Long-term effective remediation of black-odorous water via regulating calcium nitrate sustained-release

Nitrate addition is reported as a cost-effective method for remediating black-odorous water, which is mainly induced by the deficiency of electron acceptor. However, excessive release of nitrate and lack of long-term effectiveness significantly limited the application of direct nitrate dosing technology. Herein, for remediating black-odorous water, we constructed a nitrate sustained-release ecological concrete (ecoN-concrete), in which calcium nitrate (Ca(NO3)2) was dosed into concrete block to regulate the release of nitrate. The results showed that chemical oxygen demand (COD), turbidity, ammonia, phosphate, and sulfate were significantly removed in an ecoN-concrete-contained reactor fed with black-odorous water, and its removal efficiency was largely dependent on Ca(NO3)2 dosage. Meanwhile, the released nitrate was lower than 25% of its total dosage and nitrite was lower than 1.5 mg/L during 14 days remediation. After three recycles, the removal efficiencies of COD and turbidity by using ecoN-concrete were still more than 85%, indicating an excellent nitrate sustained-release performance of ecoN-concrete, which can be applied for preventing water re-blackening and re-stinking. Further investigation illustrated that the ecoN-concrete (1) decreased the abundance of Desulfovibrio, Desulfomonile, and Desulforhabdus in the phylum of Desulfobacterota to alleviate the odorous gas production and (2) significantly increased the abundance of Bacillus and Thermomonas, which utilized the released-nitrate for consuming organic matters and ammonia. This study provided an artful Ca(NO3)2 dosing strategy and long-term effective method for black-odorous water remediation.


Introduction
The blackening and stinking of waterbodies with low-flowing velocity has been emerged worldwide for decades, such as in Australia (Watts et al., 2018), Vol:.( 1234567890) India (Welling et al., 2020), and Brazil (Johannsson et al., 2020).In China, the blackening and odorization of rivers have been observed throughout the whole country since the first report in 1983 (Liang et al., 2018).According to the data released in 2019, the number of black-odorous waterbodies had increased to 2100 nationwide, which were mainly distributed in the provinces of Guangdong, Anhui, Hunan, Shandong, and Jiangsu (Cao et al., 2020;Zhu et al., 2022).Generally, black-odorous water phenomenon referred to the water with black or grey colors and malodor smells, which happened when large amounts of discharged pollutants, particularly organic compounds, exceeded carrying capacity of the waterbody (Cao et al., 2020;Zhang et al., 2022).The massively received organic pollutants, which dissolved or suspended in the water or precipitated into sediment, were firstly degraded by aerobic microorganisms, consumed excessive dissolved oxygen (DO), and induced a reductive condition (Mai et al., 2021).Subsequently, the residual organics were decomposed by anaerobic microorganisms, such as methanogens and sulfate-reducing bacteria (SRB), to form the blackening and odor compounds, such as chromophoric dissolved organic matter, ferrous sulfide (FeS), hydrogen sulfide (H 2 S), methanethiol, cadaverine, and ammonia (Liang et al., 2018;Zhang et al., 2022).Therefore, insufficient electron acceptors for organic pollutants oxidation were one of the incentive reasons for the water blackening and stinking.
For microorganisms in an aquatic environment, their common electron acceptor was DO, followed by nitrate and sulfate, and others.As depletion of DO in black-odorous water, anaerobic microorganisms (e.g., SRB) utilized sulfate as the electron acceptor to produce subsequent metabolites, such as dimethyl sulfide (DMS), hydrogen sulfide (H 2 S), and dimethyl disulfide (DMDS), which were reported as the main sources of odors (Cao et al., 2020;Wang et al., 2020).Previously, aeration was reported as the primary method for black-odorous water remediation (Yu et al., 2022).But the disadvantages of the longterm aeration technology, such as high mechanical and operating cost and stinks emission, significantly limited its large-scale application.Compared to aeration, nitrate (NO 3 − -N) addition is deemed to be a costeffective method for black-odorous water remediation (Yin et al., 2019).The added NO 3 − -N is observed to promote heterotrophic and autotrophic denitrification, during which organic pollutants, acidified volatile sulfides (AVS), and even ferrous ions are consumed as electron donors, and then repaired the water blackening and stinking (Cao et al., 2020;He et al., 2017).Finally, a shift in dominant bacterial community of the water was observed from SRB community to nitratereduction bacteria (NRB) one (He et al., 2017), which could simultaneously oxidize organic matters, inhibit sulfate reduction, and remove nitrogen.
However, insufficient NO 3 − -N could not completely oxidize the organic pollutants, sulfur, and ferrous ions, and then re-blackening and re-stinking of the water were observed (He et al., 2017).Conversely, excessively dosage of NO 3 − -N would result in the potential risk of NO 3 − -N and NO 2 − -N pollution.For example, even if after adding calcium nitrate (Ca(NO 3 ) 2 ) for 0 and 85 days, the concentration of NO 3 − -N was observed to be as high as 2298 and 253 mg/L, and the concentration of highly toxic nitrite (NO 2 − -N) even reached to 12.6-268 mg/L (Yamada et al., 2012).Simultaneously, the added NO 3 − -N sharply declined and rapidly depleted at the initial stage of experiments, which limited its effectiveness on long-term remediation of black-odorous water (Mai et al., 2021;Yin et al., 2019).In addition, the dissolved nitrate is also easily washed away by the disturbance and flow of the water.Therefore, how to dose NO 3 − -N precisely based on the characteristic of treated water or sediment has become the critical point of the NO 3 − -N addition technology.Meanwhile, it is difficult to achieve a long-term effect on black-odorous water remediation and prevent water re-blackening and re-stinking in situ by adding Ca(NO 3 ) 2 directly.
Commonly, the slopes even the bottoms of some waterbodies are covered by a hardened concrete, particularly in the urban areas.The characteristic of concrete indicated that concrete blocks have a dense structure and a controllable internal porosity via regulating the proportions of coarse aggregates, mortar matrix, and cement, so as to obtain a concrete structure with an expected water permeability (Kearsley & Wainwright, 2001).In the view of the sustainedrelease principle, we constructed a novel ecological concrete with nitrate (Ca(NO 3 ) 2 ) sustainable release properties (ecoN-concrete, or abbreviated as EC) in the current study.Firstly, the repairing performance of ecoN-concrete on black-odorous water was evaluated.Secondly, the optimal dosage of Ca(NO 3 ) 2 in the Vol.: (0123456789) ecoN-concrete and its removal efficiency of sulphate and phosphate in black-odorous water were investigated.Then, the reusability of ecoN-concrete was tested via replacing the black-odorous water repeatedly to verify the repairing performance on water re-blackening and re-stinking.Finally, the evolution of bacterial community in the black-odorous water remediated by ecoN-concrete was deciphered during a long-term experiment.

Materials and synthetic black-odorous water
The concrete blocks at different Ca(NO 3 ) 2 dosing ratios were constructed as described in literatures (Wen-jie et al. 2012;Liu et al., 2020).Briefly, calcium sulphoaluminate (CSA) cement (Anhui Conch Cement Co., Ltd., China), gravels, and natural sand (mean particle size of 1-2 cm) were purchased from local building materials market (Wuhu, China).To construct an ecological concrete (ecoN-concretes, abbreviated as EC) blocks containing various levels of Ca(NO 3 ) 2 , 34 g SAC, 150 g natural sand, and a certain amount of Ca(NO 3 ) 2 were added into a small concrete mixer, respectively.Subsequently, about 18 g pure water was added into the mixer with the waterconcrete ratio lower than 10%.After mixing evenly, the mixture was poured into a self-made mold (cylindrical mold, Φ100 × 1.5 mm).Next, raw geosynthetic materials with corresponding area were covered and fixed on the surface of the mixture to provide biofilm carriers for microorganisms.Subsequently, the mixture was vibrated for a few minutes with a small concrete vibrating tube to strengthen the compactness of the concrete and the combination between the geotextile and the concrete.Then, the concrete blocks were cured for more than 7 days by being covered with wet geotextiles at room temperature.Finally, the ecoNconcrete blocks (100 ± 2 g, dry weight) with different concentrations of Ca(NO 3 ) 2 were collected after demolding.Similarly, the concrete blocks without Ca(NO 3 ) 2 addition were also constructed and named as normal concrete (abbreviated as NC).
Synthetic black-odorous water was prepared according to the literature with minor modification (Wang et al., 2019a, b) O, 88 urea, and 600 starch.The microbial inoculum was collected from the bottom sediment of a local black-odorous water at the depth of 0.5-0.7 m, and inoculated with the volume ratio of 10%.The synthetic black-odorous water was sealed in a reactor with a volume of 20 L and anaerobically cultured at 30 °C in water bath for 10 days.

Batch tests
To identify the effect of ecoN-concrete on blackodorous water remediation, batch test A was carried out in six identical reactors (1.0 L).These reactors were equally divided into two groups.The ecoN-concrete blocks containing 0.5% Ca(NO 3 ) 2 (of the total weight) prepared as described in the "Materials and synthetic black-odorous water" section were added at the bottom of each reactor of one group.After that, 1.0 L synthetic black-odorous water was poured into each of the reactors in two groups.Subsequently, all reactors were sealed with the parafilm, on the surface of which a small hole was reserved for mechanical stirring.The reactors with ecoN-concrete addition were categorized into the ecoN-concrete group (EC group), while the reactors without ecoN-concrete were run as the control.Then, all reactors were placed in a constant temperature room (28 ± 2 °C) and stirred with a mechanical stirrer at the speed of 50 rpm.Around 10 mL water was sampled using a syringe at specific intervals for the determination of COD, NH 4 + -N, NO 3 − -N, NO 2 − -N, and turbidity.The measurements of DO, pH, and ORP were determined in situ using electrodes.
Batch test B was further conducted to evaluate the dosage effect of Ca(NO 3 ) 2 on black-odorous water remediation.Before the experiment, the ecoNconcrete blocks containing three levels of Ca(NO 3 ) 2 (0.25%, 0.50%, and 1.00% of the total weight) were prepared.The experimental procedures were carried out as described in experimental protocol I (Supporting Information, SI).Then, the optimal dosage of Ca(NO 3 ) 2 was chosen according to the different performances of ecoN-concrete blocks on blackodorous water remediation.After that, the effects of ecoN-concrete on phosphate and sulphate removal in the black-odorous water were investigated using the ecoN-concrete block containing the optimal Ca(NO 3 ) 2 dosage, and the experiment was carried out similarly to that described as batch test A.

Long-term experiment
Two long-term experimental protocols (protocols II and III) were established to investigate the reusability of ecoN-concrete and the evolution of bacterial community during black-odorous water remediation, respectively (Fig. S1).According to Protocol II (Fig. S1), three groups of identical reactors with the volume of 1.0 L were operated as follows: (1) normal concrete (NC) group: the reactor contained 1.0 L synthetic black-odorous water and a normal concrete block (100 g, prepared as described in the "Materials and synthetic black-odorous water" section), (2) EcoN-concrete (EC) group: the reactor contained 1.0 L synthetic black-odorous water and an ecoN-concrete block (100 g, 1.00% Ca(NO 3 ) 2 , prepared as described in the "Materials and synthetic black-odorous water" section), and (3) the reactors merely contained 1.0 L synthetic black-odorous water were run as the control.All reactors were sealed and operated as described in the "Batch tests" section for 10 days a cycle.At the end of each cycle, the overlying water in all reactors were sampled, analyzed, and then replaced by 1.0 L fresh black-odorous water, respectively.
Experimental protocol III was carried out to investigate the effect of concrete and ecoN-concrete on the bacterial community evolution during the blackodorous water remediation.As shown in Fig. S1, three groups of reactors were set as described in protocol II, but all reactors were continuously operated for 21 days.The bacteria of the water in reactors were collected using a sterile millipore filter (0.22 μm) for 16S rDNA Amplicon Sequencing on 0 (raw blackodorous water), 10th, and 21st day.The concrete and ecoN-concrete blocks were collected for morphological observation at the end of the experiment.

Other methods for analysis and statistical analysis
The determination of general water quality index, such as pH value, NO 3 − − N, and NO 2 − − N, were conducted according to Standard Methods (APHA, 2005).The variations in concrete morphology and the bacteria community during the long-term experiment were investigated as detailed in SI.All tests were carried out in triplicate, and the results were expressed as mean ± standard deviation.An analysis of variance (ANOVA) was used to test the significance of results, and p < 0.05 was considered to be statistically significant.

Results and discussion
Effect of ecoN-concrete on black-odorous water remediation Aliquots of ecoN-concrete (100 g, containing 0.5% Ca(NO 3 ) 2 ) were added into 1.0 L synthetic blackodorous water to evaluate its remediation effect.As shown in Fig. 1A, the concentration of COD gradually decreased in both control and ecoN-concrete groups.But COD removal efficiency in the ecoN-concrete group was significantly higher than that of the control.Specifically, COD concentration in the control group decreased from 185 mg/L to 78 mg/L after incubation of 14 days, whereas the final COD concentration in the ecoN-concrete group was significantly decreased to 44 mg/L, which was about half of the control (Fig. 1A).This indicates that ecoN-concrete addition could remarkably remediate organic pollution of black-odorous water.In addition, the final removal efficiency of turbidity made a minor difference between the control and ecoN-concrete groups; however, the turbidity removal rate of the ecoN-concrete group was significantly higher than that of the control (72 vs. 59 NTU per day in the initial stage of the experiment (0-4 days)) (Fig. 2B).Similarly, a sharper decrease of COD was also observed at the initial stage of the experiment (Fig. 1A).These data indicated that the application of ecoN-concrete could significantly improve the removal rate of COD and turbidity and shorten its remediation cycle.
Excessive NH 4 + -N was regarded as another vital pollutant in black-odorous water (Cao et al., 2020).As shown in Fig. 1C, the concentration of NH 4 + -N declined by 15 mg/L in the ecoN-concrete group after 14 days, which was 2.46-fold of that of the control.NO 3 − -N was not detected in the control group, while it gradually rose to 19 mg/L in the ecoN-concrete group indicating that Ca(NO 3 ) 2 was sustainably released from the ecoN-concrete doped with Ca(NO 3 ) 2 (Fig. 1C).As an important interme- the predecessor of some carcinogens and is commonly accumulated during denitrification (Wan et al., 2016).In this study, compared with the control, the maximum concentration of NO 2 − -N just slightly increased (0.17 vs. 0.52 mg/L, Fig. 1C) even though the concentration of NO 3 − -N reached to 19 mg/L in the ecoN-concrete group.Initially, the black-odorous water was acidic (pH < 7.0, 0 day) because of anaerobic fermentation of organic matters (Fig. 1D) (Cao et al., 2020;Yin et al., 2019).Of note, after feeding the reactors with the synthetic black-odorous water, pH value in the control group was increased to 7.53, which is likely attributed to the volatilization of acidic components, and then fluctuated around 7.51 (± 0.54) with the depletion of acidic components.In contrast, pH value in the ecoN-concrete group significantly increased and maintained up to 8.89, which might be ascribed to the release of both Ca(OH) 2 from ecoN-concrete (Huang et al., 2022;Kitamura et al., 2000) and alkalinity resulting from bio-denitrification (Xing et al., 2018).
A persistent hypoxia environment, induced by the aerobic metabolism of accumulated organic pollutants, was reported as the main incentives of water blackening and stinking (Xia et al., 2022).And indeed, a lower DO concentration in both the control and ecoN-concrete group (< 2.10 mg/L, Fig. S2A) were observed in this study.Thus, NO 3 − -N has been added into the black-odorous water and its underlying sediment for stimulating heterotrophic denitrification because that this hypoxic and organicsrich environment in black-odorous water might be favorable for boosting denitrifiers (Liu et al., 2017;Yamada et al., 2012;Yin et al., 2019).Nevertheless, previous studies depicted that the excessive release of NO (12.6-268 mg/L) posed a significant potential risk to the aquatic environment and its biota if nitrate was injected directly (Xia et al., 2022;Yamada et al., 2012).Moreover, a rapid decline or even exhaustion of NO 3 − -N was another common issue at the initial stage of the experiment in previous literatures (Mai et al., 2021;Yin et al., 2019).In this study, the addition of Ca(NO 3 ) 2 into CSA cement, on one hand, significantly decreased the capillary pore volume to form a more compact structure, improved its compressive strength and its resistance of on permeability and corrosion (Huang et al., 2022).On the other hand, the more compact structure of ecoN-concrete was favorable for locking in the added Ca(NO 3 ) 2 and maintaining a slowly and sustainably release of Ca(NO 3 ) 2 .This demonstrated that the addition of into CSA cement not only improved its compressive strength but also avoided the excessive accumulation of NO 3 − -N and NO 2 − -N and maintained a long-term remediation effect (Fig. 1).Sustainable release of NO 3 − -N stimulated the activity of heterotrophic denitrifiers and consumed organic pollutants in the blackodorous water (Fig. 1A).Moreover, a few dissolved compounds (e.g., dissolved organic matters, S 2− , and Fe 2+ ) were rapidly absorbed by the concrete (Xie et al., 2021).Simultaneously, the micro-flocculation and coprecipitation effect of Ca 2+ and Ca(OH) 2 from the ecoN-concrete quickly removed the suspended colloids and particles (such as insoluble organic matters, suspended solids), which were responsible for turbidity in the black-odorous water (Huang et al., 2022;Kitamura et al., 2000).Therefore, a drastic decrease of COD and turbidity were observed at the early stage of experiment (Fig. 1A, B).Interestingly, the concentration of NH 4 + -N not sharply declined at the early stage of the experiment but successively decreased during the experiment (Fig. 1C).Such performance might be likely resulted from a biological process rather than absorption by the concrete, and we will be addressed it in the following text.
Effect of Ca(NO 3 ) 2 dosing ratio in ecoN-concrete on black-odorous water remediation To further quantify Ca(NO 3 ) 2 dosing amount on remediation effectiveness of black-odorous water, ecoNconcretes containing 3 ratio levels of Ca(NO 3 ) 2 , 0.25% (low), 0.50% (medium), and 1.00% (high) were added into the reactors.Obviously, the final remediation effect of black-odorous water was largely dependent on the dosing ratio of Ca(NO 3 ) 2 (Fig. 2).As shown in Figs.1A and 2A, COD values were significantly decreased in all ecoN-concrete groups at the first 2 days and were obviously lower than that of the control.The variations of COD concentrations in three ecoN-concrete groups (0.25%, 0.50%, and 1.00%) were negligible at the early stage of experiment (0-8 d).After culturing for 8 days, COD concentration in the groups with low and medium Ca(NO 3 ) 2 dosing ratios was finally steady at around 56 mg/L (Fig. 2A).However, COD concentrations in the high Ca(NO 3 ) 2 dosing ratio group continuously declined with the increase of time and finally reached 15 mg/L, which were about 0.19-and 0.27-fold of that of the control and lower Ca(NO 3 ) 2 dosing ratios groups, respectively (Figs. 1A and 2A).Similarly, due to the effect of micro-flocculation and coprecipitation, turbidity of all Ca(NO 3 ) 2 dosing ecoN-concrete groups was significantly decreased in the first 2 days (Kitamura et al., 2000).But the final turbidity removal efficiencies of all ecoN-concrete groups had no significant difference and were all greater than 80%, which was significantly higher than that of the control (60%, Fig. 1B).Residual NO 3 − -N concentration was largely dependent on the Ca(NO 3 ) 2 dosing ratio of the ecoN-concrete (Fig. 2C).The final NO 3 − -N concentrations were 11, 20, and 41 mg/L following the increase of Ca(NO 3 ) 2 dosing ratios 0.25%, 0.50%, and 1.00%, respectively.Meanwhile, NO 2 − -N concentration partly raised with the increase of Ca(NO 3 ) 2 dosing ratio and the duration of experiment, but the final concentrations of all groups were lower than 1.5 mg/L (Fig. 2D).The increase of NO 2 − -N might be caused by electron competition between different denitrification steps, especially when the organic electron donor was depleted (Fig. 2A) (Pan et al., 2013).Interestingly, Figs.1C and 2E indicated that final NH 4 + -N concentration decreased with the increase of Ca(NO 3 ) 2 dosing ratio from 47 (Control), 36 (0.25%), 32 (0.50%), and 26 (1.00%) mg/L.This was contrary to the results of previous studies, which might be caused by the discrepancies in original microbial populations surviving in its respective black-odorous water and will be discussed in the following section.(Yin et al., 2019).In addition, lower pH value in control increased with the participation of ecoN-concrete, which was consistent with the aforementioned observation (Figs.1D  and 2F).However, pH variations among three ecoNconcrete groups were negligible (Fig. 2F), indicating that the increase of Ca(NO 3 ) 2 dosing ratio had insignificant effects on pH variation.
Previous studies indicated that an excessive release of NO 3 − -N and NO 2 − -N was immediately observed after a direct NO 3 − -N addition (Xia et al., 2022;Yamada et al., 2012).In this study, the maximum released NO 3 − -N amount was lower than 25% of the doped amount of NO 3 − -N in all ecoN-concrete groups after exposure for 14 days (Fig. 2C).This suggested that Vol:.( 1234567890) the release rate of NO 3 − -N was significantly slowed down for the adsorptive and locked effect of concrete on Ca(NO 3 ) 2 (Huang et al., 2022), which provided an effective way to control the excessive release of NO 3 − -N.Comparatively, at the early stage of experiment (0-2 days), a rapid release of NO 3 − -N promoted the consumption of organic pollutants by heterotrophic denitrifiers and resulted in a sharp decline of COD in all ecoN-concrete groups (Fig. 2A, C).Subsequently, the consumption rates of organic pollutants were limited by the insufficient release of NO 3 − -N from ecoN-concrete in the lower Ca(NO 3 ) 2 dosing groups (0.25% and 0.50% Ca(NO 3 ) 2 dosing ratio; Fig. 2A, C).However, an appropriate increase of Ca(NO 3 ) 2 dosing ratio (1.00%) in the ecoN-concrete could exert a positive effect on a long-term removal of COD and regulate NO 3 − -N concentrations to an acceptable range (Fig. 2A, C).However, the residual concentration of NO 3 − -N and NH 4 + -N was higher than its maximum contaminant level (MCL, 10 mg/L) of the water quality standard (Su et al., 2017).It should be noted that the higher residual NO 3 − -N and NH 4 + -N was just obtained in a fixed reaction system with adding 100 g ecoN-concrete into 1.0 L synthetic black-odorous water, which had a higher initial NH 4 + -N concentration (43 mg/L) than typical black-odorous water (Cao et al., 2020).Consequently, it is essential to regulate an optimal dosage ratio of Ca(NO 3 ) 2 to maintain a lower level of residual NO 3 − -N and NH 4 + -N in the water by preliminarily assessing the contaminants level of black-odor water.
Effect of ecoN-concrete on phosphorus and sulfate removal in black-odorous water Apart from organic and nitrogenous pollutants, sulfur and phosphorus were also considered as two important pollutants in the black-odorous water (Cao et al., 2020).As shown in Fig. 3A, SO 4 2− concentration was increased from 135 mg/L in the raw black-odorous water to 337 and 227 mg/L in the control and ecoNconcrete groups during 0 to 2 days of the experiment, respectively.It was reported that S 2− precipitated with metals ions (e.g., Fe 2+ , Mn 2+ , and Cu 2+ ), so that metal sulfides (e.g., FeS, MnS, and CuS) were formed and suspended in the overlaying water resulting in the water blackening (Liang et al., 2018).With the addition of NO 3 − -N and its consequent recovery of DO, the reduced sulfides were eventually transformed to sulfate (Figs.S2 and 3A) (Cao et al., 2020;Mai et al., 2021).Addition of NO 3 − -N also stimulated the activity of autotrophic denitrifiers, by which S 2− was oxidized to SO 4 2− and produced electrons for denitrification (Yamada et al., 2012).This transformation of S 2− might be the reason for the increase of SO 4 2− .Subsequently, SO 4 2− concentrations turned into decline and finally reached to around 186 and 14 mg/L in the control and ecoN-concrete groups, respectively.Previous literatures showed that SO 4 2− concentrations rose with the addition of NO 3 − -N and even reached to 928-4643 mg/L after NO 3 − -N was added into blackodorous sediments (Yamada et al., 2012;Yin et al., 2019).Consequently, the release of high concentration SO 4 2− resulted in another important secondary pollutant during black-odorous water remediation.In this study, the addition of ecoN-concrete remarkably decreased the peak and final concentration of SO 4 2− , which might be caused by the precipitation of Ca 2+ released from the ecoN-concrete (Kitamura et al., 2000).Similarly, a rapid decrease of TP (more than 95% of TP removal) was also observed on the second day, which was reportedly sourced from the formation of CaPO 4 and co-precipitation with Fe(OH) 3 via the release of Ca 2+ from ecoN-concrete and the oxidation of Fe 2+ in the black-odorous water (Liu et al., 2017;Tang & Pakshirajan, 2018).

Reusability of ecoN-concrete on black-odorous water remediation
The reusability of ecoN-concrete on black-odorous water remediation was investigated via renewing the black-odorous water periodically.As shown in Figs. 4 and S3, compared to the control, participation of concrete (NC and EC groups) could both improve the removal of COD, NH 4 + -N, and turbidity.Interestingly, without dosing Ca(NO 3 ) 2 , NC also played a positive role in pollutants removal due to its absorption and ions releasing (Kitamura et al., 2000;Xie et al., 2021).In the first cycle, the removal of COD, NH 4 + -N, and turbidity in black-odorous water was obviously facilitated by the participation of EC and NC, but the difference of its performance was negligible (Fig. 4).Specifically, by the end of first operation cycle, the concentration of COD and NH 4 + -N were 133 and 52 mg/L, 33 and 28 mg/L, and 16 and 26 mg/L in the control, NC, and EC groups (Fig. 4A, B), respectively.However, with the increase of operation cycle, the removal performance of pollutants (e.g., COD, NH 4 + -N, and turbidity) gradually deteriorated in the NC group, while the positive effects of EC on removal of contaminants kept at a relatively high level.For example, COD removal efficiencies in the NC group were 88%, 69%, and 64%, while those were 94%, 91%, and 87% in the EC group at the end of first, second, and third operation cycles, respectively (Fig. S3).Similar profiles were observed on the removal performance of NH 4 + -N and turbidity (Fig. 4B, C).These removal profiles demonstrated that the ecoN-concrete was characterized by its outstanding reusability after comparing with normal concrete.It is reported that organic matters and NH 4 + -N are the main pollutants in the overlying water and underlying sediment in the black-odorous waters (Cao et al., 2020;Zhu et al., 2017).The traditional ecological rehabilitation theories suggested that the concrete slope damage the ecology of soil and water (Pan et al., 2016).However, our results indicated that normal concrete revetment might be favorable for alleviate the water blacking and stinking, but this positive effect just maintained a limited time (Fig. 4).Due to the sustainable slow-release of Ca 2+ and NO 3 − -N, ecoN-concrete could maintain a higher efficiency on black-odorous water remediation (especially for the removal of COD, NH 4 + -N, and turbidity) and a lower nitrogen discharge for a long term, as compared to the NC group.

Morphological changes of concretes during the long-term experiment
The micromorphology of NC and EC before and after the long-term experiment was observed using SEM.As depicted in Fig. 5A, B, both NC and EC have a rough surface and a large number of interior pits and pores, which provided abundant specific surface area for the adsorption of pollutants, such as organic matters, NH 4 + -N, and PO 4 3− (Xie et al., 2021;Wen-jie et al. 2012).This might be the reasons for the rapid decline of COD, turbidity, NH 4 + -N, PO 4 3− at the early stage of experiment (Figs. 1, 2, and 3).Meanwhile, a compacted inner structure with some interconnected pores in the concrete could keep a slow and sustainable release rate of Ca(NO 3 ) 2 ( Huang et al., 2022 ) and provide a channel for Ca(NO 3 ) 2 releasing to the black-odorous water.In addition, the rough surface of concrete also facilitates the adhesion and growth of bacteria, especially denitrifying bacteria with a lengthy generation cycle, to form biofilms (Hill & Khan, 2008).The formation of biofilm might be another cause for keeping high COD removal efficiency at the later stage of long-term experiment (Fig. 4).After a 30-day experiment, the surface of concretes was all covered by a biofilm layer (Fig. 5C, D).However, it can be seen that the gelatinous-like layer of NC was rougher and thicker than that of the EC, which might be attributed to the accumulation of absorbed organic maters from the black-odorous water.As compared to the NC, more bacteria were adhered to Vol:. ( 1234567890) the surface of the EC.For EC, the absorbed organics were oxidized by the attached bacteria using released NO 3 − -N as electron acceptor and promoted the bacterial growth.This finally improved the water quality (Figs. 1, 2, 3, and 4).Conversely, the pollutants were accumulated on the surface of normal concrete due to lack of electron acceptor.

Microbial community evolution of black-odorous water treated with ecoN-concrete
To evaluate the effect of EC on microbial community evolution, three reactors (a NC-contained reactor, an EC-contained reactor, and an empty reactor (Blank)) were fed with identical black-odorous water and cultivated for a long-term cultivation without water renewing.The bacteria in waters were collected for highthroughput sequencing on the 1st (raw water), 10th, and 21st days, respectively.As shown in Table 1, the value of microbial community diversity indices, such as Chao1, observed OTUs, and Shannon, in both NC and EC group were significantly lower than that of the Blank.It is indicated that bacterial diversity of the black-odorous water decreased as compared to the Blank, which might be due to the screening effect of concrete and ecoN-concrete on the microorganisms, such as the variation of pH (Figs. 1 and S2) (Behnood et al., 2016).In addition, NO 3 − -N released from ecoN-concrete improved the ORP of black-odorous water, created an anoxic or aerobic environment, and inhibited survival of indigenous anaerobic bacteria (Liu et al., 2017), which further decreased the bacterial diversity of the water at the early stage (Table 1).Compared to the Blank and NC group, the continuously released NO 3 − -N from the ecoN-concrete promoted the growth of anoxic and aerobic bacteria and finally increased the diversity and richness of these bacteria (Table 1).These indices illustrated that the application of concrete or ecoN-concrete could significantly change the bacterial diversity of black-odorous water.Subsequently, the microbial populations were compared using the OTUs and shown in Venn diagrams (Fig. 6A, B).The shared OUTs were 307 among the raw water and the blank, NC, and EC groups on the 10th day, which dropped to 243 after culturing for 21 days.Simultaneously, the unique OTUs in the NC and EC group increased from 70 and 70 to 100 and 108 with the prolonging of operation time from 10 to 21 d, respectively.Taking the EC group as an example (Tables S2  and S3), the unique genera, such as, Acidibacter, Anaeromusa, and Chryseomicrobium, were involved in iron redox and organic matter degradation, due to the large amount of organic matter and iron in the black-odorous water at the early treatment stage (10 d) (Duan et al., 2022;Wu et al., 2022).However, a variety of nitrogen-related genera, such as Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Ralstonia, and Phenylobacterium, were observed in the EC group at the later stage of the experiment, which might cause by the sustainedrelease of NO 3 − -N from the ecoN-concrete (Aslam et al., 2005;Wang et al., 2022;You et al., 2021).
After annotation, the bacterial community was classified at the level of phylum.As shown in Fig. 6C and Table S4, the bacteria were categorized into 39 phyla, in which the phyla of Firmicutes, Proteobacteria, Desulfobacterota, Bacteroidota, Spirochaetota, and Cloacimonadota were the top six phyla and accounted for more than 55% of the total sequences.However, the dominant phyla ranked differently between the samples.For instance, the highest phylum was Proteobacteria in the sample of Raw (28.6%),Blank10 (30.1%),NC10 (37.5%), and Blank21 (34.0%) but was observed to be Firmicutes phylum in the group of EC10 (31.7%),NC21 (21.2%), and EC21 (29.3%).It is indicated that the bacterial community was significantly varied with the prolonging of experiment time, except for the group of Blank.Compared with the Blank, the phylum of Firmicutes significantly increased from 7.2% (Blank) to 21.2% (NC21) and 29.3% (EC21), which was only 5.9% in the Raw.It is well known that many bacteria in Firmicutes could produce endospores to resist extreme conditions (e.g., desiccation and high salinity) (Filippidou et al., 2019).In black-odorous water, these bacteria in Firmicutes survived under a resting condition (low metabolic activity) and were remarkably stimulated by the participation of concrete (especially ecoN-concrete).In addition, the relative abundance of Desulfobacterota dropped to 18.8% and 14.5% in the group of NC10 and EC10 as compared to the Raw (21.3%) and Blank10 (21.9%), respectively (Table S4).This might be caused by the rapid removal of sulfate via precipitation with the released Ca 2+ from concrete and ecoN-concrete (Fig. 3) and the inhibitory effect of released NO 3 − -N.The analysis on bacterial community was detailed at the level of genus.The detected bacteria were catalyzed into 326 genera, in which the top 30 genera accounted for more than 45% of the total sequences (Fig. 6D and Table S5).Normally, the top five genera were Bacillus, Desulfovibrio, Thermomonas, Cloacimonadaceae, and Desulfomonile, but the dominant genera varied significantly with the experiment stage and treatment.The genera of Desulfovibrio (11.04%) (Amrani et al., 2014), Cloacimonadaceae (9.21%) (Yekta et al., 2019), Tistrella (5.90%) (Shi et al., 2002), Desulfomonile (2.65%) (DeWeerd et al., 1990), Gastranaerophilales (2.64%) (Soo et al., 2014), and Thermomonas (2.04%) (Wang et al., 2019a, b) were dominated in the Raw group because the raw blackodorous water contained high levels of sulfur-and nitrogen-containing compounds and organic matters.Compared to the Blank and NC groups, the abundance of Desulfovibrio, Desulfomonile, and Desulforhabdus in most of the samples collected from EC group showed a downward trend (Table S5).These three genera all belonged to the phylum of Desulfobacterota (Fig. S4), which was reported as typical sulfate reducers to produce odorous gases in black-odorous water (Amrani et al., 2014;Cao et al., 2020;DeWeerd et al., 1990).Previous studies suggested that the activity of sulfate reducer could be inhibited by the presence of NO 3 − -N, which was released from the ecoN-concrete in this study (Fig. 1) (He et al., 2010).Therefore, these data indicated that the participation of ecoN-concrete could be helpful to alleviate the odor of black-odorous water.
Apart from the sulfur-related genera, there were two genera, Bacillus and Thermomonas, were significantly boosted in the EC groups.The most rapidly increased genus was Bacillus whose abundance was 1.14% in the Raw group.After a 10-day experiment, its abundance was 1.80% and 0.72% in the Blank and NC10 groups, but remarkably increased to 27.06% in the EC10 group, which was more than 15 and 37 folds of the Blank and NC10 groups, respectively (Table S5).As a genus of Firmicutes, Bacillus is a facultative aerobic bacterium widely found in soil and water and might keep endospores state in the raw black-odorous water (low abundance in the Raw group, Fig. 6D) in this study (Errington, 2003;Filippidou et al., 2019).Under both aerobic and anaerobic conditions, Bacillus was reported to grow using nitrate or nitrite as electron acceptor (Verbaendert et al., 2011;Yang et al., 2020).In this study, the sustainably released NO 3 − -N from ecoN-concrete provided abundant electron acceptors for the growth of Bacillus even though there were low level of DO in black-odorous water of all groups (Table S5 and Fig. 1).The significant increase of Bacillus drove heterotrophic denitrification, during which abundant organic matters were consumed as electron donors (Fig. 1A).With the decline of COD, it was more difficult for Bacillus to acquire electron donors, and hence a slight decrease of Bacillus abundance (by 3.44%) was observed in the EC group as the experimental time extended from 10 to 21 days (Fig. 6D).Simultaneously, the strains of Bacillus were also identified to utilize NH 4 + -N under aerobic conditions and were widely used for NH 4 + -N removal during wastewater treatment (Yang et al., 2020).In addition, similar to Bacillus, the second increased genus, Thermomonas, was also identified to achieve simultaneous heterotrophic nitrification-denitrification using O 2 or NO 3 − -N as electron acceptor (Langone et al., 2014;Zhou et al., 2021).On one hand, the decrease of COD, which consumed by NO 3 − -N released from Vol:. ( 1234567890) ecoN-concrete, made NH 4 + -N outcompeting with organic pollutants for electron acceptors.On the other hand, the increase of ammonia-consumed bacteria (e.g., Bacillus, Thermomonas) also favored the decline of NH 4 + -N in the water.Accordingly, high abundance of Bacillus and Thermomonas stimulating by concrete and ecoN-concrete was the main reason for the concentration decrease of both COD and NH 4 + -N in the NC and EC groups (Figs. 4 and 6D).However, it should be noted that the residual NH 4 + -N concentration was still at a high level (> 25 mg/L), indicating that additional assistive technologies (e.g., aeration) might be provided to improve the removal efficiency of NH 4 + -N, especially for black-odorous water containing high NH 4 + -N.

Conclusion
In this study, we proposed an ecoN-concrete technology via dosing Ca (NO 3 ) 2 into concrete for blackodorous water remediation, and succeed in ameliorating the excessive release of nitrate and nitrite as well as short-term effectiveness caused by a conventional direct-injection Ca (NO 3 ) 2 practice.The application of ecoN-concrete demonstrated an excellent performance in pollutants removal (COD, turbidity, NH 4 + -N, SO 4 2− , and PO 4 3− ) from black-odorous water and a sustainable NO 3 − -N release as compared to normal concrete and direct Ca (NO 3 ) 2 injection.Morphological observation and microbial community analysis revealed that the Ca (NO 3 ) 2 released from ecoN-concrete (1) decreased the phylum of Desulfobacterota to inhibit the odor production and (2) significantly increased the genera of Bacillus and Thermomonas, which consumed organic pollutants and NH 4 + -N using NO 3 − -N as an electron acceptor.

Fig. 1
Fig. 1 Effect of ecoN-concrete on black-odorous water remediation performance.Evolution of COD A, turbidity B, inorganic nitrogen C, and pH D. Error bars represent standard deviations of triplicate tests.Ca(NO 3 ) 2 dosing ratio of the ecoN-concrete was 0.5%

Fig. 2
Fig. 2 Effect of NO 3 − -N dosing ratio in ecoN-concrete on black-odorous water remediation.The evolution of COD A, turbidity B, NO 3 − -N C, NO 2 − -N D, NH 4 + -N E, and pH F. Error bars represent standard deviations of triplicate tests

Fig. 3
Fig.3Variation of sulfate A and phosphorus B in black-odorous water adding ecoN-concrete with the Ca(NO 3 ) 2 dosing ratio 1.00%

Fig. 4
Fig.4The evolution of COD A, NH 4 .+ -N B, and turbidity C during the long-term black-odorous water remediation with ecoNconcrete containing 1.00% Ca(NO 3 ) 2

Fig. 5
Fig. 5 SEM image of normal concrete A, C and ecoN-concrete B, D. A, B and C, D respectively present the raw and aged concrete during the long-term experiment

Fig. 6
Fig. 6 Effect of normal concrete (NC) and ecoN-concrete (EC) on the microbial community evolution.A, B Venn graph of microbial community at the days of 10 and 21; C, D bac-