-N and 40% of TN in 37 days when they were incubated in an ANA3 medium containing equal concentrations of NH4+-N and NO3--N (56 mg·L-1). However, the NO2--N in the medium increased to 74.2 mg·L-1. Erna et al. (2013) reported the ammoniaoxidizing bacterium consortium M1, isolated from a mangrove area, showing a removal 10

rate of 97.24% for an initial ammonia concentration of 2.17 ± 0.10 mg·L-1 over 14 days. A high efficiency of ammoxidation and relatively low accumulation of nitrite were obtained by the ammonia-oxidizing bacterium consortium in the present study compared with those consortiums. Using RNA-based RT-qPCR assays, we examined the transcript levels of several functional genes in the ammonium oxidization of the ammonia-oxidizing bacterium consortium. Fig. 1b shows that the expression level of the amoA gene was significantly higher than that of the hao and nirK genes at the early stages of cultivation but decreased with incubation time. The expression of hao and nirK transcripts had a significant increase at the middle and later stages. These results suggested that the ammonia-oxidizing bacterium consortium had simultaneous nitrification and aerobic denitrification capacity and the performance corresponded well with the changes in nitrogen content in Fig. 1a. N2O was deemed to be the most potent greenhouse gas, being approximately 200-300-fold stronger than CO2. N2O emission was observed during nitrogen removal in many wastewater treatment plants (WWTPs) (Kinh et al., 2017). In this study, we did not detect the expression of the norB transcript, which implied that the consortium, lacking nitrous oxide (N2O) generation, mainly converted NH4+-N to nitric oxide (NO) or N2 by denitrification. 3.3. Influence of carbon sources on the removal of ammonium nitrogen The data of intracellular nitrogen showed that both the growth of the ammonia-oxidizing bacterium consortium and the removal of nitrogen were clearly different when organic and inorganic carbon sources were provided (Fig. 2). In Fig. 2a, although the consortium showed rapidly grew with OC, the removal efficiency of NH4+-N was only 43.11  2.49% 11

during the first 5 days. A comparison of the initial and final TN concentrations indicated that 21.32  2.58% of NH4+-N was reduced to nitrogen gas and that 3.22  0.03% and 4.81  0.07% of NH4+-N were transformed into NO2--N and NO3--N, respectively. These data revealed that ammonium was removed by heterotrophic nitrification and denitrification and that the absence of IC might decrease activity of AOB (Torà et al., 2010). Recently, many strains capable of heterotrophic nitrification and aerobic denitrification (HNAD) nitrogen removal such as Bacillus subtilis A1, Rhodococcus sp. CPZ24 and Enterobacter cloacae CF-S27 have been isolated (Yang et al., 2011; Chen et al., 2012; Padhi et al., 2017). The consortium performed worse than Rhodococcus sp. CPZ24, and Enterobacter cloacae CFS27 performed superior to Bacillus subtilis A1 using organic carbon. The removal rate of NH4+-N was approximately 10% higher when IC was the carbon source than when OC was the carbon source, yet NH4+-N was almost entirely oxidized to NO2--N and NO3--N, in addition to being used for cell growth (Fig. 2b). The oxidization efficiency decreased with increasing NO2--N, and approximately 120 mg·L-1 NH4+-N remained in the medium at the end, which might result from the inhibition of ammoxidation by FNA. That is, this phenomenon might occur mainly because FNA leads to the elimination of NOB (Vadivelu et al., 2007). Several studies on Nitrosomonas europaea showed that certain organic compounds are available for AOB under anaerobic conditions while IC was the preferred substrate for biosynthesis (Steuernagel et al., 2018). Genomic analysis revealed that a carbonic anhydrase gene (cynT) was next to a gene for an anion transporter and only 4.6 kb from the Rubisco genes in N. europaea. CO2, the substrate for Rubisco, would be accumulated when carbonate or bicarbonate was provided, thereby promoting CO2 assimilation and metabolism of AOBs (Chain et al., 2003). Jiang et al. 12

(2015) observed that an increasing oxygen uptake activity was also associated with increased Rubisco activity. In addition, acid produced in the nitrification process could be neutralized by bicarbonate, which slowed the formation of FNA. These pathway may promote NH4+-N oxidation by AOB in IC supply. However, when OC was supplied, ammonium might be mainly removed by heterotrophic nitrification. Although heterotrophs also converted NH4+ to NO2- by the successive action of ammonia monooxy-genase (AMO) and hydroxylamine oxidoreductase (HAO), the energy required for oxidation was provided by oxidation of organic carbon, which would result in a competitive inhibition between organic carbon and ammonium on oxygen, subsequently suppressing heterotrophic nitrification (Jin et al., 2017). From the data in Fig. 1a, we found that the removal efficiency of NH4+-N was almost 100% with mixed carbon sources (OC and IC). Such a result was not affected by FNA, indicating that the heterotrophic and autotrophic bacteria in the ammonia-oxidizing bacterium consortium cooperated with each other for nitrogen removal, probably the reason for the exchange of substrates between AOB and heterotrophs. The cooperation was also observed in previous studies (Zou et al., 2014; Yong et al., 2015; Jin et al., 2017). 3.4. Inhibition of the ammonia-oxidizing bacterium consortium ammoxidation by FA and FNA The concentrations of FA and FNA were related to the ammonium or nitrite concentration, pH and temperature. They can be described with Eq 1 and Eq 2, respectively (Sun et al., 2013).

(1)

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where Kb and Kw are the ammonia base ionization constant and the ionization constant of water, respectively. An analytic expression for the change in the equilibrium constants with temperature (°C) is as follows: Kb:Kw =

. (2)

where Ka is the ionization constant of nitrous acid and was related to temperature(°C) by the equation: Ka =

.

Over the past few decades, researchers have studied the inhibitory effects of FA and FNA on nitrifying bacteria, demonstrating that 10~300 mg·L-1 FA and 0.16~2.8 mg·L-1 FNA inhibited or ceased ammoxidation by AOB and that the threshold concentrations for inhibition of NOB were much lower (Vadivelu et al., 2007; Kapoor et al., 2015). This inhibition was also confirmed in this study. As shown in Fig. 3a, a low concentration of FA (less than or equal to 27.26 mg NH3·L-1) had no effect on ammonia-oxidizing bacterium consortium activity, which was consistent with previous studies (Vadivelu et al., 2007). The removal efficiency of NH4+-N declined at 36.65 mg NH3·L-1 and continued to decrease with increasing FA. However, the ammoxidation did not completely cease, and a removal rate of 58.85  2.31% was retained at an FA concentration of approximately 90 mg NH3·L-1. The results indicated that the ammonia-oxidizing bacterium consortium was adapted to high FA concentrations. Meanwhile, the FNA concentration in the medium at the end of incubation was over 0.015 mg HNO2·L-1 when FA was 36.65 mg NH3·L-1, suggesting that the effect was probably inhibited mainly by FNA because AOB were more sensitive to FNA than FA with respect to their ammoxidation activity (Ma et al., 2017). Vadivelu et al. (2007) reported that the anabolism of NOB ceased completely at above 6.0 mg NH3-N·L-1, 14

which would result in the accumulation of the ammoxidation product NO2--N and a high FNA concentration. FNA could affect the oxygen uptake activity and energy generation by uncoupling effect and metabolism in AOB cells (Zhou et al., 2011). Studies on Nitrosomonas genus of AOB revealed that enzymes involved in DNA and protein repair and energy generation, and proteins involved in phage prevention, oxidative stress and iron transport were significantly upregulated on exposure to FNA (Laloo et al., 2018). Fig. 3b confirmed our conjecture. When FNA was 0.0101 mg HNO2·L-1, the NH4+-N removal rate was approximately 20% less than that when FNA was 0.005 mg HNO2·L-1, and the ammoxidation of the consortium was completely stopped at approximately 0.04 mg HNO2·L-1. The threshold concentration was far below those of previous studies of activated sludge systems (Vadivelu et al., 2007; Sun et al., 2013; Kinh et al., 2017), which was likely due to the AOB having a greater impact load in the activated sludge system than in our system. 3.5. Bioaugmentation of ammonia-oxidizing bacterium consortium purifying eutrophic water The

performance

of

the

ammonia-oxidizing

bacterium

consortium

during

bioaugmentation was monitored for 10 days, and the results are shown in Fig. 4. NH4+-N decreased rapidly from 1.80 ± 0.04 mg·L-1 to 0.17 ± 0.02 mg·L-1during the first 7 days, and over 80% of nitrogen was thoroughly eliminated. Such high removal was similar to other researchers’ findings. Erna et al. (2013) used the ammonia-oxidizing bacterium consortium M1, however, the result in this study takes only half the time of their experiment. The decrease in CODcr and TP revealed that the consortium grew better in the system. Only a small amount of NO2--N was detected during the experiment, and it was completely 15

removed. Meanwhile, decreases of 16.08 ± 0.81% NH4+-N, 30.73 ± 2.29% CODcr and 13.08 ± 3.12% TP were observed in the control, which might be attributed to the growth of indigenous microorganisms in landscape water. Many tests determining the ammoxidation performance of strains were performed using synthetic wastewater under ideal conditions, even by SBRs or membrane bioreactors (MBRs) (Zou et al., 2014; Zhang et al., 2015; Kinh et al., 2017), which might not exhibit the real capability of strains, indicating their poor performance in application. However, the actual water quality is complex and highly variable in natural rivers and lakes. Thus, this study, using real wastewater collected from a pond as landscape water, is more accurate and meaningful than many of these other studies. 3.6. Nitrogen removal from high-ammonium landscape water by the ammonia-oxidizing bacterium consortium Yu et al. (2012) reported that the NH4+-N concentration in the Beiyun River (Beijing, China) ranged from 5.03 mg·L-1 to 40.45 mg·L-1, which accounted for 78% of the TN and was due to the intensification of human activities and industrialization. High ammonium concentrations and low levels of carbon sources (bioavailable) have therefore become the main characteristics of domestic sewage in China due to economic structure changes (Wu et al., 2017). Thus, in this study, eutrophic wastewater with a high NH4+-N concentration (40.31 ± 0.57 mg·L-1) and a C:N ratio of 4.22 was treated by bioaugmentation with the ammonia-oxidizing bacterium consortium for approximately one month, and the results are presented in Fig. 5. The NH4+-N removal efficiency of the A and B groups (using the ammonia-oxidizing bacterium consortium) was significantly higher than that of the controls (C and D) (Fig. 5a). In particular, in the A group, NH4+-N was effectively and rapidly 16

removed within the first 18 days. The oxidization of ammonium followed zero-order reaction kinetics with a linear equation of y = -1.886x + 42.793 and R² = 0.9905 (y means the concentration of ammonium, x means the incubation time) and resulted in a maximum removal rate of 78.4 ± 0.72%. These data indicated that the ammonia-oxidizing bacterium consortium purified eutrophic water excellently and that its activities were not affected by a high ammonium concentration and a low C:N ratio, while the nitrogen removal efficiency descended slightly after 18 days and leveled off thereafter. This change might be caused by the death of AOB, resulting in a release of intracellular nitrogen into water. However, Fig. 5b shows that the concentration of TN did not increase after the first 18 days, which indicated that nitrogen removal in the A group continued. This behavior might be contributed by the indigenous bacteria coming from landscape water. Only 13.97 ± 2.82% of NH4+-N reduction was obtained in the B group (without aeration), suggesting that water oxygen content was an important limiting factor in ammonium oxidization by the consortium. The CODcr and concentrations of TP, DO, and other species further confirmed the results (Fig. 5e-g). Surprisingly, the NH4+-N concentration in the C group decreased uniformly from 39.12 ± 0.57 mg·L-1 (6 days) to 17.54 ± 0.75 mg·L-1 (30 days) (Fig. 5a), and the TN decreased from 39.68 ± 0.19 mg·L-1 (6 days) to 24.60 ± 1.14 mg·L-1 (30 days) (Fig. 5b). NO2--N and NO3--N accumulation were detected during this period, suggesting that ammonium oxidization occurred in the C group. Statistical analysis showed that the equation y = -0.9925x + 45.734 (R² = 0.9882, y means the concentration of ammonium, x means the incubation time) fit the ammoxidation kinetics, yet their speed and efficiency were low in comparison with those of the A group. Although the C group demonstrated a relatively low nitrogen 17

removal efficiency in the beginning, the ammonium nitrogen could be completely eliminated on the 46th day based on the above equation. These results revealed that AOB and NOB were present in natural water bodies, could be enriched and grew rapidly by aeration. Higher nitrite accumulation was observed during ammonium oxidization by a pure strain of AOB or a population of AOB in an activated sludge system (Zou et al., 2014; Fumasoli et al., 2017; Miao et al., 2017). In the current experiment, NO2--N was detected on the 6th day in the A group and increased gradually to the maximum concentration of 3.25 ± 0.23 mg·L-1 at day 18, thereafter decreasing rapidly to only 0.77 ± 0.07 mg·L-1 by the end of the experiment (Fig. 5c). The accumulation in this study was lower than that in previous studies. However, in the C group, NO2--N showed a continuous increase from the 8th day to the end of the experiment, with a maximum accumulation of 4.54 ± 0.28 mg·L-1. NO3--N (Fig. 5d) was eliminated from group A after 2 days of operation, yet on the 6th day, it reappeared, exhibited a nearly exponential increase and leveled off. In the C group, NO3--N was also eliminated within the first 4 days and then increased continuously with NO2-N until the end of the experiment. The NO3--N final concentration was 2.58 ± 0.07 mg·L-1. The variation tendency of CODcr, TP and pH indices coincided with NH4+-N and TN in the A and C groups during the experiment (Fig. 5e-g). However, in the B and D groups, the decreases in CODcr, TP and pH were not distinct; in contrast, DO clearly decreased (Fig. 5h). These results further demonstrate the significance of water flow and DO levels in influencing the water quality of natural water bodies. 3.7. Shifts in microbial community structure and functional microorganisms

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A total of 900307 raw tags were generated from each example of the A, B, C and D groups. After strict quality assessment and removal of low-quality reads and chimeras, an average of 177584 ± 1488 clean tags was obtained per sample. The 16S rDNA gene sequences were clustered into 2197 OTUs using 97% similarity. Good’s coverage for each sample was above 99.9%, and rarefaction curves reached saturation, suggesting that the results properly represent the bacterial components of the samples. A significant difference in community structure was found between groups with aeration (A and C) and those without aeration (B and D) based on alpha diversity statistics. The highest Shannon index and the lowest Simpson index were obtained for the example of group A, which is similar to the result obtained by Wang et al. when they compared anaerobic sludge and aerobic sludge samples from tannery WWTPs (Wang et al., 2014). These results suggested that the bacterial community structure did not significantly shift after the introduction of a specific ammonia-oxidizing bacterium consortium, while local microbial diversity could be enhanced by aeration. The microbial community structures for the samples of each group at the phylum level are displayed in Fig. 6a. A total of 34 known bacterial phyla were detected. Proteobacteria was the predominant phylum in each group and was mainly composed of Betaproteobacteria, and its proportion was much higher in the C group than in the other groups, contributing up to 58.67% ± 1.27% of the total number of OTUs, followed by Chloroflexi, Bacteroidetes, Acidobacteria and Nitrospirae. These bacterial phyla are common in eutrophic lakes (Bai et al., 2012). Based on sequence analysis of the 16S rRNA gene, almost all known AOB were classified into Betaproteobacteria and Nitrosococcus (Yong et al., 2015; Jo et al., 2017). The occurrence of Nitrospirae in all of the groups

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verified the presence of NOB in the eutrophic landscape water, with a percentage of approximately 5% for each group, suggesting that activity of NOB was not or only slightly limited due to intermittent aeration by its being associated with the gradual increase in NO3-N in the A and C groups. The result was different from observations in bioaugmentation experiments for the SBR system with activated sludge (Daims et al, 2016; Miao et al., 2017; Jo et al., 2017). The relative abundance of Cyanobacteria was notably higher in the B and D groups than in the A and C groups, which further confirmed the efficacy of reaeration technology in suppressing cyanobacterial blooms in eutrophic water. At the family level, 18 predominant families with a relative abundance of over 1% were detected in the samples. Aeromonadaceae, Anaerolineaceae, Nitrospirales_FW13, Gallionellaceae and Geobacteraceae showed lower abundance in the A and C groups than in the B and D groups. In contrast, Nitrospiraceae, Nitrosomonadaceae, Comamonadaceae and Hydrogenophilaceae were strikingly higher in abundance in the A and C groups than in the B and D groups. These differences were consistent with alpha diversity statistics. Previous studies indicated that Nitrospiraceae and Nitrosomonadaceae were predominant bacterial taxa in biological nitrogen removal and that the majority of AOB identified were members of the family Nitrosomonadaceae such as Nitrosomonas spp., Nitrosospira spp., Nitrosolobus spp. and Nitrosovibrio spp (Bai et al., 2012; Yuan et al., 2016; Pelissari et al., 2018). Comamonadaceae and Hydrogenophilaceae were observed in digested piggery wastewater treatment by Wang et al. (2017), and they could carry out denitrification (Willems and Gillis, 2005). Meanwhile, in this study, the percentages of unclassified bacterium and bacteria with an abundance sum below 1% (30.2%~34.69%) were higher than those in the samples from the SBR or MBR systems (Yuan et al., 2016; Wang et al., 20

2017), reflecting the taxonomically complex environment of natural water bodies such as that shown in the research on Dianchi Lake, China, by Bai et al. (2012). Metastat analysis was performed on the population profiles and further detected 13 predominant genera with significant shifts between groups with aeration and those without aeration, and the results are shown in Fig. 6b. Sphingobium, Comamonas and Nitrospira were the dominant populations in the A and C groups. Sphingobium and Novosphingobium, two members of the order Sphingomonadales that have been confirmed to degrade various substances (Duan et al., 2015), are widely present in freshwater systems, and their abundance was driven by high concentrations of the phosphate radical (PO4-) and the ammonium ion (NH4+) (Liu et al., 2015; Janssen et al., 2017). Liao et al. (2015) reported that Sphingobium might play important roles in the effective removal of nitrogenous organic compounds and N-nitrosamine precursors in water. The presence of Comamonas was also important for nitrogen oxidation (Yang et al., 2016; Liu et al., 2017). Conversely, Tolumonas was the most predominant population in the B and D groups, followed by Geobacter and Dechloromonas. Reportedly, these genera showed a characteristic of facultative anaerobic growth that is commonly related to the metabolism of iron and organic compounds and desulfurization in water bodies (Weber et al., 2006), yet further work elucidating the links between these bacteria and nitrogen removal by variation in the expression level of genes is necessary. 3.8. Aeration promotes growth of indigenous bacteria and their ammoxidation Currently, most research has focused on separating and identifying pure cultures of AOB (Yang et al., 2011; Chen et al., 2012; Padhi et al., 2017). However, this approach is long and arduous. Specialized bacterial consortiums were widespread in polluted environments 21

and showed excellent pollutant removal (Karamalidis er al., 2010). In this study, a bacterial consortium composed of the genera Sphingobium, Comamonas, Nitrosomonas, Thiobacillus, Nitrospira, and others was enriched from natural landscape water by aeration and exhibited high capacity and efficiency of ammonium nitrogen removal. Two molecules of oxygen are necessary for the oxidation of one ammonium to nitrate. High affinity for oxygen was observed in AOB and NOB, and the expression levels of amoA, hao, nirk and norB genes, which are involved in ammoxidation and nitrogen reduction pathways, were affected by DO levels (Yu and Chandran, 2010). In eutrophic water, the DO was exhausted by the formation of cyanobacterial blooms, which not only suppressed the growth of local nitrifying bacteria but also resulted in the failure of colonization and reproduction of exogenous AOB and NOB that had been added. Aeration was used to maintain a high level of DO in an industrial WWTP (IWTP), but it was also a good way to enrich local degrading microorganisms in the ecological restoration of lakes and rivers. This study justified the application of aeration in bioaugmentation using AOB. 4. Conclusions In the study, an ammonia-oxidizing bacterium consortium could effectively remove ammonium by heterotrophic nitrification and aerobic denitrification without N2O emission. Its activity was not affected by low concentrations of FA or FNA, but completely inhibited by 0.04 mg HNO2·L-1. In the bioaugmentation treatment of eutrophic wastewater, the consortium was excellent, but the dissolved oxygen level had an appreciable effect. The changes on the bacterial community structure were not notably after the consortium addition, local bacteria such as Sphingobium sp., Comamonas sp., Nitrosomonas sp. and Nitrospira sp. were enriched by aeration and played a vital role in nitrogen removal with 22

AOB. Acknowledgements This work was supported by the National Major Science and Technology Program for Water Pollution Control and Treatment (Grant No.2017ZX07602-001). The authors would like to thank the anonymous reviewers for their constructive and positive comments.

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Table and Figure captions Table 1 Characteristics of experimental water Fig. 1. Ammoxidation performance of the ammonia-oxidizing bacterium consortium (a) and the transcription levels of nitrogen cycle genes in the ammonia-oxidizing bacterium consortium (b). Fig. 2. Characteristics of nitrogen removal of the ammonia-oxidizing bacterium consortium with an organic carbon source (a) and an inorganic carbon source (b). Fig. 3. Effect of FA and FNA inhibition on the NH4+-N removal rate of the ammoniaoxidizing bacterium consortium. Blue diamonds and black circles are the removal efficiency of NH4+-N and the FNA concentration in the medium after 8 days of cultivation, respectively. Fig. 4. The purification of eutrophic landscape water by the ammonia-oxidizing bacterium consortium. Fig. 5. Changes in water quality in the four groups in the batch test. A group: treatment with ammonia-oxidizing bacterium consortium with aeration, B group: treatment with ammonia-oxidizing bacterium consortium without aeration, C group: only aeration, D group: neither ammonia-oxidizing bacterium consortium nor aeration. Fig. 6. Microbial community compositions of the examples from four groups at the phylum level (a) and the dominant genera with significant shifts between the four groups (b). Seven genera,

Tolumonas,

Sphingobium,

Nitrospira,

Haliangium,

Novosphingobium,

Methyloversatilis, and Bdellovibrio, were different between the A and B groups (p < 0.05), and all of these genera were different between the C and D groups (p < 0.05).

29

Table 1 Characteristics of experimental water. Characteristic

Concentration (mg·L-1) Eutrophic landscape water

High-ammonium landscape water

NH4+-N

1.80±0.04

40.31±0.57

CODcr

17.67±0.98

181.42±1.16

TN

3.09±0.02

43.02±0.86

TP

0.36±0.01

0.34±0.02

DO

4.24±0.07

4.20±0.05

pH

8.21±0.01

8.19±0.01

30

Fig. 1. Ammoxidation performance of the ammonia-oxidizing bacterium consortium (a) and the transcription levels of nitrogen cycle genes in the ammonia-oxidizing bacterium consortium (b).

Fig. 2. Characteristics of nitrogen removal of the ammonia-oxidizing bacterium consortium with an organic carbon source (a) and an inorganic carbon source (b).

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Fig. 3. Effect of FA and FNA inhibition on the NH4+-N removal rate of the ammonia-oxidizing bacterium consortium. Blue diamonds and black circles are the removal efficiency of NH4+-N and the FNA concentration in the medium after 8 days of cultivation, respectively.

Fig. 4. The purification of eutrophic landscape water by the ammonia-oxidizing bacterium consortium.

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Fig. 5. Changes in water quality in the four groups in the batch test. A group: treatment with ammoniaoxidizing bacterium consortium with aeration, B group: treatment with ammonia-oxidizing bacterium consortium without aeration, C group: only aeration, D group: neither ammonia-oxidizing bacterium consortium nor aeration.

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Fig. 6. Microbial community compositions of the examples from four groups at the phylum level (a) and the dominant genera with significant shifts between the four groups (b). Seven genera, Tolumonas, Sphingobium, Nitrospira, Haliangium, Novosphingobium, Methyloversatilis, and Bdellovibrio, were different between the A and B groups (p < 0.05), and all of these genera were different between the C and D groups (p < 0.05).

34

Highlights



AOB consortium efficiently removed ammonium by HNAD without N2O emission.



Ammoxidation was completely inhibited by 0.04 mg HNO2·L-1.



Bioaugmentation treatment using AOB consortium was feasible.



Aeration promoted ammoxidation of local bacteria.

Graphical Abstract

35