Zinc and cadmium ions are usually found in livestock breeding wastewater, and the mixed ions will have an impact on the biological nitrogen removal. Nitrification performance plays an important role in biological nitrogen removal. In order to investigate the combined effect of zinc and cadmium ions on nitrification performance and to reveal the interactions between zinc and cadmium ions, three concentration ratios of zinc and cadmium ions, as well as 18 different concentration gradients were designed with the direct equipartition ray and the dilution factor method. The effect of pollutants on the nitrification performance of biological nitrogen removal was analyzed by the nonlinear regression equation, and the concentration-addition model was conducted to probe into the relationship between the mixed pollutants and the nitrification performance. The results showed that the effect on nitrification performance increased significantly with the increase of reaction duration and pollutant concentration, which indicated that the effects are concentration-dependent and time-dependent. The concentration-addition model suggested that the interactions between zinc and cadmium ions with different concentration ratios were mainly antagonistic. However, the three concentration ratios of mixed pollutants have different interaction strengths, compared to the zinc and cadmium ions ratio of R1, the ratios of R2 and R3 are more antagonistic and have a greater impact on nitrification performance.
Research Article
Combined effect of zinc and cadmium ions on nitrification performance during the biological nitrogen removal of livestock breeding wastewater
https://doi.org/10.21203/rs.3.rs-3789580/v1
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Zinc ions
Cadmium ions
Nitrification performance
Combined effect
In recent years, with the rapid development of large-scale livestock breeding, heavy metal are widely added in livestock breeding feeds and veterinary drugs. However, the unabsorbed heavy metals are discharged through livestock urine, and the heavy metal pollution increased in livestock breeding wastewater(Feng et al. 2018; Wang et al. 2018). Studies have shown that zinc ions are one of the heavy metals with the highest frequency of exceedance in livestock breeding wastewater, and cadmium ions are detected at higher levels (Liu et al. 2020, 2023; Fu et al. 2023). The existence of zinc and cadmium ions is deleterious to the biological treatment of livestock breeding wastewater (Zhang et al. 2016, 2023b; Song et al. 2018). Consequently, it is imperative to investigate the effects of zinc and cadmium ions on biological treatment of livestock breeding wastewater.
Biological nitrogen removal is a typical approach in wastewater biological treatment. Nitrification performance is the determining factor for the effectiveness of biological nitrogen removal (Chen et al. 2019, 2021). Heavy metal ions are essential trace elements for sustaining microbial growth and play a crucial role in stimulating and enhancing microbial metabolism (Hira et al. 2012; Kimura and Isaka 2014; Chen et al. 2014). However, excessive heavy metals will lead to toxicity in microbial cells and affect the effectiveness of biological nitrogen removal (Zhang et al. 2015a; Tang et al. 2020; Fan et al. 2022). Researches showed that low concentration of zinc ions promoted nitrogen removal, but high concentrations of zinc ion prevented nitrogen removal (Chiang et al. 2012; Zhang et al. 2023a). The biological activity of ammonia oxidizing bacteria and nitrite oxidizing bacteria is improved by low concentrations of zinc ions (10 mg/L) in the wastewater treatment system (Zhang et al. 2017). However, the long-term addition of 10 mg/L zinc ions lowered the nitrogen removal rate (Cheng et al. 2019). Researches showed that when the concentration of cadmium ions was 1 mg/L and 2.5 mg/L, nitrification was inhibited by 30% and 47%, respectively. When the cadmium ion concentration was 10 mg/L, nitrate production was inhibited by 90% (Semerci and Çeçen 2009). It can be seen that zinc and cadmium ions have significant effects on the performance of biological nitrogen removal. At present, most studies on wastewater treatment mainly focused on the effect of single zinc ions and cadmium ions on biological denitrification performance. However, pollutants frequently present in the form of mixtures in wastewater, the effect of mixed pollutants are more complex and variable than those of single pollutants, and mixtures with the prolongation of the action of time, to produce the combined effect may also be different (Man et al. 2022). However, the combined effect and the interaction of zinc-cadmium ions mixtures on the nitrification performance of biological nitrogen removal have seldom been reported. Therefore, it is particularly important to study the combined effect of different concentration ratios of zinc-cadmium ions mixtures on the nitrification performance of biological nitrogen removal in livestock breeding wastewater.
Based on the above analyses, zinc and cadmium ions were selected as typical pollutants in this study, and the effect of zinc and cadmium ions on the biological nitrogen removal was characterized by the amount of nitrate-nitrogen change. Firstly, the direct equipartition ray method (Dou et al. 2011) was used to design the mixtures with different concentration ratios, and the concentration-effect curve of pollutants were fitted with the nonlinear regression equation. Then, the study analyzed the effect of mixed pollutants on nitrification performance during the biological denitrification using the concentration-addition model (Nys et al. 2018; Sousa et al. 2018), and revealed the interaction between mixed pollutants. This study hoped to provide a reference for further research into the mechanisms of the mixed pollutants on biological denitrification.
1.1 Experimental conditions
Nine sequencing batch reactors were used in the experiment. The reactors were operated for 4 cycles each day with anaerobic- aerobic alternation, which were operated in 5 phases per cycle with the help of a microcomputer time-controlled device. The operation parameters were as follows: 20 min of influent, 150 min of aeration, 120 min of mixing, 63 min of settling and 7 min of effluent. The MLSS was about 4000 mg/L. The dissolved oxygen was controlled to be 2.0-4.0 mg/L during the aerobic stage and less than 0.2 mg/L during the anoxic stage, and the pH value was 6.9-7.6.
1.2 Experimental materials and wastewater
The activated sludge of the experiment was taken from the oxidation ditch of a wastewater treatment plant, with light yellow, large flocs and good sedimentation performance. And the experiment wastewater was synthesized to simulate livestock breeding wastewater. The composition of synthetic wastewater were shown in Table 1.
Table 1 Composition of synthetic wastewater
|
Main components |
mg/L |
Trace element |
mg/L |
|
Sodium acetate trihydrate Potassium dihydrogen phosphate Ammonium chloride Sodium bicarbonate Calcium chloride dihydrate Magnesium sulfate heptahydrate Yeast extract Peptone Ethylenediaminetetraacetic acid |
800.0 34.0 92.4 220.0 94.6 94.6 5.0 5.0 10.0 |
Boric acid Sodium molybdate Potassium iodide Cobalt chloride hexahydrate Ferric chloride Zinc sulfate Manganese chloride Cadmium sulfate |
0.150 0.060 0.030 0.150 1.500 0.120 0.120 0.030 |
1.3 Analytical methods
Nitrate nitrogen concentrations was measured by ultraviolet spectrophotometry; The dissolved oxygen and pH in reactors were detected by a dissolved oxygen analyzer (Hach HQ30d). The concentration of the mixed liquor suspended solids was measured by filter paper weighting method.
1.4 Analysis of inhibition effect
When the reactors were stably, different concentration gradients of zinc sulphate and cadmium sulphate were added to reactors. The influent and effluent of the aerobic end were collected at 12-hour intervals, and the change rate of nitrate-nitrogen at the aerobic end represents the inhibition rate of pollutants on nitrification performance during the biological nitrogen removal process. The calculation of the inhibition rate is shown in Equation (1).
Where X is the inhibition rate of different concentrations of zinc ions or cadmium ions on the nitrification performance of biological nitrogen removal; A0 is the concentration of the nitrate-nitrogen at the aerobic stage without zinc or cadmium ions (the control group); Ax is the concentration of the nitrate-nitrogen of the aerobic stage at different concentrations of zinc and cadmium ions.
The concentration-effect data can be fitted the concentration-effect curve with a function, which can be used to analyze the inhibitory effect of different concentrations of zinc ions, cadmium ions and their mixtures. The Biphasic function is usually used to describe the concentration-effect curve, and the expression of the function is shown in Equation (2).
Table 2 Concentration ratios of zinc ions and cadmium ions
|
Ratio |
Percentage of zinc ions |
Percentage of cadmium ions |
|
R1 R2 R3 |
0.902 0.754 0.505 |
0.098 0.246 0.495 |
1.5 Analysis of the mixed pollutant interactions
The concentration-addition model was used to construct the concentration-effect curve to assess the interaction of the mixed pollutants, the concentration-addition model are as follows:
2.1 Effects of zinc and cadmium ions on nitrification performance of biological nitrogen removal
The effects of zinc ions on the nitrification performance is shown in Fig. 2(a). As can be seen from Fig. 2(a), the concentration-effect curves were the J-shaped curves. The results showed that when the concentration of zinc ions was less than 10 mg/L, the nitrification performance was promoted. However, when the concentration of zinc ions was more than 10 mg/L, the nitrification performance was inhibited, and the inhibitory effect gradually increased with the increasing concentration of zinc ions and the action time. Therefore, the inhibitory effect of zinc ions on the nitrification performance of biological nitrogen removal was concentration-dependent and time-dependent.
The inhibitory effect of cadmium ions on the nitrification performance of biological nitrogen removal is shown in Fig. 2b. It can be seen that the concentration-effect curve of cadmium ions on the nitrification performance showed the S-shaped curve. When the concentration of cadmium ions was 0.1~1 mg/L, the inhibition effect was not obvious, which indicated that low concentration of cadmium ions has less inhibition effect on the nitrification performance; When the concentration of cadmium ions reached 40 mg/L, the inhibition rate reached more than 98%, indicating that the high concentration of cadmium ions had a greater inhibition effect on the nitrification performance. It can be seen that the inhibitory effect of cadmium ions on the nitrification performance of biological nitrogen removal is also concentration-dependent and time-dependent.
2.2 Effects of mixtures with different ratios on nitrification performance
The effects of different ratios of zinc-cadmium mixtures on the nitrification performance are shown in Fig. 3. The concentration-effect curve of the mixtures are J-shaped curve when the ratios of zinc ions and cadmium ions was R1 and R2, and S-shaped curve was for R3. The reason may be that zinc ions had a higher proportion at the ratio of R1 and R2, and the effect of the mixtures was similar to that of the zinc ions alone. In addition, the effect of three different mixtures on the nitrification performance gradually increased with the action time, and the inhibitory effect was lower than that of the single zinc irons and cadmium irons.
2.3 Interaction of zinc ions and cadmium ions with different concentration ratios
The concentration-addition model was used to analyze the interaction of zinc ions and cadmium ions during the biological nitrogen removal of livestock breeding wastewater, and the results are shown in Fig. 4-6.
As can be seen from Fig. 4 a-d, when the ratio of zinc ions and cadmium ions was R1 and the reaction time was less than 60 h, the concentration-effect curve predicted by the concentration-addition model were higher than that of the experiment curve. The results showed that the combined effect of zinc ions and cadmium ions was lower than the sum of their individual effect, and the inhibitory effect of mixtures on the nitrification performance was weakened during biological nitrogen removal. Therefore, the inhibitory effect of mixtures was antagonistic when the reaction time was less than 60 h.
When the reaction time was 60 h and 72 h (Fig. 4 e-f), the concentration-effect curve predicted by the concentration-addition model was mainly within the 95% confidence interval of the experiment curve. The results showed that the combined effect of zinc ions and cadmium ions was equal to the sum of their individual effect. The results suggested that the inhibitory effect of the mixtures was antagonistic when the reaction time was 60 h and 72 h.
When the reaction time was 84 h (Fig. 4 e-f), the inhibitory effect of the mixtures was antagonistic during biological nitrogen removal. Maybe the proportion of zinc ions in R1 is much higher than that of cadmium ions, at the beginning of the reaction, zinc ions will dominate the competition for the active sites, reducing the toxicity of cadmium ions and producing antagonistic effects, when the competition for the active sites reaches equilibrium, the additive effect was produced, and with the progress of the reaction, the zinc ions may replace the cadmium ions bound to the active sites and produce antagonistic effects again.
When the ratio of zinc ions and cadmium ions was R2, the interaction of the mixture on the nitrification performance of biological nitrogen removal was shown in Fig. 5. As can be seen from Fig. 5, the predicted effects of concentration-addition model were higher than the experimental effects at different action times, and the difference between the predicted and experimental effects increased with the increase of concentration, indicating that the inhibitory effect of the mixture on the nitrification performance of biological nitrogen removal was mainly antagonistic at different action times, and the antagonistic effect gradually increased with the concentration of the mixture. It is possible that competition for the active sites (-SH) is more intense with the concentration of the mixture increases, and thus the antagonistic effect increases progressively with the duration of action.
As shown in Fig. 6, when the ratio of zinc ions and cadmium ions was R3, the inhibitory effect of the mixture on the nitrification performance of biological nitrogen removal was mainly antagonistic. When the reaction time reached 84 h, the inhibitory effect of the mixture on the nitrification performance was gradually transitioned from antagonistic to additive. The most possible reason is that the metal complexed with the intracellular functional groups as the reaction proceeded, and the toxicity of the complexes is less than that of zinc ions and cadmium ions. The additive effect occurs when the complexation reaction reaches equilibrium, and the chemical equilibrium of the complexation reaction becomes more favorable when the ratio of zinc ions to cadmium ions differs significantly.
It is noteworthy that the effects of the mixtures with three different concentration ratios on the nitrification performance of biological nitrogen removal were mainly antagonistic, which explains the combined effect of mixtures on the nitrification performance was lower than the action of single zinc ions or cadmium ions. This phenomenon may be attributed to the action of extracellular polymeric substances that the effect of mixed pollutants tends to produce antagonism (Wu et al. 2023). The sulfhydryl group of protein in extracellular polymeric substances will act as the active sites to bind the metal ions, leading to the change of the secondary structure of protein in extracellular polymeric substances, which affects the compactness of the extracellular polymeric substances structure (Zhang et al. 2015b). Under polymetallic interaction, different types of heavy metals s will compete for the active sites to reduce the uptake and accumulation of other metals, and resulting in antagonistic effects (Ding et al. 2015). In addition, the separate action of zinc ions and cadmium ions promotes the production and accumulation of reactive oxygen species, while zinc ions will reduce cadmium toxicity by competing with cadmium ions to reduce reactive oxygen species (Wang et al. 2019; Yu et al. 2021).
The analyses showed that the mixture toxicity interactions and the magnitude of these interactions varies, which depend on the concentration and concentration ratio of the mixture and have a certain time-dependent. In addition, the interaction factors of heavy metal mixtures are not only influenced by their chemical properties but also by environmental factors. It has been shown that different pH values and calcium ions concentrations can affect the heavy metal content and change their interactions (Zheng et al. 2023). Therefore, the toxicity mechanism of zinc ions and cadmium ions on the nitrification performance of biological nitrogen removal of aquaculture wastewater needs to be further investigated.
The inhibitory effects of zinc and cadmium ions on the nitrification performance of biological nitrogen removal in livestock breeding wastewater were different, and the concentration-effect curve exhibited J-shaped curve and S-shaped curve, but the inhibitory effects on the nitrification performance of biological nitrogen removal were both concentration-dependent and time-dependent.
During the biological nitrogen removal process in livestock breeding wastewater, with the increase of reaction time and concentration, the effect of different concentration ratios of zinc-cadmium mixtures on the nitrification performance of biological nitrogen removal was gradually enhanced, at the same time, the interaction between zinc and cadmium ions showed antagonistic effect in general, and the inhibitory effects were also dependent on the ratio, concentration and time. However, differences in the strength of antagonism among the three concentration-ratio mixtures. In R1, the proportion of zinc and cadmium ions varies greatly and the antagonistic effect is relatively weak, while in R2 and R3, the proportion of each component varies less and the antagonistic effect is relatively strong. And the combined effect of mixed pollutants was smaller, compared with the inhibitory effect of single zinc ions and cadmium ions on the nitrification performance of biological nitrogen removal.
Author contribution Pengzhan Wang: investigation, data curation, writing—original draft, funding acquisition. Jian Huang: conceptualization, methodology, writing—review and editing. Hua Zhang: writing—review and editing, funding acquisition, supervision. Tao Luo: writing—review and editing, funding acquisition, supervision. Chunhua He: methodology, investigation.
Funding information This work was supported by Anhui Provincial Key Research and Development Project (2023t07010002), National Natural Science Foundation of China （52300022）, and Natural Science Research Project of Colleges of Anhui Province (2022AH010019, KJ2021A0619, 2022AH050236).
Data availability Data will be made available on request.
Ethics approval and consent to participate Not applicable.
Consent for publication All authors have given consent to the publication of the manuscript.
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